甘氨脱氧胆酸盐诱导人正常肝细胞 HL7702凋亡及其机制的研究.doc

　　甘氨脱氧胆酸盐诱导人正常肝细胞 HL7702凋亡及其机制的研究 ：传良敏，洪华，黄文芳，杨永长，饶绍琴，邓君 【摘要】 目的 观察甘氨脱氧胆酸盐(glycodeoxycholate, GCDC)对人正常肝细胞株HL7702凋亡的影响，并探讨其凋亡机制。方法 终浓度分别为100、150、200、250μmol/L的GCDC处理HL7702细胞24h，光学显微镜观察GCDC对HL7702细胞形态的影响，AnnexinVFITC/PI双染色法检测HL7702细胞的凋亡率 ，用荧光指示剂Fluo3/AM测定HL7702细胞内钙离子浓度，RTPCR测定Bcl2、Bax基因mRNA表达水平。结果 终浓度为150μmol/L的GCDC处理HL7702细胞24h后，细胞出现典型的凋亡细胞形态学改变；100、150、200、250μmol/L的GCDC均可诱导HL7702细胞凋亡，呈剂量依赖性，凋亡率分别为(13.16±2.9)% (t=6.41)、(20.3±3.0)% (t=10.22)、(25.02±2.1)% (t=18.11)、(45.02±3.5)% (t=28.89)，较对照组(2.2±0.6)%显著升高(Plt;0.05)；GCDC能使HL7702细胞内钙离子浓度增加，且具有浓度依赖性；GCDC使细胞内Bcl2 mRNA的表达下降、Bax mRNA的表达增加。结论 GCDC可能是通过增加HL7702细胞内钙离子浓度，并进一步下调Bcl2、上调Bax表达水平，从而诱导细胞凋亡。 【关键词】 甘氨脱氧胆酸盐；细胞凋亡；钙离子；Bcl2；Bax；肝细胞 ABSTRACT: Objective To investigate the apoptosis of cultured normal human hepatocyte HL7702 cells induced by glycodeoxycholate (GCDC) and to explore its possible mechanism. Methods HL7702 cells ol/L) of GCDC. The changes of cellular morphology icroscope. The apoptosis rate of HL7702 ined by Annexin VFITC/PI double staining. The changes of HL7702 cell intracelluar [Ca2+]i ined RNA expression levels of Bcl2/Bax in HL7702 cells orphological changes ol/L GCDC for 24h; HL7702 cells could be induced to undergo apoptosis in a concentrationdependent manner after 100, 150, 200, and 250μmol/L GCDC treatment for 24 hours. The apoptosis rates arkedly higher (Plt;0.05) than those of the control group. The intracellular [Ca2+]i anner RNA RNA echanism of apoptosis might be related to increasing intracellular [Ca2+]i, doRNA and upregulating the expression of Bax mRNA. KEY I 1640培养基购自美国GIBCO公司；小牛血清购自美国GM公司；甘氨脱氧胆酸盐(GCDC)购自Sigma公司；胰酶细胞消化液购自碧云天生物技术研究所；Annexin VFITC/pI检测试剂盒购自南京凯基生物科技 发展 有限公司；钙离子测定试剂盒购自美国Biotium公司；RNA提取试剂盒、RTPCR试剂盒、DNA marker、引物合成均由北京赛北盛基因技术有限公司提供。 1.3 细胞培养 将浓度为1×105个/mL的HL7702细胞接种于含100mL/L小牛血清、青霉素100u/mL、链霉素100u/mL的RPMI 1640培养基中，在37℃、饱和湿度、50mL/L CO2培养箱中常规培养24h后换液，处理组加入GCDC，使终浓度分别为100、150、200、250μmol/L，同时设立阴性对照组(不加GCDC)，培养24h，收集各GCDC处理组和阴性对照组细胞，用于以下实验。 1.4 细胞