周期性机械应力条件下上调整合素表达促进大鼠软骨细胞功能

发布时间：2019-01-04 12:47

【摘要】：目的探讨周期性机械应力条件下上调整合素表达量对大鼠软骨细胞的影响。方法1.体外分离培养6-8d的SD大鼠软骨细胞,取第3代接种于无菌6孔板中,随机分为三组,分别为:(1)整合素上调组:将含有整合素过表达质粒的慢病毒转染至大鼠软骨细胞中(2)整合素下调组:将含有整合素低表达质粒的慢病毒转染至大鼠软骨细胞中(3)对照组:将含有整合素空白质粒的慢病毒转染至大鼠软骨细胞中。三组细胞在37℃,5%CO2条件下培养3d后,用荧光显微镜观察细胞的转染情况,Western blot法测定三组细胞整合素表达量。2.将三组细胞分别置于有、无周期性机械应力作用的培养基中培养,周期性机械应力场条件为0-200k Pa、0.1Hz。培养1h后,用Western blot法检测蛋白激酶(ERK1/2)的磷酸化水平;培养8h后,采用实时定量荧光PCR(Real-time PCR)法检测蛋白聚糖(aggrecan)、二型胶原(collagen II)基因表达的水平;加压8h/d,连续培养3d后,用CCK-8法检测细胞增殖。3.将转染过慢病毒的三组细胞分别接种于PLGA支架上,在周期性机械应力条件下(0-200k Pa、0.1Hz)培养6周(每天加压8小时),获取软骨块。观察软骨块大体形态,对软骨块湿重和GAG含量测定,HE和番红染色,二型胶原免疫组化染色。结果1.将三组转染慢病毒的软骨细胞置于荧光显微镜下观察发现,超过80%的软骨细胞已被慢病毒转染,表明三组软骨细胞具有良好的转染效率。Western blot法检测各组整合素β1蛋白的表达水平结果显示整合素过表达组中整合素β1蛋白表达量较对照组明显提高(p0.05),而整合素低表达组中整合素β1蛋白表达量较对照组明显降低(p0.05) 2.整合素过表达后,周期性机械应力场中培养的软骨细胞ERK1/2的磷酸化水平升高(P0.05),蛋白聚糖、二型胶原基因表达的水平升高(P0.05),CCK-8法检测软骨细胞增殖增加(P0.05)。而在没有周期性机械应力刺激条件下培养的三组软骨细胞之间ERK1/2的磷酸化水平没有变化(P0.05),蛋白聚糖、二型胶原基因表达的水平没有明显差异(P0.05),CCK-8法检测软骨细胞增殖无明显改变(P0.05)。3.整合素过表达组软骨块最大,湿重较其他两组显著升高(P0.05),GAG含量增多(P0.05)、二型胶原免疫组化染色阳性面积较其他两组显著升高(P0.05)。结论在周期性机械应力作用下,整合素过表达能促进大鼠软骨细胞功能,提高组织工程软骨质量。
[Abstract]:Objective to investigate the effect of upregulation of integrin expression on rat chondrocytes under cyclic mechanical stress. Method 1. The chondrocytes of SD rats were isolated and cultured for 6-8 days in vitro. The third passage was inoculated in the sterile 6-hole plate and randomly divided into three groups. The results were as follows: (1) integrin up-regulation group: lentivirus containing integrin overexpression plasmid was transfected into rat chondrocytes (2) integrin down-regulation group: lentivirus containing integrin low expression plasmid was transfected into rat soft tissue. (3) control group: lentivirus containing integrin blank plasmid was transfected into rat chondrocytes. After three groups of cells were cultured at 37 鈩，

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