人脐带Wharton's jelly间充质干细胞的生物学特性以及其与脑肿瘤干细胞共培养的实验研究
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摘要
背景与目的：
肿瘤干细胞学说这一概念的提出距今已有30多年的历史,该学说认为肿瘤组织中存在极少数具有自我更新和分化潜能的肿瘤细胞,并且是肿瘤增殖、生长、转移和复发的根源。1992年Reynolds对小鼠脑细胞的研究提出了神经干细胞(Neural stem cells, NSCs)概念,1997年Mckay在Science上发表NSCs的文章得到了公认。而2003年Singh从不同病理类型的人脑肿瘤中培养、鉴定出了一种拥有增殖、自我更新和分化能力并且表达神经干细胞表面标记物CD133的脑肿瘤干细胞(Brain Tumor Stem Cells, BTSCs)。近年来,大量研究证实,BTSCs是脑肿瘤的起源细胞,在肿瘤的发生、发展过程中起决定性作用,也是脑肿瘤对放射治疗和化学治疗产生耐受的根源所在,使得BTSCs成为治疗脑肿瘤的新靶点。而BTSCs相关分子标记物及其致瘤机制是神经肿瘤研究的热点领域,也为未来脑肿瘤的研究和临床治疗指明了方向。人们证明,移植后的NSCs具有向脑肿瘤定向迁徙的能力,能在肿瘤周边增殖并包绕脑肿瘤,从而降低脑肿瘤的侵袭性。然而宿主对NSCs移植的排斥和NSCs移植后潜在的致瘤性以及组织来源的伦理问题使得人们把目光投向了间充质干细胞(Mesenchymal Stromal Cells, MSCs)。
MSCs是来源于中胚层的多能干细胞,具有高度自我更新能力和多向分化潜能,在适宜的体内体外环境下可以分化为造血细胞以及多种组织特别是中胚层组织细胞的能力,甚至还具有跨胚层分化为神经外胚层起源组织细胞的潜能,因而具有高度的可塑性,是细胞移植和组织工程的新型种子细胞,具有广阔的应用前景。以往研究的重点放在骨髓MSCs的研究上,而其它组织中也存在有MSCs,包括动员的外周血、脐血、乳牙、脂肪、胎盘等,然而,从这些组织中得到的细胞数量有限,从而限制了其研究发展。
人脐带Wharton's jelly间充质干细胞(Mesenchymal Stromal Cells derived from Wharton's jelly cells, WJCs)满足了国际细胞治疗协会规定的MSCs的特点：如贴壁生长,自我更新,表达MSCs共有的表面标记物,能够向骨、软骨、脂肪、肌肉、神经细胞诱导分化,能够支持其它干细胞的扩增,对免疫系统有良好的耐受性,对肿瘤有定向迁徙的特性。WJCs通过其营养支持和免疫调节作用对很多临床前期的人类疾病的动物模型有治疗作用,如神经退行性疾病,肿瘤,心脏病等。而且脐带组织来源广泛,不存在伦理学问题。因此,本实验主要研究WJCs的生物学特性,以及WJCs和BTSCs在体外共培养后,WJCs是否能够影响BTSCs的生物学特性,并为细胞治疗提供理论依据。
方法：
WJCs的分离、培养：取长约10cm脐带,剥离脐带外膜并将两条脐动脉和一条脐静脉分离去除,保留Wharton's jelly,并用PBS缓冲液充分冲洗并将其剪碎至2mm×2mm×2mm大小组织块,置于T25培养瓶中,加入含有20μg/L的bFGF和体积分数为10%的FBS的DMEM/F12培养基5ml培养。
WJCs免疫表型鉴定：流式细胞仪检CD29、CD44、HLA-ABC、HLA-DR、CD34、CD45。
WJCs免疫荧光鉴定：GFAP和Nestin。
比较脐带Wharton's jelly不同时间产出原代WJCs的免疫表型变化：流式细胞仪检CD29、CD44、HLA-ABC。
CCK-8法比较Wharton's jelly不同时间产出原代WJCs增殖能力变化：绘制7天细胞生长曲线。
BTSCs的分离、培养：将新鲜的肿瘤组织用PBS缓冲液充分冲洗并去除外周坏死组织和毛细血管,在80目滤网中研碎过滤后加入2.5g/L胰酶和1.0g/L胶原酶Ⅳ37℃消化30分钟,将消化液过200滤网后制成单细胞悬液。计数活细胞,以2×105／ml终浓度接种于无血清培养基(Serum-free medium, SFM)中,置于T75培养瓶中,添加培养基至10ml培养。
BTSCs免疫表型鉴定：流式细胞仪检CD133
BTSCs免疫荧光鉴定：GFAP和Nestin
CCK-8法检测BTSCs增殖率：绘制7天细胞生长曲线。
WJCs与BTSCs共培养实验：应用不添加任何生长因子的无血清培养基将两种细胞在24孔板中进行直接共培养。第3、7天时分别离心收集孔中悬浮细胞,应用流式细胞术检测BTSCs CD133表达；贴壁细胞应用免疫荧光法检测神经干细胞标记物巢蛋白(Nestin)和神经胶质细胞标志物胶质纤维酸性蛋白(GFAP)表达；将第3天离心所得的共培养上清液(Co-culture supernatant, CCS)重悬第3代BTSCs并与正常培养悬浮的第3代BTSCs置入96孔板中,应用酶标仪检测两组细胞生长曲线的差异。
结果：
WJCs的原代培养和扩增：组织块贴壁3-5天后可零星见到单个的梭形或三角形的贴壁细胞,1周后可见贴壁组织块周围有细胞向外游出,细胞由多层逐渐变为单层,细胞由密集变为松散,体型由小到大,细胞形态多为饱满的长梭形或扁平形的成纤维样细胞,核仁明显。两周后,细胞形态变为均一的纺锤形,形态类似成纤维细胞,呈平行排列生长或旋涡状生长。
流式细胞仪检测第3代WJCs显示：高表达CD29(91.50%)；低表达CD44(36.92%)、HLA-ABC(28.10%)；不表达CD34(0.72%)、CD45(0.11%)和HLA-DR(0.80%)
对WJCs进行Nestin和GFAP免疫荧光染色后显示：未经诱导的WJCs在荧光显微镜下不发出荧光,即不表达Nestin和GFAP。
比较脐带Wharton's jelly不同时间产出原代WJCs的免疫表型变：流式细胞仪检测CD29,CD44,HLA-ABC变化无统计学意思。
CCK-8法比较Wharton's jelly不同时间产出原代WJCs增殖能力变化：不同时间Wharton's jelly产生原代WJCs的增殖曲线无差别。
BTSCs的原代培养和扩增：接种细胞1天后,倒置显微镜下观察可见大量悬浮生长、形态多样的肿瘤细胞团和一些悬浮生长的单细胞,折光性不明显。7天后可见由2-5个细胞组成大小不等的悬浮细胞球。10天后见细胞球明显增多,体积增大,形状规则且折光性强。将细胞球吹散后传代培养,大部分1天后形成新的细胞球。
BTSCs巢蛋白Nestin和GFAP免疫荧光鉴定：未经诱导的BTSCs表达Nestin而不表达GFAP。
WJCs与BTSCs共培养：倒置显微镜下可见：单独孵育WJCs 24h后用PBS冲洗24孔板,去除未贴壁的细胞,板内可见大部分呈梭形,胞核呈卵圆形,散在均匀分布生长的典型WJCs。共培养1天后,可见两种现象：一种是BTSCs能够形成典型的肿瘤干细胞球,球体圆形或椭圆形,大小不等,折光性强,每个视野能见到3-5克隆团且与贴壁的WJCs相粘连,而WJCs形态数目与WJCs阴性对照几乎无差别；第二种是BTSCs很难形成肿瘤干细胞球,多为单细胞均匀分散,漂浮在培养液里,覆盖在WJCs之上,而WJCs与WJCs阴性对照相比增殖迅速,形成很多细胞集落,胞体伸长形成细胞间连接,方向不整。BTSCs阴性对照开始形成典型的肿瘤干细胞球漂浮在培养基中并且可以滚动。
共培养3天后,可见两种现象：一种是与WJCs相连的BTSCs球体发生贴壁现象,肿瘤细胞从球体周围向外游出,形态多为星形,梭形,形成细胞突触呈放射状,并且与周围增生的WJCs形成连接,细胞体积比WJCs小很多,球体周围有一些单个的肿瘤细胞脱落漂浮在培养基中,WJCS增生明显尤其是与BTSCs连接处的WJCs形成细胞集落较多,细胞体积开始变大包体伸长；第二种是BTSCs依然分散漂浮在培养基中,形态与共培养1天时无差别,而WJCs增生明显占据视野的70%,细胞扁平宽大伸长。BTSCs阴性对照依然是典型的肿瘤干细胞球,WJCs阴性对照细胞数目变化不大,形态增大伸长,但依然是梭形。
共培养7天后,可见两种现象：一种是肿瘤干细胞球贴壁分化的细胞增多,整个球体紧贴在孔底,BTSCs与WJCs之间的链接更加紧密,WJCs与WJCs阴性对照相比包体较大扁平；第二种是BTSCs散落在WJCs之间,有的单细胞紧紧贴在孔底上,多为星形。
通过检测共培养的BTSCs的免疫表型可以得到：脑肿瘤级别高的组织分离得到的BTSCs表达CD133较高,与WJCs共培养后可以看到随着共培养天数增加,BTSCs表达CD133的量逐渐减少。
第3天离心所得的共培养上清液重悬第3代BTSCs与正常培养基培养的第3代BTSCs生长曲线的对比,在共培养上清液中培养的BTSCs比在正常培养基中培养的BTSCs的生长曲线要平。
结论：
1.成功使用组织块法在体外培养出WJCs,并能将脐带组织块充分利用获取更多间充质干细胞,以及采用酶消化法从胶质瘤组织中成功分离出BTSCs,通过换液法尽可能到纯化干细胞。
2. WJCs与BTSCs共培养后在倒置显微镜下可见脑肿瘤干细胞球随着培养时间的增加出现分解、贴壁、分化现象；贴壁的脑肿瘤干细胞免疫荧光染色GFAP和Nestin均阳性,WJCs能促使BTSCs分化。
3.恶性程度高的脑肿瘤组织培养的BTSCs表达CD133量越高,而与WJCs共培养后随着时间的变化均出现CD133表达量降低,WJCs与BTSCs体外共培养后可降低BTSCs表面标记物CD133的阳性率表达。
4.共培养上清液培养的BTSCs与正常培养基培养的BTSCs相比,增殖能力明显受到抑制,WJCs能够分泌抑制性因子产生对BTSCs的抑制作用。
Background and Objective:
The doctrine of cancer stem cells, which reckon that a small minority of tumor cells with the ability of self-renewal and potentially differentiation in tumor tissue are responsible for proliferation, growth, metastasis and recurrence of tumor, has been put forward more than 30 years ago. In 1992, Reynolds put forward the concept of neural stem cells (NSCs) based on the research on mice.5 years later, the article, wrote by Mckay concerned about NSCs, published in Science has been recognized. In 2003, Singh cultured and identified brain tumor stem cells (BTSCs) from different pathological types of human brain tumor. BTSCs has the capability of proliferation, self-renewal, differentiation and expression of neural stem cell surface marker CD133, which makes it become a new target for treatment of brain tumors. In recent years, numerous studies confirm that BTSCs is the origin of the brain tumor cells, play a decisive role in the tumor occurrence and development, and is also the roots of radiation therapy and chemotherapy tolerance.The BTSCs-related molecular markers and tumorigenic mechanism is a hot research field, but also for the future of brain tumor research and clinical treatment.It is proven that once NSCs has been transplanted into the brain tumor, it could acquire the ability of directional migration, proliferation surrounding the brain tumor, thereby reducing the invasiveness of brain tumors.However, graft-versus-host disease and the potential tumorigenicity after transplantation and ethical issues of sources of tissue make people pay more attention to the mesenchymal stem cells (MSCs).
Mesenchymal stem cells (MSCs) is derived from the number of embryos in stem cells, with a high degree of self-renewal capacity and more potential to divide, in appropriate in vivo or in vitro environment, MSCs not only differentiate into hematopoietic cells, Has divided hematopoiesis outside for a variety of organizations, especially in the cells of the embryo's ability, and even cross-layer differentiation of embryonic nerve cells of the tumor of origin potential, so they have a high degree of plasticity, is a new type of seed cells in cell transplantation and tissue engineering that have broad application prospects. Previous studies have focused on the study of bone marrow MSCs, but there are also other organizations that MSCs, including the mobilization of peripheral blood, umbilical cord blood, adipose tissue, placenta tissue and deciduous, however, these organizations have a limited number of cell restrictions on the research and development.
The study found that in recent years people from the AMT, the umbilical cord, amniotic fluid can also be extracted from a good proliferation of MSCs, which for better access to MSCs to provide a new way, and with the in-depth study found that MSCs, MSCs Can also be induced to differentiate into cardiac cells, liver cells and neurons and other cells, MSCs a variety of differentiation potential of the research staff to be more and more attention. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self-renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well-tolerated by the immune system, and they have the ability to home to tumors. WJCs are therapeutic in several different pre-clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. Whereas the source of umbilical cord is quite rich, the use of it is without ethics problem.This study is to be concentrated mainly on the research of the biological characteristics of WJCs, whether it can influence the biological characteristics of BTSCs after the two type cells co-cultured in vitro, and then provide a theoretical basis for cell therapy.
Methods:
The isolation and cultivation of WJCs:Taking about 10cm umbilical cord being removed the umbilical cord membrane, the two umbilical arteries and one umbilical vein were, and retained Wharton's jelly. Wharton's jelly is fully washed by PBS buffer and cut into pieces to the size of 2mm×2mm×2mm.
WJCs immunophenotype identified:flow cytometry CD29, CD44, HLA-ABC, HLA-DR, CD34, CD45.
WJCs immunofluorescence identified:GFAP and Nestin.
Comparison of immune phenotype of WJCs came out from Wharton's jelly at different times:flow cytometry CD29, CD44, HLA-ABC.
Comparison of Proliferation ability of WJCs came out from Wharton's jelly at different times by CCK-8:mapping the cell growth curve of 7 days.
The isolation and cultivation of BTSCs:wash fresh tumor tissue with PBS buffer and then remove the necrotic tissue and capillary from it. Filter the tissue with 80 mesh and then digest in the 2.5 g/L trypsin and 1.0 g/L collagenaseⅣat 37℃for 30 minutes. After digestion, filter the digestive juice with 200 mesh then remained single-cell suspension. Counting of living cells in a final concentration of 2×105/ml, then inoculate it in serum-free medium (SFM) and placed in T75 culture flask added 10ml culture medium into.
BTSCs immunophenotype identified:flow cytometry CD133.
BTSCs immunofluorescence identified:GFAP and Nestin.
CCK-8 assay of proliferation of BTSCs:mapping the cell growth curve of 7 days.
WJCs co-cultured experiments with BTSCs:Co-culture of two kinds of cells in 24-well plates in serum-free medium without any growth factor. Collect the centrifuged suspension cells, and then analysis expression of CD133 by flow cytometry and at day 3 and 7 respectively. Analysis adherent cells expression of Nestin and GFAP by immunofluorescence assay at day 3 and 7 respectively. Co-culture supernatant (CCS) acquired at day 3 resuspend BTSCs. Compare the differences of cell growth curve between cultured in CCS and in normal suspension by Microplate Reader.
Results:
The isolation and cultivation of WJCs:
It can be seen that a single spindle or triangular adherent cells come out from tissue being adherent after 3 to 5 days. After 1 week, adherent cells are swimming out of tissue. Cells gradually become from the multi-layer to single-layer, loose to intensive, and small to big. Cell morphology is mostly full of long spindle-shaped or flat fibroblast-like cells with prominent nucleoli. Two weeks later, it turns out to be uniform spindle cell morphology similar to fibroblasts, showing parallel growth or vortex growth.
WJCs immunophenotype identified:high expression of CD29 (91.50%), low expression of CD44 (36.92%), HLA-ABC (28.10%), not express CD34 (0.72%), CD45 (0.11%) and HLA-DR (0.80%)
WJCs immunofluorescence identified:None-induced WJCs does not express Nestin and GFAP.
Comparison of immune phenotype of WJCs came out from Wharton's jelly at different times:no difference in expression of CD29, CD44, HLA-ABC.
Comparison of Proliferation ability of MSCs came out from WJCs at different times by CCK-8:no difference in cell growth curve.
The isolation and cultivation of BTSCs:1 days after inoculation, it is observed under the inverted microscope that a large number of suspended growth and diversity of tumor cell clusters and some single-cell suspension with not obvious refraction.7 days later it can be seen suspension bulk of cells are composed by 2 to 5 the size of cells. After 10 days bulk of cell volume significantly increased with the regular shape and strong refraction. Disperse then subculture the cells, the majority of cells formed news bulk of cells 1 day later.
BTSCs immunofluorescence identified:Without induced BTSCs express Nestin but GFAP.
WJCs co-cultured experiments with BTSCs:After 24h separately incubation of WJCs in the 24-well plate, then wash plate with PBS to remove non adherent cells. It can be seen most of the spindle, homogeneous scattered growth of the typical WJCs in the bottom of the plate. After 1 day co-cultivation, it is shown two phenomena. One is that BTSCs can form a typical sphere of cells, it can be viewed 3 to 5 clone and connected with adherent WJCs, while the number and form of WJCs is almost no difference with negative control; another is that BTSCs can hardly form a typical sphere of cells, they are more dispersed as single cells, floating in the culture medium covering WJCs. Compared with negative control, WJCs rapidly proliferate and form many cell colony with the elongation of cell body connected with each other. Negative control of BTSCs began to form typical sphere of cells floating and rolling in the medium.
After 3 day co-cultivation, it is shown two phenomena. One phenomenon is that sphere of BTSCs connected with WJCs begin adherent to the plate. Tumor cells are climbing out from Sphere of BTSCs with the astral, spindle morphological, and format the radial synapse and connection with proliferation of WJCs around. Adherent BTSCs is much smaller than the WJCs. Another is that BTSCs is still scattered floating in the medium without morphology changing compared to the first day. But WJCs is proliferating obviously, becoming wider and bigger and occupying the range of 70%of the vision.
After 7 day co-cultivation, there is shown two phenomena. One phenomenon is that most of sphere of BTSCs connected with WJCs are adherent to the plate. Another is that BTSCs is scattered floating in the medium or adherent to the plate. Negative control of BTSCs and WJCs has normal morphology.
We can see the results that the higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was and CD133+in BTSCs declined when co-cultured with WJCs.
Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously.
Conclusions:
1. WJCs cultured by situ cultivation and BTSCs used enzyme digestion way respectively, and gathering the 3rd passage of WJCs though subculturing as well as BTSCs. The original WJCs came out from Wharton's jelly at different times express stable positive immunophenotype and have stable growth curve. That indicates more WJCs can be gained and used.
2. With the cocultivation days increasing, the phenomenon that tumor sphere cells began to be decomposed, adherent and differentiated observed by an inverted microscope. BTSCs in the co-cultured group expressed GFAP and Nestin when adherent and differentiated.
3. The higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was. CD133+in BTSCs declined when co-cultured with WJCs.
4. Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously. Results indicated that CD133+expression and proliferative capacity of BTSCs went down and BTSCs underwent differentiation during the co-culture in vitro.
肿瘤干细胞学说这一概念的提出距今已有30多年的历史,该学说认为肿瘤组织中存在极少数具有自我更新和分化潜能的肿瘤细胞,并且是肿瘤增殖、生长、转移和复发的根源。1992年Reynolds对小鼠脑细胞的研究提出了神经干细胞(Neural stem cells, NSCs)概念,1997年Mckay在Science上发表NSCs的文章得到了公认。而2003年Singh从不同病理类型的人脑肿瘤中培养、鉴定出了一种拥有增殖、自我更新和分化能力并且表达神经干细胞表面标记物CD133的脑肿瘤干细胞(Brain Tumor Stem Cells, BTSCs)。近年来,大量研究证实,BTSCs是脑肿瘤的起源细胞,在肿瘤的发生、发展过程中起决定性作用,也是脑肿瘤对放射治疗和化学治疗产生耐受的根源所在,使得BTSCs成为治疗脑肿瘤的新靶点。而BTSCs相关分子标记物及其致瘤机制是神经肿瘤研究的热点领域,也为未来脑肿瘤的研究和临床治疗指明了方向。人们证明,移植后的NSCs具有向脑肿瘤定向迁徙的能力,能在肿瘤周边增殖并包绕脑肿瘤,从而降低脑肿瘤的侵袭性。然而宿主对NSCs移植的排斥和NSCs移植后潜在的致瘤性以及组织来源的伦理问题使得人们把目光投向了间充质干细胞(Mesenchymal Stromal Cells, MSCs)。
MSCs是来源于中胚层的多能干细胞,具有高度自我更新能力和多向分化潜能,在适宜的体内体外环境下可以分化为造血细胞以及多种组织特别是中胚层组织细胞的能力,甚至还具有跨胚层分化为神经外胚层起源组织细胞的潜能,因而具有高度的可塑性,是细胞移植和组织工程的新型种子细胞,具有广阔的应用前景。以往研究的重点放在骨髓MSCs的研究上,而其它组织中也存在有MSCs,包括动员的外周血、脐血、乳牙、脂肪、胎盘等,然而,从这些组织中得到的细胞数量有限,从而限制了其研究发展。
人脐带Wharton's jelly间充质干细胞(Mesenchymal Stromal Cells derived from Wharton's jelly cells, WJCs)满足了国际细胞治疗协会规定的MSCs的特点：如贴壁生长,自我更新,表达MSCs共有的表面标记物,能够向骨、软骨、脂肪、肌肉、神经细胞诱导分化,能够支持其它干细胞的扩增,对免疫系统有良好的耐受性,对肿瘤有定向迁徙的特性。WJCs通过其营养支持和免疫调节作用对很多临床前期的人类疾病的动物模型有治疗作用,如神经退行性疾病,肿瘤,心脏病等。而且脐带组织来源广泛,不存在伦理学问题。因此,本实验主要研究WJCs的生物学特性,以及WJCs和BTSCs在体外共培养后,WJCs是否能够影响BTSCs的生物学特性,并为细胞治疗提供理论依据。
方法：
WJCs的分离、培养：取长约10cm脐带,剥离脐带外膜并将两条脐动脉和一条脐静脉分离去除,保留Wharton's jelly,并用PBS缓冲液充分冲洗并将其剪碎至2mm×2mm×2mm大小组织块,置于T25培养瓶中,加入含有20μg/L的bFGF和体积分数为10%的FBS的DMEM/F12培养基5ml培养。
WJCs免疫表型鉴定：流式细胞仪检CD29、CD44、HLA-ABC、HLA-DR、CD34、CD45。
WJCs免疫荧光鉴定：GFAP和Nestin。
比较脐带Wharton's jelly不同时间产出原代WJCs的免疫表型变化：流式细胞仪检CD29、CD44、HLA-ABC。
CCK-8法比较Wharton's jelly不同时间产出原代WJCs增殖能力变化：绘制7天细胞生长曲线。
BTSCs的分离、培养：将新鲜的肿瘤组织用PBS缓冲液充分冲洗并去除外周坏死组织和毛细血管,在80目滤网中研碎过滤后加入2.5g/L胰酶和1.0g/L胶原酶Ⅳ37℃消化30分钟,将消化液过200滤网后制成单细胞悬液。计数活细胞,以2×105／ml终浓度接种于无血清培养基(Serum-free medium, SFM)中,置于T75培养瓶中,添加培养基至10ml培养。
BTSCs免疫表型鉴定：流式细胞仪检CD133
BTSCs免疫荧光鉴定：GFAP和Nestin
CCK-8法检测BTSCs增殖率：绘制7天细胞生长曲线。
WJCs与BTSCs共培养实验：应用不添加任何生长因子的无血清培养基将两种细胞在24孔板中进行直接共培养。第3、7天时分别离心收集孔中悬浮细胞,应用流式细胞术检测BTSCs CD133表达；贴壁细胞应用免疫荧光法检测神经干细胞标记物巢蛋白(Nestin)和神经胶质细胞标志物胶质纤维酸性蛋白(GFAP)表达；将第3天离心所得的共培养上清液(Co-culture supernatant, CCS)重悬第3代BTSCs并与正常培养悬浮的第3代BTSCs置入96孔板中,应用酶标仪检测两组细胞生长曲线的差异。
结果：
WJCs的原代培养和扩增：组织块贴壁3-5天后可零星见到单个的梭形或三角形的贴壁细胞,1周后可见贴壁组织块周围有细胞向外游出,细胞由多层逐渐变为单层,细胞由密集变为松散,体型由小到大,细胞形态多为饱满的长梭形或扁平形的成纤维样细胞,核仁明显。两周后,细胞形态变为均一的纺锤形,形态类似成纤维细胞,呈平行排列生长或旋涡状生长。
流式细胞仪检测第3代WJCs显示：高表达CD29(91.50%)；低表达CD44(36.92%)、HLA-ABC(28.10%)；不表达CD34(0.72%)、CD45(0.11%)和HLA-DR(0.80%)
对WJCs进行Nestin和GFAP免疫荧光染色后显示：未经诱导的WJCs在荧光显微镜下不发出荧光,即不表达Nestin和GFAP。
比较脐带Wharton's jelly不同时间产出原代WJCs的免疫表型变：流式细胞仪检测CD29,CD44,HLA-ABC变化无统计学意思。
CCK-8法比较Wharton's jelly不同时间产出原代WJCs增殖能力变化：不同时间Wharton's jelly产生原代WJCs的增殖曲线无差别。
BTSCs的原代培养和扩增：接种细胞1天后,倒置显微镜下观察可见大量悬浮生长、形态多样的肿瘤细胞团和一些悬浮生长的单细胞,折光性不明显。7天后可见由2-5个细胞组成大小不等的悬浮细胞球。10天后见细胞球明显增多,体积增大,形状规则且折光性强。将细胞球吹散后传代培养,大部分1天后形成新的细胞球。
BTSCs巢蛋白Nestin和GFAP免疫荧光鉴定：未经诱导的BTSCs表达Nestin而不表达GFAP。
WJCs与BTSCs共培养：倒置显微镜下可见：单独孵育WJCs 24h后用PBS冲洗24孔板,去除未贴壁的细胞,板内可见大部分呈梭形,胞核呈卵圆形,散在均匀分布生长的典型WJCs。共培养1天后,可见两种现象：一种是BTSCs能够形成典型的肿瘤干细胞球,球体圆形或椭圆形,大小不等,折光性强,每个视野能见到3-5克隆团且与贴壁的WJCs相粘连,而WJCs形态数目与WJCs阴性对照几乎无差别；第二种是BTSCs很难形成肿瘤干细胞球,多为单细胞均匀分散,漂浮在培养液里,覆盖在WJCs之上,而WJCs与WJCs阴性对照相比增殖迅速,形成很多细胞集落,胞体伸长形成细胞间连接,方向不整。BTSCs阴性对照开始形成典型的肿瘤干细胞球漂浮在培养基中并且可以滚动。
共培养3天后,可见两种现象：一种是与WJCs相连的BTSCs球体发生贴壁现象,肿瘤细胞从球体周围向外游出,形态多为星形,梭形,形成细胞突触呈放射状,并且与周围增生的WJCs形成连接,细胞体积比WJCs小很多,球体周围有一些单个的肿瘤细胞脱落漂浮在培养基中,WJCS增生明显尤其是与BTSCs连接处的WJCs形成细胞集落较多,细胞体积开始变大包体伸长；第二种是BTSCs依然分散漂浮在培养基中,形态与共培养1天时无差别,而WJCs增生明显占据视野的70%,细胞扁平宽大伸长。BTSCs阴性对照依然是典型的肿瘤干细胞球,WJCs阴性对照细胞数目变化不大,形态增大伸长,但依然是梭形。
共培养7天后,可见两种现象：一种是肿瘤干细胞球贴壁分化的细胞增多,整个球体紧贴在孔底,BTSCs与WJCs之间的链接更加紧密,WJCs与WJCs阴性对照相比包体较大扁平；第二种是BTSCs散落在WJCs之间,有的单细胞紧紧贴在孔底上,多为星形。
通过检测共培养的BTSCs的免疫表型可以得到：脑肿瘤级别高的组织分离得到的BTSCs表达CD133较高,与WJCs共培养后可以看到随着共培养天数增加,BTSCs表达CD133的量逐渐减少。
第3天离心所得的共培养上清液重悬第3代BTSCs与正常培养基培养的第3代BTSCs生长曲线的对比,在共培养上清液中培养的BTSCs比在正常培养基中培养的BTSCs的生长曲线要平。
结论：
1.成功使用组织块法在体外培养出WJCs,并能将脐带组织块充分利用获取更多间充质干细胞,以及采用酶消化法从胶质瘤组织中成功分离出BTSCs,通过换液法尽可能到纯化干细胞。
2. WJCs与BTSCs共培养后在倒置显微镜下可见脑肿瘤干细胞球随着培养时间的增加出现分解、贴壁、分化现象；贴壁的脑肿瘤干细胞免疫荧光染色GFAP和Nestin均阳性,WJCs能促使BTSCs分化。
3.恶性程度高的脑肿瘤组织培养的BTSCs表达CD133量越高,而与WJCs共培养后随着时间的变化均出现CD133表达量降低,WJCs与BTSCs体外共培养后可降低BTSCs表面标记物CD133的阳性率表达。
4.共培养上清液培养的BTSCs与正常培养基培养的BTSCs相比,增殖能力明显受到抑制,WJCs能够分泌抑制性因子产生对BTSCs的抑制作用。
Background and Objective:
The doctrine of cancer stem cells, which reckon that a small minority of tumor cells with the ability of self-renewal and potentially differentiation in tumor tissue are responsible for proliferation, growth, metastasis and recurrence of tumor, has been put forward more than 30 years ago. In 1992, Reynolds put forward the concept of neural stem cells (NSCs) based on the research on mice.5 years later, the article, wrote by Mckay concerned about NSCs, published in Science has been recognized. In 2003, Singh cultured and identified brain tumor stem cells (BTSCs) from different pathological types of human brain tumor. BTSCs has the capability of proliferation, self-renewal, differentiation and expression of neural stem cell surface marker CD133, which makes it become a new target for treatment of brain tumors. In recent years, numerous studies confirm that BTSCs is the origin of the brain tumor cells, play a decisive role in the tumor occurrence and development, and is also the roots of radiation therapy and chemotherapy tolerance.The BTSCs-related molecular markers and tumorigenic mechanism is a hot research field, but also for the future of brain tumor research and clinical treatment.It is proven that once NSCs has been transplanted into the brain tumor, it could acquire the ability of directional migration, proliferation surrounding the brain tumor, thereby reducing the invasiveness of brain tumors.However, graft-versus-host disease and the potential tumorigenicity after transplantation and ethical issues of sources of tissue make people pay more attention to the mesenchymal stem cells (MSCs).
Mesenchymal stem cells (MSCs) is derived from the number of embryos in stem cells, with a high degree of self-renewal capacity and more potential to divide, in appropriate in vivo or in vitro environment, MSCs not only differentiate into hematopoietic cells, Has divided hematopoiesis outside for a variety of organizations, especially in the cells of the embryo's ability, and even cross-layer differentiation of embryonic nerve cells of the tumor of origin potential, so they have a high degree of plasticity, is a new type of seed cells in cell transplantation and tissue engineering that have broad application prospects. Previous studies have focused on the study of bone marrow MSCs, but there are also other organizations that MSCs, including the mobilization of peripheral blood, umbilical cord blood, adipose tissue, placenta tissue and deciduous, however, these organizations have a limited number of cell restrictions on the research and development.
The study found that in recent years people from the AMT, the umbilical cord, amniotic fluid can also be extracted from a good proliferation of MSCs, which for better access to MSCs to provide a new way, and with the in-depth study found that MSCs, MSCs Can also be induced to differentiate into cardiac cells, liver cells and neurons and other cells, MSCs a variety of differentiation potential of the research staff to be more and more attention. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self-renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well-tolerated by the immune system, and they have the ability to home to tumors. WJCs are therapeutic in several different pre-clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. Whereas the source of umbilical cord is quite rich, the use of it is without ethics problem.This study is to be concentrated mainly on the research of the biological characteristics of WJCs, whether it can influence the biological characteristics of BTSCs after the two type cells co-cultured in vitro, and then provide a theoretical basis for cell therapy.
Methods:
The isolation and cultivation of WJCs:Taking about 10cm umbilical cord being removed the umbilical cord membrane, the two umbilical arteries and one umbilical vein were, and retained Wharton's jelly. Wharton's jelly is fully washed by PBS buffer and cut into pieces to the size of 2mm×2mm×2mm.
WJCs immunophenotype identified:flow cytometry CD29, CD44, HLA-ABC, HLA-DR, CD34, CD45.
WJCs immunofluorescence identified:GFAP and Nestin.
Comparison of immune phenotype of WJCs came out from Wharton's jelly at different times:flow cytometry CD29, CD44, HLA-ABC.
Comparison of Proliferation ability of WJCs came out from Wharton's jelly at different times by CCK-8:mapping the cell growth curve of 7 days.
The isolation and cultivation of BTSCs:wash fresh tumor tissue with PBS buffer and then remove the necrotic tissue and capillary from it. Filter the tissue with 80 mesh and then digest in the 2.5 g/L trypsin and 1.0 g/L collagenaseⅣat 37℃for 30 minutes. After digestion, filter the digestive juice with 200 mesh then remained single-cell suspension. Counting of living cells in a final concentration of 2×105/ml, then inoculate it in serum-free medium (SFM) and placed in T75 culture flask added 10ml culture medium into.
BTSCs immunophenotype identified:flow cytometry CD133.
BTSCs immunofluorescence identified:GFAP and Nestin.
CCK-8 assay of proliferation of BTSCs:mapping the cell growth curve of 7 days.
WJCs co-cultured experiments with BTSCs:Co-culture of two kinds of cells in 24-well plates in serum-free medium without any growth factor. Collect the centrifuged suspension cells, and then analysis expression of CD133 by flow cytometry and at day 3 and 7 respectively. Analysis adherent cells expression of Nestin and GFAP by immunofluorescence assay at day 3 and 7 respectively. Co-culture supernatant (CCS) acquired at day 3 resuspend BTSCs. Compare the differences of cell growth curve between cultured in CCS and in normal suspension by Microplate Reader.
Results:
The isolation and cultivation of WJCs:
It can be seen that a single spindle or triangular adherent cells come out from tissue being adherent after 3 to 5 days. After 1 week, adherent cells are swimming out of tissue. Cells gradually become from the multi-layer to single-layer, loose to intensive, and small to big. Cell morphology is mostly full of long spindle-shaped or flat fibroblast-like cells with prominent nucleoli. Two weeks later, it turns out to be uniform spindle cell morphology similar to fibroblasts, showing parallel growth or vortex growth.
WJCs immunophenotype identified:high expression of CD29 (91.50%), low expression of CD44 (36.92%), HLA-ABC (28.10%), not express CD34 (0.72%), CD45 (0.11%) and HLA-DR (0.80%)
WJCs immunofluorescence identified:None-induced WJCs does not express Nestin and GFAP.
Comparison of immune phenotype of WJCs came out from Wharton's jelly at different times:no difference in expression of CD29, CD44, HLA-ABC.
Comparison of Proliferation ability of MSCs came out from WJCs at different times by CCK-8:no difference in cell growth curve.
The isolation and cultivation of BTSCs:1 days after inoculation, it is observed under the inverted microscope that a large number of suspended growth and diversity of tumor cell clusters and some single-cell suspension with not obvious refraction.7 days later it can be seen suspension bulk of cells are composed by 2 to 5 the size of cells. After 10 days bulk of cell volume significantly increased with the regular shape and strong refraction. Disperse then subculture the cells, the majority of cells formed news bulk of cells 1 day later.
BTSCs immunofluorescence identified:Without induced BTSCs express Nestin but GFAP.
WJCs co-cultured experiments with BTSCs:After 24h separately incubation of WJCs in the 24-well plate, then wash plate with PBS to remove non adherent cells. It can be seen most of the spindle, homogeneous scattered growth of the typical WJCs in the bottom of the plate. After 1 day co-cultivation, it is shown two phenomena. One is that BTSCs can form a typical sphere of cells, it can be viewed 3 to 5 clone and connected with adherent WJCs, while the number and form of WJCs is almost no difference with negative control; another is that BTSCs can hardly form a typical sphere of cells, they are more dispersed as single cells, floating in the culture medium covering WJCs. Compared with negative control, WJCs rapidly proliferate and form many cell colony with the elongation of cell body connected with each other. Negative control of BTSCs began to form typical sphere of cells floating and rolling in the medium.
After 3 day co-cultivation, it is shown two phenomena. One phenomenon is that sphere of BTSCs connected with WJCs begin adherent to the plate. Tumor cells are climbing out from Sphere of BTSCs with the astral, spindle morphological, and format the radial synapse and connection with proliferation of WJCs around. Adherent BTSCs is much smaller than the WJCs. Another is that BTSCs is still scattered floating in the medium without morphology changing compared to the first day. But WJCs is proliferating obviously, becoming wider and bigger and occupying the range of 70%of the vision.
After 7 day co-cultivation, there is shown two phenomena. One phenomenon is that most of sphere of BTSCs connected with WJCs are adherent to the plate. Another is that BTSCs is scattered floating in the medium or adherent to the plate. Negative control of BTSCs and WJCs has normal morphology.
We can see the results that the higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was and CD133+in BTSCs declined when co-cultured with WJCs.
Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously.
Conclusions:
1. WJCs cultured by situ cultivation and BTSCs used enzyme digestion way respectively, and gathering the 3rd passage of WJCs though subculturing as well as BTSCs. The original WJCs came out from Wharton's jelly at different times express stable positive immunophenotype and have stable growth curve. That indicates more WJCs can be gained and used.
2. With the cocultivation days increasing, the phenomenon that tumor sphere cells began to be decomposed, adherent and differentiated observed by an inverted microscope. BTSCs in the co-cultured group expressed GFAP and Nestin when adherent and differentiated.
3. The higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was. CD133+in BTSCs declined when co-cultured with WJCs.
4. Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously. Results indicated that CD133+expression and proliferative capacity of BTSCs went down and BTSCs underwent differentiation during the co-culture in vitro.
引文
[1]Reynolds, B. A., and Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science (Wash. DC),1992; 255:1707-1710.
[2]Mckay R. Stem cells in central nervous, Science,1997; 276 (5309):66-71.
[3]Singh SK., Clarke ID. Identification of a cancer stem cell in human brain tumors. Cancer Research,2003; 63:5821-5828.
[4]Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, Fuks Z, Kolesnick R. Tumor response to radiotherapy regulated by endothelial cell apoptosis, Science,2003; 300:1155-1159.
[5]Ehtesham M, Kabos P, Kabosova A, Neuman T, Black KL, Yu JS. The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma. Cancer Res,2002; 62 (20):5657-5663.
[6]Li S, Tokuyama T, Yamamoto J, Koide M, Yokota N, Namba H. Bystander effect-mediated gene therapy of gliomas using genetically engineered neural stem cells. Cancer Gene Ther,2005; 12:600-607.
[7]Dimitrios K, Ibrahim K.The potential use of stem cells in multiple sclerosis:An overview of the preclinical experience. Clinical Neurology and Neurosurgery,2008; 110:889-896.
[8]Deryl L. Concise. Review:Wharton's jelly-derived cells are a primitive stromal cell population [J]. Stem Cells,2008; 26:591-599.
[9]Peng HQ, Levitin-Smith M, Rochelson B, et al. Umbilical cord stricture and overcoiling are common causes of fetal demise. Pediatr Dev Pathol,2006; 9:14-19.
[10]Trevisanuto D, Doglioni N, Zanardo V, et al. Overcoiling of the umbilical cord. J Pediatr, 2007;150:112.
[11]Toren A, Nagler A, Amariglio N, et al. Successful human umbilical cord blood stem cell transplantation with conditioning in severe combined immune deficiency. Bone Marrow Transplant,1999; 23 (4):405-408.
[12]Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors. Stem Cells,2005; 23:220-229.
[13]Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003; 21:105-110.
[14]Kestendjieva S, Kyurkchiev D, Tsvetkova G, et al. Characterization of mesenchymal stem cells isolated from the human umbilical cord. Cell Biol Int,2008; 32 (7):724-732
[15]Wang Y, Pu YY, Fang L, et al. Jiangsu Daxue Xuebao (yixueban),2008;18(20):166-167.
[16]Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of the stem cells in human umbilical cord stroma:in situ and in vitro surveys. Stem Cells,2007; 25:319-331.
[17]Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton's jelly form neurons and glia. Stem Cells,2003; 21:50-60.
[18]Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica,2006; 91:1017-1026.
[19]Markov V, Kusumi K, Tadesse MG, et al, Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles. Stem Cells Dev,2007 Feb; 16(1):53-73.
[20]Weiss ML, Medicetty S, Bledsoe AR, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells,2006; 24:781-792.
[21]Fu YS, Shih YT, Cheng YC, et al. Transformation of human umbilical mesenchymal cells into neurons in vitro. J Biomed Sci,2004; 11:652-660.
[22]Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells,2004; 22:1330-1337.
[23]Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells Suppress lymphocyte Proliferation in vitro and Prolong skin graf tsurvival in vivo. Exp Hematol, 2002; 30 (1):42-48.
[24]Kao CL, Lin HT, Chen YW, et al. Fibronectin suppresses lipopolysaccharide-induced liver damage and promotes the cytoprotection abilities of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells. Transplant Proc,2007 Dec; 39(10):3444-5.
[25]Nirmalanandhan VS, Levy MS, Huth AJ, Butler DL, Effects of cell seeding density and collagen concentration on contraction kinetics of mesenchymal stem cell-seeded collagen constructs. Tissue Eng,2006 Jul; 12(7):1865-72.
[26]周长辉,田毅,杨波.人脐带沃顿胶间充质干细胞对人外周T淋巴细胞的免疫调节作用.中国组织工程研究与临床康复,2010；14(14)：2485-2491.
[27]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas. Proc Natl Acad Sci U S A, 2000; 97:12846-12851.
[28]Studeny M, Marini FC, Champlin RE, et al. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res,2002; 62:3603-3608.
[29]Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells:potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst, 2004; 96:1593-1603.
[30]Rachakatla RS, Marini F, Weiss ML, et al. Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors. Cancer Gene Ther,2007; 14: 828-835.
[31]Galli R, Binda E, Orfanelli U, et al. Isolation and characterization of tumorigenic stem-like neural precursors from human glioblastoma [J]. Cancer Res 2004; 64 (19):7011.
[32]Clark PA, Treisman DM, Ebben J, Kuo JS. Developmental signaling pathways in brain tumor-derived stem-like cells. Dev Dyn,2007 Dec; 236(12):3297-308.
[33]Nakano I, Saigusa K, Kornblum HI. BMPing off glioma stem cells. Cancer Cell,2008 Jan; 13(1):3-4.
[34]Sutter R, Yadirgi G, Marino S. Neural stem cells, tumour stem cells and brain tumours: dangerous relationships? Biochim Biophys Acta,2007 Dec; 1776(2):125-37.
[35]Sachan DS, Yatim AM. Suppression of aflatoxin B1-induced lipid abnormalities and macromolecule-adduct formation by L-carnitine. J Environ Pathol Toxicol Oncol,1992 Jul-Aug; 11(4):205-10.
[36]Ekberg S, Carlsson L, Carlsson B, et al, Plasma growth hormone pattern regulates epidermal growth factor (EGF) receptor messenger ribonucleic acid levels and EGF binding in the rat liver. Endocrinology,1989 Oct; 125(4):2158-66.
[37]Liu HM, Yang LH, Yang YJ. Schwann cell properties:3. C-fos expression, bFGF production, phagocytosis and proliferation during Wallerian degeneration. J Neuropathol Exp Neurol,1995 Jul; 54(4):487-96.
[38]Nelson R. Brain cancer stem cells may drive tumor formation [J]. Lancet Neurol,2005; 4 (1):17.
[39]Kang MK, Kang SK. Tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma [J].Stem Cells Dev,2007; 16 (5):837.
[40]Park TD, Deric M, Lin Y, et al. N2CoR pathway targeting induces brain tumor stem cell differentiation [J]. Neurology 2006; 66 (5):393.
[41]Piccirillo SG, Vescovi AL. Brain tumour stem cells:possibilities of new therapeutic st rategies [J]. Expert Opin Biol Ther,2007; 7 (8):1129.
[42]许亮,董军,兰青.干细胞标志物CD133和Nestin在不同恶性程度胶质瘤组织中的表达及其与预后的关系.中国神经肿瘤杂志,2009；7(3)：175-179.
[43]Sobolewskia K, Malkowskia A. Wharton's Jelly as a Reservoir of Peptide Growth Factors. Placenta 2005; 26:747-752.
[44]Weiss ML, Medicetty S, Bledsoe AR, etal. Human umbilical cord matrix stem cells: Preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease [J]. Stem Cells,2006; 24 (3):781-792.
[45]侯克东,卢世璧.人脐带Wharton胶中间充质干细胞的分离,培养与鉴定.解放军医学杂志,2008；33(4)：375-378.
[46]Zarling JM, Shoyab M. Oncostatin M:a growth regulator produced by differentiated histiocytic lymphoma cells. Proc Natl Acad Sci U S A,1986 Dec; 83 (24):9739-43.
[47]Bhandari RN, Riccalton LA, Lewis AL, et al. Liver tissue engineering:a role for co-culture systems in modifying hepatocyte function and viability. Tissue Eng,2001; 7(3):345-357.
[48]Zhu L, Wei W, Zheng YQ, et al. Effects and mechanisms of total glucosides of paeony on joint damage in ratcollagen-induced arthritis. Inflamm Res,2005; 54(5):211-220.
[49]Yassini PR, Stickler DL, Bloomfield SM et al. Gliomastimulated chemoattraction of endothelial cells and fibroblastsin vitro:a model for the study of glioma-induce angiogenesis. Metab Brain Dis,1994; 9(4):391-399.
[50]Lincoln DW 2nd, Phillips PG, Bove K. Estrogenerr induced Ets-1 promotes capillary formation in an in vitro tumor angiogenesis model. Breast Cancer Res Treat,2003; 78(2):167-178.
[1]Peng HQ, Levitin-Smith M, Rochelson B, et al. Umbilical cord stricture and overcoiling are common causes of fetal demise. Pediatr Dev Pathol,2006; 9:14-19.
[2]Trevisanuto D, Doglioni N, Zanardo V, et al. Overcoiling of the umbilical cord. J Pediatr, 2007; 150:112.
[3]Deryl L. Concise. Review:Wharton's jelly-derived cells are a primitive stromal cell population [J]. Stem Cells,2008; 26:591-599.
[4]Toren A, Nagler A, Amariglio N, et al. Successful human umbilical cord blood stem cell transplantation with conditioning in severe combined immune deficiency. Bone Marrow Transplant,1999; 23 (4):405-408.
[5]Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors. Stem Cells,2005; 23:220-229.
[6]Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003; 21:105-110.
[7]Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica,2006; 91:1017-1026.
[8]Friedman R, Betancur M, Boissel L, et al. Umbilical cord mesenchymal stem cells: Adjuvants for human cell transplantation. Biol Blood Marrow Transplant,2007; 13: 1477-1486.
[9]Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of the stem cells in human umbilical cord stroma:in situ and in vitro surveys. Stem Cells,2007; 25:319-331.
[10]Bieback K, Kern S, Kluter H, et al. Critical parameters for the isolationof mesenchymal stem cells from umbilical cord blood. Stem Cells,2004; 22:625-634.
[11]Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells,2006; 24:1294-1301.
[12]Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells,2007; 25:1384-1392.
[13]Lund RD, Wang S, Lu B, et al. Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease. Stem Cells,2007; 25:602-611.
[14]Gao L, Chen X, Zhang X, et al. Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34- cells in vitro. Blood Cells Mol Dis,2006; 36:322-328.
[15]Wang JF, Wang LJ, Wu YF, et al. Mesenchymal stem/progenitor cells in human umbilical cord blood as support for ex vivo expansion of CD34- hematopoietic stem cells and for chondrogenic differentiation. Haematologica,2004; 89:837-844.
[16]Ye ZQ, Burkholder JK, Qiu P, et al. Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth. Proc Natl Acad Sci U S A,1994; 91:12140-12144.
[17]Jang YK, Jung DH, Jung MH, et al. Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells. Ann Hematol,2006; 85:212-225.
[18]Goodwin HS, Bicknese AR, Chien SN, et al. Multilineage differentiation activity by cells isolated from umbilical cord blood:expression of bone, fat, and neural markers. Biol Blood Marrow Transplant,2001; 7:581-588.
[19]Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors. Stem Cells,2005; 23:220-229.
[20]Weiss ML, Medicetty S, Bledsoe AR, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells,2006; 24:781-792.
[21]Raio L, Cromi A, Ghezzi F, et al. Hyaluronan content of Wharton's jelly in healthy and Down syndrome fetuses. Matrix Biol,2005; 24:166-174.
[22]Raio L, Cromi A, Ghezzi F, et al. Hyaluronan content of Wharton's jelly in healthy and Down syndrome fetuses. Matrix Biol,2005; 24:166-174.
[23]Saito S, Ugai H, Sawai K, et al. Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro. FEBS Lett,2002; 531:389-396.
[24]Hiroyama T, Sudo K, Aoki N, et al. Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells. Cell Biol Int,2008; 32:1-7.
[25]Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003; 21:105-110.
[26]Covas DT, Siufi JL, Silva AR, et al. Isolation and culture of umbilical vein mesenchymal stem cells. Braz J Med Biol Res,2003; 36:1179-1183.
[27]Kim JW, Kim SY, Park SY, et al. Mesenchymal progenitor cells in the human umbilical cord. Ann Hematol,2004; 83:733-738.
[28]Panepucci RA, Siufi JL, Silva WA, Jr. et al. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells,2004; 22: 1263-1278.
[29]Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton's jelly form neurons and glia. Stem Cells,2003; 21:50-60.
[30]Fu YS, Shih YT, Cheng YC, et al. Transformation of human umbilical mesenchymal cells into neurons in vitro. J Biomed Sci,2004; 11:652-660.
[31]Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells,2004; 22:1330-1337.
[32]Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells. Chin Med J,2005; 118: 1987-1993.
[33]Conconi MT, Burra P, Di LR, et al. CD105+cells from Wharton's jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med,2006; 18:1089-1096.
[34]Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev,2006; 2:87-92.
[35]Hoerstrup SP, Kadner A, Breymann C, et al. Living autologous pulmonary artery conduits tissue engineered from human umbilical cord cells. Ann Thorac Surg,2002; 74:46-52.
[36]Schmidt D, Mol A, Odermatt B, et al. Engineering of biologically active living heart valve leaflets using human umbilical cord-derived progenitor cells. Tissue Eng,2006; 12: 3223-3232.
[37]Wu KH, Zhou B, Lu SH, et al. In vitro and in vivo differentiation of human umbilical cord derived stem cells into endothelial cells. J Cell Biochem,2007; 100:608-616.
[38]Cho PS, Messina DJ, Hirsh EL, et al. Immunogenicity of umbilical cord tissue derived cells. Blood,2007; 111:430-438.
[39]Carlin R, Davis D, Weiss M, et al. Expression of early transcription factors Oct4, Sox2 and Nanog by porcine umbilical cord (PUC) matrix cells. Reprod Biol Endocrinol,2006; 4: 8-21.
[40]Hoynowski SM, Fry MM, Gardner BM, et al. Characterization and differentiation of equine umbilical cord-derived matrix cells. Biochem Biophys Res Commun,2007; 362: 347-353.
[41]Fu YS, Cheng YC, Lin MY, et al. Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro:potential therapeutic application for Parkinsonism. Stem Cells,2006; 24:115-124.
[42]Borlongan CV, Hadman M, Sanberg CD, et al. Central nervous system entry of peripherally injected umbilical cord blood cells is not required for neuroprotection in stroke. Stroke,2004; 35:2385-2389.
[43]Grinnemo KH, Mansson A, Dellgren G, et al. Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infracted rat myocardium. J Thorac Cardiovasc Surg,2004; 127:1293-1300.
[44]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas. Proc Natl Acad Sci U S A, 2000; 97:12846-12851.
[45]Studeny M, Marini FC, Champlin RE, et al. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res,2002; 62:3603-3608.
[46]Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells:potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst, 2004; 96:1593-1603.
[47]Rachakatla RS, Marini F, Weiss ML, et al. Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors. Cancer Gene Ther,2007; 14: 828-835.
[48]Morrison SJ, Qian D, Jerabek L, et al. A genetic determinant that specifically regulates the frequency of hematopoietic stem cells [J]. J Immunol,2002; 168(2):635-642.
[49]Al-Hajj M, Becker MW, Wicha M, et al. Therapeutic implications of cancer stem cells [J]. Curr Opin Genet Dev,2004; 14(1):43-47.
[50]Singh SK, Clarke ID. Identification of a cancer stem cell in human brain tumors. Cancer Research,2003; 63:5821-5828.
[51]Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line [J]. Proc Natl Acad Sci U S A,2004; 101:781-786.
[52]Nelson R. Brain cancer stem cells may drive tumor formation [J]. Lancet Neurol,2005; 4 (1):17.
[53]Hemmati HD, Nakano I, Lazareff JA, et al. Cancerous stem cells can arise from pediatric brain tumors [J]. ProcNat Acad Sci USA,2003; 100(25):15178-15183.
[54]Holland EC. Glioma genesis:genetic alterations and mouse models [J]. Nat Rev Genet. 2001; 2(2):120-129.
[55]尹昌林,吕胜青,唐莉等.免疫磁珠法分选人脑胶质瘤干细胞及其培养和鉴定.中国神经肿瘤杂志[J],2007；5(4)：251-256.
[56]车晓明,崔大明,汪洋, 等.胶质母细胞瘤肿瘤干细胞体外分离培养鉴定及生物学特性的研究[J].中国临床神经科学,2007；15(6)：561-569.
[57]陈刚,秦尚阵,马廉亭,等.人胚神经干细胞体外培养和超微结构观察[J].中国临床神经外科杂志,2006；11(2)：92-95.
[58]Holland E C. Gliomagenesis:genetic alterations and mouse models [J]. Nat Rev Genet, 2001; 2(1):120-129.
[59]Su X, Gopalakrishnan V, Steams D, et al. Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation [J]. Mol Cell Biol,2006; 26(5):1666-1678.
[60]Hadnagy A, Gaboury L, Beaulieu R, et al. SP analysis may be used to identify cancer stem cell populations [J].Exp Cell Res; 2006; 312(19):3701-3710.
[61]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma[J]. Molecular Cancer,2006; 5:67-78.
[62]Salmaggi A, Boiardi A, Gelati M, et al. Glioblastomaderived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype [J]. Glia,2006; 54:850-860.
[63]Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response [J]. Nature,2006; 444:756-760.
[64]Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance [J]. Nat Rev Cancer,2005; 5:275-284.
[65]Fan X, Matsui W, Khaki L, et al. Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors [J]. Cancer Res,2006; 66:7445-7452.
[66]Imbimbo BP. Therapeutic potential of gamma-secretase inhibitors and modulators [J]. Curr Top Med Chem,2008; 8:54-61.
[67]Kanamori M, Kawaguchi T, Nigro JM, et al. Contribution of Notch signaling activation to human glioblastoma multiforme [J]. J Neurosurg,2007; 106:417-427.
[68]Clark PA, Treisman DM, Ebben J, Kuo JS. Developmental signaling pathways in brain tumor-derived stem-like cells [J]. Dev Dyn,2007; 236:3297-3308.
[69]Dihlmann S, Klein S, Doeberitz Mv MK. Reduction of beta-catenin/T-cell transcription factor signaling by aspirin and indomethacin is caused by an increased stabilization of phosphorylated beta-catenin [J]. Mol Cancer Ther,2003; 2:509-516.
[70]Hanai J, Gloy J, Karumanchi SA, et al. Endostatin is a potential inhibitor of Wnt signaling [J]. J Cell Biol,2002; 158:529-539.
[71]Piccirillo SG, Reynolds BA, Zanetti N, et al. Bone morphogenetic protein inhibit the tumorigenic potential of human brain bumor-initiating cells [J]. Nature,2006; 444:761-765.
[72]Lee J, Son MJ, Woolard K, et al. Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells[J]. Cancer Cell,2008; 13:69-80.
[73]Ehtesham M, K abos P, Gutierrez MA, et al. Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand [J]. Cancer Res,2002; 62:7170-7174.
[74]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas [J]. Proc Natl Acad Sci USA, 2000; 97(23):12846-12851.
[75]HonethG, StaflinK, KalliomakiS, et al. Chemokine-directedmigration of tumor-inhibitory neuralprogenitor cells towards an intracranially growing glioma. Exp CellRes,2006; 312(8):1265.
[76]林社裕,高树梓,刘梅.C6胶质瘤细胞体外诱导神经干细胞的迁移.解剖学杂志,2006；29(1)：44.
[77]张力,徐汉军,胡均涛.大鼠胚胎神经干细胞移植治疗C6胶质母细胞瘤.中国神经精神疾病杂志,2007；33(5)：309-11.
[78]GlassR, SynowitzM, KronenbergG, et al. Glioblastoma-induced attraction of endogenous neural precursor cells is associated with improved surviva.1 JNeurosc.i,2005; 25(10): 2637.
[2]Mckay R. Stem cells in central nervous, Science,1997; 276 (5309):66-71.
[3]Singh SK., Clarke ID. Identification of a cancer stem cell in human brain tumors. Cancer Research,2003; 63:5821-5828.
[4]Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, Fuks Z, Kolesnick R. Tumor response to radiotherapy regulated by endothelial cell apoptosis, Science,2003; 300:1155-1159.
[5]Ehtesham M, Kabos P, Kabosova A, Neuman T, Black KL, Yu JS. The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma. Cancer Res,2002; 62 (20):5657-5663.
[6]Li S, Tokuyama T, Yamamoto J, Koide M, Yokota N, Namba H. Bystander effect-mediated gene therapy of gliomas using genetically engineered neural stem cells. Cancer Gene Ther,2005; 12:600-607.
[7]Dimitrios K, Ibrahim K.The potential use of stem cells in multiple sclerosis:An overview of the preclinical experience. Clinical Neurology and Neurosurgery,2008; 110:889-896.
[8]Deryl L. Concise. Review:Wharton's jelly-derived cells are a primitive stromal cell population [J]. Stem Cells,2008; 26:591-599.
[9]Peng HQ, Levitin-Smith M, Rochelson B, et al. Umbilical cord stricture and overcoiling are common causes of fetal demise. Pediatr Dev Pathol,2006; 9:14-19.
[10]Trevisanuto D, Doglioni N, Zanardo V, et al. Overcoiling of the umbilical cord. J Pediatr, 2007;150:112.
[11]Toren A, Nagler A, Amariglio N, et al. Successful human umbilical cord blood stem cell transplantation with conditioning in severe combined immune deficiency. Bone Marrow Transplant,1999; 23 (4):405-408.
[12]Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors. Stem Cells,2005; 23:220-229.
[13]Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003; 21:105-110.
[14]Kestendjieva S, Kyurkchiev D, Tsvetkova G, et al. Characterization of mesenchymal stem cells isolated from the human umbilical cord. Cell Biol Int,2008; 32 (7):724-732
[15]Wang Y, Pu YY, Fang L, et al. Jiangsu Daxue Xuebao (yixueban),2008;18(20):166-167.
[16]Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of the stem cells in human umbilical cord stroma:in situ and in vitro surveys. Stem Cells,2007; 25:319-331.
[17]Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton's jelly form neurons and glia. Stem Cells,2003; 21:50-60.
[18]Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica,2006; 91:1017-1026.
[19]Markov V, Kusumi K, Tadesse MG, et al, Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles. Stem Cells Dev,2007 Feb; 16(1):53-73.
[20]Weiss ML, Medicetty S, Bledsoe AR, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells,2006; 24:781-792.
[21]Fu YS, Shih YT, Cheng YC, et al. Transformation of human umbilical mesenchymal cells into neurons in vitro. J Biomed Sci,2004; 11:652-660.
[22]Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells,2004; 22:1330-1337.
[23]Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells Suppress lymphocyte Proliferation in vitro and Prolong skin graf tsurvival in vivo. Exp Hematol, 2002; 30 (1):42-48.
[24]Kao CL, Lin HT, Chen YW, et al. Fibronectin suppresses lipopolysaccharide-induced liver damage and promotes the cytoprotection abilities of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells. Transplant Proc,2007 Dec; 39(10):3444-5.
[25]Nirmalanandhan VS, Levy MS, Huth AJ, Butler DL, Effects of cell seeding density and collagen concentration on contraction kinetics of mesenchymal stem cell-seeded collagen constructs. Tissue Eng,2006 Jul; 12(7):1865-72.
[26]周长辉,田毅,杨波.人脐带沃顿胶间充质干细胞对人外周T淋巴细胞的免疫调节作用.中国组织工程研究与临床康复,2010；14(14)：2485-2491.
[27]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas. Proc Natl Acad Sci U S A, 2000; 97:12846-12851.
[28]Studeny M, Marini FC, Champlin RE, et al. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res,2002; 62:3603-3608.
[29]Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells:potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst, 2004; 96:1593-1603.
[30]Rachakatla RS, Marini F, Weiss ML, et al. Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors. Cancer Gene Ther,2007; 14: 828-835.
[31]Galli R, Binda E, Orfanelli U, et al. Isolation and characterization of tumorigenic stem-like neural precursors from human glioblastoma [J]. Cancer Res 2004; 64 (19):7011.
[32]Clark PA, Treisman DM, Ebben J, Kuo JS. Developmental signaling pathways in brain tumor-derived stem-like cells. Dev Dyn,2007 Dec; 236(12):3297-308.
[33]Nakano I, Saigusa K, Kornblum HI. BMPing off glioma stem cells. Cancer Cell,2008 Jan; 13(1):3-4.
[34]Sutter R, Yadirgi G, Marino S. Neural stem cells, tumour stem cells and brain tumours: dangerous relationships? Biochim Biophys Acta,2007 Dec; 1776(2):125-37.
[35]Sachan DS, Yatim AM. Suppression of aflatoxin B1-induced lipid abnormalities and macromolecule-adduct formation by L-carnitine. J Environ Pathol Toxicol Oncol,1992 Jul-Aug; 11(4):205-10.
[36]Ekberg S, Carlsson L, Carlsson B, et al, Plasma growth hormone pattern regulates epidermal growth factor (EGF) receptor messenger ribonucleic acid levels and EGF binding in the rat liver. Endocrinology,1989 Oct; 125(4):2158-66.
[37]Liu HM, Yang LH, Yang YJ. Schwann cell properties:3. C-fos expression, bFGF production, phagocytosis and proliferation during Wallerian degeneration. J Neuropathol Exp Neurol,1995 Jul; 54(4):487-96.
[38]Nelson R. Brain cancer stem cells may drive tumor formation [J]. Lancet Neurol,2005; 4 (1):17.
[39]Kang MK, Kang SK. Tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma [J].Stem Cells Dev,2007; 16 (5):837.
[40]Park TD, Deric M, Lin Y, et al. N2CoR pathway targeting induces brain tumor stem cell differentiation [J]. Neurology 2006; 66 (5):393.
[41]Piccirillo SG, Vescovi AL. Brain tumour stem cells:possibilities of new therapeutic st rategies [J]. Expert Opin Biol Ther,2007; 7 (8):1129.
[42]许亮,董军,兰青.干细胞标志物CD133和Nestin在不同恶性程度胶质瘤组织中的表达及其与预后的关系.中国神经肿瘤杂志,2009；7(3)：175-179.
[43]Sobolewskia K, Malkowskia A. Wharton's Jelly as a Reservoir of Peptide Growth Factors. Placenta 2005; 26:747-752.
[44]Weiss ML, Medicetty S, Bledsoe AR, etal. Human umbilical cord matrix stem cells: Preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease [J]. Stem Cells,2006; 24 (3):781-792.
[45]侯克东,卢世璧.人脐带Wharton胶中间充质干细胞的分离,培养与鉴定.解放军医学杂志,2008；33(4)：375-378.
[46]Zarling JM, Shoyab M. Oncostatin M:a growth regulator produced by differentiated histiocytic lymphoma cells. Proc Natl Acad Sci U S A,1986 Dec; 83 (24):9739-43.
[47]Bhandari RN, Riccalton LA, Lewis AL, et al. Liver tissue engineering:a role for co-culture systems in modifying hepatocyte function and viability. Tissue Eng,2001; 7(3):345-357.
[48]Zhu L, Wei W, Zheng YQ, et al. Effects and mechanisms of total glucosides of paeony on joint damage in ratcollagen-induced arthritis. Inflamm Res,2005; 54(5):211-220.
[49]Yassini PR, Stickler DL, Bloomfield SM et al. Gliomastimulated chemoattraction of endothelial cells and fibroblastsin vitro:a model for the study of glioma-induce angiogenesis. Metab Brain Dis,1994; 9(4):391-399.
[50]Lincoln DW 2nd, Phillips PG, Bove K. Estrogenerr induced Ets-1 promotes capillary formation in an in vitro tumor angiogenesis model. Breast Cancer Res Treat,2003; 78(2):167-178.
[1]Peng HQ, Levitin-Smith M, Rochelson B, et al. Umbilical cord stricture and overcoiling are common causes of fetal demise. Pediatr Dev Pathol,2006; 9:14-19.
[2]Trevisanuto D, Doglioni N, Zanardo V, et al. Overcoiling of the umbilical cord. J Pediatr, 2007; 150:112.
[3]Deryl L. Concise. Review:Wharton's jelly-derived cells are a primitive stromal cell population [J]. Stem Cells,2008; 26:591-599.
[4]Toren A, Nagler A, Amariglio N, et al. Successful human umbilical cord blood stem cell transplantation with conditioning in severe combined immune deficiency. Bone Marrow Transplant,1999; 23 (4):405-408.
[5]Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors. Stem Cells,2005; 23:220-229.
[6]Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003; 21:105-110.
[7]Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica,2006; 91:1017-1026.
[8]Friedman R, Betancur M, Boissel L, et al. Umbilical cord mesenchymal stem cells: Adjuvants for human cell transplantation. Biol Blood Marrow Transplant,2007; 13: 1477-1486.
[9]Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of the stem cells in human umbilical cord stroma:in situ and in vitro surveys. Stem Cells,2007; 25:319-331.
[10]Bieback K, Kern S, Kluter H, et al. Critical parameters for the isolationof mesenchymal stem cells from umbilical cord blood. Stem Cells,2004; 22:625-634.
[11]Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells,2006; 24:1294-1301.
[12]Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells,2007; 25:1384-1392.
[13]Lund RD, Wang S, Lu B, et al. Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease. Stem Cells,2007; 25:602-611.
[14]Gao L, Chen X, Zhang X, et al. Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34- cells in vitro. Blood Cells Mol Dis,2006; 36:322-328.
[15]Wang JF, Wang LJ, Wu YF, et al. Mesenchymal stem/progenitor cells in human umbilical cord blood as support for ex vivo expansion of CD34- hematopoietic stem cells and for chondrogenic differentiation. Haematologica,2004; 89:837-844.
[16]Ye ZQ, Burkholder JK, Qiu P, et al. Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth. Proc Natl Acad Sci U S A,1994; 91:12140-12144.
[17]Jang YK, Jung DH, Jung MH, et al. Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells. Ann Hematol,2006; 85:212-225.
[18]Goodwin HS, Bicknese AR, Chien SN, et al. Multilineage differentiation activity by cells isolated from umbilical cord blood:expression of bone, fat, and neural markers. Biol Blood Marrow Transplant,2001; 7:581-588.
[19]Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors. Stem Cells,2005; 23:220-229.
[20]Weiss ML, Medicetty S, Bledsoe AR, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells,2006; 24:781-792.
[21]Raio L, Cromi A, Ghezzi F, et al. Hyaluronan content of Wharton's jelly in healthy and Down syndrome fetuses. Matrix Biol,2005; 24:166-174.
[22]Raio L, Cromi A, Ghezzi F, et al. Hyaluronan content of Wharton's jelly in healthy and Down syndrome fetuses. Matrix Biol,2005; 24:166-174.
[23]Saito S, Ugai H, Sawai K, et al. Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro. FEBS Lett,2002; 531:389-396.
[24]Hiroyama T, Sudo K, Aoki N, et al. Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells. Cell Biol Int,2008; 32:1-7.
[25]Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord. Stem Cells,2003; 21:105-110.
[26]Covas DT, Siufi JL, Silva AR, et al. Isolation and culture of umbilical vein mesenchymal stem cells. Braz J Med Biol Res,2003; 36:1179-1183.
[27]Kim JW, Kim SY, Park SY, et al. Mesenchymal progenitor cells in the human umbilical cord. Ann Hematol,2004; 83:733-738.
[28]Panepucci RA, Siufi JL, Silva WA, Jr. et al. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells,2004; 22: 1263-1278.
[29]Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton's jelly form neurons and glia. Stem Cells,2003; 21:50-60.
[30]Fu YS, Shih YT, Cheng YC, et al. Transformation of human umbilical mesenchymal cells into neurons in vitro. J Biomed Sci,2004; 11:652-660.
[31]Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells,2004; 22:1330-1337.
[32]Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells. Chin Med J,2005; 118: 1987-1993.
[33]Conconi MT, Burra P, Di LR, et al. CD105+cells from Wharton's jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med,2006; 18:1089-1096.
[34]Breymann C, Schmidt D, Hoerstrup SP. Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev,2006; 2:87-92.
[35]Hoerstrup SP, Kadner A, Breymann C, et al. Living autologous pulmonary artery conduits tissue engineered from human umbilical cord cells. Ann Thorac Surg,2002; 74:46-52.
[36]Schmidt D, Mol A, Odermatt B, et al. Engineering of biologically active living heart valve leaflets using human umbilical cord-derived progenitor cells. Tissue Eng,2006; 12: 3223-3232.
[37]Wu KH, Zhou B, Lu SH, et al. In vitro and in vivo differentiation of human umbilical cord derived stem cells into endothelial cells. J Cell Biochem,2007; 100:608-616.
[38]Cho PS, Messina DJ, Hirsh EL, et al. Immunogenicity of umbilical cord tissue derived cells. Blood,2007; 111:430-438.
[39]Carlin R, Davis D, Weiss M, et al. Expression of early transcription factors Oct4, Sox2 and Nanog by porcine umbilical cord (PUC) matrix cells. Reprod Biol Endocrinol,2006; 4: 8-21.
[40]Hoynowski SM, Fry MM, Gardner BM, et al. Characterization and differentiation of equine umbilical cord-derived matrix cells. Biochem Biophys Res Commun,2007; 362: 347-353.
[41]Fu YS, Cheng YC, Lin MY, et al. Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro:potential therapeutic application for Parkinsonism. Stem Cells,2006; 24:115-124.
[42]Borlongan CV, Hadman M, Sanberg CD, et al. Central nervous system entry of peripherally injected umbilical cord blood cells is not required for neuroprotection in stroke. Stroke,2004; 35:2385-2389.
[43]Grinnemo KH, Mansson A, Dellgren G, et al. Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infracted rat myocardium. J Thorac Cardiovasc Surg,2004; 127:1293-1300.
[44]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas. Proc Natl Acad Sci U S A, 2000; 97:12846-12851.
[45]Studeny M, Marini FC, Champlin RE, et al. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res,2002; 62:3603-3608.
[46]Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells:potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst, 2004; 96:1593-1603.
[47]Rachakatla RS, Marini F, Weiss ML, et al. Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors. Cancer Gene Ther,2007; 14: 828-835.
[48]Morrison SJ, Qian D, Jerabek L, et al. A genetic determinant that specifically regulates the frequency of hematopoietic stem cells [J]. J Immunol,2002; 168(2):635-642.
[49]Al-Hajj M, Becker MW, Wicha M, et al. Therapeutic implications of cancer stem cells [J]. Curr Opin Genet Dev,2004; 14(1):43-47.
[50]Singh SK, Clarke ID. Identification of a cancer stem cell in human brain tumors. Cancer Research,2003; 63:5821-5828.
[51]Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line [J]. Proc Natl Acad Sci U S A,2004; 101:781-786.
[52]Nelson R. Brain cancer stem cells may drive tumor formation [J]. Lancet Neurol,2005; 4 (1):17.
[53]Hemmati HD, Nakano I, Lazareff JA, et al. Cancerous stem cells can arise from pediatric brain tumors [J]. ProcNat Acad Sci USA,2003; 100(25):15178-15183.
[54]Holland EC. Glioma genesis:genetic alterations and mouse models [J]. Nat Rev Genet. 2001; 2(2):120-129.
[55]尹昌林,吕胜青,唐莉等.免疫磁珠法分选人脑胶质瘤干细胞及其培养和鉴定.中国神经肿瘤杂志[J],2007；5(4)：251-256.
[56]车晓明,崔大明,汪洋, 等.胶质母细胞瘤肿瘤干细胞体外分离培养鉴定及生物学特性的研究[J].中国临床神经科学,2007；15(6)：561-569.
[57]陈刚,秦尚阵,马廉亭,等.人胚神经干细胞体外培养和超微结构观察[J].中国临床神经外科杂志,2006；11(2)：92-95.
[58]Holland E C. Gliomagenesis:genetic alterations and mouse models [J]. Nat Rev Genet, 2001; 2(1):120-129.
[59]Su X, Gopalakrishnan V, Steams D, et al. Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation [J]. Mol Cell Biol,2006; 26(5):1666-1678.
[60]Hadnagy A, Gaboury L, Beaulieu R, et al. SP analysis may be used to identify cancer stem cell populations [J].Exp Cell Res; 2006; 312(19):3701-3710.
[61]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma[J]. Molecular Cancer,2006; 5:67-78.
[62]Salmaggi A, Boiardi A, Gelati M, et al. Glioblastomaderived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype [J]. Glia,2006; 54:850-860.
[63]Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response [J]. Nature,2006; 444:756-760.
[64]Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance [J]. Nat Rev Cancer,2005; 5:275-284.
[65]Fan X, Matsui W, Khaki L, et al. Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors [J]. Cancer Res,2006; 66:7445-7452.
[66]Imbimbo BP. Therapeutic potential of gamma-secretase inhibitors and modulators [J]. Curr Top Med Chem,2008; 8:54-61.
[67]Kanamori M, Kawaguchi T, Nigro JM, et al. Contribution of Notch signaling activation to human glioblastoma multiforme [J]. J Neurosurg,2007; 106:417-427.
[68]Clark PA, Treisman DM, Ebben J, Kuo JS. Developmental signaling pathways in brain tumor-derived stem-like cells [J]. Dev Dyn,2007; 236:3297-3308.
[69]Dihlmann S, Klein S, Doeberitz Mv MK. Reduction of beta-catenin/T-cell transcription factor signaling by aspirin and indomethacin is caused by an increased stabilization of phosphorylated beta-catenin [J]. Mol Cancer Ther,2003; 2:509-516.
[70]Hanai J, Gloy J, Karumanchi SA, et al. Endostatin is a potential inhibitor of Wnt signaling [J]. J Cell Biol,2002; 158:529-539.
[71]Piccirillo SG, Reynolds BA, Zanetti N, et al. Bone morphogenetic protein inhibit the tumorigenic potential of human brain bumor-initiating cells [J]. Nature,2006; 444:761-765.
[72]Lee J, Son MJ, Woolard K, et al. Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells[J]. Cancer Cell,2008; 13:69-80.
[73]Ehtesham M, K abos P, Gutierrez MA, et al. Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand [J]. Cancer Res,2002; 62:7170-7174.
[74]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas [J]. Proc Natl Acad Sci USA, 2000; 97(23):12846-12851.
[75]HonethG, StaflinK, KalliomakiS, et al. Chemokine-directedmigration of tumor-inhibitory neuralprogenitor cells towards an intracranially growing glioma. Exp CellRes,2006; 312(8):1265.
[76]林社裕,高树梓,刘梅.C6胶质瘤细胞体外诱导神经干细胞的迁移.解剖学杂志,2006；29(1)：44.
[77]张力,徐汉军,胡均涛.大鼠胚胎神经干细胞移植治疗C6胶质母细胞瘤.中国神经精神疾病杂志,2007；33(5)：309-11.
[78]GlassR, SynowitzM, KronenbergG, et al. Glioblastoma-induced attraction of endogenous neural precursor cells is associated with improved surviva.1 JNeurosc.i,2005; 25(10): 2637.