RNA特异腺苷脱氨酶1(p150亚型)抑制肝细胞脂质合成  

RNA-specific Adenosine Deaminase 1 p150 Isoform Inhibits Lipid Synthesis in Hepatocytes

作　　者：丁美玲[1] 冯文强[1] 张松[1] 曹海超 聂勇战[1] 

机构地区：[1]第四军医大学西京消化病医院肿瘤生物学国家重点实验室,陕西西安710032

出　　处：《现代生物医学进展》2015年第5期829-833,共5页Progress in Modern Biomedicine

基　　金：科技部重大国际合作项目(81110200)

摘　　要：目的:在油酸诱导的肝细胞脂肪变模型中,检测RNA特异腺苷脱氨酶1 p150亚型(ADAR1-p150)高表达细胞系中脂肪合成的变化。方法:利用本课题组前期摸索的油酸刺激人胚胎肝细胞L-02细胞系脂肪变的条件,q RT-PCR和Western-Blot检测油酸刺激组和对照组ADAR1-p150表达变化;将构建成功的ADAR1-p150过表达慢病毒载体GV166-ADAR1-p150及空载体病毒GV166-control感染L-02细胞,检测感染细胞中ADAR1-p150的m RNA和蛋白表达水平;通过油红O染色和BODIPY染色观察L-02 ADAR1-p150和L-02 control细胞中脂滴形成,并进一步利用高内涵系统检测其荧光强度,对脂滴合成作定量分析。结果:L-02细胞在油酸刺激后ADAR1-p150的m RNA和蛋白水平降低;成功构建ADAR1-p150过表达慢病毒载体GV166-ADAR1-p150及空载体病毒GV166-control,q RT-PCR及Western-Blot检测显示病毒转染GV166-ADAR1-p150后ADAR1-p150在细胞中的表达水平显著升高;油红O染色和BODIPY染色发现L-02 ADAR1-p150较L-02 control细胞胞质中脂滴数量减少。高内涵筛选系统检测提示L-02 ADAR1-p150组中脂滴的荧光强度明显较L-02 control组低。结论:成功构建ADAR1-p150过表达稳定转染L-02细胞系,并证实高表达ADAR1-p150能够抑制脂肪合成。Objective： To investigate the regulatory effect of RNA-specific adenosine deaminase 1 p 150 isoform （ADARI-p150） on fat synthesis in oleic acid-induced hepatic steatosis cell model. Methods： Oleic acid stimulated steatosis model in human embryonic liver cell line L-02 cells was constructed according to our previously reported protocols. ADARI-p150 expression was analyzed by qRT-PCR and Western-Blot in both control group and oleic acid （OA） group. ADARI-pl50 overexpression lentiviral vector GV166-ADARl-p150 and control lentiviral vector GV166-control were constructed to infect L-02 cell line, the mRNA and protein ex- pression levels of ADARI-p150 were detected by qRT-PCR and Western-Blot. The lipid droplets formation of L-02 ADARI-p150 and L-02 control cell lines was observed by oil red O staining and BODIPY staining. High Content Screening system was performed to detect the average fluorescence intensity of lipid droplets. Results： The mRNA and protein expression levels of ADARI-p150 were down-regu- lated in OA group as compared with that in control group. ADARI-p150 overexpression lentiviral vector GV166- ADARI-p150 and empty vector virus GV166-control were successfully constructed, qRT-PCR and Western-Blot analysis showed that ADARI-p150 ex- pression significantly increased in GV166- ADARI-p 150 treated cells. Oil Red O staining and BODIPY staining revealed that the num- ber of lipid droplets in L-02 ADARI-p 150 cells was less than that in L-02 control cells. High content screening system demonstrated that the lipid droplets fluorescence intensity of L-02 ADARI-p150 cells was significantly lower than that of L-02 control cells. Conclusions： Stable ADARI-p150 overexpressing cell line was successfully established. More importantly, high expression of ADARI-p 150 was ca- pable of inhibiting fat synthesis in L-02 cells.

关 键 词：ADAR1-p150 脂质合成 高内涵筛选系统 

分 类 号：Q78[生物学—分子生物学] R589[医药卫生—内分泌]