去甲基化酶ALKBH5高、低表达食管鳞癌细胞株的构建

Construction of esophageal squamous cell carcinoma cell lines with high and low expression of demethylase ALKBH5

目的构建高、低表达去甲基化酶ALKBH5的食管鳞癌(ESCC)细胞株。方法将生长状态良好的人源胚胎肾细胞293T于培养瓶内培养,分别记为A、B、a、b、c组,待细胞融合80%～90%后进行质粒转染,A组293T细胞转染高表达ALKBH5的质粒p LV-ALKBH5,B组293T细胞转染对照质粒p LV-con,上述2个质粒中均带有GFP蛋白和Flag标签蛋白(用于后续感染细胞的筛选); a组293T细胞转染低表达ALKBH5的质粒p LKO-shALKBH5-1,b组293T细胞转染低表达ALKBH5的质粒p LKO-shALKBH5-2,c组293T细胞转染对照质粒p LKO-shSCR,上述3个质粒中均带有嘌呤霉素抗性(用于后续感染细胞的筛选)。上述各组293T细胞质粒转染72 h后,分别收集细胞上清液,离心后分装;用A、B组细胞上清液分别感染ESCC细胞株(Eca109细胞和Kyse510细胞),采用流式细胞术筛选GFP蛋白(+)的细胞待测;用a、b、c组细胞上清液分别感染ESCC细胞株(Eca109细胞和TE-13细胞),用嘌呤霉素筛选细胞待测。采用qRT-PCR法和Western blotting法分别检测感染高、低表达ALKBH5上清液的ESCC细胞中ALKBH5 mRNA和蛋白。结果感染A组细胞上清液的Eca109细胞、Kyse510细胞中ALKBH5 mRNA和蛋白的相对表达量均显著高于感染B组细胞上清液的Eca109细胞、Kyse510细胞(P均<0. 05),感染a、b组细胞上清液的Eca109细胞、TE-13细胞中ALKBH5 mRNA和蛋白的相对表达量均显著低于感染c组细胞上清液的Eca109细胞、TE-13细胞(P均<0. 05)。结论成功构建高、低表达ALKBH5的ESCC细胞株,为进一步探讨ALKBH5在ESCC发生发展中的作用提供了细胞模型。

Objective To construct the esophageal squamous carcinoma( ESCC) cell lines with high and low expression of demethylase ALKBH5,and to provide a cellular model for further investigation of the regulation of alk B homologue5( ALKBH5) in ESCC. Methods The 293 T cells in good growth inoculated in the culture bottle were divided into the groups A,B,a,b and c,which were transfected when the cell fusionis 80% to 90%. The 293 T cells in the group A were transfected with a plasmid of up-regulated ALKBH5( p LV-ALKBH5),and group B with a control plasmid( p LV-con).Both of the above plasmids contained GFP protein and Flag tagged protein,which were used for selecting the infected cells.The 293 T cells in the groups a and b were transfected with the plasmids with down-regulated ALKBH5( p LKO-shALKBH5-1 and p LKO-shALKBH5-2),respectively,while the 293 T cells in the group c were transfected with control plasmid( p LKO-shSCR),and all of the above three plasmids contained puromycin resistance,which were used for screening the infected cells. After transfecting the 293 T cells of the three groups,the cell supernatants were separately collected. ESCC cell lines( Eca109 cells and Kyse510 cells) were infected with cell supernatants of groups A and B,respectively,and cells with GFP protein(+) were selected by flow cytometry;ESCC cell lines( Eca109 cells and TE-13 cells) were infected with cell supernatants of groups a,b and c,respectively,and cells were selected with puromycin. Finally,the mRNA and protein expression levels of ALKBH5 were detected by qRT-PCR and Western blotting. Results The relative expression levels of ALKBH5 mRNA and protein in the Eca109 cells and Kyse510 cells infected with the supernatants of group A were significantly higher than those infected with the supernatants of group B( all P < 0. 05). The relative expression levels of ALKBH5 mRNA and protein in Eca109 cells and TE-13 cells infected with supernatants of groups a and b were significantly lower than those infected with supernatants of group c( all P < 0. 05). Conclusion ESCC cell lines with high and low expression of ALKBH5 are successfully established,which provide the cell models for further study of ALKBH5 in ESCC.