周期性动态压缩应力对海藻酸钠立体培养敲减SOX9基因骨髓间充质干细胞软骨分化的实验研究

发布时间：2018-04-02 10:54

  本文选题：软骨分化　切入点：海藻酸钠　出处：《山西医科大学》2015年硕士论文

【摘要】：目的:观察周期性动态压缩应力对骨髓间充质干细胞软骨分化的作用,同时对比分析力学因素对敲减SOX9基因骨髓间充质干细胞软骨分化过程中的作用,探讨力学因素与SOX9在软骨分化过程中的重要作用。方法:取4周龄昆明小鼠骨髓间充质干细胞(C),体外培养至第三代,流式细胞仪鉴定细胞表型确定为干细胞,将构建的小鼠SOX9基因干扰的慢病毒载体体外转染小鼠骨髓间充质干细胞,构建SOX9敲减的骨髓间充质干细胞(si C)。分别将C细胞和si C细胞以1x107/ml的密度与2%海藻酸钠凝胶混匀,制成圆柱形载体。根据实验过程中对载体的处理因素的不同将试验分为8组:各组在试验的前7天均给予软骨诱导液处理。A组:C细胞加力未诱导组:在软骨诱导液诱导7天后再给予7天的加力未诱导处理;B组:C细胞加力诱导组:在软骨诱导液诱导7天后再给予7天的加力诱导处理;C组:诱导不加力组:在软骨诱导液诱导7天后再给予7天的诱导不加力处理;D组:si C细胞加力未诱导组;E组:si C细胞加力诱导组;F组:si C细胞诱导不加力组;D、E、F组的处理分别同A、B、C组。G、H组为分别为C细胞、si C细胞未加力、未诱导的对照组:诱导7天后予以普通培养液培养;实验采用Flexcell-5000基底加载系统对细胞盘施加正弦波、0.5HZ、20Kpa、2h/d压缩应力。在相关处理后采用RT-PCR分析SOX9、ROCK1及COL-Ⅱ的表达。应用免疫荧光技术对各组处理前后的COL-Ⅱ表达进行监测。结果:RT-PCR检测:SOX9及COL-Ⅱm RNA表达:B组高于C组及A组,C组较G组表达高,E组高于F组及D组,D组高于H组(P0.05)。A组与G组表达相当,B组及E组表达无显著差异(P0.05)、ROCK1m RNA:E组高于B组(P0.05)。免疫荧光检测显示B组及E组均有二型胶原免疫荧光表达。结论:周期性动态压缩应力可以促进间充质干细胞的软骨分化。周期性动态力学加载可以增加敲减SOX9基因骨髓间充质干细胞的SOX9、COL-Ⅱ的表达,降低ROCK1的表达,周期性动态压缩应力可能通过SOX9相关通路促进间充质干细胞的软骨分化。
[Abstract]:Aim: to observe the effect of cyclic dynamic compression stress on the differentiation of bone marrow mesenchymal stem cells (MSCs), and to compare the effects of mechanical factors on the differentiation of bone marrow mesenchymal stem cells (BMSCs) from SOX9 gene. Methods: bone marrow mesenchymal stem cells (BMSCs) of 4-week-old Kunming mice were cultured to the third generation in vitro and identified as stem cells by flow cytometry. Murine SOX9 gene interfering lentivirus vector was transfected into mouse bone marrow mesenchymal stem cells in vitro, and bone marrow mesenchymal stem cells (BMSCs) knocked out by SOX9 were constructed. C cells and si C cells were mixed with 2% sodium alginate gel and 1x107/ml density, respectively. The experiment was divided into 8 groups according to the different factors of treatment of the carrier during the experiment. Each group was treated with cartilage inducer on the first 7 days of the experiment. Group B: after 7 days of cartilage induction, 7 days after 7 days of fluid induction, group C: group C: group B: induced by chondrocytes: group C: group B: induced by chondrocytes: group B: group B: group B: group B: group B: induced by chondrocytes: group B: group B: after 7 days of induction of cartilage: group C: group C: induced by chondrocytes. After 7 days of induction, 7 days after induction, no additional treatment was given. Group D, group D, group D, group E, group E, group E, group E, group E, group E, group E, group E, group E, group C, group C, group C, group C, group D, group D, group F, treatment, respectively. Si C cells were not stimulated. Uninduced control group: after 7 days of induction, the medium was cultured in normal medium. The Flexcell-5000 substrate loading system was used to apply 2h / d compression stress to the cell disk. After the correlation treatment, the expressions of SOX9 roCK1 and COL- 鈪，

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