慢病毒载体构建稳定的maspin表达敲减食管癌细胞株

Construction of maspin stable knockdown esophageal carcinoma cell line using lentivirus vector

目的前期研究表明maspin在食管癌组织中表达下调。文中利用慢病毒载体介导短发夹RNA(short hairpin RNA,shRNA)感染食管癌细胞株,诱导maspin基因沉默,构建稳定的maspin表达敲减细胞株。方法合成maspin特异shRNA序列(shMas1和shMas2),构建shMas1和shMas2慢病毒表达载体;利用293FT细胞包装病毒后,感染食管癌细胞株HKESC-2;经嘌呤霉素筛选获得稳定感染细胞株。定量PCR和免疫印迹法检测干扰效果。结果成功构建shMaspin的慢病毒表达载体;与野生型HKESC-2细胞比较,获得的稳定maspin表达敲减细胞株shMas1/HK2和shMas2/HK2生长特性无明显变化,其maspin mRNA及蛋白质表达水平均明显降低,maspin mRNA敲减效率分别为37.8%及58.2%,maspin蛋白质表达的相对抑制率分别为49%和53%,差异有统计学意义(P<0.05)。结论成功构建了稳定的maspin表达敲减的食管癌细胞株shMas1/HK2和shMas2/HK2。

Objective Previous studies have shown that maspin expression is down-regulated in esophageal squamous cell carcinoma （ESCC）. The aim of this study is to construct maspin stable knockdown esophageal carcinoma cell line using lentivirus short hairpin RNA （shRNA） expression system in human esophageal carcinoma cells. Methods Two specific shRNA sequences （ shMasl and shMas2） targeting human maspin gene were cloned into lentiviral vector. The lentivirus particles were packaged using 293FT ceils and maspin specific shRNA was transmitted into HKESC-2 cells. Stable expressed cells were obtained by puromycin selection. Knock- down efficiency was evaluated by real time PCR and Western blotting. Results shMaspin lentivirus vector was successfully construc- ted. Growth characteristics had no difference between wild type HKESC-2 and maspin stable knockdown cell lines of shMasl/HK2 and shMas2/HK2. The maspin mRNA and protein in the two shMasl/HK2 and shMas2/HK2 cell lines were decreased （P 〈 0.05 ）. The relative value of maspin gene silencing were 37.8% and 58.2% and the inhibition rate of protein expression were 49% and 53% , re- spectively. Conclusion Two maspin stable knockdown ESCC cell lines （shMasl/HK2 and shMas2/HK2） were sucsessfully con-structed, and good foundation has been established for further in vitro and in vivo studies.