【摘要】 目的探讨人T-bet基因在白血病细胞株U937中的表达及其对IFN-γ分泌的影响,寻求通过免疫调节改善白血病患者机体免疫状态的可能性。方法采用电穿孔技术将重组人pIRES-eGFP-Tbet(pIRES-eGFP-hTbet)质粒转染U937细胞,RT-PCR检测转染前后U937细胞T-bet的mRNA水平;westen-blot检测表达的T-bet蛋白;ELISA检测其分泌IFN-γ的水平。试验中同时设立pIRES-eGFP质粒为对照。结果电穿孔转染U937细胞后,在荧光显微镜下观察到带荧光的细胞(转染细胞),其荧光细胞阳性率80%左右,提示转染成功;转染T-bet的U937细胞,分别经RT-PCR及westen-blot检测到T-bet的mRNA和表达的蛋白,而未经转染或转染对照质粒pIRES-eGFP的细胞,均未见目的基因的转录和蛋白表达;T-bet转染细胞的培养上清中含有较高水平的IFN-γ,而转染前及对照质粒转染的细胞IFN-γ的分泌水平极低。结论通过电穿孔方法成功将人T-bet基因转染到白血病细胞株U937中,并检测到大量IFN-γ的产生。T-bet是免疫应答调节的重要转录因子,对其研究将有助于进一步探讨T-bet基因或其表达产物作为Th1应答的调节剂用于临床血液病治疗的可能性。更多还原

【Abstract】 Objective To investigate the effects of T-bet gene in leukermic cell on the level of IFN-γ for seeking a way of improving the immune state of leukemia patient by immunomodulation. Methods pIRES-eGFP-hTbet recombinant plasmid was established, and then transfected into U937 cells by using electroporation methods. The human T-bet gene was amplified and identified by RT-PCR; the expression protein of T-bet was observed by Western blotting; the level of IFN-γ was detected by ELISA. Results After transfection, about 80% fluorescent U937 cells were detected. T-bet mRNA and protein were found in transfected T-bet U937 cells but not in negative and untransfected U937 cells. The level of IFN-γ protein was obviously high in T-bet -transfected U937 cells but was not obviously detected in negative and untransfected U937 cells. Conclusion Human T-bet transfected-U937 cell is successfully obtained and the cell secretes high level of IFN-γ. Our study may shed some light upon the clinical usage of T-bet gene or its expression product as a therapeutic reagent for induction of Th1-dominated response.更多还原