瘦素上调动脉内皮细胞环氧合酶-2及其产物的实验研究  

Experimental Studies of Leptin Up-regulate COX-2 and Its Downstream Products from Aortic Endothelial Cells

作　　者：陈岳林[1] 聂亚 方圆[1] 陈钟欣[1] 沈粤春[1] 

机构地区：[1]广州医科大学附属第一医院心内科,广东广州510120

出　　处：《现代生物医学进展》2016年第4期647-651,共5页Progress in Modern Biomedicine

基　　金：广东省自然科学基金项目(2015A030313467);广东省科技计划项目(2014A020212364);广州市科技计划项目(201510010181)

摘　　要：目的:研究瘦素对大鼠主动脉内皮细胞(RAECs)环氧化酶-2(COX-2)及其下游产物前列环素和血栓素A2表达的影响。方法:酶消化法分离RAECs;免疫荧光检测细胞Ⅷ因子的表达;实时荧光定量PCR检测不同浓度瘦素(10^(-10)M、10^(-9)M、10^(-8)M)和时间(36h、48 h)处理后对RAECs COX-2 mRNA表达的影响;Western blot检测各组COX-2蛋白的改变;酶联免疫吸附测定(ELISA)检测各组细胞上清液中6-酮前列腺素F1α(6-Keto PGF1α)和血栓素B2(TXB2)的水平。结果:酶消化法6天后见细胞呈长梭形或多边形,培养10天后细胞呈典型的"铺路石"样改变,免疫荧光检测发现Ⅷ因子在胞浆中特异性表达,内皮细胞纯度达90%。与对照组(PBS处理)相比较,瘦素处理后,各组大鼠主动脉内皮细胞COX-2 m RNA及蛋白表达量均有升高趋势,两者表达量均随浓度增加和处理时间延长而升高(P<0.05)。而6-Keto PGF1α水平升高及TXB2/6-Keto PGF1α比例降低仅见于相对高浓度瘦素(10-9、10-8M)处理后(P<0.05),但瘦素对RAECs TXB2表达无影响(P>0.05)。结论:瘦素增加RAECs炎性标志物COX-2及其下游舒张血管产物6-Keto PGF1α的表达,降低TXB2(收缩血管物质)/6-Keto PGF1α比例。COX-2可能在瘦素介导的高血压发病中起重要作用,但因其下游产物则有利于血管舒张,故选择性COX-2抑制剂可能会增加高血压风险。Objective： To study the effect of leptin on the expression levels of cyclooxygenase-2 （COX-2） and its downstream products prostacyclin and thromboxane A2 by rat aortic endothelial cells （RAECs）. Methods： The separation of RAECs was carried out by enzyme digestion method; Cytoplasm Ⅷ factor was tested by immunoflurescence; The expression of RAECs COX-2 mRNA treatmented with different concentrations（10-10 M,10-9M,10-8 M） of leptin at different time（36 h,48 h） was detected by quantificational real time polymerase chain reaction; COX-2 protein was measured by western blot; The levels of 6-Keto prostaglandin F1α （6-Keto PGF1α） and thromboxane B2（TXB2） from cell culture supemate were detected by enzyme linked irnmunosorbent assay（ELISA）. Results： Six days after followed by enzyme digestion, cells presented a shuttle or polygon shape. The cells cultured for 10days showed a typical＂paving stone＂ appearance. Cytoplasm Ⅷ factor specific expression was detected by immunofluorescence emanation, the purity of endothelial cells reached 90 %. Comparing with control group （PBS）, RAECs treated by leptin had tended to increase the expression levels of COX-2 mRNA and protein. Both of those rose with the increase of concentration and processing time （P〈0.05）. 6-Keto PGF1α increased and the proportion of TXB2/6-Keto PGF1α reduced only in relatively high concentrations of leptin （10-9 M, 10-8 M） （P〈0.05）, but leptin had no influence on TXB2 expression （P〉0.05）. Conclusions： The addition of leptin increased expression level of inflammatory marker COX-2 and its downstream vasorelaxation product 6-keto PGF1α expression, also decreased the ratio of TXB2 （vasoconstrictor）/6-Keto PGF1α in vitro. COX-2 may play an important role in the incidence of hypertension mediated by leptin. However, its downstream products are benefit to vasodilatation. Therefore, selective COX-2 inhibitors may increase the risk of hypertension.

关 键 词：瘦素 内皮细胞 环氧化酶-2 前列环素 血栓素 

分 类 号：R544.1[医药卫生—心血管疾病] R-33[医药卫生—内科学]