【作者】 刘奎利；

【导师】 石炳毅；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2009， 博士

【摘要】 间充质干细胞(MScs)是来源于发育早期中胚层的一类多能干细胞,在诱导器官移植免疫耐受中有着广阔的应用前景。目前MSCs主要来源于成人骨髓,但许多研究显示人脐带是较理想的MSCs来源。关于大鼠骨髓来源的MSCs用于移植免疫耐受及进行组织修复的研究很多,但由于没有大鼠脐带来源MSCs的相关研究,导致脐带来源的MSCs无法进行动物体内试验。本研究从大鼠脐带中分离出间充质干细胞,并对其生物学特性进行研究,找到合适的分离培养方法,并进行体外和体内试验,探讨脐带源间充质干细胞减少免疫排斥反应,诱导免疫耐受的作用。本研究第一部分采用胶原酶消化法和组织块培养法,进行贴壁传代,得到大鼠脐带源间充质干细胞,进行细胞表型分析和诱导分化。结果显示：两种方法得到的贴壁细胞均为间充质干细胞,其生物学性状与文献报导的骨髓间充质干细胞很接近。胶原酶消化法比组织块培养法的效率更高。第二部分以Lewis大鼠脾细胞为反应细胞,DA大鼠脾细胞为刺激细胞,加入来源于Wistar大鼠脐带的间充质干细胞,采用3H-胸腺嘧啶核苷掺入法,进行混合淋巴细胞培养,检测大鼠骨脐带来源充质干细胞对体外混合淋巴细胞培养的影响。结果显示脐带来源间充质干细胞不引起大鼠淋巴细胞增殖反应,但对大鼠混合淋巴细胞培养有显著的抑制作用；第三部分以DA大鼠为供体,Lewis大鼠为受体,建立大鼠异位心脏移植模型,并于术后即刻经尾静脉注入Wistar大鼠脐带来源的间充质干细胞,探讨其对大鼠同种异体心脏移植的影响。异位心脏移植后受体鼠分成2组。1、空白对照组经尾静脉注射生理盐水；2、试验组经尾静脉注射第三方大鼠脐带间充质干细胞；结果显示,移植心脏的存活时间为组1：7.5±0.5天；组2：14.8±2.1天。统计学分析显示,移植心脏存活时间组2比组1明显延长(p<0.05)。本研究结果表明,采用消化法和组织块培养法均能够从大鼠的脐带中获得间充质干细胞,消化法的效果更佳,脐带源间充质干细胞作为第三方细胞,能够在体外抑制淋巴细胞的增殖,并能够在体内延长同种异体心脏移植物的存活时间和减轻排斥反应。更多还原

【Abstract】 Mesenchymal stem cells(MSCs) are multipotential stem cells derived from mesoderm of early phase embryo. It possesses wide application perspective for inducing immunotolerance in organ transplantation. Now MSCs are mainly derived from the adult bone marrow., but many studies showed that human umbilical cord was the better sorce of mesenchymal stem cells. There are many studies that MSCs derived from rat bone marrow were used for transplantation immune tolerance and tissue repair. But because of no correlated study for MSCs derived from rat umbilical cord, study for MSCs derived from umbilical cord could not be done in vivo. In our study, we isolated MSCs from rat umbilical cord and studied their biological characteristics. At last we find a suitable method for isolating and culturing Mscs. We did experiments in vitro and in vivo to study thei effect for inducing immunotolerance and softening immunological rejection.In first part of our study, we successfully isolated MSCs from rat umbilical cord by two enzyme method and tissue mass method.Then we analyzed the cells phenotype and induced cell differentiation Results showed that adherent cells obtained through two methods were all MSCs. Their biological character were the same with MSCs’form bone marrow reported in literature. Two enzyme method was better then tissue mass method. In second part of our study, we choosed Lewis rat’s spleen cells as react cells and DA rat’s spleen cells as stimulate cells. Then MSCs form Wistar rat’s umbilical cord were added in them. Methods By the means of 3H-TdR (three tritium thymine deoxyribosid), we do the MLC(mixed lymphocyte culture) experiments to detect whether the MSCs from rat umbilical cord affected MLC. Results showed that MSCs from rat umbilical cord could not cause rats lymphproliferation response, but have a notable suppressing effect in MLC. In third part of our study, we choosed DA rat as donor and lewis rat as recipient, to establish a heterotopic model of rat heart transpalntation and after orperation immediately inject MSCs from Wistar rat umbilical cord. We investigated the influence of MSCs on allograft of rat heart transplantation In this part,all recepients were divided into two groups. In control group,recepients received normal saline via tail vein after transplantation. In experimental group Recepients received third party MSCs via tail vein. Results showed that the surviving time of transplanted hearts in control group and in experimental group was 7.5±0.5 days and 14.8±2.1 days. Statistical analysis showed that it was longer in experimental group than that in control group(p<0.05).Our research showed that we could get a plenty of MSCs from rat umbilical cord through two enzyme method and tissue mass method. Two enzyme method was better then tissue mass method. As a third party cell, MScs from umbilical cord could suppress lymphocyte proliferation in vitro, and prolong xenograft survival and soften immunological rejection in vivo.更多还原