生物技术通报
生物技術通報
생물기술통보
BIOTECHNOLOGY BULLETIN
2009年
7期
128-133
,共6页
袁成福%黄秀凝%程莉%卜友泉%刘革力%易发平%杨正梅%宋方洲
袁成福%黃秀凝%程莉%蔔友泉%劉革力%易髮平%楊正梅%宋方洲
원성복%황수응%정리%복우천%류혁력%역발평%양정매%송방주
survivin启动子%基因治疗%RNA干扰%宫颈癌
survivin啟動子%基因治療%RNA榦擾%宮頸癌
survivin계동자%기인치료%RNA간우%궁경암
Survivin gene promoter Gene therapy RNA interference Cervical cancer
研究survivin启动子驱动的小发夹RNA(shRNA)在宫颈癌SiHa细胞中的特异性表达.PCR扩增survivin启动子,构建以survivin启动子驱动增强绿色荧光蛋白(EGFP)表达的真核表达载体pEGFP-C1/Surv,并检测survivin启动子的活性;构建针对EGFP的shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6,并检测shRNA在U6启动子驱动下的干扰效果;用有活性的survivin启动子取代pGenesil-1-EGFP-shRNA/U6载体中的U6启动子,从而获得新的shRNA真核表达载体pGenesil-1-EGFP-shRNA/Surv;将pGenesil-1-EGFP-shRNA/Surv转染到宫颈癌SiHa细胞及正常人脐静脉内皮HuVEC细胞,观察EGFP在两种细胞中的表达变化.结果表明,成功扩增survivin启动子并验证该启动子具有活性;成功构建EGFP shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6,并验证shRNA在U6启动子驱动下具有较好的干扰效果;成功构建pGenesil-1-EGFP-shRNA/Surv载体,分别转染SiHa及HuVEC细胞,在SiHa 细胞中观察到较少的绿色荧光蛋白表达,而在HuVEC细胞中观察到较多的绿色荧光蛋白表达.survivin启动子能驱动shRNA在宫颈癌SiHa细胞中特异性表达,而在正常HuVEC细胞中不表达.
研究survivin啟動子驅動的小髮夾RNA(shRNA)在宮頸癌SiHa細胞中的特異性錶達.PCR擴增survivin啟動子,構建以survivin啟動子驅動增彊綠色熒光蛋白(EGFP)錶達的真覈錶達載體pEGFP-C1/Surv,併檢測survivin啟動子的活性;構建針對EGFP的shRNA真覈錶達載體pGenesil-1-EGFP-shRNA/U6,併檢測shRNA在U6啟動子驅動下的榦擾效果;用有活性的survivin啟動子取代pGenesil-1-EGFP-shRNA/U6載體中的U6啟動子,從而穫得新的shRNA真覈錶達載體pGenesil-1-EGFP-shRNA/Surv;將pGenesil-1-EGFP-shRNA/Surv轉染到宮頸癌SiHa細胞及正常人臍靜脈內皮HuVEC細胞,觀察EGFP在兩種細胞中的錶達變化.結果錶明,成功擴增survivin啟動子併驗證該啟動子具有活性;成功構建EGFP shRNA真覈錶達載體pGenesil-1-EGFP-shRNA/U6,併驗證shRNA在U6啟動子驅動下具有較好的榦擾效果;成功構建pGenesil-1-EGFP-shRNA/Surv載體,分彆轉染SiHa及HuVEC細胞,在SiHa 細胞中觀察到較少的綠色熒光蛋白錶達,而在HuVEC細胞中觀察到較多的綠色熒光蛋白錶達.survivin啟動子能驅動shRNA在宮頸癌SiHa細胞中特異性錶達,而在正常HuVEC細胞中不錶達.
연구survivin계동자구동적소발협RNA(shRNA)재궁경암SiHa세포중적특이성표체.PCR확증survivin계동자,구건이survivin계동자구동증강록색형광단백(EGFP)표체적진핵표체재체pEGFP-C1/Surv,병검측survivin계동자적활성;구건침대EGFP적shRNA진핵표체재체pGenesil-1-EGFP-shRNA/U6,병검측shRNA재U6계동자구동하적간우효과;용유활성적survivin계동자취대pGenesil-1-EGFP-shRNA/U6재체중적U6계동자,종이획득신적shRNA진핵표체재체pGenesil-1-EGFP-shRNA/Surv;장pGenesil-1-EGFP-shRNA/Surv전염도궁경암SiHa세포급정상인제정맥내피HuVEC세포,관찰EGFP재량충세포중적표체변화.결과표명,성공확증survivin계동자병험증해계동자구유활성;성공구건EGFP shRNA진핵표체재체pGenesil-1-EGFP-shRNA/U6,병험증shRNA재U6계동자구동하구유교호적간우효과;성공구건pGenesil-1-EGFP-shRNA/Surv재체,분별전염SiHa급HuVEC세포,재SiHa 세포중관찰도교소적록색형광단백표체,이재HuVEC세포중관찰도교다적록색형광단백표체.survivin계동자능구동shRNA재궁경암SiHa세포중특이성표체,이재정상HuVEC세포중불표체.
It was to investigate the specific expression of shRNA drived by survivin gene promoter in SiHa cells. The survivin gene promoter was amplified by PCR and pEGFP-C1/Surv plasmid was constructed in which the EGFP was drived by survivin gene promoter, and the activity of survivin gene promoter was detected. shRNA eukaryotic expression vector pGenesil-1-EGFP-shRNA/U6 targeting EGFP was constructed,and the RNA interferencing effect of shRNA drived by U6 promoter was detected. shRNA eukaryotic expression vector pGenesil-1-EGFP-shRNA/Surv in which the U6 promoter was replaced by surviving gene promoter. SiHa and HuVEC cells were transfected by pGenesil-1-EGFP-shRNA/Surv plasmid and the expression of EGFP in two cell lines was observed . Results showed that the survivin gene promoter was amplified successfully and its activity was verified. The pGenesil-1-EGFP-shRNA/U6 plasmid was constructed successfully, and the RNA interferencing effect of shRNA drived by U6promoter was verified as good as expected. The pGenesil-1-EGFP-shRNA/Surv vector was constructed successfully and transfected into SiHa and HuVEC cell lines. After 72 h, there was less green fluorescence in SiHa cells, whereas there was more green fluorescence in HuVEC cells. Therefore,it proved that the survivin gene promoter can drive the specific expression of shRNA in SiHa cell line,which can not happened in normal HuVEC cell line.
