补体复合物在大鼠骨性关节炎模型中表达的研究

发布时间：2018-01-03 07:03

  本文关键词：补体复合物在大鼠骨性关节炎模型中表达的研究　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 骨性关节炎 补体 补体复合物 软骨 滑膜

【摘要】：背景及目的:骨性关节炎(OA)在全世界范围内都被人们所熟知,主要累及膝关节,是老人下肢致残的主要危险因素。国内外众多学者多年来进行大量不懈的研究,但其确切的病因及发病机制仍不甚明了。OA治疗主要针对三个方面:减轻疼痛、改善功能、延缓或阻止疾病进展,但现阶段仍缺乏早期有效的治疗,治疗方法主要包括非药物保守治疗、非甾体类抗炎药缓解疼痛、氨基糖苷类等营养软骨药以及关节润滑剂透明质酸钠。但这些治疗均不针对发病病理机制,不能很好的控制疾病进展,最终患者不得不进行关节置换,不仅给患者造成负担,也给国家卫生健康系统造成巨大经济负担。因此深入探究OA病理损伤机制,早期针对病因预防及治疗至关重要。越来越多的研究表明OA中,滑膜是低级别的,滑膜炎不是造成软骨缺损的主要原因。早在上世纪就有研究指出OA滑膜及软骨中存在补体系统的激活,但是补体终末复合物MAC在滑膜及软骨中是否都能最终形成,滑膜和软骨谁起主要的病理角色仍不甚明了。本实验通过建立大鼠骨性关节炎模型探讨补体复合物MAC是否在OA软骨及滑膜中起一定的病理作用,是否是OA进展中的主要病理因素之一。方法:第一部分动物模型建立及标本检测1)动物模型建立及病理评分:将30只SD大鼠随机分为对照组、4周组、8周组、12周组。手术组每组10只,取大鼠后肢一侧膝关节行前交叉韧带切断加部分内侧半月板切除术制造骨性关节炎模型;另一侧仅切开皮肤暴露关节腔作为对照组。手术后4周,8周及12周取两侧膝关节软骨行大体观察、HE染色、番红O-固绿染色、甲苯胺蓝染色,估计软骨退变程度以及进行组织病理学的Mankin评分,滑膜仅行HE染色后行病理评分。2)应用免疫组化、Western Blot及Elisa技术检测软骨及滑膜中MAC沉积情况。第二部分体外软骨细胞培养及补体攻击实验1)软骨细胞原代培养及鉴定:原代培养大鼠软骨细胞,应用甲苯胺蓝染色及Ⅱ型胶原免疫荧光鉴定软骨细胞。2)补体攻击实验及免疫荧光检测MAC形成:取不同浓度血清攻击软骨细胞,并行免疫荧光检测MAC在软骨细胞表面沉积情况。结果:第一部分动物模型建立及标本检测1)动物模型建立及病理评分:与对照组相比,手术组大鼠膝关节软骨4周,8周及12周后呈现软骨粗糙,糜烂,裂隙形成,缺损及骨赘形成等早中晚期OA表现。Mankin评分:对照组(0.11±0.17)分,4周组(3.89±1.31)分,8周组(8.17±1.67)分,12周组(12.89±1.05)分,各组间相比均有统计学差异(p0.01),损伤逐渐加重。滑膜评分:对照组:(0.00±0.00)分,1周组:(3.47±0.25)分,2周组:(4.58±0.51)分,4周组:(5.17±0.75)分,8周组:(4.17±0.75)分,12周组:(2.83±0.75)分,各组间有统计学意义(p0.01),炎症逐渐降低。2)免疫组化软骨细胞细胞膜周围出现环形MAC沉积,滑膜中没有MAC。蛋白质印迹发现实验组有MAC条带,对照组没有。应用Elisa法检测后,可见OA软骨加入血清后随着孵育时间延长,MAC量逐渐增加,而滑膜与PBS则趋势相近。第二部分体外软骨细胞培养及补体攻击实验1)软骨细胞原代培养及鉴定:软骨细胞生长良好,呈三角形、多边形或长梭形。甲苯胺蓝染色后可见软骨细胞呈多边形、三角形、长梭形或类圆形,软骨细胞基质被染成浅蓝色,细胞核染成深蓝。Ⅱ型胶原免疫荧光可见细胞质染成红色。细胞呈现多边形或类圆形。2)补体攻击实验及免疫荧光检测MAC形成:补体攻击软骨细胞后,免疫荧光可见软骨细胞失去正常长梭形、多边形或三角形的形态,细胞变圆,细胞质减少,MAC沉积在细胞膜表面,呈一圈分布。结论:应用膝关节行前交叉韧带切断加部分内侧半月板切除术可成功制造出早中晚期骨性关节炎模型。补体复合物主要作用于软骨而非滑膜,OA中滑膜炎是低级别的,补体系统在OA进展中扮演着重要的病理角色。
[Abstract]:Background and objective: osteoarthritis (OA) in the world are well known, mainly involving the knee, were the main risk factors of the elderly lower limb disability. Many scholars at home and abroad to study a large number of years of unremitting, but the exact etiology and pathogenesis is still not very clear.OA treatment mainly for three a: to relieve pain, improve function, delay or prevent the progression of the disease, but it is lack of effective early treatment, the treatment methods included conservative treatment of non drug, non steroidal anti-inflammatory drugs to alleviate the pain, aminoglycosides and joint cartilage nutrition medicine lubricant with sodium hyaluronate. But these treatments are not according to the pathological pathogenesis, progress of disease control is not good, finally had to patients with joint replacement, not only a burden to the patients, but also caused a huge economic burden to the national health system. So deep Study on Mechanism of OA injury, early prevention and treatment for essential cause. More and more studies show that OA in synovium is low level, the main reason is not caused by synovitis of cartilage defect. In the early studies have pointed out the existence of the complement system activation of OA in synovium and articular cartilage, but the terminal complement complex MAC in the synovium and cartilage can be formed, the cartilage and synovium of who the main pathological role is still unclear. In this experiment, through the establishment of rat osteoarthritis model to complement complex MAC whether a pathological role in OA cartilage and synovium, whether is one of the main pathological factors in the progression of OA. Methods: in the first part of the animal model establishment and test specimens of 1) animal model establishment and pathological score: 30 SD rats were randomly divided into control group, 4 week group, 8 week group, 12 week group. The operation group with 10 rats in each group of rats Hind knee joint anterior cruciate ligament transection and partial medial meniscectomy manufacturing osteoarthritis model; the other side is only skin incision exposed articular cavity as the control group. 4 weeks after surgery, 8 weeks and 12 weeks on both sides of knee articular cartilage for gross observation, HE staining, safranin O- fast green staining, toluidine blue staining. To estimate the degree of cartilage degeneration and pathological Mankin score, HE staining was only synovial pathological score.2) immunohistochemistry, Western Blot and Elisa MAC deposition technique for the detection of cartilage and synovium. The second part in soft bone cell culture and complement attack Experiment 1) primary chondrocytes culture and identification: the original cultured rat chondrocytes, toluidine blue staining and type II collagen immunofluorescence identification of chondrocyte.2) complement attack experiment and immunofluorescence detection of MAC formation: different concentrations of serum cartilage attack 缁嗚優,骞惰�屽厤鐤�鑽у厜妫，

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