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  

Adenovirus expression vector mediated gene silencing in suspension cells

作　　者：王淳[1] 方文刚[2] 李波[2] 

机构地区：[1]辽宁中医药大学基础医学院卫生生物教研室,辽宁沈阳110032 [2]中国医科大学基础医学院发育生物教研室卫生部细胞生物学重点实验室,辽宁沈阳110001

出　　处：《基础医学与临床》2012年第2期226-232,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(30600270)

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-shuttle-scramble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/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 adenovi- ruses were packaged and amplified in HEK 293 ceils. Human small cell lung cancer ceils 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 con- structed. The titer of these recombinant adenovirus particles was 1.5 x 1011 , 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 liposomemedia ted method.

关 键 词：RNA干扰 SHRNA 腺病毒载体 悬浮细胞 

分 类 号：R346[医药卫生—基础医学]