利用腺病毒载体介导的RNA干扰技术在悬浮细胞中获得基因沉默效应

摘要

目的 利用重组腺病毒载体携带短发夹RNA (shRNA),使难于转染的悬浮细胞达到较高的目的基因沉默效率.方法 分别将含有人胎盘生长因子(PLGF)的干扰序列及阴性对照序列(scramble)连入pRNAT-H1.1/Adeno载体,获得重组穿梭质粒；将线性化的重组穿梭质粒与腺病毒骨架重组；将重组子转染HEK 293细胞,经过3轮病毒包装,收获病毒颗粒并测定滴度.病毒颗粒感染悬浮细胞——人小细胞肺癌细胞(NCI-H 250和NCI-H 209),通过实时定量PCR方法鉴定靶基因沉默效应,并利用肿瘤细胞重建基质膜侵袭实验进行功能鉴定.结果 成功构建带有靶基因干扰序列及scramble、空载体的3种腺病毒重组子(AD-shuttle-shPLGF、AD-shuttlescramble和AD-shuttle).经过HEK 293细胞包装,最终获得3株病毒颗粒,病毒滴度分别为1.5×1011、1.6×1011和1.6×1011.NCI-H 250和NCI-H 209细胞感染腺病毒颗粒48 h后,大部分细胞表达GFP,感染率接近100％；且两株细胞感染AD-shuttle-shPLGF病毒48 h后,PLGF mRNA表达水平降低(P＜0.01),肿瘤细胞侵袭能力明显下降(P＜0.01).结论 携带shRNA的腺病毒表达载体可以代替电转、脂质体介导等方法,使难于转染细胞的目的基因达到有效沉默.%Objective Using recombinant adenovirus to make gene silencing in suspension cell. Methods Placen-tal growth factor (PLGF) silencing target sequence and scramble sequence were inserted into shuttle plasmid pR-NAT-H1. 1/Adenb respectively to gain recombinant shuttle vectors. Then the two recombinant shuttle vectors and empty vector were co-transformed with pAdeasy-1 through homologous recombination. These recombinant adenovi-ruses were packaged and amplified in HEK 293 cells. Human small cell lung cancer cells NCI-H 250 and NCI-H 209, were infected by recombinant adenovirus. And the expression of target gene was detected by real - time PCRand cancer cells' invation function was analyzed by Transwell cancer cell migrate test. Results Three kinds of re-combinant adenovirus vectors ( AD-shuttle-shPLGF, AD-shuttle-scramble and AD-shuttle) were successfully constructed. The titer of these recombinant adenovirus particles was 1. 5 x 10" , 1. 6 x 1011 and 1. 6 x 1011 respective-ly. Efficient and specific silencing of PLGF gene was found in NCI-H 250 and NCI-H 209 cells after adenovirus transfection 48 h(P<0.01), and a significant reduction of migration was observed in target gene silenced cancer cells ( P < 0. 01). Conclusions The target gene was efficiently silenced in suspension cells and the result provides a basis for further experiments on difficultly transfected cells by other methods like power switch or liposome-media-ted method.