拉伸应变对破骨细胞凋亡的影响及其作用机制的研究
发布时间：2019-07-03 14:11
【摘要】：研究背景和目的 骨是人体重要的承重和运动器官，骨组织始终处于外界力学环境中。骨既能承受外界力学载荷，又能随着外界力学载荷的变化改变自身的形态和功能。骨重建是成熟骨组织的一种重要替换机制。力学载荷是通过骨重建过程对骨的结构和功能产生调节作用。生理状态下，骨吸收和骨形成之间形成一种动态平衡，骨在功能需要的地方发生骨形成，在不需要的地方发生骨吸收。骨重建过程包括了破骨细胞的破骨作用和成骨细胞的成骨作用。破骨细胞是来源于骨髓造血干细胞从单核巨噬细胞系早期的分化过程中分支出来。破骨细胞是骨重建的过程的直接参与者，并在骨重建的起始阶段起到关键作用。破骨细胞的活性变化和凋亡变化都会对骨重建过程有重要影响。但是，力学载荷是否对破骨细胞凋亡起作用目前尚不清楚。 本研究试图通过细胞生物学和分子生物学的方法来研究基底拉伸应变对破骨细胞凋亡的影响，并初步探索基底拉伸应变对破骨细胞凋亡影响的作用机制。 研究内容 (1)破骨细胞的诱导培养及鉴定。 (2)基底拉伸应变对破骨细胞凋亡的影响的研究。 (3)基底拉伸应变对破骨细胞凋亡影响的机制的初步研究。 研究方法 (1)利用来自小鼠的破骨前体细胞株RAW264.7细胞，采用含有两种细胞因子浓度为50ng/mL的小鼠重组可溶性核因子кB受体活化因子配基(receptor activatorof NF-кB ligand，RANKL)和50ng/mL的巨噬细胞集落刺激因子(macrophage-colonystimulating factor，M-CSF)的DMEM(Dulbecco minimum essential medium)培养基，诱导培养实验用的成熟破骨细胞。利用多种方法鉴定诱导的细胞是否具有成熟破骨细胞活性：首先采用光学显微镜下直接观察和TRAP染色后观察诱导的破骨细胞，又采用了甲苯胺蓝染色和扫描电子显微镜观察破骨细胞形成的骨吸收陷窝的方法鉴定破骨细胞。 (2)对破骨细胞施加生理强度2500με和病理强度5000με的基底拉伸应变，加载条件采用连续3d，1次/d，每次1h，加载频率为0.5HZ。检测拉伸应变是否对破骨细胞凋亡有影响：对加载后的破骨细胞采用hoechst染色，Annexin结合实验，caspase-3活性测定实验等方法检测破骨细胞的凋亡情况。 (3)对破骨细胞施加生理强度2500με和病理强度5000με的基底拉伸应变，，加载条件采用1次/d，每次1h，连续3d，加载频率为0.5HZ。检测线粒体通路是否参与拉伸应变对破骨细胞凋亡的调节中：对加载后的破骨细胞进行JC-1染色，采用线粒体膜电位测定实验测定线粒体膜电位，免疫印迹实验测定破骨细胞Bcl-2、caspase-3的表达和细胞色素C的释放。 研究结果 (1)诱导培养3d后，光学显微镜下有体积增大的多核细胞出现，形态不规则并且边缘模糊。诱导培养7d后，多核细胞体积进一步增大，数量增多。抗酒石酸酸性磷酸酶(TRAP)染色多核巨细胞的胞核呈蓝色，胞质特异性染色呈红色。骨片骨吸收陷窝甲苯胺蓝染色结果显示，骨片上有边缘清晰的骨吸收陷窝形成，有骨吸收陷窝处因染料填入而呈甲苯胺蓝浓染的蓝紫色。扫描电子显微镜观察结果显示薄骨片上有明显的骨吸收陷窝形成，边缘清晰，内部凹陷且底面凹凸不平。 (2)对破骨细胞施加基底拉伸应变3d后，hoechst染色结果显示，两组加载组细胞和一组对照组细胞均有细胞凋亡发生，其中凋亡的多核破骨细胞呈现蓝色致密浓染的胞核。计数后得出三组破骨细胞的凋亡率，对照组破骨细胞的凋亡率为21.45%±0.99%，2500με载荷加载组破骨细胞的凋亡率为15.34%±1.50%，与对照组相比显著下降(p＜0.05)，5000με载荷加载组破骨细胞的凋亡率为21.95%±1.06%，与对照组相比无显著差异(p＞0.05)。 Annexin结合实验的结果显示，对照组破骨细胞的早期凋亡率为11.80%，2500με载荷加载组破骨细胞的早期凋亡率为7.54%，与对照组相比显著下降，5000με载荷加载组破骨细胞的凋亡率为10.30%，与对照组相比下降并不明显。 最后，caspase-3活性测定实验结果显示，2500με载荷加载组相对对照组的caspase-3活性为0.7046±0.0148，与对照组相比显著下降(p＜0.05)，5000με载荷加载组相对对照组的caspase-3活性为1.1244±0.0602，与对照组相比稍有上升(p＜0.05)。 (3)对破骨细胞施加基底拉伸应变3d后发现，线粒体膜电位测定实验结果显示，与对照组相比，2500με载荷加载组破骨细胞的线粒体膜电位显著增高(p＜0.05)，表明破骨细胞线粒体膜的去极化过程被阻止，5000με载荷加载组破骨细胞的线粒体膜电位与对照组相比无明显差异(p＞0.05)。 免疫印迹实验结果显示，与对照组相比，2500με载荷加载组破骨细胞的Bcl-2表达明显大幅度增高(p＜0.05)，5000με载荷加载组破骨细胞的Bcl-2表达同样增高(p＜0.05)，但幅度并不没有2500με载荷加载组的那么大。同时，与对照组相比，2500με载荷加载组破骨细胞的胞浆细胞色素C含量显著减少(p＜0.05)，暗示细胞色素C的释放被抑制，而5000με载荷加载组破骨细胞的胞浆细胞色素C含量与对照组相比并无明显变化(p＞0.05)。最后，2500με载荷加载组破骨细胞的caspase-3表达显著减少(p＜0.05)，而5000με载荷加载组破骨细胞的caspase-3表达与对照组相比并无明显变化(p＞0.05)。 结论 (1)小鼠单核/巨噬细胞系RAW264.7细胞在含有50ng/mL的RANKL和50ng/mL的M-CSF的DMEM培养基诱导下，可以分化出具有典型生物学活性的成熟破骨细胞。 (2)拉伸应变的加载能够影响破骨细胞的凋亡。目前为止，未见此类相关报道。本研究发现，对破骨细胞施加2500με的四点弯曲基底拉伸应变能够明显抑制破骨细胞的凋亡，而5000με的加载对破骨细胞凋亡的影响并不明显。 (3)线粒体通路参与到破骨细胞凋亡的力学响应过程中。同样，尚未发现有类似研究。
[Abstract]:Study Background and Purpose Bone is an important bearing and moving organ of the human body, and the bone tissue is always in the external mechanical environment The bone can not only bear the external mechanical load, but also can change its shape and work with the change of the external mechanical load. The bone reconstruction is an important replacement for mature bone tissue. The mechanical load is made by the structure and function of the bone through the bone reconstruction process. in a physiological state, there is a dynamic balance between bone resorption and bone formation, bone formation occurs where that function is desire, bone resorption occurs in unwanted places, The bone remodeling process includes the osteoclast osteoclast and the osteogenesis of the osteoblast. Used. Osteoclasts are derived from the early differentiation of bone marrow hematopoietic stem cells from the early differentiation of mononuclear macrophages. The osteoclast is a direct participant in the process of bone reconstruction and plays a key role in the initial stage of bone reconstruction. The changes of the activity of osteoclast and the changes of apoptosis will play an important role in the process of bone reconstruction. In response, that effect of mechanical load on the apoptosis of osteoclasts is still unclear. The effect of tensile strain on the apoptosis of osteoclasts was studied by the methods of cell biology and molecular biology, and the effect of the tensile strain of the substrate on the apoptosis of osteoclast was studied. With a mechanism. Study content (1) Osteoclasts Induction of culture and identification. (2) The tensile strain of the substrate is fine to the osteoclast. The study of the effect of the cell apoptosis. (3) the base tensile strain is used for osteoclast. dead A preliminary study of the mechanism was carried out. Methods (1) were used to make use of RAW264.7 cells from the bone-breaking precursor cells of the mouse, and the macrophage colony-stimulating factor (RANKL) and the macrophage colony-stimulating factor of 50 ng/ mL (macrophage-colonyst) containing two kinds of mouse recombinant soluble nuclear factor, which contains two kinds of cytokine concentrations of 50 ng/ mL, were used. imulating factor,M-CSF)鐨凞MEM(Dulbecco minimum essential medium)鍩瑰吇鍩
本文编号：2509456
[Abstract]:Study Background and Purpose Bone is an important bearing and moving organ of the human body, and the bone tissue is always in the external mechanical environment The bone can not only bear the external mechanical load, but also can change its shape and work with the change of the external mechanical load. The bone reconstruction is an important replacement for mature bone tissue. The mechanical load is made by the structure and function of the bone through the bone reconstruction process. in a physiological state, there is a dynamic balance between bone resorption and bone formation, bone formation occurs where that function is desire, bone resorption occurs in unwanted places, The bone remodeling process includes the osteoclast osteoclast and the osteogenesis of the osteoblast. Used. Osteoclasts are derived from the early differentiation of bone marrow hematopoietic stem cells from the early differentiation of mononuclear macrophages. The osteoclast is a direct participant in the process of bone reconstruction and plays a key role in the initial stage of bone reconstruction. The changes of the activity of osteoclast and the changes of apoptosis will play an important role in the process of bone reconstruction. In response, that effect of mechanical load on the apoptosis of osteoclasts is still unclear. The effect of tensile strain on the apoptosis of osteoclasts was studied by the methods of cell biology and molecular biology, and the effect of the tensile strain of the substrate on the apoptosis of osteoclast was studied. With a mechanism. Study content (1) Osteoclasts Induction of culture and identification. (2) The tensile strain of the substrate is fine to the osteoclast. The study of the effect of the cell apoptosis. (3) the base tensile strain is used for osteoclast. dead A preliminary study of the mechanism was carried out. Methods (1) were used to make use of RAW264.7 cells from the bone-breaking precursor cells of the mouse, and the macrophage colony-stimulating factor (RANKL) and the macrophage colony-stimulating factor of 50 ng/ mL (macrophage-colonyst) containing two kinds of mouse recombinant soluble nuclear factor, which contains two kinds of cytokine concentrations of 50 ng/ mL, were used. imulating factor,M-CSF)鐨凞MEM(Dulbecco minimum essential medium)鍩瑰吇鍩
本文编号：2509456
本文链接：https://www.wllwen.com/xiyixuelunwen/2509456.html
最近更新
教材专著
