The Oxidasive Stress And Proliferlation Effect Of Leptin And Its Mechanism In HUVECs

Part one The oxidative stress and the proliferlation effect of leptin in HUVECs Object: We observed the effect of leptin on the change of ROS production and the proliferlation of HUVECs.Method: (1 )We used collagenase enzyme digestion for HUVECs isolating culture from human newborn's umbilical vein and factor VI antibody mediated immunity immunofluorescence to identify cells; (3) We tested the production of ROS in HUVECs after incubation with leptin; (4) MTT chromometry was used to measure the growth rate and inhibition ratio of HUVECs after incubation with leptin and different inhibitors.Result: (1) More than 95% of the cells cultured from collagenase enzyme digestion were identified to be endothelial cells ; (2) Leptin can increase ROS production markedly, (VS control, P <0.01) , which can be inhibited by PKC inhibitor and DPI; (3) Leptin can dramatically increase HUVECs proliferlation, and the growth rate was about 36% compared with control. While such effect can be inhibited by PKC inhibitor and DPI too, the inhibition ratios were 37%,43% respectively (VS leptin, P<0.01).Conclusion: (1) The cultured cells in vitro were pure enough to be used in experiments after identification ; (2 ) Leptin can promoted the ROS production, it meant that leptin could cause the oxidative stress in HUVECs ; (3) Leptin can promote the proliferlation of HUVECs through PKC dependent way.Part Two The Mechanism of Leptin-induced Oxidasive Stress in HUVECsObject: We observed whether the expression of p-PKC and NADPH oxidase key subuint NOX₂,NOX₄ changed and how p-PKC and NOX₂,NOX₄ interact each other during the oxidative stress caused by leptin in HUVECs. And then we explored the mechanism of leptin-induced oxidative stress in HUVECs.Method: Western blotting was used to tested the p-PKC level and RT-PCR was used to measured the mRNA expression of NOX₂,NOX₄ in HUVECs which were incubated with different concentrations of leptin and different inhibitors.Result: (1) Leptin can dramatically increase p-PKC expression (VS control, P <0.01) ; (2) Leptin can markedly increase NOX₂,NOX₄ mRNA expression (VS control, P <0.01) , which can be inhibited by PKC inhibitor.Conclusion: (1) Leptin can dramatically increase p-PKC expression ; (2) Leptin can upregulate of NADPH oxidase mRNA expression in HUVECs through PKC dependent way.