转化生长因子β1小干扰RNA对小鼠血吸虫病肝纤维化的抑制作用  

Inhibitory effect of specific small interference RNA targeted against transforming growth factorβ1 in mice with hepatic fibrosis infected with Schistosoma japonicum

作　　者：王鲁文 陈辉[2] 焦方舟 杨凡[1] 李汛[1] 张海月[1] Wang Luwen;Chen Hui;Jiao Fangzhou;Yang Fan;Li Xun;Zhang Haiyue(Department of Infectious Diseases,Renmin Hospital of Wuhan University,Wuhan 430060,China;Institute of Infectious Diseases,Hubei Center for Disease Control and Prevention,Wuhan 430079,China)

机构地区：[1]武汉大学人民医院感染科,430060 [2]湖北省疾病预防控制中心传染病研究所,武汉430079

出　　处：《中华传染病杂志》2019年第9期545-551,共7页Chinese Journal of Infectious Diseases

基　　金：湖北省卫生计生委科研基金(WJ2017X004)。

摘　　要：目的观察转化生长因子(transforming growth factorβ1,TGFβ1)小干扰RNA对小鼠血吸虫病肝纤维化的抑制作用。方法设计3条针对TGFβ1不同靶位的短发夹RNA(short hairpin RNA,shRNA),以及1条无关对照序列(HK),克隆至pGenesil-1质粒中。30只雄性BALB/c小鼠随机分为正常组、模型组、无关序列对照组、序列1治疗组、序列2治疗组、序列3治疗组各5只。建立血吸虫病肝纤维化小鼠模型。测定各组小鼠肝组织羟脯氨酸含量;苏木精-伊红染色与Masson染色观察肝组织病理变化;实时荧光定量聚合酶链反应和蛋白质印迹法分别检测肝组织内TGFβ1、母亲信号蛋白同源物(mothers against decapentaplegic homolog,Smad)3、Smad 7、α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)的mRNA和蛋白质表达水平。计量资料组间比较采用两独立样本t检验。结果所有治疗组小鼠的肝组织纤维化病理改变较模型组均得到明显改善;序列1治疗组、序列2治疗组和序列3治疗组小鼠肝组织羟脯氨酸含量较模型组均降低(t=14.870、7.097、10.741,均P<0.01)。所有治疗组肝脏内TGFβ1、Smad 3和α-SMA mRNA表达水平(模型组比序列1组t=3.235、5.141、10.026,模型组比序列2组t=3.396、5.145、4.951,模型组比序列3组t=3.511、5.429、6.485)与蛋白质表达水平(模型组比序列1组t=8.847、8.044、10.746,模型组比序列2组t=9.709、7.484、10.847,模型组比序列3组t=9.672、8.766、11.508)较模型组均下降(均P<0.01),而Smad 7的mRNA与蛋白质表达水平较模型组均升高(序列1组比模型组t=11.742、11.211,序列2组比模型组t=14.446、13.736,序列3组比模型组t=10.892、10.908,均P<0.01)。结论TGFβ1的RNA干扰能有效抑制小鼠血吸虫肝纤维化的发生,其机制可能通过抑制肝内TGFβ1、Smad 3和α-SMA的表达,促进Smad 7表达,从而下调肝星状细胞的激活信号,减少细胞内胶原合成,发挥抗血吸虫肝纤维化作用。Objective To investigate the inhibitory effect of small interference RNA(siRNA)targeted against transforming growth factorβ1(TGFβ1)in mice with hepatic fibrosis infected with Schistosoma japonicum.Methods Three short hairpin RNAs(shRNA)targeting different positions of TGFβ1 and one unrelated control sequence(HK)were designed and cloned to a plasmid pGenesil-1 respectively to obtain four recombinant expression vectors.Thirty male BALB/c mice were randomly divided into six groups,including normal group,model group,control group(pGenesil-HK)and three treatment groups(pGenesil-TGFβ1-m1,pGenesil-TGFβ1-m2 and pGenesil-TGFβ1-m3)and each group had five mice.The hepatic fibrosis animal models infected with Schistosoma japonicum were constructed.The levels of hydroxyproline(HYP)in liver tissue were examined by biochemistry.Liver histopathology was examined by hematoxylin-eosin and Masson staining.The mRNA expression and protein expression levels of TGFβ1,mothers against decapentaplegic homolog(Smad)3,Smad 7 andα-smooth muscle actin(α-SMA)in the livers were detected by quantitative real time polymerase chain reaction(RT-qPCR)and Western blot.Two independent samples t test was used to compare the measurement data between groups.Results The liver fibrogenesis was obviously improved in all treatment groups compared with model group.The levels of HYP of liver tissue in all treatment groups were significantly lower than that in model group(t=14.870,7.097 and 10.741,respectively,all P<0.01).The mRNA expression levels of TGFβ1,Smad 3 andα-SMA(model group vs pGenesil-TGFβ1-m1 group,t=3.235,5.141 and 10.026,respectively;model group vs pGenesil-TGFβ1-m2 group,t=3.396,5.145 and 4.951,respectively;model group vs pGenesil-TGFβ1-m3 group,t=3.511,5.429 and 6.485,respectively)and protein(model group vs pGenesil-TGFβ1-m1 group,t=8.847,8.044 and 10.746,respectively;model group vs pGenesil-TGFβ1-m2 group,t=9.709,7.484 and 10.847,respectively;model group vs pGenesil-TGFβ1-m3 group,t=9.672,8.766 and 11.508,respectively)were sig

关 键 词：转化生长因子Β1 RNA 小分子干扰 血吸虫 日本 肝纤维化 

分 类 号：R73[医药卫生—肿瘤]