Wnt3a蛋白调控大鼠骨髓间充质干细胞向成骨细胞分化的研究
发布时间：2019-03-23 19:32
【摘要】：目的:Wnt信号通路是近年发现的对于细胞生物学行为具有重要调节作用的一个新的研究热点。现有研究表明无论是经典的Wnt/β-catenin/TCF(LEF1)通路,还是非经典的Wnt通路对于具有多向分化潜能的间充质干细胞的骨发生都具有重要的调节作用。本研究在于探索经典Wnt/β-catenin/TCF(LEF1)通路对大鼠骨髓来源的间充质干细胞在体外成骨分化中的调节作用。 方法:新生SD大白鼠6只,由华中科技大学同济医院试验动物中心提供,用于制备骨髓间充质干细胞。Wnt3a蛋白为PeproTech公司产品。细胞学体外对照观察:采用全骨髓体外分离法培养骨髓间充质干细胞,选取传至第3代的骨髓间充质干细胞,按5×10~7 L~(-1)接种后各分为3组,分别加入含10~8 mol/L的地塞米松、10 mol/Lβ-甘油磷酸盐、50 mg/L维生素C的DMEM成骨诱导培养基2.0 mL。A组在诱导分化后的第8天起加入浓度10mg/LWnt3a,连续3天;B组在诱导分化后的第18天起加入浓度10mg/LWnt3a,连续3天。C组为对照组,在分化的全过程不加入Wnt3a蛋白。主要观察指标:倒置显微镜观察间充质干细胞生长和增殖情况,流式细胞仪检测间充质干细胞表面标志物的表达,碱性磷酸酶染色检测向成骨细胞分化情况,碱性磷酸酶定量分析结果。 结果:第3代骨髓间充质干细胞的恶CD44阳性率为94.88%,CD90的阳性率为94.37%,CD45为0.95%。加入成骨诱导剂后7d,经诱导的骨髓基质干细胞呈碱性磷酸酶阳性。在第11天,检测A组的碱性磷酸酶的活性较B、C组高(P0.01)。第21日,检测B组的碱性磷酸酶活性较C组低(P0.01)。 结论:大鼠骨髓来源间充质干细胞体的外成骨性能受Wnt3a信号通路调节,并且这种调节依赖于被作用细胞的分化阶段的,在间充质干细胞进入分化期后,Wnt3a所介导的经典Wnt信号通路对于间充质干细胞向成骨细胞分化具有促进作用,但当到成骨细胞发育成熟的晚期,Wnt3a介导的经典Wnt信号通路的作用由促进变为抑制成骨细胞最终发育成熟。
[Abstract]:Aim: Wnt signaling pathway is a new research hotspot in recent years, which plays an important role in the regulation of cell biological behavior. Existing studies have shown that both the classical Wnt/ 尾-catenin/TCF (LEF1) pathway and the non-classical Wnt pathway play an important role in the osteogenesis of mesenchymal stem cells with multipotential differentiation. The aim of this study was to explore the role of classical Wnt/ 尾-catenin/TCF (LEF1) pathway in the regulation of bone marrow derived mesenchymal stem cells (MSCs) in osteogenic differentiation in vitro. Methods: six newborn SD rats, provided by Experimental Animal Center of Tongji Hospital, Huazhong University of Science and Technology, were used to prepare bone marrow mesenchymal stem cells. Wnt3a protein was produced by PeproTech Company. Cytological comparative observation in vitro: bone marrow mesenchymal stem cells were cultured by whole bone marrow isolation in vitro. Bone marrow mesenchymal stem cells passed to the third generation were selected and divided into three groups according to 5 脳 10 ~ 7 L ~ (- 1) inoculation, and the cells were divided into 3 groups according to 5 脳 10 ~ 7 L ~ (- 1). The DMEM osteogenic induction medium containing 10 ~ 8 mol/L dexamethasone, 10 mol/L 尾-glycerophosphate and 50 mg/L vitamin C was added to the DMEM osteogenic induction medium 2.0 mL.A on the 8th day after differentiation, for 3 consecutive days, and the concentration was 10 mg / L / L Wnt3a on the 8th day after differentiation. The concentration of 10 mg / L Wnt3a was added to group B on the 18th day after differentiation, for 3 consecutive days. Group C was the control group, and no Wnt3a protein was added in the whole process of differentiation. Main outcome measures: the growth and proliferation of mesenchymal stem cells were observed by inverted microscope, the expression of surface markers of mesenchymal stem cells was detected by flow cytometry, and the differentiation of mesenchymal stem cells into osteoblasts was detected by alkaline phosphatase staining. Quantitative analysis of alkaline phosphatase. Results: the positive rates of malignant CD44, CD90 and CD45 were 94.88%, 94.37% and 0.95% respectively in the third generation of bone marrow mesenchymal stem cells. On the 7th day after adding osteogenic inducer, the induced bone marrow stromal stem cells showed alkaline phosphatase positive. On the 11th day, the activity of alkaline phosphatase in group A was higher than that in group B and C (P0.01). On the 21st day, the activity of alkaline phosphatase in group B was lower than that in group C (P0.01). Conclusion: the osteogenesis of rat bone marrow derived mesenchymal stem cell bodies is regulated by Wnt3a signaling pathway, and this regulation is dependent on the differentiation stage of the affected cells. After mesenchymal stem cells enter the differentiation phase, the bone forming ability of mesenchymal stem cells is regulated by the differentiation of mesenchymal stem cells. The classical Wnt signaling pathway mediated by Wnt3a promotes the differentiation of mesenchymal stem cells into osteoblasts, but at the late stage of osteoblast maturation, Wnt3a-mediated classical Wnt signaling pathway changes from promoting to inhibiting the ultimate maturation of osteoblasts.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2446156
[Abstract]:Aim: Wnt signaling pathway is a new research hotspot in recent years, which plays an important role in the regulation of cell biological behavior. Existing studies have shown that both the classical Wnt/ 尾-catenin/TCF (LEF1) pathway and the non-classical Wnt pathway play an important role in the osteogenesis of mesenchymal stem cells with multipotential differentiation. The aim of this study was to explore the role of classical Wnt/ 尾-catenin/TCF (LEF1) pathway in the regulation of bone marrow derived mesenchymal stem cells (MSCs) in osteogenic differentiation in vitro. Methods: six newborn SD rats, provided by Experimental Animal Center of Tongji Hospital, Huazhong University of Science and Technology, were used to prepare bone marrow mesenchymal stem cells. Wnt3a protein was produced by PeproTech Company. Cytological comparative observation in vitro: bone marrow mesenchymal stem cells were cultured by whole bone marrow isolation in vitro. Bone marrow mesenchymal stem cells passed to the third generation were selected and divided into three groups according to 5 脳 10 ~ 7 L ~ (- 1) inoculation, and the cells were divided into 3 groups according to 5 脳 10 ~ 7 L ~ (- 1). The DMEM osteogenic induction medium containing 10 ~ 8 mol/L dexamethasone, 10 mol/L 尾-glycerophosphate and 50 mg/L vitamin C was added to the DMEM osteogenic induction medium 2.0 mL.A on the 8th day after differentiation, for 3 consecutive days, and the concentration was 10 mg / L / L Wnt3a on the 8th day after differentiation. The concentration of 10 mg / L Wnt3a was added to group B on the 18th day after differentiation, for 3 consecutive days. Group C was the control group, and no Wnt3a protein was added in the whole process of differentiation. Main outcome measures: the growth and proliferation of mesenchymal stem cells were observed by inverted microscope, the expression of surface markers of mesenchymal stem cells was detected by flow cytometry, and the differentiation of mesenchymal stem cells into osteoblasts was detected by alkaline phosphatase staining. Quantitative analysis of alkaline phosphatase. Results: the positive rates of malignant CD44, CD90 and CD45 were 94.88%, 94.37% and 0.95% respectively in the third generation of bone marrow mesenchymal stem cells. On the 7th day after adding osteogenic inducer, the induced bone marrow stromal stem cells showed alkaline phosphatase positive. On the 11th day, the activity of alkaline phosphatase in group A was higher than that in group B and C (P0.01). On the 21st day, the activity of alkaline phosphatase in group B was lower than that in group C (P0.01). Conclusion: the osteogenesis of rat bone marrow derived mesenchymal stem cell bodies is regulated by Wnt3a signaling pathway, and this regulation is dependent on the differentiation stage of the affected cells. After mesenchymal stem cells enter the differentiation phase, the bone forming ability of mesenchymal stem cells is regulated by the differentiation of mesenchymal stem cells. The classical Wnt signaling pathway mediated by Wnt3a promotes the differentiation of mesenchymal stem cells into osteoblasts, but at the late stage of osteoblast maturation, Wnt3a-mediated classical Wnt signaling pathway changes from promoting to inhibiting the ultimate maturation of osteoblasts.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【共引文献】
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