山东医药Issue(7)：20-23,4.DOI:10.3969/j.issn.1002-266X.2016.07.007
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
摘要
Abstract
Objective To establish the bladder cancer cell subline stably expressing and silencing cell adhesion mol-ecule 1 (CADM1).Methods We cultured and collected the bladder cancer cell line T24.①Establishment of T24 over-expressing CADM1.T24 cells were divided into the groups 1, 2 and 3.The full length of CADM1 was amplified by real-time PCR and was connected with pHBLV-IRES -ZsGreen-PGK-puro lentiviral vector ( group 1 ) , a vector control group (group 2) and a blank group (group 3).Virus was packaged in 293T cells after enzyme digestion and the sequencing was correct, and viruses after being concentrated were used to infect T24 cells.②Establishment 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 pHBLV-U6-shRNA-ZsGreen-Puro lentiviral vector, respectively ( groups A, B and C), pHBLV-U6-ZsGreen-Puro (group D) and a blank group (group E).The connected vector was identified using double-enzyme digestion, virus was packaged in 293T 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 pro-tein 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 and 1.00 ±0.03, respectively.CADM1 mRNA and protein in the group A was decreased as compared with that of groups B, C, D and E (all P<0.05).Conclusion The stable bladder cancer cell lines T24 overexpressing or silencing CADM1 were successfully established.关键词
膀胱肿瘤/膀胱癌细胞株/细胞黏附分子1/基因沉默Key words
urinary bladder neoplasms/urinary bladder carcinoma cell line/cell adhesion molecule 1/gene silencing分类
医药卫生引用本文复制引用
刘莉,杨永姣,陈业刚,王尚任,刘晓强,陈少峰,孙光..过表达及沉默细胞黏附分子1基因膀胱癌细胞株的构建[J].山东医药,2016,(7):20-23,4.基金项目
天津市自然科学基金重点项目（12JCZDJC23700）。 （）
