磷离子对三维培养下人骨髓间充质干细胞的影响

Effect of phosphorus ions on human bone marrow mesenchymal stem cells under three-dimensional culture

背景:以往研究是关于含磷复合物对动物源性或人源性其他干细胞的影响,三维情况下磷离子对人骨髓间充质干细胞作用研究尚未见。目的:探讨磷离子对三维培养条件下人骨髓间充质干细胞的影响。方法:实验分为6组,将人骨髓间充质干细胞接种在三维聚苯乙烯培养支架上,细胞分别在无血清生长培养基和添加4,8 mmol/L磷离子的无血清生长培养基中培养21 d;在二维聚苯乙烯培养板上培养的细胞作为相应的对照组。CCK8法检测细胞增殖情况,RT-PCR检测成骨分化标记性基因的表达,茜素红染色检测人骨髓间充质干细胞成骨分化生成的矿化结节。结果与结论:培养4,7,14,21 d,相对于二维的细胞培养,磷离子的促细胞生长作用在三维聚苯乙烯培养支架上表现更明显(P<0.05)。相对与三维培养对照组,培养7,14 d时,胶原蛋白Ⅰ基因的表达在4,8 mmol/L磷离子三维培养组显著增加(P<0.05);培养14 d时,碱性磷酸酶基因的表达在4,8 mmol/L磷离子三维培养组显著增加(P<0.05);培养14,21 d时,骨钙素基因的表达在4,8 mmol/L磷离子三维培养组显著增加(P<0.05)。培养21 d时,在4,8 mmol/L磷离子三维培养组可生成矿化结节,在三维培养对照组无矿化结节生成。结果说明,磷离子可以促进人骨髓间充质干细胞的增殖和成骨分化;相对于二维的细胞培养,磷离子的促生长作用在三维聚苯乙烯培养支架上表现更明显。

BACKGROUND：Previous researches have focused on the effect of phosphorus compounds on stem cels from animals or from human. But there is no study on the effect of phosphorus ions on human bone marrow mesenchymal stem cels under three-dimensional culture. OBJECTIVE：To explore the effect of phosphorus ions on human bone marrow mesenchymal stem cels under three-dimensional culture. METHODS：There were six groups in the experiment. Human bone marrow mesenchymal stem cels were inoculated in three-dimensional polystyrene scaffolds and then subjected to serum-free growth medium （group 3-GM） or serum-free growth medium containing 4 mmol/L （group 3-4P）, 8 mmol/L （group 3-8P） phosphorus ions for 21 days, respectively. Cels cultured on the two-dimensional polystyrene scaffolds were used as control groups （groups 2-GM, 2-4P, 2-8P）. Celular proliferation was examined by cel counting kit-8; the mRNAexpressions of osteogenic marker genes were assessed by RT-PCR; the formation of mineralized nodules for the osteogenic differentiation of human bone marrow mesenchymal stem cels was examined by Alizarin red staining. RESULTS AND CONCLUSION：Compared with the two-dimensional culture, the growth of human bone marrow mesenchymal stem cels induced by phosphorus ions were more obvious in the three-dimensional polystyrenes scaffolds at days 4, 7, 14 and 21 （P 〈 0.05）. Compared with the group 3-GM, the mRNA expression of colagen type I in groups 3-4P and 3-8P increased more significantly at days 7 and 14 （P 〈 0.05）; the mRNA expression of alkaline phosphatase in groups 3-4P and 3-8P increased more significantly at day 14 （P 〈 0.05）; the mRNA expression of osteocalcin in groups 3-4P and 3-8P increased more significantly at days 14 and 21 （P 〈 0.05）. Mineralized nodules were formed in groups 3-4P and 3-8P but not in group 3-GM at day 21. So we concluded that phosphorus ions can promote proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cels in three-dimensional polystyrenes scaffolds. Compared with the two-dimensional cel culture, the promoting growth effect of phosphorus ions on human bone marrow mesenchymal stem cels in three dimensional polystyrenes scaffolds are more obvious.