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脑胶质瘤因其发生部位——中枢神经系统缺乏淋巴组织,且可分泌免疫抑制因子如TGF-β使其免疫治疗显得十分困难.为了探索脑胶质瘤有效的免疫治疗途径,本文研究了经脂质体途径将TNF-α基因转染人胶质瘤细胞U251-SP后其表面粘附分子表达的变化及其对免疫效应细胞LAK细胞杀伤敏感性的影响.首先将人TNF-α基因插人SV40衍生的载体中,构建成pcDV-TNF-α,采用反相蒸馏法将载体用阳离子脂质体包裹,然后在体外将其与U251-SP胶质瘤细胞共育24h后.检测上清中TNF-α的分泌水平,发现9d内培养上清中含有较高浓度的TNF-α,转染后第5d,TNF-α的分泌水平最高.用间接免疫荧光法对胶质瘤细胞表面ICAM-1表达水平进行流式细胞仪分析表明:未经处理的U251-SP细胞表面未检测出有ICAM-1表达;外源性TNF-α处理后,U251-SP细胞表面ICAM-1表达水平增加,但48h后,表面ICAM-1消失;TNF-α.基因转染后3d,U251-SP细胞表面ICAM-1表达明显增加,且ICAM-1可持续表达达9d以上.取正常人外周血经Ficoll-hypaque密度梯度离心分离出单个核细胞,体外经IL-2诱导5d后,制备出LAK细胞,分别观察经外源性TNF-α基因转染U251-SP细胞对LAK细胞敏感性的变化,结果表明,外源性TNF-α处理后1d,U251-SP细胞对LAK细胞杀伤敏感性明显高于对照组,但处理后3d、5d、9d
Glioma because of its location - the central nervous system, the lack of lymphoid tissue, and can secrete immunosuppressive factors such as TGF-β immunotherapy is very difficult.In order to explore the glioma effective immunotherapy approach, this study After transfection of TNF-α gene into human glioma U251-SP by liposome, the expression of adhesion molecules on the surface of human glioma cells and its effect on the killing effect of LAK cells were observed.Firstly, the human TNF-α gene Inserted into SV40-derived vector, constructed into pcDV-TNF-α, using reverse-phase method the carrier with cationic liposomes, and then in vitro with U251-SP glioma cells for 24 hours after the test The level of TNF-α secreted in Qing dynasty was higher than that of the control group, and the highest concentration of TNF-α was found in the culture supernatant within 9 days after transfection. The secretion level of TNF-α was the highest on the 5th day after transfection. Indirect immunofluorescence The expression of ICAM-1 was analyzed by flow cytometry. The results showed that ICAM-1 expression was not detected on untreated U251-SP cells. After exogenous TNF-α treatment, the expression of ICAM-1 on U251-SP cells But after 48h, the surface ICAM-1 disappeared; and after 3 days of TNF-α gene transfection, U251-SP was fine The expression of ICAM-1 on the cell surface was significantly increased, and the ICAM-1 expression persisted for more than 9 days. Mononuclear cells were isolated from normal human peripheral blood by Ficoll-hypaque density gradient centrifugation and induced by IL-2 for 5 days. To investigate the sensitivity of U251-SP cells to LAK cells transfected with exogenous TNF-α gene. The results showed that the sensitivity of U251-SP cells to LAK cell killing was significantly higher than that of U251-SP cells Higher than the control group, but after the treatment 3d, 5d, 9d