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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 p1 50亚型（ ADAR1-p1 50）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， qRT-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 mRNA 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 mRNA 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 GV1 66-control， qRT-PCR 及 Western-Blot 检测显示病毒转染 GV1 66- ADAR1 -p1 50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-p1 50组中脂滴的荧光强度明显较 L-02 control 组低。结论：成功构建 ADAR1-p1 50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。

英文摘要:

Objective:To investigate the regulatory effect of RNA-specific adenosine deaminase 1 p150 isoform (ADAR1 -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. ADAR1 -p150 expression was analyzed by qRT-PCR and Western-Blot in both control group and oleic acid (OA) group. ADAR1-p1 50 overexpression lentiviral vector GV166-ADAR1 -p150 and control lentiviral vector GV166-control were constructed to infect L-02 cell line, the mRNA and protein expression levels of ADAR1 -p150 were detected by qRT-PCR and Western-Blot. The lipid droplets formation of L-02 ADAR1 -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 ADAR1-p1 50 were down-regulated in OA group as compared with that in control group. ADAR1-p1 50 overexpression lentiviral vector GV1 66- ADAR1 -p150 and empty vector virus GV166-control were successfully constructed. qRT-PCR and Western-Blot analysis showed that ADAR1 -p150 expression significantly increased in GV166- ADAR1 -p150 treated cells. Oil Red O staining and BODIPY staining revealed that the number of lipid droplets in L-02 ADAR1 -p150 cells was less than that in L-02 control cells. High content screening system demonstrated that the lipid droplets fluorescence intensity of L-02 ADAR1-p1 50 cells was significantly lower than that of L-02 control cells.Conclusion:Stable ADAR1 -p150 overexpressing cell line was successfully established. More importantly, high expression of ADAR1-p1 50 was capable of inhibiting fat synthesis in L-02 cells.

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