核转录因子Gli在胆道闭锁肝上皮间充质转化中的调控作用  

The role of Gli in the regulation of hepatic epithelial mesenchymal transition in biliary atresia

作　　者：汪俊祥[1] 蒲思宇 王琦[1] 张一茂 靳曙光[1] Wang Junxiang;Pu Siyu;Wang Qi;Zhang Yimao;Jin Shuguang(Department of Pediatric Surgery,West China Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西医院小儿外科,成都610041

出　　处：《中华小儿外科杂志》2021年第9期823-829,共7页Chinese Journal of Pediatric Surgery

摘　　要：目的研究胆道闭锁肝纤维化过程中Shh信号通路核转录因子Gli1/Gli2在肝上皮-间充质转化(epithelial mesenchymal transition,EMT)中的调控作用,为阐明胆道闭锁肝纤维化的机制提供依据。方法选择2018年1月至2018年12月经手术证实为胆道闭锁Ⅲ型患儿的肝活检组织标本10例作为胆道闭锁组,年龄在1~3月龄。以相同年龄段无肝胆系统畸形死亡新生儿尸检肝组织5例作为正常对照组。应用实时荧光定量聚合酶链(RT-qPCR)反应及蛋白质印迹法检测肝组织中Gli1/Gli2、EMT关键调控因子Snail/Slug及EMT特征性细胞因子的表达。另将2周龄BALB/c小鼠处死后分离肝内胆管上皮细胞mIBEC,按处理方式不同分为正常对照组、干扰对照组、过表达对照组、Gli1-shRNA组、Gli1过表达组、Gli2-shRNA组、Gli2过表达组。其中,正常对照组加入等体积培养基,干扰对照组加入空白慢病毒,过表达对照组加入空白腺病毒,Gli1-shRNA组应用Gli1-shRNA沉默Gli1,Gli2-shRNA组应用Gli2-shRNA沉默Gli2,Gli1过表达组应用Gli1腺病毒过表达Gli1,Gli2过表达组应用Gli2腺病毒过表达Gli2。应用RNA干扰技术研究Shh信号通路关键转录因子Gli1/Gli2表达对EMT过程的调控作用。采用单因素方差分析和LSD-t检验对数据进行统计学分析。结果RT-qPCR检测结果显示,胆道闭锁组患儿肝组织Gli2、Snail、Vimentin、α-SMA mRNA相对表达量分别为10.96±6.21、7.37±4.09、8.85±2.37和7.35±4.09,较对照组肝组织4.46±1.24、3.34±0.95、4.49±2.07和3.38±0.93均明显升高,组间比较,差异有统计学意义(P<0.05)。胆道闭锁组患儿肝组织E-cadherin相对表达量为3.00±1.29,较对照组4.96±1.91明显降低,组间差异亦有统计学意义(P<0.05)。应用RNA干扰技术沉默Gli2基因后,Gli2-shRNA组小鼠肝内胆管上皮细胞mIBEC中Snail、Vimentin、α-SMA mRNA相对表达量分别为2.23±0.67、3.11±1.07和2.74±0.61,较干扰对照组5.21±0.88、3.67±1.04和3.46±1.24Objective To explore the regulatory role of Gli1/Gli2,a nuclear transcription factor of Shh signaling pathway,in the process of epithelial mesenchymal transition(EMT)of biliary atresia(BA)hepatic fibrosis and provide rationales for elucidating the mechanism of liver fibrosis caused by BA.Methods Liver biopsies of children with BA typeⅢconfirmed by operation were selected.The age range was 1-3 months.Autopsy liver tissues of deceased neonates without digestive tract malformation in the same age group were adopted as normal control.Real-time polymerase chain reaction(PCR)and Western blot were utilized for detecting the expression of Gli1/Gli2,Snail/Slug and characteristic cytokines of EMT.Two-week-old BALB/c mice were sacrificed and the intrahepatic bile duct epithelial cells(mIBEC)were isolated and divided into normal control group,interference control group,overexpression control group,Gli1-shRNA group,Gli1 overexpression group,Gli2-shRNA group and Gli2 overexpression group.Then RNA interference technology was employed for examining the regulatory effect of Gli1/Gli2 expression on EMT process in intrahepatic bile duct epithelial cells of mIBEC.Results RT-qPCR results showed that the expressions of Gli2,Snail,Vimentin,andα-SMA mRNA in the liver tissues of biliary atresia group were 10.96±6.21,7.37±4.09,8.85±2.37,and 7.35±4.09,respectively,which were significantly higher than those of the control group at 4.46±1.24,3.34±0.95,4.49±2.07,3.38±0.93,and the difference between groups was statistically significant(P<0.05).The expression of E-cadherin in the liver tissue of children in the biliary atresia group was 3.00±1.29,which was significantly lower than that of the control group at 4.96±1.91,and the difference between the groups was also statistically significant.After silencing Gli2 gene by RNA interference,the expressions of Snail,Vimentin,andα-SMA mRNA in mIBEC in the Gli2-shRNA group were 2.23±0.67,3.11±1.07,2.74±0.61,which were significantly reduced compared with the interference control group 5

关 键 词：胆道闭锁 上皮-间充质转化 信号通路 RNA干扰 

分 类 号：R726.5[医药卫生—儿科]