【摘要】：研究背景和目的 骨是人體重要的承重和運(yùn)動(dòng)器官，骨組織始終處于外界力學(xué)環(huán)境中。骨既能承受外界力學(xué)載荷，又能隨著外界力學(xué)載荷的變化改變自身的形態(tài)和功能。骨重建是成熟骨組織的一種重要替換機(jī)制。力學(xué)載荷是通過(guò)骨重建過(guò)程對(duì)骨的結(jié)構(gòu)和功能產(chǎn)生調(diào)節(jié)作用。生理狀態(tài)下，骨吸收和骨形成之間形成一種動(dòng)態(tài)平衡，骨在功能需要的地方發(fā)生骨形成，在不需要的地方發(fā)生骨吸收。骨重建過(guò)程包括了破骨細(xì)胞的破骨作用和成骨細(xì)胞的成骨作用。破骨細(xì)胞是來(lái)源于骨髓造血干細(xì)胞從單核巨噬細(xì)胞系早期的分化過(guò)程中分支出來(lái)。破骨細(xì)胞是骨重建的過(guò)程的直接參與者，并在骨重建的起始階段起到關(guān)鍵作用。破骨細(xì)胞的活性變化和凋亡變化都會(huì)對(duì)骨重建過(guò)程有重要影響。但是，力學(xué)載荷是否對(duì)破骨細(xì)胞凋亡起作用目前尚不清楚。 本研究試圖通過(guò)細(xì)胞生物學(xué)和分子生物學(xué)的方法來(lái)研究基底拉伸應(yīng)變對(duì)破骨細(xì)胞凋亡的影響，并初步探索基底拉伸應(yīng)變對(duì)破骨細(xì)胞凋亡影響的作用機(jī)制。 研究?jī)?nèi)容 (1)破骨細(xì)胞的誘導(dǎo)培養(yǎng)及鑒定。 (2)基底拉伸應(yīng)變對(duì)破骨細(xì)胞凋亡的影響的研究。 (3)基底拉伸應(yīng)變對(duì)破骨細(xì)胞凋亡影響的機(jī)制的初步研究。 研究方法 (1)利用來(lái)自小鼠的破骨前體細(xì)胞株RAW264.7細(xì)胞，采用含有兩種細(xì)胞因子濃度為50ng/mL的小鼠重組可溶性核因子кB受體活化因子配基(receptor activatorof NF-кB ligand，RANKL)和50ng/mL的巨噬細(xì)胞集落刺激因子(macrophage-colonystimulating factor，M-CSF)的DMEM(Dulbecco minimum essential medium)培養(yǎng)基，誘導(dǎo)培養(yǎng)實(shí)驗(yàn)用的成熟破骨細(xì)胞。利用多種方法鑒定誘導(dǎo)的細(xì)胞是否具有成熟破骨細(xì)胞活性：首先采用光學(xué)顯微鏡下直接觀察和TRAP染色后觀察誘導(dǎo)的破骨細(xì)胞，又采用了甲苯胺藍(lán)染色和掃描電子顯微鏡觀察破骨細(xì)胞形成的骨吸收陷窩的方法鑒定破骨細(xì)胞。 (2)對(duì)破骨細(xì)胞施加生理強(qiáng)度2500με和病理強(qiáng)度5000με的基底拉伸應(yīng)變，加載條件采用連續(xù)3d，1次/d，每次1h，加載頻率為0.5HZ。檢測(cè)拉伸應(yīng)變是否對(duì)破骨細(xì)胞凋亡有影響：對(duì)加載后的破骨細(xì)胞采用hoechst染色，Annexin結(jié)合實(shí)驗(yàn)，caspase-3活性測(cè)定實(shí)驗(yàn)等方法檢測(cè)破骨細(xì)胞的凋亡情況。 (3)對(duì)破骨細(xì)胞施加生理強(qiáng)度2500με和病理強(qiáng)度5000με的基底拉伸應(yīng)變，，加載條件采用1次/d，每次1h，連續(xù)3d，加載頻率為0.5HZ。檢測(cè)線粒體通路是否參與拉伸應(yīng)變對(duì)破骨細(xì)胞凋亡的調(diào)節(jié)中：對(duì)加載后的破骨細(xì)胞進(jìn)行JC-1染色，采用線粒體膜電位測(cè)定實(shí)驗(yàn)測(cè)定線粒體膜電位，免疫印跡實(shí)驗(yàn)測(cè)定破骨細(xì)胞Bcl-2、caspase-3的表達(dá)和細(xì)胞色素C的釋放。 研究結(jié)果 (1)誘導(dǎo)培養(yǎng)3d后，光學(xué)顯微鏡下有體積增大的多核細(xì)胞出現(xiàn)，形態(tài)不規(guī)則并且邊緣模糊。誘導(dǎo)培養(yǎng)7d后，多核細(xì)胞體積進(jìn)一步增大，數(shù)量增多�？咕剖崴嵝粤姿崦�(TRAP)染色多核巨細(xì)胞的胞核呈藍(lán)色，胞質(zhì)特異性染色呈紅色。骨片骨吸收陷窩甲苯胺藍(lán)染色結(jié)果顯示，骨片上有邊緣清晰的骨吸收陷窩形成，有骨吸收陷窩處因染料填入而呈甲苯胺藍(lán)濃染的藍(lán)紫色。掃描電子顯微鏡觀察結(jié)果顯示薄骨片上有明顯的骨吸收陷窩形成，邊緣清晰，內(nèi)部凹陷且底面凹凸不平。 (2)對(duì)破骨細(xì)胞施加基底拉伸應(yīng)變3d后，hoechst染色結(jié)果顯示，兩組加載組細(xì)胞和一組對(duì)照組細(xì)胞均有細(xì)胞凋亡發(fā)生，其中凋亡的多核破骨細(xì)胞呈現(xiàn)藍(lán)色致密濃染的胞核。計(jì)數(shù)后得出三組破骨細(xì)胞的凋亡率，對(duì)照組破骨細(xì)胞的凋亡率為21.45%±0.99%，2500με載荷加載組破骨細(xì)胞的凋亡率為15.34%±1.50%，與對(duì)照組相比顯著下降(p＜0.05)，5000με載荷加載組破骨細(xì)胞的凋亡率為21.95%±1.06%，與對(duì)照組相比無(wú)顯著差異(p＞0.05)。 Annexin結(jié)合實(shí)驗(yàn)的結(jié)果顯示，對(duì)照組破骨細(xì)胞的早期凋亡率為11.80%，2500με載荷加載組破骨細(xì)胞的早期凋亡率為7.54%，與對(duì)照組相比顯著下降，5000με載荷加載組破骨細(xì)胞的凋亡率為10.30%，與對(duì)照組相比下降并不明顯。 最后，caspase-3活性測(cè)定實(shí)驗(yàn)結(jié)果顯示，2500με載荷加載組相對(duì)對(duì)照組的caspase-3活性為0.7046±0.0148，與對(duì)照組相比顯著下降(p＜0.05)，5000με載荷加載組相對(duì)對(duì)照組的caspase-3活性為1.1244±0.0602，與對(duì)照組相比稍有上升(p＜0.05)。 (3)對(duì)破骨細(xì)胞施加基底拉伸應(yīng)變3d后發(fā)現(xiàn)，線粒體膜電位測(cè)定實(shí)驗(yàn)結(jié)果顯示，與對(duì)照組相比，2500με載荷加載組破骨細(xì)胞的線粒體膜電位顯著增高(p＜0.05)，表明破骨細(xì)胞線粒體膜的去極化過(guò)程被阻止，5000με載荷加載組破骨細(xì)胞的線粒體膜電位與對(duì)照組相比無(wú)明顯差異(p＞0.05)。 免疫印跡實(shí)驗(yàn)結(jié)果顯示，與對(duì)照組相比，2500με載荷加載組破骨細(xì)胞的Bcl-2表達(dá)明顯大幅度增高(p＜0.05)，5000με載荷加載組破骨細(xì)胞的Bcl-2表達(dá)同樣增高(p＜0.05)，但幅度并不沒(méi)有2500με載荷加載組的那么大。同時(shí)，與對(duì)照組相比，2500με載荷加載組破骨細(xì)胞的胞漿細(xì)胞色素C含量顯著減少(p＜0.05)，暗示細(xì)胞色素C的釋放被抑制，而5000με載荷加載組破骨細(xì)胞的胞漿細(xì)胞色素C含量與對(duì)照組相比并無(wú)明顯變化(p＞0.05)。最后，2500με載荷加載組破骨細(xì)胞的caspase-3表達(dá)顯著減少(p＜0.05)，而5000με載荷加載組破骨細(xì)胞的caspase-3表達(dá)與對(duì)照組相比并無(wú)明顯變化(p＞0.05)。 結(jié)論 (1)小鼠單核/巨噬細(xì)胞系RAW264.7細(xì)胞在含有50ng/mL的RANKL和50ng/mL的M-CSF的DMEM培養(yǎng)基誘導(dǎo)下，可以分化出具有典型生物學(xué)活性的成熟破骨細(xì)胞。 (2)拉伸應(yīng)變的加載能夠影響破骨細(xì)胞的凋亡。目前為止，未見(jiàn)此類(lèi)相關(guān)報(bào)道。本研究發(fā)現(xiàn)，對(duì)破骨細(xì)胞施加2500με的四點(diǎn)彎曲基底拉伸應(yīng)變能夠明顯抑制破骨細(xì)胞的凋亡，而5000με的加載對(duì)破骨細(xì)胞凋亡的影響并不明顯。 (3)線粒體通路參與到破骨細(xì)胞凋亡的力學(xué)響應(yīng)過(guò)程中。同樣，尚未發(fā)現(xiàn)有類(lèi)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

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