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人重型肝炎相关基因Fas、TNFR1真核表达载体和microRNA表达载体的构建及其体外干预效应的初步研究
인중형간염상관기인Fas、TNFR1진핵표체재체화microRNA표체재체적구건급기체외간예효응적초보연구
Construction of the Eukaryotic Expression Vectors and the microRNA Expression Plasmids of Human Fas and TNFR1 Gene and Their Biological Effects in vitro

万方数据

华中科技大学学报（医学版）華中科技大學學報（醫學版） 화중과기대학학보（의학판）
ACTA UNIVERSITATIS MEDICINAE TONGJI
2010年 1期 50-54 ,共5页

高随%习东%郭健文%严伟明%罗小平%宁琴

고수%습동%곽건문%엄위명%라소평%저금

Fas%TNFR1%microRNA%细胞凋亡%重型肝炎 Fas%TNFR1%microRNA%細胞凋亡%重型肝炎
Fas%TNFR1%microRNA%세포조망%중형간염
Fas%TNFR1%microRNA%apoptosis%fulminant hepatitis

目的构建人Fas(hFas)和人TNFR1(hTNFR1)基因的真核表达质粒pcDNA3.0-hFas、pcDNA3.0-hTNFR1和microRNA(miRNA)干扰质粒p-hFasmiRNA、 p-hTNFR1miRNA,初步验证其在体外细胞系对Fas和TNFR1基因表达的干预效应.方法从人肝癌细胞HepG2细胞中获得模板cDNA,通过PCR扩增出hFas和hTNFR1全长片段,将目的片段通过T载体过渡克隆至表达载体pcDNA3.0,得到重组质粒pcDNA3.0-hFas和pcDNA3.0-hTNFR1.利用miRNA设计软件,针对Fas和TNFR1基因分别设计3对pre-microRNA(pre-miRNA)序列,通过T4连接酶将pre-miRNA克隆至pcDNA6.2-GW/EmGFP-miRNA表达载体,同时构建非相关干扰质粒.构建成功的p-hFasmiRNA1、p-hFasmiRNA2、p-hFasmiRNA3和p-hTNFR1miRNA1、p-hTNFR1miRNA2、p-hTNFR1miRNA3分别与pcDNA3.0-hFas和pcDNA3.0-hTNFR1共转染至人293T细胞,通过Real-time PCR和Western blot检测转染48 h后对基因表达的干预效应.结果通过测序鉴定表明Fas和TNFR1基因的真核表达载体和miRNA干扰质粒均构建成功. Real-time PCR结果显示干预组的Fas和TNFR1基因的表达与对照组比较明显减少,抑制效率分别可达到87%和80%.Western blot结果也同样证实干预组的Fas和TNFR1基因的蛋白表达量与对照组比较明显减少.结论成功构建了hFas和hTNFR1的真核表达载体和microRNA干扰质粒,并初步证实构建的microRNA干扰质粒在细胞水平对hFas和hTNFR1的表达具有特异性的抑制效应.
목적 구건인Fas(hFas)화인TNFR1(hTNFR1)기인적진핵표체질립pcDNA3.0-hFas、pcDNA3.0-hTNFR1화microRNA(miRNA)간우질립p-hFasmiRNA、 p-hTNFR1miRNA,초보험증기재체외세포계대Fas화TNFR1기인표체적간예효응.방법 종인간암세포HepG2세포중획득모판cDNA,통과PCR확증출hFas화hTNFR1전장편단,장목적 편단통과T재체과도극륭지표체재체pcDNA3.0,득도중조질립pcDNA3.0-hFas화pcDNA3.0-hTNFR1.이용miRNA설계연건,침대Fas화TNFR1기인분별설계3대pre-microRNA(pre-miRNA)서렬,통과T4련접매장pre-miRNA극륭지pcDNA6.2-GW/EmGFP-miRNA표체재체,동시구건비상관간우질립.구건성공적p-hFasmiRNA1、p-hFasmiRNA2、p-hFasmiRNA3화p-hTNFR1miRNA1、p-hTNFR1miRNA2、p-hTNFR1miRNA3분별여pcDNA3.0-hFas화pcDNA3.0-hTNFR1공전염지인293T세포,통과Real-time PCR화Western blot검측전염48 h후대기인표체적간예효응.결과통과측서감정표명Fas화TNFR1기인적진핵표체재체화miRNA간우질립균구건성공. Real-time PCR결과현시간예조적Fas화TNFR1기인적표체여대조조비교명현감소,억제효솔분별가체도87%화80%.Western blot결과야동양증실간예조적Fas화TNFR1기인적단백표체량여대조조비교명현감소.결론 성공구건료hFas화hTNFR1적진핵표체재체화microRNA간우질립,병초보증실구건적microRNA간우질립재세포수평대hFas화hTNFR1적표체구유특이성적억제효응.
Objective To construct the eukaryotic expression plasmids of human Fas and TNFR1 gene(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and microRNA(miRNA)expression plasmid of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA,and to investigate their inhibitory effects in vitro.Methods The eukaryotic expression plasmids of human Fas and TNFR1 gene were constructed(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and have been shown successfully to express hFas and hTNFR1 protein.miRNA expression plasmids of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA complimentary to the sequence responsible for hFas and hTNFR1 respective were constructed,meanwhile irrelevant miRNA plasmid was used as a control.By respective co-transfection of p-hFasmiRNA and pcDNA3.0-hFas,p-hTNFR1 miRNA and pcDNA3.0-hTNFR1 expression construct into 293T cells,the inhibition of hFas and hTNFR1 expression was analyzed by real-time PCR and Western blot.Results The experiments showed the significant inhibitory effect of p-hFasmiRNA on hFas and p-hTNFR1miRNA on hTNFR1 expression at 48 h post-transfection both at RNA level and protein level as well in 293T cell lines with the inhibitory efficiency being as high as 87% for hFas and 80% for hTNFR1,respectively.Conclusion The p-hFasmiRNA and p-hTNFR1miRNA were constructed successfully,and it was verified that they could specifically inhibit the hFas and hTNFR1 expression at the cellular level.