CAJ | 학술논문

目的构建慢病毒载体表达小干扰RNA(small interfering RNA,siRNA)以沉默转化生长因子-β(TGF-β)早期反应基因(TIEG),观察其对糖尿病大鼠肾组织中Smad信号蛋白通路负反馈调节因子Smad7及对细胞外基质主要成分Ⅳ型胶原的影响作用.方法选取体莺200～230 g的SD雄性大鼠15只,分为3组,对照组5只,其余10只用链脲佐菌素(STZ)诱导制成糖尿病大鼠模型,随机分为TIEGsiRNA治疗组(5只)和空载体组(5只),治疗组和空载体组于成模后分别予TIEGsiRNA病毒液和空载体液尾静脉注射,治疗4周后实时PCR检测大鼠肾组织内TIEG的表达水平;Westernblot和免疫组化检测Smad7蛋白表达水平;免疫组化检测Ⅳ型胶原蛋白表达水平;MASSON染色检测胶原生成.单因素ANOVA方差分析,方差不齐用Kruskal-Wallis秩和检验.结果免疫组化半定量结果示Smad7蛋白在TIEGsiRNA治疗组(3.2±0.2)%较空载体组(1.7±0.5)%表达明显增多,Western-blot也同样验证了免疫组化结果(F=7.934,P=0.006),IV型胶原蛋白在TIEGsiRNA治疗组[(3.7±0.4)%]较空载体组[(4.8±0.4)0%]表达明显减少,MASSON染色半定量结果示纤维化程度在TIEGsiRNA治疗组[(3.5±0.4)%]较空载体组[(4.4±0.4)%]减少,差异均有统计学意义(P<0.01).结论 TIEGsiRNA可以通过上调糖尿病大鼠肾组织内Smad信号蛋白通路负反馈调节因子Smad7减少肾脏纤维化来延缓糖尿病进程.
목적 구건만병독재체표체소간우RNA(small interfering RNA,siRNA)이침묵전화생장인자-β(TGF-β)조기반응기인(TIEG),관찰기대당뇨병대서신조직중Smad신호단백통로부반궤조절인자Smad7급대세포외기질주요성분Ⅳ형효원적영향작용.방법 선취체앵200～230 g적SD웅성대서15지,분위3조,대조조5지,기여10지용련뇨좌균소(STZ)유도제성당뇨병대서모형,수궤분위TIEGsiRNA치료조(5지)화공재체조(5지),치료조화공재체조우성모후분별여TIEGsiRNA병독액화공재체액미정맥주사,치료4주후실시PCR검측대서신조직내TIEG적표체수평;Westernblot화면역조화검측Smad7단백표체수평;면역조화검측Ⅳ형효원단백표체수평;MASSON염색검측효원생성.단인소ANOVA방차분석,방차불제용Kruskal-Wallis질화검험.결과 면역조화반정량결과시Smad7단백재TIEGsiRNA치료조(3.2±0.2)%교공재체조(1.7±0.5)%표체명현증다,Western-blot야동양험증료면역조화결과(F=7.934,P=0.006),IV형효원단백재TIEGsiRNA치료조[(3.7±0.4)%]교공재체조[(4.8±0.4)0%]표체명현감소,MASSON염색반정량결과시섬유화정도재TIEGsiRNA치료조[(3.5±0.4)%]교공재체조[(4.4±0.4)%]감소,차이균유통계학의의(P<0.01).결론 TIEGsiRNA가이통과상조당뇨병대서신조직내Smad신호단백통로부반궤조절인자Smad7감소신장섬유화래연완당뇨병진정.
Objective To investigate the effect small interfere RNA of TGF-βinducible early gene to the negative feedback factor Smad7 in the Smad signal pathway and collagen IV which is the primary composition of deposition of extracellular matrix in the kidnev of diabetic rats.To provide a new target spot for diabetic nephropathy treatment. Methods 15 male SD rats(weight 200-300g)were randomly assigned to 3 groups.Ten Sprague-Dawley rats injected with streptozotocin(STZ)were ramdomly devided into TIEGsi-R group and TIEGsi-C group,Other five normal rats were used as control.Each of the TIEGsi-R group and TIEGsi-C group were injected with TIEGsi-R and TIEGsi-C respectively via the tail vein. 4 weeks after the treatment.TIEG expression levels were determined by fluorescence quantitative PCR.The expression of Smad7 protein was detected by immunohistochemistry and western blotting,and Collagen IV protein was detected by immunohistochemistry only.Renal fibrosis wag also assessed by Masson staining.All the datas were expressed in mean±standard deviation.We used one way ANOVA when compared between groops,Kruskal-Wallis rank test when there was heterogeneity of variance.Results Compared to TIEGsi-C group,TIEGsi-R group effectively increased expression of Smad7 protein which wag detected by immunohistochemical semi-quantification((3.2±0.2)%vs(4.8±0.4)%)and western-blot method(F=7.934,P=0.006),and downregulated expression of Collagen IV protein((3.7±0.4)%vs(4.8±0.4)%)and alleviated fibrosis which was detected by MASSON staining semi-quantification results.All the differences had statistical significance(P<0.01).Conclusions TIEGsiRNA may be useful in preventing the progression of diabetic renal fibrosis through upregulating negative feedback factor Smad7 in the Smad signal pathway.