硫酸钙对人骨髓基质干细胞向成骨细胞转化时基因表达的影响

目的 观察硫酸钙(CS)在人骨髓基质干细胞(BMSCs)向成骨细胞转化过程中对成骨基因表达的影响,探讨硫酸钙修复骨缺损可能的生物学机制.方法 制备硫酸钙浸提的成骨诱导液(实验组)与常规的成骨诱导液(对照组),分别加入人BMSCs培养瓶(各3例),使其向成骨细胞的诱导分化,注意观察细胞的分化和生长.培养至第7天时进行RNA的抽提、纯化和质量检测,并合成cDNA,采用成骨功能基因芯片分别检测实验组和对照组中各种成骨基因的变化.结果 实验组和对照组细胞均生长良好,缓慢增殖,但实验组向成骨细胞分化的趋势要明显较对照组好.成骨基因芯片共检测到89种基因,其中有23种基因表达改变显著(Fold change＞2,P＜0.05).表达上调超过2倍的基因包括:AMELY、BMP2、COL4A3、COMP、EGF、FLT1、IGF1、ITGA2、MMP10、MMP2、TGFB2、TGFBR1、VDR和VEGFA.表达下调超过2倍的有:COL2A1、COL15A1、COL1A1、COL1A2、COL5A1、CSF2、FGF1、ITGA3和MMP8.结论 硫酸钙促进了人BMSCs向成骨细胞转化的过程,这种作用与硫酸钙促进成骨基因表达上调、合成活性因子增加相关,说明硫酸钙具有潜在的骨诱导活性,可以作为良好的骨修复替代材料,促进细胞的骨修复能力.

Effect of calcium sulfate on gene expression in differentiation of human BMSCs into osteoblasts

Objective To investigate the effect of calcium sulfate (CS) on the osteogenic gene expression in the differentiation process from human bone marrow stromal cells (BMSCs) to osteoblasts, and explore its possible biological mechanism in repairing bone defects. Methods CS-dissolved (test group) and conventional (control group) osteoblast-induced fluids were added into human BMSCs culture flasks (3 in each team) respectively for induction and differentiation to osteoblasts, and the cells were observed under a microscope until the 7th day. RNA was extracted from the cultured cells, and purification and quality tests were performed. After cDNA was synthesized, osteogenesis polymerase chain reaction (PCR) microarrays were used to examine the differently expressed osteogenic genes between two groups. Results We found that cells in both groups grew well and proliferated gradually. However, the osteogenic differentiation potential in test group was significantly better than in control group. The microarrays detected 89 kinds of genes, including 23 which changed significantly in expression ( Fold change ＞ 2,P ＜ 0. 05 ). The genes up-regulated more than 2 times included AMELY, BMP2, COL4A3, COMP, EGF, FLT1, IGF1,ITGA2, MMP10, MMP2, TGFB2, TGFBR1, VDR and VEGFA, while the genes down-regulated more than 2 times included COL2A1, COL15A1, COL1A1, COL1A2, COL5A1, CSF2, FGF1, ITGA3 and MMP8. Conclusion CS promoted the transforming process from human BMSCs to osteoblasts, which may be related to the up-regulation of osteoblastic gene expression and the increased synthesis of active factors.This suggested that CS has potential osteoinductive activity and can improve the bone repairing capacity of cells when used as a kind of ideal substitute bone repairing material.