摘要
目的研究慢病毒携带的短发夹状干扰RNA(short hairp in RNA,shRNA)对宫颈癌Cask i细胞中人乳头瘤病毒(hum an pap illom a virus,HPV)16型E6表达的抑制作用。方法将靶向HPV16型E6的shRNA表达序列克隆到改建的慢病毒表达载体PLL3.7中,经限制性酶切鉴定和测序后,与3种包装质粒混合,以L ipofectam ine 2000包裹后转染293FT细胞,72 h后收取含病毒的上清液并感染宫颈癌Cask i细胞,感染48 h后加入G418筛选阳性克隆。每天对阳性细胞克隆进行细胞计数;提取细胞总mRNA,进行RT-PCR反应。结果与对照组相比HPV16型E6的表达降低70%,宫颈癌Cask i细胞生长速度减慢50%。结论慢病毒携带的短发夹状干扰RNA能干扰宫颈癌细胞中HPV16型E6的表达并有抑制癌细胞生长的作用。
Objective To investigate if lentivirus mediated short hairpin RNA （shRNA） could constantly inhibit HPV16 E6 expression in cervical cancer Caski cells. Methods Short hairpin RNA targeting to HPV16 E6 was cloned to lentivirus work vector （ lentilox RNAi vector PLL3.7 ）. The recombined vector was identified by restriction enzyme analysis and sequencing. Work vector and three package plasmids were cotransfected to 293FT cells with the help of lipofectamine2000. Lentivirus was collected after 72 h, and was added to cultured Caski cells. Caski cells were screened by G418 at 48 h after infection. Cells were counted every day and total mRNA was extracted and underwent RT-PCR. Results Expression level of HPV16 E6 in Caski cells decreased by 70% as compared with controls. Cells growth velocity was reduced to 50% of controls. Conclusion Short hairpin RNA mediated by lentivirus can inhibit expression of HPV16 E6 and growth velocity of cervical cancer cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第11期1218-1220,共3页
Journal of Third Military Medical University
基金
第三军医大学青年科研基金资助项目(2003)~~
关键词
宫颈癌
慢病毒
RNA干扰
人乳头瘤病毒
cervical cancer
lentivirus
RNA interfering
human papilloma virus HPV
