干扰ALDH1A1表达的shRNA载体成功转染宫颈癌HeLa细胞及意义

The Success and Significance of shRNA of ALDH1A1 Gene Transfection to HeLa Cell Lines

目的:采用shRNA转染构建干扰乙醛脱氢酶-1(ALDH1A1)表达的宫颈癌HeLa细胞,以进一步探讨ALDH1A1与宫颈癌细胞的生物学特性之间的关系。方法:体外培养宫颈癌HeLa细胞,加入梯度浓度G418溶液进行筛选,确定最佳筛选浓度;取对数生长期细胞,采用Lipofectamine 2000脂质体将4种干扰重组质粒(ALDH1A1-1719、ALDH1A1-574、ALDH1A1-740、ALDH1A1-921,其中有一种具最佳干扰效果)及1种阴性对照质粒(shNC)转染宫颈癌HeLa细胞。5种重组质粒与Lipofectamine 2000脂质体的比例均为0.2μg:1μL,空白对照组中仅加入Lipofectamine2000脂质体。6 h后换液,24 h后荧光显微镜下观察荧光表达情况。结果:确定G418的最佳筛选浓度为400 mg/L;5种重组质粒均成功转染入HeLa细胞中。其中ALDH1A1-1719的转染效率为4.67%;ALDH1A1-574的转染效率为1.16%;ALDH1A1-740的转染效率为12.45%;ALDH1A1-921的转染效率为3.42%;shNC组的转染效率为1.02%。结论:干扰ALDH1A1表达的shRNA载体转染宫颈腺癌HeLa细胞成功可行且效果好。

Objective ：To construct the interfering effects of ALDH1A1 expression in HeLa cells with shRNA transfec- tion, and next to explore the relationship between ALDH1A1 and the biological characteristics of HeLa cells, nethods：HeLa cells line were cultured in vitro. To determine the best screening concentration with different concentration of G418 solution. ALDH1A1 interfering of shRNA （including ALDH1A1-1719,ALDH1A1-574,ALDH1A1-740,ALDH1A1-921 ,and one of them has the best effective） and negative control recombinant plasmids （shNC） were transfected into human cervical HeLa ceils line using Lipofectamine 2000 reagent. All of the ratio of shRNA to Lipo were 0.2 μg：l μL,and the blank control group was only plused with Lipo 2000. The medium was changed after 6 hours, and observing the results of transfecfion under fluorc-scent microscope after 24 hours. Results：The best screening concentration of G418 was 400 mg/L,and five kinds of recombinant plasmids were successfully transfected into HeLa ceils. And the transfection effective of ALDH1A1-1719,ALDH1A1-574, ALDH1A 1-740, ALDH1A1-921, shNC were 4.67%, 1.16%, 12.45%, 3.42%, 1.02%, respectively. Conclusions ：The shRNA of interfering ALDH 1A 1 expression transfection to HeLa cells was feasible and the effect was good.