中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2008年
12期
930-934
,共5页
习东%李黎%高随%朱传龙%郭健文%王鸣%罗小平%宁琴
習東%李黎%高隨%硃傳龍%郭健文%王鳴%囉小平%寧琴
습동%리려%고수%주전룡%곽건문%왕명%라소평%저금
肝功能衰竭%RNA%CHO细胞%fg12凝血酶原酶
肝功能衰竭%RNA%CHO細胞%fg12凝血酶原酶
간공능쇠갈%RNA%CHO세포%fg12응혈매원매
Liver failure%RNA%CHO cells%fgl2/fibroleukin
目的 探索人fg12凝血酶原酶(hfg12)短干扰RNA(siRNA)在体外对hfg12凝血酶原酶基因表达的干预效应.方法 构建产生hfg12短发夹状RNA(shRNA)的载体p-hfg12shRNA,将其与hfg12-增强型绿色荧光蛋白(EGFP)融合基因表达质粒pEGFP-hfg12共转染到中国仓鼠卵母细胞(CHO细胞),转染48 h后,倒置荧光显微镜下观察EGFP在CHO细胞中的表达,并通过流式细胞仪检测荧光细胞阳性率.将P-hfg12shRNA和hfg12表达质粒pcDNA3.1-hfg12共转染到CHO细胞,通过实时荧光定量PCR和免疫组织化学检测hfg12基因转录和蛋白表达水平的变化.分为4组:共转染p-hfg12shRNA和pcDNA3.1-hfg12为干预组,共转染无关序列shRNA表达质粒和pcDNA3.1-hfg12为非相关干预组,仅转染pcDNA3.1-hfgl2为未干预组,不转染任何质粒为空白组.结果 在CHO细胞中,含p-hfg12shRNA的干预组较未干预组和非相关干预组的绿色荧光强度明显减弱,荧光细胞数明显减少;荧光表达阳性细胞率:干预组α为6.5%±0.2%,未干预组0t为40.1%±1.8%,两组比较,x<,2>=8.056,P<0.01,差异有统计学意义.抑制效率达85.5%.hfg12shRNA显著抑制了hfg12 mRNA和蛋白质的表达,干预组hfg12 mRNA水平为0.11±0.01,未干预组为0.84±0.06,两组比较,F=10.7,P<0.01,差异有统计学意义.结论 p-hfg12shRNA可在体外高效特异地抑制hfg12基因转录和蛋白的表达,为进一步的体内干扰实验奠定了基础.
目的 探索人fg12凝血酶原酶(hfg12)短榦擾RNA(siRNA)在體外對hfg12凝血酶原酶基因錶達的榦預效應.方法 構建產生hfg12短髮夾狀RNA(shRNA)的載體p-hfg12shRNA,將其與hfg12-增彊型綠色熒光蛋白(EGFP)融閤基因錶達質粒pEGFP-hfg12共轉染到中國倉鼠卵母細胞(CHO細胞),轉染48 h後,倒置熒光顯微鏡下觀察EGFP在CHO細胞中的錶達,併通過流式細胞儀檢測熒光細胞暘性率.將P-hfg12shRNA和hfg12錶達質粒pcDNA3.1-hfg12共轉染到CHO細胞,通過實時熒光定量PCR和免疫組織化學檢測hfg12基因轉錄和蛋白錶達水平的變化.分為4組:共轉染p-hfg12shRNA和pcDNA3.1-hfg12為榦預組,共轉染無關序列shRNA錶達質粒和pcDNA3.1-hfg12為非相關榦預組,僅轉染pcDNA3.1-hfgl2為未榦預組,不轉染任何質粒為空白組.結果 在CHO細胞中,含p-hfg12shRNA的榦預組較未榦預組和非相關榦預組的綠色熒光彊度明顯減弱,熒光細胞數明顯減少;熒光錶達暘性細胞率:榦預組α為6.5%±0.2%,未榦預組0t為40.1%±1.8%,兩組比較,x<,2>=8.056,P<0.01,差異有統計學意義.抑製效率達85.5%.hfg12shRNA顯著抑製瞭hfg12 mRNA和蛋白質的錶達,榦預組hfg12 mRNA水平為0.11±0.01,未榦預組為0.84±0.06,兩組比較,F=10.7,P<0.01,差異有統計學意義.結論 p-hfg12shRNA可在體外高效特異地抑製hfg12基因轉錄和蛋白的錶達,為進一步的體內榦擾實驗奠定瞭基礎.
목적 탐색인fg12응혈매원매(hfg12)단간우RNA(siRNA)재체외대hfg12응혈매원매기인표체적간예효응.방법 구건산생hfg12단발협상RNA(shRNA)적재체p-hfg12shRNA,장기여hfg12-증강형록색형광단백(EGFP)융합기인표체질립pEGFP-hfg12공전염도중국창서란모세포(CHO세포),전염48 h후,도치형광현미경하관찰EGFP재CHO세포중적표체,병통과류식세포의검측형광세포양성솔.장P-hfg12shRNA화hfg12표체질립pcDNA3.1-hfg12공전염도CHO세포,통과실시형광정량PCR화면역조직화학검측hfg12기인전록화단백표체수평적변화.분위4조:공전염p-hfg12shRNA화pcDNA3.1-hfg12위간예조,공전염무관서렬shRNA표체질립화pcDNA3.1-hfg12위비상관간예조,부전염pcDNA3.1-hfgl2위미간예조,불전염임하질립위공백조.결과 재CHO세포중,함p-hfg12shRNA적간예조교미간예조화비상관간예조적록색형광강도명현감약,형광세포수명현감소;형광표체양성세포솔:간예조α위6.5%±0.2%,미간예조0t위40.1%±1.8%,량조비교,x<,2>=8.056,P<0.01,차이유통계학의의.억제효솔체85.5%.hfg12shRNA현저억제료hfg12 mRNA화단백질적표체,간예조hfg12 mRNA수평위0.11±0.01,미간예조위0.84±0.06,량조비교,F=10.7,P<0.01,차이유통계학의의.결론 p-hfg12shRNA가재체외고효특이지억제hfg12기인전록화단백적표체,위진일보적체내간우실험전정료기출.
Objective Our previous studies have shown that an anti-sense plasmid to mouse fibrinogen like protein 2 (mfgl2) significantly reduced mfgl2 expression in vivo, markedly ameliorated inflammatory infiltration, fibrin deposition and hepatoeyte necrosis, prolonged the survival time period and elevated the survival rate in Balb/cJ mice with murine hepatitis virus type 3 (MHV-3) induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in the inhibi-tory application of hfgl2 expression, which has been reported to be involved in a variety of disease develop-ments including fulminant viral hepatitis, acute rejection of allo/xeno transplantation and fetal loss syndrome. Methods A plasmid named p-hfgl2shgNA complimentary to the sequence responsible for bfgl2 was constructed; meanwhile irrelevant shRNA plasmid with a random combination of the p-hfgl2shRNA se-quence was used as a control. A plasmid named pEGFP-hfg12 expressing hfg12-EGFP fusion protein was also constructed for the screening of the effect of p-hfg12shRNA on the bfgl2 expression. By cotransfection of the constructed p-hfg12shRNA and pEGFP-hfg12 or pcDNA3, 1-hfg12 expression plasmids into CHO cells, the inhibition of hfg12 expression by hfg12shRNA was analyzed by direct observation through fluo-rescent microscopy, FACS, real time PCR and immunohistochemistry staining. Results The experiments showed a significant inhibitory effect of p-hfgl2shRNA on hfgl2 expression at 48 h post-cotransfecfion by observation of green fluorescent cells, FACS, real time PCR and immunohistochemistry staining, with the inhibitory efficiency reaching as high as 85.5%. Conclusion The study demonstrated that p-hfgl2shRNA successfully interfered with hfgl2 expression in vitro. This provides a foundation to further explore its application in vivo.
