瘦素对人脑血管平滑肌细胞活性及ROS表达的影响

EFFECT OF LEPTIN ON THE ACTIVITY OF HUMAN BRAIN VASCULAR SMOOTH MUSCLE CELLS AND THE EXPRESSION OF REACTIVE OXYGEN SPECIES

目的探讨瘦素对人脑血管平滑肌细胞(HBVSMCs)活性及活性氧(ROS)表达水平的影响。方法体外培养HBVSMCs,使用不同浓度瘦素干预HBVSMCs,采用CCK-8法检测瘦素对细胞增殖活性的影响,蛋白免疫印迹(Western blot)检测细胞中增殖细胞核抗原(PCNA)和Ki-67的表达水平,流式细胞术检测瘦素对细胞周期的影响,DCFH-DA探针法检测细胞内ROS水平。结果CCK-8检测显示,不同浓度瘦素处理组培养48和72 h后HBVSMCs的增殖活性明显升高(F=117.818、160.349,P<0.001);Western blot检测显示,不同浓度瘦素处理组培养48 h后HBVSMCs中PCNA和Ki-67的表达水平明显上调(F=290.000、898.769,P<0.001);流式细胞术检测显示,不同浓度瘦素处理组G0/G1期细胞比例降低(F=264.180,P<0.001),S期和G2/M期细胞比例升高(F=160.033、380.234,P<0.001);DCFH-DA探针法检测显示,不同浓度瘦素处理组HBVSMCs内ROS水平明显升高(F=394.025,P<0.001);以上各指标变化均具有浓度依赖性。结论瘦素能够促进HBVSMCs的增殖活性,并诱导细胞中ROS的表达。

Objective To investigate the effect of leptin on the activity of human brain vascular smooth muscle cells(HBVSMCs)and the expression of reactive oxygen species(ROS).Methods HBVSMCs were cultured in vitro and then treated with different concentrations of leptin.CCK-8 assay was used to observe the effect of leptin on the proliferative activity of cells;Western blot was used to measure the expression of proliferating cell nuclear antigen(PCNA)and Ki-67;flow cytometry was used to observe the effect of leptin on cell cycle;the DCFH-DA probe method was used to measure the level of ROS in cells.ResultsThe results of CCK-8 assay showed that HBVSMCs treated with different concentrations of leptin had a significant increase in proliferative activity after 48 and 72 hours of culture(F=117.818,160.349;P<0.001).The results of Western blot showed that HBVSMCs treated with different concentrations of leptin had significantly upregulated expression of PCNA and Ki-67 after 48 hours of culture(F=290.000,898.769;P<0.001).The results of flow cytometry showed that HBVSMCs treated with different concentrations of leptin had a significant reduction in the proportion of cells in G0/G1 phase(F=264.180,P<0.001)and a significant increase in the proportion of cells in S phase or G2/M phase(F=160.033,380.234;P<0.001).The results of the DCFH-DA probe method showed that HBVSMCs treated with different concentrations of leptin had a significant increase in the level of ROS(F=394.025,P<0.001).All the changes above were concentration-dependent.Conclusion Leptin can promote the proliferative activity of HBVSMCs and induce the expression of ROS in cells.