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目的:探讨磷离子对人骨髓来源的间充质干细胞(Human bone marrow mesenchymal stem cells,BM-h MSCs)增殖及成骨分化的影响。方法:从Scien Cell实验室购买的BM-hMSCs分别在无血清生长培养基(对照组)和添加4mmol(4P组)、8mmol(8P组)磷离子的无血清生长培养基中培养21天,通过CCK8比色法评估细胞的增殖情况;RT-PCR检测成骨分化标记性基因胶原蛋白Ⅰ、骨钙素、碱性磷酸酶的表达水平;茜素红染色检测BM-hMSCs成骨分化产生的矿化结节。结果:在培养4、7、14天时,4P和8P组中BM-hMSCs的增殖都明显高于对照组,且8P组中BM-h MSCs的增殖高于4P组;培养21天时,4P和8P组中BM-hMSCs的增殖明显低于对照组。与对照组相比,在培养7天时,4P和8P组OC的表达下调,而14、21天时,OC的表达上调。4P和8P组中ALP的表达水平与对照组无明显差异。7天时,4P、8P组ColⅠ的表达水平均明显高与对照组;14天时,8P组ColⅠ的表达水平与对照组表达无明显差异;21天时,4P、8P组中ColⅠ的表达水平都均与对照组表达无明显差异。培养21天时,4P、8P组矿化结节的形成明显增加。结论:磷离子在早期可以促进BM-hMSCs的增殖,晚期诱导其成骨分化。
Objective: To investigate the effect of phosphorus ion on proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (BM-h MSCs). METHODS: BM-hMSCs purchased from Scien Cell Laboratories were cultured for 21 days in serum-free growth medium (control group) and serum-free growth medium supplemented with 4 mmol (4P group) and 8 mmol (8P group) The proliferation of BM-hMSCs was evaluated by CCK8 colorimetric assay. The expression of osteogenic differentiation marker gene collagen Ⅰ, osteocalcin and alkaline phosphatase was detected by RT-PCR. Alizarin red staining was used to detect the mineralization of BM-hMSCs Nodules. RESULTS: At 4, 7 and 14 days, the proliferation of BM-hMSCs in 4P and 8P groups was significantly higher than that in control group. The proliferation of BM-h MSCs in 8P group was higher than that in 4P group. When cultured for 21 days, 4P and 8P The proliferation of BM-hMSCs in the group was significantly lower than that in the control group. Compared with the control group, the OC expression in 4P and 8P groups was down-regulated at 7 days of culture, while the OC expression was up-regulated on 14 and 21 days. The expression of ALP in 4P and 8P groups had no significant difference with that in control group. At 7 days, the expression of ColⅠ in 4P and 8P groups was significantly higher than that in control group. On day 14, the expression level of ColⅠ in 8P group was not significantly different from that in control group. On day 21, the expression levels of ColⅠ in 4P and 8P group were The control group showed no significant difference. When cultured for 21 days, the formation of mineralized nodules in 4P and 8P groups increased significantly. Conclusion: Phosphorus can promote the proliferation of BM-hMSCs in early stage and induce osteogenic differentiation in late stage.