转分化和未转分化人脐带间充质干细胞在脑损伤修复中的比较研究
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摘要
创伤性脑损伤(traumatic brain injury, TBI)是一种严重影响人们生活质量的中枢神经系统损伤。大多数TBI病人长期遭受认知、运动和感觉等功能缺陷后遗症困扰,给患者、患者家庭和社会带来了沉重的负担。尽管科研工作者在如何治疗TBI后遗症方面进行了长期不懈地努力,然而截止目前仍然没有找到有效的治疗方法,特别是认知功能缺陷(cognitive deficits)仍然是困扰医疗界的一个难题。
为了攻克TBI后遗症,科学家们尝试了各种各样的方法,越来越多的证据表明细胞移植是其中最有应用前景的治疗手段之一。目前有多种细胞被用于临床和实验研究。理论上来说,胚胎干细胞(embryonic stem cells, ESCs)是细胞移植的最佳种子细胞,但是由于受伦理道德和胚胎组织来源不足等因素的的限制,ESCs很难在实验研究和临床上应用推广。目前在实验研究和临床上使用得最为广泛的种子细胞是骨髓间充质干细胞(bone marrow stromal cells, BMSCs),然而BMSCs的增殖和分化能力会随着供者年龄的增长而显著下降,而且BMSCs的获取过程是一个疼痛的有创操作过程。所以寻找新的干细胞替代来源仍然是十分必要的。近年来一种新的细胞引起了科研工作者的广泛关注,这种细胞叫人脐带间充质干细胞(human umbilical mesenchymal stem cells, HUMSCs) HUMSCs不是来源于脐带血而是来源于脐带间充质的干细胞,相对于ESCs或BMSCs, HUMSCs具有以下优点：(1)无伦理道德争议；(2)来源丰富,体外增殖能力强；(3)获取方便,无痛无创。HUMSCs的以上优点引起了细胞移植方面科研工作者的极大兴趣。
近来,有些团队报道HUMSCs可分泌神经营养因子(neurotrophins),并且对中枢神经系统疾病表现出治疗作用。另一方面,有些实验室报道HUMSCs在体外可分化为神经样细胞,但是受到另外一些团体的质疑。前不久,我们用Hermann等的方法在体外成功地将HUMSCs转分化为具有神经干细胞(neural stem cells)特点的脐带间充质干细胞源神经球(human umbilical mesenchymal stem cells-derived neurospheres, HUMSC-NSs),我们接着将这些HUMSC-NSs移植到脊髓全横断大鼠模型中,10周后,一些HUMSC-NSs在体内分化为神经样细胞并对运动功能恢复表现出了促进作用。那么,转分化和未转分化的脐带间充质干细胞到底谁的效果更好呢?据我们了解,目前还没有在相同条件下的比较研究报道。事实上,关于间充质干细胞移植前是否需要转分化一直存在争议。所以,进行间充质干细胞转分化前后的比较研究是迫切需要的。
基于以上背景,本课题选用HUMSCs为本研究的种子细胞,首先建立HUMSCs的分离和培养方法,并研究脐带间充质干细胞的生物学特性,为临床治疗的种子细胞刷选提供理论依据。其次,用改良的两步法对HUMSCs向神外胚层细胞方向进行转分化,检测HUMSCs是否能跨胚层转分化为神经样细胞。最后,将HUMSCs-NSs和HUMSCs移植到TBI大鼠模型上,对他们的治疗效果进行比较,以确认移植前的转分化是否有必要。同时探讨细胞移植治疗脑损伤的可能机制。考虑到将HUMSCs-NSs和HUMSCs移植到大鼠脑内是异种移植,我们还对HUMSCs-NSs和HUMSCs进行了免疫排斥检测。本论文包括以下三个早节：
第一章人脐带间充质干细胞的分离、培养和鉴定
目的：建立分离、培养人脐带基质干细胞(HUMSCs)的方法,了解其生物学特性。
方法：采用胶原酶消化法从足月妊娠健康新生儿的脐带,通过贴壁法获得原代培养的HUMSCs,并进行传代扩增,并对其一般形态和体外扩增能力进行观察分析,同时通过流式细胞检测对其免疫表型进行鉴定。所有数据采用均数±标准误(x±SE)表示,并用统计软件SPSS13.0进行统计分析。用单因素ANOVA分析对P1至P25代HUMSCs的倍增时间进行进行统计分析,P<0.05为差异有统计意义。
结果：用胶原酶消化法从脐带中成功地分离培养出了HUMSCs。光镜观察见HUMSCs具备典型的MSCs的形态学特征。流式检测结果显示：HUMSCs高表达间充质标记物CD73(99.18%), CD90(97.90%), CD105(96.05%)和粘附分子CD29(99.12%), CD44(99.97%), CD166(99.11%);低表达造血系细胞表面标记物CD34(0.05%),CD45(0.91%),血管内皮性抗原CD31(0.02%),人类白细胞抗原HLA-DR(0.17%)和细胞表面标记物CD19(0.10%)。传代培养显示HUMSCs具备较强的体外扩增能力,第1-25代HUMSCs之间的倍增时间波动于26.74～27.68小时之间,经单因素ANOVA统计分析,结果显示第1-25代HUMSCs倍增时间之间的差异无显著性(F值=0.056,P值=0.785),说明整体而言每代细胞倍增时间无显著差异,细胞增殖能力不因传代而下降。
结论：本研究通过胶原酶消化法成功建立了HUMSCs体外培养体系。HUMSCs具有较强的体外扩增能力,多次传代后增殖特性稳定,细胞免疫表型与典型MSCs相似,符合国际细胞治疗协会的间充质干细胞标准,是一种理想的成体干细胞,有望成为细胞移植的理想种子细胞。
第二章人脐带间充质干细胞向神经外胚层样细胞的转分化研究
目的：建立HUMSCs——HUMSC-NSs——神经样细胞的两步转分化方法。通过以上两步转分化法,首先为体内移植治疗TBI的研究提供无伦理道德争议神经干细胞源,其次对HUMSCs在体外是否能转分化为神经样细胞进行验证。此外,在体外对转分化的HUMSCs(即HUMSC-NSs)和未转分化的HUMSCs的神经营养因子分泌能力进行比较,为在体内研究移植前的转分化是否有必要提供一些实验依据。
方法：首先用神经培养基(neurobasal medium),表皮生长因子(epidermal growth factor, EGF),碱性成纤维生长因子(basic fibroblast growth factor, bFGF),神经培养添加剂B27在体外将HUMSCs诱导为HUMSC-NSs,并对HUMSC-NSs是否表达神经干细胞标记物nestin进行免疫细胞化学检测。然后进一步用neurobasal medium, N2,胎牛血清,马血清和全反式维甲酸对HUMSC-NSs进行终末诱导分化,并用免疫细胞化学和Western blot对终末诱导分化结果进行检验。最后采用Elisa对HUMSCs条件培养基和HUMSC-NSs条件培养基中的BDNF和NT-3蛋白水平进行检测。所有数据采用均数±标准误(x±SE)表示,并用统计软件SPSS13.0进行统计分析。对HUMSCs条件培养基和HUMSC-NSs条件培养基中的BDNF和NT-3ELISA检测结果,采用t检验进行比较分析,以P<0.05为差异有统计意义。
结果：通过本实验方案在体外成功地将HUMSCs诱导为具有神经干细胞特点的HUMSC-NSs,经免疫细胞化学检测HUMSC-NSs为nestin强阳性,而且HUMSC-NSCs具有自我更新能力。HUMSC-NSs经终末诱导分化14天后,表达β-tubulin Ⅲ、GalC、GFAP和MAP2ab神经标记物的细胞比例分别为：19.2±3.0%、27±2%、42.8±3.8%和4.4±1.8%。而作为对照的未转分化的HUMSCs不表达以上神经标记物。Elisa检测结果显示：HUMSCs条件培养基中的BDNF蛋白水平(128.75±9.22pg/ml)显著高于HUMSC-NSs条件培养基中的BDNF蛋白水平(81.36±8.39pg/ml)(t=3.802, P=0.003)。而且HUMSCs条件培养基中的NT-3蛋白水平(97.79±8.18pg/m1)也显著高于HUMSC-NSs条件培养基中的NT-3蛋白水平(67.53±8.45pg/ml)(t=2.574,P=0.028)。
结论：通过本实验方案成功建立了HUMSCs向神经外胚层样细胞转分化的两步诱导方案。HUMSC-NSs具备大部分神经系统来源NSCs的典型特征,为NSCs的获取提供了新的来源。HUMSCs在体外可最终分化为神经样细胞,说明HUMSCs在体外可实现跨胚层转化。转分化在提高HUMSCs向神经样细胞分化能力的同时却降低HUMSCs分泌神经营养因子的能力,所以HUMSCs和HUMSCs-NSs谁的效果更好,还有待体内实验验证。
第三章转分化和未转分化人脐带间充质干细胞在脑损伤后认知功能修复中的比较研究
目的：在TBI大鼠模型上,比较HUMSCs和HUMSC-NSs治疗效果,以确定间充质干细胞移植前的转分化是否有意义,同时探讨细胞移植治疗TBI的机制。
方法：改良自由落体打击装置制作成年SD大鼠TBI模型。实验动物随机分为4组：HUMSCs组、HUMSC-NSs组、Matrigel组和假手术组。损伤7天后,将10μLDiI标记的HUMSCs和HUMSC-NSs Matrigel细胞悬液(含1×105个细胞)用微量注射器分别移植到HUMSCs移植组和HUMSC-NSs组受损海马内,损伤对照组注射10μLMatrigel,假手术组未接受任何移植,移植后不给动物使用任何免疫抑制剂。细胞移植术后24至28天采用Morris水迷宫实验每天对各组动物进行认知功能评价。并从组织形态学修复(大体形态学观察、H&E染色和空洞分析),免疫排斥反应、细胞在体内的分化情况以及各组动物海马组织提取液中的BDNF和NT-3蛋白水平这几方面对HUMSCs和HUMSC-NSs进行综合比较。实验中的所有数据采用均数±标准误(x±SE)表示,并用统计软件SPSS13.0进行统计分析。对各实验组间空间学习记忆能力的比较采用重复测量资料的方差分析方法；为分析每一个时间点各组动物在的认知功能是否有差别,我们采用方差分析(one-way ANOVA)进行组间的多重比较,组间的多重比较采用LSD检验,方差不齐采用Welch法校正F检验及Dunnett T3多重比较。各实验组脑组织空洞分析采用单因素方差分析(one-way ANOVA),组间的多重比较采用LSD检验,方差不齐采用Welch法校正F检验及Dunnett T3多重比较。对HUMSCs移植组和HUMSCs-NSs移植组两组间每种神经细胞标记物的比较分析采用t检验。体内海马提取液中的BDNF和NT-3蛋白水平的比较采用单因素方差分析(one-way ANOVA),组间的多重比较采用LSD检验,方差不齐采用Welch法校正F检验及Dunnett T3多重比较。以P<0.05为差异有统计意义。
结果：认知功能恢复情况：重复测量方差分析结果显示各组老鼠整体上显示出显著的分组效应(F=150.602,P<0.001)和时间效应(F=97.706,P<0.001)以及分组与时间的交互效应(F=4.125,P=0.001),说明分组因素和时间因素对各组动物空间学习记忆能力有影响,组间差异大小在不同时间点不一样。进一步用单因素方差分析(one-way ANOVA)对每一个时间点各组动物在的认知功能进行组间的多重比较。发现从移植术后第25天开始两个细胞移植组每天测得的逃避潜伏期明显比Matrigel组短(P<0.05),说明移植术后第25天开始细胞移植组的空间学习记忆能力强于Matrigel组。两个细胞移植组之间相比较,在细胞移植后第27天开始,HUMSCs组每天测得逃避潜伏期比HUMSC-NSs组短(P<0.001),表明随着时间的推延,HUMSCs组大鼠的空间学习记忆能力恢复得更好。
组织修复情况：脑组织大体形态学观察显示除假手术组脑组织中几乎没有缺损灶外,另外三个组脑组织均存在不同程度的缺损灶。两个细胞移植组缺损灶较Matrigel组小。相对而言,HUMSCs组的脑组织缺损灶似乎比HUMSC-NSs组小。进一步用Cavalieri法测量计算空洞体积百分率,我们发现假手术组、Matrigel组、HUMSCs组和HUMSC-NSs组的空洞体积百分率分别为：0.64±0.10%、11.38±0.67%、5.49±0.58%和8.15±0.47%。方差分析结果显示F值=2.570,P值=0.083。可认为各组老鼠脑组织空洞体积百分率存在显著差异。用LSD法进行两两比较,发现3个脑损伤组脑组织空洞体积百分率都显著大于假手术组(P值<0.001)。HUMSCs组和HUMSC-NSs组的空洞体积百分率都显著小于Matrigel组空洞体积百分率(P值<0.001)。而且,HUMSCs组和HUMSC-NSs组的空洞体积百分率之间比较亦有显著性差异(P值<0.05)。
免疫排斥情况：无论是HUMSCs还是HUMSC-NSs植入大鼠体内28天后都没有表现出任何明显地免疫排斥反应现象,如炎细胞浸润,血管套形成等。组织切片用CD4(T辅助细胞表面标记物)、CD8(T杀伤细胞表面标记物)、CDllb(巨噬细胞表面标记物)和MPO(白细胞表面标记物)等抗体进行检测,结果显示：组织切片中以上抗体的免疫反应几乎都可以忽略不计。进一步说明HUMSCs和HUMSC-NSs植入体内后不会引起免疫排斥反应。
移植细胞分化情况：细胞移植28天后,HUMSCs组和HUMSC-NSs组海马内仍有DiI阳性HUMSCs或HUMSC-NSs存在。大部分HUMSCs或HUMSC-NSs位于病变周围靠近正常组织,也有一些移植细胞向损伤病灶中心迁移。免疫组织化学染色后计数并计算HUMSCs组和HUMSC-NSs组中DiI/β-tubulin Ⅲ, DiI/GalC, DiI/GFAP, DiI/MAP2ab双阳性细胞占DiI阳性细胞得比例。结果发现,在HUMSCs组DiI/β-tubulin Ⅲ, DiI/GalC, DiI/GFAP和DiI/MAP2ab双阳性细胞占DiI阳性细胞得比例分别为：5.71±0.52%,3.61±0.41%,5.62±0.60%和3.39±0.23%。而在HUMSC-NSs组DiI/β-tubulin Ⅲ, DiI/GalC, DiI/GFAP和DiI/MAP2ab双阳性细胞占DiI阳性细胞得比例分别为：6.90±0.66%,4.01±0.43%,6.51±0.70%和3.12±0.28%。统计结果显示HUMSCs组和HUMSC-NSs组的分化结果没有统计差异：DiI/β-tubulin Ⅲ(t=-1.418, P=0.178), DiI/GalC(t=-0.674, P=0.512), DiI/GFAP(t=-0.967, P=0.350)和DiI/MAP2ab(t=0.761, P=0.459)。说明无论是HUMSCs还是HUMSC-NSs在植入体内后,绝大大部分都没有向神经外胚层细胞分化,二者分化结果无统计差异。
各组海马提取液中BDNF和NT-3蛋白水平检测结果：在假手术组、Matrigel组、HUMSCs组和HUMSC-NSs组中的BDNF水平分别为：59.07±6.06pg/ml,130.80±10.40pg/ml,269.40±14.00pg/ml,190.47±15.24pg/ml。而相对应的各组NT-3蛋白水平分别为：23.54±3.38pg/ml,48.96±4.74pg/ml,128.97±13.46pg/ml和78.55±7.81pg/ml。方差分析显示各组的BDNF (F值=67.754,P值<0.001)和NT-3(F值=30.003, P值<0.001)蛋白水平存在显著差异。用Dunntt T3检验进行多重比较。结果显示与假手术组相比,三个海马损伤组中的BDNF水平均显著增高(P值<0.001)。而且,HUMSCs组和HUMSC-NSs组中的BDNF水平也显著高于Matrigel组(HUMSCs组与Matrigel group相比P值<0.001；HUMSC-NSs组与Matrigel group相比,P值=0.025)。HUMSCs组和HUMSC-NSs组的BDNF水平之间也存在显著差异(P值=0.007)。有趣地是各实验组海马提取液中的NT-3蛋白水平的表现模式与BDNF相似。与假手术组比较,Matrigel组(P值=0.002)、HUMSCs组(P值<0.001)和HUMSC-NSs组(P值<0.001)中的NT-3蛋白水平都显著增高。而且,HUMSCs组(P值<0.001)和HUMSC-NSs组(P值=0.026)中的NT-3蛋白水平较Matrigel组显著增高。HUMSCs组和HUMSC-NSs组的NT-3蛋白水平之间亦存在显著差异(P值=0.026)。
结论：转分化的HUMSCs和未转分化的HUMSCs对TBI后的认知功能恢复都有促进作用,但是未转分化的HUMSCs效果更好,而且转分化后HUMSCs分泌神经营养因子的能力显著降低,所以移植前的转分化可能不是必需的。同时,绝大多数植入细胞在体内未分化为神经样细胞,说明体外的转分化难以在体内实现,这也提示细胞替代作用不大可能是TBI后认知功能恢复的机制。认知功能恢复与神经营养因子水平成正相关,提示神经营养因子的保护作用可能是TBI后的认知功能恢复的机制之一
Traumatic brain injury (TBI) is a common disease that severely affects quality of life. Most patients suffer from long-term disabilities in cognition, movement and sensation. Despite large efforts, there is no effective treatment for TBI, especially for cognitive deficits.
There are numerous strategies being investigated in the treatment of TBI. There is accumulating evidence that suggest cellular transplantation is one of the most promising options. A number of different cell types are being investigated for seeding. In theory, embryonic stem cells (ESCs) are the best choice in this regard, but their use in clinical applications is hindered by moral and ethical concerns, as well as the scarcity of fetal tissue. Bone marrow stromal cells (BMSCs) are currently the most widely used seeding cells for both experimental and clinical studies. Unfortunately, the cell number and proliferation/differentiation capacity of BMSCs significantly decrease with age. Furthermore, the harvesting of BMSCs involves an invasive and painful procedure. Thus, the search for possible alternative MSCs is ongoing. In recent years, human umbilical mesenchymal stem cells (HUMSCs) were discovered and have attracted extensive attention as they have several advantages over ESCs or BMSCs:(a) no ethical or moral concerns;(b) rich sources and very rapid expansion in vitro; and (c) easily available with noninvasive and painless procedure. These advantages have excited great interest in the scientific community in the use of HUMSCs for cell-based therapies in central nervous system (CNS) injury.
Recently, a number of groups reported that implanted untransdifferentiated HUMSCs secreted neurotrophins, which provided a therapeutic benefit to CNS diseases. Conversely, some groups showed that transdifferentiated HUMSCs differentiated into neural-like cells in vitro. Most recently, using a protocol described by Hermann et al., we successfully transdifferentiated HUMSCs into neurospheres, with the major characteristics of neural stem cells. We subsequently transplanted the HUMSC-derived neurospheres (HUMSC-NSs) into a complete transection model of spinal cord injury in rats. After10weeks, we found that some HUMSC-NSs differentiated into neural-like cells, with therapeutic benefits to locomotor functional recovery. Thus, we decided to test which would be better to transplant for CNS injury, transdifferentiated or untransdifferentiated HUMSCs. To our knowledge, no comparative study has been undertaken with the same conditions, even though there has always been an unresolved controversy about whether it is necessary to transdifferentiate mesenchymal stem cells (MSCs) prior to transplantation.
Based on the above research background, we use HUMSCs as seeding cells in this study. Firstly, we established the protocol to isolate and culture HUMSCs and investigated the basic characteriscs of HUMSCs. Secondly, to establish the protocol to induce HUMSCs into HUMSC-NSs and then into neural-like cells. Finally, We conducted a comprehensive comparison of cognitive functional recovery, tissue structure, neural differentiation and neurotrophin secretion in a rat model of TBI. As the transplantation was a xenotransplantation, we also looked at immune rejection without immunosuppressants.This study includes three chapters:
Chapter Ⅰ. Isolatiton, culture and identification of human umbilical mesenchymal stem cells
Objective:to establish the protocol to isolation, culture and identification of HUMSCs and investigate the basic characteriscs of HUMSCs.
Methods:to obtain HUMSCs by using collagen digestion protocol, and also the morphological, proliferation ability and phenotypic charateristics were investigated.
Results:we obtained HUMSCs successfully by using collagen digestion method. HUMSCs express the marker of MSCs and some adhesion molecules phenotype, but not express hematopoietic and endothelial phenotypes. The results are in accordance with the criteria of MSCs by The International Society for Cellular Therapy. Moreover, HUMSCs have strong proliferation ability, and the proliferation rates have no differences between passage1and25, and the doubling time of cells is26.74-27.68h.
Conclusions:we have successfully established a protocol to isolate and culture HUMSCs. HUMSCs have the similar morphologies and phenotype with BMSCs, and also have very strong proliferation ability. It is a promising cell types for future studies and application.
Chapter Ⅱ. Transdifferentiation of human umbilical mesenchymal stem cells into neuroectodermal-like cells
Objective:to establish the protocol to induce HUMSCs differente into HUMSC-NSs and then into neural-like cells.By the above protocol, we firstly want to obtain a new source of neural stem cells, and secondly to test the neuronal differentiation ability of HUMSCs. And also, to get some data of HUMSCs and HUMSC-NSs in neurotrophinc serection. It will provide new experimental data for the following research in vivo.
Methods:The transdifferentiation protocols included two steps. Firstly, the HUMSCs were induced in neurospheres induction medium:neurobasal medium supplemented with20ng/ml epidermal growth factor (EGF),20ng/ml basic fibroblast growth factor (bFGF) and B27(1:50).3-4days later, small HUMSC-NSs could be observed. Secondly, to investigate the terminal differentiation of HUMSC-NSs, HUMSC-NSs were induced in neurobasal medium supplemented with0.5μmol/L all-trans-retinoic acid,1%FBS,5%horse serum,1%N2supplement. Cells were differentiated for two weeks. We added fresh N2every3days and change medium every6days. The results of transdifferentiation including the two steps were examined using immunofluorescence and western blot. And also, elisa was performed to quantify the protein levels of BDNF and NT-3in HUMSCs and HUMSC-NSs conditioned medium.
Results:HUMSCs were induced into HUMSC-NSs successfully. More importantly, HUMSC-NSs displayed most of the chanracteristics of neural stem cells and could differentiate into neural-like cells in vitro. However the BDNF and NT-3levels of HUMSC-NSs conditioned medium was lower than that of HUMSCs conditioned medium.
Conclusions:we successfully established the protocol to induce HUMSCs to differentiate into HUMSC-NSs, and then into neural-like cells, showing HUMSCs could be transdifferentiated into neuroectodermal-like cells in vitro. We find new source for neural stem cells. But in vitro transdifferentiattion procedures could weaken the secretion characteristics of cells. Therefore, who is more efficacious between transdifferentiated and untransdifferentiated HUMSCs is still need to evaluate in vivo.
Chapter III. Comparison of the effects of transdifferentiated and untransdifferentiated human umbilical mesenchymal stem cells on cognitive functional recovery after rat traumatic brain injury
Objective:to determine the necessity of transdifferentiation of MSCs prior to transplantation and investigate the mechanisms for grafted cells promoting functional recovery in animals suffered TBI.
Methods:TBI animal models were established in adult rat using a weight-drop device. For the different treatments, the rats were divided into four experimental groups:(a) Sham group,(b) Matrigel group,(c) HUMSCs group and (d) HUMSC-NSs group. Seven days after TBI, rats received cellular transplantation. For HUMSCs group,10μl Matrigel containing1×105HUMSCs were injected into the injured hippocampus; for HUMSC-NSs group,10μl Matrigel containing1×105HUMSC-NSs were injected; for Matrigel group, only10μl Matrigel was injected; Sham group received no injection. The overall animals did not receive any immunosuppressant. We compared cognitive functional recovery, tissue structure, neuronal differentiation, and neurotrophin secretion between groups using Morris water maze test, cavitation analysis, immunochemistry and Western blot, and enzyme-linked immunosorbent assay.
Results:Firstly, HUMSCs significantly improved cognitive functional recovery compared with HUMSC-NSs. Secondly, the percentage cavitation of the brain in the HUMSCs group is significantly smaller than that of HUMSC-NSs group. Thirdly there was no obvious immune response in the HUMSCs or HUMSC-NSs groups. Fourthly, most of the grafted HUMSC-NSs remain undifferentiated like HUMSCs, with very few differentiating into neural-like cells. Finally, HUMSCs secreted higher levels of BDNF and NT-3than HUMSC-NSs in vivo.
Conclusions:HUMSCs are more efficacious to cognitive functional recovery and tissue structural protection than HUMSC-NSs, following implantation in TBI in rats. Therefore in vitro neuronal transdifferentiation of HUMSCs may not be necessary prior to their transplantation. Cellular replacement is unlikely the mechanism for grafted cells promoting functional recovery in animals suffered TBI and neuroprotection by neurotrophins may be one of the reasons for grafted cells promoting functional recovery in animals suffered TBI.
为了攻克TBI后遗症,科学家们尝试了各种各样的方法,越来越多的证据表明细胞移植是其中最有应用前景的治疗手段之一。目前有多种细胞被用于临床和实验研究。理论上来说,胚胎干细胞(embryonic stem cells, ESCs)是细胞移植的最佳种子细胞,但是由于受伦理道德和胚胎组织来源不足等因素的的限制,ESCs很难在实验研究和临床上应用推广。目前在实验研究和临床上使用得最为广泛的种子细胞是骨髓间充质干细胞(bone marrow stromal cells, BMSCs),然而BMSCs的增殖和分化能力会随着供者年龄的增长而显著下降,而且BMSCs的获取过程是一个疼痛的有创操作过程。所以寻找新的干细胞替代来源仍然是十分必要的。近年来一种新的细胞引起了科研工作者的广泛关注,这种细胞叫人脐带间充质干细胞(human umbilical mesenchymal stem cells, HUMSCs) HUMSCs不是来源于脐带血而是来源于脐带间充质的干细胞,相对于ESCs或BMSCs, HUMSCs具有以下优点：(1)无伦理道德争议；(2)来源丰富,体外增殖能力强；(3)获取方便,无痛无创。HUMSCs的以上优点引起了细胞移植方面科研工作者的极大兴趣。
近来,有些团队报道HUMSCs可分泌神经营养因子(neurotrophins),并且对中枢神经系统疾病表现出治疗作用。另一方面,有些实验室报道HUMSCs在体外可分化为神经样细胞,但是受到另外一些团体的质疑。前不久,我们用Hermann等的方法在体外成功地将HUMSCs转分化为具有神经干细胞(neural stem cells)特点的脐带间充质干细胞源神经球(human umbilical mesenchymal stem cells-derived neurospheres, HUMSC-NSs),我们接着将这些HUMSC-NSs移植到脊髓全横断大鼠模型中,10周后,一些HUMSC-NSs在体内分化为神经样细胞并对运动功能恢复表现出了促进作用。那么,转分化和未转分化的脐带间充质干细胞到底谁的效果更好呢?据我们了解,目前还没有在相同条件下的比较研究报道。事实上,关于间充质干细胞移植前是否需要转分化一直存在争议。所以,进行间充质干细胞转分化前后的比较研究是迫切需要的。
基于以上背景,本课题选用HUMSCs为本研究的种子细胞,首先建立HUMSCs的分离和培养方法,并研究脐带间充质干细胞的生物学特性,为临床治疗的种子细胞刷选提供理论依据。其次,用改良的两步法对HUMSCs向神外胚层细胞方向进行转分化,检测HUMSCs是否能跨胚层转分化为神经样细胞。最后,将HUMSCs-NSs和HUMSCs移植到TBI大鼠模型上,对他们的治疗效果进行比较,以确认移植前的转分化是否有必要。同时探讨细胞移植治疗脑损伤的可能机制。考虑到将HUMSCs-NSs和HUMSCs移植到大鼠脑内是异种移植,我们还对HUMSCs-NSs和HUMSCs进行了免疫排斥检测。本论文包括以下三个早节：
第一章人脐带间充质干细胞的分离、培养和鉴定
目的：建立分离、培养人脐带基质干细胞(HUMSCs)的方法,了解其生物学特性。
方法：采用胶原酶消化法从足月妊娠健康新生儿的脐带,通过贴壁法获得原代培养的HUMSCs,并进行传代扩增,并对其一般形态和体外扩增能力进行观察分析,同时通过流式细胞检测对其免疫表型进行鉴定。所有数据采用均数±标准误(x±SE)表示,并用统计软件SPSS13.0进行统计分析。用单因素ANOVA分析对P1至P25代HUMSCs的倍增时间进行进行统计分析,P<0.05为差异有统计意义。
结果：用胶原酶消化法从脐带中成功地分离培养出了HUMSCs。光镜观察见HUMSCs具备典型的MSCs的形态学特征。流式检测结果显示：HUMSCs高表达间充质标记物CD73(99.18%), CD90(97.90%), CD105(96.05%)和粘附分子CD29(99.12%), CD44(99.97%), CD166(99.11%);低表达造血系细胞表面标记物CD34(0.05%),CD45(0.91%),血管内皮性抗原CD31(0.02%),人类白细胞抗原HLA-DR(0.17%)和细胞表面标记物CD19(0.10%)。传代培养显示HUMSCs具备较强的体外扩增能力,第1-25代HUMSCs之间的倍增时间波动于26.74～27.68小时之间,经单因素ANOVA统计分析,结果显示第1-25代HUMSCs倍增时间之间的差异无显著性(F值=0.056,P值=0.785),说明整体而言每代细胞倍增时间无显著差异,细胞增殖能力不因传代而下降。
结论：本研究通过胶原酶消化法成功建立了HUMSCs体外培养体系。HUMSCs具有较强的体外扩增能力,多次传代后增殖特性稳定,细胞免疫表型与典型MSCs相似,符合国际细胞治疗协会的间充质干细胞标准,是一种理想的成体干细胞,有望成为细胞移植的理想种子细胞。
第二章人脐带间充质干细胞向神经外胚层样细胞的转分化研究
目的：建立HUMSCs——HUMSC-NSs——神经样细胞的两步转分化方法。通过以上两步转分化法,首先为体内移植治疗TBI的研究提供无伦理道德争议神经干细胞源,其次对HUMSCs在体外是否能转分化为神经样细胞进行验证。此外,在体外对转分化的HUMSCs(即HUMSC-NSs)和未转分化的HUMSCs的神经营养因子分泌能力进行比较,为在体内研究移植前的转分化是否有必要提供一些实验依据。
方法：首先用神经培养基(neurobasal medium),表皮生长因子(epidermal growth factor, EGF),碱性成纤维生长因子(basic fibroblast growth factor, bFGF),神经培养添加剂B27在体外将HUMSCs诱导为HUMSC-NSs,并对HUMSC-NSs是否表达神经干细胞标记物nestin进行免疫细胞化学检测。然后进一步用neurobasal medium, N2,胎牛血清,马血清和全反式维甲酸对HUMSC-NSs进行终末诱导分化,并用免疫细胞化学和Western blot对终末诱导分化结果进行检验。最后采用Elisa对HUMSCs条件培养基和HUMSC-NSs条件培养基中的BDNF和NT-3蛋白水平进行检测。所有数据采用均数±标准误(x±SE)表示,并用统计软件SPSS13.0进行统计分析。对HUMSCs条件培养基和HUMSC-NSs条件培养基中的BDNF和NT-3ELISA检测结果,采用t检验进行比较分析,以P<0.05为差异有统计意义。
结果：通过本实验方案在体外成功地将HUMSCs诱导为具有神经干细胞特点的HUMSC-NSs,经免疫细胞化学检测HUMSC-NSs为nestin强阳性,而且HUMSC-NSCs具有自我更新能力。HUMSC-NSs经终末诱导分化14天后,表达β-tubulin Ⅲ、GalC、GFAP和MAP2ab神经标记物的细胞比例分别为：19.2±3.0%、27±2%、42.8±3.8%和4.4±1.8%。而作为对照的未转分化的HUMSCs不表达以上神经标记物。Elisa检测结果显示：HUMSCs条件培养基中的BDNF蛋白水平(128.75±9.22pg/ml)显著高于HUMSC-NSs条件培养基中的BDNF蛋白水平(81.36±8.39pg/ml)(t=3.802, P=0.003)。而且HUMSCs条件培养基中的NT-3蛋白水平(97.79±8.18pg/m1)也显著高于HUMSC-NSs条件培养基中的NT-3蛋白水平(67.53±8.45pg/ml)(t=2.574,P=0.028)。
结论：通过本实验方案成功建立了HUMSCs向神经外胚层样细胞转分化的两步诱导方案。HUMSC-NSs具备大部分神经系统来源NSCs的典型特征,为NSCs的获取提供了新的来源。HUMSCs在体外可最终分化为神经样细胞,说明HUMSCs在体外可实现跨胚层转化。转分化在提高HUMSCs向神经样细胞分化能力的同时却降低HUMSCs分泌神经营养因子的能力,所以HUMSCs和HUMSCs-NSs谁的效果更好,还有待体内实验验证。
第三章转分化和未转分化人脐带间充质干细胞在脑损伤后认知功能修复中的比较研究
目的：在TBI大鼠模型上,比较HUMSCs和HUMSC-NSs治疗效果,以确定间充质干细胞移植前的转分化是否有意义,同时探讨细胞移植治疗TBI的机制。
方法：改良自由落体打击装置制作成年SD大鼠TBI模型。实验动物随机分为4组：HUMSCs组、HUMSC-NSs组、Matrigel组和假手术组。损伤7天后,将10μLDiI标记的HUMSCs和HUMSC-NSs Matrigel细胞悬液(含1×105个细胞)用微量注射器分别移植到HUMSCs移植组和HUMSC-NSs组受损海马内,损伤对照组注射10μLMatrigel,假手术组未接受任何移植,移植后不给动物使用任何免疫抑制剂。细胞移植术后24至28天采用Morris水迷宫实验每天对各组动物进行认知功能评价。并从组织形态学修复(大体形态学观察、H&E染色和空洞分析),免疫排斥反应、细胞在体内的分化情况以及各组动物海马组织提取液中的BDNF和NT-3蛋白水平这几方面对HUMSCs和HUMSC-NSs进行综合比较。实验中的所有数据采用均数±标准误(x±SE)表示,并用统计软件SPSS13.0进行统计分析。对各实验组间空间学习记忆能力的比较采用重复测量资料的方差分析方法；为分析每一个时间点各组动物在的认知功能是否有差别,我们采用方差分析(one-way ANOVA)进行组间的多重比较,组间的多重比较采用LSD检验,方差不齐采用Welch法校正F检验及Dunnett T3多重比较。各实验组脑组织空洞分析采用单因素方差分析(one-way ANOVA),组间的多重比较采用LSD检验,方差不齐采用Welch法校正F检验及Dunnett T3多重比较。对HUMSCs移植组和HUMSCs-NSs移植组两组间每种神经细胞标记物的比较分析采用t检验。体内海马提取液中的BDNF和NT-3蛋白水平的比较采用单因素方差分析(one-way ANOVA),组间的多重比较采用LSD检验,方差不齐采用Welch法校正F检验及Dunnett T3多重比较。以P<0.05为差异有统计意义。
结果：认知功能恢复情况：重复测量方差分析结果显示各组老鼠整体上显示出显著的分组效应(F=150.602,P<0.001)和时间效应(F=97.706,P<0.001)以及分组与时间的交互效应(F=4.125,P=0.001),说明分组因素和时间因素对各组动物空间学习记忆能力有影响,组间差异大小在不同时间点不一样。进一步用单因素方差分析(one-way ANOVA)对每一个时间点各组动物在的认知功能进行组间的多重比较。发现从移植术后第25天开始两个细胞移植组每天测得的逃避潜伏期明显比Matrigel组短(P<0.05),说明移植术后第25天开始细胞移植组的空间学习记忆能力强于Matrigel组。两个细胞移植组之间相比较,在细胞移植后第27天开始,HUMSCs组每天测得逃避潜伏期比HUMSC-NSs组短(P<0.001),表明随着时间的推延,HUMSCs组大鼠的空间学习记忆能力恢复得更好。
组织修复情况：脑组织大体形态学观察显示除假手术组脑组织中几乎没有缺损灶外,另外三个组脑组织均存在不同程度的缺损灶。两个细胞移植组缺损灶较Matrigel组小。相对而言,HUMSCs组的脑组织缺损灶似乎比HUMSC-NSs组小。进一步用Cavalieri法测量计算空洞体积百分率,我们发现假手术组、Matrigel组、HUMSCs组和HUMSC-NSs组的空洞体积百分率分别为：0.64±0.10%、11.38±0.67%、5.49±0.58%和8.15±0.47%。方差分析结果显示F值=2.570,P值=0.083。可认为各组老鼠脑组织空洞体积百分率存在显著差异。用LSD法进行两两比较,发现3个脑损伤组脑组织空洞体积百分率都显著大于假手术组(P值<0.001)。HUMSCs组和HUMSC-NSs组的空洞体积百分率都显著小于Matrigel组空洞体积百分率(P值<0.001)。而且,HUMSCs组和HUMSC-NSs组的空洞体积百分率之间比较亦有显著性差异(P值<0.05)。
免疫排斥情况：无论是HUMSCs还是HUMSC-NSs植入大鼠体内28天后都没有表现出任何明显地免疫排斥反应现象,如炎细胞浸润,血管套形成等。组织切片用CD4(T辅助细胞表面标记物)、CD8(T杀伤细胞表面标记物)、CDllb(巨噬细胞表面标记物)和MPO(白细胞表面标记物)等抗体进行检测,结果显示：组织切片中以上抗体的免疫反应几乎都可以忽略不计。进一步说明HUMSCs和HUMSC-NSs植入体内后不会引起免疫排斥反应。
移植细胞分化情况：细胞移植28天后,HUMSCs组和HUMSC-NSs组海马内仍有DiI阳性HUMSCs或HUMSC-NSs存在。大部分HUMSCs或HUMSC-NSs位于病变周围靠近正常组织,也有一些移植细胞向损伤病灶中心迁移。免疫组织化学染色后计数并计算HUMSCs组和HUMSC-NSs组中DiI/β-tubulin Ⅲ, DiI/GalC, DiI/GFAP, DiI/MAP2ab双阳性细胞占DiI阳性细胞得比例。结果发现,在HUMSCs组DiI/β-tubulin Ⅲ, DiI/GalC, DiI/GFAP和DiI/MAP2ab双阳性细胞占DiI阳性细胞得比例分别为：5.71±0.52%,3.61±0.41%,5.62±0.60%和3.39±0.23%。而在HUMSC-NSs组DiI/β-tubulin Ⅲ, DiI/GalC, DiI/GFAP和DiI/MAP2ab双阳性细胞占DiI阳性细胞得比例分别为：6.90±0.66%,4.01±0.43%,6.51±0.70%和3.12±0.28%。统计结果显示HUMSCs组和HUMSC-NSs组的分化结果没有统计差异：DiI/β-tubulin Ⅲ(t=-1.418, P=0.178), DiI/GalC(t=-0.674, P=0.512), DiI/GFAP(t=-0.967, P=0.350)和DiI/MAP2ab(t=0.761, P=0.459)。说明无论是HUMSCs还是HUMSC-NSs在植入体内后,绝大大部分都没有向神经外胚层细胞分化,二者分化结果无统计差异。
各组海马提取液中BDNF和NT-3蛋白水平检测结果：在假手术组、Matrigel组、HUMSCs组和HUMSC-NSs组中的BDNF水平分别为：59.07±6.06pg/ml,130.80±10.40pg/ml,269.40±14.00pg/ml,190.47±15.24pg/ml。而相对应的各组NT-3蛋白水平分别为：23.54±3.38pg/ml,48.96±4.74pg/ml,128.97±13.46pg/ml和78.55±7.81pg/ml。方差分析显示各组的BDNF (F值=67.754,P值<0.001)和NT-3(F值=30.003, P值<0.001)蛋白水平存在显著差异。用Dunntt T3检验进行多重比较。结果显示与假手术组相比,三个海马损伤组中的BDNF水平均显著增高(P值<0.001)。而且,HUMSCs组和HUMSC-NSs组中的BDNF水平也显著高于Matrigel组(HUMSCs组与Matrigel group相比P值<0.001；HUMSC-NSs组与Matrigel group相比,P值=0.025)。HUMSCs组和HUMSC-NSs组的BDNF水平之间也存在显著差异(P值=0.007)。有趣地是各实验组海马提取液中的NT-3蛋白水平的表现模式与BDNF相似。与假手术组比较,Matrigel组(P值=0.002)、HUMSCs组(P值<0.001)和HUMSC-NSs组(P值<0.001)中的NT-3蛋白水平都显著增高。而且,HUMSCs组(P值<0.001)和HUMSC-NSs组(P值=0.026)中的NT-3蛋白水平较Matrigel组显著增高。HUMSCs组和HUMSC-NSs组的NT-3蛋白水平之间亦存在显著差异(P值=0.026)。
结论：转分化的HUMSCs和未转分化的HUMSCs对TBI后的认知功能恢复都有促进作用,但是未转分化的HUMSCs效果更好,而且转分化后HUMSCs分泌神经营养因子的能力显著降低,所以移植前的转分化可能不是必需的。同时,绝大多数植入细胞在体内未分化为神经样细胞,说明体外的转分化难以在体内实现,这也提示细胞替代作用不大可能是TBI后认知功能恢复的机制。认知功能恢复与神经营养因子水平成正相关,提示神经营养因子的保护作用可能是TBI后的认知功能恢复的机制之一
Traumatic brain injury (TBI) is a common disease that severely affects quality of life. Most patients suffer from long-term disabilities in cognition, movement and sensation. Despite large efforts, there is no effective treatment for TBI, especially for cognitive deficits.
There are numerous strategies being investigated in the treatment of TBI. There is accumulating evidence that suggest cellular transplantation is one of the most promising options. A number of different cell types are being investigated for seeding. In theory, embryonic stem cells (ESCs) are the best choice in this regard, but their use in clinical applications is hindered by moral and ethical concerns, as well as the scarcity of fetal tissue. Bone marrow stromal cells (BMSCs) are currently the most widely used seeding cells for both experimental and clinical studies. Unfortunately, the cell number and proliferation/differentiation capacity of BMSCs significantly decrease with age. Furthermore, the harvesting of BMSCs involves an invasive and painful procedure. Thus, the search for possible alternative MSCs is ongoing. In recent years, human umbilical mesenchymal stem cells (HUMSCs) were discovered and have attracted extensive attention as they have several advantages over ESCs or BMSCs:(a) no ethical or moral concerns;(b) rich sources and very rapid expansion in vitro; and (c) easily available with noninvasive and painless procedure. These advantages have excited great interest in the scientific community in the use of HUMSCs for cell-based therapies in central nervous system (CNS) injury.
Recently, a number of groups reported that implanted untransdifferentiated HUMSCs secreted neurotrophins, which provided a therapeutic benefit to CNS diseases. Conversely, some groups showed that transdifferentiated HUMSCs differentiated into neural-like cells in vitro. Most recently, using a protocol described by Hermann et al., we successfully transdifferentiated HUMSCs into neurospheres, with the major characteristics of neural stem cells. We subsequently transplanted the HUMSC-derived neurospheres (HUMSC-NSs) into a complete transection model of spinal cord injury in rats. After10weeks, we found that some HUMSC-NSs differentiated into neural-like cells, with therapeutic benefits to locomotor functional recovery. Thus, we decided to test which would be better to transplant for CNS injury, transdifferentiated or untransdifferentiated HUMSCs. To our knowledge, no comparative study has been undertaken with the same conditions, even though there has always been an unresolved controversy about whether it is necessary to transdifferentiate mesenchymal stem cells (MSCs) prior to transplantation.
Based on the above research background, we use HUMSCs as seeding cells in this study. Firstly, we established the protocol to isolate and culture HUMSCs and investigated the basic characteriscs of HUMSCs. Secondly, to establish the protocol to induce HUMSCs into HUMSC-NSs and then into neural-like cells. Finally, We conducted a comprehensive comparison of cognitive functional recovery, tissue structure, neural differentiation and neurotrophin secretion in a rat model of TBI. As the transplantation was a xenotransplantation, we also looked at immune rejection without immunosuppressants.This study includes three chapters:
Chapter Ⅰ. Isolatiton, culture and identification of human umbilical mesenchymal stem cells
Objective:to establish the protocol to isolation, culture and identification of HUMSCs and investigate the basic characteriscs of HUMSCs.
Methods:to obtain HUMSCs by using collagen digestion protocol, and also the morphological, proliferation ability and phenotypic charateristics were investigated.
Results:we obtained HUMSCs successfully by using collagen digestion method. HUMSCs express the marker of MSCs and some adhesion molecules phenotype, but not express hematopoietic and endothelial phenotypes. The results are in accordance with the criteria of MSCs by The International Society for Cellular Therapy. Moreover, HUMSCs have strong proliferation ability, and the proliferation rates have no differences between passage1and25, and the doubling time of cells is26.74-27.68h.
Conclusions:we have successfully established a protocol to isolate and culture HUMSCs. HUMSCs have the similar morphologies and phenotype with BMSCs, and also have very strong proliferation ability. It is a promising cell types for future studies and application.
Chapter Ⅱ. Transdifferentiation of human umbilical mesenchymal stem cells into neuroectodermal-like cells
Objective:to establish the protocol to induce HUMSCs differente into HUMSC-NSs and then into neural-like cells.By the above protocol, we firstly want to obtain a new source of neural stem cells, and secondly to test the neuronal differentiation ability of HUMSCs. And also, to get some data of HUMSCs and HUMSC-NSs in neurotrophinc serection. It will provide new experimental data for the following research in vivo.
Methods:The transdifferentiation protocols included two steps. Firstly, the HUMSCs were induced in neurospheres induction medium:neurobasal medium supplemented with20ng/ml epidermal growth factor (EGF),20ng/ml basic fibroblast growth factor (bFGF) and B27(1:50).3-4days later, small HUMSC-NSs could be observed. Secondly, to investigate the terminal differentiation of HUMSC-NSs, HUMSC-NSs were induced in neurobasal medium supplemented with0.5μmol/L all-trans-retinoic acid,1%FBS,5%horse serum,1%N2supplement. Cells were differentiated for two weeks. We added fresh N2every3days and change medium every6days. The results of transdifferentiation including the two steps were examined using immunofluorescence and western blot. And also, elisa was performed to quantify the protein levels of BDNF and NT-3in HUMSCs and HUMSC-NSs conditioned medium.
Results:HUMSCs were induced into HUMSC-NSs successfully. More importantly, HUMSC-NSs displayed most of the chanracteristics of neural stem cells and could differentiate into neural-like cells in vitro. However the BDNF and NT-3levels of HUMSC-NSs conditioned medium was lower than that of HUMSCs conditioned medium.
Conclusions:we successfully established the protocol to induce HUMSCs to differentiate into HUMSC-NSs, and then into neural-like cells, showing HUMSCs could be transdifferentiated into neuroectodermal-like cells in vitro. We find new source for neural stem cells. But in vitro transdifferentiattion procedures could weaken the secretion characteristics of cells. Therefore, who is more efficacious between transdifferentiated and untransdifferentiated HUMSCs is still need to evaluate in vivo.
Chapter III. Comparison of the effects of transdifferentiated and untransdifferentiated human umbilical mesenchymal stem cells on cognitive functional recovery after rat traumatic brain injury
Objective:to determine the necessity of transdifferentiation of MSCs prior to transplantation and investigate the mechanisms for grafted cells promoting functional recovery in animals suffered TBI.
Methods:TBI animal models were established in adult rat using a weight-drop device. For the different treatments, the rats were divided into four experimental groups:(a) Sham group,(b) Matrigel group,(c) HUMSCs group and (d) HUMSC-NSs group. Seven days after TBI, rats received cellular transplantation. For HUMSCs group,10μl Matrigel containing1×105HUMSCs were injected into the injured hippocampus; for HUMSC-NSs group,10μl Matrigel containing1×105HUMSC-NSs were injected; for Matrigel group, only10μl Matrigel was injected; Sham group received no injection. The overall animals did not receive any immunosuppressant. We compared cognitive functional recovery, tissue structure, neuronal differentiation, and neurotrophin secretion between groups using Morris water maze test, cavitation analysis, immunochemistry and Western blot, and enzyme-linked immunosorbent assay.
Results:Firstly, HUMSCs significantly improved cognitive functional recovery compared with HUMSC-NSs. Secondly, the percentage cavitation of the brain in the HUMSCs group is significantly smaller than that of HUMSC-NSs group. Thirdly there was no obvious immune response in the HUMSCs or HUMSC-NSs groups. Fourthly, most of the grafted HUMSC-NSs remain undifferentiated like HUMSCs, with very few differentiating into neural-like cells. Finally, HUMSCs secreted higher levels of BDNF and NT-3than HUMSC-NSs in vivo.
Conclusions:HUMSCs are more efficacious to cognitive functional recovery and tissue structural protection than HUMSC-NSs, following implantation in TBI in rats. Therefore in vitro neuronal transdifferentiation of HUMSCs may not be necessary prior to their transplantation. Cellular replacement is unlikely the mechanism for grafted cells promoting functional recovery in animals suffered TBI and neuroprotection by neurotrophins may be one of the reasons for grafted cells promoting functional recovery in animals suffered TBI.
引文
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