CAJ | 학술논문

目的用RNA干扰技术,分别以转化生长因子(TGF)β 1、基质金属蛋白酶抑制剂(TIMP)-1和TIMP-2为靶基因,设计并构建针对TGF β 1、TIMP-1和TIMP-2基因的小干扰RNA(siRNA)真核表达载体,并在体外检测其对大鼠肝星状细胞株(HSC-T6)的TGF β 1、TIMP-1和TIMP-2基因表达的抑制情况.方法设计合成TGF β 1、TIMP-1和TIMP-2的siRNA并与含绿色荧光蛋白的pGenesil-1载体连接,构建siRNA真核表达载体,并测序鉴定.体外转染HSC-T6细胞,观察转染效率,并用荧光定量PCR以及Western blot分析对目的基因的抑制效率.组间比较用方差分析,两两比较用q检验.结果成功构建了针对TGF β 1、TIMP-1和TIMP-2基因的siRNA真核表达载体.体外成功转染HSC-T6细胞,转染后的细胞TGF β 1、TIMP-1及TIMP-2 mRNA表达分别下调63.4%±8.0%,64.5%±9.0%,55.0%±17.0%(F值分别为17.55、128.42、210.36,P值均＜0.01),TGF β 1、TIMP-1及TIMP-2蛋白表达分别下降57.8%±3.0%,55.1%±5.0%,49.3%±1.0%(F值分别为130.75、159.09、35.72,P值均＜0.01).结论成功构建了针对TGF β 1、TIMP-1和TIMP-2基因的siRNA真核表达载体;将重组载体成功转染入体外培养的HSC-T6细胞,并显著抑制了目的基因的表达;为进一步研究其在体抑制表达提供了实验工具.
목적 용RNA간우기술,분별이전화생장인자(TGF)β 1、기질금속단백매억제제(TIMP)-1화TIMP-2위파기인,설계병구건침대TGF β 1、TIMP-1화TIMP-2기인적소간우RNA(siRNA)진핵표체재체,병재체외검측기대대서간성상세포주(HSC-T6)적TGF β 1、TIMP-1화TIMP-2기인표체적억제정황.방법 설계합성TGF β 1、TIMP-1화TIMP-2적siRNA병여함록색형광단백적pGenesil-1재체련접,구건siRNA진핵표체재체,병측서감정.체외전염HSC-T6세포,관찰전염효솔,병용형광정량PCR이급Western blot분석대목적 기인적억제효솔.조간비교용방차분석,량량비교용q검험.결과 성공구건료침대TGF β 1、TIMP-1화TIMP-2기인적siRNA진핵표체재체.체외성공전염HSC-T6세포,전염후적세포TGF β 1、TIMP-1급TIMP-2 mRNA표체분별하조63.4%±8.0%,64.5%±9.0%,55.0%±17.0%(F치분별위17.55、128.42、210.36,P치균＜0.01),TGF β 1、TIMP-1급TIMP-2단백표체분별하강57.8%±3.0%,55.1%±5.0%,49.3%±1.0%(F치분별위130.75、159.09、35.72,P치균＜0.01).결론 성공구건료침대TGF β 1、TIMP-1화TIMP-2기인적siRNA진핵표체재체;장중조재체성공전염입체외배양적HSC-T6세포,병현저억제료목적 기인적표체;위진일보연구기재체억제표체제공료실험공구.
Objective To construct the siRNA eukaryotic expression vectors targeting on TGF β1,TIMP-1 and TIMP-2 and to investigate the inhibitory efficiency of target genes expression on rat hepatic stellate cell in vitro. Methods The siRNA cDNA sequences ofTGF β 1, TIMP-1 and TIMP-2 were designed,synthesized and inserted into plasmid pGenesil-1 respectively to generate eukaryotic expression plasmids.The plasmids were transfected into HSC T6 cells in vitro and the inhibitory efficiency of target genes expression was observed with real-time PCR and Western blot. Results The eukaryotic expression vectors were constructed successfully. The expressions of TGF β1 mRNA, TIMP-1 mRNA and TIMP-2mRNA in siRNAtransfected groups were decreased by 63.4% ± 8.0%, 64.5% ± 9.0% and 55.0% ± 17.0% respectively and the expressions of TGF β1 protein, TIMP-1 protein and TIMP-2 protein were decreased by 57.8% ± 3.0%,55.1% ± 5.0%, 49.3% ± 1.0% respectively as compared to the control groups. Conclusions The siRNA eukaryotic expression vectors constructed targeting on TGF β1, TIMP-1 and TIMP-2 could reduce the expressions of target genes and they might be able to used for the exploration of new anti-fibrosis drugs genetically.