【摘要】： 間充質(zhì)干細胞(MScs)是來源于發(fā)育早期中胚層的一類多能干細胞,在誘導器官移植免疫耐受中有著廣闊的應用前景。目前MSCs主要來源于成人骨髓,但許多研究顯示人臍帶是較理想的MSCs來源。關于大鼠骨髓來源的MSCs用于移植免疫耐受及進行組織修復的研究很多,但由于沒有大鼠臍帶來源MSCs的相關研究,導致臍帶來源的MSCs無法進行動物體內(nèi)試驗。本研究從大鼠臍帶中分離出間充質(zhì)干細胞,并對其生物學特性進行研究,找到合適的分離培養(yǎng)方法,并進行體外和體內(nèi)試驗,探討臍帶源間充質(zhì)干細胞減少免疫排斥反應,誘導免疫耐受的作用。 本研究第一部分采用膠原酶消化法和組織塊培養(yǎng)法,進行貼壁傳代,得到大鼠臍帶源間充質(zhì)干細胞,進行細胞表型分析和誘導分化。結果顯示：兩種方法得到的貼壁細胞均為間充質(zhì)干細胞,其生物學性狀與文獻報導的骨髓間充質(zhì)干細胞很接近。膠原酶消化法比組織塊培養(yǎng)法的效率更高。第二部分以Lewis大鼠脾細胞為反應細胞,DA大鼠脾細胞為刺激細胞,加入來源于Wistar大鼠臍帶的間充質(zhì)干細胞,采用3H-胸腺嘧啶核苷摻入法,進行混合淋巴細胞培養(yǎng),檢測大鼠骨臍帶來源充質(zhì)干細胞對體外混合淋巴細胞培養(yǎng)的影響。結果顯示臍帶來源間充質(zhì)干細胞不引起大鼠淋巴細胞增殖反應,但對大鼠混合淋巴細胞培養(yǎng)有顯著的抑制作用；第三部分以DA大鼠為供體,Lewis大鼠為受體,建立大鼠異位心臟移植模型,并于術后即刻經(jīng)尾靜脈注入Wistar大鼠臍帶來源的間充質(zhì)干細胞,探討其對大鼠同種異體心臟移植的影響。異位心臟移植后受體鼠分成2組。1、空白對照組經(jīng)尾靜脈注射生理鹽水；2、試驗組經(jīng)尾靜脈注射第三方大鼠臍帶間充質(zhì)干細胞；結果顯示,移植心臟的存活時間為組1：7.5±0.5天；組2：14.8±2.1天。統(tǒng)計學分析顯示,移植心臟存活時間組2比組1明顯延長(p0.05)。 本研究結果表明,采用消化法和組織塊培養(yǎng)法均能夠從大鼠的臍帶中獲得間充質(zhì)干細胞,消化法的效果更佳,臍帶源間充質(zhì)干細胞作為第三方細胞,能夠在體外抑制淋巴細胞的增殖,并能夠在體內(nèi)延長同種異體心臟移植物的存活時間和減輕排斥反應。
[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 鹵0.5 days. Group 2: 14.8 鹵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.
【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392

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