620nm非相干红光对大鼠骨髓间充质干细胞的光生物调节作用

发布时间：2018-06-24 04:58

  本文选题：骨髓间充质干细胞 + 非相干光　； 参考：《华中科技大学》2011年博士论文

【摘要】：目的：(1)获得均一、稳定、可靠的大鼠骨髓间充质干细胞；(2)探讨620nm发光二极管(Light Emitting Diodes, LED)非相干光对大鼠骨髓间充质干细胞在增殖和成骨分化方面的影响；(3)研究在低能量光照促进的细胞增殖过程中mTOR信号蛋白的变化情况；(4)研究在低能量光照促进的成骨分化中昼夜节律基因的变化情况。 方法：(1)密度梯度离心法结合贴壁法分离间充质干细胞,体外扩增,镜下观察细胞形态,用流式细胞仪检测其表面标记物,分别作间充质干细胞的成骨、成脂肪和成软骨诱导培养,采用von kossa染色、油红O染色和阿利新蓝染色鉴定细胞的三向分化能力； (2)获取的骨髓间充质干细胞分别在普通培养基和成骨诱导培养基中培养,两种培养基中的间充质干细胞均分为四组,用620nm LED非相干光分别以0、1、2和4J/cm2的剂量进行照射,照射后采用CCK-8和EdU标记观察细胞增殖情况,采用ALP比活性检测、von kossa染色和多种成骨标志基因(Coll a 1,Alpl,Bglap and Runx2) RT-PCR来评估细胞成骨分化的情况； (3) 620nm LED非相干光照射分别以0、0.5、1和2J/cm2的剂量照射间充质干细胞,CCK-8检测细胞增殖情况,Western-blot检测总mTOR和磷酸化mTOR蛋白含量； (4) 620nm LED非相干光照射分别以0、1、2和4J/cm2的剂量照射成骨诱导培养基中间充质干细胞,von kossa染色观察细胞成骨分化情况,RT-PCR检测多个核心昼夜节律基因的表达(Cry1、Cry2、Per1、Per2、Clock、BMAL1). 结果：(1)获取的骨髓间充值干细胞呈长梭形,贴壁生长,流式细胞仪检测显示CD29、CD44高表达,CD34低表达在经过成骨、成脂肪和成软骨诱导培养后,von kossa染色示有大量钙结节生成,油红O染色示有胞浆内大量脂滴形成,阿利新蓝染色示细胞外基质中含有大量Ⅱ型胶原 (2)普通培养基中的间充质干细胞增殖加速,而成骨诱导基中的间充质干细胞无明显增殖,成骨诱导基中的间充质细胞成熟加速,而普通培养基中的间充质干细胞没有分化迹象； (3)细胞在光照下有明显的增殖,总mTOR的水平没有明显变化,但mTOR的磷酸化水平增高； (4)受光照的细胞有更多的钙结节生成,Cry2、Per2、Clock的表达水平有不同程度的上升。 结论：(1)密度梯度离心法结合贴壁法可获得均一、稳定、可靠的间充质干细胞； (2)低能量非相干光可以促进普通培养基中的间充质干细胞增殖,不能促进其向成骨分化,但可促进成骨诱导基中的间充质干细胞成骨分化并使其增殖速度减缓； (3)低能量非相干光可促进间充质干细胞增殖并激活mTOR信号通路； (4)昼夜节律基因可能参与调控低能量非相干光诱导的间充质干细胞成骨分化。
[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，

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