脑组织微环境及神经甾体对脐带间充质干细胞分化的影响
|
摘要
神经系统损伤及神经退行性疾病所造成的神经功能缺损一直是困扰人类健康的难题,也是神经科学领域研究的焦点和热点。长期以来的研究表明,神经细胞本身缺乏再生能力。迄今为止,已有的药物治疗只是对症治疗,而不能从根本上解决神经细胞缺失的问题,更无法治愈该类疾病。
干细胞在特定微环境条件下,能够被诱导分化成为多种组织细胞,通过移植给受者参与组织的再生与修复,为临床医学提供了一个新的充满希望的治疗手段,可用于治疗修复一些曾经被人类认为是不可再生的组织器官。干细胞技术的不断发展,为治疗甚至治愈神经损伤及神经退行性疾病提供了可能。目前研究较多的干细胞类型包括胚胎干细胞、神经干细胞、间充质干细胞。
间充质干细胞相对于其他干细胞,如胚胎干细胞和神经干细胞等具有多方面的优势,因此备受关注。多项研究表明,间充质干细胞对神经退行性疾病、神经免疫性疾病、神经系统损伤性疾病和脑血管疾病都有积极的治疗意义。脐带由于具有采集方便,免疫原性低,病毒污染率低,不涉及社会、伦理及法律方面争议等优点,可以作为间充质干细胞的理想来源。
目前,脐带间充质干细胞体外神经分化的方法主要有化学试剂和神经营养因子诱导法,但化学试剂如丁基羟基苯甲醚,二甲亚砜、β-巯基乙醇等或多或少对细胞都会产生细胞毒性；而细胞因子bFGF、EGF、BDNF等由于是大分子蛋白质不易通过血脑屏障,因此限制了它们进一步应用于临床。
神经甾体(neurosteroids)是一类由神经系统的神经元或神经胶质细胞合成、代谢产生的具有神经活性甾类物质的总称,主要包括孕烯醇酮(pregnenolone, PREG)、孕酮(progesterone, PROG)及别孕烯醇酮(allopregnanolone, AP)等。大量研究显示,神经甾体不仅参与调节神经系统的发育并对神经系统的功能产生影响；对神经系统具有营养、保护以及促进神经发生和神经元存活作用；同时,神经甾体作为小分子物质易通过血脑屏障而发挥作用。已有文献报道,孕酮及其代谢物别孕烯醇酮促进干细胞的增殖与分化。
目的：采用组织块法培养人脐带间充质干细胞,通过应用含脑组织的培养液来模拟脑内微环境,考察脐带间充质干细胞是否具有向神经细胞分化的能力。在此基础上,采用高效液相色谱-质谱(HPLC-MS)方法对细胞分化过程中主要神经甾体的合成代谢水平的变化进行研究。随后,探讨其中具有作用潜力的孕酮在模拟脑内微环境中对脐带间充质干细胞定向分化为神经细胞的影响,并进一步探讨其作用机制,以期为临床治疗神经损伤和神经变性病提供新的思路和依据。
方法：
1人脐带间充质干细胞的分离纯化及生物学特性
取足月、健康的顺产胎儿脐带,PBS冲洗,剥除动、静脉血管,分离获得wharton's jelly (华尔通)胶,充分剪碎至约1mm3大小,置于含有10%胎牛血清(FBS),100U/mL青霉素,100U/mL链霉素的DMEM/F12培养基中,在体积分数为5%的CO2培养箱中37℃培养。培养获得人脐带间充质干细胞(hUMSCs),传代、纯化、扩增。倒置显微镜下观察细胞形态变化。取生长状态良好的第3代hUMSCs,用0.25%的胰酶/EDTA消化,制成单细胞悬液,分别加入抗人的HLA-DR-FITC、CD34-PE. CD45-PC5、CD29-FITC和CD105-PE单克隆抗体,流式细胞术检测细胞表面标志物。将生长状态良好的第3代hUMSCs以2×104/孔的密度接种在6孔板内,以DMEM/F12含10%FBS,10-6M地塞米松,50mg/L抗坏血酸,100mg/L1-甲基-3-异丁基-黄嘌呤,100U/ml青霉素和100U/mL链霉素对hUMSCs进行成脂诱导。以DMEM/F12含10%FBS,50mg/L抗坏血酸,10-8M地塞米松,10mmol/Lp-甘油磷酸,100U/mL青霉素和100U/mL链霉素对hUMSCs进行成骨诱导。14d后,采用油红O染色检测脂肪滴,用VonKossa染色检测钙化基质沉淀。
2脑组织微环境对hUMSCs分化的影响
SD雄性大鼠断头取出脑组织,置于冰上,称取湿重,按150g/L加入DMEM/F12培养基,用匀浆器在冰浴中将脑组织匀浆,低温高速离心(5000g,10min)后吸取上清液,得到含脑组织的培养液(脑组织匀浆上清)。将培养的第3代hUMSCs制成单细胞悬液,以1×105/mL的细胞密度接种于24孔板中,制备细胞爬片,孔内预先铺有经多聚赖氨酸处理的盖玻片,待细胞贴壁伸展后加入诱导液(脑组织匀浆上清+100U/mL青霉素+100U/mL链霉素),倒置显微镜下观察细胞形态变化。于培养后的第3d,取出盖玻片进行免疫细胞化学染色鉴定神经干细胞标志物nestin,神经元表面标志物NSE及胶质细胞表面标志物GFAP的表达,并进行甲苯胺兰染色鉴定尼氏体。
3hUMSCs在脑组织微环境分化过程中主要神经甾体及3β-HSD的变化
将培养的生长状态良好的第3代hUMSCs制成单细胞悬液,调整细胞密度以1×105/mL的细胞密度接种于24孔板中,待细胞贴壁伸展后去掉培养基,进行诱导培养。分为对照组(DMEM/F12+10%FBS)和脑组织匀浆上清组。分别于培养的0、1、3、7d收集细胞培养液,1000r·min-1离心5min,收集上清液,用乙酸乙酯/正己烷萃取,以固相萃取法进行纯化,加入内标甲睾酮,样品和标准品与2-硝基-4-三氟甲基苯肼(2-nitro-4-trifluoromethyphenylhydrazine,2NFPH)衍生化,采用高效液相色谱-质谱(HPLC-MS)联用技术测定孕烯醇酮(PREG)、孕酮(PROG)和别孕烯醇酮(AP)的含量。采用免疫细胞化学染色法检测3β-羟基甾醇脱氢酶(3β-HSD)的表达变化。
4孕酮在脑组织微环境中对hUMSCs分化的影响
取生长状态良好的第3代hUMSCs,按1×106/孔密度接种于6孔板。待细胞贴壁伸展后去掉培养基,进行诱导培养。对照组培养基为脑组织匀浆上清,实验组在脑组织匀浆上清中加入不同浓度的PROG,使终浓度分别为0.1μM、1μM、10μM。在倒置显微镜下观察细胞的形态变化。培养3d后,RT-PCR检测细胞NSE表达情况。在RT-PCR得到PROG促进分化的最佳浓度的基础上,进一步应用流式细胞术对分化后细胞NSE、nestin、GFAP的表达进行检测。
5孕酮对hUMSCs分化过程中BDNF水平的影响
取生长状态良好的第3代hUMSCs,1×105/mL的密度种植于24孔板中,待细胞伸展后,弃去培养基,加入诱导培养基。分为①对照组：DMEM/F12+10%FBS;②脑组织匀浆上清组；(DPROG (1μM)+脑组织匀浆上清组。分别于培养后的0、1、3、7d收取培养液,1000r·min-1,离心,取上清,用酶联免疫法测检脑源性神经营养因子(BDNF)的水平。
结果：
1hUMSCs的培养与鉴定
人脐带wharton's jelly胶组织块培养约3-5d后,倒置显微镜下可见组织块间隙散在分布的长条索状纺锤型细胞,2w左右细胞达到80-90%融合,呈放射状或漩涡状分布。经传代培养后细胞增殖迅速,可得到纯化的成纤维样细胞,细胞约4-5d即可达到80%-90%融合。流式细胞仪检测人脐带间充质干细胞高表达CD29、CD44、CD105等间充质干细胞标志,不表达CD34、CD45等造血细胞标志和人类白细胞抗原HLA-DR。以成脂培养基诱导培养14d后,大部分细胞转变为扁平、肥大、内含大量可被油红着色的多角形脂肪细胞；以成骨分化方案诱导培养14d后,细胞逐渐转变为多角形或不规则形,可见VonKossa染色阳性细胞。
2hUMSCs在模拟脑内微环境中向神经细胞分化
hUMSCs在脑组织匀浆上清中培养约12h后,细胞形态即发生变化,细胞质向核收缩,胞体变小,呈不规则形或圆形。培养72h,部分细胞可见两个或多个突起伸出,类似神经细胞,有些细胞呈简单的双极或多极,细胞之间有简单的交联。经免疫细胞化学染色后在镜下可观察到：多数细胞分别呈nestin、NSE及GFAP阳性表达。甲苯胺蓝染色后镜下可观察到胞质中存在着深蓝色颗粒状的尼氏小体。
3hUMSCs在模拟脑内微环境中向神经细胞分化过程中主要神经甾体及3β-HSD的变化
在基础培养基组,PROG浓度低于最低定量限。脑匀浆上清培养组细胞培养上清液中PROG的含量随着培养时间的延长显著升高,0d：1.78±0.50ng/mL；1d：2.88±0.29ng/mL；3d：7.51±0.65ng/mL；7d：10.56±0.65ng/mL,差异显著(P<0.05)。基础培养基中可检测到PREG,随着hUMSCs培养时间的延长,PREG水平未见显著变化(P>0.05)；在脑组织匀浆上清中可检测到PREG,随着hUMSCs培养时间的延长,PREG水平逐渐降低,0d：14.74±0.63ng／mL；ld：12.34±0.56ng／mL；3d：10.62±0.32ng／mL；7d：8.61±0.50ng／mL,差异显著(P<0.05)。基础培养基与脑组织匀浆上清培养组的AP浓度均低于最低定量限。
与对照组相比,hUMSCs在脑匀浆上清中培养的细胞中显示3β-HSD表达,且随着培养时间的增加,表达增强。
4孕酮在体外模拟脑内微环境中对hUMSCs向神经细胞分化的影响
PROG处理后,hUMSCs的形态与单纯脑组织匀浆上清培养相比变化更加明显,神经细胞的特征形态出现的时间更早,并且细胞之间发生明显的交联。
RT-PCR结果显示,与含脑组织匀浆上清培养液培养相比,加入PROG处理后可促进hUMSCs向神经元分化,且1μM为最适宜浓度。流式细胞术进一步检测了1μMPROG处理3d后细胞NSE、nestin、GFAP的表达,结果显示,1μMPROG处理组中NSE表达显著高于对照组,58.40±8.22%vs22.15±1.41%(P<0.05),nestin表达显著低于对照组,24.60±2.86%vs35.07±1.95%(P<0.05),GFAP表达无显著性差异,8.0±1.05%vs7.07±0.64%(P>0.05)。
5孕酮对hUMSCs向神经细胞分化过程时微环境中BDNF水平的影响
与对照组相比,脑匀浆上清培养组与PROG处理组中细胞培养上清液中BDNF的浓度均显著升高(P<0.05),而PROG处理后BDNF的浓度与单纯脑匀浆上清培养相比也有显著升高(P<0.05)。
结论：
1应用组织块贴壁培养法能够从脐带wharton's jelly胶中培养出大量增殖能力较强、具有多向分化能力的成纤维样细胞,其高表达间充质细胞标志,不表达造血细胞标志和人类白细胞抗原,证实了脐带可作为MSCs的重要来源。
2hUMSCs在脑组织微环境下培养,显示神经细胞形态。分化后的细胞经nestin、GFAP及NSE免疫染色和尼氏体染色均表达阳性,提示,hUMSCs在脑组织微环境中分化为神经细胞,其分化方式可能是先分化为神经干细胞再向神经元或神经胶质细胞分化。
3在脑组织微环境培养hUMSCs过程中,PROG随着培养时间的延长,生成显著增多,PREG水平显著降低。说明分化后细胞能够合成、分泌神经甾体,具有功能性神经细胞的特性。同时提示PROG可能在脑组织微环境促进hUMSCs(?)神经分化过程中扮演了角色。
4PROG处理可以提高hUMSCs在脑组织微环境中的神经分化速度,并且促进hUMSCs向神经元方向分化,这对该类干细胞移植用于相关神经系统疾病的治疗具有重要意义。
5脑组织微环境及其中的PROG可显著增加分化中的hUMSCs合成BDNF的能力。对BDNF的调节可能是微环境及PROG促进hUMSCs神经分化的机制之一。
Nerve functional impairment caused by nervous system injury and neurodegenerative diseases has been a problem that troubled the human health. It is also a focus that scientists dedicated to study. For a long time, it is generally believed that the nerve cells of the nervous system lack the ability to regenerate. To date, drug treatment is only symptomatic treatment, but cannot fundamentally solve the lack of nerve cells.
With the development of stem cells technology, it provided a chance to treat nerve injury and neurodegenerative diseases. The stem cells in specific microenvironmental conditions can be induced to differentiate into a variety of tissues and cells. Through transplantation to recipients, stem cells participate in tissue regeneration and repair. It provides a new promising therapeutic means for clinical medicine.
Mesenehymal stem cells(MSCs) seem to be a more promising one regarding their advantages over ESC and other type of adult stem cells. A number of studies have shown, mesenchymal stem cells in neurodegenerative diseases, nervous autoimmune disease, neurological impairment and cerebral vascular disease have a positive therapeutic significance. For with convenient collection, low immunogenicity and viral contamination rate, the umbilical cord can be used as an ideal source of mesenchymal stem cells.
At present, the neural differentiation methods of umbilical cord mesenchymal stem cells in vitro are main chemical molecules and neurotrophic factor induction method. But, chemical molecules such as dimethyl sulfoxide, β-mercaptoethanol and butylated hydroxyanisole are more or less toxic. Neurotrophic factors with large molecular weight, such as FGF and BDNF, do not easily passing the blood-brain barrier and causing untoward side effects. Thus those methods are limited for use in vivo.
Progesterone (PROG) is a hormone secreted by ovaries and placenta. PROG is also synthesized in the brain. PROG is an important hormone and has a variety of beneficial effects. PROG has been shown to improve behavioral and functional recovery and to reduce inflammation, oxidative damage, cerebral edema, and neural cell death. PROG is required for the maintenance and the differentiation of primary hippocampal/cortical/striatal neurons in vitro.
Objective:In the present study, human umbilical cord mesenchymal stem cells (hUMSCs) are derived form the umbilical cords by tissue culture method. hUMSCs was cultured in rat brain tissue extrects to Investigate if hUMSCs could have neural differentiation ability in mimic microenvironme-nts of brain. On this basis, HPLC-MS assay was applied to investigate PROG, PREG and AP levels. Afterwards, we explore the effect of PROG on neuronal differentiation of hUMSCs in mimic microenvironments of brain, urthermore explore its mechanism. The purpose of this paper is to provide new ideas and basis for treatment ervous system injury and neurodegenerative diseases.
Methods:
1. Isolation, culture, and identification of hUMSCs
Human umbilical cords were obtained from full-term caesarian section births and were rinsed twice by phosphate-buffered saline (PBS) to wash off blood. After removal of blood vessels (one vein and two arteries), the Wharton's jelly was stripped carefully and cut into pieces of1mm3, which were then cultured in DMEM/Ham's F12medium (1:1) supplemented with10%FBS plus100U/mL penicillin and streptomycin. The cultures were incubated at37℃with5%CO2. The medium was changed every3days after the initial seeding. When the cultures reached80-90%confluence, the cells were subcultured.
To analyze MSC surface markers, hUMSCs from the third passages were incubated with antibodies against human CD105, CD44, CD29, CD45, CD34and HLA-DR. The cells were then assessed by the FACSCalibur flow cytometer.
Assay of adipogenic and osteogenic differentiation were performed after plating the cells into6-well plates at a density of2x104cells. For adipogenic differentiation,10-6M dexamethasone,100mg/L isobutylmethylxanthine,50mg/L ascorbic acid were added to the growth medium. For osteogenic differentiation,10-8M dexamethasone,50mg/L ascorbic acid and10mmol/L β-glycerophosphate were added to the growth medium. After two weeks of induction, cells were fixed with4%paraformaldehyde and stained with Oil red O to view lipid vacuoles in adipocytes and VonKossa staining to view calcium deposition in osteocytes.
2. Influence of brain tissue extracts on hUMSCs
SD rat brains were obtained immediately following craniotomy and were placed on ice. The brains were measured and homogenized in DMEM/F12(150mg/mL). After homogenization, the samples were incubated on ice for10min. The homogenates were centrifuged twice at5000×g at4℃for15min. The supernatants were filtered with a0.22μm filter and stored at-70℃.
hUMSCs from the third passages were cultured in gelatine-coated24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, fresh medium with brain tissue extracts was added. Phase-contrast microscope was used to record morphology changes. After cultured72h, immunocytochemistry was used to detect the expression of nestin、NSE and GFAP. Toluidin blue staining was carried out for detection of Nissl body.
3. The level changes of main neurosteroid and3β-HSD of hUMSCs cultured in brain tissue extracts
hUMSCs from the third passages were cultured in gelatine-coated24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, change the medium. Control group:(DMEM/F12+10%FBS), test group:brain tissue extracts. The culture media were collected at0,1,3,7d, centrifugate5minutes at1000r·min-1. The supernatant were mixed with methyltesterone(MT). Then, samples were isolated using ethylacetate/hexane and wre further purified by solid phase extraction(SPE). Thereafter, samples were derivatized with2-nitro -4-trfluo-romethylphenylhydrazine(2NFPH) and determinated by high performance liquid chromatography-mass spectrometry(HPLC-MS). Immu-nocytochemistry was used to detect the expression of3β-HSD.
4. Influence of progesterone treatment on hUMSCs cultured in brain tissue extracts
hUMSCs from the third passages were cultured in6-well plates at a density of1×106cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, change the medium. Control group: brain tissue extracts, test group:0.0μM、1μM、10μM progesterone in brain tissue extracts. Phase-contrast microscope was used to record morphology changes. After cultured72h, PT-PCR was carried out to detect the expression of NSE. Flow cytometry method was used to verify the yesukts.
5. Influence of progesterone treatment on BDNF level of hUMSCs cultured in brain tissue extracts
hUMSCs from the third passages were cultured in24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80%confluence, change the medium. The experiment was divided into3groups:①DMEM/F12+10%FBS②brain tissue extracts③PROG(1μM) in brain tissue extracts. The culture media were collected at0,1,3,7d, centrifugate5minutes at1000r·min-1. The supematant was determinated using Enzyme-Linked ImmunoSorbent Assay (ELISA).
6. Data and statistics
Satatistic was performed with SPSS13.0for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA. Significance was considered when P<0.05.
Results:
1. Isolation, culture, and identification of hUMSCs
Three to five days after primary culture, adherent cells with a homogenous fibroblastic morphology came out of fragments of Wharton's jelly. Two weeks after plating, when the cells reached80%-90%confluence, they were detached with0.25%trypsin/EDTA solution and were passaged at a ratio of1:3. After subcultured, the cells proliferate rapidly. The cells can reach80%-90%confluence in4to5days. FACS revealed that hUMSCs expressed putative markers of MSCs, which included CD29, CD44and CD105, but not hematopoietic andendothelial cell markers, such as CD34, CD45. They also did not express human leukocyte antigen HLA-DR. After being exposed to adipogenic differentiation medium for two weeks, most of fibroblastic cells changed into large, flattened appearance with accumulated lipid vacuoles, which stained red with Oil Red O. In osteogenic differentiation medium for two weeks, these cells changed into multi-angular or irregular appearance with calcium in cytoplasm detected by VonKossa.
2. Neuronal differentiation of hUMSCs in brain tissue extracts
After cultured in brain tissue extracts for12h, we observed that some cells had undergone morphological changes. Initially, the cytoplasm retracted towards the nucleus, and then cells bodies became increasingly retractil. After72h incubation, most cells had a simple bipolar or multipolar shape, similar to neuron. A simple cross-linking between cells. At72h, neuron specific enolase (NSE)-positive cells、nestin-positive cells and GFAP-positive cells were observed in the cultures. We observed that deep blue granular Nissl body in cytoplasm after Toluidin biue staining.
3. The changes of main neurosteroids and3β-HSD in neuronal differentiation of hUMSCs
With the increasing time of culture, the level of PROG gradually increased, Od:1.78±0.50ng/mL; Id:2.88±0.29ng/mL;3d:7.51±0.65ng/mL;7d:10.56±0.65ng/mL (P<0.05). The level of PREG gradually decreased, Od:14.74±0.63ng/mL; Id:12.34±0.56ng/mL;3d:10.62±0.32ng/mL;7d:8.61±0.50ng/mL (P<0.05). The level of AP was lower than LLOQ.
Compared with control group, the cells cultured in brain tissue extracts showed expression of3β-HSD. With the increasing time of culture, expression enchanced.
4. The effect of progesterone on neuronal differentiation of hUMSCs in brain tissue extracts
By analyzing the expression of NSE by PT-PCR, we found that PROG could increase the hUMSCs'differentiation into neuron-like cells and1μM of PROG has the best effect on the differentiation. This result was also confirmed by flow cytometry analysis.
5. The effect of progesterone on BDNF level in neuronal differentiation of hUMSCs
Compared with control group, the level of BDNF was significantly increased in brain tissue extracts group and PROG group(P<0.05).
Compared with brain tissue extracts group, the level of BDNF was significantly increased after treatment with PROG (P<0.05).
Conclusion:
1. MSCs derived from human umbilical cord could be easily isolated cultured and passaged in vitro. hUMSCs could differentiate into fat and osteoblast under permissive conditions. HUCMSCs expressed putative markers of MSCs, which included CD29, CD44and CD105, but not hematopoietic andendothelial cell markers, such as CD34, CD45. They also did not express human leukocyte antigen HLA-DR. So, umbilical cord was a rich source of MSCs.
2. Brain tissue microenvironment can promote hUMSCs differentiation into neuron-like cells. The level of PROG and PREG changed during differentiation. PROG production significantly increased and PREG levels reduced. It showed that the cells had mature nerve cell function. The cells could synthesis and secret neurotransmitters.
3. PROG treatment could improve the neural differentiation rate of hUMSCs in brain tissue microenvironment and promot hUMSCs to differentiate into neurons
4. During hUMSCs cultured in brain tissue microenvironment and PROG treatment, the level of BDNF in cell culture medium improved. After PROG treatment, the level of BDNF improved more significantly. It suggested that increase of BDNF level maby one of the mechanisms of neural differentiation of hUMSCs cultured in brain tissue microenvironment. It also maybe one of the mechanisms that PROG promoted neural differentiation.
干细胞在特定微环境条件下,能够被诱导分化成为多种组织细胞,通过移植给受者参与组织的再生与修复,为临床医学提供了一个新的充满希望的治疗手段,可用于治疗修复一些曾经被人类认为是不可再生的组织器官。干细胞技术的不断发展,为治疗甚至治愈神经损伤及神经退行性疾病提供了可能。目前研究较多的干细胞类型包括胚胎干细胞、神经干细胞、间充质干细胞。
间充质干细胞相对于其他干细胞,如胚胎干细胞和神经干细胞等具有多方面的优势,因此备受关注。多项研究表明,间充质干细胞对神经退行性疾病、神经免疫性疾病、神经系统损伤性疾病和脑血管疾病都有积极的治疗意义。脐带由于具有采集方便,免疫原性低,病毒污染率低,不涉及社会、伦理及法律方面争议等优点,可以作为间充质干细胞的理想来源。
目前,脐带间充质干细胞体外神经分化的方法主要有化学试剂和神经营养因子诱导法,但化学试剂如丁基羟基苯甲醚,二甲亚砜、β-巯基乙醇等或多或少对细胞都会产生细胞毒性；而细胞因子bFGF、EGF、BDNF等由于是大分子蛋白质不易通过血脑屏障,因此限制了它们进一步应用于临床。
神经甾体(neurosteroids)是一类由神经系统的神经元或神经胶质细胞合成、代谢产生的具有神经活性甾类物质的总称,主要包括孕烯醇酮(pregnenolone, PREG)、孕酮(progesterone, PROG)及别孕烯醇酮(allopregnanolone, AP)等。大量研究显示,神经甾体不仅参与调节神经系统的发育并对神经系统的功能产生影响；对神经系统具有营养、保护以及促进神经发生和神经元存活作用；同时,神经甾体作为小分子物质易通过血脑屏障而发挥作用。已有文献报道,孕酮及其代谢物别孕烯醇酮促进干细胞的增殖与分化。
目的：采用组织块法培养人脐带间充质干细胞,通过应用含脑组织的培养液来模拟脑内微环境,考察脐带间充质干细胞是否具有向神经细胞分化的能力。在此基础上,采用高效液相色谱-质谱(HPLC-MS)方法对细胞分化过程中主要神经甾体的合成代谢水平的变化进行研究。随后,探讨其中具有作用潜力的孕酮在模拟脑内微环境中对脐带间充质干细胞定向分化为神经细胞的影响,并进一步探讨其作用机制,以期为临床治疗神经损伤和神经变性病提供新的思路和依据。
方法：
1人脐带间充质干细胞的分离纯化及生物学特性
取足月、健康的顺产胎儿脐带,PBS冲洗,剥除动、静脉血管,分离获得wharton's jelly (华尔通)胶,充分剪碎至约1mm3大小,置于含有10%胎牛血清(FBS),100U/mL青霉素,100U/mL链霉素的DMEM/F12培养基中,在体积分数为5%的CO2培养箱中37℃培养。培养获得人脐带间充质干细胞(hUMSCs),传代、纯化、扩增。倒置显微镜下观察细胞形态变化。取生长状态良好的第3代hUMSCs,用0.25%的胰酶/EDTA消化,制成单细胞悬液,分别加入抗人的HLA-DR-FITC、CD34-PE. CD45-PC5、CD29-FITC和CD105-PE单克隆抗体,流式细胞术检测细胞表面标志物。将生长状态良好的第3代hUMSCs以2×104/孔的密度接种在6孔板内,以DMEM/F12含10%FBS,10-6M地塞米松,50mg/L抗坏血酸,100mg/L1-甲基-3-异丁基-黄嘌呤,100U/ml青霉素和100U/mL链霉素对hUMSCs进行成脂诱导。以DMEM/F12含10%FBS,50mg/L抗坏血酸,10-8M地塞米松,10mmol/Lp-甘油磷酸,100U/mL青霉素和100U/mL链霉素对hUMSCs进行成骨诱导。14d后,采用油红O染色检测脂肪滴,用VonKossa染色检测钙化基质沉淀。
2脑组织微环境对hUMSCs分化的影响
SD雄性大鼠断头取出脑组织,置于冰上,称取湿重,按150g/L加入DMEM/F12培养基,用匀浆器在冰浴中将脑组织匀浆,低温高速离心(5000g,10min)后吸取上清液,得到含脑组织的培养液(脑组织匀浆上清)。将培养的第3代hUMSCs制成单细胞悬液,以1×105/mL的细胞密度接种于24孔板中,制备细胞爬片,孔内预先铺有经多聚赖氨酸处理的盖玻片,待细胞贴壁伸展后加入诱导液(脑组织匀浆上清+100U/mL青霉素+100U/mL链霉素),倒置显微镜下观察细胞形态变化。于培养后的第3d,取出盖玻片进行免疫细胞化学染色鉴定神经干细胞标志物nestin,神经元表面标志物NSE及胶质细胞表面标志物GFAP的表达,并进行甲苯胺兰染色鉴定尼氏体。
3hUMSCs在脑组织微环境分化过程中主要神经甾体及3β-HSD的变化
将培养的生长状态良好的第3代hUMSCs制成单细胞悬液,调整细胞密度以1×105/mL的细胞密度接种于24孔板中,待细胞贴壁伸展后去掉培养基,进行诱导培养。分为对照组(DMEM/F12+10%FBS)和脑组织匀浆上清组。分别于培养的0、1、3、7d收集细胞培养液,1000r·min-1离心5min,收集上清液,用乙酸乙酯/正己烷萃取,以固相萃取法进行纯化,加入内标甲睾酮,样品和标准品与2-硝基-4-三氟甲基苯肼(2-nitro-4-trifluoromethyphenylhydrazine,2NFPH)衍生化,采用高效液相色谱-质谱(HPLC-MS)联用技术测定孕烯醇酮(PREG)、孕酮(PROG)和别孕烯醇酮(AP)的含量。采用免疫细胞化学染色法检测3β-羟基甾醇脱氢酶(3β-HSD)的表达变化。
4孕酮在脑组织微环境中对hUMSCs分化的影响
取生长状态良好的第3代hUMSCs,按1×106/孔密度接种于6孔板。待细胞贴壁伸展后去掉培养基,进行诱导培养。对照组培养基为脑组织匀浆上清,实验组在脑组织匀浆上清中加入不同浓度的PROG,使终浓度分别为0.1μM、1μM、10μM。在倒置显微镜下观察细胞的形态变化。培养3d后,RT-PCR检测细胞NSE表达情况。在RT-PCR得到PROG促进分化的最佳浓度的基础上,进一步应用流式细胞术对分化后细胞NSE、nestin、GFAP的表达进行检测。
5孕酮对hUMSCs分化过程中BDNF水平的影响
取生长状态良好的第3代hUMSCs,1×105/mL的密度种植于24孔板中,待细胞伸展后,弃去培养基,加入诱导培养基。分为①对照组：DMEM/F12+10%FBS;②脑组织匀浆上清组；(DPROG (1μM)+脑组织匀浆上清组。分别于培养后的0、1、3、7d收取培养液,1000r·min-1,离心,取上清,用酶联免疫法测检脑源性神经营养因子(BDNF)的水平。
结果：
1hUMSCs的培养与鉴定
人脐带wharton's jelly胶组织块培养约3-5d后,倒置显微镜下可见组织块间隙散在分布的长条索状纺锤型细胞,2w左右细胞达到80-90%融合,呈放射状或漩涡状分布。经传代培养后细胞增殖迅速,可得到纯化的成纤维样细胞,细胞约4-5d即可达到80%-90%融合。流式细胞仪检测人脐带间充质干细胞高表达CD29、CD44、CD105等间充质干细胞标志,不表达CD34、CD45等造血细胞标志和人类白细胞抗原HLA-DR。以成脂培养基诱导培养14d后,大部分细胞转变为扁平、肥大、内含大量可被油红着色的多角形脂肪细胞；以成骨分化方案诱导培养14d后,细胞逐渐转变为多角形或不规则形,可见VonKossa染色阳性细胞。
2hUMSCs在模拟脑内微环境中向神经细胞分化
hUMSCs在脑组织匀浆上清中培养约12h后,细胞形态即发生变化,细胞质向核收缩,胞体变小,呈不规则形或圆形。培养72h,部分细胞可见两个或多个突起伸出,类似神经细胞,有些细胞呈简单的双极或多极,细胞之间有简单的交联。经免疫细胞化学染色后在镜下可观察到：多数细胞分别呈nestin、NSE及GFAP阳性表达。甲苯胺蓝染色后镜下可观察到胞质中存在着深蓝色颗粒状的尼氏小体。
3hUMSCs在模拟脑内微环境中向神经细胞分化过程中主要神经甾体及3β-HSD的变化
在基础培养基组,PROG浓度低于最低定量限。脑匀浆上清培养组细胞培养上清液中PROG的含量随着培养时间的延长显著升高,0d：1.78±0.50ng/mL；1d：2.88±0.29ng/mL；3d：7.51±0.65ng/mL；7d：10.56±0.65ng/mL,差异显著(P<0.05)。基础培养基中可检测到PREG,随着hUMSCs培养时间的延长,PREG水平未见显著变化(P>0.05)；在脑组织匀浆上清中可检测到PREG,随着hUMSCs培养时间的延长,PREG水平逐渐降低,0d：14.74±0.63ng／mL；ld：12.34±0.56ng／mL；3d：10.62±0.32ng／mL；7d：8.61±0.50ng／mL,差异显著(P<0.05)。基础培养基与脑组织匀浆上清培养组的AP浓度均低于最低定量限。
与对照组相比,hUMSCs在脑匀浆上清中培养的细胞中显示3β-HSD表达,且随着培养时间的增加,表达增强。
4孕酮在体外模拟脑内微环境中对hUMSCs向神经细胞分化的影响
PROG处理后,hUMSCs的形态与单纯脑组织匀浆上清培养相比变化更加明显,神经细胞的特征形态出现的时间更早,并且细胞之间发生明显的交联。
RT-PCR结果显示,与含脑组织匀浆上清培养液培养相比,加入PROG处理后可促进hUMSCs向神经元分化,且1μM为最适宜浓度。流式细胞术进一步检测了1μMPROG处理3d后细胞NSE、nestin、GFAP的表达,结果显示,1μMPROG处理组中NSE表达显著高于对照组,58.40±8.22%vs22.15±1.41%(P<0.05),nestin表达显著低于对照组,24.60±2.86%vs35.07±1.95%(P<0.05),GFAP表达无显著性差异,8.0±1.05%vs7.07±0.64%(P>0.05)。
5孕酮对hUMSCs向神经细胞分化过程时微环境中BDNF水平的影响
与对照组相比,脑匀浆上清培养组与PROG处理组中细胞培养上清液中BDNF的浓度均显著升高(P<0.05),而PROG处理后BDNF的浓度与单纯脑匀浆上清培养相比也有显著升高(P<0.05)。
结论：
1应用组织块贴壁培养法能够从脐带wharton's jelly胶中培养出大量增殖能力较强、具有多向分化能力的成纤维样细胞,其高表达间充质细胞标志,不表达造血细胞标志和人类白细胞抗原,证实了脐带可作为MSCs的重要来源。
2hUMSCs在脑组织微环境下培养,显示神经细胞形态。分化后的细胞经nestin、GFAP及NSE免疫染色和尼氏体染色均表达阳性,提示,hUMSCs在脑组织微环境中分化为神经细胞,其分化方式可能是先分化为神经干细胞再向神经元或神经胶质细胞分化。
3在脑组织微环境培养hUMSCs过程中,PROG随着培养时间的延长,生成显著增多,PREG水平显著降低。说明分化后细胞能够合成、分泌神经甾体,具有功能性神经细胞的特性。同时提示PROG可能在脑组织微环境促进hUMSCs(?)神经分化过程中扮演了角色。
4PROG处理可以提高hUMSCs在脑组织微环境中的神经分化速度,并且促进hUMSCs向神经元方向分化,这对该类干细胞移植用于相关神经系统疾病的治疗具有重要意义。
5脑组织微环境及其中的PROG可显著增加分化中的hUMSCs合成BDNF的能力。对BDNF的调节可能是微环境及PROG促进hUMSCs神经分化的机制之一。
Nerve functional impairment caused by nervous system injury and neurodegenerative diseases has been a problem that troubled the human health. It is also a focus that scientists dedicated to study. For a long time, it is generally believed that the nerve cells of the nervous system lack the ability to regenerate. To date, drug treatment is only symptomatic treatment, but cannot fundamentally solve the lack of nerve cells.
With the development of stem cells technology, it provided a chance to treat nerve injury and neurodegenerative diseases. The stem cells in specific microenvironmental conditions can be induced to differentiate into a variety of tissues and cells. Through transplantation to recipients, stem cells participate in tissue regeneration and repair. It provides a new promising therapeutic means for clinical medicine.
Mesenehymal stem cells(MSCs) seem to be a more promising one regarding their advantages over ESC and other type of adult stem cells. A number of studies have shown, mesenchymal stem cells in neurodegenerative diseases, nervous autoimmune disease, neurological impairment and cerebral vascular disease have a positive therapeutic significance. For with convenient collection, low immunogenicity and viral contamination rate, the umbilical cord can be used as an ideal source of mesenchymal stem cells.
At present, the neural differentiation methods of umbilical cord mesenchymal stem cells in vitro are main chemical molecules and neurotrophic factor induction method. But, chemical molecules such as dimethyl sulfoxide, β-mercaptoethanol and butylated hydroxyanisole are more or less toxic. Neurotrophic factors with large molecular weight, such as FGF and BDNF, do not easily passing the blood-brain barrier and causing untoward side effects. Thus those methods are limited for use in vivo.
Progesterone (PROG) is a hormone secreted by ovaries and placenta. PROG is also synthesized in the brain. PROG is an important hormone and has a variety of beneficial effects. PROG has been shown to improve behavioral and functional recovery and to reduce inflammation, oxidative damage, cerebral edema, and neural cell death. PROG is required for the maintenance and the differentiation of primary hippocampal/cortical/striatal neurons in vitro.
Objective:In the present study, human umbilical cord mesenchymal stem cells (hUMSCs) are derived form the umbilical cords by tissue culture method. hUMSCs was cultured in rat brain tissue extrects to Investigate if hUMSCs could have neural differentiation ability in mimic microenvironme-nts of brain. On this basis, HPLC-MS assay was applied to investigate PROG, PREG and AP levels. Afterwards, we explore the effect of PROG on neuronal differentiation of hUMSCs in mimic microenvironments of brain, urthermore explore its mechanism. The purpose of this paper is to provide new ideas and basis for treatment ervous system injury and neurodegenerative diseases.
Methods:
1. Isolation, culture, and identification of hUMSCs
Human umbilical cords were obtained from full-term caesarian section births and were rinsed twice by phosphate-buffered saline (PBS) to wash off blood. After removal of blood vessels (one vein and two arteries), the Wharton's jelly was stripped carefully and cut into pieces of1mm3, which were then cultured in DMEM/Ham's F12medium (1:1) supplemented with10%FBS plus100U/mL penicillin and streptomycin. The cultures were incubated at37℃with5%CO2. The medium was changed every3days after the initial seeding. When the cultures reached80-90%confluence, the cells were subcultured.
To analyze MSC surface markers, hUMSCs from the third passages were incubated with antibodies against human CD105, CD44, CD29, CD45, CD34and HLA-DR. The cells were then assessed by the FACSCalibur flow cytometer.
Assay of adipogenic and osteogenic differentiation were performed after plating the cells into6-well plates at a density of2x104cells. For adipogenic differentiation,10-6M dexamethasone,100mg/L isobutylmethylxanthine,50mg/L ascorbic acid were added to the growth medium. For osteogenic differentiation,10-8M dexamethasone,50mg/L ascorbic acid and10mmol/L β-glycerophosphate were added to the growth medium. After two weeks of induction, cells were fixed with4%paraformaldehyde and stained with Oil red O to view lipid vacuoles in adipocytes and VonKossa staining to view calcium deposition in osteocytes.
2. Influence of brain tissue extracts on hUMSCs
SD rat brains were obtained immediately following craniotomy and were placed on ice. The brains were measured and homogenized in DMEM/F12(150mg/mL). After homogenization, the samples were incubated on ice for10min. The homogenates were centrifuged twice at5000×g at4℃for15min. The supernatants were filtered with a0.22μm filter and stored at-70℃.
hUMSCs from the third passages were cultured in gelatine-coated24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, fresh medium with brain tissue extracts was added. Phase-contrast microscope was used to record morphology changes. After cultured72h, immunocytochemistry was used to detect the expression of nestin、NSE and GFAP. Toluidin blue staining was carried out for detection of Nissl body.
3. The level changes of main neurosteroid and3β-HSD of hUMSCs cultured in brain tissue extracts
hUMSCs from the third passages were cultured in gelatine-coated24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, change the medium. Control group:(DMEM/F12+10%FBS), test group:brain tissue extracts. The culture media were collected at0,1,3,7d, centrifugate5minutes at1000r·min-1. The supernatant were mixed with methyltesterone(MT). Then, samples were isolated using ethylacetate/hexane and wre further purified by solid phase extraction(SPE). Thereafter, samples were derivatized with2-nitro -4-trfluo-romethylphenylhydrazine(2NFPH) and determinated by high performance liquid chromatography-mass spectrometry(HPLC-MS). Immu-nocytochemistry was used to detect the expression of3β-HSD.
4. Influence of progesterone treatment on hUMSCs cultured in brain tissue extracts
hUMSCs from the third passages were cultured in6-well plates at a density of1×106cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, change the medium. Control group: brain tissue extracts, test group:0.0μM、1μM、10μM progesterone in brain tissue extracts. Phase-contrast microscope was used to record morphology changes. After cultured72h, PT-PCR was carried out to detect the expression of NSE. Flow cytometry method was used to verify the yesukts.
5. Influence of progesterone treatment on BDNF level of hUMSCs cultured in brain tissue extracts
hUMSCs from the third passages were cultured in24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80%confluence, change the medium. The experiment was divided into3groups:①DMEM/F12+10%FBS②brain tissue extracts③PROG(1μM) in brain tissue extracts. The culture media were collected at0,1,3,7d, centrifugate5minutes at1000r·min-1. The supematant was determinated using Enzyme-Linked ImmunoSorbent Assay (ELISA).
6. Data and statistics
Satatistic was performed with SPSS13.0for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA. Significance was considered when P<0.05.
Results:
1. Isolation, culture, and identification of hUMSCs
Three to five days after primary culture, adherent cells with a homogenous fibroblastic morphology came out of fragments of Wharton's jelly. Two weeks after plating, when the cells reached80%-90%confluence, they were detached with0.25%trypsin/EDTA solution and were passaged at a ratio of1:3. After subcultured, the cells proliferate rapidly. The cells can reach80%-90%confluence in4to5days. FACS revealed that hUMSCs expressed putative markers of MSCs, which included CD29, CD44and CD105, but not hematopoietic andendothelial cell markers, such as CD34, CD45. They also did not express human leukocyte antigen HLA-DR. After being exposed to adipogenic differentiation medium for two weeks, most of fibroblastic cells changed into large, flattened appearance with accumulated lipid vacuoles, which stained red with Oil Red O. In osteogenic differentiation medium for two weeks, these cells changed into multi-angular or irregular appearance with calcium in cytoplasm detected by VonKossa.
2. Neuronal differentiation of hUMSCs in brain tissue extracts
After cultured in brain tissue extracts for12h, we observed that some cells had undergone morphological changes. Initially, the cytoplasm retracted towards the nucleus, and then cells bodies became increasingly retractil. After72h incubation, most cells had a simple bipolar or multipolar shape, similar to neuron. A simple cross-linking between cells. At72h, neuron specific enolase (NSE)-positive cells、nestin-positive cells and GFAP-positive cells were observed in the cultures. We observed that deep blue granular Nissl body in cytoplasm after Toluidin biue staining.
3. The changes of main neurosteroids and3β-HSD in neuronal differentiation of hUMSCs
With the increasing time of culture, the level of PROG gradually increased, Od:1.78±0.50ng/mL; Id:2.88±0.29ng/mL;3d:7.51±0.65ng/mL;7d:10.56±0.65ng/mL (P<0.05). The level of PREG gradually decreased, Od:14.74±0.63ng/mL; Id:12.34±0.56ng/mL;3d:10.62±0.32ng/mL;7d:8.61±0.50ng/mL (P<0.05). The level of AP was lower than LLOQ.
Compared with control group, the cells cultured in brain tissue extracts showed expression of3β-HSD. With the increasing time of culture, expression enchanced.
4. The effect of progesterone on neuronal differentiation of hUMSCs in brain tissue extracts
By analyzing the expression of NSE by PT-PCR, we found that PROG could increase the hUMSCs'differentiation into neuron-like cells and1μM of PROG has the best effect on the differentiation. This result was also confirmed by flow cytometry analysis.
5. The effect of progesterone on BDNF level in neuronal differentiation of hUMSCs
Compared with control group, the level of BDNF was significantly increased in brain tissue extracts group and PROG group(P<0.05).
Compared with brain tissue extracts group, the level of BDNF was significantly increased after treatment with PROG (P<0.05).
Conclusion:
1. MSCs derived from human umbilical cord could be easily isolated cultured and passaged in vitro. hUMSCs could differentiate into fat and osteoblast under permissive conditions. HUCMSCs expressed putative markers of MSCs, which included CD29, CD44and CD105, but not hematopoietic andendothelial cell markers, such as CD34, CD45. They also did not express human leukocyte antigen HLA-DR. So, umbilical cord was a rich source of MSCs.
2. Brain tissue microenvironment can promote hUMSCs differentiation into neuron-like cells. The level of PROG and PREG changed during differentiation. PROG production significantly increased and PREG levels reduced. It showed that the cells had mature nerve cell function. The cells could synthesis and secret neurotransmitters.
3. PROG treatment could improve the neural differentiation rate of hUMSCs in brain tissue microenvironment and promot hUMSCs to differentiate into neurons
4. During hUMSCs cultured in brain tissue microenvironment and PROG treatment, the level of BDNF in cell culture medium improved. After PROG treatment, the level of BDNF improved more significantly. It suggested that increase of BDNF level maby one of the mechanisms of neural differentiation of hUMSCs cultured in brain tissue microenvironment. It also maybe one of the mechanisms that PROG promoted neural differentiation.
引文
1 Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. Stem Cells,2001,19(3):193-204
2 Friedenstein AJ, Gorskaja JF, Kulagina NN.Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol,1976, 4(5):267-74
3 Guo L, Yin F, Meng H, et al. Differentiation of mesenchymal stem cells into dopaminergic neuron-like cells in virto. Biomed Environ Sci,2005, 18(1):36-42
4 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuron cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113(12):1701-1710
5 Baksh D, Song L, Tuan RS.Adult mesenchymal stem cells characterization differentiation and application in cell and gene therapy. J cell Mol Med, 2004,8(3):301-306
6 Ortiz-Gonzalez XR, Keene CD, Verfaillie CM, et al. Neural induction of adult bone marrow and umbilical cord stem cells. Curr Neurovasc Res, 2004, 1(3):207-13
7 Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol,2000, 16(2):247-56
8 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res,2000, 61(4):364-70
9 Mueller SM, Glowacki J. Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem,2001,82(4):583-590
10 Rao MS, Mattson MP. Stem cells and aging:expanding the possibilities. Mech Ageing Dev,2001,122(7):713-34
11 Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma;in situ and in vitro surveys. Stem Cells,2007,25(2): 319-331
12 Sarugaser R, Lickorish D, Baksh D, et al. Human Umbilical Cord Perivascular (HUCPV) Cells:A Source of Mesenchymal Progenitors. Stem Cells,2005,23(2):220-229
13 Lee KD. Applications of mesenchymal stem cells:an updated review. Chang Gung Med J,2008,31(3):228-236
14 Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells,2006,24(4):1030-1041
15 Li Y, McIntosh K, Chen J, et al. Allogeneic bone marrow stromal cells promote glial-axonal remodeling without immunologic sensitization after stroke in rats. Exp Neurol,2006,198(2):313-325
16 Spaggiari GM, Capobianco A, Abdelrazik H, et al. Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production:role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood,2008,111(3):1327-1333
17迟志华,张洹.脐血间充质干细胞研究进展.国际生物医学工程杂志,2006,29(1)：29-34
18 Bieback K, Kern S, Kluter H, et al. Critical parameters for the isolation of mesenchymal stem cell from umbilical cord blood. Stem cells,2004,22(4): 625-634
19 McElreavey KD, Irvine AI, Ennis KT, et al. Isolation,culture and characterization of fibroblast-like cells derived from the Wharton's jelly portionof human umbilical cord. Biochem Soc Trans,1991,19(1):29(s)
20 Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchyal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003,21(1):105-110
21 Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular(HUCPV) cells:a source of mesenchymal progenitors. Stem Cell,2005,23(2):220-229
22 Guo L, Yin F, Meng H, et al. Differentiation of mesenchymal stem cells into dopaminergic neuron-like cells in virto. Biomed Environ Sci,2005, 18(1):36-42
23 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuron cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113(12):1701-1710
24 Baksh D, Song L, Tuan RS.Adult mesenchymal stem cells characterization differentiation and application in cell and gene therapy. J cell Mol Med, 2004,8(3):301-306
25 Jones BJ, McTaggart SJ. Immunosuppression by mesenchymal stromal cells:from culture to clinic. Exp Hematol,2008,36(6):733-741
1 Ostenfeld T, Svendsen CN. Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells.2004,22(5):798-811
2邓方阁,张秀英,王心蕊,等.体外模拟心肌微环境中人骨髓间充质干细胞向心肌细胞的分化.吉林大学学报(医学版),2007,33(2)：257-259
3王显振,蔡文清,宋永周,等.膀胱匀浆上清在大鼠骨髓间充质干细胞诱导分化为平滑肌样细胞中的作用.中国组织工程研究与临床康复,2008,12(8)：1422-1425
4 Jang YY, Collector MI, Baylin SB, et al. Hematopoictic stem cells convert into liver cells within days without fusion. Nat Cell Biol,2004,6(6): 532-539
5 Choi KS, Shin JS, Lee JJ, et al. In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract. Biochem Biophys Res Commun,2005,330(4):1299-305
6 Jin K MX, Batteur S, Sun Y, et al. Induction of neuronal markers in bone marrow cells:differential effects of growth factors and patterns of intracellular expression. Exp Neurol,2003,184:78-89
7 Jiang YH, Scutt G, Goltzman D, et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci,2003, 100(1):1854-1860
8 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113(12):1701-1710
9 Belegu V, Oudega M, Gary DS, et al. Restoring function after spinal cord injury:promoting sponatneous regneration with stem cells and activity-based therapies. Neurosurg Clin N Am,2007,18(1):143-168
10 Kelly S, Bliss TM, Shah AK, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci,2004,101(32):11839-11844
11 Zhang SC, Wernig M, Duncan ID, et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol,2001,19(12):1129-1133
12 Schuldiner M, Eiges R, Eden A, et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res,2001,913(2):201-205
13 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113:1701-1710
14 Munoz-Elias G, Woodbury D, Black IB. Marrow stromal cells, mitosis, and neuronal differentiation:stem cell and precursor functions. Stem Cells, 2003,21(4):437-448
15 Lu D, Mahmood A, Wang L, et al. Adult bone marrow strmal cells administration intravenously to rats after traumatic brain into brain and improve neurological outcome. Neuroreport,2001,12:559-563
16 Gurgo RD, Bedi KS, Nurcombe V. Current concepts in central nervous system regeneration. J Clin Neurosci,2002,9(6):613-617
17 Isacson O. The production and use of cells as therapeutic agents in neurodegenerative diseases. Lancet Neurol,2003,2:417-424
18 Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood,2002,99(10):3838-3843
19 Le Blanc K, ammik C, Rosendahl K, et al. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol,2003,31(10):890-896
20 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke,2001,34(4):1005-1011
21 Chen J, Li Y, Katakowski M, et al. Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat. J Neurosci Res,2003,73(6):778-86
22 Pavlichenko N, Sokolova I, Vijde S, et al. Mesenchymal stem cells transplantation could be beneficial for treatment of experimental ischemic stroke in rats. Brain Res,2008,1233:203-13
23 Park HJ, Lee PH, Bang OY, et al. Mesenchymal stem cells therapy exerts neuroprotection in a progressive animal model of Parkinson's disease. J Neurochem,2008,107(1):141-51
24 Slavin S, Kurkalli BG, Karussis D. The potential use of adult stem cells for the treatment of multiple sclerosis and other neurodegenerative disorders. Clin Neurol Neurosurg,2008,110(9):943-946
25 Wang JM, Patrick B, Johnston, et al.The Neurosteroid Allopregnanolone Promotes Proliferation of Rodent and Human Neural Progenitor Cells and Regulates Cell-Cycle Gene and Protein Expression. The Journal of Neuroscience,2005,25(19):4706-4718
26 Crigler L, Robey RC, Asawachaicharn A, et al. Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Exp Neurol,2006,198 (1):54-64
27 Suzuki M, Wright LS, Marwah P, et al. Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex. Proc Natl Acad Sci,2004,101(9):3202-7
28 Huang W, Miao ZN, Chen L, et al. Brain-derived neurotrophic factor, ciliary neurotrophic factory and their combination for in vitro differentiation of human umbilical blood-derived mesenchymal stem cells into nerve-like cells. Journal of Clinical Rehabilitative Tissue Engineering Research,2010,14(19):3606-3610
29 Suzuki M, DNelson A, Joshua B, et al. Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex. European Journal of Neuroscience,2006,24(3):645-653
30 Shetty A K. Progenitor cells from the CA3 region of the embryonic day 19 rat hippocampus generate region-specific neuronal phenotypes in vitro. Hippocampus,2004,128(4):807-817
31 Whittemore S R, Morassutti D J, Walters W M, et al. Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell population. Exp Cell Res,1999, 252(1):75-95
1 Ibanez C, Guennoun R, Liere P, et al. Developmental expression of genes involved in neurosteroidogenesis:3β-hydroxysteroid-dehydrogenase/Δ5-Δ4-isomerase in the rat brain. Endocrinology,2003,144:2902-2911
2 Zwain IH, Yen SC, et al. Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain. Endocrinology,1999,140:385-3843
3 Charalampopoulos I, Remboutsika E, Margioris AN, et al. Neurosteroids as modulators of neurogenesis and neuronal survival. Trends Endocrinol Metab,2008,19:300-307
4 Hirst JJ, Palliser HK, Yates DM, et al. Neurosteroids in the fetus and neonate:potential protective role in compromised pregnancies. Neurochem Int,2008,52:602-610
5 Keller EA, Zamparini A, Borodinsky LN, et al. Role of allopregnanolone on cerebellar granule cells neurogenesis. Brain Res Dev Brain Res,2004, 153:13-17
6 Lockhart EM, Warner DS, Pearlstein RD, et al. Allopregnanolone attenuates N-methyl-D-aspartate-induced excitotoxicity and apoptosis in the human NT2 cell line in culture. Neurosci Lett,2002,328:33-36
7 Mellon SH. Neurosteroid regulation of central nervous system development. Pharmacol Ther,2007,116:107-124
8 Sakamoto H, Ukena K, Tsutsui K, et al. Effects of progesterone synthesized de novo in the developing purkinje cell on its dendritic growth and synaptogenesis. J Neurosci,2001,21:6221-6232
9 Schumacher M, Guennoun R, Stein DG, et al. Progesterone:therapeutic opportunities for neuroprotection and myelin repair. Pharmacol Ther,2007, 116:77-106
10 Wagner CK. Progesterone receptors and neural development:a gap between bench and bedside? Endocrinology,2008,149:2743-2749
11 Wang JM, Johnston PB, Ball BG, et al. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J Neurosci,2005,25: 4706-4718
12于洋,薛改,吴红海,等.Aβ25-35对大鼠皮质神经元神经甾体水平的影响.中国药理学通报,2010,26(6)：241-244
13 Liere P, Akwa Y, Weill-Engerer S, et al. Validation of an analytical procedure to measure trace amount of neurosteroids in rat brain tissue by gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl, 2000,739(2):301-312
14郭再玉,姜晓丹,徐如祥,等.骨髓基质细胞源性神经干细胞体外分化及电生理特性的研究.中华神经医学杂志,2005,4(6)：545-550
15杨慧民,张苏明,郑荣远.骨髓间充质干细胞诱导分化为神经样细胞的离子通道：是否有电生理特性?中国组织工程研究与临床康复, 2009,13(40)：7881-7884
16陈剑荣,徐如祥,姜晓丹,等.兔骨髓源性神经干细胞分化为成熟神经元样细胞的特征研究.中国临床康复,2004,8(16)：30242-3043
17 Baulieu EE, Robel P, Schumacher M. Neurosteroids:A New Regulatory Function in the Nervous System. J Soc Biol,1999,193(3):285-92
18 Guennoun R, Benmessahel Y, Delespierre B, et al. Progesterone stimulates Krox-20 gene expression in Schwann cells. Brain Res Mol Brain Res, 2001,90:75-82
19 Chan JR, Rodriguez-Waitkus PM, Ng BK, et al. Progesterone synthesized by Schwann cells during myelin formation regulates neuronal gene expression. Mol Biol Cell,2000,11:2283-2295
20 Ghoumari AM, banez C, El-Etr M, et al. Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum. J Neurochem,2003,86:848-859
21 Ghoumari AM, Baulieu EE, Schumacher M. Progesterone increases oligodendroglial cell proliferation in rat cerebellar slice cultures. Neuroscience,2005,135:47-58
22 Gago N, Akwa Y, Sananes N, et al. Progesterone and the oligodendroglial lineage:stage-dependentbiosynthesis and metabolism. Glia,2001,36: 295-308
23 Gago N, El-Etr M, Sananes N, et al.3alpha,5alpha-Tetrahydro-progesterone (allopregnanolone) and gamma-aminobutyric acid:autocrine /paracrine interactions in the control of neonatal PSA-NCAM+progenitor proliferation. J Neurosci Res,2004,78:770-783
24 De Nicola AF, Gonzalez SL, Labombarda F, et al. Progesterone treatment of spinal cord injury:Effects on receptors, neurotrophins, and myelination. J Mol Neurosci,2006,28:3-15
25 Tsutsui K, Mellon SH. Neurosteroids in the Brain Neuron:Biosynthesis, Action and MEdicinal Impact on Neurodegenerative Disease. Cent Nerv Syst Agents in Med Chem,2006,6:73-82
1 VanLandingham JW, Cutler SM, Virmani S, et al. The enantiomer of progesterone acts as a molecular neuroprotectant after traumatic brain injury. Neuropharmacology,2006,51(6):1078-1085
2 Cutler SM, VanLandingham JW, Murphy AZ, et al. Slow-release and injected progesterone treatments enhance acute recovery after traumatic brain injury. Pharmacol Biochem Behav,2006,84(3):420-428
3 Guo Q, Sayeed I, Baronne LM, et al. Progesterone administration modulates AQP4 expression and edema after traumatic brain injury in male rats. Exp Neurol,2006,198(2):469-478
4 Brewer GJ, Torricelli JR, Evege EK, et al. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serumfree medium combination. J Neurosci Res,1993,35(5):567-576
5 Wang JM, Johnston PB, Ball BG, et al. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J Neurosci,2005,25(19): 4706-4718
6 Gilmer LK, Roberts KN, Scheff SW. Efficacy of progesterone following a moderate unilateral cortical contusion injury. J Neurotrauma,2008,25(6): 593-602
7 Ogata T, Nakamura Y, Tsuji K, et al. Steroid hormones protect spinal cord neurons from glutamate toxicity. Neuroscience,1993,55:445-449
8 Gonzalez Deniselle MC, Lopez-Costa JJ, Saavedra JP, et al. Progesterone neuroprotection in the Wobbler mouse,a genetic model of spinal cord motor neuron disease. Neurobiol Dis,2002,11:457-468
9 Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science,2000,290: 1775-1779
10 Einstein O, Ben-Hur T. The changing face of neural stem cell therapy in neurologic diseases. Alch Neurol,2008,65(4):452-456
11 Chen J, Chop M, Li Y. Neuroprotective effects of progesterone after transient middle cerebral aetery occlusion in rat. J Neurol Sci,1999, 171(1):24-30
12 Roof RL, Duvdevani R, Stein DG. Gender influences outcome of brain injury:progesterone plays s protective role. Brain Res,1993,607(1-2): 333-336
13 Asbury ET, Fritts ME, Horton JE, et al. Progesterone facilitates the acquisition of avoidance learning and protests against subcotical neuronal death following prefrontal cortex ablation in the rat. Behav Brain Res, 1998,97(1-2):99-106
14 Morali G, Letechipia-Vallejo G, Lopez-Loeza E, et al. Post-ischemic administration of progesterone in rats exerts neuroprotective effects on the hippocampus. Neurosci Lett,2005,382(3):268-290
15 Thoman AJ, Nockels RP, Pan HQ, et al. Progesterone is neuroprotective after acute experimental spinal cord trauma in rats. Spine,1999,24(20): 2134-2138
16 Sakamoto H, Ukena K, Tsutsui K. Effects of progesterone synthesized de novo in the developing Purkinje cell on its dendritic growth and synaptogenesis. J Neurosci,2001,21(16):6221-6232
17 Sakamoto H, Ukena K, Tsutsui K. Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats. Neurosci Lett,2002,322(2):111-115
18 Gibson CL, Gray LJ, Bath PM, et al. Progesterone for the treatment of experimental brain injury:a systematic review. Brain,2008,131:318-328
19 Singh M, Sumien N, Kyser C, et al. Estrogens and progesterone as neuron protectants:what animal models teach us. Front Biosci,2008,13: 1083-9
20 Stein DG. Progesterone exerts neuroprotective effects after brain injury. Brain Res Rev,2008,57:386-397
21 Stein DG, Wright DW, Kellermann AL. Does progesterone have neuroprotective properties? Ann Emerg Med,2008,51:164-172
22 Wright DW, Kellermann AL, Hertzberg VS, et al. ProTECT:a randomized clinical trial of progesterone for acutetraumatic brain injury. Ann Emerg Med,2007,49:391-402
23 Xiao G, Wei J, Yan W, et al. Improved outcomes from the administration of progesterone for patients with acute severe traumatic brain injury:a randomized controlled trial. Crit Care,2008,12:R61
24 Lopez-Gonzalez R, Camacho-Arroyo I, Velasco I, et al. Progesterone and 17b-Estradiol Increase Differentiation of Mouse Embryonic Stem Cells to Motor Neurons. IUBMB Life,2011,63(10):930-939
25 Diaz NF, Diaz-Martinez NE, Velasco I, et al. Progesterone increases dopamine neurone number indifferentiat-Ing mouse embryonic stem cells. J Neuroendocrinol,2009,21:730-736
1陈平安,董晓先,冷水龙.黄体酮诱导骨髓间充质干细胞分化为神经元样细胞实验研究.实用医学杂志,2004,20(6)：606-607
2 De Nicola AF, Gonzalez SL, Labombarda F, et al. Progesterone treatment of spinal cord injury:Effects on receptors, neurotrophins, and myelination. Journal of Molelcular Neuroscience,2006,28:3-15
3 Gonzalez SL, Labombarda F, Gonzalez Deniselle MC, et al. Progester-one up-regulates neuronal brainderived neurotrophic factor expression in the injured spinal cord. Neuroscience,2004,125:605-614
4 Gonzalez SL, Labombarda F, Gonzalez Deniselle MC, et al. Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain derived neurotrophic factor in motoneurons. J Steroid Biochem Mol Biol, 2005,94:143-149
5 Gonzalez Deniselle MC, Garay L, Gonzalez S, et al. Progesterone modulates brain-derived neurotrophic factor and choline acetyltransferase in degenerating Wobbler motoneurons. Experimental Neurology,2007, 203:406-414
6 Kaur P, Jodhka PK, Underwood WA, et al. Progesterone increases brain-derived neuroptrophic factor expression and protects against glutamate toxicity in a mitogen-activated protein kinase and phosphoinositide-3 kinase dependent manner in cerebral cortical explants. Journal of Neuroscience Research,2007,85:2441-2449.
7 Cai W, Zhu Y, Furuya K, et al. Two different molecular mechanisms underlying progesterone neuroprotection against ischemic brain damage. Neurosei,2001,21(17):6706-6717
8王海珍,刘呜,赵萃瑜,等.BDNF的克隆及pTAT/HA-BDNF重组表达载体的构建.神经损伤与功能重建,2008,3(9)：304-306
9 Shumsky JS, Tobias CA, Tumolo M, et al. Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT-3 into a spinal cord injury site is associated with limited recovery offunction. Exp Neurol, 2003,1(184):114-30
10 Poo MM. Neurotrophins as synaptic modulators. Nat Rev Neurosci,2001, 2:24-32
11 Lim JY, Park SI, Oh JH, et al. Brain-derived neu-rotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways. J Neurosci Res,2008,86(10):2168-78
12 Chen ZY, Bath K, McEwen B, et al. Impact of genetic variant BDNF on brain structure and function. Novartis Found Symp,2008,28(9):180-188
13 Spedding M, Gressens P. Neurotrophins and cytokines in neuronal plasticity. Novartis Found Symp,2008,28(9):222-233
14 Johnson MA, Weick JP, Pearce RA, et al. Functional neural development from human embryonic stem cells:accelerated synaptic activity via astrocyte cocul-ture. J Neurosci,2007,27(12):3069-77
15 Alfimova MV, Golimbet VE, Barkhatova AN, et al. The role of genotype-environment interactions in the development of symptoms of anxiety and depression related to the disease burden for family. Zh Nevrol Psikhiatr Im SS Korsakova,2009,109(12):50-54
16 Jakeman LB, Wei P, Guan Z, et al.Brain derived neurotrophic factor timulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury. Exp Neurol,2001,154:170-184
17 Coumans JV, Lin TT, Dai HN, et al. Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins. J Neurosci,2001,21: 9334-9344
18 Chaldakov GN, Tonchev AB, Aloe L. NGF and BDNF:from nerves to adipose tissue, from neurokines to metabokines. Riv Psichiatr,2009,44(2): 79-87
19 Tapia-Aranciba L, Rage F, Givalois L, et al. Physiology of BDNF:focus on hypothalamic function. Front Neuroendocrinol,2004,25(2):77-107
20许期年,金钧,苗宗宁.脑源性神经营养因子促进骨髓间充质干细胞分化为神经元样细胞的实验研究.苏州大学学报(医学版),2007,27(4)：520-523
21 Huang EJ, Reichardt LF. Trk receptors:roles in neuronal signal transduction. Annu Rev Biochem,2003,72:609-642
22 Gomes C, Smith SC, Youssef MN, et al. RNA polymerase 1 driven transcription as a mediator of BDNF-induced neurite out growth. J Biol Chem,2011,286(6):4357-4363
23 Lee JG, Shin BS, You YS, et al. Decreased serum brain-derived neurotrophic factor levels in elderly korean with dementia. Psychiatry Investig,2009,6(4):299-305
24 Castren E, Rantamaki T. Role of brain-derived neurotrophic factor in the aetiology of depression:implications for pharmacological treatment. CNS Drugs,2010,24(1):1-7
25 Chen XG, Li Y, Wang L, et al. Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology,2002, 22(4):275-9
26 Gonzalez SL, Labombarda F, Gonzalez Deniselle MC, et al. Progesterone up-regulates neuronal brain-derived neurotrophic factor expression in the injured spinal cord. Neuroscience,2004,125:605-614
27 Gonzalez SL, Labombarda F, Deniselle MC, et al. Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain-derived neurotrophic factor in motoneurons. J Steroid Biochem Mol Biol,2005,94:143-149
28 Scharfman HE, Maclusky NJ. Similarities between actions of estrogen and BDNF in the hippocampus:coincidence or clue? Trends Neurosci,2005, 28:79-85
29 Li Y, Chen J, Chen XG, Wang L, et al. Human marrow stromal cell therapy for stroke in rat:neurotrophins and functional recovery. Neurology,2002, 59(4):515-523
30 Song S, Kamath S, Mosquera D, et al. Expression of brain natriuretic peptide by human bone marrow stromal cells. Exp Neurol,2004,185(1): 191-197
31 Zhang XM, Basch RS. Mmortalized multipotential mesenchymal cells and the hematopoietic microenvironment. J Hematother Stem Cell Res,2001, 10:125-140
32 Sensebe L, Deschaseaux M, Li J, et al. The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment. Stem Cells,1997,15:133-143
1 Reynolds BA, Weiss S. Generation of neurons and astrocytes from iso2 lated cell of the adult mammalian central nervous system. Science,1992, 255 (5052):1707-1710
2 Richards LJ, Kilpatrick TJ, Bartlett PF. De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci,1992, 89(18):8591-8595
3 Kelly CM, Zitetlow R, Dunnett SB, et al. The effects of various concentrations of FGE-2 on the proliferation and neuronal yield of murine embryonic neural precursor cells in vitro.Cell Transplant,2003,12(3): 215-223
4姚忠祥,孙俞,刘建军,等.碱性成纤维细胞生长因子促进脑室管膜前下区细胞增殖的实验研究.中国临床康复,2004,8(4)：664-665
5 Fiore M, Aca V, Amendola T, et al. Brain NGF and EGF adminis tration improves passive avoidance response and stimulates brain precursor cells in aged male mice. Phys iol Behav,2002,77:437-443
6余剑,曾进胜,盛文利,等.表皮生长因子对脑梗死大鼠脑室管膜下区神经干细胞迁徙和分化的影响.中华医学杂志,2004,84(23)：1965-1967
7 Sofroniew MV, Howe CL, Mobley WC. Never growth factor signaling, neuroprotection, and neural repair. Ann Rev Neurosci,2001,24: 1217-1281
8 Benoit BO, Savarese T, Joly M, et al. Neurotrophin channeling of neural progenitor cell differentiation. J Neurobiol,2001,46(4):265-280
9 Frielingsdorf H, Simpson DR, Thal LJ, et al. Nerve growth factor promotes survival of new neurons in the adult hippo-campus. Neurobiol Dis,2007,26(1):47-55
10 Mondal D, Pradhan L, LaRussa VF. Signal transduction pathways involved in the lineage-differentiation of NSCs:can the knowledge gained from blood be used in the brain? Cancer Invest,2004,22(6):925-943
11 Pagano SF, Impagnatiello F, Girelli M, et al. Isolation and characterization of neural stem cells from the adult human olfactory bulb. Stem cells,2000, 18(4):295-299
12 Molne M, Studer L, Tabar V, et al. Early cortical precursors do not undergo LIF-mediated astrocytic differentiation. J Neurosci Res,2000,59(3): 301-11
13 Katic M, Kahn CR. The role of insulin and insulin-like growth factor-1 signaling in longevity. Cell Mol Life Sci,2005,62(2):3420-3431
14 Juul A. IGF-I measurements.Epidemiological and clinical aspects. Front Horm Res,2005,33(1):45-67
15 Ercole AJ, Dai Z, Xing Y, et al. Brain growth redardation due to the expression of human insulin-like growth factor binding protein-1 in transgenic mice:an in vivo model for the analysis of IGF function in the brain. Brain Res Dev Brain Res,1994,82(2):213-2221
16 Aberg MA, Aberg ND, Hedbackerh, et al. Peripheral infusion of IGF-1 selectively induces neurogenesis in the adult rat rappocampus. J Neurosci, 2000,20(8):2896-930
17 Morishita E, Masuda S, Nagao M, et al. Erythropoietin recaptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamateinduced neuronal death. Neuroscience,1997,76(1):105-116
18 Sakanaka M, Wen TC, Matsuda S, et al. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci,1998,95(8):4635-4640
19 Wang L, Zhang ZG, Wang Y, et al. Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats. Stroke,2004,35(7):1732-1737
20 Lee SM, Nguyen TH, Park MH, et al. EPO receptor-mediated ERK kinase and NF-kappaB activation in erythropoietin promoted differentiation of astrocytes. Biochem Biophys Res Commun,2004,320(4):1087-1095
21张引成,王贵和,张政华.神经节苷酯M1介导神经生长因子对运动神经元再生的影响.西安医科大学学报,2001,22(5)：459-462
22 Rusnati M, Urbinati C, Tanghetti E, et al. Cell membrance GM1 ganglioside is a functional coreceptor for fibroblast growth factor 2. Proc Natl Acad Sci,2002,99(7):4307-72
23赵新利,周国胜,张新中等.GM-1对离体大鼠神经干细胞增殖和分化的影响.医学信息手术学分册,2006,19(2)：57-59
24 EmaM, Faloon P, ZhangWJ, et al. Combinatorial effects of Flk1 and Tall on vascular and hematopoietic development in the mouse. Genes Dev, 2003,17(3):380-393
25 Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. Comp Neurol,2000,425(4):479-494
26 Schwarz Q, Gu C, Fujisawa H, et al. Vascular endothelial growth factor controls neuronalmigration and cooperates with Sema3 A to pattern distinct compartments of the facial nerve. Genes Dev,2004,18(22):2822-2834
27 Sondell M, Lundborg G, Kanje M. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing csell survival and Schwann cell p roli-feration in the peripheral nervous system. Neuroscience,1999,19(14):5731-5740
28 Sun Y, J in K, Childs JT, et al. Vascular endothelial growth factor-B (VEGFB) stimulates neurogenesis:evidence from knockoutmice and growth factor administration. Dev Biol,2006,289(2):329-335
29 Le Bras B, Barallobre MJ, Homman-Ludiye J, et al. VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain. Nat Neurosci,2006,9(3):340-348
30 Maurer MH, Tripps WKC, Feldmann RE, et al. Expression of vascular endothelial growth factor and its receptor in rat neural stem cells. Neurosci Lett,2003,344(3):165-168
31 Meng H, Zhang Z, Zhang R, et al. Biphasic effects of exogenous VEGF on VEGF expression of adult neural progenitors. Neurosci Lett,2006,393 (23):97-101
32 Kim BK, Kim SE, Shim JH, et al. Neurogenic effect of vascular endothelial growth factor during germ layer formation of human embryonic stem cells. FEBS Lett,2006,580(25):5869-5874
33 Jakeman LB, Wei P, Guan Z, et al. Brain derived neurotrophic factor timulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury. Exp Neurol,2001,154:170-184
34 Chaldakov GN, Tonchev AB, Aloe L. NGF and BDNF:from nerves to adipose tissue, from neurokines to metabokines. Riv Psichiatr,2009,44(2): 79-87
35 Alfimova MV, Golimbet VE Barkhatova AN, et al. The role of genotype-environment interactions in the development of symptoms of anxiety and depression related to the disease burden for family. Zh Nevrol Psikhiatr Im SS Korsakova,2009,109(12):50-54
36 Lee JG, Shin BS, You YS, et al. Decreased serum brain-derived neurotrophic factor levels in elderly korean with dementia. Psychiatry Investig,2009,6(4):299-305
37 Castren E, Rantamaki T. Role of brain-derived neurotrophic factor in the aetiology of depression:implications for pharmacological treatment. CNS Drugs,2010,24(1):1-7
38余震,陈莉芬,唐玲,等.腺病毒介导的脑源性神经营养因子对大鼠脑出血后内源性神经干细胞分化的影响.第三军医大学学报,2010,32(15)：1628-1631
39李听松,蒋莉,陈恒胜,等.脑源性神经营养因子促进海马神经干细胞的存活、增殖及向神经元分化.细胞生物学杂志,2008,30：387-391
40王景周,高唱.神经递质与神经干细胞的增殖与分化.国外医学：脑血管疾病分册,2003,11(3)：121-123
41 Watkins JC. L-glutamate as a central neurotransmitter:looking back. Biochem Soc Trans,2000,28(4):297-309
42 Mattson MP, Kater SB. Calcium regulation of neurite elon-gation and growth cone motility. J Neurosci,1987,7(12):4034-4043
43 Simon DK, Prusky GT, O'Leary DD, et al. N-methyl-D-aspartate receptor antagonists disrupt the formation of a mammalian neural map. Proc Natl Acad Sci,1992,89(22):10593-10597
44 Rossi DJ, Slater NT. The developmental onset of NMD A receptorchannel activity during neuronal migration. Neuropharmacology,1993,32(11): 1239-1248
45 Rakic P, Komuro H. The role of receptor/channel activity in neuronal cell migration. J Neurobiol,1995,26(3):299-315
46 Suzuki M, Aaron D, Joshua B, et al. Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex. Eur J Neurosci,2006,24(3):645-653
47 Poulsen FR, Blaabjerg M, Montero M, et al. Glutamate receptor antagonists and growth factors modulate dentate granule cell neurogenesis in organotypic, rat hippocampal slice cultures. Brain Res,2005,27(1-2): 35-49
48 Kitayama T, Yoneyama M, Tamaki K, et al. Regulation of neuronal differentiation by N-methyl-D-aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus. J Neurosci Res, 2004,24(4):259-276
49 Ma W, Maric D, Li BS, et al. Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation. EurJ Neurosci,2000,12(4):1227-1240
50 Zhou C, Wen ZX, Shi DM, et al. Muscarinic acetylcholine receptors involved in the regulation of neural stem cell proliferation and differentiation in vitro. Cell Biol Int,2004,28(1):63-67
51 Contestabile A. Roles of NMDA receptor activity and nitric oxide production in brain development. Brain Res Rev,2000,32(2-3):476-509
52 Shibuki K. An electrochemical microprobe for detecting nitric oxide release in brain tissue. Neurosci Res,1990,9(1):69-76
53 Cheng A, Chan SL, Milhavet O, et al. p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells. J Biol Chem,2001, 276(46):43320-43327
54 Merrill JE, Ignarro LJ, Sherman MP, et al. Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol,1993, 151(4):2132-2141
55 Boje KM, Arora PK. Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res,1992,587(2): 250-256
56 Cheng A, Wang S, Cai J, et al. Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentation in the mammalian brain. Dev Biol,2003,258(2):319-333
57 Covacu R, Danilov Al, Rasmussen BS, et al. Nitric oxide exposure diverts neural stem cell fate from neurogenesis towards astrogliogenesis. Stem Cells,2006,24(12):2792-800
58 Haas H, Panula P. The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci,2003,4(2):121-130
59 Molina-Hernandez A, Nunez A, Sierra JJ, et al. Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes. Neuropharmacology,2001,41(8):928-934
60 Molina-Hernandez A, Velasco I. Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptors. Neurochem,2008,106(2):706-717
61曹江北,李云峰.γ-氨基丁酸受体系统在脑缺血诱发神经元再生过程中的调节作用.国外医学药学分册,2006,2(33)：89-92
62 Tozuka Y, Fukuda S, Namba T, et al. GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron, 2005,47(6):803-815
63 Ge S, Goh EL, Sailor KA, et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature,2006,439(7076): 589-593
64 Overstreet-Wadiche LS, Bensen AL, Westbrook GL, et al. Delayed development of adult-generated granule cells in dentate gyrus. J Neurosci, 2006,26(8):2326-2334
65 Jelitai M, Anderova M, Marko K, et al. Role of gammaaminobutyric acid in early neuronal development:studies with an embryonic neuroectodermal stem cell clone. J Neurosci Res,2004,76(6):801-811
66 Wang LP, Kempermann G, Kettenmann H. A subpopulation of precursor cells in the mouse dentate gyrus receives synaptic GABA ergic input. Mol Cell Neurosci,2005,29(2):181-189
67 Carrasco MA, Castro P, Sepulveda FJ, et al. Regulation of glycinergic and GABAergic synaptogenesis by brain-derived neurotrophic factor in developing spinal neurons. Neuroscience,2007,145(2):484-494
68 Ryan SK, Shotts LR, Hong SK, et al. Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey. Dev Neurobiol,2007,67(2):173-188
69 Carrasco MA, Castro PA, Sepulveda FJ, et al. Anti-homeostatic synaptic plasticity of glycine receptor function after chronic strychnine in developing cultured mouse spinal neurons. J Neurochem,2007,100(5): 1143-1154
70 Cuevas ME, Carrasco MA, Fuentes Y, et al. The presence of glia stimulates the appearance of glycinergic synaptic transmission in spinal cord neurons. Mol Cell Neurosci,2005,28(4):770-778
71 Caraiscos VB, Bonin RP, Newell JG, et al. Insulin increases the potency of glycine at ionotropic glycine receptors. Mol Pharmacol,2007,71(5): 1277-1287
72 Kimura K, Matsumoto N, Kitada M, et al. Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells in vitro. J Neurocytol,2004,33(4):465-476
73 Aguayo LG, van Zundert B, Tapia JC, et al. Changes on the properties of glycine receptors during neuronal development. Brain Res Brain Res Rev, 2004,47(1-3):33-45
74 Tapia JC, Cardenas AM, Nualart F, et al. Neurite outgrowth in developing mouse spinal cord neurons is modulated by glycine receptors. Neuroreport, 2000,11(11):3007-3010
75 Mercer A, Ronnholm H, Holmberg J, et al. PACAP promotes neural stem cell proliferation in adult mouse brain. Journal of Neuroscience Research, 2004,76:205-215
76 Eser D, Schule C, Baghai TC, et al. Neuroactive steroids and affective disorders. Pharmacol Biochem Behav,2006,84 (4):656-666
77 Karishma KK, Herbert J. Dehydroepiandrosterone (DHEA) stimulates neurogenesis in the hippocampus of the rat, promotes survival of newly formed neurons and prevents corticosterone-induced suppression. Eur J Nrurosci,2002,16(3):445-453
78 Suzuki M, Wright LS, Marwah P, et al. Mitotic and neurogenic effects of dehydroepiandrosterone(DHEA)on human neural stem cell cultures derived from the fetal cortex. Pans,2004,101(9):3202-3207
79 Azizi H, Mehrjardi NZ, Shahbazi E, et al. Dehydroepiandrosterone Stimulates Neurogenesis in Mouse Embryonal Carcinoma Cell and Human Embryonic Stem Cell-Derived Neural Progenitors and Induces Dopaminergic Neurons. Stem Cells and Development,2010,19(6): 809-819
80 Wang JM, Johnston PB, Ball BG, et al. Allopregnanolone Promotes Proliferation of Rodent and Human Neural Progenitor Cells and Regulates Cell-Cycle Gene and Protein Expression. The Journal of Neuroscience, 2005,25(19):4706-4718
81 McEwen B, Akama K, Alves S, et al. Tracking the estrogen receptor in neurons:Implications for estrogen-induced synapse formation. Proc Natl Acad Sci,2001,98(13):7093-7100
82 Brannvall K, Korhonen L, Lindholm D. Estrogen-Receptor-Dependent Regulation of Neural Stem Cell Proliferation and Differentiation. Mol Cell Neurosci,2002,21(3):512-520
83 Yo Kishi, Jun Takahashi, Masaomi Koyanagi, et al. Estrogen Promotes Differentiation and Survival of Dopaminergic Neurons Derived from Human Neural Stem Cells. Journal of Neuroscience Research,2005,79: 279-286
84 Shen Q, Gode S K, Jin L, et al. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science,2004,304(5675): 1338-1340
85 Ullian EM, Sapperstein SK, Christopherson KS, et al. Control of synapse number by glia. Science,2001,291:657-661
86 Christopherson KS, Ullian EM, Stokes CC, et al. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell,2005, 120:421-433
87 Kornyei Z, Szlavik V, Szabo B, et al. Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis. Glia,2005,49:430-444
88 Song HJ, Charles FS, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature,2002,417(2):39-44
89 Jacques TS, Relvas JB, Nishimura S, et al. Neural precursor cell chain migration and division are regulated through different betal integrins. Development,1998,125:3167
90 Amoutenx MC, Cunningham BA, Edelman GM, et al. NCAM binding inhibits the proliferation of hippocampal progenitor cells and promotes their differentiation to a neuronal phenotype. Journal of Neuroscience, 2000,20(10):3631
91 Horie N, Moriya T, Mitome M, et al. Lowered glucose suppressed the proliferation and increased the differentiation of murine neural stem cells in vitro. FEBS Lett,2004,571(3):237-242
92 Xu SZ, Shan CJ, bullock L, et al. Pb2+ reduces PKCs and NF-κB in vitro. Cell Biol Toxicol,2006,22:189-198
93 Emmanouil EN, Goret NM, Kerameos FC, et al. Anterior neural tube malformations induced after alltrans retinoic acid administration in white rat embryos. I. Macroscopical observations. Morphologie,2000,84(264): 5-11
94 Yohei O, Takuya S, Gen S, et al. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol,2004,275(1):124-142
95 Li L, Milner LA, Deng Y, et al. The human homolog of rat jagged-1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch-1. Immunity,1998,8(1):43-55
96 Morrison SJ, Perez SE, Qiao Z, et al. Transien Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell,2000,101(5):499-510
97 Sun Z, Andersson R. NF-κB activation and inhibition:a review. Shock, 2002,18:99-106
98 Brazel CY, Limke TL, Osborne JK, et al. Sox2 exp ression defines a heterogeneous population of neurosphere forming cells in the adult murine brain. Aging Cell,2005,4:197-207
99 Widera D, Mikenberg I, ElversM, et al. Tumor necrosis factor alpha trigers proliferation of adult neural stem cells via IKK/NF-κB signaling. BMC Neurosci,2006,7:64
100Kaltschmidt B, UherekM, Wellmann H, et al. Inhibition of NF-κB potentiates amyloid-β-mediated neuronal apoptosis. Proc Natl Acad Sci USA,1999,96(16):9409-9414
101Harada J, Foley M, Moskowitz MA, et al. Sphingosine-1-phosphate induces proliferation and morphological changes of neural p rogenitorcells. J Neurochem,2004,88:1026-1039
102Shingo T, Sorokan ST, Shimazaki T, et al. Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci,2001,21:9733-6743
103Harris TJ, Peifer M. Deeisions:beta-ceatenin chooses between adhesion and transcription. Trends Cell Biol,2005,15:234-7
104Reya T, Clevers H. Wnt signaling in stem cells and cancer. Nature,2005, 434:843-50
105Huelsken J, Birehmeier W. New aspects of Wnt signaling pathways in higher vertbrates. Curr Opin Genet Dev,2001,11:547-53
106Peifer M, polakis P. Wnt signaling in oncogenesis and embryogenesisa look outside the nucleus. Seienee,2000,287:1606-9
107Eastman Q, Grossehedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biol,1999,11:233-40
108Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol,1998,14:59-88
109Hirabayashi Y, Cotoh Y. Stage-dependent fate determination of neural precursor cells in mouse forebrain. Neurosci Res,2005,51(4):331-336
110Muroyama Y, Kondoh H, Takada S. Wnt proteins promote neuronal differentiation in neural stem cell culture. Biochemical and Biophysical Research Communications,2004,313:915-921
111Yu JM, Kim JH, Song GS, et al. Inerease in proliferation and differentiation of neural progenitor cells isolated from postoatal and adult mice brain by Wnt-3a and Wnt-5a. Mol Cell Biochem,2006,288:17-28
112Ostenfeld T, Caldwell M A, Prowse KR, et al. Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth folloeing transplantion. Exp Neurol,2000,164:215-226
113Wagner J, Akerud P, Castro DS, et al. Induction of a midbrain dopaminergic phenotype in Nurrl-overexpressing neural stem cells by type 1 astrocytes. Nature Biotech,1999,17:653-659
114Li XK, Guo AC, Zuo PP. Survival and differentiation of transplanted neural stem cells in mice brain with MPTP-induced Parkinson disease. Acta Parmacol Sin,2003,24(12):1192-1198
115Akiyama Y, Honmou O, Kato T, et al. Transplantation of clonal neural precursor cells from adult human brain establishes functional peripheral myelin in the rat spinal cord. Exp Neurol,2001,167:27-39
116Vacanti MP, Leonard JK, Dore B, et al. Tissue-engineered spinal cord. Transplantation Proceedings,2001,33:592-598
117Yagita Y, Kitagawa K, Ohtsuki T, et al. Neurogenesis by progenitors in the ischemic adult rat hippocampus. Stroke,2001,32:1890-1896
118Veizovic T, Beech JS, Stroemer P, et al. Resolution of stroke deficits followinf contralateral grafts of conditionally immortal neuroepithelial stem cells. Stroke,2001,32:1012-1019
119Freeman TB, Cicchetti F, Hauser RA, et al. Transplanted fetal striatum in Huntington's disease:phenotypic development and lack of pathology. Proc Natl Acad Sci USA,2000,97:13877
2 Friedenstein AJ, Gorskaja JF, Kulagina NN.Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol,1976, 4(5):267-74
3 Guo L, Yin F, Meng H, et al. Differentiation of mesenchymal stem cells into dopaminergic neuron-like cells in virto. Biomed Environ Sci,2005, 18(1):36-42
4 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuron cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113(12):1701-1710
5 Baksh D, Song L, Tuan RS.Adult mesenchymal stem cells characterization differentiation and application in cell and gene therapy. J cell Mol Med, 2004,8(3):301-306
6 Ortiz-Gonzalez XR, Keene CD, Verfaillie CM, et al. Neural induction of adult bone marrow and umbilical cord stem cells. Curr Neurovasc Res, 2004, 1(3):207-13
7 Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol,2000, 16(2):247-56
8 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res,2000, 61(4):364-70
9 Mueller SM, Glowacki J. Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem,2001,82(4):583-590
10 Rao MS, Mattson MP. Stem cells and aging:expanding the possibilities. Mech Ageing Dev,2001,122(7):713-34
11 Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma;in situ and in vitro surveys. Stem Cells,2007,25(2): 319-331
12 Sarugaser R, Lickorish D, Baksh D, et al. Human Umbilical Cord Perivascular (HUCPV) Cells:A Source of Mesenchymal Progenitors. Stem Cells,2005,23(2):220-229
13 Lee KD. Applications of mesenchymal stem cells:an updated review. Chang Gung Med J,2008,31(3):228-236
14 Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells,2006,24(4):1030-1041
15 Li Y, McIntosh K, Chen J, et al. Allogeneic bone marrow stromal cells promote glial-axonal remodeling without immunologic sensitization after stroke in rats. Exp Neurol,2006,198(2):313-325
16 Spaggiari GM, Capobianco A, Abdelrazik H, et al. Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production:role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood,2008,111(3):1327-1333
17迟志华,张洹.脐血间充质干细胞研究进展.国际生物医学工程杂志,2006,29(1)：29-34
18 Bieback K, Kern S, Kluter H, et al. Critical parameters for the isolation of mesenchymal stem cell from umbilical cord blood. Stem cells,2004,22(4): 625-634
19 McElreavey KD, Irvine AI, Ennis KT, et al. Isolation,culture and characterization of fibroblast-like cells derived from the Wharton's jelly portionof human umbilical cord. Biochem Soc Trans,1991,19(1):29(s)
20 Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchyal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003,21(1):105-110
21 Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular(HUCPV) cells:a source of mesenchymal progenitors. Stem Cell,2005,23(2):220-229
22 Guo L, Yin F, Meng H, et al. Differentiation of mesenchymal stem cells into dopaminergic neuron-like cells in virto. Biomed Environ Sci,2005, 18(1):36-42
23 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuron cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113(12):1701-1710
24 Baksh D, Song L, Tuan RS.Adult mesenchymal stem cells characterization differentiation and application in cell and gene therapy. J cell Mol Med, 2004,8(3):301-306
25 Jones BJ, McTaggart SJ. Immunosuppression by mesenchymal stromal cells:from culture to clinic. Exp Hematol,2008,36(6):733-741
1 Ostenfeld T, Svendsen CN. Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells.2004,22(5):798-811
2邓方阁,张秀英,王心蕊,等.体外模拟心肌微环境中人骨髓间充质干细胞向心肌细胞的分化.吉林大学学报(医学版),2007,33(2)：257-259
3王显振,蔡文清,宋永周,等.膀胱匀浆上清在大鼠骨髓间充质干细胞诱导分化为平滑肌样细胞中的作用.中国组织工程研究与临床康复,2008,12(8)：1422-1425
4 Jang YY, Collector MI, Baylin SB, et al. Hematopoictic stem cells convert into liver cells within days without fusion. Nat Cell Biol,2004,6(6): 532-539
5 Choi KS, Shin JS, Lee JJ, et al. In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract. Biochem Biophys Res Commun,2005,330(4):1299-305
6 Jin K MX, Batteur S, Sun Y, et al. Induction of neuronal markers in bone marrow cells:differential effects of growth factors and patterns of intracellular expression. Exp Neurol,2003,184:78-89
7 Jiang YH, Scutt G, Goltzman D, et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci,2003, 100(1):1854-1860
8 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113(12):1701-1710
9 Belegu V, Oudega M, Gary DS, et al. Restoring function after spinal cord injury:promoting sponatneous regneration with stem cells and activity-based therapies. Neurosurg Clin N Am,2007,18(1):143-168
10 Kelly S, Bliss TM, Shah AK, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci,2004,101(32):11839-11844
11 Zhang SC, Wernig M, Duncan ID, et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol,2001,19(12):1129-1133
12 Schuldiner M, Eiges R, Eden A, et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res,2001,913(2):201-205
13 Dezawa M, Kanno H, Hoshino M, et al. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest,2004,113:1701-1710
14 Munoz-Elias G, Woodbury D, Black IB. Marrow stromal cells, mitosis, and neuronal differentiation:stem cell and precursor functions. Stem Cells, 2003,21(4):437-448
15 Lu D, Mahmood A, Wang L, et al. Adult bone marrow strmal cells administration intravenously to rats after traumatic brain into brain and improve neurological outcome. Neuroreport,2001,12:559-563
16 Gurgo RD, Bedi KS, Nurcombe V. Current concepts in central nervous system regeneration. J Clin Neurosci,2002,9(6):613-617
17 Isacson O. The production and use of cells as therapeutic agents in neurodegenerative diseases. Lancet Neurol,2003,2:417-424
18 Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood,2002,99(10):3838-3843
19 Le Blanc K, ammik C, Rosendahl K, et al. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol,2003,31(10):890-896
20 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke,2001,34(4):1005-1011
21 Chen J, Li Y, Katakowski M, et al. Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat. J Neurosci Res,2003,73(6):778-86
22 Pavlichenko N, Sokolova I, Vijde S, et al. Mesenchymal stem cells transplantation could be beneficial for treatment of experimental ischemic stroke in rats. Brain Res,2008,1233:203-13
23 Park HJ, Lee PH, Bang OY, et al. Mesenchymal stem cells therapy exerts neuroprotection in a progressive animal model of Parkinson's disease. J Neurochem,2008,107(1):141-51
24 Slavin S, Kurkalli BG, Karussis D. The potential use of adult stem cells for the treatment of multiple sclerosis and other neurodegenerative disorders. Clin Neurol Neurosurg,2008,110(9):943-946
25 Wang JM, Patrick B, Johnston, et al.The Neurosteroid Allopregnanolone Promotes Proliferation of Rodent and Human Neural Progenitor Cells and Regulates Cell-Cycle Gene and Protein Expression. The Journal of Neuroscience,2005,25(19):4706-4718
26 Crigler L, Robey RC, Asawachaicharn A, et al. Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Exp Neurol,2006,198 (1):54-64
27 Suzuki M, Wright LS, Marwah P, et al. Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex. Proc Natl Acad Sci,2004,101(9):3202-7
28 Huang W, Miao ZN, Chen L, et al. Brain-derived neurotrophic factor, ciliary neurotrophic factory and their combination for in vitro differentiation of human umbilical blood-derived mesenchymal stem cells into nerve-like cells. Journal of Clinical Rehabilitative Tissue Engineering Research,2010,14(19):3606-3610
29 Suzuki M, DNelson A, Joshua B, et al. Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex. European Journal of Neuroscience,2006,24(3):645-653
30 Shetty A K. Progenitor cells from the CA3 region of the embryonic day 19 rat hippocampus generate region-specific neuronal phenotypes in vitro. Hippocampus,2004,128(4):807-817
31 Whittemore S R, Morassutti D J, Walters W M, et al. Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell population. Exp Cell Res,1999, 252(1):75-95
1 Ibanez C, Guennoun R, Liere P, et al. Developmental expression of genes involved in neurosteroidogenesis:3β-hydroxysteroid-dehydrogenase/Δ5-Δ4-isomerase in the rat brain. Endocrinology,2003,144:2902-2911
2 Zwain IH, Yen SC, et al. Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain. Endocrinology,1999,140:385-3843
3 Charalampopoulos I, Remboutsika E, Margioris AN, et al. Neurosteroids as modulators of neurogenesis and neuronal survival. Trends Endocrinol Metab,2008,19:300-307
4 Hirst JJ, Palliser HK, Yates DM, et al. Neurosteroids in the fetus and neonate:potential protective role in compromised pregnancies. Neurochem Int,2008,52:602-610
5 Keller EA, Zamparini A, Borodinsky LN, et al. Role of allopregnanolone on cerebellar granule cells neurogenesis. Brain Res Dev Brain Res,2004, 153:13-17
6 Lockhart EM, Warner DS, Pearlstein RD, et al. Allopregnanolone attenuates N-methyl-D-aspartate-induced excitotoxicity and apoptosis in the human NT2 cell line in culture. Neurosci Lett,2002,328:33-36
7 Mellon SH. Neurosteroid regulation of central nervous system development. Pharmacol Ther,2007,116:107-124
8 Sakamoto H, Ukena K, Tsutsui K, et al. Effects of progesterone synthesized de novo in the developing purkinje cell on its dendritic growth and synaptogenesis. J Neurosci,2001,21:6221-6232
9 Schumacher M, Guennoun R, Stein DG, et al. Progesterone:therapeutic opportunities for neuroprotection and myelin repair. Pharmacol Ther,2007, 116:77-106
10 Wagner CK. Progesterone receptors and neural development:a gap between bench and bedside? Endocrinology,2008,149:2743-2749
11 Wang JM, Johnston PB, Ball BG, et al. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J Neurosci,2005,25: 4706-4718
12于洋,薛改,吴红海,等.Aβ25-35对大鼠皮质神经元神经甾体水平的影响.中国药理学通报,2010,26(6)：241-244
13 Liere P, Akwa Y, Weill-Engerer S, et al. Validation of an analytical procedure to measure trace amount of neurosteroids in rat brain tissue by gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl, 2000,739(2):301-312
14郭再玉,姜晓丹,徐如祥,等.骨髓基质细胞源性神经干细胞体外分化及电生理特性的研究.中华神经医学杂志,2005,4(6)：545-550
15杨慧民,张苏明,郑荣远.骨髓间充质干细胞诱导分化为神经样细胞的离子通道：是否有电生理特性?中国组织工程研究与临床康复, 2009,13(40)：7881-7884
16陈剑荣,徐如祥,姜晓丹,等.兔骨髓源性神经干细胞分化为成熟神经元样细胞的特征研究.中国临床康复,2004,8(16)：30242-3043
17 Baulieu EE, Robel P, Schumacher M. Neurosteroids:A New Regulatory Function in the Nervous System. J Soc Biol,1999,193(3):285-92
18 Guennoun R, Benmessahel Y, Delespierre B, et al. Progesterone stimulates Krox-20 gene expression in Schwann cells. Brain Res Mol Brain Res, 2001,90:75-82
19 Chan JR, Rodriguez-Waitkus PM, Ng BK, et al. Progesterone synthesized by Schwann cells during myelin formation regulates neuronal gene expression. Mol Biol Cell,2000,11:2283-2295
20 Ghoumari AM, banez C, El-Etr M, et al. Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum. J Neurochem,2003,86:848-859
21 Ghoumari AM, Baulieu EE, Schumacher M. Progesterone increases oligodendroglial cell proliferation in rat cerebellar slice cultures. Neuroscience,2005,135:47-58
22 Gago N, Akwa Y, Sananes N, et al. Progesterone and the oligodendroglial lineage:stage-dependentbiosynthesis and metabolism. Glia,2001,36: 295-308
23 Gago N, El-Etr M, Sananes N, et al.3alpha,5alpha-Tetrahydro-progesterone (allopregnanolone) and gamma-aminobutyric acid:autocrine /paracrine interactions in the control of neonatal PSA-NCAM+progenitor proliferation. J Neurosci Res,2004,78:770-783
24 De Nicola AF, Gonzalez SL, Labombarda F, et al. Progesterone treatment of spinal cord injury:Effects on receptors, neurotrophins, and myelination. J Mol Neurosci,2006,28:3-15
25 Tsutsui K, Mellon SH. Neurosteroids in the Brain Neuron:Biosynthesis, Action and MEdicinal Impact on Neurodegenerative Disease. Cent Nerv Syst Agents in Med Chem,2006,6:73-82
1 VanLandingham JW, Cutler SM, Virmani S, et al. The enantiomer of progesterone acts as a molecular neuroprotectant after traumatic brain injury. Neuropharmacology,2006,51(6):1078-1085
2 Cutler SM, VanLandingham JW, Murphy AZ, et al. Slow-release and injected progesterone treatments enhance acute recovery after traumatic brain injury. Pharmacol Biochem Behav,2006,84(3):420-428
3 Guo Q, Sayeed I, Baronne LM, et al. Progesterone administration modulates AQP4 expression and edema after traumatic brain injury in male rats. Exp Neurol,2006,198(2):469-478
4 Brewer GJ, Torricelli JR, Evege EK, et al. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serumfree medium combination. J Neurosci Res,1993,35(5):567-576
5 Wang JM, Johnston PB, Ball BG, et al. The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J Neurosci,2005,25(19): 4706-4718
6 Gilmer LK, Roberts KN, Scheff SW. Efficacy of progesterone following a moderate unilateral cortical contusion injury. J Neurotrauma,2008,25(6): 593-602
7 Ogata T, Nakamura Y, Tsuji K, et al. Steroid hormones protect spinal cord neurons from glutamate toxicity. Neuroscience,1993,55:445-449
8 Gonzalez Deniselle MC, Lopez-Costa JJ, Saavedra JP, et al. Progesterone neuroprotection in the Wobbler mouse,a genetic model of spinal cord motor neuron disease. Neurobiol Dis,2002,11:457-468
9 Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science,2000,290: 1775-1779
10 Einstein O, Ben-Hur T. The changing face of neural stem cell therapy in neurologic diseases. Alch Neurol,2008,65(4):452-456
11 Chen J, Chop M, Li Y. Neuroprotective effects of progesterone after transient middle cerebral aetery occlusion in rat. J Neurol Sci,1999, 171(1):24-30
12 Roof RL, Duvdevani R, Stein DG. Gender influences outcome of brain injury:progesterone plays s protective role. Brain Res,1993,607(1-2): 333-336
13 Asbury ET, Fritts ME, Horton JE, et al. Progesterone facilitates the acquisition of avoidance learning and protests against subcotical neuronal death following prefrontal cortex ablation in the rat. Behav Brain Res, 1998,97(1-2):99-106
14 Morali G, Letechipia-Vallejo G, Lopez-Loeza E, et al. Post-ischemic administration of progesterone in rats exerts neuroprotective effects on the hippocampus. Neurosci Lett,2005,382(3):268-290
15 Thoman AJ, Nockels RP, Pan HQ, et al. Progesterone is neuroprotective after acute experimental spinal cord trauma in rats. Spine,1999,24(20): 2134-2138
16 Sakamoto H, Ukena K, Tsutsui K. Effects of progesterone synthesized de novo in the developing Purkinje cell on its dendritic growth and synaptogenesis. J Neurosci,2001,21(16):6221-6232
17 Sakamoto H, Ukena K, Tsutsui K. Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats. Neurosci Lett,2002,322(2):111-115
18 Gibson CL, Gray LJ, Bath PM, et al. Progesterone for the treatment of experimental brain injury:a systematic review. Brain,2008,131:318-328
19 Singh M, Sumien N, Kyser C, et al. Estrogens and progesterone as neuron protectants:what animal models teach us. Front Biosci,2008,13: 1083-9
20 Stein DG. Progesterone exerts neuroprotective effects after brain injury. Brain Res Rev,2008,57:386-397
21 Stein DG, Wright DW, Kellermann AL. Does progesterone have neuroprotective properties? Ann Emerg Med,2008,51:164-172
22 Wright DW, Kellermann AL, Hertzberg VS, et al. ProTECT:a randomized clinical trial of progesterone for acutetraumatic brain injury. Ann Emerg Med,2007,49:391-402
23 Xiao G, Wei J, Yan W, et al. Improved outcomes from the administration of progesterone for patients with acute severe traumatic brain injury:a randomized controlled trial. Crit Care,2008,12:R61
24 Lopez-Gonzalez R, Camacho-Arroyo I, Velasco I, et al. Progesterone and 17b-Estradiol Increase Differentiation of Mouse Embryonic Stem Cells to Motor Neurons. IUBMB Life,2011,63(10):930-939
25 Diaz NF, Diaz-Martinez NE, Velasco I, et al. Progesterone increases dopamine neurone number indifferentiat-Ing mouse embryonic stem cells. J Neuroendocrinol,2009,21:730-736
1陈平安,董晓先,冷水龙.黄体酮诱导骨髓间充质干细胞分化为神经元样细胞实验研究.实用医学杂志,2004,20(6)：606-607
2 De Nicola AF, Gonzalez SL, Labombarda F, et al. Progesterone treatment of spinal cord injury:Effects on receptors, neurotrophins, and myelination. Journal of Molelcular Neuroscience,2006,28:3-15
3 Gonzalez SL, Labombarda F, Gonzalez Deniselle MC, et al. Progester-one up-regulates neuronal brainderived neurotrophic factor expression in the injured spinal cord. Neuroscience,2004,125:605-614
4 Gonzalez SL, Labombarda F, Gonzalez Deniselle MC, et al. Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain derived neurotrophic factor in motoneurons. J Steroid Biochem Mol Biol, 2005,94:143-149
5 Gonzalez Deniselle MC, Garay L, Gonzalez S, et al. Progesterone modulates brain-derived neurotrophic factor and choline acetyltransferase in degenerating Wobbler motoneurons. Experimental Neurology,2007, 203:406-414
6 Kaur P, Jodhka PK, Underwood WA, et al. Progesterone increases brain-derived neuroptrophic factor expression and protects against glutamate toxicity in a mitogen-activated protein kinase and phosphoinositide-3 kinase dependent manner in cerebral cortical explants. Journal of Neuroscience Research,2007,85:2441-2449.
7 Cai W, Zhu Y, Furuya K, et al. Two different molecular mechanisms underlying progesterone neuroprotection against ischemic brain damage. Neurosei,2001,21(17):6706-6717
8王海珍,刘呜,赵萃瑜,等.BDNF的克隆及pTAT/HA-BDNF重组表达载体的构建.神经损伤与功能重建,2008,3(9)：304-306
9 Shumsky JS, Tobias CA, Tumolo M, et al. Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT-3 into a spinal cord injury site is associated with limited recovery offunction. Exp Neurol, 2003,1(184):114-30
10 Poo MM. Neurotrophins as synaptic modulators. Nat Rev Neurosci,2001, 2:24-32
11 Lim JY, Park SI, Oh JH, et al. Brain-derived neu-rotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways. J Neurosci Res,2008,86(10):2168-78
12 Chen ZY, Bath K, McEwen B, et al. Impact of genetic variant BDNF on brain structure and function. Novartis Found Symp,2008,28(9):180-188
13 Spedding M, Gressens P. Neurotrophins and cytokines in neuronal plasticity. Novartis Found Symp,2008,28(9):222-233
14 Johnson MA, Weick JP, Pearce RA, et al. Functional neural development from human embryonic stem cells:accelerated synaptic activity via astrocyte cocul-ture. J Neurosci,2007,27(12):3069-77
15 Alfimova MV, Golimbet VE, Barkhatova AN, et al. The role of genotype-environment interactions in the development of symptoms of anxiety and depression related to the disease burden for family. Zh Nevrol Psikhiatr Im SS Korsakova,2009,109(12):50-54
16 Jakeman LB, Wei P, Guan Z, et al.Brain derived neurotrophic factor timulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury. Exp Neurol,2001,154:170-184
17 Coumans JV, Lin TT, Dai HN, et al. Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins. J Neurosci,2001,21: 9334-9344
18 Chaldakov GN, Tonchev AB, Aloe L. NGF and BDNF:from nerves to adipose tissue, from neurokines to metabokines. Riv Psichiatr,2009,44(2): 79-87
19 Tapia-Aranciba L, Rage F, Givalois L, et al. Physiology of BDNF:focus on hypothalamic function. Front Neuroendocrinol,2004,25(2):77-107
20许期年,金钧,苗宗宁.脑源性神经营养因子促进骨髓间充质干细胞分化为神经元样细胞的实验研究.苏州大学学报(医学版),2007,27(4)：520-523
21 Huang EJ, Reichardt LF. Trk receptors:roles in neuronal signal transduction. Annu Rev Biochem,2003,72:609-642
22 Gomes C, Smith SC, Youssef MN, et al. RNA polymerase 1 driven transcription as a mediator of BDNF-induced neurite out growth. J Biol Chem,2011,286(6):4357-4363
23 Lee JG, Shin BS, You YS, et al. Decreased serum brain-derived neurotrophic factor levels in elderly korean with dementia. Psychiatry Investig,2009,6(4):299-305
24 Castren E, Rantamaki T. Role of brain-derived neurotrophic factor in the aetiology of depression:implications for pharmacological treatment. CNS Drugs,2010,24(1):1-7
25 Chen XG, Li Y, Wang L, et al. Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology,2002, 22(4):275-9
26 Gonzalez SL, Labombarda F, Gonzalez Deniselle MC, et al. Progesterone up-regulates neuronal brain-derived neurotrophic factor expression in the injured spinal cord. Neuroscience,2004,125:605-614
27 Gonzalez SL, Labombarda F, Deniselle MC, et al. Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain-derived neurotrophic factor in motoneurons. J Steroid Biochem Mol Biol,2005,94:143-149
28 Scharfman HE, Maclusky NJ. Similarities between actions of estrogen and BDNF in the hippocampus:coincidence or clue? Trends Neurosci,2005, 28:79-85
29 Li Y, Chen J, Chen XG, Wang L, et al. Human marrow stromal cell therapy for stroke in rat:neurotrophins and functional recovery. Neurology,2002, 59(4):515-523
30 Song S, Kamath S, Mosquera D, et al. Expression of brain natriuretic peptide by human bone marrow stromal cells. Exp Neurol,2004,185(1): 191-197
31 Zhang XM, Basch RS. Mmortalized multipotential mesenchymal cells and the hematopoietic microenvironment. J Hematother Stem Cell Res,2001, 10:125-140
32 Sensebe L, Deschaseaux M, Li J, et al. The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment. Stem Cells,1997,15:133-143
1 Reynolds BA, Weiss S. Generation of neurons and astrocytes from iso2 lated cell of the adult mammalian central nervous system. Science,1992, 255 (5052):1707-1710
2 Richards LJ, Kilpatrick TJ, Bartlett PF. De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci,1992, 89(18):8591-8595
3 Kelly CM, Zitetlow R, Dunnett SB, et al. The effects of various concentrations of FGE-2 on the proliferation and neuronal yield of murine embryonic neural precursor cells in vitro.Cell Transplant,2003,12(3): 215-223
4姚忠祥,孙俞,刘建军,等.碱性成纤维细胞生长因子促进脑室管膜前下区细胞增殖的实验研究.中国临床康复,2004,8(4)：664-665
5 Fiore M, Aca V, Amendola T, et al. Brain NGF and EGF adminis tration improves passive avoidance response and stimulates brain precursor cells in aged male mice. Phys iol Behav,2002,77:437-443
6余剑,曾进胜,盛文利,等.表皮生长因子对脑梗死大鼠脑室管膜下区神经干细胞迁徙和分化的影响.中华医学杂志,2004,84(23)：1965-1967
7 Sofroniew MV, Howe CL, Mobley WC. Never growth factor signaling, neuroprotection, and neural repair. Ann Rev Neurosci,2001,24: 1217-1281
8 Benoit BO, Savarese T, Joly M, et al. Neurotrophin channeling of neural progenitor cell differentiation. J Neurobiol,2001,46(4):265-280
9 Frielingsdorf H, Simpson DR, Thal LJ, et al. Nerve growth factor promotes survival of new neurons in the adult hippo-campus. Neurobiol Dis,2007,26(1):47-55
10 Mondal D, Pradhan L, LaRussa VF. Signal transduction pathways involved in the lineage-differentiation of NSCs:can the knowledge gained from blood be used in the brain? Cancer Invest,2004,22(6):925-943
11 Pagano SF, Impagnatiello F, Girelli M, et al. Isolation and characterization of neural stem cells from the adult human olfactory bulb. Stem cells,2000, 18(4):295-299
12 Molne M, Studer L, Tabar V, et al. Early cortical precursors do not undergo LIF-mediated astrocytic differentiation. J Neurosci Res,2000,59(3): 301-11
13 Katic M, Kahn CR. The role of insulin and insulin-like growth factor-1 signaling in longevity. Cell Mol Life Sci,2005,62(2):3420-3431
14 Juul A. IGF-I measurements.Epidemiological and clinical aspects. Front Horm Res,2005,33(1):45-67
15 Ercole AJ, Dai Z, Xing Y, et al. Brain growth redardation due to the expression of human insulin-like growth factor binding protein-1 in transgenic mice:an in vivo model for the analysis of IGF function in the brain. Brain Res Dev Brain Res,1994,82(2):213-2221
16 Aberg MA, Aberg ND, Hedbackerh, et al. Peripheral infusion of IGF-1 selectively induces neurogenesis in the adult rat rappocampus. J Neurosci, 2000,20(8):2896-930
17 Morishita E, Masuda S, Nagao M, et al. Erythropoietin recaptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamateinduced neuronal death. Neuroscience,1997,76(1):105-116
18 Sakanaka M, Wen TC, Matsuda S, et al. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci,1998,95(8):4635-4640
19 Wang L, Zhang ZG, Wang Y, et al. Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats. Stroke,2004,35(7):1732-1737
20 Lee SM, Nguyen TH, Park MH, et al. EPO receptor-mediated ERK kinase and NF-kappaB activation in erythropoietin promoted differentiation of astrocytes. Biochem Biophys Res Commun,2004,320(4):1087-1095
21张引成,王贵和,张政华.神经节苷酯M1介导神经生长因子对运动神经元再生的影响.西安医科大学学报,2001,22(5)：459-462
22 Rusnati M, Urbinati C, Tanghetti E, et al. Cell membrance GM1 ganglioside is a functional coreceptor for fibroblast growth factor 2. Proc Natl Acad Sci,2002,99(7):4307-72
23赵新利,周国胜,张新中等.GM-1对离体大鼠神经干细胞增殖和分化的影响.医学信息手术学分册,2006,19(2)：57-59
24 EmaM, Faloon P, ZhangWJ, et al. Combinatorial effects of Flk1 and Tall on vascular and hematopoietic development in the mouse. Genes Dev, 2003,17(3):380-393
25 Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. Comp Neurol,2000,425(4):479-494
26 Schwarz Q, Gu C, Fujisawa H, et al. Vascular endothelial growth factor controls neuronalmigration and cooperates with Sema3 A to pattern distinct compartments of the facial nerve. Genes Dev,2004,18(22):2822-2834
27 Sondell M, Lundborg G, Kanje M. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing csell survival and Schwann cell p roli-feration in the peripheral nervous system. Neuroscience,1999,19(14):5731-5740
28 Sun Y, J in K, Childs JT, et al. Vascular endothelial growth factor-B (VEGFB) stimulates neurogenesis:evidence from knockoutmice and growth factor administration. Dev Biol,2006,289(2):329-335
29 Le Bras B, Barallobre MJ, Homman-Ludiye J, et al. VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain. Nat Neurosci,2006,9(3):340-348
30 Maurer MH, Tripps WKC, Feldmann RE, et al. Expression of vascular endothelial growth factor and its receptor in rat neural stem cells. Neurosci Lett,2003,344(3):165-168
31 Meng H, Zhang Z, Zhang R, et al. Biphasic effects of exogenous VEGF on VEGF expression of adult neural progenitors. Neurosci Lett,2006,393 (23):97-101
32 Kim BK, Kim SE, Shim JH, et al. Neurogenic effect of vascular endothelial growth factor during germ layer formation of human embryonic stem cells. FEBS Lett,2006,580(25):5869-5874
33 Jakeman LB, Wei P, Guan Z, et al. Brain derived neurotrophic factor timulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury. Exp Neurol,2001,154:170-184
34 Chaldakov GN, Tonchev AB, Aloe L. NGF and BDNF:from nerves to adipose tissue, from neurokines to metabokines. Riv Psichiatr,2009,44(2): 79-87
35 Alfimova MV, Golimbet VE Barkhatova AN, et al. The role of genotype-environment interactions in the development of symptoms of anxiety and depression related to the disease burden for family. Zh Nevrol Psikhiatr Im SS Korsakova,2009,109(12):50-54
36 Lee JG, Shin BS, You YS, et al. Decreased serum brain-derived neurotrophic factor levels in elderly korean with dementia. Psychiatry Investig,2009,6(4):299-305
37 Castren E, Rantamaki T. Role of brain-derived neurotrophic factor in the aetiology of depression:implications for pharmacological treatment. CNS Drugs,2010,24(1):1-7
38余震,陈莉芬,唐玲,等.腺病毒介导的脑源性神经营养因子对大鼠脑出血后内源性神经干细胞分化的影响.第三军医大学学报,2010,32(15)：1628-1631
39李听松,蒋莉,陈恒胜,等.脑源性神经营养因子促进海马神经干细胞的存活、增殖及向神经元分化.细胞生物学杂志,2008,30：387-391
40王景周,高唱.神经递质与神经干细胞的增殖与分化.国外医学：脑血管疾病分册,2003,11(3)：121-123
41 Watkins JC. L-glutamate as a central neurotransmitter:looking back. Biochem Soc Trans,2000,28(4):297-309
42 Mattson MP, Kater SB. Calcium regulation of neurite elon-gation and growth cone motility. J Neurosci,1987,7(12):4034-4043
43 Simon DK, Prusky GT, O'Leary DD, et al. N-methyl-D-aspartate receptor antagonists disrupt the formation of a mammalian neural map. Proc Natl Acad Sci,1992,89(22):10593-10597
44 Rossi DJ, Slater NT. The developmental onset of NMD A receptorchannel activity during neuronal migration. Neuropharmacology,1993,32(11): 1239-1248
45 Rakic P, Komuro H. The role of receptor/channel activity in neuronal cell migration. J Neurobiol,1995,26(3):299-315
46 Suzuki M, Aaron D, Joshua B, et al. Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex. Eur J Neurosci,2006,24(3):645-653
47 Poulsen FR, Blaabjerg M, Montero M, et al. Glutamate receptor antagonists and growth factors modulate dentate granule cell neurogenesis in organotypic, rat hippocampal slice cultures. Brain Res,2005,27(1-2): 35-49
48 Kitayama T, Yoneyama M, Tamaki K, et al. Regulation of neuronal differentiation by N-methyl-D-aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus. J Neurosci Res, 2004,24(4):259-276
49 Ma W, Maric D, Li BS, et al. Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation. EurJ Neurosci,2000,12(4):1227-1240
50 Zhou C, Wen ZX, Shi DM, et al. Muscarinic acetylcholine receptors involved in the regulation of neural stem cell proliferation and differentiation in vitro. Cell Biol Int,2004,28(1):63-67
51 Contestabile A. Roles of NMDA receptor activity and nitric oxide production in brain development. Brain Res Rev,2000,32(2-3):476-509
52 Shibuki K. An electrochemical microprobe for detecting nitric oxide release in brain tissue. Neurosci Res,1990,9(1):69-76
53 Cheng A, Chan SL, Milhavet O, et al. p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells. J Biol Chem,2001, 276(46):43320-43327
54 Merrill JE, Ignarro LJ, Sherman MP, et al. Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol,1993, 151(4):2132-2141
55 Boje KM, Arora PK. Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res,1992,587(2): 250-256
56 Cheng A, Wang S, Cai J, et al. Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentation in the mammalian brain. Dev Biol,2003,258(2):319-333
57 Covacu R, Danilov Al, Rasmussen BS, et al. Nitric oxide exposure diverts neural stem cell fate from neurogenesis towards astrogliogenesis. Stem Cells,2006,24(12):2792-800
58 Haas H, Panula P. The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci,2003,4(2):121-130
59 Molina-Hernandez A, Nunez A, Sierra JJ, et al. Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes. Neuropharmacology,2001,41(8):928-934
60 Molina-Hernandez A, Velasco I. Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptors. Neurochem,2008,106(2):706-717
61曹江北,李云峰.γ-氨基丁酸受体系统在脑缺血诱发神经元再生过程中的调节作用.国外医学药学分册,2006,2(33)：89-92
62 Tozuka Y, Fukuda S, Namba T, et al. GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron, 2005,47(6):803-815
63 Ge S, Goh EL, Sailor KA, et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature,2006,439(7076): 589-593
64 Overstreet-Wadiche LS, Bensen AL, Westbrook GL, et al. Delayed development of adult-generated granule cells in dentate gyrus. J Neurosci, 2006,26(8):2326-2334
65 Jelitai M, Anderova M, Marko K, et al. Role of gammaaminobutyric acid in early neuronal development:studies with an embryonic neuroectodermal stem cell clone. J Neurosci Res,2004,76(6):801-811
66 Wang LP, Kempermann G, Kettenmann H. A subpopulation of precursor cells in the mouse dentate gyrus receives synaptic GABA ergic input. Mol Cell Neurosci,2005,29(2):181-189
67 Carrasco MA, Castro P, Sepulveda FJ, et al. Regulation of glycinergic and GABAergic synaptogenesis by brain-derived neurotrophic factor in developing spinal neurons. Neuroscience,2007,145(2):484-494
68 Ryan SK, Shotts LR, Hong SK, et al. Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey. Dev Neurobiol,2007,67(2):173-188
69 Carrasco MA, Castro PA, Sepulveda FJ, et al. Anti-homeostatic synaptic plasticity of glycine receptor function after chronic strychnine in developing cultured mouse spinal neurons. J Neurochem,2007,100(5): 1143-1154
70 Cuevas ME, Carrasco MA, Fuentes Y, et al. The presence of glia stimulates the appearance of glycinergic synaptic transmission in spinal cord neurons. Mol Cell Neurosci,2005,28(4):770-778
71 Caraiscos VB, Bonin RP, Newell JG, et al. Insulin increases the potency of glycine at ionotropic glycine receptors. Mol Pharmacol,2007,71(5): 1277-1287
72 Kimura K, Matsumoto N, Kitada M, et al. Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells in vitro. J Neurocytol,2004,33(4):465-476
73 Aguayo LG, van Zundert B, Tapia JC, et al. Changes on the properties of glycine receptors during neuronal development. Brain Res Brain Res Rev, 2004,47(1-3):33-45
74 Tapia JC, Cardenas AM, Nualart F, et al. Neurite outgrowth in developing mouse spinal cord neurons is modulated by glycine receptors. Neuroreport, 2000,11(11):3007-3010
75 Mercer A, Ronnholm H, Holmberg J, et al. PACAP promotes neural stem cell proliferation in adult mouse brain. Journal of Neuroscience Research, 2004,76:205-215
76 Eser D, Schule C, Baghai TC, et al. Neuroactive steroids and affective disorders. Pharmacol Biochem Behav,2006,84 (4):656-666
77 Karishma KK, Herbert J. Dehydroepiandrosterone (DHEA) stimulates neurogenesis in the hippocampus of the rat, promotes survival of newly formed neurons and prevents corticosterone-induced suppression. Eur J Nrurosci,2002,16(3):445-453
78 Suzuki M, Wright LS, Marwah P, et al. Mitotic and neurogenic effects of dehydroepiandrosterone(DHEA)on human neural stem cell cultures derived from the fetal cortex. Pans,2004,101(9):3202-3207
79 Azizi H, Mehrjardi NZ, Shahbazi E, et al. Dehydroepiandrosterone Stimulates Neurogenesis in Mouse Embryonal Carcinoma Cell and Human Embryonic Stem Cell-Derived Neural Progenitors and Induces Dopaminergic Neurons. Stem Cells and Development,2010,19(6): 809-819
80 Wang JM, Johnston PB, Ball BG, et al. Allopregnanolone Promotes Proliferation of Rodent and Human Neural Progenitor Cells and Regulates Cell-Cycle Gene and Protein Expression. The Journal of Neuroscience, 2005,25(19):4706-4718
81 McEwen B, Akama K, Alves S, et al. Tracking the estrogen receptor in neurons:Implications for estrogen-induced synapse formation. Proc Natl Acad Sci,2001,98(13):7093-7100
82 Brannvall K, Korhonen L, Lindholm D. Estrogen-Receptor-Dependent Regulation of Neural Stem Cell Proliferation and Differentiation. Mol Cell Neurosci,2002,21(3):512-520
83 Yo Kishi, Jun Takahashi, Masaomi Koyanagi, et al. Estrogen Promotes Differentiation and Survival of Dopaminergic Neurons Derived from Human Neural Stem Cells. Journal of Neuroscience Research,2005,79: 279-286
84 Shen Q, Gode S K, Jin L, et al. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science,2004,304(5675): 1338-1340
85 Ullian EM, Sapperstein SK, Christopherson KS, et al. Control of synapse number by glia. Science,2001,291:657-661
86 Christopherson KS, Ullian EM, Stokes CC, et al. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell,2005, 120:421-433
87 Kornyei Z, Szlavik V, Szabo B, et al. Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis. Glia,2005,49:430-444
88 Song HJ, Charles FS, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature,2002,417(2):39-44
89 Jacques TS, Relvas JB, Nishimura S, et al. Neural precursor cell chain migration and division are regulated through different betal integrins. Development,1998,125:3167
90 Amoutenx MC, Cunningham BA, Edelman GM, et al. NCAM binding inhibits the proliferation of hippocampal progenitor cells and promotes their differentiation to a neuronal phenotype. Journal of Neuroscience, 2000,20(10):3631
91 Horie N, Moriya T, Mitome M, et al. Lowered glucose suppressed the proliferation and increased the differentiation of murine neural stem cells in vitro. FEBS Lett,2004,571(3):237-242
92 Xu SZ, Shan CJ, bullock L, et al. Pb2+ reduces PKCs and NF-κB in vitro. Cell Biol Toxicol,2006,22:189-198
93 Emmanouil EN, Goret NM, Kerameos FC, et al. Anterior neural tube malformations induced after alltrans retinoic acid administration in white rat embryos. I. Macroscopical observations. Morphologie,2000,84(264): 5-11
94 Yohei O, Takuya S, Gen S, et al. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol,2004,275(1):124-142
95 Li L, Milner LA, Deng Y, et al. The human homolog of rat jagged-1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch-1. Immunity,1998,8(1):43-55
96 Morrison SJ, Perez SE, Qiao Z, et al. Transien Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell,2000,101(5):499-510
97 Sun Z, Andersson R. NF-κB activation and inhibition:a review. Shock, 2002,18:99-106
98 Brazel CY, Limke TL, Osborne JK, et al. Sox2 exp ression defines a heterogeneous population of neurosphere forming cells in the adult murine brain. Aging Cell,2005,4:197-207
99 Widera D, Mikenberg I, ElversM, et al. Tumor necrosis factor alpha trigers proliferation of adult neural stem cells via IKK/NF-κB signaling. BMC Neurosci,2006,7:64
100Kaltschmidt B, UherekM, Wellmann H, et al. Inhibition of NF-κB potentiates amyloid-β-mediated neuronal apoptosis. Proc Natl Acad Sci USA,1999,96(16):9409-9414
101Harada J, Foley M, Moskowitz MA, et al. Sphingosine-1-phosphate induces proliferation and morphological changes of neural p rogenitorcells. J Neurochem,2004,88:1026-1039
102Shingo T, Sorokan ST, Shimazaki T, et al. Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci,2001,21:9733-6743
103Harris TJ, Peifer M. Deeisions:beta-ceatenin chooses between adhesion and transcription. Trends Cell Biol,2005,15:234-7
104Reya T, Clevers H. Wnt signaling in stem cells and cancer. Nature,2005, 434:843-50
105Huelsken J, Birehmeier W. New aspects of Wnt signaling pathways in higher vertbrates. Curr Opin Genet Dev,2001,11:547-53
106Peifer M, polakis P. Wnt signaling in oncogenesis and embryogenesisa look outside the nucleus. Seienee,2000,287:1606-9
107Eastman Q, Grossehedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biol,1999,11:233-40
108Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol,1998,14:59-88
109Hirabayashi Y, Cotoh Y. Stage-dependent fate determination of neural precursor cells in mouse forebrain. Neurosci Res,2005,51(4):331-336
110Muroyama Y, Kondoh H, Takada S. Wnt proteins promote neuronal differentiation in neural stem cell culture. Biochemical and Biophysical Research Communications,2004,313:915-921
111Yu JM, Kim JH, Song GS, et al. Inerease in proliferation and differentiation of neural progenitor cells isolated from postoatal and adult mice brain by Wnt-3a and Wnt-5a. Mol Cell Biochem,2006,288:17-28
112Ostenfeld T, Caldwell M A, Prowse KR, et al. Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth folloeing transplantion. Exp Neurol,2000,164:215-226
113Wagner J, Akerud P, Castro DS, et al. Induction of a midbrain dopaminergic phenotype in Nurrl-overexpressing neural stem cells by type 1 astrocytes. Nature Biotech,1999,17:653-659
114Li XK, Guo AC, Zuo PP. Survival and differentiation of transplanted neural stem cells in mice brain with MPTP-induced Parkinson disease. Acta Parmacol Sin,2003,24(12):1192-1198
115Akiyama Y, Honmou O, Kato T, et al. Transplantation of clonal neural precursor cells from adult human brain establishes functional peripheral myelin in the rat spinal cord. Exp Neurol,2001,167:27-39
116Vacanti MP, Leonard JK, Dore B, et al. Tissue-engineered spinal cord. Transplantation Proceedings,2001,33:592-598
117Yagita Y, Kitagawa K, Ohtsuki T, et al. Neurogenesis by progenitors in the ischemic adult rat hippocampus. Stroke,2001,32:1890-1896
118Veizovic T, Beech JS, Stroemer P, et al. Resolution of stroke deficits followinf contralateral grafts of conditionally immortal neuroepithelial stem cells. Stroke,2001,32:1012-1019
119Freeman TB, Cicchetti F, Hauser RA, et al. Transplanted fetal striatum in Huntington's disease:phenotypic development and lack of pathology. Proc Natl Acad Sci USA,2000,97:13877