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  本文選題：IL-35 + 轉染��； 參考：《天津醫(yī)科大學》2012年碩士論文

【摘要】：目的：探討IL-35基因轉染小鼠骨髓間充質干細胞的可行性,及其通過體內和體外實驗對小鼠免疫功能的影響。 方法：(1)利用體外細胞培養(yǎng)技術將Balb/c小鼠的骨髓間充質干細胞自脛骨和股骨中分離、純化和培養(yǎng),在地塞米松,胰島素,吲哚美辛和3-異丁基-1-甲基黃嘌呤(IBMX)的作用下進行成脂肪誘導,在地塞米松,β-甘油磷酸鈉和維生素C的作用下進行成骨誘導。流式細胞技術檢測所分離和培養(yǎng)的間充質干細胞的CD105, CD90, CD44, CD45, CD34, SCA-1情況,對成脂誘導和成骨誘導的細胞分別進行油紅O染色和茜素紅染色；(2)IL-35基因表達質粒載體和綠熒光蛋白表達載體(L6)通過陽離子脂質體轉染法對小鼠骨髓充質干細胞進行體外轉染,倒置熒光顯微鏡觀察間充質干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(lián)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)經鼠尾靜脈將IL-35基因轉染的間充質干細胞注射于BALB/c小鼠,ELISA檢測細胞轉染后血清中IL-35的濃度,流式細胞技術檢測轉染后小鼠外周血CD3+細胞、CD4+T細胞、CD8+T細胞、CD4+CD25+Treg比例；(4)分離BALB/c小鼠和C57BL/6小鼠脾臟單個核細胞,進行單向混合淋巴細胞培養(yǎng),觀察IL-35轉染的MSC細胞對培養(yǎng)體系細胞增殖的影響,以流式細胞技術檢測培養(yǎng)體系CD3+細胞、CD4+T細胞、CD8+T細胞、CD4+CD25+Treg比例。 結果：(1)利用全骨髓貼壁法可成功獲得小鼠骨髓間充質干細胞,分離和擴增后的細胞形態(tài)呈長梭型,旋渦狀生長,細胞陽性表達CD105, CD90, CD44和SCA-1,陰性表達CD45和CD34；第3代間充質干細胞經誘導后可向成脂細胞和成骨細胞分化。(2)L6體外轉染間充質干細胞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MSC細胞的轉染效率約27.40%,ELISA可檢測到IL-35轉染的MSC細胞上清液中有IL-35的表達；(3)IL-35轉染的MSC經鼠尾靜脈注入小鼠后,其血清中可檢測到IL-35的表達；注入IL-35轉染的MSC組小鼠外周血CD4+CD25+Treg比例(5.24±0.61%)升高,與其他各組組比較,P0.05,差異具有統(tǒng)計學意義；同時,注入IL-35轉染的MSC組小鼠外周血CD4+T比例(41.72±4.75%)降低,與生理鹽水組比較差異有統(tǒng)計學意義(P0.05)。(4)IL-35轉染的MSC細胞能上調同種異體混合淋巴細胞反應體系中的CD4+CD25+Treg (4.35±0.57%)水平,下調CD3+細胞(29.65±4.44%)的水平,下調CD4+T細胞(14.47±3.86%)水平,與其他各組兩兩比較差異均具有統(tǒng)計學意義(P0.05)。 結論：小鼠骨髓間充質干細胞可通過全骨髓貼壁法進行體外分離和培養(yǎng),間充質干細胞具有多向分化潛能性,可向成脂肪細胞及成骨細胞分化,并陽性表達CD105, CD90, CD44和SCA-1, IL-35轉染的MSC能誘導小鼠CD4+CD25+Treg的增殖和分化,并直接或間接抑制CD3+細胞、CD4+T細胞的活化水平,從而對免疫移植耐受的建立產生積極的影響。
[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 鹵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 鹵4.75%). Compared with normal saline group, IL-35 transfected). (cells could up-regulate CD4 CD25 Treg (4.35 鹵0.57%), down-regulate CD3 cells (29.65 鹵4.44%) and down-regulate CD4 T cells (14.47 鹵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.
【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】：2012
【分類號】：R392

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本文編號：2077206