冲击波诱导人骨髓间充质干细胞向成骨细胞分化的机制
发布时间：2018-07-27 13:41
【摘要】： 随着骨组织工程研究的不断发展,种子细胞的来源、培养、诱导、增殖、分化等研究成为近年来骨组织工程进行研究的热点。间充质干细胞具有能大量增殖、分化为多种组织细胞和表达外源目的基因等优点,并且可以在特定的条件下定向向成骨细胞分化,因其可能成为用于临床的组织工程化骨种子细胞而受到越来越多的关注。 近年来体外冲击波疗法正被广泛的用于治疗骨不连、骨折延迟愈合和股骨头坏死以及运动系统慢性劳损性疾病,并取得了很好的疗效。人们发现,机械物理刺激促使间充质干细胞分化。冲击波作为一种机械物理刺激能够促使间充质干细胞向成骨细胞分化,但这一过程不是自发完成的,其中可能涉及到多条信号通路的调控,其中MAPK通路可能起着极其重要的调节作用,但具体通过MAPK传导路途径的ERK、JNK以及p38MAPK通路中哪一条通路参与以及各通路在诱导成骨分化过程中的作用尚不十分清楚。本试验通过前期获得最佳冲击波(工作电压为8.5kv,作用频次为120次)作用于分离培养后的人骨髓间充质干细胞,设立空白对照组,并分别加入ERK、JNK以及p38MAPK通路抑制剂,通过western blot检测ERK、JNK以及p38MAPK的磷酸化以及MAPK通路作用底物AP-1(c-fos和c-jun mRNA)的表达。用MTT法检测细胞增殖,用成骨特性鉴定的方法(钙-钴法染色检测碱性磷酸酶的表达及其活性、Von Kossa染色检测钙结节和钙沉积量的检测)判断冲击波是否诱导人骨髓间充质干细胞向成骨细胞分化,研究ERK、JNK、p38 MAPK通路在体外冲击波诱导下人骨髓间充质干细胞向成骨细胞分化过程中的作用。 本研究发现在冲击波诱导人骨髓间充质干细胞诱导向成骨细胞分化过程中,通过Western blot方法证实ERK、JNK以及p38MAPK通路均早期磷酸化激活,提示ERK、JNK以及p38MAPK通路均参与了成骨细胞增殖、分化。应用ERK、JNK以及p38MAPK通路相应的阻断剂研究结果表明:ERK通路促进人骨髓间充质干细胞增殖,抑制ERK通路后细胞增殖作用明显降低。抑制JNK通路后向成骨细胞分化作用虽有降低,但不具有统计学意义;对细胞增殖未见有明显作用。抑制p38MAPK通路后MAPK作用底物AP-1 (c-fos和c-jun mRNA)表达明显受抑制,成骨鉴定证实向成骨细胞分化作用明显降低,并呈剂量依赖性;低浓度的p38MAPK抑制剂反而促进细胞增殖。应用阻断剂后行同样条件进行诱导,提示p38MAPK通路是冲击波诱导人骨髓间充质干细胞诱导向成骨细胞分化的主要途径。
[Abstract]:With the development of bone tissue engineering, the research on the origin, culture, induction, proliferation and differentiation of seed cells has become the focus of bone tissue engineering research in recent years. Mesenchymal stem cells (MSCs) have the advantages of proliferation, differentiation into various histocytes and expression of exogenous target genes. Mesenchymal stem cells can differentiate into osteoblasts under specific conditions. More and more attention has been paid to its potential as tissue engineered bone seed cells for clinical use. In recent years, extracorporeal shock wave therapy has been widely used in the treatment of nonunion, delayed union of fracture, necrosis of femoral head and chronic strain disease of motor system. Mechanical physical stimulation has been found to induce differentiation of mesenchymal stem cells. Shock wave, as a mechanical physical stimulation, can induce mesenchymal stem cells to differentiate into osteoblasts, but this process is not spontaneous and may involve the regulation of multiple signaling pathways. The MAPK pathway may play an extremely important role in regulating osteogenesis, but it is not clear which one of the ERKN JNK pathways and which p38MAPK pathway is involved in the process of osteogenic differentiation through the MAPK pathway, and the role of each pathway in the process of osteogenic differentiation is not well understood. In this experiment, the optimal shock wave (working voltage 8.5 kv, action frequency 120 times) was obtained for isolation and culture of human bone marrow mesenchymal stem cells, blank control group was established, and ERKN JNK and p38MAPK pathway inhibitor were added respectively. The phosphorylation of p38MAPK and the expression of AP-1 (c-fos and c-jun mRNA) in MAPK pathway were detected by western blot. Cell proliferation was detected by MTT assay. The osteogenic characteristics of human bone marrow mesenchymal stem cells (BMSCs) were determined by the method of identification of osteogenic characteristics (detection of alkaline phosphatase expression by calcium cobalt staining and detection of calcium nodules and calcium deposition by Von Kossa staining) in order to determine whether shock wave induced the differentiation of human bone marrow mesenchymal stem cells into osteoblasts. To study the role of ERKN JNKN p38 MAPK pathway in the differentiation of human bone marrow mesenchymal stem cells into osteoblasts induced by extracorporeal shock wave. In the course of inducing osteoblast differentiation from human bone marrow mesenchymal stem cells (BMSCs) induced by shock wave, it was confirmed by Western blot method that early phosphorylation activation of ERKN JNK and p38MAPK pathway, both ERKN JNK and p38MAPK pathway were involved in osteoblast proliferation. To divide. The results showed that p38MAPK pathway promoted the proliferation of human bone marrow mesenchymal stem cells and inhibited the proliferation of human bone marrow mesenchymal stem cells after inhibition of ERK pathway. The inhibitory effect of JNK pathway on osteoblast differentiation was decreased, but there was no significant difference between the two groups, and there was no obvious effect on the proliferation of osteoblasts. The expression of AP-1 (c-fos and c-jun mRNA) in the substrate of MAPK was obviously inhibited after inhibiting the p38MAPK pathway. Osteogenic identification showed that the differentiation into osteoblasts was obviously decreased in a dose-dependent manner, and the low concentration of p38MAPK inhibitor could promote the proliferation of cells. The same condition was used to induce human bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts under the same conditions. The results indicated that p38MAPK pathway was the main pathway of inducing human bone marrow mesenchymal stem cells to differentiate into osteoblasts by shock wave.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329
本文编号：2148010
[Abstract]:With the development of bone tissue engineering, the research on the origin, culture, induction, proliferation and differentiation of seed cells has become the focus of bone tissue engineering research in recent years. Mesenchymal stem cells (MSCs) have the advantages of proliferation, differentiation into various histocytes and expression of exogenous target genes. Mesenchymal stem cells can differentiate into osteoblasts under specific conditions. More and more attention has been paid to its potential as tissue engineered bone seed cells for clinical use. In recent years, extracorporeal shock wave therapy has been widely used in the treatment of nonunion, delayed union of fracture, necrosis of femoral head and chronic strain disease of motor system. Mechanical physical stimulation has been found to induce differentiation of mesenchymal stem cells. Shock wave, as a mechanical physical stimulation, can induce mesenchymal stem cells to differentiate into osteoblasts, but this process is not spontaneous and may involve the regulation of multiple signaling pathways. The MAPK pathway may play an extremely important role in regulating osteogenesis, but it is not clear which one of the ERKN JNK pathways and which p38MAPK pathway is involved in the process of osteogenic differentiation through the MAPK pathway, and the role of each pathway in the process of osteogenic differentiation is not well understood. In this experiment, the optimal shock wave (working voltage 8.5 kv, action frequency 120 times) was obtained for isolation and culture of human bone marrow mesenchymal stem cells, blank control group was established, and ERKN JNK and p38MAPK pathway inhibitor were added respectively. The phosphorylation of p38MAPK and the expression of AP-1 (c-fos and c-jun mRNA) in MAPK pathway were detected by western blot. Cell proliferation was detected by MTT assay. The osteogenic characteristics of human bone marrow mesenchymal stem cells (BMSCs) were determined by the method of identification of osteogenic characteristics (detection of alkaline phosphatase expression by calcium cobalt staining and detection of calcium nodules and calcium deposition by Von Kossa staining) in order to determine whether shock wave induced the differentiation of human bone marrow mesenchymal stem cells into osteoblasts. To study the role of ERKN JNKN p38 MAPK pathway in the differentiation of human bone marrow mesenchymal stem cells into osteoblasts induced by extracorporeal shock wave. In the course of inducing osteoblast differentiation from human bone marrow mesenchymal stem cells (BMSCs) induced by shock wave, it was confirmed by Western blot method that early phosphorylation activation of ERKN JNK and p38MAPK pathway, both ERKN JNK and p38MAPK pathway were involved in osteoblast proliferation. To divide. The results showed that p38MAPK pathway promoted the proliferation of human bone marrow mesenchymal stem cells and inhibited the proliferation of human bone marrow mesenchymal stem cells after inhibition of ERK pathway. The inhibitory effect of JNK pathway on osteoblast differentiation was decreased, but there was no significant difference between the two groups, and there was no obvious effect on the proliferation of osteoblasts. The expression of AP-1 (c-fos and c-jun mRNA) in the substrate of MAPK was obviously inhibited after inhibiting the p38MAPK pathway. Osteogenic identification showed that the differentiation into osteoblasts was obviously decreased in a dose-dependent manner, and the low concentration of p38MAPK inhibitor could promote the proliferation of cells. The same condition was used to induce human bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts under the same conditions. The results indicated that p38MAPK pathway was the main pathway of inducing human bone marrow mesenchymal stem cells to differentiate into osteoblasts by shock wave.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 王芳;奶山羊骨髓间充质干细胞的分离培养及向雄性生殖细胞诱导分化[D];西北农林科技大学;2011年
，本文编号：2148010
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