暨南大学学报（自然科学与医学版）
暨南大學學報（自然科學與醫學版）
기남대학학보（자연과학여의학판）
JOURNAL OF JINAN UNIVERSITY（NATURAL SCIENCE & MEDICINE EDITION）
2015年
3期
239-245
,共7页
陶燕%付生军%洪梅%王志平%马宝良%卢建中
陶燕%付生軍%洪梅%王誌平%馬寶良%盧建中
도연%부생군%홍매%왕지평%마보량%로건중
载体构建%survivin%短发卡状 RNA(shRNA)%膀胱癌%细胞凋亡
載體構建%survivin%短髮卡狀 RNA(shRNA)%膀胱癌%細胞凋亡
재체구건%survivin%단발잡상 RNA(shRNA)%방광암%세포조망
vector construction%survivin%short hairpin RNA(shRNA)%bladder cancer%cell apoptosis
目的：构建 survivin 基因特异性 shRNA 慢病毒干扰载体，转染膀胱癌 EJ 细胞，研究 survivin 基因在膀胱癌细胞株中的表达抑制情况，观察 survivin shRNA 慢病毒载体对 EJ 细胞凋亡的影响．方法：以 survivin 基因为靶标设计 shRNA 干扰序列，克隆至 pSIH1-H1-copGFP 慢病毒载体．干扰载体鉴定正确后转染膀胱癌细胞株 EJ 细胞，荧光显微镜下观察 GFP 表达情况，实时荧光定量 PCR 检测 survivin 基因 mRNA 含量变化，Western blotting 法检测 sur-vivin 蛋白表达，Annexin V-FITC ／PI 双染法检测 EJ 细胞凋亡情况．结果：PCR 扩增鉴定、DNA 测序证实 survivin 慢病毒干扰载体构建成功；EJ 细胞经干扰载体处理后，EJ 细胞 survivin 基因 mRNA 水平下调了73.33％，蛋白表达受到显著抑制，EJ 细胞凋亡率达到24.39％．结论：成功构建了靶向 survivin 基因的 shRNA 重组慢病毒干扰载体，可以显著降低转染细胞 survivin 基因的表达水平，并有效提高膀胱癌细胞凋亡率．
目的：構建 survivin 基因特異性 shRNA 慢病毒榦擾載體，轉染膀胱癌 EJ 細胞，研究 survivin 基因在膀胱癌細胞株中的錶達抑製情況，觀察 survivin shRNA 慢病毒載體對 EJ 細胞凋亡的影響．方法：以 survivin 基因為靶標設計 shRNA 榦擾序列，剋隆至 pSIH1-H1-copGFP 慢病毒載體．榦擾載體鑒定正確後轉染膀胱癌細胞株 EJ 細胞，熒光顯微鏡下觀察 GFP 錶達情況，實時熒光定量 PCR 檢測 survivin 基因 mRNA 含量變化，Western blotting 法檢測 sur-vivin 蛋白錶達，Annexin V-FITC ／PI 雙染法檢測 EJ 細胞凋亡情況．結果：PCR 擴增鑒定、DNA 測序證實 survivin 慢病毒榦擾載體構建成功；EJ 細胞經榦擾載體處理後，EJ 細胞 survivin 基因 mRNA 水平下調瞭73.33％，蛋白錶達受到顯著抑製，EJ 細胞凋亡率達到24.39％．結論：成功構建瞭靶嚮 survivin 基因的 shRNA 重組慢病毒榦擾載體，可以顯著降低轉染細胞 survivin 基因的錶達水平，併有效提高膀胱癌細胞凋亡率．
목적：구건 survivin 기인특이성 shRNA 만병독간우재체，전염방광암 EJ 세포，연구 survivin 기인재방광암세포주중적표체억제정황，관찰 survivin shRNA 만병독재체대 EJ 세포조망적영향．방법：이 survivin 기인위파표설계 shRNA 간우서렬，극륭지 pSIH1-H1-copGFP 만병독재체．간우재체감정정학후전염방광암세포주 EJ 세포，형광현미경하관찰 GFP 표체정황，실시형광정량 PCR 검측 survivin 기인 mRNA 함량변화，Western blotting 법검측 sur-vivin 단백표체，Annexin V-FITC ／PI 쌍염법검측 EJ 세포조망정황．결과：PCR 확증감정、DNA 측서증실 survivin 만병독간우재체구건성공；EJ 세포경간우재체처리후，EJ 세포 survivin 기인 mRNA 수평하조료73.33％，단백표체수도현저억제，EJ 세포조망솔체도24.39％．결론：성공구건료파향 survivin 기인적 shRNA 중조만병독간우재체，가이현저강저전염세포 survivin 기인적표체수평，병유효제고방광암세포조망솔．
Aim:To construct an effective survivin gene shRNA lentiviral vector,and evaluate its effects on apoptosis of bladder cancer cell line EJ.Methods:The shRNA template of survivin was cloned into pSIH1-H1-copGFP shRNA vectors.The recombinant plasmid was identificated by agarose gel elec-trophoresis and sequencing analysis;then it was transfected into the EJ cells.The mRNA and protein lev-els were measured using real-time quantitative PCR and Western blotting assay respectively,and the ap-optosis of EJ cells was measured by Annexin V-FITC /PI double staining assay.Results:The survivin gene shRNA lentivirus vector plasmid was successfully constructed.Agarose gel electrophoresis and se-quencing analysis confirmed the correct of plasmid construction.After shRNA plasmid transfection,the mRNA levels of survivin was significantly suppressed(suppression rate 73.33%,P <0.05),and the pro- <br> tein expression was decreased significantly.The apoptosis rate in the shRNA treated group was dramatic-ally increased when compared to the control group.Conclusion:The survivin shRNA lentiviral vector sig-nificantly suppressed survivin expression and induced apoptosis of bladder cancer EJ cells.
