人羊膜及脐带来源间充质干细胞对创伤性脑损伤治疗潜力的生物学特性比较
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摘要
创伤性脑损伤(Traumatic brain injury, TBI)是一个世界性的公共卫生问题。TBI不仅具有较高的患病率和致死率,而且是继发性癫痫发病的一个重要原因,严重影响患者的生活质量。在我国,TBI是40岁以下成年人创伤性死亡的主要原因。即使有幸存活下来一部分TBI也将造成严重的神经功能缺失和行为能力的障碍,给家庭和社会造成极大的负担。目前临床主要采用对症支持疗法,另外采用积极的院前复苏、快速分流、重症监护也有助于降低死亡率,但这些治疗方法仍不能达到令人满意的效果。
TBI在病理生理学上可分为原发性脑损伤和继发性脑损伤两个阶段。首先原发性的机械性创伤会对局部的神经组织造成损害,而损伤本身随后又会触发一连串的级联反应,包括神经组织缺血、继发于弥漫性轴索损伤的Wallerian变性、兴奋毒性、钙稳态失调、线粒体功能障碍和自由基介导的损伤等。继发性的脑损伤常发生在头部受伤后的数小时到数天的时间内。而且TBI之后神经元的丧失不仅仅发生在损伤局部而且可以弥散到其它脑区。局灶性损伤典型的特征是损伤区周围的灰质内或灰白质交界处的出血。这种局灶性的神经元死亡有两种机制,迅速的细胞坏死和进程较慢的细胞调亡。弥漫性损伤则主要发生在海马区神经元,海马区的神经元对TBI的反应尤为敏感,在所有致命的TBI患者中有超过80%存在海马区神经元的大量丧失,即使在没有颅高压存在的情况下亦是如此。这造成的直接后果便是导致实验动物或患者记忆和认知功能的损害。
当前对TBI治疗方法的研究主要基于以上对继发性脑损伤的病理生理、分子和细胞机制的认识,包括占位性病变的早期发现和清除防止二次损伤,并尝试通过药物来减轻生化和细胞级联反应造成的损害。人们普遍认为,成年人在损伤后刺激中枢神经系统再生将可能需要一个或多个以下的进程：细胞更换、提供神经营养因子、促进轴突导向和去除抑制轴突生长的因素、细胞内信号传导的调控、桥接和材料、和/或调节的免疫反应。
自从20年前哺乳动物大脑的神经元和星形胶质细胞具有自我更新的能力被证实以来,对中枢神经系统损伤的研究热点逐渐转入对神经生物学的研究,例如细胞替代疗法已经成为研究和商业活动的一大焦点。越来越多的证据表明,基手干细胞移植的治疗方法可能是用于治疗TBI后功能障碍的最有前途的治疗策略之一。细胞替代疗法以如下的治疗理念为基础,即创伤或疾病所引起的神经功能缺失可以通过引入新的细胞来替代丧失的神经元和胶质细胞,或者所引进细胞通过神经营养作用来增加受损的神经细胞的存活、可塑性和功能的恢复来发挥作用。迄今为止各种类型的干细胞,包括胚胎干细胞,神经干细胞和间充质干细胞目前正在被用于移植研究来治疗中枢神经系统损伤。
胚胎干细胞(embryonic stem cells, ESC)移植对大鼠的脑损伤功能恢复有治疗作用,但是移植后的肿瘤形成限制了它在人体当中的应用。另外胚胎干细胞的应用存在明显道德伦理障碍和胎儿组织来源不足等问题。最新的研究显示诱导多能干细胞induced pluripotent stem cells, iPS)具有ESC的全能性,但是它同样存在体内形成肿瘤的危险。神经干细胞(Neural stem cells, NSC)是一个用来移植治疗脑损伤理想的种子细胞,它既可以分化成神经细胞又可以分泌多种神经营养因子,但是大量神经干细胞的来源问题目前为止仍然限制了它的应用。人间充质干细胞(mesenchymal stem cells, MSC)被认为是一个很好作为脑损伤移植治疗的备选细胞。在实验和临床研究中骨髓间充质干细胞(bone marrow mesenchymal stem cells, BM-MSC)是目前使用最广泛的种子细胞。然而抽取骨髓是一个具有高度侵入性的过程中,存在疼痛和感染的风险,而且其分化的潜能和扩增能力都会随着年龄的增加而降低。因此寻找BM-MSC的替代细胞已经成为一个研究方向。来自胎盘,胎膜,羊水或胎儿组织细胞在细胞数量、扩张潜力和分化能力上都较成体组织来源的间充质干细胞更高。而且它们当中的一些细胞已经被用来研究治疗神经退行性疾病和神经创伤性疾病。
尽管胎盘来源的某些间充质干细胞已经被用末研究其对实验动物某些神经系统疾病模型的治疗作用,但是目前仍不清楚围生期组织来源的各种细胞对神经系统疾病的治疗作用是否相同。在本研究中,我们将重点比较人羊膜来源间充质干细胞(amniotic membrane mesenchymal stem cells, AM-MSC)和脐带华通胶来源的间充质干细胞(umbilical cord Wharton's jelly mesenchymal stem cells, WJ-MSC)的生物学特性,比较它们的形态、体外扩增能力、免疫表型、和多能性。鉴于神经干细胞是用来移植治疗TBI一个最理想的种子细胞,我们还比较了这两种间充质干细胞的体外神经分化潜能,哪种细胞越容易的分化成神经干细胞,它就越适合作为治疗TBI的种子细胞。
细胞移植治疗TBI,除了细胞替代以外,移植细胞提供的旁分泌功能也起到一定的作用。MSC被移植到脑损伤区域后可以提高损伤局部的神经营养因子浓度,这对受损细胞的存活和分化具有重要的意义。本实验中我们使用人细胞因子抗体芯片检测两种来源间充质干细胞的507个细胞因子的表达。同时在两种间充质干细胞的神经干的诱导过程中,我们在体外检测了对神经系统发育和重建具有关键作用的5个神经营养因子。它们是脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)、神经生长因子(nerve growth factor, NGF)、神经营养因子3(neurotrophin3, NT-3)、胶质细胞源性神经营养因子(glial cell derived neurotrophic factor, GDNF)和睫状神经营养因子(ciliary neurotrophic factor, CNTF)。这一结果将在一定程度上对于TBI损伤修复中种子细胞的选择提供-定的参考,尤其是在神经营养因子分泌方面。同时这也反映神经干细胞诱导过程对不同细胞分泌神经营养因子能力的影响。
另一方面移植入体内干细胞的存活能力也是干细胞移植治疗需要考虑的重要方面。间充质干细胞的移植治疗效果往往被植入细胞的大量损失所限制,细胞的死亡是由于损伤部位的恶劣微环境所引发。本研究中我们通过过氧化氢和去血清培养两种方式来模拟过氧化应激和局部的缺血缺氧环境,并比较了两种来源间充质干细胞体外的抗凋亡能力。抗凋亡能力强的干细胞更适合体内移植治疗TBI.
本研究包括三个部分：
第一部分AM-MSC和WJ-MSC的分离、培养和鉴定
目的：建立一种简单有效的分离培养AM-MSC和WJ-MSC的方法,观察其形态,检测其表面抗原标记的表达情况,观察培养过程中核型的变化,比较两种细胞体外增殖能力,观察其表面的细微结构,并检测它们中胚层多向分化能力。
方法：对与羊膜细胞的分离我们采用2.4U/mL中性蛋白酶、1.0mg/mL胶原酶A和0.01mg/mL脱氧核糖核酸酶依次消化来分离；对于华通胶细胞的分离我们采用Ⅱ型胶原酶和0.125%胰酶/EDTA依次消化的方法来分离。通过光学显微镜对细胞进行形态学观察,并通过原子力显微镜(atomic force microscope, AFM)对细胞表面的细微结构进行扫描。运用流式细胞术对两种分离得到的细胞的表面抗原进行检测,并通过免疫细胞化学的方法对其表达神经干细胞标志的情况进行检测。通过测定体外累积群体倍增来对两种细胞体外增殖能力进行比较,并对体外培养的细胞进行核型分析。我们对两种MSC的中胚层多向分化能力进行的检测,包括向骨细胞、脂肪细胞和软骨细胞的分化能力。
结果：通过上述的方法我们成功的分离培养出AM-MSC和WJ-MSC.两种细胞都呈现成纤维细胞样贴壁生长,相比之下WJ-MSC形态更加细长并具有更加强的折光性。对细胞表面抗原检测,我们发现两种细胞的表达情况相类似,它们表达间充质干细胞的表面抗原(CD13、CD29、CD44、CD73、CD90和CD105),不表达血液和内皮系统的表面抗原(CD19、CD31、CD34和CD45),表达Ⅰ类抗原HLA-ABC但不表达Ⅱ类抗原HLA-DR。通过对两种细胞向中胚层多向分化能力的检测,发现两种细胞都能够向骨细胞、脂肪细胞和软骨细胞方向分化。以上结果与国际细胞治疗学会对间充质干细胞的定义和标准相符合,这说明我们从上述组织中分离出的细胞符合间充质干细胞的特性。免疫细胞化学检测结果显示两种细胞都强烈表达波形蛋白(100.00±0.00%vs.100.00±0.00%),少量的AM-MSC和WJ-MSC表达nestin (28.73±2.97%vs.19.22±2.96%, t=2265, P=0.053)±sox2(20.58±2.43%vs.21.11±2.51%,t=0.154, P=0.881)和Musashil (10.17±3.10%vs.12.62±2.58%,t=0.609,P=0.559),两组之间nestin在AM-MSC中阳性率较高但差异无统计学意义。AFM扫描结果显示,AM-MSC具有更强的粘附能力,在细胞的生长过程中边缘胞体有更长的伪足伸出,这提示其可能具有更强的迁移能力。体外扩增实验结果显示,WJ-MSC具有更高的体外增殖能力(F=817.948,P<0.001)。在细胞培养的各个阶段我们都对细胞进行了核型分析,未发现体外培养对其正常核型的产生影响。
结论：我们成功分离出AM-MSC和WJ-MSC,分离出的细胞符合MSC的标准,包括表面抗原的表达情况和中胚层多向分化的能力。AM-MSC具有更强的粘附能力,并可能有更强的体内迁移能力；而WJ-MSC则具有更强的体外增殖能力。两种细胞在体外培养条件下都能保持正常的二倍体核型。
第二部分AM-MSC和WJ-MSC向神经干细胞分化能力的比较
目的：建立一种诱导AM-MSC和WJ-MSC分化为神经干细胞的方法。通过这种方法我们想找到神经干细胞的新的来源,另外我们比较两种来源间充质干细胞向神经干细胞的分化能力。检测在诱导过程中神经营养因子表达量的变化情况。
方法：间充质干细胞向神经干细胞分化的方法如下：AM-MSC和WJ-MSC培养在神经干细胞诱导培养基中,培养基含有KnockOutTM DMEM/F-12基础培养基、20ng/mL人上皮细胞生长因子(human epidermal growth factor, EGF)、20ng/mL人成纤维细胞细胞生长因子(human beta fibroblastic growth factor, bFGF)、神经干细胞添加剂StemPro(?) NSC SFM和GlutaMAXTM-Ⅰ添加剂(1：100)同时含有1%的青链霉素,在37℃和5%CO2置于25cm2超低粘附力表面的培养瓶中。每3天更换一次新鲜培养基。诱导10天后,收集神经球通过实时定量PCR和酶联免疫吸(enzyme-linked immunoadsordent assay,ELISA)附实验来检测神经营养因子的表达水平。
结果：AM-MSC和WJ-MSC可以被诱导成神经球(一种神经干细胞的生长方式),羊膜来源的神经干细胞(amnion derived neural stem cells,AM-NSC)和脐带华通胶来源的神经干细胞(umbilical cord Warton's jelly derived neural stem cells,AM-NSC).而且这种诱导得到的神经球具有神经干细胞的大部分特征。免疫细胞化学结果显示AM-MSC和WJ-MSC经过诱导之后表达三种神经干细胞的标志。经过神经干细胞诱导培养基诱导之后与未诱导的间充质干细胞相比,神经干细胞的标志表达明显上调。细胞免疫化学具体结果如下：AM-NSC与AM-MSC相比表现更强的nestin.sox2和musashi荧光信号(nestin,92.05±2.75%vs.28.73±2.97%,P<0.001；sox2,70.17±3.16%vs.20.58±2.43%,P<0.001； Musashil,66.94±3.62%vs.10.17±3.10%,P<0.001)；WJ-NSC与WJ-MSC相比表现更强的nestin、sox2和musashi荧光信号(nestin,83.57±2.60%vs.19.22±2.96%,P<0.001；sox2,71.17±3.63%vs.21.11±2.51%,P<0.001；Musashi,64.15±3.81%vs.12.62±2.58%,P<0.001).有意思的是诱导后AM-NSC与WJ-NSC相比表现更强的mestin荧光信号但sox2和musashi未见显著差异(nestin,92.1±2.82%vs.83.6±22.52%,P=0.0497:sox2,70.2±3.26%vs.71.2±3.54%, P=0.816:Musashil,67.0±3.67%vs.64.0±3.89%,P=0.559).实时定量PCR结果和Western blot结果与免疫细胞化学结果一致。
我们对两种来源细胞的间充质干细胞在诱导前后分泌神经营养因子的能力的变化情况进行的检测。我们检测了BDNF、GDNF、NT-3、CNTF和NGF。具体的ELISA结果如下：AM-MSC分泌因子的情况,BDNF,109.51±15.26pg/mL, GDNF32.85±14.21pg/mL,NT-327.43±11.91pg/mL,CNTF39.62±13.56pg/mL和NGF21.46±9.83pg/mL；诱导之后AM-NSC分泌因子情况,BDNF,477.39±39.95pg/mL,GDNF101.01±11.67pg/mL,NT-3206.33±26.36pg/mL,CNTF160.48±22.69pg/mL和NGF185.23±23.59pg/m。WJ-MSC分泌因子情况,BDNF,241.69±25.90pg/mL,GDNF16.21±10.01pg/mL,NT-3172.35±25.20pg/mL,CNTF34.37±11.42pg/mL和NGF105.59±18.24pg/mL；诱导之后WJ-NSC分泌因子情况,BDNF,333.66±31.59pg/mL,GDNF93.64±19.17pg/mL,NT-3122.46±20.02pg/mL,CNTF231.28±22.07pg/mL和NGF32.76±10.17pg/mL。组间比较结果显示诱导前WJ-MSC与AM-MSC相比表达高的BDNF(P=0.006).NT-3(P<0.001)和NGF(P=0.002)。而有意思的是诱导后AM-NSC却比WJ-NSC表达更高的BDNF(P=0.003).NT-3(P=0.015)、 CNTF(P=0.014)和NGF(P=0.009)。实时定量PCR的结果与上述的ELISA结果一致。
结论：我们成功的将AM-MSC和WJ-MSC诱导为AM-NSC和WJ-NSC。这些诱导得到的NSC与未诱导的间充质干细胞相比表达更高的NSC标志。诱导之前WJ-MSC和AM-MSC相比表达更多的BDNF、NT-3和NGF,然而诱导之后AM-NSC却比WJ-NSC表达更高的BDNF、NT-3、CNTF和NGF。由此可见来自新生儿附属组织不同部位的间充质干细胞对同一神经干细胞诱导方法的反应不完全相同,相比之下AM-MSC对神经干细胞诱导的反应更有利于神经营养因子的分泌。
第三部分AM-MSC和WJ-MSC体外细胞因子抗体芯片检测和抗凋亡能力的检测
目的：我们运用人细胞因子抗体芯片对两种来源间充质干细胞分泌细胞因子的能力进行检测。通过过氧化氢和去血清培养诱导AM-MSC和WJ-MSC凋亡,并检测其抗凋亡能力。
方法：在本研究中我们使用人细胞因子抗体芯片(Human L-507Array, RayBiotech Inc.)检测两种间充干细胞所分泌的507种细胞因子。我们用化学发光的方法来探测膜上的信号强度,信号的强度通过光密度计来进行定量。对于数值差别在两倍以上的因子,我们对其进行统计学分析。我们选用阳性对照对来自各张膜的数值进行标准化。本实验中采用2mmol/L的H202和去血清培养来诱导细胞凋亡。诱导凋亡的细胞以末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记法(TUNEL)和Annexin V和propidine碘(PI)染色来进行测定。
结果：细胞因子抗体芯片的结果如下：与WJ-MSC相比AM-MSC中检测到有23种细胞因子高表达差异倍数在两倍以上；而在WJ-MSC中有49种细胞因子相对AM-MSC高表达差异倍数在两倍以上。在AM-MSC中高表达的23种细胞因子中,有4种因子差异有统计学意义；而在WJ-MSC中两倍以上高表达的49种因子,统计结果显示差异没有统计学意义。在AM-MSC中上调的4种因子为白细胞介素(interleukin,IL)或者白细胞介素的受体。具体的细胞因子如下：IL-6(t=3.546,P=0.038),IL-13R alpha2(t=3.336,P=0.045),IL-12p70(t=3.743,P=0.033),IL-22R(t=3.187,P=0.0498)。
H202诱导凋亡后,TUNEL法检测AM-MSC和WJ-MSC细胞凋亡率(27.94±2.31%vs.35.70±2.27%,t=2.401,P=0.043),Annexin V/PI染色方法检测细胞凋亡率(31.31±2.05%vs.39.52±2.65%,t=2.448,P,=0.040)；去血清培养诱导凋亡后,Annexin V/PI染色方法检测细胞凋亡率(8.23±0.91%vs.11.71±1.13%,t=2.396,P=0.043).结果显示AM-MSC无论在过氧化氢还是去血清培养诱导的细胞凋亡中都比WJ-MSC显示更强的抗凋亡能力。
结论：与WJ-MSC相比AM-MSC表达更多的细胞因子,这些细胞因子主要为细胞介素,它们可能与细胞的增殖和炎症反应有关。AM-MSC比WJ-MSC具有更强的抗凋亡能力。
Traumatic brain injury (TBI) is a worldwide public health problem. It not only has conciderable morbility and mortality, but is a major course of epilepsy, and functional impairments severely affect the quality of life of a patient. In China, TBI is responsible for a significant proportion of all traumatic death in individuals yunger than40years old. And in survivors, TBI can lead to severe neurological and behavioral disabilities, great family strain and high cost to society. Despite large efforts, there is currently no specific treatment available for TBI other than supportive care, but aggressive prehospital resuscitation, rapid triage, and intensive care have reduced mortality rates.
One way in which TBI can be classified is by either primary or secondary brain injury. In addition to local neuronal destruction resulting from the mechanical primary insult, TBI also induces a progressive cascade of delayed secondary events triggered by the trauma that contribute to neuronal death, including ischemia, Wallerian degeneration secondary to diffuse axonal injury, excitotoxicity, dysregulation of calcium homeostasis, mitochondrial dysfunction, and free radical-mediated damage. Secondary TBI develop over a period of hours or days after the initial impact to the head. The patterns of neuronal loss following these events in TBI are both focal and diffuse. Focal damage is typically seen around hemorrhagic lesions, such as contusions within the gray matter or at gray-white junctions. Such focal neuronal death may occur by both rapid necrotic and slower apoptotic mechanisms. Among diffuse injury sites, the hippocampus is known to be especially vulnerable in humans, with neuronal loss occurring in>80%of fatal TBI, even in the absence of elevated intracranial pressure. These hippocampal changes correlate with the profound memory impairment seen in both human and animal models of TBI.
The current therapeutic approach in research to the treatment of TBI is based on a relatively new understanding of the pathophysiological, molecular, and cellular mechanisms causing secondary brain damage, and includes early detection and evacuation of mass lesions, prevention of secondary insults, and attempts to pharmacologically attenuate damaging biochemical and cellular cascades. It is generally believed that the stimulation of regeneration in the injured adult CNS will likely require one or more of the following processes:cellular replacement, neurotrophic factor delivery, promotion of axonal guidance and removal of growth inhibition, manipulation of intracellular signaling, bridging and artificial substrates, and/or modulation of the immune response.
Since the demonstration nearly20years ago that the mammalian brain has the capacity for self-renewal of neurons and astrocytes, there has been a gradual paradigm shift in neurobiology research such that cell replacement strategies have become a major focus of research and commercial activity. And increasing evidence suggests that stem cell-based transplantation therapies may be one of the most promising therapeutic strategies for treating functional impairments following TBI. Cell replacement therapies have been based on the concept that neurological function lost to injury or disease can be improved by introducing new cells that can replace lost neurons or glial cells, or via trophic support to the surviving cells to increase survival, plasticity, and functional recovery. To date various types of stem cells, including embryonic stem cells, neural stem cells and mesenchymal stem cells are currently being investigated for transplantation.
Transplantation of embryonic stem cells into the injured rat brain can provide functional recovery, however tumor formation raises serious safety concerns about their transplantation use in humans. At the same time, their use in clinical applications is hindered by moral and ethical concerns, as well as the scarcity of fetal tissue. The latest researches show that induced pluripotent stem cells (iPS) have the pluripotency of embryonic stem cells, but it also exist the danger of forming tumors in vivo. Neural stem or progenitor cells (NSC) are also a good candidate to be transplanted into the injured brain, but the production of the NSC is a thorny issue for the use for thansplantation. Human mesenchymal stem cells (MSC) are considered good candidate for transplantation into the injuried brain of the patients. Bone marrow mesenchymal stem cells are currently the most widely used seeding cells for both experimental and clinical studies. However harvesting bone marrow is a highly invasive procedure, and the number, differentiation potential, and maximum life span of mesenchymal stem cells from bone marrow decline with increasing age. Thus, the search for possible alternative MSC is ongoing. Cells derived from the placenta, membranes, amniotic fluid or fetal tissues are higher in number, expansion potential and differentiation abilities compared with mesenchymal stem cells from adult tissues. And some of them have been utilized in the treatment of neurodegenerative and neural traumatic diseases.
Although mesenchymal derived from derived from perinatal tissues have been used in the therapies for a variety of neurological diseases in the experimental animal models, it is mot clear whether the cells from different part of the perinatal tissues have the parallel effect on the treatment of neurological diseases. Here we focus our study on the comparison of the biological characteristics of mesenchymal stem cells from human amniotic membrane (AM-MSC) and umbilical cord Wharton's jelly (WJ-MSC) with respect to their morphology, expansion kinetics, immunophenotype, multipotency. Since NSC are the idealest seed cells to transplant for the cell replacemental treatment for TBI, the capacity of AM-MSC and WJ-MSC to differentiate into neural stem cells were determined in vitro, the easier MSC can differentiat into neural stem cells, the better MSC could be considered as candidate for cell-based seed cells for treatment of TBI.
Neurologic benefit resulting from human MSC treatment of nervous system injuries may be due, at least in part, to the increase of growth factors in the ischemic tissue, and neurotrophic factors play crucial roles in the differentiation and survival of neural cells. Human cytokine antibody arrays (Human L-507Array, RayBiotech Inc.) were used to detect the expression of507cytokines in the cells from two sources of MSC. At the same time, the five important neurotrophic factors which play crucial role in the development and central nervous system restoration, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin3(NT-3), glial cell derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), were detected during neural stem cell induction in vitro. These data may in some degree reflect the neurotrophic benefit of MSC on the treatment of TBI between the AM-MSC and WJ-MSC as well as between the MSC and NSC derived from MSC, which may attribute to the selection of seed cells to the cell-based therapy for TBI.
The survival capacity of MSCs in host tissues in conditions of ischemia or ischemic reperfusion is another important property to be considered. The use of an MSC graft approach is limited by the fact that most of the transplanted MSC are readily lost, potentially triggered by the ischemic or ischemia-reperfusion environment in vivo. In our study, we investigated the anti-apoptosis ability of these MSC towards oxidative stress induced by hydrogen peroxide (H2O2) or serum deprivation.
The study includes three chapters:
Chapter Ⅰ Isolation and characterization of MSC from amniotic membrane and umbilical Wharton's jelly
Objective:To establish a simplified culture system to isolate AM-MSC and WJ-MSC, observe their morphology, and investigate the expression of neural stem cell markers and mesenchymal stem cell markers, their karyotype, proliferative capacities and their multi-lineage differentiation capacities, scan their surfacial fine structure.
Methods:Amniotic membrane was sequentially digested in2.4U/mL dispase,1.0mg/mL Collagenase A and0.01mg/mL DNase; and Wharton's jelly was digested in collagenase type Ⅱ followed by further digestion with0.125%trypsin/EDTA. The morphology was observed by an optic microscope and their surfacial fine structures were scaned by an atomic force microscope, the immunophenotype were tested by Immunocytochemistry or flow cytometry. Before we determined their proliferative capacities, their charyotypes were analysed. The differentiation potential to osteogenic, adipogenic and chondrogenic cells were detected.
Results:AM-MSC and WJ-MSC were successfully isolated and cultured by the protocols mentioned in the methods section. AM-MSC and WJ-MSC both express the markers of MSC (CD13, CD29, CD44, CD73, CD90and CD105), but not express hematopoietic and endothelial phenotypes (CD19, CD31, CD34and CD45), and they both express HLA-ABC, not HLA-DR, and pocess osteogenic, adipogenic and chondrogenic differentiation potential. The results are in accordance with the criteria of MSC by The International Society for Cellular Therapy. Two kinds of MSC both strongly expressed vimentin (100.00±0.00%vs.100.00±0.00%), while few AM-MSC and WJ-MSC expressed nestin (28.73±2.97%vs.19.22±2.96%,t=2.265, P=0.053), sox2(20.58±2.43%vs.21.11±2.51%, t=0.154, P=0.881) and Musashi1(10.17±3.10%vs.12.62±2.58%,t=0.609, P=0.559). These data were confirmed by quantitative real-time PCR and Western blot analysis. AM-MSC possess stronger adherent capacity to the surface compared with WJ-MSC in culture scanning by atomic force microscope, While WJ-MSC have higher praliferative potential (F=817.948, P<0.001). Normal chariotypes we observed throughout the whole culture in vitro.
Conclusion:we have successfully isolate and culture AM-MSC and WJ-MSC, and characterized the cells based on a set of criteria proposed by the International Society for Cellular Therapy. Moreover the two kinds of cells expressed low levels of neural stemness markers. AM-MSC showed stronger adherent capacity to the cultured surface and stronger anti-apoptosis ability compared with WJ-MSC in culture, While WJ-MSC have higher praliferative potential.
Chapter Ⅱ Determination of the capacity of AM-MSC and WJ-MSC for neural stem cells differentiation
Objective:to establish the protocol to induce AM-MSC and WJ-MSC into neural stem cells. By the above protocol, we firstly want to obtain a new source of neural stem cells, and secondly to compare the neuronal differentiation ability of AM-MSC and WJ-MSC. And the expressing change of NT was investigated during the neural stem induction.
Methods:The transdifferentiation protocols:the AM-MSC and WJ-MSC were induced in neural stem cell differentiation medium, composed of KnockOutTM DMEM/F-12Basal Medium supplemented with20ng/mL human epidermal growth factor (EGF),20ng/mL bFGF, StemPro(?) NSC SFM Supplement and GlutaMAXTM-Ⅰ Supplement (1:100) containing1%penicillin/streptomycin at37℃with5%CO2,and plated in Ultra-Low Attachment25cm2culture flasks. The medium was changed every3days. Ten days after induction, neurospheres were collected to determine levels of neurotrophic factors by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) analysis.
Results:AM-MSC and WJ-MSC could be induced into neurosphere-like aggregates, a growth form of neural stem cells, successfully (AM-NSC and WJ-NSC respectively). More importantly, these neural stem cells displayed most of the chanracteristics of neural stem cells. Both AM-MSC and WJ-MSC were examined for expression of the three neural stemness markers by immunocytochemistry after neural stem cell differentiation. After differentiation, the fluorescent signal for the stemness markers of NSC derived from MSC significantly upregulated than that of undifferentiated MSC, and a similar results were obtained from the two kinds of MSC. The details are as follows, AM-NSC showed a higher fluorescent signals for nestin, sox2and musashi1than that of undifferentiated AM-MSC (nestin,92.05±2.75%vs. 28.73±2.97%,P<0.001；sox2,70.17±3.16%vs.20.58±2.43%,P<0.001； Musashil,66.94±3.62%vs.10.17±3.10%,P<0.001)；WJ-NSC showed a higher fluorescent signals for nestin,sox2and musashi1than that of undifferentiated WJ-MSC(nestin,83.57±2.60%vs.19.22±2.96%,P<0.001；sox2,71.17±3.63%vs.21.11±2.51%,P<0.001；Musashi,64.15±3.81%vs.12.62±2.58%,P<0.001). Intrestingly,AM-NSC showed a higher fluorescent signal for the nestin but parallel signal for sox2and musashi than that of WJ-NSC(nestin,92.1±2.82%vs.83.6±22.52%,P=0.0497；sox2,70.2±3.26%vs.71.2±3.54%,P=0.816；Musashil,67.0±3.67%vs.64.0±3.89%,P=0.559).These immunocytochemical data were confirmed by quantitative real-time PCR and Western blot analysis.
Secreted levels of BDNF,GDNF,NT-3,CNTF and NGF in both populations of MSC to NSC were detected.For AM-MSC,the secreted level of BDNF was BDNF,109.51±15.26pg/mL,GDNF32.85±14.21pg/mL,NT-327.43±11.91pg/mL, CNTF39.62±13.56pg/mL and NGF21.46±9.83pg/mL.After differentiation,for AM-NSC,the secreted level of BDNF was BDNF,477.39±39.95pg/mL,GDNF101.01±11.67pg/mL,NT-3206.33±26.36pg/mL,CNTF160.48±22.69pg/mL and NGF185.23±23.59pg/m.For WJ-MSC,the secreted level of BDNF was BDNF,241.69±25.90pg/mL,GDNF16.21±10.01pg/mL,NT-3172.35±25.20pg/mL, CNTF34.37±11.42pg/mL and NGF105.59±18.24pg/mL.After differentiation, for AM-NSC,the secreted level of BDNF was BDNF,333.66±31.59pg/mL,GDNF93.64±19.17pg/mL,NT-3122.46±20.02pg/mL,CNTF231.28±22.07pg/mL and NGF32.76±10.17pg/gmL.There were significant differences between AM-MSC and WJ-MSC in BDNF(P=0.006),NT-3(P<0.001)and NGF(P=0.002)；between AM-NSC and WJ-NSC in BDNF(P=0.003),NT-3(P=0.015),CNTF(P=0.014) and NGF (P=0.009).These ELISA data were confirmed by quantitative real-time PCR.
Conclusion:we successfully established protocols to induce both AM-MSC and WJ-MSC to differentiate into AM-NSC and WJ-NSC in vitro,with AM-NSC expressing higher level of nestin.Before induction,undifferentiated WJ-MSC secret higher levels of BDNF,NT-3and NGF than those of AM-MSC.However after differentiation, the secretions of BDNF, NT-3, CNTF and NGF of differentiated AM-MSC were significantly higher than those of differentiated WJ-MSC. These findings suggested that MSC from different part of placent perssess different reaction to this neural stem cells introduction protocol, and considering the secreting of NTs, AM-NSC may more suitable for transplantation for the treatment of TBI than undifferentiated AM-MSC.
Chapter Ⅲ Cytokine antibody array detection and anti-apoptosis abilities of AM-MSC and WJ-MSC in vitro
Objective:To investigate the cytokine secreting capacity, a Cytokine antibody array was used to detect the expression of507cytokines in the cells from two sources of MSC. To detecte their anti-apoptosis abilities in vitro, apoptosis was triggered by H2O2and serum deprivation, and the rate of apoptosis was counted.
Methods:Total protein was extracted from the AM-MSC and WJ-MSC using a tissue protein extraction reagent (Kangchen, China). Human cytokine antibody arrays (Human L-507Array, RayBiotech Inc.) were used to detect the expression of507cytokines in the cells from two sources of MSC. The signal from the membrane was detected with a chemiluminescene imaging system. The signal intensity was quantified by densitometry. The value altered by twofold or more was statistically significant. A positive control was used to normalize the results from different membranes. Apoptosis triggered by2mmol/L H2O2and serum deprivation. Apoptosis in MSC were identified by nuclear positive staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) and Annexin V and propidine iodide (PI) staining after H2O2or serum deprivation induction.
Results:Results of the cytokine antibody array detection:23cytokines in the AM-MSC were detected by cytokine antibody array which were two fold up regulated than that of WJ-MSC; while49cytokines in the WJ-MSC were detected by cytokine antibody array which were two fold up regulated than that of AM-MSC. And expression levels of4cytokines were changed significantly in AM-MSC compared to WJ-MSC; while there was no significant difference in the cytokines that increased by twofold in WJ-MSC compared with AM-MSC. And the4cytokines up regulated in AM-MSC contain interleukins or their receptor that were considered as inflammatory factors. The details are as follows, IL-6(t=3.546, P=0.038), IL-13R alpha2(t=3.336, P=0.045), IL-12p70(t=3.743, P=0.033), IL-22R (t=3.187, P=0.0498).
Results of the anti-apoptosis ability detection:AM-MSC showed significantly stronger anti-apoptosis ability not only to the induction by H2O2but by serum deprivation, which were detected by TUNEL and Annexin V/PI staining compared with WJ-MSC in vitro (P<0.05).
Conclusion:Cytokines expressed in AM-MSC increased compared with those in WJ-MSC, and these cytokines were growth factors and interleukins, which are related with regulation of cell proliferation and inflammatory response. AM-MSC showed stronger anti-apoptosis ability compared with WJ-MSC in culture.
TBI在病理生理学上可分为原发性脑损伤和继发性脑损伤两个阶段。首先原发性的机械性创伤会对局部的神经组织造成损害,而损伤本身随后又会触发一连串的级联反应,包括神经组织缺血、继发于弥漫性轴索损伤的Wallerian变性、兴奋毒性、钙稳态失调、线粒体功能障碍和自由基介导的损伤等。继发性的脑损伤常发生在头部受伤后的数小时到数天的时间内。而且TBI之后神经元的丧失不仅仅发生在损伤局部而且可以弥散到其它脑区。局灶性损伤典型的特征是损伤区周围的灰质内或灰白质交界处的出血。这种局灶性的神经元死亡有两种机制,迅速的细胞坏死和进程较慢的细胞调亡。弥漫性损伤则主要发生在海马区神经元,海马区的神经元对TBI的反应尤为敏感,在所有致命的TBI患者中有超过80%存在海马区神经元的大量丧失,即使在没有颅高压存在的情况下亦是如此。这造成的直接后果便是导致实验动物或患者记忆和认知功能的损害。
当前对TBI治疗方法的研究主要基于以上对继发性脑损伤的病理生理、分子和细胞机制的认识,包括占位性病变的早期发现和清除防止二次损伤,并尝试通过药物来减轻生化和细胞级联反应造成的损害。人们普遍认为,成年人在损伤后刺激中枢神经系统再生将可能需要一个或多个以下的进程：细胞更换、提供神经营养因子、促进轴突导向和去除抑制轴突生长的因素、细胞内信号传导的调控、桥接和材料、和/或调节的免疫反应。
自从20年前哺乳动物大脑的神经元和星形胶质细胞具有自我更新的能力被证实以来,对中枢神经系统损伤的研究热点逐渐转入对神经生物学的研究,例如细胞替代疗法已经成为研究和商业活动的一大焦点。越来越多的证据表明,基手干细胞移植的治疗方法可能是用于治疗TBI后功能障碍的最有前途的治疗策略之一。细胞替代疗法以如下的治疗理念为基础,即创伤或疾病所引起的神经功能缺失可以通过引入新的细胞来替代丧失的神经元和胶质细胞,或者所引进细胞通过神经营养作用来增加受损的神经细胞的存活、可塑性和功能的恢复来发挥作用。迄今为止各种类型的干细胞,包括胚胎干细胞,神经干细胞和间充质干细胞目前正在被用于移植研究来治疗中枢神经系统损伤。
胚胎干细胞(embryonic stem cells, ESC)移植对大鼠的脑损伤功能恢复有治疗作用,但是移植后的肿瘤形成限制了它在人体当中的应用。另外胚胎干细胞的应用存在明显道德伦理障碍和胎儿组织来源不足等问题。最新的研究显示诱导多能干细胞induced pluripotent stem cells, iPS)具有ESC的全能性,但是它同样存在体内形成肿瘤的危险。神经干细胞(Neural stem cells, NSC)是一个用来移植治疗脑损伤理想的种子细胞,它既可以分化成神经细胞又可以分泌多种神经营养因子,但是大量神经干细胞的来源问题目前为止仍然限制了它的应用。人间充质干细胞(mesenchymal stem cells, MSC)被认为是一个很好作为脑损伤移植治疗的备选细胞。在实验和临床研究中骨髓间充质干细胞(bone marrow mesenchymal stem cells, BM-MSC)是目前使用最广泛的种子细胞。然而抽取骨髓是一个具有高度侵入性的过程中,存在疼痛和感染的风险,而且其分化的潜能和扩增能力都会随着年龄的增加而降低。因此寻找BM-MSC的替代细胞已经成为一个研究方向。来自胎盘,胎膜,羊水或胎儿组织细胞在细胞数量、扩张潜力和分化能力上都较成体组织来源的间充质干细胞更高。而且它们当中的一些细胞已经被用来研究治疗神经退行性疾病和神经创伤性疾病。
尽管胎盘来源的某些间充质干细胞已经被用末研究其对实验动物某些神经系统疾病模型的治疗作用,但是目前仍不清楚围生期组织来源的各种细胞对神经系统疾病的治疗作用是否相同。在本研究中,我们将重点比较人羊膜来源间充质干细胞(amniotic membrane mesenchymal stem cells, AM-MSC)和脐带华通胶来源的间充质干细胞(umbilical cord Wharton's jelly mesenchymal stem cells, WJ-MSC)的生物学特性,比较它们的形态、体外扩增能力、免疫表型、和多能性。鉴于神经干细胞是用来移植治疗TBI一个最理想的种子细胞,我们还比较了这两种间充质干细胞的体外神经分化潜能,哪种细胞越容易的分化成神经干细胞,它就越适合作为治疗TBI的种子细胞。
细胞移植治疗TBI,除了细胞替代以外,移植细胞提供的旁分泌功能也起到一定的作用。MSC被移植到脑损伤区域后可以提高损伤局部的神经营养因子浓度,这对受损细胞的存活和分化具有重要的意义。本实验中我们使用人细胞因子抗体芯片检测两种来源间充质干细胞的507个细胞因子的表达。同时在两种间充质干细胞的神经干的诱导过程中,我们在体外检测了对神经系统发育和重建具有关键作用的5个神经营养因子。它们是脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)、神经生长因子(nerve growth factor, NGF)、神经营养因子3(neurotrophin3, NT-3)、胶质细胞源性神经营养因子(glial cell derived neurotrophic factor, GDNF)和睫状神经营养因子(ciliary neurotrophic factor, CNTF)。这一结果将在一定程度上对于TBI损伤修复中种子细胞的选择提供-定的参考,尤其是在神经营养因子分泌方面。同时这也反映神经干细胞诱导过程对不同细胞分泌神经营养因子能力的影响。
另一方面移植入体内干细胞的存活能力也是干细胞移植治疗需要考虑的重要方面。间充质干细胞的移植治疗效果往往被植入细胞的大量损失所限制,细胞的死亡是由于损伤部位的恶劣微环境所引发。本研究中我们通过过氧化氢和去血清培养两种方式来模拟过氧化应激和局部的缺血缺氧环境,并比较了两种来源间充质干细胞体外的抗凋亡能力。抗凋亡能力强的干细胞更适合体内移植治疗TBI.
本研究包括三个部分：
第一部分AM-MSC和WJ-MSC的分离、培养和鉴定
目的：建立一种简单有效的分离培养AM-MSC和WJ-MSC的方法,观察其形态,检测其表面抗原标记的表达情况,观察培养过程中核型的变化,比较两种细胞体外增殖能力,观察其表面的细微结构,并检测它们中胚层多向分化能力。
方法：对与羊膜细胞的分离我们采用2.4U/mL中性蛋白酶、1.0mg/mL胶原酶A和0.01mg/mL脱氧核糖核酸酶依次消化来分离；对于华通胶细胞的分离我们采用Ⅱ型胶原酶和0.125%胰酶/EDTA依次消化的方法来分离。通过光学显微镜对细胞进行形态学观察,并通过原子力显微镜(atomic force microscope, AFM)对细胞表面的细微结构进行扫描。运用流式细胞术对两种分离得到的细胞的表面抗原进行检测,并通过免疫细胞化学的方法对其表达神经干细胞标志的情况进行检测。通过测定体外累积群体倍增来对两种细胞体外增殖能力进行比较,并对体外培养的细胞进行核型分析。我们对两种MSC的中胚层多向分化能力进行的检测,包括向骨细胞、脂肪细胞和软骨细胞的分化能力。
结果：通过上述的方法我们成功的分离培养出AM-MSC和WJ-MSC.两种细胞都呈现成纤维细胞样贴壁生长,相比之下WJ-MSC形态更加细长并具有更加强的折光性。对细胞表面抗原检测,我们发现两种细胞的表达情况相类似,它们表达间充质干细胞的表面抗原(CD13、CD29、CD44、CD73、CD90和CD105),不表达血液和内皮系统的表面抗原(CD19、CD31、CD34和CD45),表达Ⅰ类抗原HLA-ABC但不表达Ⅱ类抗原HLA-DR。通过对两种细胞向中胚层多向分化能力的检测,发现两种细胞都能够向骨细胞、脂肪细胞和软骨细胞方向分化。以上结果与国际细胞治疗学会对间充质干细胞的定义和标准相符合,这说明我们从上述组织中分离出的细胞符合间充质干细胞的特性。免疫细胞化学检测结果显示两种细胞都强烈表达波形蛋白(100.00±0.00%vs.100.00±0.00%),少量的AM-MSC和WJ-MSC表达nestin (28.73±2.97%vs.19.22±2.96%, t=2265, P=0.053)±sox2(20.58±2.43%vs.21.11±2.51%,t=0.154, P=0.881)和Musashil (10.17±3.10%vs.12.62±2.58%,t=0.609,P=0.559),两组之间nestin在AM-MSC中阳性率较高但差异无统计学意义。AFM扫描结果显示,AM-MSC具有更强的粘附能力,在细胞的生长过程中边缘胞体有更长的伪足伸出,这提示其可能具有更强的迁移能力。体外扩增实验结果显示,WJ-MSC具有更高的体外增殖能力(F=817.948,P<0.001)。在细胞培养的各个阶段我们都对细胞进行了核型分析,未发现体外培养对其正常核型的产生影响。
结论：我们成功分离出AM-MSC和WJ-MSC,分离出的细胞符合MSC的标准,包括表面抗原的表达情况和中胚层多向分化的能力。AM-MSC具有更强的粘附能力,并可能有更强的体内迁移能力；而WJ-MSC则具有更强的体外增殖能力。两种细胞在体外培养条件下都能保持正常的二倍体核型。
第二部分AM-MSC和WJ-MSC向神经干细胞分化能力的比较
目的：建立一种诱导AM-MSC和WJ-MSC分化为神经干细胞的方法。通过这种方法我们想找到神经干细胞的新的来源,另外我们比较两种来源间充质干细胞向神经干细胞的分化能力。检测在诱导过程中神经营养因子表达量的变化情况。
方法：间充质干细胞向神经干细胞分化的方法如下：AM-MSC和WJ-MSC培养在神经干细胞诱导培养基中,培养基含有KnockOutTM DMEM/F-12基础培养基、20ng/mL人上皮细胞生长因子(human epidermal growth factor, EGF)、20ng/mL人成纤维细胞细胞生长因子(human beta fibroblastic growth factor, bFGF)、神经干细胞添加剂StemPro(?) NSC SFM和GlutaMAXTM-Ⅰ添加剂(1：100)同时含有1%的青链霉素,在37℃和5%CO2置于25cm2超低粘附力表面的培养瓶中。每3天更换一次新鲜培养基。诱导10天后,收集神经球通过实时定量PCR和酶联免疫吸(enzyme-linked immunoadsordent assay,ELISA)附实验来检测神经营养因子的表达水平。
结果：AM-MSC和WJ-MSC可以被诱导成神经球(一种神经干细胞的生长方式),羊膜来源的神经干细胞(amnion derived neural stem cells,AM-NSC)和脐带华通胶来源的神经干细胞(umbilical cord Warton's jelly derived neural stem cells,AM-NSC).而且这种诱导得到的神经球具有神经干细胞的大部分特征。免疫细胞化学结果显示AM-MSC和WJ-MSC经过诱导之后表达三种神经干细胞的标志。经过神经干细胞诱导培养基诱导之后与未诱导的间充质干细胞相比,神经干细胞的标志表达明显上调。细胞免疫化学具体结果如下：AM-NSC与AM-MSC相比表现更强的nestin.sox2和musashi荧光信号(nestin,92.05±2.75%vs.28.73±2.97%,P<0.001；sox2,70.17±3.16%vs.20.58±2.43%,P<0.001； Musashil,66.94±3.62%vs.10.17±3.10%,P<0.001)；WJ-NSC与WJ-MSC相比表现更强的nestin、sox2和musashi荧光信号(nestin,83.57±2.60%vs.19.22±2.96%,P<0.001；sox2,71.17±3.63%vs.21.11±2.51%,P<0.001；Musashi,64.15±3.81%vs.12.62±2.58%,P<0.001).有意思的是诱导后AM-NSC与WJ-NSC相比表现更强的mestin荧光信号但sox2和musashi未见显著差异(nestin,92.1±2.82%vs.83.6±22.52%,P=0.0497:sox2,70.2±3.26%vs.71.2±3.54%, P=0.816:Musashil,67.0±3.67%vs.64.0±3.89%,P=0.559).实时定量PCR结果和Western blot结果与免疫细胞化学结果一致。
我们对两种来源细胞的间充质干细胞在诱导前后分泌神经营养因子的能力的变化情况进行的检测。我们检测了BDNF、GDNF、NT-3、CNTF和NGF。具体的ELISA结果如下：AM-MSC分泌因子的情况,BDNF,109.51±15.26pg/mL, GDNF32.85±14.21pg/mL,NT-327.43±11.91pg/mL,CNTF39.62±13.56pg/mL和NGF21.46±9.83pg/mL；诱导之后AM-NSC分泌因子情况,BDNF,477.39±39.95pg/mL,GDNF101.01±11.67pg/mL,NT-3206.33±26.36pg/mL,CNTF160.48±22.69pg/mL和NGF185.23±23.59pg/m。WJ-MSC分泌因子情况,BDNF,241.69±25.90pg/mL,GDNF16.21±10.01pg/mL,NT-3172.35±25.20pg/mL,CNTF34.37±11.42pg/mL和NGF105.59±18.24pg/mL；诱导之后WJ-NSC分泌因子情况,BDNF,333.66±31.59pg/mL,GDNF93.64±19.17pg/mL,NT-3122.46±20.02pg/mL,CNTF231.28±22.07pg/mL和NGF32.76±10.17pg/mL。组间比较结果显示诱导前WJ-MSC与AM-MSC相比表达高的BDNF(P=0.006).NT-3(P<0.001)和NGF(P=0.002)。而有意思的是诱导后AM-NSC却比WJ-NSC表达更高的BDNF(P=0.003).NT-3(P=0.015)、 CNTF(P=0.014)和NGF(P=0.009)。实时定量PCR的结果与上述的ELISA结果一致。
结论：我们成功的将AM-MSC和WJ-MSC诱导为AM-NSC和WJ-NSC。这些诱导得到的NSC与未诱导的间充质干细胞相比表达更高的NSC标志。诱导之前WJ-MSC和AM-MSC相比表达更多的BDNF、NT-3和NGF,然而诱导之后AM-NSC却比WJ-NSC表达更高的BDNF、NT-3、CNTF和NGF。由此可见来自新生儿附属组织不同部位的间充质干细胞对同一神经干细胞诱导方法的反应不完全相同,相比之下AM-MSC对神经干细胞诱导的反应更有利于神经营养因子的分泌。
第三部分AM-MSC和WJ-MSC体外细胞因子抗体芯片检测和抗凋亡能力的检测
目的：我们运用人细胞因子抗体芯片对两种来源间充质干细胞分泌细胞因子的能力进行检测。通过过氧化氢和去血清培养诱导AM-MSC和WJ-MSC凋亡,并检测其抗凋亡能力。
方法：在本研究中我们使用人细胞因子抗体芯片(Human L-507Array, RayBiotech Inc.)检测两种间充干细胞所分泌的507种细胞因子。我们用化学发光的方法来探测膜上的信号强度,信号的强度通过光密度计来进行定量。对于数值差别在两倍以上的因子,我们对其进行统计学分析。我们选用阳性对照对来自各张膜的数值进行标准化。本实验中采用2mmol/L的H202和去血清培养来诱导细胞凋亡。诱导凋亡的细胞以末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记法(TUNEL)和Annexin V和propidine碘(PI)染色来进行测定。
结果：细胞因子抗体芯片的结果如下：与WJ-MSC相比AM-MSC中检测到有23种细胞因子高表达差异倍数在两倍以上；而在WJ-MSC中有49种细胞因子相对AM-MSC高表达差异倍数在两倍以上。在AM-MSC中高表达的23种细胞因子中,有4种因子差异有统计学意义；而在WJ-MSC中两倍以上高表达的49种因子,统计结果显示差异没有统计学意义。在AM-MSC中上调的4种因子为白细胞介素(interleukin,IL)或者白细胞介素的受体。具体的细胞因子如下：IL-6(t=3.546,P=0.038),IL-13R alpha2(t=3.336,P=0.045),IL-12p70(t=3.743,P=0.033),IL-22R(t=3.187,P=0.0498)。
H202诱导凋亡后,TUNEL法检测AM-MSC和WJ-MSC细胞凋亡率(27.94±2.31%vs.35.70±2.27%,t=2.401,P=0.043),Annexin V/PI染色方法检测细胞凋亡率(31.31±2.05%vs.39.52±2.65%,t=2.448,P,=0.040)；去血清培养诱导凋亡后,Annexin V/PI染色方法检测细胞凋亡率(8.23±0.91%vs.11.71±1.13%,t=2.396,P=0.043).结果显示AM-MSC无论在过氧化氢还是去血清培养诱导的细胞凋亡中都比WJ-MSC显示更强的抗凋亡能力。
结论：与WJ-MSC相比AM-MSC表达更多的细胞因子,这些细胞因子主要为细胞介素,它们可能与细胞的增殖和炎症反应有关。AM-MSC比WJ-MSC具有更强的抗凋亡能力。
Traumatic brain injury (TBI) is a worldwide public health problem. It not only has conciderable morbility and mortality, but is a major course of epilepsy, and functional impairments severely affect the quality of life of a patient. In China, TBI is responsible for a significant proportion of all traumatic death in individuals yunger than40years old. And in survivors, TBI can lead to severe neurological and behavioral disabilities, great family strain and high cost to society. Despite large efforts, there is currently no specific treatment available for TBI other than supportive care, but aggressive prehospital resuscitation, rapid triage, and intensive care have reduced mortality rates.
One way in which TBI can be classified is by either primary or secondary brain injury. In addition to local neuronal destruction resulting from the mechanical primary insult, TBI also induces a progressive cascade of delayed secondary events triggered by the trauma that contribute to neuronal death, including ischemia, Wallerian degeneration secondary to diffuse axonal injury, excitotoxicity, dysregulation of calcium homeostasis, mitochondrial dysfunction, and free radical-mediated damage. Secondary TBI develop over a period of hours or days after the initial impact to the head. The patterns of neuronal loss following these events in TBI are both focal and diffuse. Focal damage is typically seen around hemorrhagic lesions, such as contusions within the gray matter or at gray-white junctions. Such focal neuronal death may occur by both rapid necrotic and slower apoptotic mechanisms. Among diffuse injury sites, the hippocampus is known to be especially vulnerable in humans, with neuronal loss occurring in>80%of fatal TBI, even in the absence of elevated intracranial pressure. These hippocampal changes correlate with the profound memory impairment seen in both human and animal models of TBI.
The current therapeutic approach in research to the treatment of TBI is based on a relatively new understanding of the pathophysiological, molecular, and cellular mechanisms causing secondary brain damage, and includes early detection and evacuation of mass lesions, prevention of secondary insults, and attempts to pharmacologically attenuate damaging biochemical and cellular cascades. It is generally believed that the stimulation of regeneration in the injured adult CNS will likely require one or more of the following processes:cellular replacement, neurotrophic factor delivery, promotion of axonal guidance and removal of growth inhibition, manipulation of intracellular signaling, bridging and artificial substrates, and/or modulation of the immune response.
Since the demonstration nearly20years ago that the mammalian brain has the capacity for self-renewal of neurons and astrocytes, there has been a gradual paradigm shift in neurobiology research such that cell replacement strategies have become a major focus of research and commercial activity. And increasing evidence suggests that stem cell-based transplantation therapies may be one of the most promising therapeutic strategies for treating functional impairments following TBI. Cell replacement therapies have been based on the concept that neurological function lost to injury or disease can be improved by introducing new cells that can replace lost neurons or glial cells, or via trophic support to the surviving cells to increase survival, plasticity, and functional recovery. To date various types of stem cells, including embryonic stem cells, neural stem cells and mesenchymal stem cells are currently being investigated for transplantation.
Transplantation of embryonic stem cells into the injured rat brain can provide functional recovery, however tumor formation raises serious safety concerns about their transplantation use in humans. At the same time, their use in clinical applications is hindered by moral and ethical concerns, as well as the scarcity of fetal tissue. The latest researches show that induced pluripotent stem cells (iPS) have the pluripotency of embryonic stem cells, but it also exist the danger of forming tumors in vivo. Neural stem or progenitor cells (NSC) are also a good candidate to be transplanted into the injured brain, but the production of the NSC is a thorny issue for the use for thansplantation. Human mesenchymal stem cells (MSC) are considered good candidate for transplantation into the injuried brain of the patients. Bone marrow mesenchymal stem cells are currently the most widely used seeding cells for both experimental and clinical studies. However harvesting bone marrow is a highly invasive procedure, and the number, differentiation potential, and maximum life span of mesenchymal stem cells from bone marrow decline with increasing age. Thus, the search for possible alternative MSC is ongoing. Cells derived from the placenta, membranes, amniotic fluid or fetal tissues are higher in number, expansion potential and differentiation abilities compared with mesenchymal stem cells from adult tissues. And some of them have been utilized in the treatment of neurodegenerative and neural traumatic diseases.
Although mesenchymal derived from derived from perinatal tissues have been used in the therapies for a variety of neurological diseases in the experimental animal models, it is mot clear whether the cells from different part of the perinatal tissues have the parallel effect on the treatment of neurological diseases. Here we focus our study on the comparison of the biological characteristics of mesenchymal stem cells from human amniotic membrane (AM-MSC) and umbilical cord Wharton's jelly (WJ-MSC) with respect to their morphology, expansion kinetics, immunophenotype, multipotency. Since NSC are the idealest seed cells to transplant for the cell replacemental treatment for TBI, the capacity of AM-MSC and WJ-MSC to differentiate into neural stem cells were determined in vitro, the easier MSC can differentiat into neural stem cells, the better MSC could be considered as candidate for cell-based seed cells for treatment of TBI.
Neurologic benefit resulting from human MSC treatment of nervous system injuries may be due, at least in part, to the increase of growth factors in the ischemic tissue, and neurotrophic factors play crucial roles in the differentiation and survival of neural cells. Human cytokine antibody arrays (Human L-507Array, RayBiotech Inc.) were used to detect the expression of507cytokines in the cells from two sources of MSC. At the same time, the five important neurotrophic factors which play crucial role in the development and central nervous system restoration, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin3(NT-3), glial cell derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), were detected during neural stem cell induction in vitro. These data may in some degree reflect the neurotrophic benefit of MSC on the treatment of TBI between the AM-MSC and WJ-MSC as well as between the MSC and NSC derived from MSC, which may attribute to the selection of seed cells to the cell-based therapy for TBI.
The survival capacity of MSCs in host tissues in conditions of ischemia or ischemic reperfusion is another important property to be considered. The use of an MSC graft approach is limited by the fact that most of the transplanted MSC are readily lost, potentially triggered by the ischemic or ischemia-reperfusion environment in vivo. In our study, we investigated the anti-apoptosis ability of these MSC towards oxidative stress induced by hydrogen peroxide (H2O2) or serum deprivation.
The study includes three chapters:
Chapter Ⅰ Isolation and characterization of MSC from amniotic membrane and umbilical Wharton's jelly
Objective:To establish a simplified culture system to isolate AM-MSC and WJ-MSC, observe their morphology, and investigate the expression of neural stem cell markers and mesenchymal stem cell markers, their karyotype, proliferative capacities and their multi-lineage differentiation capacities, scan their surfacial fine structure.
Methods:Amniotic membrane was sequentially digested in2.4U/mL dispase,1.0mg/mL Collagenase A and0.01mg/mL DNase; and Wharton's jelly was digested in collagenase type Ⅱ followed by further digestion with0.125%trypsin/EDTA. The morphology was observed by an optic microscope and their surfacial fine structures were scaned by an atomic force microscope, the immunophenotype were tested by Immunocytochemistry or flow cytometry. Before we determined their proliferative capacities, their charyotypes were analysed. The differentiation potential to osteogenic, adipogenic and chondrogenic cells were detected.
Results:AM-MSC and WJ-MSC were successfully isolated and cultured by the protocols mentioned in the methods section. AM-MSC and WJ-MSC both express the markers of MSC (CD13, CD29, CD44, CD73, CD90and CD105), but not express hematopoietic and endothelial phenotypes (CD19, CD31, CD34and CD45), and they both express HLA-ABC, not HLA-DR, and pocess osteogenic, adipogenic and chondrogenic differentiation potential. The results are in accordance with the criteria of MSC by The International Society for Cellular Therapy. Two kinds of MSC both strongly expressed vimentin (100.00±0.00%vs.100.00±0.00%), while few AM-MSC and WJ-MSC expressed nestin (28.73±2.97%vs.19.22±2.96%,t=2.265, P=0.053), sox2(20.58±2.43%vs.21.11±2.51%, t=0.154, P=0.881) and Musashi1(10.17±3.10%vs.12.62±2.58%,t=0.609, P=0.559). These data were confirmed by quantitative real-time PCR and Western blot analysis. AM-MSC possess stronger adherent capacity to the surface compared with WJ-MSC in culture scanning by atomic force microscope, While WJ-MSC have higher praliferative potential (F=817.948, P<0.001). Normal chariotypes we observed throughout the whole culture in vitro.
Conclusion:we have successfully isolate and culture AM-MSC and WJ-MSC, and characterized the cells based on a set of criteria proposed by the International Society for Cellular Therapy. Moreover the two kinds of cells expressed low levels of neural stemness markers. AM-MSC showed stronger adherent capacity to the cultured surface and stronger anti-apoptosis ability compared with WJ-MSC in culture, While WJ-MSC have higher praliferative potential.
Chapter Ⅱ Determination of the capacity of AM-MSC and WJ-MSC for neural stem cells differentiation
Objective:to establish the protocol to induce AM-MSC and WJ-MSC into neural stem cells. By the above protocol, we firstly want to obtain a new source of neural stem cells, and secondly to compare the neuronal differentiation ability of AM-MSC and WJ-MSC. And the expressing change of NT was investigated during the neural stem induction.
Methods:The transdifferentiation protocols:the AM-MSC and WJ-MSC were induced in neural stem cell differentiation medium, composed of KnockOutTM DMEM/F-12Basal Medium supplemented with20ng/mL human epidermal growth factor (EGF),20ng/mL bFGF, StemPro(?) NSC SFM Supplement and GlutaMAXTM-Ⅰ Supplement (1:100) containing1%penicillin/streptomycin at37℃with5%CO2,and plated in Ultra-Low Attachment25cm2culture flasks. The medium was changed every3days. Ten days after induction, neurospheres were collected to determine levels of neurotrophic factors by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) analysis.
Results:AM-MSC and WJ-MSC could be induced into neurosphere-like aggregates, a growth form of neural stem cells, successfully (AM-NSC and WJ-NSC respectively). More importantly, these neural stem cells displayed most of the chanracteristics of neural stem cells. Both AM-MSC and WJ-MSC were examined for expression of the three neural stemness markers by immunocytochemistry after neural stem cell differentiation. After differentiation, the fluorescent signal for the stemness markers of NSC derived from MSC significantly upregulated than that of undifferentiated MSC, and a similar results were obtained from the two kinds of MSC. The details are as follows, AM-NSC showed a higher fluorescent signals for nestin, sox2and musashi1than that of undifferentiated AM-MSC (nestin,92.05±2.75%vs. 28.73±2.97%,P<0.001；sox2,70.17±3.16%vs.20.58±2.43%,P<0.001； Musashil,66.94±3.62%vs.10.17±3.10%,P<0.001)；WJ-NSC showed a higher fluorescent signals for nestin,sox2and musashi1than that of undifferentiated WJ-MSC(nestin,83.57±2.60%vs.19.22±2.96%,P<0.001；sox2,71.17±3.63%vs.21.11±2.51%,P<0.001；Musashi,64.15±3.81%vs.12.62±2.58%,P<0.001). Intrestingly,AM-NSC showed a higher fluorescent signal for the nestin but parallel signal for sox2and musashi than that of WJ-NSC(nestin,92.1±2.82%vs.83.6±22.52%,P=0.0497；sox2,70.2±3.26%vs.71.2±3.54%,P=0.816；Musashil,67.0±3.67%vs.64.0±3.89%,P=0.559).These immunocytochemical data were confirmed by quantitative real-time PCR and Western blot analysis.
Secreted levels of BDNF,GDNF,NT-3,CNTF and NGF in both populations of MSC to NSC were detected.For AM-MSC,the secreted level of BDNF was BDNF,109.51±15.26pg/mL,GDNF32.85±14.21pg/mL,NT-327.43±11.91pg/mL, CNTF39.62±13.56pg/mL and NGF21.46±9.83pg/mL.After differentiation,for AM-NSC,the secreted level of BDNF was BDNF,477.39±39.95pg/mL,GDNF101.01±11.67pg/mL,NT-3206.33±26.36pg/mL,CNTF160.48±22.69pg/mL and NGF185.23±23.59pg/m.For WJ-MSC,the secreted level of BDNF was BDNF,241.69±25.90pg/mL,GDNF16.21±10.01pg/mL,NT-3172.35±25.20pg/mL, CNTF34.37±11.42pg/mL and NGF105.59±18.24pg/mL.After differentiation, for AM-NSC,the secreted level of BDNF was BDNF,333.66±31.59pg/mL,GDNF93.64±19.17pg/mL,NT-3122.46±20.02pg/mL,CNTF231.28±22.07pg/mL and NGF32.76±10.17pg/gmL.There were significant differences between AM-MSC and WJ-MSC in BDNF(P=0.006),NT-3(P<0.001)and NGF(P=0.002)；between AM-NSC and WJ-NSC in BDNF(P=0.003),NT-3(P=0.015),CNTF(P=0.014) and NGF (P=0.009).These ELISA data were confirmed by quantitative real-time PCR.
Conclusion:we successfully established protocols to induce both AM-MSC and WJ-MSC to differentiate into AM-NSC and WJ-NSC in vitro,with AM-NSC expressing higher level of nestin.Before induction,undifferentiated WJ-MSC secret higher levels of BDNF,NT-3and NGF than those of AM-MSC.However after differentiation, the secretions of BDNF, NT-3, CNTF and NGF of differentiated AM-MSC were significantly higher than those of differentiated WJ-MSC. These findings suggested that MSC from different part of placent perssess different reaction to this neural stem cells introduction protocol, and considering the secreting of NTs, AM-NSC may more suitable for transplantation for the treatment of TBI than undifferentiated AM-MSC.
Chapter Ⅲ Cytokine antibody array detection and anti-apoptosis abilities of AM-MSC and WJ-MSC in vitro
Objective:To investigate the cytokine secreting capacity, a Cytokine antibody array was used to detect the expression of507cytokines in the cells from two sources of MSC. To detecte their anti-apoptosis abilities in vitro, apoptosis was triggered by H2O2and serum deprivation, and the rate of apoptosis was counted.
Methods:Total protein was extracted from the AM-MSC and WJ-MSC using a tissue protein extraction reagent (Kangchen, China). Human cytokine antibody arrays (Human L-507Array, RayBiotech Inc.) were used to detect the expression of507cytokines in the cells from two sources of MSC. The signal from the membrane was detected with a chemiluminescene imaging system. The signal intensity was quantified by densitometry. The value altered by twofold or more was statistically significant. A positive control was used to normalize the results from different membranes. Apoptosis triggered by2mmol/L H2O2and serum deprivation. Apoptosis in MSC were identified by nuclear positive staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) and Annexin V and propidine iodide (PI) staining after H2O2or serum deprivation induction.
Results:Results of the cytokine antibody array detection:23cytokines in the AM-MSC were detected by cytokine antibody array which were two fold up regulated than that of WJ-MSC; while49cytokines in the WJ-MSC were detected by cytokine antibody array which were two fold up regulated than that of AM-MSC. And expression levels of4cytokines were changed significantly in AM-MSC compared to WJ-MSC; while there was no significant difference in the cytokines that increased by twofold in WJ-MSC compared with AM-MSC. And the4cytokines up regulated in AM-MSC contain interleukins or their receptor that were considered as inflammatory factors. The details are as follows, IL-6(t=3.546, P=0.038), IL-13R alpha2(t=3.336, P=0.045), IL-12p70(t=3.743, P=0.033), IL-22R (t=3.187, P=0.0498).
Results of the anti-apoptosis ability detection:AM-MSC showed significantly stronger anti-apoptosis ability not only to the induction by H2O2but by serum deprivation, which were detected by TUNEL and Annexin V/PI staining compared with WJ-MSC in vitro (P<0.05).
Conclusion:Cytokines expressed in AM-MSC increased compared with those in WJ-MSC, and these cytokines were growth factors and interleukins, which are related with regulation of cell proliferation and inflammatory response. AM-MSC showed stronger anti-apoptosis ability compared with WJ-MSC in culture.
引文
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