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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。
OBJECTIVE: To investigate the effects of osteoprotegerin (OPG) as a starting factor on the changes of the number of human endothelial cells and the production of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO). Methods: Immortalized human endothelial cells were cultured routinely. After treated with OPG at different concentrations (0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 100.0 ng / ml, Counting kit-8 (cell count kit, CCK-8) to detect the number of cells and NO detection kit detection of NO production. The level of eNOS protein was detected by immunocytochemical staining, and the quantitative expression of eNOS gene was detected by real-time quantitative PCR. Results: Compared with the control group (0.759 ± 0.067), the number of cells in OPG group was (1.091 ± 0.200), P <0.05 (P <0.05), and the number of cells in OPG group was (1.03 ± 0.152) ) And 100.0 ng / ml OPG group were (1.231 ± 0.192) P <0.01.8.0 ng / ml. The NO production in OPG group was (102.224 ± 0.612) μmol / ml, NO production in OPG group was (117.183 ± 0.552) μmol / L and NO production in OPG group was 100.0 ng / ml (128.216 ± 0.492) μmol / L (all P <0.01). Meanwhile, the expression of eNOS protein and eNOS mRNA were significantly increased in a concentration-dependent manner in 6.0 ng / ml, 8.0 ng / ml, 10.0 ng / ml and 100.0 ng / ml OPG groups (all P <0.01). CONCLUSION: OPG can promote the proliferation of endothelial cells and may have some significance in the process of anti-atherosclerosis, which is positively correlated with the concentration gradient.