热休克蛋白47-shRNA调节肝星状细胞膜受体对日本血吸虫鼠肝纤维化的影响  

Regulation of hepatic stellate cell membrane receptors by Hsp47-shRNA affects hepatic fibrosis in mice with Schistosoma japonicum infection

作　　者：邓菊红[1] 陶然 宋启琴 孔红言 焦云桃 黄加权[2] DENG Ju-hong;TAO Ran;SONG Qi-qin;KONG Hong-yan;JIAO Yun-tao;HUANG Jia-quan(Department of Internal Medicine,Liyuan Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China;Department of Infection,Tong]i Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属梨园医院内科,武汉430077 [2]华中科技大学同济医学院附属同济医院感染科,武汉430030

出　　处：《中国寄生虫学与寄生虫病杂志》2018年第4期317-324,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：湖北省血吸虫病防治项目基金(No.XF2012-17);国家"十二五"科技重大专项(No.2014ZX10005001)~~

摘　　要：目的了解热休克蛋白47-短发夹RNA(HSP47-sh RNA)调节肝星状细胞(HSCs)膜受体对日本血吸虫鼠肝纤维化的影响。方法建立日本血吸虫鼠肝纤维化模型,感染前(健康对照组)和感染后第4、9、14周,分别随机取5只小鼠,提取肝脏组织总RNA,采用RT-PCR检测HSP47 mRNA相对表达水平,蛋白质印迹(Western blotting)检测HSP47蛋白表达情况,用RM-6240BD型多道生理信号系统动态检测模型鼠门静脉压力,流式细胞术检测HSCs膜内皮素受体(ETAR和ETBR)的平均荧光强度(MFI),免疫组织化学法检测感染前和感染后14周的模型鼠肝组织膜受体表达情况,采用线性相关分析方法分析各时间点的HSP47 mRNA相对表达水平与门静脉压力、ETAR和ETBR相对表达量的关系。用HSP47-sh RNA质粒转染小鼠成纤维细胞NIH/3T3细胞和血吸虫肝纤维化模型鼠,并设阴性对照组(空质粒)和空白对照组(PBS),每组12只血吸虫感染模型鼠。采用RT-PCR检测NIH/3T3细胞转染0、24、48 h及模型鼠干预至第14周的HSP47 mRNA、Ⅰ型和Ⅲ型胶原mRNA相对表达水平,Western blotting检测对应蛋白的表达情况,流式细胞术检测膜受体(ET、TGF-β及PDGF的受体)的MFI。两组间均数的比较采用Student-t检验,多组间采用单因素方差分析。结果模型鼠感染日本血吸虫后第4、9、14周,RT-PCR结果显示,HSP47 mRNA相对表达水平分别为0.592±1.031、1.253±2.101和0.651±1.532,较感染前(0.253±0.120)均升高(P<0.01);Western blotting结果显示,HSP47蛋白表达情况与mRNA变化一致;门静脉压力分别为(3.010±0.250)、(8.850±0.63)和(12.390±0.830)mm Hg,感染后第14周与感染前的(2.350±0.180)mm Hg比较,差异有统计学意义(P<0.01)。流式细胞术检测结果显示,ETAR的MFI分别为3 245±186、6 108±213和8 784±257,高于感染前的2 104±232(P<0.01);ETBR的MFI分别为3 812±158、4 524±197和5 554±156,高于感染前的2 091±237(P<0.01)。相关性分析结果显示,HSP47 mRNA相对表达水平�Objective To investigate the effect of hepatic stellate cell（HSC） membrane receptor regulation by heat shock protein 47（HSP47）-sh RNA on the Schistosoma japonicum-induced liver fibrosis in mice. Methods The mouse model of Schistosoma japonicum-induced liver fibrosis was established. Total RNA was extracted from liver tissues before infection and at weeks 4, 9 and 14 after infection（ n = 5 mice in each group）. The mRNA expression and protein levels of HSP47 were assessed by RT-PCR and Western blotting, respectively. The RM-6240 BD multichannel physiological signals were used to measure the portal vein pressure of mice, flow cytometry was used to de-tect the mean fluorescence intensity（MFI） of endothelin receptor（ETAR and ETBR） in HSC membranes, and immunohistochemistry was used to detect the expression of membrane receptors in liver before and at week 14 after infection. The relationships between the expression of HSP47 and portal vein pressure as well as expression of ETAR and ETBR in HSC were analyzed using the linear correlation analysis method. The NIH/3 T3 cells and mouse model of Schistosoma japonicum-induced liver fibrosis（n = 12） were transfected with HSP47-sh RNA expression plasmid, accompanied by a negative control group（control plasmid） and a blank control group（PBS group）. RT-PCR was used to detect the mRNA expression of HSP47, type Ⅰ collagen and type Ⅲ collagen in NIH/3 T3 cells at 0, 24 and48 h after transfection and in the mouse model at week 14. Their protein levels were determined by Western blotting. MFI of the membrane receptors for ET, TGF-β and PDGF was detected by flow cytometry. Data were analyzed by student＇s t-test for comparisons between groups, and by ANOVA for comparisons among groups. Results RT-PCT results showed that the relative expression levels of HSP47 mRNA were 0.592 ± 1.031, 1.253 ± 2.101 and 0.651 ± 1.532,respectively, in the mouse model at weeks 4, 9 and 14 after infection, all significantly increased compared with that be-fore infec

关 键 词：HSP47 肝纤维化 日本血吸虫 SHRNA干扰 内皮素受体 

分 类 号：R383.24[医药卫生—医学寄生虫学]