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设计与鉴定负性协同刺激分子人B7-H1基因有效RNAi干扰位点,探讨RNAi阻断B7-H1分子表达对肺癌细胞株H1299生物学功能的影响。采用流式细胞术分析B7-H1基因在人细胞株H1299表面表达特性,应用生物信息学方法分析该基因并设计其RNAi干扰的探针序列,构建含有该干扰序列的重组腺病毒载体,转染细胞株,免疫荧光方法分析细胞表面B7-H1的表达变化,在此基础上,应用RNAi转染细胞和T细胞共培养体系分析B7-H1表达受抑后对T细胞效应的影响,CCK-8掺入法和ELISA法分析T细胞增殖和细胞因子产生的变化。与对照组相比,三个B7-H1RNA干扰序列对B7-H1分子的表达均有一定的抑制效应,其中217-245位干扰序列使细胞表面目的分子表达强度降为45.4%,734-762靶点组目的分子表达强度则降为51.3%,427-455序列对B7-H1分子表达的干扰效应最为显著,使其表达降至18.9%。T细胞功能研究结果显示,抑制效应较强的RNAi作用T细胞后对T细胞负性调控能力弱,细胞增殖能力和细胞因子产生比其他各组强(P<0.05)。人B7-H1基因427-455位进行RNAi干扰具有显著效果,可有效调节靶基因的表达和生物学功能。
Design and identify the negative costimulatory molecule B7-H1 gene effective RNAi interference sites, to explore the RNAi blocking B7-H1 expression of lung cancer cell line H1299 biological function. The expression of B7-H1 gene on human cell line H1299 was analyzed by flow cytometry. The gene was analyzed by bioinformatics method and its RNAi interference probe was designed. The recombinant adenovirus vector containing the interference sequence was constructed and transfected Cell lines and immunofluorescence assay were used to analyze the expression of B7-H1 on the cell surface. On the basis of this, the effect of suppression of B7-H1 expression on T cell responses was analyzed by co-culture system of RNAi and T cells. CCK-8 Analysis of T cell proliferation and cytokine production by incorporation and ELISA. Compared with the control group, the three B7-H1 RNA interference sequences had a certain inhibitory effect on the expression of B7-H1 molecule, in which 217-245 disruption sequence reduced the expression level of the target molecule on the cell surface to 45.4%, and the target of 734-762 The target molecule expression intensity of the target group was reduced to 51.3%. The interference effect of the 427-455 sequence on the expression of B7-H1 molecule was the most significant, and its expression was reduced to 18.9%. The results of T cell function showed that RNAi-induced T cells with strong inhibitory effect showed weak T-cell negative regulatory ability, and cell proliferation and cytokine production were stronger than other groups (P <0.05). Human B7-H1 gene 427-455 for RNAi interference has a significant effect, can effectively regulate the target gene expression and biological function.