發(fā)布時(shí)間：2018-06-24 04:58

  本文選題：骨髓間充質(zhì)干細(xì)胞 + 非相干光��； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：目的：(1)獲得均一、穩(wěn)定、可靠的大鼠骨髓間充質(zhì)干細(xì)胞；(2)探討620nm發(fā)光二極管(Light Emitting Diodes, LED)非相干光對(duì)大鼠骨髓間充質(zhì)干細(xì)胞在增殖和成骨分化方面的影響；(3)研究在低能量光照促進(jìn)的細(xì)胞增殖過(guò)程中mTOR信號(hào)蛋白的變化情況；(4)研究在低能量光照促進(jìn)的成骨分化中晝夜節(jié)律基因的變化情況。 方法：(1)密度梯度離心法結(jié)合貼壁法分離間充質(zhì)干細(xì)胞,體外擴(kuò)增,鏡下觀察細(xì)胞形態(tài),用流式細(xì)胞儀檢測(cè)其表面標(biāo)記物,分別作間充質(zhì)干細(xì)胞的成骨、成脂肪和成軟骨誘導(dǎo)培養(yǎng),采用von kossa染色、油紅O染色和阿利新藍(lán)染色鑒定細(xì)胞的三向分化能力； (2)獲取的骨髓間充質(zhì)干細(xì)胞分別在普通培養(yǎng)基和成骨誘導(dǎo)培養(yǎng)基中培養(yǎng),兩種培養(yǎng)基中的間充質(zhì)干細(xì)胞均分為四組,用620nm LED非相干光分別以0、1、2和4J/cm2的劑量進(jìn)行照射,照射后采用CCK-8和EdU標(biāo)記觀察細(xì)胞增殖情況,采用ALP比活性檢測(cè)、von kossa染色和多種成骨標(biāo)志基因(Coll a 1,Alpl,Bglap and Runx2) RT-PCR來(lái)評(píng)估細(xì)胞成骨分化的情況； (3) 620nm LED非相干光照射分別以0、0.5、1和2J/cm2的劑量照射間充質(zhì)干細(xì)胞,CCK-8檢測(cè)細(xì)胞增殖情況,Western-blot檢測(cè)總mTOR和磷酸化mTOR蛋白含量； (4) 620nm LED非相干光照射分別以0、1、2和4J/cm2的劑量照射成骨誘導(dǎo)培養(yǎng)基中間充質(zhì)干細(xì)胞,von kossa染色觀察細(xì)胞成骨分化情況,RT-PCR檢測(cè)多個(gè)核心晝夜節(jié)律基因的表達(dá)(Cry1、Cry2、Per1、Per2、Clock、BMAL1). 結(jié)果：(1)獲取的骨髓間充值干細(xì)胞呈長(zhǎng)梭形,貼壁生長(zhǎng),流式細(xì)胞儀檢測(cè)顯示CD29、CD44高表達(dá),CD34低表達(dá)在經(jīng)過(guò)成骨、成脂肪和成軟骨誘導(dǎo)培養(yǎng)后,von kossa染色示有大量鈣結(jié)節(jié)生成,油紅O染色示有胞漿內(nèi)大量脂滴形成,阿利新藍(lán)染色示細(xì)胞外基質(zhì)中含有大量Ⅱ型膠原 (2)普通培養(yǎng)基中的間充質(zhì)干細(xì)胞增殖加速,而成骨誘導(dǎo)基中的間充質(zhì)干細(xì)胞無(wú)明顯增殖,成骨誘導(dǎo)基中的間充質(zhì)細(xì)胞成熟加速,而普通培養(yǎng)基中的間充質(zhì)干細(xì)胞沒(méi)有分化跡象； (3)細(xì)胞在光照下有明顯的增殖,總mTOR的水平?jīng)]有明顯變化,但mTOR的磷酸化水平增高； (4)受光照的細(xì)胞有更多的鈣結(jié)節(jié)生成,Cry2、Per2、Clock的表達(dá)水平有不同程度的上升。 結(jié)論：(1)密度梯度離心法結(jié)合貼壁法可獲得均一、穩(wěn)定、可靠的間充質(zhì)干細(xì)胞； (2)低能量非相干光可以促進(jìn)普通培養(yǎng)基中的間充質(zhì)干細(xì)胞增殖,不能促進(jìn)其向成骨分化,但可促進(jìn)成骨誘導(dǎo)基中的間充質(zhì)干細(xì)胞成骨分化并使其增殖速度減緩； (3)低能量非相干光可促進(jìn)間充質(zhì)干細(xì)胞增殖并激活mTOR信號(hào)通路； (4)晝夜節(jié)律基因可能參與調(diào)控低能量非相干光誘導(dǎo)的間充質(zhì)干細(xì)胞成骨分化。
[Abstract]:Objective: (1) to obtain homogeneous, stable and reliable rat bone marrow mesenchymal stem cells (BMSCs), (2) to investigate the effects of light emitting diode (620nm) incoherent light on the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). (3) to study the changes of mTOR signal protein in the process of cell proliferation stimulated by low energy light, and (4) to study the changes of circadian rhythm gene in osteogenic differentiation induced by low energy light. Methods: (1) Mesenchymal stem cells were isolated by density gradient centrifugation combined with adherent method. The morphology of mesenchymal stem cells was observed under microscope. The surface markers of mesenchymal stem cells were detected by flow cytometry and used as osteoblasts of mesenchymal stem cells. The differentiation ability of the cells was evaluated by von kossa staining, oil red O staining and alimine blue staining. (2) Bone marrow mesenchymal stem cells were cultured in normal culture medium and osteoblast induction medium, and the mesenchymal stem cells were divided into four groups. The cell proliferation was observed by CCK-8 and Edu labeling after irradiation with incoherent light of 620nm at the doses of 0J / cm ~ 2 and 4J / cm ~ 2, respectively. The specific activity of ALP was assayed by von kossa staining and various osteoblastic marker genes (Colla1 / Alpllap and Runx2) RT-PCR were used to evaluate the osteogenic differentiation of the cells. (3) the proliferation of mesenchymal stem cells (MSCs) was measured by Western-blot, and the total mTOR and phosphorylated mTOR protein were detected by Western-blot. (4) 620nm LED incoherent light irradiation was used to observe the osteoblast differentiation in osteoblast induced medium by von kossa staining. The expression of several core circadian rhythmic genes (Cry1, Cry2PER1, Per1, Per2P1, ClockBMAL1) was detected by RT-PCR. Results: (1) the bone marrow filled stem cells were spindle-shaped and adherent to the wall. Flow cytometry showed that the high expression of CD29 and CD44 and the low expression of CD34 were found in osteogenic, adipogenic and chondrogenic culture, and von kossa staining showed that a large number of calcium nodules were formed. Oil red O staining showed a large number of lipid droplets in the cytoplasm, and Alixin blue staining showed the proliferation of mesenchymal stem cells in the extracellular matrix containing a large number of type 鈪，

本文編號(hào)：2060117