大鼠脐带来源间充质干细胞生物学特性及其对移植免疫耐受作用的研究
发布时间：2018-10-18 21:09
【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(p0.05)。 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。
[Abstract]:Mesenchymal stem cell (MScs) is a kind of pluripotent stem cells derived from mesoderm in early stage of development. At present, MSCs mainly comes from adult bone marrow, but many studies show that human umbilical cord is an ideal source of MSCs. There are many studies on rat bone marrow-derived MSCs for transplantation of immune tolerance and tissue repair. However, there is no related research on MSCs derived from rat umbilical cord, so the MSCs derived from umbilical cord can not be tested in vivo. In this study, mesenchymal stem cells (MSCs) were isolated from rat umbilical cord and their biological characteristics were studied. The suitable isolation and culture methods were found, and in vitro and in vivo experiments were carried out to explore the reduction of immune rejection by umbilical cord derived mesenchymal stem cells. The effect of inducing immune tolerance. In the first part of this study, collagenase digestion and tissue mass culture were used to obtain mesenchymal stem cells derived from rat umbilical cord. The results showed that the adherent cells obtained by both methods were mesenchymal stem cells, and their biological characteristics were close to those of bone marrow mesenchymal stem cells reported in the literature. Collagenase digestion was more efficient than tissue culture. In the second part, the spleen cells of Lewis rats were used as reaction cells, and the spleen cells of DA rats were used as stimulating cells. The mesenchymal stem cells derived from the umbilical cord of Wistar rats were added to the mesenchymal stem cells. The mixed lymphocytes were cultured by 3H-thymidine incorporation. The effects of mesenchymal stem cells derived from rat bone umbilical cord on the culture of mixed lymphocytes in vitro were detected. The results showed that mesenchymal stem cells derived from umbilical cord did not induce lymphocyte proliferation in rats, but had significant inhibitory effects on mixed lymphocyte culture in rats. The third part consisted of DA rats as donor and Lewis rats as receptors. A rat model of heterotopic heart transplantation was established and mesenchymal stem cells (MSCs) derived from umbilical cord of Wistar rats were injected into caudal vein immediately after operation to investigate the effect of mesenchymal stem cells (MSCs) on allogeneic heart transplantation in rats. The recipient mice were divided into two groups: 1, the blank control group was injected with normal saline via tail vein; 2, the third party umbilical cord mesenchymal stem cells were injected into the experimental group through tail vein; the results showed that the survival time of the transplanted heart was 1: 7.5 卤0.5 days. Group 2: 14.8 卤2.1 days. Statistical analysis showed that the survival time of transplanted heart was significantly longer in group 2 than in group 1 (p0.05). The results showed that mesenchymal stem cells could be obtained from the umbilical cord of rats by digestion and tissue mass culture, and the effect of digestion was better. Mesenchymal stem cells derived from umbilical cord were used as third party cells. It can inhibit the proliferation of lymphocytes in vitro, prolong the survival time of cardiac allografts and alleviate the rejection in vivo.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
本文编号：2280371
[Abstract]:Mesenchymal stem cell (MScs) is a kind of pluripotent stem cells derived from mesoderm in early stage of development. At present, MSCs mainly comes from adult bone marrow, but many studies show that human umbilical cord is an ideal source of MSCs. There are many studies on rat bone marrow-derived MSCs for transplantation of immune tolerance and tissue repair. However, there is no related research on MSCs derived from rat umbilical cord, so the MSCs derived from umbilical cord can not be tested in vivo. In this study, mesenchymal stem cells (MSCs) were isolated from rat umbilical cord and their biological characteristics were studied. The suitable isolation and culture methods were found, and in vitro and in vivo experiments were carried out to explore the reduction of immune rejection by umbilical cord derived mesenchymal stem cells. The effect of inducing immune tolerance. In the first part of this study, collagenase digestion and tissue mass culture were used to obtain mesenchymal stem cells derived from rat umbilical cord. The results showed that the adherent cells obtained by both methods were mesenchymal stem cells, and their biological characteristics were close to those of bone marrow mesenchymal stem cells reported in the literature. Collagenase digestion was more efficient than tissue culture. In the second part, the spleen cells of Lewis rats were used as reaction cells, and the spleen cells of DA rats were used as stimulating cells. The mesenchymal stem cells derived from the umbilical cord of Wistar rats were added to the mesenchymal stem cells. The mixed lymphocytes were cultured by 3H-thymidine incorporation. The effects of mesenchymal stem cells derived from rat bone umbilical cord on the culture of mixed lymphocytes in vitro were detected. The results showed that mesenchymal stem cells derived from umbilical cord did not induce lymphocyte proliferation in rats, but had significant inhibitory effects on mixed lymphocyte culture in rats. The third part consisted of DA rats as donor and Lewis rats as receptors. A rat model of heterotopic heart transplantation was established and mesenchymal stem cells (MSCs) derived from umbilical cord of Wistar rats were injected into caudal vein immediately after operation to investigate the effect of mesenchymal stem cells (MSCs) on allogeneic heart transplantation in rats. The recipient mice were divided into two groups: 1, the blank control group was injected with normal saline via tail vein; 2, the third party umbilical cord mesenchymal stem cells were injected into the experimental group through tail vein; the results showed that the survival time of the transplanted heart was 1: 7.5 卤0.5 days. Group 2: 14.8 卤2.1 days. Statistical analysis showed that the survival time of transplanted heart was significantly longer in group 2 than in group 1 (p0.05). The results showed that mesenchymal stem cells could be obtained from the umbilical cord of rats by digestion and tissue mass culture, and the effect of digestion was better. Mesenchymal stem cells derived from umbilical cord were used as third party cells. It can inhibit the proliferation of lymphocytes in vitro, prolong the survival time of cardiac allografts and alleviate the rejection in vivo.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
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