国际妇产科学杂志
國際婦產科學雜誌
국제부산과학잡지
JOURNAL OF INTERNATIONAL OBSTETRICS AND GYNECOLOGY
2014年
4期
366-369
,共4页
沈青丽%何耀娟%郭凯敏%叶明%林仲秋
瀋青麗%何耀娟%郭凱敏%葉明%林仲鞦
침청려%하요연%곽개민%협명%림중추
宫颈肿瘤%癌%DNA结合蛋白质类%人乳头瘤病毒16%RNA干扰%逆转录病毒科
宮頸腫瘤%癌%DNA結閤蛋白質類%人乳頭瘤病毒16%RNA榦擾%逆轉錄病毒科
궁경종류%암%DNA결합단백질류%인유두류병독16%RNA간우%역전록병독과
Uterine cervical neoplasms%Carcinoma%DNA -binding proteins%Human papillomavirus 16%RNA interference%Retroviridae
目的:建立针对人信号传导与转录激活因子3(STAT3)的小发夹RNA(shRNA)逆转录病毒表达载体,建立稳定感染的人乳头瘤病毒16(HPV16)阳性宫颈癌细胞系,以进一步探讨STAT3在HPV16阳性宫颈癌发生和发展中的作用。方法:设计合成3条靶向STAT3基因的小干扰RNA(siRNA),从中选择1条具有最佳干扰效果的siRNA,构建STAT3-shRNA载体pSUPERretro-puro-STAT3,用293FT细胞包装质粒产生STAT3 shRNA逆转录病毒,感染HPV16阳性的宫颈癌细胞株SiHa和CaSki细胞后,通过嘌呤霉素筛选2周,约3 d传代1次,建立稳定细胞株,稳定沉默STAT3的细胞株命名为SiHa/STAT3-sh和CaSki/STAT3-sh。通过实时定量逆转录聚合酶链反应(qRT-PCR)和蛋白质印迹(Western blotting)分别从mRNA和蛋白水平检测STAT3的表达量。结果:经RT-PCR和Western blotting实验证实,在SiHa/STAT3-sh和CaSki/STAT3-sh细胞中,STAT3 mRNA的抑制率达99%以上(P<0.01),STAT3总蛋白抑制率达70%以上(P<0.01),磷酸化STAT3蛋白的抑制率达78%以上(P<0.01)。结论:稳定沉默STAT3的HPV16阳性宫颈癌细胞株建立成功。
目的:建立針對人信號傳導與轉錄激活因子3(STAT3)的小髮夾RNA(shRNA)逆轉錄病毒錶達載體,建立穩定感染的人乳頭瘤病毒16(HPV16)暘性宮頸癌細胞繫,以進一步探討STAT3在HPV16暘性宮頸癌髮生和髮展中的作用。方法:設計閤成3條靶嚮STAT3基因的小榦擾RNA(siRNA),從中選擇1條具有最佳榦擾效果的siRNA,構建STAT3-shRNA載體pSUPERretro-puro-STAT3,用293FT細胞包裝質粒產生STAT3 shRNA逆轉錄病毒,感染HPV16暘性的宮頸癌細胞株SiHa和CaSki細胞後,通過嘌呤黴素篩選2週,約3 d傳代1次,建立穩定細胞株,穩定沉默STAT3的細胞株命名為SiHa/STAT3-sh和CaSki/STAT3-sh。通過實時定量逆轉錄聚閤酶鏈反應(qRT-PCR)和蛋白質印跡(Western blotting)分彆從mRNA和蛋白水平檢測STAT3的錶達量。結果:經RT-PCR和Western blotting實驗證實,在SiHa/STAT3-sh和CaSki/STAT3-sh細胞中,STAT3 mRNA的抑製率達99%以上(P<0.01),STAT3總蛋白抑製率達70%以上(P<0.01),燐痠化STAT3蛋白的抑製率達78%以上(P<0.01)。結論:穩定沉默STAT3的HPV16暘性宮頸癌細胞株建立成功。
목적:건립침대인신호전도여전록격활인자3(STAT3)적소발협RNA(shRNA)역전록병독표체재체,건립은정감염적인유두류병독16(HPV16)양성궁경암세포계,이진일보탐토STAT3재HPV16양성궁경암발생화발전중적작용。방법:설계합성3조파향STAT3기인적소간우RNA(siRNA),종중선택1조구유최가간우효과적siRNA,구건STAT3-shRNA재체pSUPERretro-puro-STAT3,용293FT세포포장질립산생STAT3 shRNA역전록병독,감염HPV16양성적궁경암세포주SiHa화CaSki세포후,통과표령매소사선2주,약3 d전대1차,건립은정세포주,은정침묵STAT3적세포주명명위SiHa/STAT3-sh화CaSki/STAT3-sh。통과실시정량역전록취합매련반응(qRT-PCR)화단백질인적(Western blotting)분별종mRNA화단백수평검측STAT3적표체량。결과:경RT-PCR화Western blotting실험증실,재SiHa/STAT3-sh화CaSki/STAT3-sh세포중,STAT3 mRNA적억제솔체99%이상(P<0.01),STAT3총단백억제솔체70%이상(P<0.01),린산화STAT3단백적억제솔체78%이상(P<0.01)。결론:은정침묵STAT3적HPV16양성궁경암세포주건립성공。
Objective:Constructing retroviral vector of small hairpin RNA ( shRNA ) targeting human transducer and activator of transcription 3(STAT3),establishing stable infected HPV16 positive cervical cancer cell lines,to further explore the role of STAT3 in the development of HPV16 positive cervical cancer. Methods:Design and synthetise three siRNA sequences targeting STAT3,then select one with best gene interference effect,and construct STAT3-shRNA vector,pSUPERretro-puro-STAT3. The recombinant plasmid was transfected into packaging cell line 293FT and then infected into HPV16 positive cervical cancer cell line SiHa and CaSki cells. Cells were selected with puromycin for 2 weeks,passaged around every 3 days,and named with SiHa/STAT3-sh and CaSki/STAT3-sh. Expression of STAT3 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. Results:The knockdown of the expression of STAT3 mRNA was over 99%( P<0 . 01 ) , that of the expression of STAT3 total protein was over 70% ( P<0 . 01 ) and that of the expression of STAT3 phosphorylated protein was over 78% (P<0.01) in both SiHa/STAT3-sh and CaSki/STAT3-sh cells. Conclusions:HPV16 positive cervical cancer cell lines with stable silence of STAT3 were successfully established.