本文選題：IL-35 + CD4~+CD25~+調節(jié)性T細胞��； 參考：《天津醫(yī)科大學》2011年碩士論文

【摘要】：目的：探討IL-35質粒載體體內基因轉染的可行性及其對小鼠免疫功能的影響。 方法：(1)鑒定格拉斯哥大學惠贈的IL-35基因表達質粒載體(pSecTag2-IL35)。設計目的基因IL-35的上下游引物,通過聚合酶鏈式反應(PCR),擴增目的基因IL-35；雙酶切質粒載體,采用瓊脂糖凝膠電泳法檢測目的核酸片段分子量的大小；(2)IL-35基因表達質粒載體和另一種綠熒光蛋白表達載體陽離子脂質體轉染法體外轉染人腎上皮細胞系293細胞,倒置熒光顯微鏡觀察293細胞綠熒光表達水平評估細胞轉染情況,流式細胞術(FCM)分析細胞的轉染效率,酶聯免疫吸附法(ELISA)檢測細胞轉染后上清液中IL-35的濃度,分析IL-35基因表達情況：(3)采用鼠尾靜脈流體力學轉染技術將IL-35基因表達質粒載體和PBS緩沖液分別體內轉染BALB/c小鼠,流式細胞技術檢測轉染后小鼠外周血及脾臟CD4+CD8+T細胞、CD4-CD8+T細胞、CD3-CD16+NK細胞、CD4+CD25+Treg比例；(4)利用密度梯度離心法分離純化體內轉染IL-35基因表達質粒載體的BALB/c(H-2d)小鼠和C57BL/6(H-2b)小鼠脾臟單個核細胞,分別以經絲裂霉素處理的BALB/c和C57BL/6小鼠脾細胞作為刺激細胞,體內轉染后的未經絲裂霉素處理的BALB/c小鼠脾細胞作為反應細胞,進行單向混合淋巴細胞培養(yǎng),觀察IL-35對培養(yǎng)體系細胞增殖的影響,以流式細胞技術檢測培養(yǎng)體系CD4+CD8T細胞、CD4-CD8+T細胞、CD4+CD25+Treg比例。 結果：(1)通過瓊脂糖凝膠電泳的檢測,目的核酸片段的分子量大小均正確；(2)體外轉染后倒置熒光顯微鏡可以觀察到綠熒光蛋白表達,流式細胞術分析293細胞的轉染效率在35.30%,ELISA可檢測到IL-35基因表達的質粒載體轉染的細胞上清液中有IL-35的表達；(3)體內轉染IL-35可引起小鼠外周血及脾臟CD4+CD25+Treg比例的上調,外周血CD4+CD8T細胞、CD3-CD16+NK細胞比例的下調；(4)IL-35能上調同種異體混合淋巴細胞反應體系中的CD4+CD25+Treg水平,下調CD4+CD8-T細胞比例的水平。導致這些變化的原因可能是被轉染的小鼠表達了IL-35,引起了脾臟及外周血中CD4+CD25+Treg比例的上調,增強了Treg免疫調節(jié)的作用。 結論：IL-35質粒載體體內轉染能誘導小鼠CD4+CD25+調節(jié)性T細胞增殖和分化,并間接或直接抑制CD4+T細胞的活化水平,抑制NK細胞的增殖和分化,從而對免疫移植耐受的建立產生積極的影響。
[Abstract]:Objective: To investigate the feasibility of in vivo gene transfection of IL-35 plasmid vector and its effect on immune function in mice.
Methods: (1) identify the IL-35 gene expression plasmid vector (pSecTag2-IL35) of University of Glasgow, design the upstream and downstream primers of the target gene IL-35, amplify the target gene IL-35 by polymerase chain reaction (PCR), double enzyme cut plasmid vector, and use agarose gel electrophoresis to detect the molecular weight of the target nucleic acid fragment; (2) IL-35 base The transfection of human renal epithelial cell line 293 cells was transfected in vitro with the expression plasmid vector and another green fluorescent protein expression vector transfected by cationic liposome. Inverted fluorescence microscope was used to observe the cell transfection of 293 cells, and the transfection efficiency of the cells was analyzed by flow cytometry (FCM), and the enzyme linked immunosorbent assay (ELISA) was used to detect the cell transfection efficiency. The concentration of IL-35 in the supernatant after cell transfection was used to analyze the expression of IL-35 gene: (3) IL-35 gene expression plasmid vector and PBS buffer solution were transfected into BALB/c mice in vivo by the tail vein fluid mechanics transfection technique, and the transfected mouse peripheral blood and spleen CD4+CD8+T cells, CD4-CD8+T cells and CD3-CD16+NK fine were detected by flow cytometry. Cell and CD4+CD25+Treg ratio; (4) the splenic mononuclear cells of BALB/c (H-2d) and C57BL/6 (H-2b) mice transfected with IL-35 gene expression plasmid were isolated and purified by density gradient centrifugation. The BALB/c and C57BL/6 mouse splenocytes treated with mitomycin as the stimulating cells were treated with no mitomycin treatment after transfection in vivo. The BALB/c mouse splenocytes were used as reactive cells to conduct unidirectional mixed lymphocyte culture and observe the effect of IL-35 on the cell proliferation of the culture system. Flow cytometry was used to detect the CD4+CD8T cells, CD4-CD8+T cells and CD4+CD25+Treg ratio.
Results: (1) the molecular weight of the target nucleic acid fragment was correct by agarose gel electrophoresis. (2) the expression of green fluorescent protein could be observed by inverted fluorescence microscope after transfection in vitro, and the transfection efficiency of 293 cells by flow cytometry was 35.30%. ELISA could detect the cell supernatant transfected by plasmid vector expressing IL-35 gene. The expression of IL-35 in the liquid; (3) in vivo transfection of IL-35 can cause the up regulation of CD4+CD25+Treg ratio in peripheral blood and spleen, the down regulation of CD4+CD8T cells in peripheral blood and the proportion of CD3-CD16+NK cells; (4) IL-35 can increase the level of CD4+CD25+ Treg in the allogenic mixed lymphocyte reaction system and reduce the level of CD4+CD8-T cell ratio. Some of the changes may be due to the expression of IL-35 in the transfected mice, which leads to the increase in the proportion of CD4+CD25+Treg in the spleen and peripheral blood, enhancing the role of Treg immunoregulation.
Conclusion: the transfection of IL-35 plasmid in vivo can induce the proliferation and differentiation of CD4+CD25+ regulatory T cells in mice, and indirectly or directly inhibit the activation level of CD4+T cells, inhibit the proliferation and differentiation of NK cells, and thus have a positive effect on the establishment of immune transplantation tolerance.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【參考文獻】

相關期刊論文 前5條

1 ;IL-10-Producing Type 1 Regulatory T Cells and Allergy[J];Cellular & Molecular Immunology;2007年04期

2 錢其軍,吳孟超,岑信棠;腫瘤生物治療的新策略——基因-病毒治療[J];第二軍醫(yī)大學學報;2002年05期

3 沈二霞;吳長有;;CD4~+CD25~+調節(jié)性T細胞對B細胞免疫應答的抑制作用[J];國際免疫學雜志;2006年04期

4 王繼納;朱同玉;;藥物在誘導臨床移植免疫耐受中的應用[J];中國臨床藥學雜志;2007年05期

5 李冬妹;胡永秀;;CD4~+CD25~+ Treg細胞與移植免疫耐受[J];微生物學免疫學進展;2007年02期

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