IP-10基因过量表达对MA-10小鼠Leydig肿瘤细胞类固醇合成及细胞增殖的影响

Effects of Overexpression of IP-10Gene on MA-10 Mouse Leydig Tumor Cell Steroidoge-nesis and Cell Proliferation

背景与目的:研究在体外培养的MA_10小鼠Leydig肿瘤细胞中,IP_10基因的过表达对细胞类固醇合成以及细胞增殖的影响作用。材料与方法:采用细胞转染实验,将含有IP_10基因cDNA的重组质粒转入MA_10小鼠Leydig肿瘤细胞中,用Westernblotting法检测细胞IP_10基因的过表达,用放免分析(Radioimmunoassay,RIA)方法检测IP_10基因过表达对MA_10细胞孕酮合成的影响,用3H_Thymidine掺入DNA合成实验研究IP_10基因过表达对MA_10细胞增殖的作用。结果:实验数据表明,成功地在MA_10细胞中过量表达了IP_10蛋白;MA_10细胞内转染的IP_10基因的过量表达可显著抑制8_bromo_cAMP(0.2mmol/L)诱导的孕酮生成,加入1.0μgIP_10重组质粒转染细胞时,可以使8_bromo_cAMP诱导的孕酮的合成水平由对照组的(38.5±1.7)ng/mL显著降低到(23.2±1.5)ng/ml(1.5×105cells/ml) ,且抑制作用随所用IP_10重组质粒的浓度增加而增加,它的抑制作用可能是通过减少了类固醇合成急性调节蛋白StAR基因的表达而达到的。3H_Thymidine掺入实验结果还显示,IP_10基因在转染的MA_10细胞中过量表达能够显著抑制MA_10细胞的增殖。结论:过量表达的IP_10基因对MA_10小鼠Leydig肿瘤细胞的类固醇合成及生长具有显著的抑制作用,此结果提示我们可以考虑使用转入IP_10基因的?

BACKGROUND&AIM: To determine whether an overexpression of IP-10by transfection experiments in MA-10cells has any effect on cell growth and progesterone synthesis. MATERIAL AND METHODS: We cloned the complete IP-10cDNA in a mammalian expression vector with CMV promoter,pcDNA3.1D/V5-His-TOPO and transfected MA-10cells.We checked the expression with rat IP-10antibody and V5antibody using Western blotting.The effects of overexpression of IP-10gene on cell growth were checked by [ 3 H]thymidine incorporation experiment.The progesterone synthesis was checked by RIA method. RESULTS: Results showed that IP-10protein secreted in the medium was30～40fold more in the IP-10transfectants over the basal levels as estimated by Western blotting.Transfection of MA-10cells with IP-10decreased8-bromo-cAMP-induced progesterone synthesis from(38.5±1.7)ng/ml(1.5×10 5 cells/ml)of control cells to(23.2±1.5)ng/ml in transfected cells(P<0.01).8-bromo-cAMP(0.2mmol/L)induced StAR D1mRNA and decreased30%～40%by transfection with IP-10.Transfection of IP-10gene also significantly decreased insulin-like growth factor(IGF-I,100ng/ml)induced [ 3 H] thymidine incorporation into DNA. CONCLUSION: IP-10inhibits StAR D1expression,decreases progesterone synthesis and inhibits cell proliferation.IP-10can be used as gene therapy for prostate cancer due to its antiangiogenic effects and its inhibitory effects on steroidogenesis.