剪切力对体外培养人软骨细胞代谢的影响

发布时间：2018-05-09 08:14

  本文选题：软骨细胞 + 应力　； 参考：《武汉大学》2011年博士论文

【摘要】：目的： 随着我国经济的高速发展,人口预期寿命逐步延长,我国骨关节炎的发病人数也呈高增长趋势。在我国75岁以上人群中,骨关节炎的发病率为70%；每年由于骨关节炎导致的劳动力丧失和药物治疗产生的费用庞大,给患者及其家庭带来了巨大的痛苦。 随着生活水平的不断提高,体育爱好者也越来越多,而不恰当的体育运动也很容易引起关节软骨的损伤,关节软骨损伤在膝关节疾病患者中比较常见。 关节软骨损伤的治疗方法目前主要有关节清理、钻孔,自体骨膜或软骨膜移植,微骨折(Microfracture),自体软骨细胞移植(Autologous chondrocyte implantation, ACI)和马赛克成形术(Mosaicplasty)等,其中后3种治疗方法的疗效已得到了公认。为了进行自体软骨细胞移植(ACI)和寻求使软骨细胞保持正常表型以便进行力学干预实验,均需进行体外人关节软骨细胞的分离,培养和鉴定。本课题第一部分选用人关节软骨进行体外培养,探讨了分离及培养人关节软骨细胞的方法。虽然力学作用于软骨细胞的机制还未明了,但一定的力学作用对关节软骨细胞有着促进其分泌软骨基质并修复关节软骨的作用已得到大家认可。本课题第二部分实验试图通过将不同程度的间断流体剪切力作用于人关节软骨细胞,观测软骨细胞分泌蛋白多糖和胶原蛋白的能力变化,探讨人关节软骨细胞体外培养适宜的力学条件,以及间断流体剪切力对软骨基质代谢、软骨细胞增殖的影响,为以后进行关节软骨损伤的修复和康复锻炼提供直接的依据。 方法： 第一部分实验以手术中获得的人关节软骨为材料,通过改良二步酶消化法进行人关节软骨细胞的提取,采用含小牛血清的高糖型DMEM培养基进行培养,采用倒置显微镜观察细胞生长情况,用计数板进行细胞计数,绘制细胞生长曲线；将获得的人关节软骨细胞经过原代培养增殖后行甲苯胺蓝染色检测软骨细胞表型,MTT比色法估计细胞增殖情况。 第二部分实验以第一部分实验获得的第二代人软骨细胞为材料,将第二代软骨细胞按摇床作用时不同的转速分为：控制组(空白对照组)；低转速组(20转/分)；中等转速组(40转/分)；高转速组(60转/分),共4组；来模拟关节软骨细胞所受到不同大小的剪切力。空白对照组置于5%CO2,37℃培养箱中静止培养；每天将不同转速组人软骨细胞置于摇床上按设定转速作用2h(相当于给予间断剪切力作用),每天两次,隔天更换培养液。 通过MTT法检测细胞增殖,甲苯胺蓝染色判断软骨细胞表型,软骨细胞爬片Ⅱ型胶原免疫组化检测,Rt-PCR法检测聚集蛋白聚糖(Aggrecan) mRNA及Ⅱ型胶原(TypeⅡCollagen) mRNA表达来评估不同大小间断剪切力对人软骨细胞增殖和分泌蛋白多糖功能的作用。 实验结果显示： 1.运用改良二步酶消化法消化人关节软骨具有高效率的同时细胞收获量也较大,获得的原代人关节软骨细胞成活率高。 2.原代人关节软骨细胞经过高糖DMEM培养基培养可以获得软骨表型完好的人关节软骨细胞,能正常分泌蛋白多糖,细胞附壁良好,可以作为力学作用的材料细胞。 3.一定强度间断剪切力能够显著影响人关节软骨细胞的代谢、增殖活动,使其合成聚集蛋白聚糖(Aggrecan)及Ⅱ型胶原(TypeⅡCollagen)增多。 4.本实验的结果与临床和基础研究对于骨关节炎和软骨损伤的认识有较强的关联性,有必要进一步深入研究。研究结果还提示在软骨损伤的治疗和康复阶段使用非负重的关节锻炼方式(即间断剪切力作用)可能得到更好的结果。
[Abstract]:Objective:
With the rapid development of China's economy, the life expectancy of the population is gradually extended, and the number of osteoarthritis in China is also increasing. The incidence of osteoarthritis is 70% in the population over 75 years of age in our country. The cost of labor loss and drug treatment caused by osteoarthritis is huge and brought to the patients and their families. Great pain.
With the continuous improvement of living standards, sports enthusiasts are becoming more and more, and inappropriate sports can also easily cause joint cartilage damage. Articular cartilage damage is more common in patients with knee joint diseases.
At present, the treatment of articular cartilage injury mainly includes joint cleaning, drilling, autogenous periosteal or periosteal transplantation, micro fracture (Microfracture), autologous chondrocyte transplantation (Autologous chondrocyte implantation, ACI) and mosaic plasty (Mosaicplasty). The effect of the latter 3 methods has been recognized. The body cartilage cell transplantation (ACI) and the search for the cartilage cells to maintain normal phenotype in order to carry out the mechanical intervention experiments, the separation, culture and identification of human articular chondrocytes in vitro are needed. The first part of this topic is to culture human articular cartilage in vitro, and explore the methods of separating and cultivating human articular chondrocytes. The mechanism of chondrocytes is not clear, but the effect of certain mechanical action on articular cartilage cells to promote the secretion of cartilage matrix and repair articular cartilage has been recognized. The second part of the experiment is trying to observe the cartilage cell by using different degrees of intermittent fluid shear force on human articular cartilage cells. The changes in the ability of secreting proteoglycan and collagen to explore the suitable mechanical conditions for the culture of human articular cartilage cells in vitro, and the effect of intermittent fluid shear force on cartilage matrix metabolism and chondrocyte proliferation, provide a direct basis for the repair and rehabilitation of articular cartilage injury.
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本文编号：1865251