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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 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 detect the changes in adipogenesis in an ADAR1-p150-overexpressing cell line induced by oleic acid in hepatocyte steatosis model. Methods: By using the oleic acid stimulated steatosis of L-02 cell line of human embryonic liver cells, the expression of ADAR1-p150 in oleic acid stimulation group and control group were detected by q RT-PCR and Western-Blot. The successful ADAR1-p150 overexpression lentiviral vector GV166-ADAR1-p150 and the empty vector GV166-control infection of L-02 cells, detection of infected cells ADAR1-p150 m RNA and protein expression levels; by oil red O staining and BODIPY The lipid droplets in L-02 ADAR1-p150 and L-02 control cells were stained and their fluorescence intensities were further detected by a high-content system. The lipid droplets were quantitatively analyzed. Results: The mRNA and protein levels of ADAR1-p150 in L-02 cells decreased after oleic acid stimulation. The ADAR1-p150 overexpression lentiviral vector GV166-ADAR1-p150 and empty vector GV166-control, q RT-PCR and The results of Western-Blot showed that the expression of ADAR1-p150 in GV166-ADAR1-p150 cells was significantly increased. The results of oil red O staining and BODIPY staining showed that the expression of ADAR1-p150 in L-02 control cells Reduce the number of lipid droplets. The high content screening system showed that the fluorescence intensity of lipid droplets in L-02 ADAR1-p150 group was significantly lower than that in L-02 control group. Conclusion: ADAR1-p150 overexpression was successfully constructed and transfected into L-02 cell line, and confirmed that ADAR1-p150 overexpression can inhibit lipogenesis.