摘要
目的:探讨bcl-2基因在阻塞性黄疸大鼠肝细胞凋亡中的调控作用。 方法:健康(?)Wistar大鼠,进腹,游离出胆总管,于肝门部结扎胆总管并切断,远端再予以结扎。进腹后不结扎切断胆总管作为阴性对照。结扎后3,7,14,21d处死大鼠,每组8只。采用胶原酶原位肝灌注法获取体外培养正常大鼠及阻塞性黄疸大鼠肝细胞。分别收取正常大鼠及胆总管结扎大鼠肝细胞1×10~9/L^(-1),用Trizol试剂盒一步法提取大鼠肝细胞总RNA,行RT-PCR扩增;用PCR扩增bcl-2,和对照β-actin基因片段;分离的正常大鼠及胆总管结扎14 d大鼠的肝细胞行原代培养,培养板内置小飞片,使用100μmol·L^(-1)甘氨鹅脱氧胆酸钠(GCDC)作用于两组肝细胞24h后,用FCM法分析肝细胞的凋亡比率,用TUNEL技术进行肝细胞凋亡的原位检测。 结果:体外培养正常大鼠及结扎3d大鼠肝细胞bcl-2 mRNA的表达为阴性,仅见β-actin条带;胆总管结扎大鼠体外培养肝细胞在结扎后7d即可见肝细胞内bcl-2 mRNA的表达,其吸光度A值为0.13±0.15;结扎后14、21d也可检测其表达,bcl-2 mRNA吸光度在结扎14d达高峰,为0.56±0.21,且与结扎7、21d相比,两者差异有显著性(P<0.05);100μmol·L^(-1)的GCDC作用正常大鼠肝细胞后,凋亡明显增加,凋亡率为34.9±2.9％;
AIM:To explore the regulating mechanism of bcl-2 gene in liver cell apoptosis in experimental obstructive jaundice.METHODS: The peritoneal cavity of male Wistar rats was opened, and the common bile duct was double ligated and cut between the ligatures. Controls underwent a sham operation that was consisted of exposure, but not ligation, of the common bile duct. Experimental obstructive jaundice (BDL) was induced by double ligation of the bile duct at 3, 7, 14 and 21 days after operation. Every group consisted of 8 rats. In normal and experimental rats after 7, 14, 21 d, hepatocytes were isolated by in situ collagenase perfusion and primary culture was set up. Total cellular RNA was isolated using the standard acid guanidinium thiocyanate-phenolchlorofonm extraction method and further purified by cesiumchloride gradient centrifugation. bcl-2 mRNA was detected by RT-PCR in all the cells. After added with 100 μmol· L-1 GCDC for 24 h, the cells were evaluated by FCM and TUNEL.RESULTS: Normal rat hepatocytes did not express bcl-2 by RT-PCR. bcl-2 expression was assessed at 7, 14, 21 days of BDL. The expression was the highest at 14-day's BDL rats, compared with the rats at 7 and 21-day' s BDL The difference was significantly (P< 0.05). After added with 100 μmol · L-1 GCDC for 24 h, the apoptosis Irate of hepatocytes in normal rats increased obviously to 34.9 ± 2.9 %. The apoptosis rate of hepatocytes in 14-day BDL rats was 15.6 ± 2.1 %. After added with 100 μmol · L-1 GCDC for 24 h, the apoptosis rate of hepatocytes in BDL rats was not significantly higher than that in control (P> 0.05) .There was a significant difference between GCDC-induced normal and BDL hepatocytes ( P < 0.05).CONCLUSION: Hepatocytes in bile duct-ligated rats express bcl-2, which is an adaptive phenomenon to resist apoptosis by bile salts.
出处
《世界华人消化杂志》
CAS
2002年第3期324-327,共4页
World Chinese Journal of Digestology
