尼古丁对大鼠骨髓间充质干细胞软骨分化的干预作用研究

目的 从细胞水平证实尼古丁对大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)诱导分化为软骨细胞的干预作用.方法 大鼠BMSCs传代至第3代,以海藻酸钠微球生物支架进行BMSCs三维培养.加入转化生长因子β1(TGF-β1)进行诱导分化,在细胞分化过程中,分别给予0.1、1、10、100μmol/L尼古丁干预,设对照组.第28天收获细胞,采用四甲基偶氮唑蓝(MTT)法检测细胞代谢活力、阿利新蓝特殊染色检测细胞中氨基葡聚糖的含量、实时荧光定量PCR检测软骨分化标志基因(COL2A1、Aggrecan、COL1A1、COL10A1)的表达情况.结果 诱导培养28 d后,MTT法结果示不同浓度尼古丁干预后的各组细胞代谢活力与对照组细胞相比,没有明显改变;当尼古丁作用浓度分别为0、0.1、1、10和100μmol/L时,其BMSCs海藻酸钠微球的阳性染色区域占切片的百分比分别为90.1％、76.5％、64.8％、44.1％、4.5％;与对照组比较,尼古丁能显著抑制软骨诱导分化下的BMSCs细胞中Aggrecan、COL2A1的表达,同时促进COL1A1、COL10A1的表达(P均<0.05),并且存在浓度依赖性.结论 尼古丁能够明显抑制大鼠BMSCs的软骨分化,导致其软骨分化质量差.

Effect of nicotine on chondrogenic differentiation of rat bone mesenchymal stem cells

Objective To explore the effect of nicotine on the differentiation of rat bone mesenchymal stem cells (BMSCs) at the cellular level. Methods The rat BMSCs were first replicated under monolayer culture condition until the third generation, and then suspended in the aqueous sodium alginate beads with high density for 3D culture. BMSCs were treated with nicotine at concentrations of 0.1, 1, 10, 100μmol/L while TGF-β1 was added to induce differentiation. A control group was set up. After 28 days, the viability of the cells was detected by MTT assay. The algineta beads sections were stained for glycosaminoglycan with alcian blue, and the expression levels of chondrogenesis related genes, including COL2A1, Aggrecan, COL1A1, COL10A1 were detected by q-PCR. Results Nicotine did not affect viability of BMSCs at any indicated concentration. After BMSCs treated with nicotine at different concentrations (0, 0.1, 1, 10, 100μmol/L) for 4 weeks, the proportion of the area stained with alcian blue in the section was 90.1％, 76.5％, 64.8％, 44.1％ and 4.5％ respectively. Nicotine significantly decreased the expression of COL2A1 and Aggrecan, and significantly increased the expression of COL1A1 and COL10A1 in a concentration-dependent manner during chondrogenic differentiation of rat BMSCs on day 28 as compared with control group (P<0.05 for all). Conclusion These results suggest that nicotine suppresses chondrogenic potential of rat BMSCs, leading to failure of chondrogenic differentiation in rat BMSCs.