外源性IL-10转染大鼠DC细胞诱导角膜移植免疫耐受的研究

发布时间：2018-04-07 16:41

本文选题：树突状细胞　切入点：IL-10　出处：《辽宁医学院》2012年硕士论文

【摘要】：目的 1、研究IL-10基因修饰后的大鼠树突状细胞（DC）的表型及其生物学特性。 2、建立大鼠穿透性角膜移植模型，探讨外源性IL-10转染的大鼠树突状细胞在诱导同种异体角膜移植免疫耐受中的作用。方法 1、以含IL-10基因的重组腺病毒载体体外转染大鼠骨髓来源的DC，实验分3组，培养6天的DC加入IL-10-GFP-Adenovirus转染48h后，继续培养至第12天，记做IL-10-GFP-DC组；培养6天的DC加入GFP-Adenovirus转染48h后，继续培养至第12天，记做GFP-DC组；不加任何处理，继续培养至第12天，记做12-DC组。Western blot测定转染后各组DC中IL-10蛋白的表达，流式细胞仪检测各组DC表面抗原CD83、CD86分子的表达情况，混合淋巴细胞反应法测定各组DC刺激同种异体T细胞增殖的能力。 2、动物实验分5组，阴性对照组：受体SD大鼠6只，未做任何处理。余4组以SD大鼠为受体、Wistar大鼠为供体建立同种异体角膜移植模型，每组20只，分别为：阳性对照组：术前3天受体大鼠尾静脉注射等量PBS；GFP-DC组：术前3天受体大鼠尾静脉注射2×106个转染48hGFP-Adenovirus的DC细胞；8-DC组：术前3天受体大鼠尾静脉注射2×106个培养8天的DC细胞；IL-10-GFP-DC组：术前3天受体大鼠尾静脉注射2×106个转染IL-10-GFP-Adenovir-us48h的DC细胞。每日观察角膜植片的状态，记录植片的生存时间，组织病理切片HE染色观察角膜植片的病理变化，，RT-PCR方法检测各组术眼角膜细胞因子IL-10、TGF-β及IL-2的变化，ELISA方法检测各组术眼房水中细胞因子IL-4、IFN-γ的变化。结果 1、IL-10基因修饰组DC可检测到IL-10高表达，表面抗原CD83、CD86低表达，其刺激T淋巴细胞增殖水平较其他各组低，差异具有统计学意义。GFP-DC组与12-DC组细胞表型及功能无差异。 2、IL-10-GFP-DC组角膜植片的存活时间显著延长，HE染色示植片的水肿、炎性细胞浸润的程度轻，RT-PCR及ELISA方法检测IL-10-GFP-DC组细胞因子IL-10、TGF-β、 IL-4表达较其他各组高，而IL-2、IFN-γ的表达较其他各组低，差异具有统计学意义。结论 1、转染IL-10基因可抑制树突状细胞的成熟，为研究未成熟DC诱导同种异体移植免疫耐受奠定了基础。 2、IL-10基因修饰的未成熟树突状细胞能抑制角膜移植排斥反应，诱导免疫耐受的形成，延长角膜植片的存活时间。其中Th1/Th2偏离及TGF-β的高表达是诱导角膜移植免疫耐受的机制之一。
[Abstract]:Purpose1. The phenotype and biological characteristics of IL-10 gene modified rat dendritic cells (DC) were studied.2. To establish a rat model of penetrating keratoplasty and to investigate the role of dendritic cells transfected with exogenous IL-10 in inducing corneal allograft tolerance.Method1. The DC derived from rat bone marrow was transfected with recombinant adenovirus vector containing IL-10 gene in vitro. The DC was cultured for 6 days after transfection with IL-10-GFP-Adenovirus for 48 h, then continued to be cultured for 12 days, recorded as IL-10-GFP-DC group, and DC for 6 days was added into GFP-Adenovirus for 48 hours.On the 12th day of culture, the cells were recorded as GFP-DC group, and then cultured for 12 days without any treatment. The expression of IL-10 protein was measured by 12-DC group. Western blot, and the expression of CD83 CD86 molecule on the surface antigen of DC in each group was detected by flow cytometry.The ability of DC to stimulate the proliferation of allogeneic T cells was measured by mixed lymphocyte reaction.2. Animal experiment was divided into 5 groups, negative control group: 6 SD rats without any treatment.In the remaining 4 groups, SD rats were used as recipients and Wistar rats were used as donors to establish corneal allograft models with 20 rats in each group.Positive control group: 3 days before operation the recipient rats were injected with the same amount of PBSGFP-DC group: 3 days before operation, 2 脳 106 48hGFP-Adenovirus transfected DC cells were injected into the tail vein of 2 脳 10 6 DC cells: 3 days before operation, 2 脳 106 culture cells were injected into the tail vein of the recipient rats.IL-10-GFP-DC group for 8 days: 2 脳 106 IL-10-GFP-Adenovir-us48h transfected DC cells were injected into the tail vein of recipient rats 3 days before operation.Observe the state of corneal grafts daily and record the survival time of the grafts.The pathological changes of corneal grafts were observed by HE staining in histopathological sections. The changes of corneal cytokines IL-10 TGF- 尾 and IL-2 were detected by RT-PCR. The changes of IL-4 and IFN- 纬 in aqueous humor were detected by Elisa.Result1the high expression of IL-10 and the low expression of CD83 + CD86 could be detected in DC modified with IL-10 gene, and the level of T lymphocyte proliferation stimulated by IL-10 gene modified DC was lower than that of other groups. There was no difference in phenotype and function between GFP-DC group and 12-DC group.(2) the survival time of corneal grafts in IL-10-GFP-DC group was significantly longer than that in other groups. The degree of inflammatory cell infiltration and edema of corneal grafts were significantly prolonged in IL-10-GFP-DC group. RT-PCR and ELISA methods were used to detect the expression of IL-10 TGF- 尾 and IL-4 in IL-10-GFP-DC group, but the expression of IL-2mIFN- 纬 in IL-10-GFP-DC group was lower than that in other groups.The difference is statistically significant.Conclusion1. Transfection of IL-10 gene can inhibit the maturation of dendritic cells, which lays a foundation for the study of allograft tolerance induced by immature DC.2the immature dendritic cells modified with IL-10 gene can inhibit the rejection of corneal transplantation, induce the formation of immune tolerance and prolong the survival time of corneal grafts.Th1/Th2 deviation and high expression of TGF- 尾 are one of the mechanisms of inducing corneal transplantation tolerance.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.65

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