摘要目的 研究胆道闭锁(biliary atresia,BA)患儿肝组织中RhoA、Rac1及Cdc42表达情况与肝纤维化的关系,探讨其在BA发病中可能的作用机制.方法 选取胆管扩张症患儿肝组织(胆扩组)15例,BA患儿肝组织(BA组)15例,BA发展至肝硬化接受肝移植患儿自体肝活检组织(移植组)10例,采用HE观察肝组织标本纤维化程度;免疫组化染色检测RhoA、Rac1及Cdc42在肝组织中的蛋白表达情况;实时荧光定量聚合酶链式反应(qRT-PCR)方法检测肝组织中RhoA、Rac1及Cdc42基因表达情况.结果 ①HE染色:胆扩组偶见少许纤维组织增生;BA组汇管区增宽,纤维组织增生、桥接纤维化现象普遍,可见假小叶形成;移植组假小叶显著;②免疫组化:胆扩组RhoA、Rac1及Cdc42均表达为弱阳性,BA组及肝移植组患儿RhoA、Rac1及Cdc42蛋白在肝细胞、汇管区胆管上皮细胞中均为阳性表达;③半定量分析:RhoA(0.1419±0.0683、0.2203±0.0476和0.2295±0.0623),Rac1(0.1587±0.0072、0.1970±0.0242和0.1993±0.0270)和Cdc42 (0.887±0.0270、0.1397±0.0360和0.1519±0.0168)在三组蛋白表达水平之间比较差异有统计学意义(P＜0.05),且三组中BA组及肝移植组RhoA、Rac1及Cdc42蛋白表达水平明显高于胆扩组(P＜0.05);移植组RhoA、Rac1及Cdc42蛋白含量与BA组,表达差异无统计学意义(P＞0.05);④qRT-PCR:RhoA[0.3350(0.2525～0.6350)、1.5200(1.4375～2.1700)和1.9350 (1.7950～2.3925)],Rac1 [0.2800 (0.1450～0.3025)、0.8200(0.7275～0.9300)和0.8000(0.7725～0.9825)],Cdc42[0.5950(0.4375～0.7125)、1.8950(1.8675～2.1925)和1.9900(1.8675～2.2050)],三组中mRNA表达水平比较差异有统计学意义(P＜0.017),同时在这三组中,BA组及肝移植组RhoA、Rac1及Cdc42含量表达明显高于胆扩组(P＜0.017).结论 RhoA、Rac1及Cdc42在BA患儿肝组织呈高水平表达,表明其可能参与并促进BA肝纤维进程.

abstractsObjective To explore the expressions of RhoA,Rac1 and Cdc42 in liver tissues of children with biliary atresia (BA).Methods Samples were collected from patients with congenital biliary dilatation (CBD group,n =15),BA (BA group,n =15) and BA children undergoing liver transplantation (LT) due to liver failure (LT group,n =10).Hematoxylin & eosin (HE) staining was used for observing the degrees of liver fibrosis;the expressions of RhoA,Rac1 and Cdc42 in liver tissues were detected by immunohistochemical staining;Quantitative real-time polymerase chain reaction (qRT-PCR) was used for testing the mRNA expression levels of RhoA,Rac1 and Cdc42.Results (1) HE staining:less fiber cell hyperplasia in CBD group;Widened portal tracts,portal fibrosis and bridging fibrosis and pseudo-lobules were seen in BA group;The widened portal area was obvious,portal fibrosis was heavier and bridging fibrosis generally formed,pseudo-lobular was remarkable in LT group.(2) Immunohistochemistry:the expressions of RhoA,Rac1 and Cdc42 were weakly positive in CBD group.The positive expression of RhoA,Rac1 and Cdc42 protein in cytoplasm of hepatocytes and bile duct epithelial cells in BA and LT groups.Semi-quantitative analysis:the results of RhoA,Rac1 and Cdc42 protein among three groups were:RhoA (0.1419 ± 0.0683,0.2295 ±0.0623,0.2203 ± 0.0476),Rac1 (0.1587 ± 0.0072,0.1970 ± 0.0242,0.1993 ± 0.0270) and Cdc42 (0.887 ± 0.0270,0.1397 ± 0.0360,0.1519 ± 0.0168) indicating significant differences (P＜ 0.05);Rho protein expressions were significantly higher in BA and LT groups than that in CBD group (P＜0.05);No significant difference in three proteins existed between BA and LT groups (P＞ 0.05);(3) qRT-PCR:the mRNA expression levels of RhoA,Rac1 and Cdc42 in three groups were RhoA [0.3350(0.2525-0.6350),1.5200(1.4375-2.1700) & 1.9350(1.7950-2.3925)],Rac1[0.2800(0.1450-0.3025),0.8200 (0.7275-0.9300) & 0.8000 (0.7725-0.9825)],Cdc42 [0.5950 (0.4375-0.7125),1.8950(1.8675-2.1925)& 1.9900(1.8675-2.2050)].Significant differences existed among three groups(P＜0.017).Furthermore,the mRNA expression levels of RhoA,Rac1 and Cdc42 were higher in BA and LT groups than those of CBD group (P＜0.017).Conclusions The expressions of RhoA,Rac1 and Cdc42 are up-regulated in BA patients.It suggests that RhoA,Rac1 and Cdc42 may be involved in the process of liver fibrosis.