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目的:观察外源给予H2O2对C2C12细胞中UII/UT系统表达的影响。方法:培养小鼠肌原细胞系C2C12细胞株,应用胎盘蓝染色和测定细胞培养液中LDH的含量检测H2O2对细胞损伤的影响,应用放射免疫的方法检测细胞培养液和裂解液中UII的含量,应用RT-PCR法测定C2C12中UT mRNA的表达,应用Western Blot检测不同浓度H2O2对肌原细胞系C2C12中UT蛋白表达的影响。结果:外源给予不同浓度的H2O2,C2C12细胞裂解液中UII的含量各组间无明显差异,但增加了细胞孵育液中UII的含量,10-4 M和10-3 M时分别增加了83.1%(p<0.01)和94.5%(P<0.01)。低浓度H2O2(0.5×10-6 M,10-6 M,10-5 M,10-4 M)刺激明显增加了UT m RNA的表达,而高浓度的H2O2(10-3M)刺激反而使UT mRNA的表达降低。高浓度的H2O2(10-6 M,10-5M,10-4 M,10-3M)刺激使UT蛋白表达分别增加了200.1%、255.6%、111.1%和100.1%(均p<0.05)。结论:H2O2作为T2DM发病因素之一的活性氧族,可能是T2DM时骨骼肌组织中UII/UT系统表达增加的一个因素。
Objective: To observe the effect of exogenous administration of H2O2 on the expression of UII / UT system in C2C12 cells. Methods: The mouse myoblast cell line C2C12 was cultured. The effect of H2O2 on cell injury was detected by placental staining and LDH assay in cell culture medium. Radioimmunoassay was used to detect the content of UII in cell culture medium and lysate The expression of UT mRNA in C2C12 cells was detected by RT-PCR. The effects of different concentrations of H2O2 on the expression of UT protein in C2C12 myoblasts were detected by Western Blot. RESULTS: Exogenous administration of H2O2 resulted in no significant difference in the contents of UII in lysates of C2C12 cells, but increased the content of UII in the cell incubation solution and increased by 83.1 at 10-4 M and 10-3 M, respectively % (p <0.01) and 94.5% (p <0.01). Stimulation with low concentrations of H2O2 (0.5 × 10-6 M, 10-6 M, 10-5 M, 10-4 M) significantly increased the expression of UT m RNA, whereas high concentrations of H2O2 (10-3 M) stimulated UT mRNA expression decreased. High concentrations of H2O2 (10-6 M, 10-5 M, 10-4 M, 10-3 M) increased UT protein expression by 200.1%, 255.6%, 111.1% and 100.1%, respectively (all p <0.05). CONCLUSION: H2O2, an active oxygen species that is one of the pathogenesis of T2DM, may be a factor of UII / UT system expression increase in skeletal muscle tissue during T2DM.