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

Effect of Wnt3a signaling molecules on the activity of telomerase during the osteogenic differentiation of bone marrow mesenchymal stem cells in rats

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(P<0.05),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(P<0.05)。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。

Objective To explore the effect of Wnt3a signaling molecules on the activity of telomerase during the osteogenic differentiation of rat bone marrow mesenchymal stem cells （ BMSCs） in vitro.Methods BMSCs of the femur of Sprague Dawley rats were isolated using the density gradient centrifugation combined with adherence screening method, and then cultured in vitro. After passaging, cell surface markers CD29, CD44, and CD45 were detected using morphology and flow cytometry.The cells were screened and identified.Cells were cultured with osteogenic inducing medium combined with different concentrations of Wnt3a （5 ng/mL, 25 ng/mL, and 100 ng/mL） , in order to induce the differentiation of BMSCs.the activity of ALP was detected to evaluate the capability of osteogenic differentiation of BMSCs.The activity of telomerase during the differentiation of BMSCs into osteoblasts with the intervention of different concentrations of Wnt3a was detected using telomeric repeat amplification protocol-enzyme-linked immunosorbent assay （TRAP-ELISA）.Results At the 3rdpassage of BMSCs, cells with high uniformity were obtained.The results of flow cytometry showed that the expression of cell surface markers CD29 and CD44 was high, while CD45 had low expression. After 7-day culture of BMSCs with osteogenic inducing medium combined with different concentrations of Wnt3a, the results of ALP activity detection revealed that, compared with the ordinary osteogenic inducing medium, the osteogenic inducing medium containing 5 ng/mL and 25 ng/mL Wnt3a could significantly increase ALP activity （P＆lt;0.05）.On the contrary, the medium＆amp;nbsp;containing 100 ng/mL Wnt3a could significantly inhibit ALP activity （P ＆lt;0.05）.Compared with that in ordinary osteogenic inducing medium, no significant difference of telomerase activity in the osteogenic inducing medium containing Wnt3a was observed.Conclusion Lower dose （5ng/mL and 25ng/mL） of Wnt3a molecules can promote the osteogenic differentiation of BMSCs, while higher dose （100 ng/mL） of Wnt3a molecules can inhibit the osteogenic differentiation.The activity of telomerase in BMSCs is weak, and it gradually weakens during the osteogenic differentiation till disappearing.Wnt3a signaling molecules cannot effectively stimulate the activity of telomerase in BMSCs.