Leptin对HUVECs的氧化应激和增殖作用

目的观察Leptin对脐静脉内皮细胞（HuvEcs）的氧化应激作用及增殖的影响及其有关机制的探讨。方法体外胶原酶法消化分离培养HUVECs进行免疫荧光鉴定;观察Leptin对HUVECs的活性氧簇（ROS）水平的变化的影响;运用MTT比色法测定leptin及抑制剂对HU—VECs增殖的影响,并分别测定增殖率及抑制率。结果胶原酶消化法分离培养的内皮细胞经纯度高;Leptin能显著增加HUVECs内活性氧簇的产量。（与对照组比较,P〈0．01）,上述变化可以被NADPH氧化酶抑制剂-二苯基碘（DPI）和PKC抑制剂（PKCinhibitor）所抑制;Leptin能显著促进HUVECs增殖,与对照组比较,增殖率达36％,运用DPI和PKC抑制剂可以明显抑制leptin对HUVECs的增殖作用．DPI的抑制率达37％,PKC抑制剂的抑制率达43％,（与对照组比较,P均〈0．01）。结论体外消化分离培养的脐静脉内皮细胞纯度较高可以成功运用于实验;Leptin能提高HUVECs内ROS的产量,即leptin可以促进内皮细胞的氧化应激反应;Leptin可以通过PKC途径提高ROS水平促进HUVECs增殖。

Oxidative stress and proliferation effect of Leptin on HUVECs

OBJECT We observed the effect of leptin on the change of ROS production and the proliferation of HUVECs and investigated relevant mechanism. METHODS We used collagenase enzyme digestion for HUVECs isolating culture from human newborn＇s umbilical vein and factor Ⅷ antibody mediated immunity immunofluoreseence to identify cells; We tested the production of ROS in HUVECs after incubation with leptin; MTT colorimetry was used to measure the growth rate and inhibition ratio of HUVECs after incubation with leptin and different inhibitors. RESULTS More than 95 % of the cells cultured from eollagenase enzyme digestion were identified to be endothelial cells ; Leptin can increase ROS production markedly, （ vs control, P 〈 0.01）, which can be inhibited by PKC inhibitor and DPI; Leptin can dramatically increase HUVECs proliferation, 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 The cultured cells in vitro were pure enough to be used in experiments after identification; Leptin can promoted the ROS production, it meant that leptin could cause the oxidative stress in HUVECs; Leptin can promote the proliferation of HUVECs through PKC dependent way.