雌二醇对大鼠肝细胞胆汁分泌及细胞骨架再建的影响

Effects of β-estradiol on bile secretion and cytoskeletal reorganization of hepatocytes in rats

目的 探讨雌激素对大鼠肝细胞胆汁分泌及细胞骨架再建的影响。方法 从F344大鼠肝脏分离肝细胞 ,行单层培养或肝细胞球状体三维培养 ,培养基中加入雌二醇 ,行位相差显微镜观察 ;进行肝细胞荧光素排泌实验及Rhodamine Phallodin染色观察 ,细胞间粘连相关蛋白质和胆汁分泌相关的Pgp糖蛋白测定采用免疫印记杂交。 结果 雌二醇 (E2 )使单层培养的肝细胞间毛细胆管样结构显著扩张 ,三维培养的肝细胞球状体中央出现空泡样改变 ,肌动蛋白在肝毛细胆管壁及球状体空泡壁周围聚集 ,荧光代谢物在扩张的毛细胆管及空泡中聚集 ,E2促进Pgp和RhoA和radixin的表达 ,其水平与空泡形成率相一致。结论 E2促进大鼠三维培养的肝细胞胆汁分泌 ,在RhoA和radixin介导下肌动蛋白重新分布 ,细胞骨架再建 ,调节紧密连接功能和聚集 ,导致毛细胆管样结构的扩张和三维培养时球状体空泡样结构的形成。

Objective To investigate the influence of β-estradiol (E2) le secretion and cytoskeletal reorganization of hepatocytes in rats. Methods After the hepatocytes were extracted from F344 rats, they were cultured in monolayer or with type Ⅰ-C collagen matrix in William's E medium in which E2 was added. Then the cells were observed with phase-contract microscopy. The cytoskeletal reorganization was determined by Rhodamine-Phallodin staining. The function of bile canaliculi was assessed by observation of the transcellular transport of fluorescin diacetate by fluorescent microscopy. The levels of expression of proteins such as junction complex proteins, ERM proteins (ezrin, radixin and moesin), small GTPase proteins (rho A, rac and cdc42) and Pgp were detected by Western blot. Results There was dilatation of hepatocytic bile canalicular structure after treatment with E2. Fluorescence was accumulated in the dilated bile canaliculi and actins accumulated at the edge of bile canaliculi. The levels of proteins and their expression were increased after use of E2, which is corresponded to cyst formation. Conclusions E2 increases bile secretion and induces dilatation of bile canaliculi and cyst structure formed in the hepatocytes spheroids. After application of fluorescin diacetate in the bile canaliculi or in the cysts, the fluorescence gets stronger to form long-term accumulation. Actins accumulates at the edge of bile canaliculi or hepatocytic spheroid cysts. E2 increases the levels of rho A and radixin, induce accumulation of actin and junction complex proteins, cytoskeletal reorganization and cyst formation.