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 （short hairpin 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 将含有人胎盘生长因子（placental growth factor，PLGF）干扰序列的76 nt的寡核苷酸序列连入含有绿色荧光蛋白基因（GFP）的腺病毒穿梭质粒pRNAT-H1.1/Adeno中，获得重组穿梭质粒。重组穿梭质粒经限制性内切酶PmeⅠ线性化、去磷酸化回收后与腺病毒骨架pAdeasy-1共电转入BJ5183感受态细胞。同源重组后经限制性内切酶PacⅠ酶切鉴定，挑选阳性克隆并大量获得重组质粒。PacⅠ酶切质粒后转染HEK 293细胞，经过三轮病毒包装，收获病毒颗粒，经OD260方法和TCID50方法测定病毒滴度。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 NCI-H 250和NCI-H 209细胞在感染腺病毒颗粒48 h后，大部分细胞表达GFP，感染率接近100%；两株细胞感染腺病毒48 h后，PLGF mRNA表达水平明显下降，且靶基因沉默的肿瘤细胞侵袭能力明显下降。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。

Adenovirus expression vector mediated gene silencing in suspension cells

Objective Using recombinant adenovirus to make gene silencing in suspension cell.Methods Placental growth factor(PLGF) silencing target sequence and scramble sequence were inserted into shuttle plasmid pRNAT-H1.1/Adeno 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 adenoviruses 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 PCR and cancer cells’ invation function was analyzed by Transwell cancer cell migrate test.Results Three kinds of recombinant adenovirus vectors(AD-shuttle-shPLGF,AD-shuttle-scramble and AD-shuttle) were successfully constructed.The titer of these recombinant adenovirus particles was 1.5×1011,1.6×1011 and 1.6×1011 respectively.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-mediated method.