【作者】 王华；

【导师】 刘世清；

【作者基本信息】 武汉大学 ， 外科学， 2011， 博士

【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。更多还原

【Abstract】 Objective:Along with our country economy developed at top speed and life-span of population is gradually prolong, the number of patients who suffered osteoarthritis is increased. In our country, the morbidity of osteoarthritis is about 70% in population older than 75. In our country every year the osteoarthritis causes the labor force loses and the expense which produces with the pharmacological treatment to be huge, it also has brought the huge pain for the patient and their family. The number of who . like sports is increased by enhanced of quality of life, but the inappropriate movement is very easy to cause articular cartilage’s injury. The articular cartilage injury is quite common in the patient who suffered knee joint disease.The method of therapy injury of cartilage are contain joint arthroscope, drilling, microfracture, autologous chondrocyte implantation, mosaicplasty. For the aim to ACI and let chondrocyte keep its phenotype, so in the first party of my study we have selected the human articular cartilage to carry on the experiment, and we discussion raises and separates the human cartilage cell’s method. Although the difficult mechanics function not to have understood in cartilage cell’s mechanism, But certain mechanics function has to the articular cartilage cell promotes its secretion cartilage matrix and repairs articular cartilage’s function to obtain everybody approval. In the second party of my study the experiment attempts through the different size interrupted cutting devoting to use in the person articular cartilage cell, observed the change of chondrocyte secretion protein polysaccharide and collagen, Interrupted shearing force to cartilage matrix metabolism, the effect of chondrocyte proliferation. It will help us later carry on the recovery exercise,for us the articular cartilage will damage provides the direct basis.Method:The first part of experiment is human articular cartilage which obtains by the surgery in is a material, disappears through two enzymes Person articular cartilage cell’s extraction, used high suger DMEM which contain FBS to culture the .chondrocyte, Uses the inverted microscope observation cell growth, Carries on the cell counting with the counting board, than plan cell growth curve. We will gong obtain the human articular cartilage cell undergoes the original generation of raise to multiply next the toluidine blue dyeing examination cartilage cell table. MTT color method estimate cell multiplication situation.The seconds parts of experiments is used the second generation of human chondrocyte which obtains by the first part of experiment. Then let the seconds generation of human chondrocyte arrange to 4 group:control group, low speed group(20rpm), middle speed group(40rpm) and high speed group(60rpm).Simulates the articular cartilage cell to receive the different size the shearing force. The blank control group puts in 5%CO2,37℃incubator to static cultivation. Every day puts the different rotational group human chondrocyte on the rotational table to affect according to the hypothesis rotational speed 2h. (is equal in giving interrupted shearing force), daily two times, previous day replaced the nutrient fluid.Through MTT examination cell multiplication, Rt-PCR examination Aggrecan mRNA and TypeⅡCollagen, the mRNA expression appraises the different size interrupted shearing force to the person cartilage cell multiplication and the secretion protein polysaccharide function.Result:1. Digests the human chondrocyte using two step enzyme digestion method to have the high efficiency at the same time cell harvest to be also big, the original generation of human chondrocyte survival rate which obtains is high.2. The original generation of human chondrocyte undergoes the sugar DMEM culture medium raise to be possible to obtain the cartilage phenotype complete human chondrocyte, it can secrete the protein polysaccharide normally, the cell frozen to the wall is good, may take mechanics function the material.3. The certain intensity interrupted shearing force can obviously affect the human chondrocyte’s metabolism, the multiplication activity, causes its synthesis Aggrecan and TypeⅡCollagen increases.4. This experiment’s result and clinical and the basic research the understanding which damages regarding the osteoarthritis and the cartilage has the strong relatedness, it is the necessary further deep research. The findings also prompt the treatment which and the recovery stage use nonnegative heavy joint damages in the cartilage exercise (i.e. interrupted shearing force), it possibly obtains a better result.更多还原