骨保护素不同层面对内皮细胞的影响

Effect of osteoprotegerin on endothelial cells

目的:观察骨保护素(OPG)作为始动因素对人内皮细胞的数量变化及内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)和一氧化氮(NO)产生的影响。方法:常规培养永生化的人内皮细胞,以不同浓度(0、0.5、1.0、2.0、4.0、6.0、8.0、10.0及100.0 ng/ml,共9组)的OPG作用细胞48 h后,用细胞计数试剂盒-8(cell count kit,CCK-8)来检测细胞的数量及NO检测试剂盒检测NO的产量。用免疫细胞化学染色法检测eNOS蛋白表达的水平,实时定量PCR检测eNOS基因的定量表达。结果:与对照组的细胞数(0.759±0.067)相比,8.0 ng/ml OPG组的细胞数为(1.091±0.200),P<0.05,10.0 ng/ml OPG组的细胞数为(1.103±0.152)及100.0 ng/ml OPG组的细胞数为(1.231±0.192),均P<0.01。8.0 ng/ml OPG组NO生成量为(102.224±0.612)μmol/L(P<0.05),10.0 ng/ml OPG组NO生成量为(117.183±0.552)μmol/L和100.0 ng/ml OPG组NO生成量为(128.216±0.492)μmol/L(均P<0.01)。同时eNOS蛋白及eNOS mRNA的表达量在6.0 ng/ml、8.0 ng/ml、10.0 ng/ml及100.0 ng/ml OPG组均显著地呈浓度依赖性增加(均P<0.01)。结论:OPG具有促进内皮细胞增殖效应,可能在抗动脉粥样硬化过程中有一定意义,且与浓度梯度呈明显的正相关。

AIM：To observe the effects of osteoprotegerin （OPG） as an initiating factor on the changes in the number of endothelial cells and on nitric oxide synthase （eNOS） and nitric oxide （NO）. METHODS： Immortalized human endothelial cells were conventionally cultured and then treated with different concentrations of OPG （0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 100.0 ng/ml, total nine groups） for 48 h. Number of cells was detected with the cell count kit （CCK-8）. Nitric oxide production was detected with nitric oxide detection kit, eNOS protein expressions was examined with immunocytochemical method and the level of eNOS mRNA was assayed with realtime quantitative PCR. RESULTS： Compared with the number （0.759±0.067） in control group, the number of endothelial cells gradually increased with the increase of OPG within the physiological concentration range. The increased number of cells in 8.0 ng/ml group and higher concentration groups were statistically significant, （1.091±0.200） in 8.0 ng/ml group （P〈0.05）, （1.103±0.152） in 10.0 ng/ml group （P〈0.01） and （1.231±0.192） in 100.0 ng/ml group （P〈0.01）. NO increased synchronously with the proliferation of endothelial cells and also reached statistical significance in 8.0 ng/ml and higher groups （102.224±0.612） in 8.0 ng/ml group （P〈0.05）, （117.183 ± 0.552） in 10.0 ng/ml group （P〈0.01） and （128216±0.492） in 100.0 ng/ml group （P〈0.01）. eNOS protein and mRNA expression in 6.0 ng/ml, 8.0 ng/ml, 100 ng/ml and 1000 ng/ml groups increased significantly in a concentration dependent manner （P〈0.01）. CONCLUSION： OPG within the human physiological concentration range promotes endothelial cell proliferation. During the process of antiatherosclerosis, OPG may promote endothelial cell repair and the effect is positively correlated with the OPG concentration gradient.