Wnt3a联合骨形态发生蛋白2对骨髓间充质干细胞增殖及成骨分化的影响
发布时间：2018-08-24 20:25
【摘要】：目的观察经典Wnt/β-catenin以及骨形态发生蛋白(BMP)信号通路对大鼠骨髓来源的间充质干细胞体外增殖以及向成骨方向分化的影响。 方法应用密度梯度离心联合贴壁筛选法分离培养骨髓间充质干细胞并分成四组:对照组,Wnt组,BMP组,Wnt3a和BMP-2联合组,在不同时间点用细胞计数试剂盒-8(CCK-8)法检测细胞增殖活性,碱性磷酸酶(ALP)活性定量测定、Von Kossa染色观察细胞向成骨分化情况和基质矿化程度。反转录聚合酶链式反应(RT-PCR)检测成骨特异性标志物表达。 结果培养第7d,CKK-8测吸光度值联合组为0.845,Wnt组为0.738,明显高于对照组0.409(P0.05)。培养第6d和第12d,ALP活性吸光度值联合组为63.8和144.3,BMP-2组为40.8和104.1,均明显高于对照组7.3和18.9(P0.05)。培养14d后,联合组骨钙素mRNA表达量高于对照组,而Runx2及Osterix表达量与其他组相比增高不显著。培养3w后,Von Kossa染色显示联合组钙结节数量及大小均高于对照组。 结论Wnt3a因子和骨形态发生蛋白2联合诱导能有效地促进骨髓间充质干细胞增殖及向成骨方向分化,两者具有协同作用。
[Abstract]:Objective to investigate the effects of classical Wnt/ 尾 -catenin and bone morphogenetic protein (BMP) signaling pathway on the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. Methods Bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation combined with adherent screening and were divided into four groups: control group, Wnt group, BMP group, Wnt3a group and BMP-2 group. Cell proliferation activity was detected by cell count kit 8 (CCK-8) at different time points. Alkaline phosphatase (ALP) activity was measured quantitatively by Von Kossa staining to observe the osteogenic differentiation and the degree of matrix mineralization. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of osteogenic specific markers. Results on the 7th day of culture, the absorbance value of CKK-8 in the combined group was 0.738, which was significantly higher than that in the control group (0.409) (P0.05). The absorbance values of ALP activity in the combined group were 40.8 and 104.1, which were significantly higher than those in the control group (7.3 and 18.9, P0.05). After 14 days of culture, the expression of osteocalcin mRNA in the combined group was higher than that in the control group, while the expression of Runx2 and Osterix in the combined group was not significantly higher than that in the other groups. After 3 weeks of culture, Von Kossa staining showed that the number and size of calcium nodules in the combined group were higher than those in the control group. Conclusion combined induction of Wnt3a factor and bone morphogenetic protein 2 can effectively promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2201944
[Abstract]:Objective to investigate the effects of classical Wnt/ 尾 -catenin and bone morphogenetic protein (BMP) signaling pathway on the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. Methods Bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation combined with adherent screening and were divided into four groups: control group, Wnt group, BMP group, Wnt3a group and BMP-2 group. Cell proliferation activity was detected by cell count kit 8 (CCK-8) at different time points. Alkaline phosphatase (ALP) activity was measured quantitatively by Von Kossa staining to observe the osteogenic differentiation and the degree of matrix mineralization. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of osteogenic specific markers. Results on the 7th day of culture, the absorbance value of CKK-8 in the combined group was 0.738, which was significantly higher than that in the control group (0.409) (P0.05). The absorbance values of ALP activity in the combined group were 40.8 and 104.1, which were significantly higher than those in the control group (7.3 and 18.9, P0.05). After 14 days of culture, the expression of osteocalcin mRNA in the combined group was higher than that in the control group, while the expression of Runx2 and Osterix in the combined group was not significantly higher than that in the other groups. After 3 weeks of culture, Von Kossa staining showed that the number and size of calcium nodules in the combined group were higher than those in the control group. Conclusion combined induction of Wnt3a factor and bone morphogenetic protein 2 can effectively promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 李慧武 ,戴\戎 ,汤亭亭 ,郁朝锋 ,严孟宁 ,李旭 ,楼觉人;人骨形态发生蛋白-2基因转染人脂肪源性基质细胞的异位成骨作用[J];中华实验外科杂志;2005年05期
，本文编号：2201944
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