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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 To study the mechanism of chenodeoxycholic acid damage to hepatocytes by primary culture of hepatocytes. Methods Primary cultured hepatocytes of SD rats were cultured in short term, and different concentrations of glycochenodeoxycholate (GCDC) were added to detect the proportion of apoptotic cells and necrotic hepatocytes by flow cytometry. After biotinylated dUTP labeled apoptotic cells. Bcl 2 gene expression was detected by immunohistochemical staining in the livers of SD rats induced by common bile duct ligation. After cultured with 100 μmol / L LGCDC and different concentrations of fructose, the ratio of apoptotic cells was detected by the same method of flow cytometry. Results The apoptotic rates of hepatocytes increased with the increase of GCDC concentration and the duration of action (29% -62%, P <0.01). The rate of apoptotic cells was Correlation (r = 0 45 71, P> 0 0 5). Bcl 2 expression was negative in the early (3 and 7 days) after common bile duct ligation, and was positive in 14 days after ligation. Under the same conditions, the apoptosis rate of hepatocytes in GCDC-treated group with 100 μmol / L GCDC was lower than that in normal control group (P <0.05). With the increase of fructose concentration, the apoptosis rate of hepatocytes decreased gradually from (50 ± 4)% to (28 ± 4)%. The fructose concentration was closely related to the rate of hepatocyte apoptosis (r = 0 9772, P <0.01). Conclusion GCDC induces hepatocyte apoptosis. The body antagonizes GCDC-induced apoptosis by expressing the Bcl2 gene. Fructose has a protective effect on GCDC-induced hepatocyte apoptosis.