摘要
目的研究基质金属蛋白酶组织抑制因子(TIMP)-1 siRNA、TIMP-2 siRNA对CCl4诱导的肝纤维化大鼠肝星状细胞(HSC)中smad 2/3/4蛋白表达的影响。方法采用前期构建的TIMP-1 siRNA、TIMP-2 siRNA治疗后肝纤维化大鼠的肝脏组织作为研究对象,用免疫组织化学、蛋白质印迹法(Western blot)及Real-time PCR法检测smad2、smad3、smad4的蛋白表达和相应的mRNA表达水平,并用正置荧光显微镜计数免疫荧光双标记TUNEL阳性细胞和α-平滑肌肌动蛋白(α-SMA)阳性细胞。多组间均数比较采用ANOVA方差分析,均数两两比较采用SNK检验。结果免疫组织化学结果显示TIMP-1 siRNA组、TIMP-2 siRNA组中smad2、smad3、smad4的蛋白表达量较模型组、阴性对照组明显减少(P值均<0.05)。Western blot结果也显示相同趋势,TIMP-1siRNA组、TIMP-2siRNA组中smad2、smad3、smad4的蛋白表达量较模型组、阴性对照组显著减少(P值均<0.01)。TIMP-1siRNA组、TIMP-2siRNA组smad2、smad3、smad4的mRNA表达量较模型组、阴性对照组明显减少(P值均<0.05)。免疫荧光显示TIMP-1 siRNA组(0.0143±0.0024)、TIMP-2 siRNA组(0.0107±0.0044)活化HSC的凋亡指数较模型组(0)、阴性对照组(0.0024±0.0024)增加(P值均<0.05)。结论TIMP-1 siRNA、TIMP-2 siRNA促进了活化HSC的凋亡,对smads蛋白的表达量有明显抑制作用。
Objective To study the effect of tissue inhibitor of metalloproteinases(TIMP)-1 siRNA and TIMP-2 siRNA on the expression of smad2/3/4 protein in CCl4-induced liver fibrosis rat hepatic stellate cells(HSC).Methods Rat’s liver tissues with liver fibrosis after treatment with pre-built TIMP-1siRNA and TIMP-2 siRNA were used as the research subjects.Immunohistochemistry,Western blotting and real-time PCR were used to detect the protein and corresponding mRNA expression levels on smad2/3/4.TUNEL andα-smooth muscle actin(α-SMA)positive cells were quantified by double-labeled immunofluorescence.Analysis of variance(ANOVA)was used to compare the means between multiple groups,and the SNK test was used for the pairwise comparison of means.Results The results of immunohistochemistry showed that the protein expressions of smad2,smad3,and smad4 in the TIMP-1 siRNA group and TIMP-2 siRNA group were significantly reduced than those of the model and the negative control group(P<0.05).In addition,Western blotting results had also shown the same trend.The protein expression of smad2,smad3,and smad4 in the TIMP-1siRNA group and TIMP-2siRNA group were significantly reduced than those of the model and the negative control group(P<0.01).The mRNA expression of smad2,smad3,and smad4 in TIMP-1siRNA group and TIMP-2siRNA group was significantly reduced than those of the model and negative control group(P<0.05).Immunofluorescence showed that the apoptosis of activated HSC in the TIMP-1 siRNA group(0.0143±0.0024)and TIMP-2 siRNA group(0.0107±0.0044)was increased than those of the model(0)and the negative control group(0.0024±0.0024,P<0.05).Conclusion TIMP-1 siRNA and TIMP-2 siRNA promote the apoptosis of activated HSCs.In addition,it also has a significant inhibitory effect on the expression of smad protein.
作者
程雪
张敏
薛莹
孙航
刘杞
石小枫
Cheng Xue;Zhang Min;Xue Ying;Sun Hang;Liu Qi;Shi Xiaofeng(Key Laboratory of Molecular Biology for Infectious Diseases,Institute for Viral Hepatitis,the Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China;Department of Infectious Diseases,the Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2020年第9期753-759,共7页
Chinese Journal of Hepatology
基金
国家自然科学基金(81270503)。
