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目的探讨 RhoGTP 酶在肝窦内皮细胞转分化过程中的调控作用,及其对血吸虫病性鼠肝窦毛细血管化病变的影响和可能机制。方法采用腹部敷贴法感染血吸虫尾蚴建立血吸虫病性肝纤维化模型,于13周吡喹酮顿服杀虫,14周追加 hydroxyfasudil。13周时正常对照组(A 组,10只)与血吸虫病组(B 组,6只),16、19周和21周后(B 组)、杀虫实验对照组(C 组)、血吸虫病+hydroxyfasudil 组(D 组)、杀虫+hydroxyfasudil 组(E 组)各6只)剖腹取肝组织。分别作 HE、Masson染色和透射电镜观察;免疫组化检测:p-膜联蛋白、CTGF、CD31、Col和 LN;Western 印迹检测:p-膜联蛋白、CTGF、RhoA、Col和 LN 蛋白表达;逆转录-聚合酶链反应(RT-PCR)检测:CTGF、RhoA、ROCK mRNA 水平。结果与 A 组相比,动态观察 B 组肝组织 RhoA、ROCK及 CTGF mRNA 表达水平明显上升,p-膜联蛋白、CTGF、RhoA、ColIV 和 LN 蛋白表达量也增高。给予干预措施后,与 C 组及其它组相比,E 组 CTGF mRNA 表达于16周显著下调,同时显示 CTGF、LN、ColIV 蛋白水平也降低。与 B 组相比,D 组、E 组 p-膜联蛋白降低,16周时最为显著,但 D 组于19周开始 p-膜联蛋白表达量渐恢复到原水平,其中 E 组明显低于 D 组。肝窦的超微结构观察,于21周时 C 组分别与 B、D 组相比,浸润的炎性细胞明显减少,Disse 腔内胶原纤维有所减少,但肝窦内皮细胞窗孔、肝窦内皮下基底膜差异无统计学意义。E 组与 C 组比较肝细胞器形态明显恢复,可见窗孔,未见基底膜。结论 RhoGTP酶在调控血吸虫病性鼠肝窦内皮细胞转分化过程中,通过上调 CTGF 和 p-膜联蛋白发挥作用,抑制RhoGTP 酶信号通路有可能为防治肝窦毛细血管化提供新的有效靶点。
Objective To investigate the regulatory effect of RhoGTPase on the process of transdifferentiation of sinusoidal endothelial cells and its effect on schistosomiasis of schistosoma japonicum and its possible mechanism. Methods Schistosoma japonicum hepatic fibrosis was induced by abdominal application of schistosomula cercariae. After 13 weeks of treatment, praziquantel was used to kill insects and hydroxyfasudil was added for 14 weeks. At 13 weeks, the control group (group A, 10) and schistosomiasis group (group B, 6), 16, 19 and 21 weeks after treatment (group B), insecticide control group (group C), schistosomiasis + hydroxyfasudil group (group D) and insecticide + hydroxyfasudil group (group E) 6). The expression of p-annexin, CTGF, RhoA, Col and LN were detected by Western blotting. The expression of p-annexin, CTGF, CD31, Col and LN were detected by immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) detection: CTGF, RhoA, ROCK mRNA levels. Results Compared with group A, the expressions of RhoA, ROCK and CTGF mRNA in group B were significantly increased, and the expressions of p-annexin, CTGF, RhoA, ColIV and LN were also increased. Compared with group C and other groups, CTGF mRNA expression in group E was significantly down-regulated at 16 weeks after intervention, and at the same time, the protein levels of CTGF, LN and ColIV were also decreased. Compared with group B, the expression of p-annexin in group D and group E decreased at 16 weeks, but the expression of p-annexin in group D gradually returned to its original level at week 19, and the level in group E was significantly lower than that in group E Group D The ultrastructure of hepatic sinusoids showed that infiltrating inflammatory cells were significantly decreased in Group C compared with those in Groups B and D at 21 weeks, while Collagen fibers in Disse cavity were decreased. However, the diameters of hepatic sinusoidal endothelial cells There was no significant difference in the subendothelial basement membrane. E group and C group compared with liver cell morphology was significantly restored, visible window, no basement membrane. Conclusion RhoGTPase may play an important role in the prevention and treatment of hepatic sinusoidogenesis by regulating the expression of CTGF and p-annexin in regulation of schistosomal transdifferentiation .