IL-35基因转染小鼠骨髓间充质干细胞对小鼠免疫功能的研究

发布时间：2018-06-28 07:24

本文选题：IL-35 + 转染　；参考：《天津医科大学》2012年硕士论文

【摘要】：目的：探讨IL-35基因转染小鼠骨髓间充质干细胞的可行性,及其通过体内和体外实验对小鼠免疫功能的影响。方法：(1)利用体外细胞培养技术将Balb/c小鼠的骨髓间充质干细胞自胫骨和股骨中分离、纯化和培养,在地塞米松,胰岛素,吲哚美辛和3-异丁基-1-甲基黄嘌呤(IBMX)的作用下进行成脂肪诱导,在地塞米松,β-甘油磷酸钠和维生素C的作用下进行成骨诱导。流式细胞技术检测所分离和培养的间充质干细胞的CD105, CD90, CD44, CD45, CD34, SCA-1情况,对成脂诱导和成骨诱导的细胞分别进行油红O染色和茜素红染色；(2)IL-35基因表达质粒载体和绿荧光蛋白表达载体(L6)通过阳离子脂质体转染法对小鼠骨髓充质干细胞进行体外转染,倒置荧光显微镜观察间充质干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)经鼠尾静脉将IL-35基因转染的间充质干细胞注射于BALB/c小鼠,ELISA检测细胞转染后血清中IL-35的浓度,流式细胞技术检测转染后小鼠外周血CD3+细胞、CD4+T细胞、CD8+T细胞、CD4+CD25+Treg比例；(4)分离BALB/c小鼠和C57BL/6小鼠脾脏单个核细胞,进行单向混合淋巴细胞培养,观察IL-35转染的MSC细胞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CD3+细胞、CD4+T细胞、CD8+T细胞、CD4+CD25+Treg比例。结果：(1)利用全骨髓贴壁法可成功获得小鼠骨髓间充质干细胞,分离和扩增后的细胞形态呈长梭型,旋涡状生长,细胞阳性表达CD105, CD90, CD44和SCA-1,阴性表达CD45和CD34；第3代间充质干细胞经诱导后可向成脂细胞和成骨细胞分化。(2)L6体外转染间充质干细胞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MSC细胞的转染效率约27.40%,ELISA可检测到IL-35转染的MSC细胞上清液中有IL-35的表达；(3)IL-35转染的MSC经鼠尾静脉注入小鼠后,其血清中可检测到IL-35的表达；注入IL-35转染的MSC组小鼠外周血CD4+CD25+Treg比例(5.24±0.61%)升高,与其他各组组比较,P0.05,差异具有统计学意义；同时,注入IL-35转染的MSC组小鼠外周血CD4+T比例(41.72±4.75%)降低,与生理盐水组比较差异有统计学意义(P0.05)。(4)IL-35转染的MSC细胞能上调同种异体混合淋巴细胞反应体系中的CD4+CD25+Treg (4.35±0.57%)水平,下调CD3+细胞(29.65±4.44%)的水平,下调CD4+T细胞(14.47±3.86%)水平,与其他各组两两比较差异均具有统计学意义(P0.05)。结论：小鼠骨髓间充质干细胞可通过全骨髓贴壁法进行体外分离和培养,间充质干细胞具有多向分化潜能性,可向成脂肪细胞及成骨细胞分化,并阳性表达CD105, CD90, CD44和SCA-1, IL-35转染的MSC能诱导小鼠CD4+CD25+Treg的增殖和分化,并直接或间接抑制CD3+细胞、CD4+T细胞的活化水平,从而对免疫移植耐受的建立产生积极的影响。
[Abstract]:Aim: to investigate the feasibility of IL-35 gene transfection into mouse bone marrow mesenchymal stem cells (BMSCs) and the effect of IL-35 gene transfection on mouse immune function in vivo and in vitro. Methods: (1) Bone marrow mesenchymal stem cells from Balb / c mice were isolated, purified and cultured from tibia and femur by cell culture in vitro. Indomethacin and 3-isobutyl -1-methylxanthine (IBMX) were induced by adipogenesis and osteogenesis induced by dexamethasone, sodium 尾 -glycerophosphate and vitamin C. Flow cytometry was used to detect CD105, CD90, CD44, CD45, CD34, SCA-1 of mesenchymal stem cells isolated and cultured. Oil red O staining and alizarin red staining were performed on adipogenic and osteogenic cells. (2) IL-35 gene expression plasmid and green fluorescent protein expression vector (L6) were transfected into mouse bone marrow mesenchymal stem cells by cationic liposome transfection in vitro, and the green fluorescence expression level of mesenchymal stem cells was observed by inverted fluorescence microscope. The transfection efficiency was analyzed by flow cytometry (FCM), the concentration of IL-35 in supernatant was detected by enzyme-linked immunosorbent assay (Elisa), and the expression of IL-35 gene was analyzed. (3) Interleukin-35 gene transfected mesenchymal stem cells were injected into BALB / c mice via tail vein to detect the concentration of IL-35 in serum by Elisa, and the percentage of CD4 / CD8 T cells to CD25 Treg in peripheral blood of the transfected mice was detected by flow cytometry. (4) spleen mononuclear cells were isolated from BALB _ (r-c) mice and C57BL / 6 mice. The effects of IL-35 transfected MSC cells on the proliferation of cultured cells were observed. The percentage of CD 4 CD 25 Treg in CD 3 cells and CD 4 T cells and CD 8 T cells were detected by flow cytometry. Results: (1) Mouse bone marrow mesenchymal stem cells were successfully obtained by whole bone marrow adherent method. The cells isolated and amplified showed long fusiform shape, spiral growth, positive expression of CD105, CD90, CD44 and SCA-1, negative expression of CD45 and CD34; The third generation of mesenchymal stem cells could differentiate into adipogenic cells and osteoblasts after induction. (2) after L6 was transfected into mesenchymal stem cells in vitro, the expression of green fluorescent protein could be observed by inverted fluorescence microscope. Flow cytometry analysis showed that IL-35 expression could be detected in the supernatant of MSCs transfected with IL-35 by Elisa. (3) IL-35 expression could be detected in serum of MSC transfected with IL-35 after injected into mouse tail vein. The percentage of CD4 CD25 Treg in peripheral blood of MSCs transfected with IL-35 was increased (5.24 卤0.61%), which was significantly higher than that of other groups (P 0.05), and the percentage of CD4 T in peripheral blood of MSCs transfected with IL-35 was decreased (41.72 卤4.75%). Compared with normal saline group, IL-35 transfected). (cells could up-regulate CD4 CD25 Treg (4.35 卤0.57%), down-regulate CD3 cells (29.65 卤4.44%) and down-regulate CD4 T cells (14.47 卤3.86%). Compared with other groups, the difference was statistically significant (P0.05). Conclusion: mouse bone marrow mesenchymal stem cells can be isolated and cultured in vitro by whole bone marrow adherent method. Mesenchymal stem cells have the potential to differentiate into adipoblasts and osteoblasts. MSC transfected with positive expression of CD105, CD90, CD44 and SCA-1, IL-35 could induce the proliferation and differentiation of mouse CD4 CD25 Treg, and directly or indirectly inhibit the activation of CD 4 T cells in CD3 cells, thus exerting 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】：2012
【分类号】：R392

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