本文選題：樹突狀細(xì)胞　切入點(diǎn)：IL-10　出處：《遼寧醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的 1、研究IL-10基因修飾后的大鼠樹突狀細(xì)胞（DC）的表型及其生物學(xué)特性。 2、建立大鼠穿透性角膜移植模型，探討外源性IL-10轉(zhuǎn)染的大鼠樹突狀細(xì)胞在誘導(dǎo)同種異體角膜移植免疫耐受中的作用。 方法 1、以含IL-10基因的重組腺病毒載體體外轉(zhuǎn)染大鼠骨髓來(lái)源的DC，實(shí)驗(yàn)分3組，培養(yǎng)6天的DC加入IL-10-GFP-Adenovirus轉(zhuǎn)染48h后，繼續(xù)培養(yǎng)至第12天，記做IL-10-GFP-DC組；培養(yǎng)6天的DC加入GFP-Adenovirus轉(zhuǎn)染48h后，繼續(xù)培養(yǎng)至第12天，記做GFP-DC組；不加任何處理，繼續(xù)培養(yǎng)至第12天，記做12-DC組。Western blot測(cè)定轉(zhuǎn)染后各組DC中IL-10蛋白的表達(dá)，流式細(xì)胞儀檢測(cè)各組DC表面抗原CD83、CD86分子的表達(dá)情況，混合淋巴細(xì)胞反應(yīng)法測(cè)定各組DC刺激同種異體T細(xì)胞增殖的能力。 2、動(dòng)物實(shí)驗(yàn)分5組，陰性對(duì)照組：受體SD大鼠6只，未做任何處理。余4組以SD大鼠為受體、Wistar大鼠為供體建立同種異體角膜移植模型，每組20只，分別為：陽(yáng)性對(duì)照組：術(shù)前3天受體大鼠尾靜脈注射等量PBS；GFP-DC組：術(shù)前3天受體大鼠尾靜脈注射2×106個(gè)轉(zhuǎn)染48hGFP-Adenovirus的DC細(xì)胞；8-DC組：術(shù)前3天受體大鼠尾靜脈注射2×106個(gè)培養(yǎng)8天的DC細(xì)胞；IL-10-GFP-DC組：術(shù)前3天受體大鼠尾靜脈注射2×106個(gè)轉(zhuǎn)染IL-10-GFP-Adenovir-us48h的DC細(xì)胞。每日觀察角膜植片的狀態(tài)，記錄植片的生存時(shí)間，組織病理切片HE染色觀察角膜植片的病理變化，，RT-PCR方法檢測(cè)各組術(shù)眼角膜細(xì)胞因子IL-10、TGF-β及IL-2的變化，ELISA方法檢測(cè)各組術(shù)眼房水中細(xì)胞因子IL-4、IFN-γ的變化。 結(jié)果 1、IL-10基因修飾組DC可檢測(cè)到IL-10高表達(dá)，表面抗原CD83、CD86低表達(dá)，其刺激T淋巴細(xì)胞增殖水平較其他各組低，差異具有統(tǒng)計(jì)學(xué)意義。GFP-DC組與12-DC組細(xì)胞表型及功能無(wú)差異。 2、IL-10-GFP-DC組角膜植片的存活時(shí)間顯著延長(zhǎng)，HE染色示植片的水腫、炎性細(xì)胞浸潤(rùn)的程度輕，RT-PCR及ELISA方法檢測(cè)IL-10-GFP-DC組細(xì)胞因子IL-10、TGF-β、 IL-4表達(dá)較其他各組高，而IL-2、IFN-γ的表達(dá)較其他各組低，差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)論 1、轉(zhuǎn)染IL-10基因可抑制樹突狀細(xì)胞的成熟，為研究未成熟DC誘導(dǎo)同種異體移植免疫耐受奠定了基礎(chǔ)。 2、IL-10基因修飾的未成熟樹突狀細(xì)胞能抑制角膜移植排斥反應(yīng)，誘導(dǎo)免疫耐受的形成，延長(zhǎng)角膜植片的存活時(shí)間。其中Th1/Th2偏離及TGF-β的高表達(dá)是誘導(dǎo)角膜移植免疫耐受的機(jī)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- 緯 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- 緯 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.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R779.65

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