中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2009年
40期
2847-2852
,共6页
程文%苏筠%马宏青%位志峰%高建平%张征宇%葛京平%景抗震%徐锋
程文%囌筠%馬宏青%位誌峰%高建平%張徵宇%葛京平%景抗震%徐鋒
정문%소균%마굉청%위지봉%고건평%장정우%갈경평%경항진%서봉
端粒酶逆转录酶%联合RNA干扰%基因,hTR%基因,hTERT%膀胱移行细胞癌
耑粒酶逆轉錄酶%聯閤RNA榦擾%基因,hTR%基因,hTERT%膀胱移行細胞癌
단립매역전록매%연합RNA간우%기인,hTR%기인,hTERT%방광이행세포암
Human telomerase reverse transcriptase%Combining RNAi%hTR geng%hTERT gene%Transitional cell cancer of bladder
目的 探索联合RNA干扰TR及TERT[小型干扰RNA(siRNA)、人端粒酶RNA(hTR)及人端粒酶逆转录酶(hTERT)]基因对膀胱移行细胞癌细胞株BIU-87端粒酶活性及其增殖的影响.方法 根据hTRmRNA和hTERTmRNA的序列,分别设计合成3对不同的且能干扰TR和TERT基因表达的siRNA并筛选出最高效抑制序列(pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ),构建以hTR、hTERT基因为靶点的联合RNA干扰装置pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ(脂质体法)并将其转染BIU-87细胞株,采用荧光定量PCR分析hTR及hTERT mRNA表达,TRAP-ELISA检测端粒酶活性,MTT法检测细胞增殖.结果 分别及联合RNA干扰hTR及hTERT基因后,pRNAT-TERT-Ⅲ,pRNAT-TR-Ⅲ及pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ的联合干扰可以特异性抑制BIU-87细胞株中TERT和TR表达(分别减少pRNAT-TERT-Ⅲ TERTmRNA:67%、pRNAT-TR-Ⅲ TRmRNA:41%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TRmRNA:57%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TERTmRNA:70%.P＜0.05).且膀胱移行细胞癌细胞株BIU-87的生长和端粒酶活性均明显受到抑制,以联合RNA干扰尤为明显(pRNAT-TERT-Ⅲ:1.80±0.014,pRNAT-TR-Ⅲ:1.95±0.016,pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ:1.25±0.012,P＜0.05).结论 联合RNA干扰hTERT及hTR基因能更明显抑制膀胱移行细胞癌BIU-87细胞株的生长,为其临床应用奠定了基础.
目的 探索聯閤RNA榦擾TR及TERT[小型榦擾RNA(siRNA)、人耑粒酶RNA(hTR)及人耑粒酶逆轉錄酶(hTERT)]基因對膀胱移行細胞癌細胞株BIU-87耑粒酶活性及其增殖的影響.方法 根據hTRmRNA和hTERTmRNA的序列,分彆設計閤成3對不同的且能榦擾TR和TERT基因錶達的siRNA併篩選齣最高效抑製序列(pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ),構建以hTR、hTERT基因為靶點的聯閤RNA榦擾裝置pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ(脂質體法)併將其轉染BIU-87細胞株,採用熒光定量PCR分析hTR及hTERT mRNA錶達,TRAP-ELISA檢測耑粒酶活性,MTT法檢測細胞增殖.結果 分彆及聯閤RNA榦擾hTR及hTERT基因後,pRNAT-TERT-Ⅲ,pRNAT-TR-Ⅲ及pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ的聯閤榦擾可以特異性抑製BIU-87細胞株中TERT和TR錶達(分彆減少pRNAT-TERT-Ⅲ TERTmRNA:67%、pRNAT-TR-Ⅲ TRmRNA:41%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TRmRNA:57%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TERTmRNA:70%.P＜0.05).且膀胱移行細胞癌細胞株BIU-87的生長和耑粒酶活性均明顯受到抑製,以聯閤RNA榦擾尤為明顯(pRNAT-TERT-Ⅲ:1.80±0.014,pRNAT-TR-Ⅲ:1.95±0.016,pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ:1.25±0.012,P＜0.05).結論 聯閤RNA榦擾hTERT及hTR基因能更明顯抑製膀胱移行細胞癌BIU-87細胞株的生長,為其臨床應用奠定瞭基礎.
목적 탐색연합RNA간우TR급TERT[소형간우RNA(siRNA)、인단립매RNA(hTR)급인단립매역전록매(hTERT)]기인대방광이행세포암세포주BIU-87단립매활성급기증식적영향.방법 근거hTRmRNA화hTERTmRNA적서렬,분별설계합성3대불동적차능간우TR화TERT기인표체적siRNA병사선출최고효억제서렬(pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ),구건이hTR、hTERT기인위파점적연합RNA간우장치pRNAT-TR-Ⅲ、pRNAT-TERT-Ⅲ(지질체법)병장기전염BIU-87세포주,채용형광정량PCR분석hTR급hTERT mRNA표체,TRAP-ELISA검측단립매활성,MTT법검측세포증식.결과 분별급연합RNA간우hTR급hTERT기인후,pRNAT-TERT-Ⅲ,pRNAT-TR-Ⅲ급pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ적연합간우가이특이성억제BIU-87세포주중TERT화TR표체(분별감소pRNAT-TERT-Ⅲ TERTmRNA:67%、pRNAT-TR-Ⅲ TRmRNA:41%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TRmRNA:57%、pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ TERTmRNA:70%.P＜0.05).차방광이행세포암세포주BIU-87적생장화단립매활성균명현수도억제,이연합RNA간우우위명현(pRNAT-TERT-Ⅲ:1.80±0.014,pRNAT-TR-Ⅲ:1.95±0.016,pRNAT-TERT-Ⅲ、pRNAT-TR-Ⅲ:1.25±0.012,P＜0.05).결론 연합RNA간우hTERT급hTR기인능경명현억제방광이행세포암BIU-87세포주적생장,위기림상응용전정료기출.
Objective To investigate the influence of combining RNAi-hTR plus hTERT genes upon the telomerase activity of bladder cancer BIU-87 cell line and provide new methods and evidence for RNAi in gene therapy of bladder transitional cell cancer.Methods Three hTR-specific double-stranded siRNAs and 3 hTERT-speeific double-stranded siRNAs were designed targeting different regions of hTR and hTERT mRNA.siRNAs (systems-PhTR-siRNA,PhTERT-siRNA and combining systems-PhTR plus PhTERT-siRNA) were transfeeted into bladder transitional cancer BIU-87 cell line.And hTR and hTERT mRNA expression were determined by fluorescence quantitative RT-PCR while telomerie repeat amplification protocol (TRAP) was applied to detect the telomerase activity and the growth inhibition of BIU-87 cells detected by MTT assay.Results RNAi-pRNAT-hTERT-Ⅲ,RNAi-pRNAT-hTR-Ⅲ and combining RNAi-hTR plus hTERT could inhibit the expression of hTERT and hTR mRNA in bladder cancer BIU-87 cell lines by RNAi-pRNAT-hTERT-Ⅲ hTERTmRNA 67%,RNAi-pRNAT-hTR-Ⅲ hTRmRNA 41% and pRNAT-hTR-Ⅲ hTRmRNA:57%,pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ hTERTmRNA:70% (P＜0.05).The growth of bladder cancer BIU-87 ceil was inhibited and telomerase activity considerably decreased,especially in combining RNAi-hTR and hTERT.Conclusion hTR-siRNA,hTERT-siRNA and combing siRNA hTR plus hTERT have been successfully designed and constructed.They can suppress specifically and effectively both hTR and hTERT mRNA expression and telomerase activity,especially in combining siRNA-hTR+hTERT.Combining siRNA-hTR plus hTERT are needed to explore its clinical applications.
