Peng Dong-xian, He Yuan-li, Qiu Li-wen
Department of Gynecology and Obstetrics, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 Apr;30(4):859-62.
To construct a lentiviral expression vector for short hairpin RNA (shRNA) of human survivin gene, and assess its gene silencing effect in human ectopic endometrial cells.
Human survivin gene shRNA sequence was designed using a software available on-line. The synthesized shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin shRNA, which was confirmed by PCR and DNA sequence analysis. The packaging 293T cells were cotransfected with LV-survivin shRNA, pHelper 1.0 and pHelper 2.0, and the titer of the lentivirus was determined. The recombinant lentivirus was injected into human ectopic endometrial cells and the survivin mRNA expression was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in comparison with that in the non-transfected and blank vector-transfected human ectopic endometrial cells.
PCR analysis and DNA sequencing confirmed correct insertion of the shRNA sequence into the lentiviral vector. The titer of virus after packaging was 8x10(8) U/ml. Survivin mRNA expression in human ectopic endometrial cells transfected by LV-survivin shRNA was significantly inhibited compared with those in the non-transfected and empty vector transfected human ectopic endometrial cells (P<0.01), and no significant difference was found between the latter two groups.
The lentiviral shRNA vector of survivin gene constructed can effectively inhibit the expression of survivin gene in human ectopic endometrial cells in vitro. This vector provides a tool for investigating the role of survivin gene in the occurrence and progression of endometriosis and for searching new therapeutic targets.
构建人survivin基因短发夹RNA（shRNA）慢病毒表达载体，并评估其对人异位子宫内膜细胞的基因沉默效果。
利用在线软件设计人survivin基因shRNA序列。将合成的shRNA序列克隆到pGCL-GFP载体中构建LV-survivin shRNA，通过PCR和DNA序列分析进行鉴定。将包装293T细胞与LV-survivin shRNA、pHelper 1.0和pHelper 2.0共转染，测定慢病毒滴度。将重组慢病毒注入人异位子宫内膜细胞，采用半定量逆转录-聚合酶链反应（RT-PCR）检测survivin mRNA表达，并与未转染及空载体转染的人异位子宫内膜细胞进行比较。
PCR分析和DNA测序证实shRNA序列正确插入慢病毒载体。包装后的病毒滴度为8×10⁸ U/ml。与未转染及空载体转染的人异位子宫内膜细胞相比，LV-survivin shRNA转染的人异位子宫内膜细胞中survivin mRNA表达明显受到抑制（P<0.01），而后两组之间无显著差异。
构建的survivin基因慢病毒shRNA载体可有效抑制人异位子宫内膜细胞中survivin基因的表达。该载体为研究survivin基因在子宫内膜异位症发生发展中的作用及寻找新的治疗靶点提供了工具。
