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转化生长因子βⅠ型受体基因的靶向小干扰RNA抑制肝星状细胞合成胶原的体外研究
전화생장인자βⅠ형수체기인적파향소간우RNA억제간성상세포합성효원적체외연구
Effect of small interfering RNA targeting transforming growth factor β receptor Ⅰ gene on the collagen synthesis of hepatic stellate cells in vitro

万方数据

中华传染病杂志中華傳染病雜誌 중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2010年 1期 5-9 ,共5页

俞富军%楼迪栋%林镯%董培红%陈永平

유부군%루적동%림탁%동배홍%진영평

受体%转化生长因子β%RNA%小分子干扰%肝硬化%星形细胞%质粒%胶原受體%轉化生長因子β%RNA%小分子榦擾%肝硬化%星形細胞%質粒%膠原
수체%전화생장인자β%RNA%소분자간우%간경화%성형세포%질립%효원
Receptors%transforming growth factor beta%RNA%small interfering%Liver cirrhosis%Astrocytes%Plasmids%Collagen

目的观察大鼠转化生长因子βⅠ型受体(TβR Ⅰ)基因的靶向小干扰RNA(siRNA)表达质粒对肝星状细胞(HSC)合成胶原的影响.方法根据大鼠Tβ R Ⅰ的基因序列,设计并构建3个大鼠TβR Ⅰ基因的靶向siRNA表达质粒,以脂质体作转染试剂,将siRNA表达质粒分别转染至HSC-T6细胞中,RT-PCR和Western印迹技术分析TβRⅠ mRNA及蛋白表达水平,噻唑蓝(MTT)法检测细胞增殖,放射免疫法测HA、PCⅢ含量.采用LSD法进行统计学处理.结果酶切证实siRNA的目的基因片段已成功克隆入载体中.与空白对照组比较,转染siRNA表达质粒后,3组HSC-T6细胞TβR Ⅰ mRNA表达水平均抑制,其中以psiRNA2组抑制作用最强(psiRNA1组:t=7.354,P＜0.01;psiRNA2组:t=9.214,P＜0.01;psiRNA3组:t=5.967,P＜0.01).3组TβRⅠ蛋白表达水平均降低,以psiRNA2组降低最明显(psiRNA1组:t=6.324,P＜0.01;psiRNA2组:t=8.741,P＜0.01;psiRNA3组:t=4.128,P＜0.01).3组HSC-T6细胞增殖活性均下降,合成胶原均减少,以psiRNA2组最明显;而转染无关siRNA组无明显变化.结论构建的TβR Ⅰ siRNA表达质粒可抑制HSC-T6细胞合成胶原,为肝纤维化基因治疗提供新的靶点.
목적 관찰대서전화생장인자βⅠ형수체(TβR Ⅰ)기인적파향소간우RNA(siRNA)표체질립대간성상세포(HSC)합성효원적영향.방법 근거대서Tβ R Ⅰ적기인서렬,설계병구건3개대서TβR Ⅰ기인적파향siRNA표체질립,이지질체작전염시제,장siRNA표체질립분별전염지HSC-T6세포중,RT-PCR화Western인적기술분석TβRⅠ mRNA급단백표체수평,새서람(MTT)법검측세포증식,방사면역법측HA、PCⅢ함량.채용LSD법진행통계학처리.결과 매절증실siRNA적목적 기인편단이성공극륭입재체중.여공백대조조비교,전염siRNA표체질립후,3조HSC-T6세포TβR Ⅰ mRNA표체수평균억제,기중이psiRNA2조억제작용최강(psiRNA1조:t=7.354,P＜0.01;psiRNA2조:t=9.214,P＜0.01;psiRNA3조:t=5.967,P＜0.01).3조TβRⅠ단백표체수평균강저,이psiRNA2조강저최명현(psiRNA1조:t=6.324,P＜0.01;psiRNA2조:t=8.741,P＜0.01;psiRNA3조:t=4.128,P＜0.01).3조HSC-T6세포증식활성균하강,합성효원균감소,이psiRNA2조최명현;이전염무관siRNA조무명현변화.결론 구건적TβR Ⅰ siRNA표체질립가억제HSC-T6세포합성효원,위간섬유화기인치료제공신적파점.
Objective To observe the effect of small interfering RNA(siRNA)expression plasmids targeting transforming growth factor p receptor(TαR)Ⅰ gene on the collagen synthesis of hepatic stellate cells(HSCs).Methods Three siRNA expression plasmids were designed and constructed according to TBR Ⅰ sequence.Then the plasmids were transfected into HSC-T6 using 1ipofectamine2000 reagent. The mRNA and protein expressions of TβR Ⅰ were analyzed by reverse transcription polymerase chain reaction(RT-PCR)and Western blot technique, respectively. The cell proliferation was detected using methylthiazo-lyldiphenyl-tetrazolium bromide(MTT)methods. Concentrations of haluronic acid and type Ⅲ pro-collagen in the supernatants were determined by radioimmunoassay. The data were analyzed using least significant difference(LSD).Results Three recombinant plasmids expressing siRNAs were successfully constructed and confirmed by restriction enzyme assay. Compared with the blank control,all the three recombinant plasmids could inhibit the expressions of TβR Ⅰ mRNA,of which plasmid expressing siRNA2 exhibited the strongest inhibitory effect(psiRNA1 group:t=7.354,P＜0.01;psiRNA2 group:t=9.214,P＜0.01;psiRNA3 group:t=5.967,P＜0.01).The expressions of TβR Ⅰ protein were also reduced by all the three recombinant plasmids,of which the plasmid expressing siRNA2 showed the strongest inhibitory effect(psiRNA1 group: t=6.324,P＜0.01;psiRNA2 group:t=8.741,P＜0.01;psiRNA3 group:t=4.128,P＜0.01).The proliferation activity and collagen synthesis of HSCs also decreased in all three HSC groups treated with recombinant plasmids, of which, again, plasmid expressing siRNA2 exhibited the strongest inhibitory effect. However, no significant change was observed in HSCs transfected with non-related siRNA. Conclusion Recombinant plasmids targeting TβR I can inhibit collagen synthesis, which suggests a novel target for gene therapy of liver fibrosis.