两种来源人胎盘间充质干细胞CD200的差异表达对小鼠皮肤移植免疫调节作用的研究

发布时间：2018-06-18 01:04

本文选题：胎盘间充质干细胞 + CD200　；参考：《宁夏医科大学》2015年硕士论文

【摘要】：目的研究两种来源的人胎盘间充质干细胞CD200的表达差异在小鼠皮肤移植免疫调节中的作用及作用机制,为临床上选择更好的抑制免疫排斥的干细胞提供一定理论依据。方法分离两种来源细胞;流式细胞术检测间充质干细胞表面标志分子等的表达情况;流式细胞抗体中和高表达CD200分子的细胞、携带CD200基因的腺病毒载体及腺病毒空载体分别转染低表达CD200分子的细胞,将等量PBS及上述处理过的细胞通过尾静脉注射入将ICR乳鼠皮肤移植至C57BL/6背部建立的皮肤移植免疫排斥模型小鼠体内,统计各组皮肤存活时间;术后第7天,HE染色法观察小鼠移植部位炎性细胞浸润情况;RT-PCR法检测小鼠脾脏中IL-17、IFN-γ、TNF-α、IL-10基因的相对表达量;眼眶静脉取血,检测各组小鼠血液中白细胞的含量;ELISA法检测血液中相应免疫因子的含量。结果胎儿侧来源细胞CD200阳性率明显高于母体侧。与PBS组比较:m PMSC组和f PMSC组移植皮肤存活时间均明显延长;与f PMSC+anti-CD200组比较:f PMSC组移植皮肤存活时间明显延长;与m PMSC+腺病毒空载体组比较:m PMSC+CD200腺病毒组移植皮肤存活时间明显延长。HE结果显示:细胞移植组炎性细胞的浸润数量明显少于PBS组。RT-PCR结果显示:与PBS组比较:m PMSC组IL-17、IFN-γ、TNF-α的相对表达量,f PMSC组TNF-α的相对表达量明显降低,f PMSC组IL-10的相对表达量明显升高;与m PMSC组比较:f PMSC组IFN-γ、TNF-α的相对表达量均明显降低;与f PMSC组比较:f PMSC+Anti-CD200组IL-17、TNF-α的相对表达量,f PMSC+Anti-CD200组IFN-γ的相对表达量均明显升高;与m PMSC+CD200腺病毒载体转染组比较:m PMSC+腺病毒空载体转染组TNF-α、IFN-γ的相对表达量均明显升高,m PMSC+腺病毒空载体转染组IL-17的相对表达量明显升高,m PMSC+腺病毒空载体转染组IL-10的相对表达量明显降低。ELISA结果与RT-PCR结果相符。结论f PMSC抑制小鼠皮肤移植免疫排斥作用优于m PMSC;与其更强的抑制免疫细胞的增殖、分化以及成熟,从而更有效的调节免疫因子IL-17、IFN-γ、TNF-α、IL-10等的分泌有关;f PMSC更适于临床上作为抑制皮肤移植免疫排斥的种子细胞。
[Abstract]:Objective to study the effect and mechanism of CD200 expression of two kinds of human placental mesenchymal stem cells in the immunomodulation of mouse skin transplantation, and to provide a theoretical basis for clinical selection of better stem cells that inhibit immune rejection. Methods two kinds of derived cells were isolated, the expression of surface markers of mesenchymal stem cells was detected by flow cytometry, and the cells with high expression of CD200 molecules were detected by flow cytometry. Adenovirus vector carrying CD200 gene and empty adenovirus vector were transfected into cells with low expression of CD200 molecule, respectively. The same amount of PBS and the above treated cells were injected into the skin of ICR mice through caudal vein and transplanted into the C57BL / 6 mice. The skin survival time of each group was counted. On the 7th day after operation, the infiltration of inflammatory cells was observed by HE staining method and the relative expression of IL-17, IFN- 纬, TNF- 伪, IL-10 gene in the spleen of mice was detected by RT-PCR, and blood was collected from orbital vein. The content of WBC in blood of each group was determined by Elisa. Results the positive rate of CD200 in fetal side was significantly higher than that in maternal side. Compared with the PBS group, the survival time of the skin grafts in the two groups were significantly prolonged, and the survival time of the skin grafts in the FPMSC anti-CD200 group was significantly longer than that in the fPMSC anti-CD200 group. Compared with the empty vector group of mPMSC adenovirus, the survival time of skin grafts in the adenovirus group was significantly prolonged. The results showed that the infiltration of inflammatory cells in the cell transplantation group was significantly less than that in the PBS group. RT-PCR results showed that: compared with the PBS group, the survival time of the 10% m PMSC was significantly longer. The relative expression of TNF- 伪 in fPMSC group was significantly lower than that in fPMSC group. Compared with mPMSC group, the relative expression of IFN- 纬 TNF- 伪 and IL-17 TNF- 伪 in FPMSC Anti-CD200 group and FPMSC Anti-CD200 group and IFN- 纬 expression in fPMSC Anti-CD200 group were significantly lower than those in mPMSC group, and the relative expression of IFN- 纬 in fPMSC Anti-CD200 group was significantly higher than that in fPMSC group. Compared with the mPMSC CD200 adenovirus vector transfection group, the relative expression of TNF- 伪 ~ + IFN- 纬 in the empty adenovirus vector transfection group was significantly higher than that in the mPMSC adenovirus empty vector transfection group. The relative expression of IL-17 in the mPMSC adenovirus empty vector transfection group was significantly higher than that in the mPMSC adenovirus empty vector transfection group. The relative expression of IL-10 was significantly decreased. The results of Elisa were consistent with that of RT-PCR. Conclusion the immunosuppressive effect of fPMSC on skin graft rejection in mice is better than that on mPMSC.Conclusion fPMSC can inhibit the proliferation, differentiation and maturation of immune cells more strongly than that of mPMSCC. Therefore, it is more effective to regulate the secretion of IL-17, IFN- 纬, TNF- 伪, IL-10 and so on, which is more suitable for clinical application as seed cells for inhibiting skin transplantation immune rejection.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R644

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