过表达及沉默细胞黏附分子1基因膀胱癌细胞株的构建  

Establishment of bladder cancer cell line overexpressing and silencing cell adhesion molecule 1

作　　者：刘莉[1,2] 杨永姣 陈业刚[1,2] 王尚任 刘晓强[1,2] 陈少峰[1,2] 孙光[1,2] 

机构地区：[1]天津医科大学第二医院,天津300211 [2]天津市泌尿外科研究所

出　　处：《山东医药》2016年第7期20-23,共4页Shandong Medical Journal

基　　金：天津市自然科学基金重点项目(12JCZDJC23700)

摘　　要：目的构建过表达及沉默细胞黏附分子1(CADM1)基因的膀胱癌细胞株。方法培养并收集膀胱癌细胞株T24。1过表达CADM1基因T24细胞构建:将T24细胞分为1、2、3组,real-time PCR扩增CADM1基因全长,与慢病毒载体p HBLV-IRES-Zs Green-PGK-puro进行连接(1组),并设载体对照组(2组)和空白对照(3组);经酶切及测序鉴定正确后在293T细胞中包装病毒,病毒浓缩后感染T24细胞。2沉默CADM1基因T24细胞构建:将T24细胞分为A、B、C、D、E组,设计3条针对CADM1基因的干扰序列(siRNA1/2/3)分别与载体p HBLV-U6-shRNA-ZsGreen-Puro进行连接(A、B、C组),D组与p HBLV-U6-Zs Green-Puro进行连接,E组为空白对照;经双酶切、PCR及测序鉴定连接载体,在293T细胞中包装病毒,病毒浓缩后感染T24细胞。采用real-time PCR检测T24细胞中的CADM1 mRNA,Western blotting法检测CADM1蛋白。结果过表达和沉默CADM1基因的慢病毒载体及细胞株均正确构建。1、2、3组CADM1 mRNA相对表达量分别为4.84±0.02、1.12±0.03、1.00±0.00,CADM1蛋白相对表达量分别为4.53±0.03、1.05±0.04、1.00±0.01,1组CADM1 mRNA及蛋白相对表达量高于2、3组(P均<0.05)。A、B、C、D、E组CADM1 mRNA相对表达量分别为0.28±0.05、0.98±0.02、0.74±0.01、0.99±0.01、1.00±0.00,CADM1蛋白相对表达量分别为0.33±0.04、0.86±0.12、0.67±0.07、1.00±0.04、1.00±0.03,A组CADM1 mRNA及蛋白相对表达量低于B、C、D、E组(P均<0.05)。结论成功构建了过表达及沉默CADM1基因的膀胱癌细胞株T24。Objective To establish the bladder cancer cell subline stably expressing and silencing cell adhesion molecule 1（ CADM1）. Methods We cultured and collected the bladder cancer cell line T24. 1 Establishment of T24 overexpressing CADM1. T24 cells were divided into the groups 1,2 and 3. The full length of CADM1 was amplified by realtime PCR and was connected with p HBLV-IRES-Zs Green-PGK-puro lentiviral vector（ group 1）,a vector control group（ group 2） and a blank group（ group 3）. Virus was packaged in 293 T cells after enzyme digestion and the sequencing was correct,and viruses after being concentrated were used to infect T24 cells. 2 Establishment of T24 stably silencing CADM1. T24 cells were divided into the groups A,B,C,D and E. Three sequences（ siRNA1 /2 /3） interfering CADM1 gene were designed and were connected with p HBLV-U6-shRNA-Zs Green-Puro lentiviral vector,respectively（ groups A,B and C）,p HBLV-U6-Zs Green-Puro（ group D） and a blank group（ group E）. The connected vector was identified using double-enzyme digestion,virus was packaged in 293 T cells,and virus of enrichment was used to infect T24 cells. CADM1 mRNA and protein were assessed by real-time PCR and Western blotting. Results The recombinant lentiviral vectors and cell lines of expressing and silencing of CADM1 were correctly established. The relative expression levels of CADM1 mRNA in the groups 1,2,and 3 were 4. 84 ± 0. 02,1. 12 ± 0. 03,1. 00 ± 0. 00,and the relative expression levels of CADM1 protein in the groups 1,2,and 3 were 4. 53 ± 0. 03,1. 05 ± 0. 04 and 1. 00 ± 0. 01,respectively. CADM1 mRNA and protein in the group 1 was enhanced effectively as compared with that of the groups 2 and 3（ all P〈0. 05）. The relative expression levels of CADM1 mRNA in the groups A,B,C,D and E were 0. 28 ± 0. 05,0. 98 ± 0. 02,0. 74 ± 0. 01,0. 99 ± 0. 01,1. 00 ± 0. 00,and the relative expression levels of CADM1 protein in the groups A,B,C,D and E were 0. 33 ± 0. 04,0. 86 ± 0. 12,0. 67 ± 0. 07,1. 00 ± 0. 04

关 键 词：膀胱肿瘤 膀胱癌细胞株 细胞黏附分子1 基因沉默 

分 类 号：R737.14[医药卫生—肿瘤]