神经生长因子对山羊骨髓间充质干细胞增殖及向成骨分化的调节作用
发布时间：2018-11-15 11:30
【摘要】： 目的体外培养山羊骨髓间充质干细胞(Bone marrow Mesenchymal stem cells,BMSCs)并向成骨方向进行诱导,通过加入神经生长因子(Nerve growth factor,NGF),研究NGF在成骨培养基中对山羊BMSCs增殖及分化的影响。为阐明NGF促进牵张成骨术(Distraction osteogenesis, DO)中骨形成的机理提供实验依据,希望所建立的实验方法能为将来进一步的临床和科研工作建立稳定的细胞模型,从而使牵张成骨术中促进牵张区新骨生成,解决临床问题提供一种可能。 方法抽取山羊骨髓,利用Percoll分离液进行密度梯度离心结合贴壁法分离山羊BMSCs,体外培养扩增,观察其形态学变化。体外培养至第三代后,首先用流式细胞仪检测细胞表面标志物,以鉴定细胞纯度,排除了杂质细胞的干扰;再将第三代BMSCs分为A、B、C、D四组,A组(空白对照组):用含10%胎牛血清的低糖DMEM常规培养液培养。B组(成骨诱导对照组):成骨诱导液为常规培养液中加入10-8mol/L地塞米松、50mg/L维生素C、10mmol/Lβ-甘油磷酸钠。C组(NGF组):常规培养液中加入终浓度为100ng/ml的NGF。D组(实验组):成骨诱导液中加入终浓度为100ng/ml的NGF。于第3,5,7天利用MTT法测定各培养组细胞增殖活性,第14天检测各组细胞碱性磷酸酶(ALP)活性和骨钙素(OC)水平、第21天应用Von Kossa染色的方法检测钙结节的形成,比较各组细胞成骨性能,观察NGF对山羊BMSCs增殖及向成骨分化的影响。 结果1.利用Percoll分离液进行密度梯度离心结合贴壁法分离、纯化山羊BMSCs,是一种简便可行的方法,可获得较高的纯度,流式细胞术测得CD90、CD105为强阳性表达,CD34、CD45为阴性。 2.MTT法检测细胞增殖实验中,A组和C组细胞增殖率相似,B组和D组细胞增殖率相似,但B、D两组明显低于A、C两组,差异有显著性意义(p0.05)。 3.各组细胞成骨性能检测结果:A组和C组ALP和OC表达均无明显差异(p0.05),B组和D组ALP和OC表达明显高于A组和C组,且D组较B组表达水平增高,差异有显著性意义(p0.05)。A、C两组细胞Von Kossa染色阴性,未见明显的钙化结节出现;B组细胞Von Kossa染色阳性,可见黑色钙化结节,D组较成骨诱导对照组钙化结节数目明显增多,面积增大。 结论密度梯度离心结合贴壁法是分离、纯化山羊BMSCs的简便实用的方法,传至第三代即可获得较高的纯度。单纯使用NGF对山羊BMSCs并无明显促进增殖及分化的作用,但NGF在山羊BMSCs向成骨细胞分化过程中具有明显的促进作用。这为加快骨形成,进一步进行相关的临床研究提供了实验基础。
[Abstract]:Objective to culture goat bone marrow mesenchymal stem cells (Bone marrow Mesenchymal stem cells,BMSCs) in vitro and induce them to osteogenesis. By adding nerve growth factor (Nerve growth factor,NGF), the effect of NGF on the proliferation and differentiation of goat BMSCs in osteoblasts was studied. In order to elucidate the mechanism of NGF in promoting bone formation in distraction osteogenesis (Distraction osteogenesis, DO), it is hoped that the established experimental method can establish a stable cell model for further clinical and scientific research in the future. So that distraction osteogenesis can promote the formation of new bone in distraction zone and provide a possibility to solve clinical problems. Methods Goat bone marrow was extracted and BMSCs, was isolated by density gradient centrifugation and adherent method. The morphological changes of goat BMSCs, were observed. After the third passage in vitro, the cell surface markers were detected by flow cytometry in order to identify the cell purity and eliminate the interference of impurity cells. The third generation of BMSCs was divided into four groups, Group A (blank control group) was cultured with low glucose DMEM medium containing 10% fetal bovine serum. Group B (osteogenic control group): 10-8mol/L dexamethasone and 50mg/L vitamin C were added to osteogenic medium. 10mmol/L 尾 -glycerophosphate. C group (NGF group): NGF.D group with 100ng/ml final concentration in conventional culture medium (experimental group): NGF. with 100ng/ml final concentration in osteoblast inducer The proliferative activity of the cultured cells was measured by MTT method on the 7th day, the alkaline phosphatase (ALP) activity and osteocalcin (OC) level were measured on the 14th day, and the formation of calcium nodules was detected by Von Kossa staining on the 21st day. The effects of NGF on the proliferation and osteogenic differentiation of goat BMSCs were observed. Result 1. It is a simple and feasible method to purify goat BMSCs, by density gradient centrifugation combined with adherent method. The purity of goat BMSCs, can be obtained by flow cytometry. The strong positive expression of CD90,CD105 and negative expression of CD34,CD45 were detected by flow cytometry. The cell proliferation rate of group A and group C was similar to that of group B and group D by 2.MTT assay, but the cell proliferation rate of group B and D was significantly lower than that of group A and C (p0.05). 3. There was no significant difference in the expression of ALP and OC between group A and group C (p0. 05), B and group D were significantly higher than those in group A and C, and the expression of ALP and OC in group D was higher than that in group B. The difference was significant (p0.05). Von Kossa staining was negative and no calcified nodules were found in the two groups. In group B, the number and area of calcified nodules were significantly increased and the number of calcified nodules in group D was significantly higher than that in the control group. Conclusion density gradient centrifugation combined with adherent method is a simple and practical method for the separation and purification of goat BMSCs. NGF alone did not promote the proliferation and differentiation of goat BMSCs, but NGF could promote the differentiation of goat BMSCs into osteoblasts. This provides experimental basis for accelerating bone formation and further related clinical research.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2333189
[Abstract]:Objective to culture goat bone marrow mesenchymal stem cells (Bone marrow Mesenchymal stem cells,BMSCs) in vitro and induce them to osteogenesis. By adding nerve growth factor (Nerve growth factor,NGF), the effect of NGF on the proliferation and differentiation of goat BMSCs in osteoblasts was studied. In order to elucidate the mechanism of NGF in promoting bone formation in distraction osteogenesis (Distraction osteogenesis, DO), it is hoped that the established experimental method can establish a stable cell model for further clinical and scientific research in the future. So that distraction osteogenesis can promote the formation of new bone in distraction zone and provide a possibility to solve clinical problems. Methods Goat bone marrow was extracted and BMSCs, was isolated by density gradient centrifugation and adherent method. The morphological changes of goat BMSCs, were observed. After the third passage in vitro, the cell surface markers were detected by flow cytometry in order to identify the cell purity and eliminate the interference of impurity cells. The third generation of BMSCs was divided into four groups, Group A (blank control group) was cultured with low glucose DMEM medium containing 10% fetal bovine serum. Group B (osteogenic control group): 10-8mol/L dexamethasone and 50mg/L vitamin C were added to osteogenic medium. 10mmol/L 尾 -glycerophosphate. C group (NGF group): NGF.D group with 100ng/ml final concentration in conventional culture medium (experimental group): NGF. with 100ng/ml final concentration in osteoblast inducer The proliferative activity of the cultured cells was measured by MTT method on the 7th day, the alkaline phosphatase (ALP) activity and osteocalcin (OC) level were measured on the 14th day, and the formation of calcium nodules was detected by Von Kossa staining on the 21st day. The effects of NGF on the proliferation and osteogenic differentiation of goat BMSCs were observed. Result 1. It is a simple and feasible method to purify goat BMSCs, by density gradient centrifugation combined with adherent method. The purity of goat BMSCs, can be obtained by flow cytometry. The strong positive expression of CD90,CD105 and negative expression of CD34,CD45 were detected by flow cytometry. The cell proliferation rate of group A and group C was similar to that of group B and group D by 2.MTT assay, but the cell proliferation rate of group B and D was significantly lower than that of group A and C (p0.05). 3. There was no significant difference in the expression of ALP and OC between group A and group C (p0. 05), B and group D were significantly higher than those in group A and C, and the expression of ALP and OC in group D was higher than that in group B. The difference was significant (p0.05). Von Kossa staining was negative and no calcified nodules were found in the two groups. In group B, the number and area of calcified nodules were significantly increased and the number of calcified nodules in group D was significantly higher than that in the control group. Conclusion density gradient centrifugation combined with adherent method is a simple and practical method for the separation and purification of goat BMSCs. NGF alone did not promote the proliferation and differentiation of goat BMSCs, but NGF could promote the differentiation of goat BMSCs into osteoblasts. This provides experimental basis for accelerating bone formation and further related clinical research.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前9条
1 梁伟;苟三怀;齐进;魏立;欧阳跃平;刘岩;;低强度脉冲超声波对体外培养的兔骨髓基质细胞成骨作用的影响[J];第二军医大学学报;2007年06期
2 张伟;于立明;吴亚东;刘麒麟;;神经生长因子受体(p75)在下颌骨骨折愈合中的表达及意义的研究[J];口腔医学研究;2007年03期
3 王恩群;张菊会;刘彦普;周树夏;;骨缝牵张成骨的比较组织学研究[J];临床口腔医学杂志;2008年01期
4 艾春媚;魏泉德;吴怡;崔燎;;羊胎素对体外骨髓基质细胞的增殖和向成骨细胞分化的影响[J];时珍国医国药;2007年02期
5 李彦,刘彦普,邓军红;牵引成骨速率适应组织功能性变化的实验[J];中国临床康复;2005年14期
6 魏波;陈日景;;胰岛素生长因子对骨髓基质细胞增殖和向成骨细胞分化的调节[J];中国组织工程研究与临床康复;2007年24期
7 张炜真,于世凤,郑麟蕃;白细胞介素1调节成骨细胞分化的实验研究[J];中华口腔医学杂志;1996年05期
8 宓宝杰;王相利;;神经生长因子与其受体的信号转导[J];中国煤炭工业医学杂志;2007年06期
9 杨小荣;方煌;罗永湘;;重组人骨形成蛋白2与骨诱导剂对大鼠骨髓间充质干细胞增殖分化的影响[J];中国修复重建外科杂志;2007年02期
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