摘要
目的 利用原代培养肝细胞 ,研究鹅脱氧胆酸损害肝细胞机制。方法 SD大鼠肝细胞原代短期培养 ,分别加入不同浓度的甘氨鹅脱氧胆酸 (glycochenodeoxycholate ,GCDC)后 ,流式细胞术检测凋亡细胞和坏死肝细胞的比例 ;抽提DNA电泳 ;涂片后生物素 dUTP标记凋亡细胞。胆总管结扎SD大鼠肝组织免疫组织化学染色检测Bcl 2基因表达。肝细胞与 10 0 μmol/LGCDC和不同浓度的果糖培养后 ,同法流式细胞术检测凋亡细胞的比率。结果 加入GCDC后肝细胞的凋亡率随着GCDC的浓度增加和作用时间的延长而增加 (2 9%到 6 2 % ,P <0 0 1) ;坏死细胞率和凋亡细胞率之间无相关性 (r=0 45 71,P >0 0 5 )。胆总管结扎后早期 (3、7d)Bcl 2表达阴性 ,结扎 14d后Bcl 2表达阳性。相同条件下 ,10 0 μmol/L的GCDC作用胆总管结扎组肝细胞的凋亡率较正常对照组肝细胞低(P <0 0 5 )。随着果糖浓度的提高 ,肝细胞的凋亡率逐渐降低 (5 0± 4) %到 (2 8± 4) %。果糖浓度与肝细胞凋亡率密切相关 (r=0 9772 ,P <0 0 1)。结论 GCDC诱发肝细胞凋亡。机体通过表达Bcl 2基因拮抗GCDC诱导的凋亡。果糖对GCDC诱发肝细胞凋亡有保护作用。
Objective[WT5”BZ] Our aim was to determine if the bile salt induces hepatocytes apoptosis in primary culture.[WT5”HZ]Methods[WT5”BZ] Rat's hepatocytes in primary culture were added with 25, 50, 100, 200,and 300 μmol/L of glyco chenodeoxycholate (GCDC).Cells were evaluated by DNA PI staining FACS and terminal deoxynucleotidyl transferase madiated nick end labeling (TUNEL) and by DNA electrophoresis.The apoptosis rates were compared between normal hepatocytes with those added with different density of fructose, and hepatocytes of rats with bile duct obstruction. The Bcl 2 expressions were also tested by immuno histochemistry of liver tissue.[WT5”HZ]Results[WT5”BZ] The hepatocytes treated with GCDC have a high apoptotic rates than the control, 2 9% to 62%, P <0 01,and the rate was found proportional to the concentration of GCDC and the incubation time. The TUNEL showed that apoptotic hepatocytes have less cell volume and stained with brown by Biotin 11 dUTP. DNA ladder of bile salt treated hepatocytes were showed in agarose gel electrophoresis.There is no relationship between apoptosis rate and recrosis rate ( r =0 4571, P >0 05). Fructose inhibited GCDC induced hepatocytes apoptosis from (50±4)% to (28±4)%. 14 days after bile duct ligation, the hepatocyte expressed bcl 2.[WT5”HZ]Conclusions[WT5”BZ] The mechanisms of hepatic injury during obstructive jaundice is related to bile salt caused hepatocytes apoptosis. Fructose inhibited GCDC induced apoptosis in a concentration dependent manner. The obstructive jaundice induced the expression of bcl 2 to protect hepatocytes injury by bile salts.
出处
《中华普通外科杂志》
CSCD
2000年第1期32-35,共4页
Chinese Journal of General Surgery
基金
1998年北京市科技新星计划资助
