Wnt3a信号分子对大鼠骨髓间充质干细胞成骨分化过程中端粒酶活性的影响

发布时间：2018-05-12 08:21

本文选题：wnta + 骨髓间充质干细胞　；参考：《中国骨质疏松杂志》2014年01期

【摘要】：目的探索Wnt3a信号分子对体外培养的大鼠骨髓间充质干细胞成骨分化过程中端粒酶活性的影响。方法采用密度梯度离心联合贴壁筛选法分离培养SD大鼠股骨骨髓间充质干细胞。传代后通过形态学和流式细胞学检测细胞表面标志物CD29、CD44、CD45,筛选并鉴定细胞。采用成骨诱导培养基联合不同浓度的wnt3a(5 ng/mL、25 ng/mL、100 ng/mL)诱导大鼠骨髓间充质干细胞分化,以碱性磷酸酶活性检测评价其成骨分化能力。通过TRAP-ELISA端粒酶活性检测法检测在不同浓度的wnt3a干预下诱导骨髓间充质干细胞向成骨细胞分化过程中端粒酶活性的表达。结果骨髓间充质干细胞传至第3代后可获得均一性较高的细胞,流式细胞学检测细胞表面标志物CD29、CD44高表达,CD45低表达。成骨诱导培养基联合不同浓度的wnt3a诱导大鼠BMSCs 7 d后,碱性磷酸酶(Alkaline Phosphatase,ALP)活性检测结果:与普通成骨诱导培养基相比,含有5 ng/mL及25 ng/mL wnt3a的成骨诱导培养基可显著增加其碱性磷酸酶(ALP)活性(P0.05),而含有100 ng/mL wnt3a的成骨诱导培养基则明显抑制其碱性磷酸酶(ALP)活性(P0.05)。与普通成骨诱导培养基相比,联合诱导培养基诱导骨髓间充质干细胞成骨分化过程中,端粒酶活性的改变无明显的统计学差异。结论小剂量(5 ng/mL、25 ng/mL)的wnt3a分子能够促进体外培养的骨髓间充质干细胞成骨分化,而大剂量(100 ng/mL)的wnt3a分子则抑制其成骨分化。骨髓间充质干细胞具有较弱的端粒酶活性,成骨分化过程中其活性逐渐减弱,直至消失;wnt3a信号分子刺激并不能有效激活其端粒酶活性。
[Abstract]:Objective to investigate the effect of Wnt3a signaling molecules on telomerase activity during osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro. Methods the bone marrow mesenchymal stem cells of SD rats were isolated and cultured by density gradient centrifugation and adherent screening. After passage, the cell surface markers CD29, CD44 and CD45 were detected by morphology and flow cytology to screen and identify the cells. Bone marrow mesenchymal stem cells (BMSCs) were induced by osteoblast induction medium combined with different concentrations of wnt3a(5 ng / mL1 25 ng / mL1. The osteogenic differentiation ability was evaluated by alkaline phosphatase activity test (ALP activity) in rat bone marrow mesenchymal stem cells (BMSCs). Telomerase activity of bone marrow mesenchymal stem cells (BMSCs) induced by different concentrations of wnt3a was detected by TRAP-ELISA telomerase activity assay during the differentiation of bone marrow mesenchymal stem cells into osteoblasts. Results the bone marrow mesenchymal stem cells (BMSCs) could obtain high homogeneity after the third passage. The high expression of CD29 + CD44 and the low expression of CD45 were detected by flow cytometry. The activity of alkaline phosphatase Alkaline phosphatase (ALP) was detected after 7 days of BMSCs induced by osteogenic medium combined with different concentrations of wnt3a in rats. The results showed that the activity of ALP was higher than that of normal osteoblast induction medium. The osteogenic medium containing 5 ng/mL and 25 ng/mL wnt3a could significantly increase the activity of alkaline phosphatase (ALP), while the osteogenic medium containing 100 ng/mL wnt3a significantly inhibited the activity of alkaline phosphatase (ALP). There was no significant difference in telomerase activity during osteogenic differentiation of bone marrow mesenchymal stem cells induced by combined culture medium. Conclusion low dose of wnt3a (5 ng / mL) can promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro, while high dose of wnt3a (100 ng / mL) can inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cells had weak telomerase activity, which decreased gradually during osteogenic differentiation, until the signal molecular stimulation of WNT3a could not effectively activate the telomerase activity of bone marrow mesenchymal stem cells.
【作者单位】：解放军第309医院全军骨科中心骨内科;
【分类号】：R329

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