【作者】 王军；

【导师】 陈虎； 张斌；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2012， 博士

【摘要】 研究目的间充质干细胞（mesenchymal stem cells，MSCs）具有再生修复、免疫调控、支持造血等重要功能，近年来已成为干细胞研究及应用领域的热点，主要用于治疗免疫性、退行性及损伤性等多种难治性疾病。而脐带（umbilical cord，UC）中MSCs含量丰富，细胞增殖能力强，受病毒等病原微生物污染的风险低，非常适宜体外规模化扩增、冻存及临床应用，而且脐带作为胎儿娩出后的“废弃物”，取材方便，对产妇及新生儿没有任何风险，不存在伦理学问题。因此，人脐带间充质干细胞（human umbilical cord-derived mesenchymal stem cells，hUC-MSCs）有望成为可以规模化生产的“干细胞治疗药物”。当然，对于细胞治疗来说，安全性是首要关注和亟需研究的问题，特别是非人灵长类动物（non-human primates，NHPs）在亲缘关系上和人类最为接近，在组织结构、病理生理、免疫调节和能量代谢等方面与人类高度近似，其研究价值明显优于其他种属的实验动物。本课题旨在研究UC-MSCs输注食蟹猴（Macacafascicularis）的毒性及主要免疫学指标的影响，并建立食蟹猴脐带间充质干细胞（Macaca fascicularis umbilical cord-derived mesenchymal stem cells，mUC-MSCs）的培养及鉴定方法，为UC-MSCs的同种异体临床应用提供非人灵长类动物的试验支持。研究内容与方法1.人脐带间充质干细胞重复静脉输注食蟹猴4周的毒性试验及主要免疫学指标的影响。试验选用食蟹猴24只，随机分为4组，分别为阴性对照组、溶剂对照组、低剂量组（2×106个/kg）、高剂量组（2×107个/kg）。每周静脉输注1次，连续5次，试验期间进行临床观察、体重、体温、心电图、血细胞计数、凝血功能、血液生化、T淋巴细胞亚群（CD3、CD4、CD8及CD4/CD8）、CD4+CD25+调节性T细胞、Th1/Th2细胞因子（IFN-γ、IL2、IL-4、IL-6、IL-5、TNF）、T淋巴细胞增殖试验、免疫球蛋白G及hUC-MSCs抗体等指标的监测。2.食蟹猴脐带间充质干细胞的培养及生物学特性。通过剖宫产获取食蟹猴脐带，经过机械粉碎、酶学消化、贴壁培养传代，获得食蟹猴脐带MSCs，观察细胞形态、绘制生长曲线、流式细胞术测定细胞周期及免疫表型、并鉴定其多向分化潜能。3.食蟹猴脐带间充质干细胞单次静脉输注食蟹猴的毒性试验及主要免疫学指标的影响。试验选用食蟹猴3只，单次静脉输注mUC-MSCs（2×106个/kg）后，进行临床观察、体重、体温、心电图、血细胞计数、凝血功能、血液生化、T淋巴细胞亚群、CD4+CD25+调节性T细胞、Th1/Th2细胞因子、免疫球蛋白G等指标的动态监测。研究结果1. hUC-MSCs重复静脉输注对食蟹猴的一般生理活动、体重、体温、心电图、血常规、凝血功能、生化等指标均未见有毒理学意义的不可逆的异常改变，注射局部也无明显刺激性。可能的毒性反应为PLT、血清TP、无机P的降低（P≤0.05）。hUC-MSCs重复静脉输注对食蟹猴的T淋巴细胞亚群、T淋巴细胞增殖能力、CD4+CD25+调节性T细胞、血清IgG等指标均未发生有统计学意义的影响，仅在个别时间点观察到IFN-γ、IL-4、IL-6与阴性对照组有统计学差异，在3只动物体内检测到hUC-MSCs抗体。2. mUC-MSCs形态呈梭形，第3代以后细胞的免疫表型显示高表达CD29、CD73、CD90、CD105、CD166，不表达或低表达CD14、CD31、CD34、CD45、CD40、CD80、CD86、HLA-DR；在特定的体外诱导条件下，mUC-MSCs可以向骨、脂肪、软骨分化，细胞周期检测显示G0/G1期细胞>90%。3. mUC-MSCs单次静脉输注对食蟹猴的一般生理活动、体重、体温、心电图、凝血功能均未见有毒理学意义的异常改变。给药后RBC、HGB、HCT有所下降，而Retic有所升高（P≤0.05）；药后Glb有所下降，A/G有所升高（P≤0.05）；输注mUC-MSCs后D3、D5、D7、D8的CD4+细胞与药前比较显著升高，药后D7的CD8+细胞与药前比较有所下降，而药后D3、D5、D7、D8的CD4+/CD8+比例与药前比较亦显著升高（P≤0.05）；药后D4的CD4+CD25+调节性T细胞较药前有所升高（P≤0.05）；仅在个别时间点观察到IL-4、IL-6、TNF的改变有统计学差异。给药前后T淋巴细胞增殖能力及IgG均未见有统计学差异的改变。结论1.本试验成功建立了食蟹猴脐带MSCs的分离培养及鉴定方法，发现mUC-MSCs在细胞形态、免疫表型、多向分化潜能等方面与目前报道的人脐带MSCs相似，为同种异体脐带MSCs的临床前研究及应用提供了非人灵长类动物的研究基础。2.本试验从异种及同种异体两种模式证明了脐带MSCs静脉输注的安全性，在本试验条件下，hUC-MSCs重复静脉输注食蟹猴的安全剂量为2.0×107个/kg，该剂量是临床拟用剂量的20倍。3.本试验证实hUC-MSCs静脉输注对健康食蟹猴的外周血T淋巴细胞亚群、T淋巴细胞增殖能力、CD4+CD25+调节性T细胞、血清IgG、Th1/Th2细胞因子等免疫学指标的影响微乎其微。但mUC-MSCs静脉输注对食蟹猴T淋巴细胞亚群、调节性T细胞还是有所影响的，说明MSCs异种输注和同种异体输注对免疫系统的影响是有区别的。更多还原

【Abstract】 ObjectivesMesenchymal stem cells (MSCs)，which have many important functions such asregeneration repair, immunoregulation and hematopoietic support, have becomehotspot in the field of stem cell research and application in recent years. MSCs aremainly used in the treatment of immune diseases, degenerative diseases, damage andother refractory diseases. Umbilical cord-derived MSCs (UC-MSCs) have strongability of proliferation with low risk of infection of pathogenic microorganisms suchas the virus, and UC-MSCs are very suitable for large scale expansion andfreeze-stored in vitro and clinical application. Moreover, umbilical cords as the"waste" after birth, can be gained conveniently without any risks to puerperas andnewborns and without ethics problems. Therefore, human umbilical cord-derivedmesenchymal stem cells (hUC-MSCs) can be large-scalely produced as a kind of"stem cell medicine".Of course, for cell therapy, safety is the primary concern and needs to beresearched. Especially, non-human primates (NHPs) are the closest to human ingenetic relationship, tissue organization, pathophysiology, immunoregulation andmetabolism, and so the research value of NHPs is much better than other species ofexperiment animals. The present study was aimed to evaluate toxicity andimmunological effects of UC-MSCs administration in macaca fascicularis, and toestablish methods to isolate, culture and identify macaca fascicularis umbilicalcord-derived mesenchymal stem cells (mUC-MSCs), and to provide NHPs testsupport for the clinical application of allogeneic UC-MSCs.Contents and Methods1. Toxicity study and immunological effects of hUC-MSCs repeatedly intravenousadministrations in macaca fascicularis for4weeks.24monkeys were randomlydivided into4groups: the negative control group, the solvent control group, the hUC-MSCs low dose group (2x106cells/kg), and the hUC-MSCs high dose group(2x10~7cells/kg). The monkeys were administrated once a week for5times. During thetest, clinical observations were performed and body weights, body temperatures,electrocardiograms, blood cell counts, coagulation parameter, biochemistry, Tlymphocyte subsets（CD3, CD4, CD8and CD4/CD8）, CD4+CD25+regulatory Tcells, Th1/Th2cytokines（IFN-γ, IL2, IL-4, IL-6, IL-5, TNF）, T lymphocyteproliferation test, IgG and antibody of hUC-MSCs were monitored.2. To isolate and culture mUC-MSCs and to analyze their biological characteristics.We obtained the umbilical cord of macaca fascicularis by caesarean section, and thenumbilical cord was cut into pieces with scissors and was digested with collagenase. SomUC-MSCs were isolated from umbilical cord and subcultured. They were analyzedon morphology, growth curve, immunophenotype, cell cycle and multi-directionaldifferentiation potential.3. Toxicity study and immunological effects of mUC-MSCs (2x106cells/kg) singleadministration in macaca fascicularis. After3monkeys were administrated once,clinical observations were performed and body weights, body temperatures,electrocardiograms, blood cell counts, coagulation parameter, biochemistry, Tlymphocyte subsets, regulatory T cells, Th1/Th2cytokines, T lymphocyteproliferation test and IgG were monitored.Results1.Repeatedly intravenous administrations of hUC-MSCs had no toxicologicallysignificant and irreversible effects on the general physical activity, body weights,body temperatures, electrocardiograms, blood cell counts, coagulation parameter andbiochemistry in monkeys, and there were no apparent changes in injection local. Thepossible toxic reactions were reduction of PLT, serum TP, and P（P≤0.05）. Afteradministrations, there were no statistically significant differences in T lymphocytesubsets, regulatory T cells, T lymphocyte proliferation test between experimentalgroups and control ones. Only at individual time points，there were statisticallysignificant differences in IFN-γ, IL-4and IL-6. Antibodies to hUC-MSCs were detected only in3monkeys.2. The shape of mUC-MSCs was fusiform. After the third passage, immunophenotypeof mUC-MSCs was CD29, CD73, CD90, CD105, CD166positive and CD14, CD31,CD34, CD45, CD40, CD80, CD86, HLA-DR negative. In special inductionconditions, mUC-MSCs could differentiate into osteocytes, adipocytes andchondrocytes in vitro. The cell cycle analyzed by flow cytometry showed that morethan90percent cells were in G0/G1phase.3.Single administration of mUC-MSCs had no toxicologically significant effects onthe general physical activity, body weights, body temperatures, electrocardiogramsand coagulation parameter in monkeys. After administration, RBC, HGB and HCTdeclined, and Retic increased（P≤0.05）. And we found that Glb declined and A/Gincreased after administration （P≤0.05）. Moreover, after administration, thepercentage of CD4+T cells and the ratio of CD4+/CD8+increased significantly（P≤0.05）. On the fourth day after administration, CD4+CD25+Treg increased（P≤0.05）. Before and after administration, T lymphocyte proliferation test and IgGhad no significant differences. Only at individual time points，there were statisticallysignificant changes in TNF, IL-4and IL-6.Conclusions1.This study successfully set up the methods to isolate, culture and identifymUC-MSCs, and showed that mUC-MSCs were similar to hUC-MSCs inmorphology, immunophenotype and multi-directional differentiation potential, andprovided NHPs test support for the clinical application of allogeneic UC-MSCs.2.This study proved the safety of intravenous administration of UC-MSCs based onxenogeneic and allogeneic patterns. In the present experimental condition, the safedosage of hUC-MSC of repeatedly intravenous administrations was2.0x10~7cells/kg,which was20times as much as the dose of clinical therapy.3.The study confirmed intravenous administrations of hUC-MSCs had little effects onT lymphocyte subsets in peripheral blood, T lymphocyte proliferation ability,CD4+CD25+regulatory T cells, IgG, Th1/Th2cytokines of healthy monkeys. But administration of mUC-MSC could effect T lymphocyte subsets and regulatory Tcells, which showed that there were maybe differences between xenogeneic infusionand allogeneic infusion of MSCs.更多还原