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肝窦内皮细胞(SEC)凋亡是肝脏疾病发生、发展的重要因素之一。在胆汁淤积症中,疏水性胆汁酸甘氨鹅去氧胆酸(GCDC)直接诱发肝细胞凋亡,而亲水性胆汁酸熊去氧胆酸(UDCA)能防止诱发肝细胞凋亡。目的:研究GCDC引起原代培养大鼠SEC损害和凋亡、UDCA对GCDC诱导凋亡的抑制作用以及涉及caspase-8的作用机制。方法:采用胶原酶A原位灌注和Percoll分离液(25%和50%)梯度离心法分离Wistar大鼠SEC,SEC于添加了EGM-2因子的EBM-2培养基中培养(5%CO2,37℃)3d,然后在含有GCDC和(或)UDCA的新鲜培养液中继续培养4h。以原位末端标记(TUNEL)技术检测SEC凋亡情况。以FLICE/caspase-8比色蛋白酶检测试剂盒测定caspase-8的表达情况。结果:相差显微镜下,培养3d时的SEC呈纺锤状并基本融合。而在与GCDC孵育的过程中SEC趋于皱缩,孵育4h时最终从培养皿脱落。SEC与不同浓度的GCDC(50～250μmol/L)孵育1h后,TUNEL染色阳性的细胞数量开始增加,4h后几乎所有细胞呈TUNEL染色阳性(P<0.05);对照组SEC仅少数呈TUNEL染色阳性。在原代培养SEC与终浓度100μmol/LGCDC培养液中加入UDCA(50～200μmol/L)可明显减少TUNEL染色阳性的SEC,且抑制作用呈剂量依赖性(P<0.05);200μmol/LUDCA存在时,GCDC引起SEC凋亡的水平是GCDC单独孵育4h时的20%。SEC与250μmol/LGCDC孵育2h,SEC中caspase-8表达明显增加,而加入200μmol/LUDCA则几乎可完全阻止caspase-8表达增加。结论:UDCA能阻止GCDC诱发的原代培养SEC凋亡,此作用至少部分是通过抑制GCDC上调SEC中caspase-8的表达而实现的。
Apoptosis of hepatic sinusoidal endothelial cells (SEC) is one of the important factors in the occurrence and development of liver diseases. In cholestasis, hydrophobic bile acid glycyrrhetinic acid (GCDC) induces hepatocyte apoptosis directly, whereas hydrophilic bile acid ursodeoxycholic acid (UDCA) prevents the induction of hepatocyte apoptosis. OBJECTIVE: To study the effects of GCDC on SEC injury and apoptosis induced by primary cultured rat, the inhibitory effect of UDCA on GCDC induced apoptosis and the mechanism of caspase-8. METHODS: Wistar rats were isolated by collagenase A in situ perfusion and Percoll separation (25% and 50%) gradient centrifugation. SEC was cultured in EBM-2 medium supplemented with EGM-2 (5% 37 ° C) for 3 days and then cultured for another 4 hours in fresh medium containing GCDC and / or UDCA. SEC apoptosis was detected by TUNEL. The expression of caspase-8 was detected by FLICE / caspase-8 colorimetric proteinase assay kit. Results: Under phase contrast microscopy, SEC was spindle-shaped and essentially fused during 3 days of culture. In the course of incubation with GCDC, the SEC tended to shrink, eventually shedding from the culture dish after incubation for 4 hours. After incubated with different concentration of GCDC (50 ~ 250μmol / L) for SEC for 1h, TUNEL positive cells began to increase, almost all cells showed TUNEL staining after 4h (P <0.05); only a few TUNEL positive cells in control group . The addition of UDCA (50 ~ 200μmol / L) to primary culture SEC and the final concentration of 100μmol / L CDC could significantly reduce TUNEL-stained SEC, and the inhibitory effect was dose-dependent (P <0.05) The level of GCDC-induced SEC apoptosis was 20% at 4 h of GCDC alone. SEC incubated with 250μmol / LGCDC for 2h, caspase-8 expression in SEC significantly increased, while adding 200μmol / LUDCA almost completely prevent the expression of caspase-8 increased. Conclusion: UDCA can prevent GCDC-induced primary culture SEC apoptosis, at least in part by inhibiting GCDC up-regulation of caspase-8 expression in SEC.