纤维连接蛋白1经PI3K/Akt途径调控肝星状细胞活化影响胆道闭锁肝纤维化  

Mechanism of FN1 promoting hepatic stellate cell activation via PI3K/Akt signaling pathway in hepatic fibrosis of biliary atresia

作　　者：杨仁杰 张聪 陈灵芝 郑启鹏 李梦迪 王琼 祁滢伊 赵方园 薛文帆 詹江华[2] Yang Renjie;Zhang Cong;Chen Lingzhi;Zhen Qipeng;Li Mengdi;Wang Qiong;Qi Yingyi;Zhao Fangyuan;Xue Wenfan;Zhan Jianghua(Tianjin Medical University,Tianjin 300070,China;Department of General Surgery,Tianjin Children's Hospital,Tianjin 300134,China;Children's Hospital,Capital Institute of Pediatrics,Beijing 100020,China;Wuhan Children's Hospital,Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430016,China;Department of Pediatric Surgery,Xinjiang Yili Friendship Hospital,Yili 835000,China)

机构地区：[1]天津医科大学研究生院,天津300070 [2]天津市儿童医院普外科,天津300134 [3]首都儿科研究所附属儿童医院,北京100020 [4]华中科技大学同济医学院附属武汉儿童医院,武汉430016 [5]新疆伊犁州友谊医院小儿外科,伊犁835000

出　　处：《中华小儿外科杂志》2023年第2期114-124,共11页Chinese Journal of Pediatric Surgery

基　　金：新疆维吾尔自治区自然科学基金(2019D01A12);新疆维吾尔自治区自然科学基金(2021D01A38)。

摘　　要：目的研究纤维连接蛋白1(fibronectin 1,FN1)在胆道闭锁(biliary atresia,BA)肝纤维化进程中的作用,阐述其作用机制与调控细胞凋亡肌醇磷脂-3-激酶/蛋白激酶B(PI3K/Akt)通路相关因子的关系。方法利用基因数据库数据对FN1在肝纤维化中的作用进行生物信息学分析。收集2017年4月至2020年10月在天津市儿童医院普外科手术时留取的患儿肝组织标本31例,其中因非肝胆疾病导致死亡的婴儿肝脏组织样本3例、先天性胆总管扩张症(congenital biliary dilatation,CBD)5例和BA 23例;根据日本Ohkuma’’s肝纤维化分级标准对所有肝组织样本进行分级处理,按照纤维化程度又将BA分两组:轻度(Ⅰ~Ⅱ级)、重度(Ⅲ~Ⅳ级);应用免疫组织化学检测FN1、PI3K、p-Akt和肝星状细胞活化标志物α-SMA在肝组织中的表达情况。分析以上蛋白与肝纤维化分级的相关性,利用qRT-PCR检测肝组织FN1 mRNA的表达水平并进行统计学分析。结果生物信息分析结果显示FN1在肝脏中表达量较高,是调控小鼠肝纤维化的核心基因之一。FN1在BA肝组织中的表达情况:(1)免疫组织化学检测显示,FN1表达于BA肝细胞的细胞质以及肝细胞外间隙,半定量分析结果显示,FN1的表达水平与肝纤维化程度呈正相关(rs=0.938,P<0.01),其在BA重度肝纤维化组表达明显高于轻度肝纤维化组[(0.1358±0.0416)比(0.0548±0.0095),t=-6.035,P<0.001];(2)qRT-PCR定量分析显示BA重度肝纤维化组FN1 mRNA的表达水平亦显著高于轻度肝纤维化组,为(1.038±0.2643)ng/ml比(0.5221±0.2365)ng/ml,P<0.01。采用Pearson相关分析显示,PI3K与PI3K/Akt通路上下游基因FN1(r=0.981,P<0.01)和α-SMA(r=0.988,P<0.01)在BA肝纤维化中的表达都呈现显著正相关;同样,p-Akt与该通路上下游基因FN1(r=0.946,P<0.01)和α-SMA(r=0.971,P<0.01)在BA肝纤维化中的表达也都呈现显著正相关。结论BA肝组织中FN1随着肝纤维化程度的加重而增加,并经PI3K/Akt信号途径参�Objective To explore the role of fibronectin 1(FN1)in the progression of hepatic fibrosis in biliary atresia(BA)and to elucidate its acting mechanism in relation to factors involved in the regulation of apoptotic inositol-3-kinase/protein kinase B(PI3K/Akt)pathway.Methods The role of FN1 in liver fibrosis was analyzed by bioinformatics using gene database data.Thirty-one liver tissue samples were collected from 3 infants who died from non-hepatobiliary diseases,5 infants with congenital biliary dilatation(CBD)and 23 BA infants.All liver tissue samples were graded according to the Japanese Ohkuma's liver fibrosis grading standard.According to the degree of fibrosis,BA was divided into two groups:mild fibrosis(Ⅰ~Ⅱgrade)and severe fibrosis(Ⅲ~Ⅳgrade).The expressions of FN1,PI3K,p-Akt and hepatic stellate cell activation markerα-SMA in liver tissue were detected by immunohistochemistry and the correlation between the above proteins and grade of hepatic fibrosis examined.Quantitative real-time polymerase chain reaction(qRT-PCR)was employed for detecting the expression level of FN1mRNA in liver tissues.Results Bioinformatic analysis revealed that FN1 was highly expressed in liver and it was one of core genes regulating hepatic fibrosis in mice.Expression of FN1 in BA liver tissue:Immunohistochemistry indicated that FN1 was expressed in cytoplasm and extracellular space of BA hepatocytes.The results of semi-quantitative analysis showed that the expression level of FN1 was positively correlated with severity of hepatic fibrosis(rs=0.938,P<0.01).And the expression of FN1 was significantly higher in severe BA group than that in mild BA group[(0.1358±0.0416)vs(0.0548±0.0095),t=-6.035,P<0.001].QRT-PCR quantitative analysis indicated that the expression level of FN1 was also significantly higher mRNA in BA severe hepatic fibrosis group than that in mild hepatic fibrosis group[(1.0530±0.2510)vs(0.5221±0.2365)ng/ml,P<0.01].Pearson's correlation analysis showed that the expression of PI3K was significantly positively

关 键 词：胆道闭锁 肝纤维化 纤维连接蛋白1 PI3K/AKT 肝星状细胞 

分 类 号：R726.5[医药卫生—儿科]