Bcl-2基因在阻塞性黄疸大鼠肝细胞凋亡中作用.doc

Bcl-2基因在阻塞性黄疸大鼠肝细胞凋亡中作用 　【摘要】 　　目的：探讨Bcl-2基因对阻塞性黄疸大鼠肝细胞损害中细胞凋亡的调控作用。方法：采用胶原酶原位肝灌注法获取对照组大鼠肝细胞及阻塞性黄疸实验组术后3、7、14、21 d大鼠的肝细胞，分别用RT-PCR检测Bcl-2 mRNA的表达。分离对照组及实验组14 d组大鼠的肝细胞行原代培养后,使用100 μmol/L甘氨鹅脱氧胆酸钠（GCDC）作用于两组肝细胞24 h后，用FCM法测定肝细胞的凋亡比率，用TUNEL技术进行肝细胞凋亡的原位检测。结果：（1）对照组大鼠肝细胞无Bcl-2 mRNA表达，实验组术后7、14、21 d组大鼠肝细胞均有Bcl-2 mRNA表达；（2）GCDC作用两组大鼠肝细胞后,实验组比对照组肝细胞凋亡明显减少（Plt; 0.001）。结论：在阻塞性黄疸过程中，大鼠肝细胞通过表达Bcl-2基因是抑制胆盐致肝细胞的凋亡作用的途径之一。 【关键词】 凋亡　黄疸　阻塞性　 基因　Bcl-2　大鼠　Wistar 　　Role of Bcl-2 gene in apoptosis of hepatocytes in rats with obstructive jaundice 【ABSTRACT】 Objective: To explore the regulating mechanism of Bcl-2 gene in hepatocyte injury caused by obstructive jaundice in rats by comparing apoptotic rate. Methods: Normal rats and bile duct-ligated 7 d, 14 d, 21 d rats’ hepatocytes were isolated by in situ collagenase perfusion and primary culture. （1）Bcl-2 mRNA was detected by RT-PCR in all cells; （2）After normal rat and bile duct-ligated 14d rat hepatocytes were added 100 μmol/L GCDC and kept for 24 hours, cells were evaluated by FCM and TUNEL. Results:（1）Normal rat hepatocytes did not express Bcl-2 by RT-PCR technique. Bcl-2 was expressed in 7-,14-,21- day BDL rats.（2）After adding 100 μmol/L GCDC and keeping for 24 hours, the apoptosis of bile duct-ligated rat hepatocytes significantly decreased compared with that of normal rat hepatocytes. Conclusions: Bile duct-ligated rat hepatocytes expressed Bcl-2.Hepatocellular expression of Bcl-2 during obstructive jaundice is an adaptive phenomenon to resist apoptosis by bile salts. 【KEY WORDS】 Hepatocyte apoptosis·Jaundice, obstructive ·Genes, Bcl-2·Rat, Wastar 　　阻塞性黄疸因胆汁淤积引起胆盐升高,造成肝损害。本研究通过观察正常大鼠及阻塞性黄疸大鼠体外培养肝细胞Bcl-2 mRNA的表达情况，研究其与细胞凋亡的关系,探讨胆盐致肝细胞损害的调控机制。　　 1　材料与方法 1.1　实验动物及分组　健康雄性Wistar大鼠50只，随机分为5组，每组10只，其中4组为实验组，1组为对照组。全部实验组大鼠均手术游离出胆总管，于肝门部结扎胆总管并切断，远端再予结扎。分别于结扎3 、7 、14 和21 d后处死1组。对照组手术不结扎切断胆总管，14 d处死。 1.2　大鼠肝细胞的分离与培养　采用胶原酶原位肝灌注法［1］获取肝细胞，4%台盼蓝染色判断肝细胞存活率，细胞计数板记数细胞浓度。细胞于合成培养基（RPMI 1640 80 ml，新生小牛血清20 ml，青霉素100 U/ml，链霉素100 μg/ml，胰岛素0.1 U/ml，