利用Tet-Off表达系统构建鞘磷脂合酶可调控高表达的Huh7肝癌细胞

Constructing the regulative overexpression of sphingomyelin synthase in Huh7 hepatoma cells by Tet-Off expression system

目的利用Tet-Off表达系统构建鞘磷脂合酶(sphingomyelin synthase,SMS)可被Dox调控高表达的Huh7细胞,为进一步探讨SMS活性升高致动脉粥样硬化的机制打下基础(建立细胞模型)。方法首先将Tet-Off调控质粒转染Huh7细胞构建Huh7-tTA细胞,然后再将含外源性人SMS基因(hSMS)的PTREE2hyg质粒转染Huh7-tTA细胞,经分离纯化成功构建可调控表达hSMS的Huh7单克隆细胞(命名为SMS细胞,SMS1和SMS2)。为了便于检测外源性hSMS,利用FLAG标记hSMS(SMS-FLAG)。结果 SMS细胞表达SMS-FLAG受不同剂量Dox调控,SMS mRNA测定结果显示:0 ng/mL Dox剂量组较0.01 ng/mL和100 ng/mL Dox组均有显著升高(P<0.05和P<0.001);0.01 ng/mL Dox组较100 ng/mL Dox组也有显著升高(P<0.05)。SMS蛋白测定结果相似于SMS mRNA结果。SMS酶活性测定结果显示0 ng/mL Dox组显著高于100 ng/mL Dx组(P<0.001),而对照组(Huh7-tTA)1、00 ng/mL Dox组和野生型Huh7细胞组之间差异均无统计学意义(P>0.05)。结论利用Tet-Off体系成功构建SMS高表达可调控Huh7细胞,SMS细胞SMS高表达受Dox剂量调控。

Objective Tet-Off expression system is a new technology which not only express exogenous genes but also can control their expression level through doxycycline（Dox） treatment.In this study,we used this system and prepared Huh7 cells（a human hepatoma cell） which can express controllable sphingomyelin synthase（SMS）.These cells would be used for further investigation of the relationship between elevated SMS activity and proatherogenesis.Methods Firstly,we constructed Huh7-tTA cells by transfecting with a pTet-Off in Huh7 cells.Then Huh7-tTA cells were transfected with PTREE2hyg plasmid ligated with exogenous human SMS gene.We isolated two cell lines with adjustable SMS1 or SMS2 expression,named as SMS1 and SMS2 cells.In order to facilitate detection of exogenous hSMS,we added a FLAG tag at the C terminal of both SMS1 and SMS2.Results The present study showed that SMS mRNA expression in 0 ng/mL Dox group increased significantly compared to in 0.01 and 100 ng/mL Dox group（P0.05,P0.001,respectively）.The results of SMS protein expression assay were similar to SMS mRNA expression.The SMS activity in 0 ng/mL Dox group increased siginificantly compared to those in 100 ng/mL Dox group（P0.001）.There were not siginificant differences in the SMS activity between control group（Huh7-tPA）,100 ng/mL Dox group and wild type group（Huh7,P0.05）.Conclusions We successfully created two cell lines which overexpressed SMS1 or SMS2,respectively,by using the Tet-Off expression system.The SMS expression levels can be adjusted by Dox treatment.