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目的:建立人脐静脉内皮细胞与人成骨细胞共育系,观察人脐静脉内皮细胞在共育系中对人成骨细胞功能的影响,进一步探讨人脐静脉内皮细胞对成骨细胞骨形成能力的影响。方法:采用二维共培养模型建立人内皮细胞/成骨细胞(1∶1)共育系,运用噻唑蓝比色法(MTT),分别在1d、3d、5d、7d时观察人脐静脉内皮细胞与成骨细胞单体培养以及共育系中的增殖情况;同时,运用蛋白免疫印迹方法(In cell western blot)分别测定人成骨细胞单体培养和共育系中骨保护素/破骨细胞分化因子配体(OPG/RANKL)蛋白的表达情况。采用方差分析比较不同状态下细胞增殖能力和蛋白表达的差异性。结果:从第3天开始,共育系中人成骨细胞的增殖能力明显优于成骨细胞单体培养;两者比较差异有统计学意义(P<0.05);于细胞培养第8天时分别测定人成骨细胞单体培养组和共育系中OPG/RANKL蛋白的表达,观察到共育系中(10.82±1.08)OPG蛋白表达量明显优于人成骨细胞单体培养组(6.65±0.43),两者比较差异有统计学意义(P<0.05);而共育系中(7.33±0.22)RANKL蛋白的表达含量与人成骨细胞单体培养(7.37±0.85)时表达无差异。结论:人脐静脉内皮细胞与人成骨细胞具有良好的相容性,两种细胞之间具有相互协同效应;人脐静脉内皮细胞可能通过促进OPG蛋白的表达来促进成骨细胞骨形成的作用。
OBJECTIVE: To establish human umbilical vein endothelial cells and human osteoblast co-culture system to observe the effect of human umbilical vein endothelial cells on human osteoblast function in a co-education system and further explore the effect of human umbilical vein endothelial cells on osteoblast formation The impact of ability. Methods: Human endothelial cell / osteoblast (1: 1) co-culture system was established by two-dimensional co-culture model. Thyrozolium colorimetry (MTT) Cells and osteoblast monolayer culture, and proliferation in co-culture. In the meantime, osteosarcoma cell proliferation and osteoclast differentiation in human osteoblasts were measured by using In cell western blot Cell differentiation factor ligand (OPG / RANKL) protein expression. Variance analysis was used to compare the differences in cell proliferation and protein expression under different conditions. Results: From day 3, the proliferation ability of human osteoblasts in co-culture group was significantly better than that in osteoblast monolayer culture. The difference was statistically significant (P <0.05). On day 8 of cell culture, The expression of OPG / RANKL in human osteoblast monolayer and co-culture lines was measured. The expression of OPG protein in co-cultured line was significantly higher than that in human osteoblast monolayer (6.65 ± 0.43). There was significant difference between the two groups (P <0.05). However, there was no difference in the expression of RANKL protein (7.33 ± 0.22) between the co-cultured and cultured human osteoblasts (7.37 ± 0.85). CONCLUSION: The human umbilical vein endothelial cells have good compatibility with human osteoblasts and the two cells have synergistic effects. Human umbilical vein endothelial cells may promote osteoblastic bone formation by promoting the expression of OPG protein .