阻断钙调磷酸酶／激活T细胞核因子通路对聚甲基丙烯酸甲酯颗粒抑制骨祖细胞向成骨细胞分化的影响

Effect of blockade of calcineurin/nuclear factor of activated T cells paths by VIVIT peptide on inhibition of osteoprogenitor differentiation by polymethylmethacrylate particles

目的探讨VIVIT肽阻断钙调磷酸酶（Cn）／激活T细胞核因子（NFAT）信号通路对聚甲基丙烯酸甲酯（PMMA）颗粒抑制骨祖细胞向成骨细胞分化的影响。方法体外分离培养Sprague-Drawlev大鼠胎鼠颅骨原代细胞（包含大量骨祖细胞），根据处理条件不同分为4组：对照组、PMMA组、PMMA／VIVIT组和VIVIT组。细胞培养2、4、7和14d用MTT法检测细胞增殖情况，7d和14d用碱性磷酸酶（ALP）定量反映细胞分化；细胞培养14d后茜素红染色观察细胞矿化，RT-PCR法观察ALP、骨钙素、Ⅰ型胶原、Fra-2（与成骨细胞分化有关的转录因子）、NFATc1的基因表达，Western Blot法检测细胞核和细胞质中NFATc1蛋白的表达。结果PMMA组较对照组NFATc1基因和蛋白表达增加，并伴有NFATc1蛋白转位入核明显增加，成骨细胞分化、矿化和相关基因表达明显降低。PMMA／VIVIT组较PMMA组细胞分化、矿化和相关基因表达增加，但低于VIVIT组，NFATc1基因表达降低，转位入核的NFATc1蛋白明显减少。各组细胞增殖差异均无统计学意义（P〉0．05）。结论PMMA颗粒抑制骨祖细胞向成骨细胞分化与Cn／NFAT信号通路激活有关，VIVIT肽阻断Cn／NFAT信号通路可促进PMMA颗粒抑制的骨祖细胞向成骨细胞分化。

Objective To investigate the effect of blockade of calcineurin/nuclear factor of activated T cells （Cn/NFAT） paths by Valine Isoleueine Valine Isoleueine Threonine （VIVIT） peptide on the inhibition of osteoprogenitor differentiation by polymethylmethacrylate （PMMA） particles. Methods The rat calvaria （RC） cells were isolated and cultured in vitro in four groups： 1 ） supplemented with neither PMMA particles nor 11R-VIVIT peptide （control）; 2） supplemented with PMMA particles only （PMMA）; 3） supplemented with both PMMA particles and 11R-VIVIT peptide （PMMA/VIVIT）; and 4） supplemented with 11R-VIVIT peptide only （VIVIT） . 3-（4, 5-dimethylthiazol-2-yl） -2, 5-diphenyl tetrazolium bromide （MTT） test was conducted at 2, 4, 7, 14 days to assess the proliferation ofosteoblasts. The alkaline phosphatase （ALP） activity was measured at days 7 and 14 and alizarin red staining was performed at day 14 to assess the differentiation and mineralization of RC cells respectively. The mRNA expressions of ALP osteocalcin （OC）, collagen Ⅰ （Col Ⅰ）, los related antigen-2 （Fra-2） and NFATc1 extracted from the cells were detected by RT-PCR. The protein expression of NFATc 1 in cytosol and nucleus were detected by Western Blot. Results Compared with the control group, the gene and protein expressions of NFATcl increased while the ALP activity, mineralization and the gene expressions of ALP, OC, Col Ⅰ, Fra-2 decreased significantly in the PMMA group. Compared with the PMMA group, the ALP activity, mineralization and the expressions of osteogenesis related gene increased while the protein expression of NFATcl in nucleus decreased in PMMA/VIVIT group. However, the cell proliferation showed no significant difference between groups. Conclusions Cn/NFAT paths play a critical role in the inhibition of osteoprogenitor differentiation by PMMA particles. Blockade of Cn/NFAT paths by VIVIT peptide may increase osteoprogenitor differentiation and mineralization inhibited by the PMMA particles.