IL-35基因转染及其对小鼠免疫功能的影响

发布时间：2018-05-27 01:35

本文选题：IL-35 + CD4~+CD25~+调节性T细胞　；参考：《天津医科大学》2011年硕士论文

【摘要】：目的：探讨IL-35质粒载体体内基因转染的可行性及其对小鼠免疫功能的影响。方法：(1)鉴定格拉斯哥大学惠赠的IL-35基因表达质粒载体(pSecTag2-IL35)。设计目的基因IL-35的上下游引物,通过聚合酶链式反应(PCR),扩增目的基因IL-35；双酶切质粒载体,采用琼脂糖凝胶电泳法检测目的核酸片段分子量的大小；(2)IL-35基因表达质粒载体和另一种绿荧光蛋白表达载体阳离子脂质体转染法体外转染人肾上皮细胞系293细胞,倒置荧光显微镜观察293细胞绿荧光表达水平评估细胞转染情况,流式细胞术(FCM)分析细胞的转染效率,酶联免疫吸附法(ELISA)检测细胞转染后上清液中IL-35的浓度,分析IL-35基因表达情况：(3)采用鼠尾静脉流体力学转染技术将IL-35基因表达质粒载体和PBS缓冲液分别体内转染BALB/c小鼠,流式细胞技术检测转染后小鼠外周血及脾脏CD4+CD8+T细胞、CD4-CD8+T细胞、CD3-CD16+NK细胞、CD4+CD25+Treg比例；(4)利用密度梯度离心法分离纯化体内转染IL-35基因表达质粒载体的BALB/c(H-2d)小鼠和C57BL/6(H-2b)小鼠脾脏单个核细胞,分别以经丝裂霉素处理的BALB/c和C57BL/6小鼠脾细胞作为刺激细胞,体内转染后的未经丝裂霉素处理的BALB/c小鼠脾细胞作为反应细胞,进行单向混合淋巴细胞培养,观察IL-35对培养体系细胞增殖的影响,以流式细胞技术检测培养体系CD4+CD8T细胞、CD4-CD8+T细胞、CD4+CD25+Treg比例。结果：(1)通过琼脂糖凝胶电泳的检测,目的核酸片段的分子量大小均正确；(2)体外转染后倒置荧光显微镜可以观察到绿荧光蛋白表达,流式细胞术分析293细胞的转染效率在35.30%,ELISA可检测到IL-35基因表达的质粒载体转染的细胞上清液中有IL-35的表达；(3)体内转染IL-35可引起小鼠外周血及脾脏CD4+CD25+Treg比例的上调,外周血CD4+CD8T细胞、CD3-CD16+NK细胞比例的下调；(4)IL-35能上调同种异体混合淋巴细胞反应体系中的CD4+CD25+Treg水平,下调CD4+CD8-T细胞比例的水平。导致这些变化的原因可能是被转染的小鼠表达了IL-35,引起了脾脏及外周血中CD4+CD25+Treg比例的上调,增强了Treg免疫调节的作用。结论：IL-35质粒载体体内转染能诱导小鼠CD4+CD25+调节性T细胞增殖和分化,并间接或直接抑制CD4+T细胞的活化水平,抑制NK细胞的增殖和分化,从而对免疫移植耐受的建立产生积极的影响。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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