静磁场对SD乳鼠髁突软骨细胞和鼻中隔软骨细胞软骨形成的影响研究

发布时间：2018-04-25 12:32

本文选题：髁突软骨细胞 + 鼻中隔软骨细胞　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]建立静磁场作用下SD乳鼠原代下颌髁突软骨细胞(Mandibular Condylar Chondroc-yte,MCC)和鼻中隔软骨细胞(Nasal Septal Chondrocyte,NSC)体外培养模型,观察静磁场对髁突软骨细胞和鼻中隔软骨细胞软骨形成的影响及差异,为进一步深入研究静磁场作用下Flrt-2对髁突软骨和鼻中隔软骨形成过程的影响及内部调控机制提供指导,为后续探讨静磁场对颌面部生长发育的影响提供理论基础。[方法]取出生1～3天的SD乳鼠髁突软骨和鼻中隔软骨,用机械分离和酶联消化法获取原代细胞,在相同培养条件下进行原代培养和软骨细胞鉴定。对两种细胞分别加载280mT静磁场,通过阿辛蓝染色检测静磁场对两种软骨细胞糖胺聚糖(Glycosaminoglycan,GAG)表达差异的影响;通过CCK-8(Cell Counting Kit-8,CCK-8)比色法描绘软骨细胞生长曲线,检测静磁场对两种软骨细胞增殖的影响差异;划痕实验观察静磁场作用下两种细胞划痕24h后细胞迁移的能力;蛋白质印迹法(WesternBlot,WB)检测加载静磁场后两种软骨细胞Flrt-2和N-cadherin蛋白的表达差异。[结果]采用相同的消化方式及培养条件成功培养出原代髁突软骨细胞和鼻中隔软骨细胞,Ⅱ型胶原免疫组化染色和阿辛蓝染色细胞鉴定结果均呈阳性。两种软骨细胞均分为空白组和磁场组,在大部分细胞贴壁后,磁场组细胞载280mT磁场继续培养。四组软骨细胞在第1、3、5、8天时进行阿辛蓝染色,染色结果均呈阳性,鼻中隔软骨细胞糖胺聚糖表达量及软骨结节形成能力均高于髁突软骨细胞,两种细胞加载磁场组糖胺聚糖表达量及软骨结节形成能力均较空白组细胞高;四组软骨细胞CCK-8比色法检测出两种软骨细胞的生长曲线基本一致,鼻中隔软骨细胞的增殖能力较髁突软骨细胞强,加载静磁场后两种软骨细胞增殖能力均增强;24h划痕实验结果显示鼻中隔软骨细胞迁移能力强于髁突软骨细胞,磁场组细胞迁移能力强于空白组细胞。两种细胞在培养第1、3、5、8天时对Flrt-2、N-cadherin蛋白表达进行检测,发现在软骨细胞凝集期,Flrt-2蛋白表达下调,N-cadherin上调;凝集期过后Flrt-2蛋白表达上调,N-cadherin下调。加载静磁场后,两种软骨细胞Flrt-2蛋白表达增加,N-cadherin蛋白表达则下降。[结论]1.鼻中隔软骨细胞糖胺聚糖表达及软骨结节形成能力均高于髁突软骨细胞,加载静磁场后两种软骨细胞糖胺聚糖表达及软骨结节形成能力均较空白组高。2.鼻中隔软骨细胞增殖和迁移能力均强于髁突软骨细胞,加载静磁场后两种软骨细胞增殖和迁移能力均增强。3.软骨细胞凝集期,Flrt-2蛋白表达下调,N-cadherin上调;凝集期过后Flrt-2蛋白表达上调,N-cadherin下调。加载静磁场后,两种软骨细胞Flrt-2蛋白表达增加,N-cadherin蛋白表达则下降。
[Abstract]:[objective] to establish the model of primary mandibular condylar chondroc-chondroc-chondroc (Condylar) and nasal septal chondrocyte (NSCC) cultured in neonatal SD rats under the action of magnetostatic magnetic field, and to observe the effect and difference of magnetic field on cartilage formation of condylar chondrocytes and nasal septal chondrocytes. In order to further study the effect of Flrt-2 on the formation of condylar cartilage and nasal septal cartilage and provide guidance for further study of the effect of magnetostatic magnetic field on the growth and development of maxillofacial region, the theoretical basis is provided for the further study of the effect of magnetostatic magnetic field on the growth and development of maxillofacial region. [methods] the condylar cartilage and nasal septal cartilage of SD rats were taken out for 3 days. Primary cells were obtained by mechanical separation and enzyme-linked digestion. Primary culture and chondrocyte identification were carried out under the same culture conditions. The effect of 280mT static magnetic field on the expression of glycosaminoglycan Gage in two kinds of chondrocytes was detected by acinyl blue staining, and the growth curve of chondrocytes was described by CCK-8(Cell Counting Kit-8 CCK-8 colorimetry. The effect of static magnetic field on the proliferation of two chondrocytes was examined, and the migration ability of the two cells after 24 hours of scratch was observed by scratch test. The expression of Flrt-2 and N-cadherin protein in chondrocytes was detected by Western blotblotWB after static magnetic field loading. [results] the primary condylar chondrocytes and nasal septal chondrocytes were successfully cultured in the same digestion mode and culture conditions. The two kinds of chondrocytes were divided into blank group and magnetic field group. After most of the cells were adhered to the wall, the magnetic field of the cells loaded with 280mT in the magnetic field group continued to grow. The four groups of chondrocytes were stained with acinyl blue on the 1st and 3rd day. The results showed that the expression of glycosaminoglycan and the ability of forming chondrocytes in nasal septal chondrocytes were higher than those in condylar chondrocytes. The expression of glycosaminoglycan and the ability of forming chondrocytes in the two groups were higher than those in the blank group, and the growth curves of the two chondrocytes were similar by CCK-8 colorimetry. The proliferation ability of nasal septal chondrocytes was stronger than that of condylar chondrocytes. The results of 24 h scratch test showed that the migration ability of nasal septal chondrocytes was stronger than that of condylar chondrocytes. The migration ability of the cells in the magnetic field group was stronger than that in the blank group. The expression of Flrt-2N-cadherin protein was detected on the 8th day of culture in two kinds of cells. It was found that the expression of Flrt-2 protein was down-regulated in chondrocyte agglutination phase, and the expression of Flrt-2 protein was up-regulated after the agglutination phase, and the expression of N-cadherin was down-regulated. The expression of Flrt-2 protein in the two chondrocytes increased and the expression of N-cadherin decreased after static magnetic field loading. [conclusion] 1. The expression of glycosaminoglycan and the ability of forming cartilage nodules in nasal septal chondrocytes were higher than those in condylar chondrocytes. The proliferation and migration ability of nasal septal chondrocytes was stronger than that of condylar chondrocytes. The expression of Flrt-2 protein in chondrocytes was down-regulated and the expression of Flrt-2 protein was up-regulated after the agglutination stage. The expression of Flrt-2 protein in the two chondrocytes increased and the expression of N-cadherin decreased after static magnetic field loading.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782

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