核转录因子Gli在恒河猴轮状病毒致胆道闭锁小鼠肝脏上皮间充质化中的调控作用

The role of nuclear transcription factor Gli in the regulation of hepatic epithelial mesenchymal transition in biliary atresia mice caused by Rhesus monkey rotavirus infection

目的研究sonic hedgehog(Shh)信号通路核转录因子Gli1/Gli2在恒河猴轮状病毒(RRV)感染致胆道闭锁小鼠肝脏上皮间充质化(EMT)中的调控作用。方法通过RRV构建胆道闭锁小鼠模型, 根据RNA干扰技术对Shh信号通路关键转录因子Gli1/Gli2表达调控的不同, 将实验小鼠分为正常组、模型组、Gli1过表达组、Gli1 shRNA组、Gli2过表达组、Gli2 shRNA组, 应用实时荧光定量聚合酶链反应及蛋白质印迹法检测实验小鼠肝脏组织中EMT关键调控因子Snail/Slug及EMT特征性细胞因子波形蛋白(Vimentin)、平滑肌肌动蛋白(α-SMA)、钙黏蛋白(E-cadherin)mRNA及蛋白的表达;对不同分组中的小鼠肝脏组织分别行苏木精-伊红染色和Masson染色观察记录肝纤维组织表达面积百分比。采用单因素方差分析和LSD-t检验进行数据分析。结果 Gli2过表达组、Gli2 shRNA组及模型组Snail、Slug、Vimentin、α-SMA、E-cadherin mRNA相对表达量分别为15.13±3.40、5.48±0.46、8.78±1.06、12.40±2.18和3.06±0.53;3.73±1.16、5.62±1.75、3.56±1.06、3.88±1.16和10.51±1.83;8.13±1.27、5.32±0.98、5.05±0.98、4.02±0.77和5.12±1.60。与模型组相比, Gli2过表达组Snail、Vimentin、α-SMA的mRNA表达明显升高, E-cadherin的mRNA表达明显降低, 差异均有统计学意义(t=4.53、5.29、8.12、-2.13, 均P<0.05);与模型组相比, Gli2 shRNA组Snail、Vimentin的mRNA表达明显降低, E-cadherin的mRNA表达明显升高, 差异均有统计学意义(t=-2.86、-2.12、5.62, 均P<0.05)。Gli2过表达组、Gli2 shRNA组及模型组Snail、Slug、Vimentin、α-SMA、E-cadherin蛋白灰度比值分别为2.02±0.39、0.31±0.08、0.95±0.17、1.07±0.17和0.42±0.06;0.53±0.13、0.40±0.18、0.20±0.04、0.28±0.07和1.09±0.31;0.70±0.15、0.42±0.22、0.64±0.13、0.81±0.11和0.42±0.09。与模型组相比, Gli2过表达组Snail、Vimentin、α-SMA蛋白灰度比值明显升高, 差异均有统计学意义(t=12.71、4.28、3.70, 均P<0.05);与模型组相比, Gli2 shRNA组Vimentin、α-SMA表达明显降低, E-cadherin表达明显升高, 差异均有统计学意义(t=-6.14、-7.57、5.96, 均P<0.05)。Gli1过表达组及Gli1 shRNA组小鼠肝脏组织未见明确EMT特征细胞因子表达变化趋势。正常组、模型组、Gli1过表达组、Gli1 shRNA组、Gli2过表达组、Gli2 shRNA组肝脏纤维组织表达面积百分比分别为(1.03±0.58)%、(33.02±11.39)%、(39.81±5.67)%、(26.06±1.29)%、(49.81±8.57)%和(17.55±0.66)%。Gli2过表达组及Gli2 shRNA组与模型组肝脏纤维组织表达面积百分比相比, 差异均有统计学意义(t=3.21、-2.96, 均P<0.05);Gli1过表达组及Gli1 shRNA组与模型组肝脏纤维组织表达面积百分比相比, 差异均无统计学意义(均P>0.05)。结论 Shh信号通路在胆道闭锁小鼠肝纤维化中起重要作用, Shh信号通路关键转录因子Gli2可显著调控胆道闭锁小鼠肝脏EMT过程。

Objective To investigate the role of nuclear transcription factor Gli1/Gli2 of the sonic hedgehog(Shh)signaling pathway in the hepatic epithelial mesenchymal transition(EMT)of biliary atresia mice caused by Rhesus rotavirus(RRV)infection.Methods The biliary atresia model in mice was generated by RRV infection.Mice were divided into normal group,model group,Gli1 overexpression group,Gli1 shRNA group,Gli2 overexpression group and Gli2 shRNA group.Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the mRNA and protein expressions of regulatory factors for EMT(Snail/Slug)and characteristic cytokines of EMT[Vimentin,α-smooth muscle actin(α-SMA),E-cadherin]in mouse liver tissues.Additionally,hematoxylin-eosin staining and Masson staining were performed to calculate the percentage of liver fibrous tissue expression area.The data were analyzed by One-Way ANOVA and LSD-t test.Results The relative mRNA expression of Snail,Slug,Vimentin,α-SMA and E-cadherin in Gli2 overexpression group,Gli2 shRNA group and model group were 15.13±3.40,5.48±0.46,8.78±1.06,12.40±2.18 and 3.06±0.53;3.73±1.16,5.62±1.75,3.56±1.06,3.88±1.16 and 10.51±1.83;8.13±1.27,5.32±0.98,5.05±0.98,4.02±0.77 and 5.12±1.60.Compared with those of the model group,mRNA levels of Snail,Vimentin andα-SMA were significantly higher in Gli2 overexpression group,while that of E-cadherin was significantly lower(t=4.53,5.29,8.12,-2.13;all P<0.05);compared with those of the model group,mRNA levels of Snail and Vimentin in Gli2 shRNA group significantly decreased,while that of E-cadherin significantly increased(t=-2.86,-2.12,5.62;all P<0.05).In Gli2 overexpression group,Gli2 shRNA group and model group,the protein levels of Snail,Slug,Vimentin,α-SMA and E-cadherin were 2.02±0.39,0.31±0.08,0.95±0.17,1.07±0.17 and 0.42±0.06;0.53±0.13,0.40±0.18,0.20±0.04,0.28±0.07 and 1.09±0.31;0.70±0.15,0.42±0.22,0.64±0.13,0.81±0.11 and 0.42±0.09.Compared with those of the model group,protein levels of Snail,Vimentin andα-SMA were significantly higher in Gli2 overexpression group(t=12.71,4.28,3.70;all P<0.05);compared with those of the model group,protein levels of Vimentin andα-SMA in Gli2 shRNA group significantly decreased,while that of E-cadherin significantly increased(t=-6.14,-7.57,5.96;all P<0.05).However,no significant change trend were detected in expression levels of characteristic cytokines of EMT between Gli1 overexpression group and Gli1 shRNA group.The area percentage of liver fiber expression in normal group,model group,Gli1 overexpression group,Gli1 shRNA group,Gli2 overexpression group and Gli2 shRNA group were(1.03±0.58)%,(33.02±11.39)%,(39.81±5.67)%,(26.06±1.29)%,(49.81±8.57)%and(17.55±0.66)%,respectively.Besides,in terms of percentage of area expressed in liver fiber tissue,the Gli2 overexpression group and Gli2 shRNA group were statistically significant compared with the model group(t=3.21,-2.96;all P<0.05),while the Gli1 overexpression group and Gli1 shRNA group were not statistically significant compared with the model group(all P>0.05).Conclusions The Shh signaling pathway plays an important role in liver fibrosis in mice with biliary atresia.Gli2,a key transcription factor of Shh signaling pathway,can significantly regulate liver EMT process in mice with biliary atresi.