本文關(guān)鍵詞：補(bǔ)體復(fù)合物在大鼠骨性關(guān)節(jié)炎模型中表達(dá)的研究　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨性關(guān)節(jié)炎 補(bǔ)體 補(bǔ)體復(fù)合物 軟骨 滑膜

【摘要】：背景及目的:骨性關(guān)節(jié)炎(OA)在全世界范圍內(nèi)都被人們所熟知,主要累及膝關(guān)節(jié),是老人下肢致殘的主要危險(xiǎn)因素。國(guó)內(nèi)外眾多學(xué)者多年來進(jìn)行大量不懈的研究,但其確切的病因及發(fā)病機(jī)制仍不甚明了。OA治療主要針對(duì)三個(gè)方面:減輕疼痛、改善功能、延緩或阻止疾病進(jìn)展,但現(xiàn)階段仍缺乏早期有效的治療,治療方法主要包括非藥物保守治療、非甾體類抗炎藥緩解疼痛、氨基糖苷類等營(yíng)養(yǎng)軟骨藥以及關(guān)節(jié)潤(rùn)滑劑透明質(zhì)酸鈉。但這些治療均不針對(duì)發(fā)病病理機(jī)制,不能很好的控制疾病進(jìn)展,最終患者不得不進(jìn)行關(guān)節(jié)置換,不僅給患者造成負(fù)擔(dān),也給國(guó)家衛(wèi)生健康系統(tǒng)造成巨大經(jīng)濟(jì)負(fù)擔(dān)。因此深入探究OA病理?yè)p傷機(jī)制,早期針對(duì)病因預(yù)防及治療至關(guān)重要。越來越多的研究表明OA中,滑膜是低級(jí)別的,滑膜炎不是造成軟骨缺損的主要原因。早在上世紀(jì)就有研究指出OA滑膜及軟骨中存在補(bǔ)體系統(tǒng)的激活,但是補(bǔ)體終末復(fù)合物MAC在滑膜及軟骨中是否都能最終形成,滑膜和軟骨誰起主要的病理角色仍不甚明了。本實(shí)驗(yàn)通過建立大鼠骨性關(guān)節(jié)炎模型探討補(bǔ)體復(fù)合物MAC是否在OA軟骨及滑膜中起一定的病理作用,是否是OA進(jìn)展中的主要病理因素之一。方法:第一部分動(dòng)物模型建立及標(biāo)本檢測(cè)1)動(dòng)物模型建立及病理評(píng)分:將30只SD大鼠隨機(jī)分為對(duì)照組、4周組、8周組、12周組。手術(shù)組每組10只,取大鼠后肢一側(cè)膝關(guān)節(jié)行前交叉韌帶切斷加部分內(nèi)側(cè)半月板切除術(shù)制造骨性關(guān)節(jié)炎模型;另一側(cè)僅切開皮膚暴露關(guān)節(jié)腔作為對(duì)照組。手術(shù)后4周,8周及12周取兩側(cè)膝關(guān)節(jié)軟骨行大體觀察、HE染色、番紅O-固綠染色、甲苯胺藍(lán)染色,估計(jì)軟骨退變程度以及進(jìn)行組織病理學(xué)的Mankin評(píng)分,滑膜僅行HE染色后行病理評(píng)分。2)應(yīng)用免疫組化、Western Blot及Elisa技術(shù)檢測(cè)軟骨及滑膜中MAC沉積情況。第二部分體外軟骨細(xì)胞培養(yǎng)及補(bǔ)體攻擊實(shí)驗(yàn)1)軟骨細(xì)胞原代培養(yǎng)及鑒定:原代培養(yǎng)大鼠軟骨細(xì)胞,應(yīng)用甲苯胺藍(lán)染色及Ⅱ型膠原免疫熒光鑒定軟骨細(xì)胞。2)補(bǔ)體攻擊實(shí)驗(yàn)及免疫熒光檢測(cè)MAC形成:取不同濃度血清攻擊軟骨細(xì)胞,并行免疫熒光檢測(cè)MAC在軟骨細(xì)胞表面沉積情況。結(jié)果:第一部分動(dòng)物模型建立及標(biāo)本檢測(cè)1)動(dòng)物模型建立及病理評(píng)分:與對(duì)照組相比,手術(shù)組大鼠膝關(guān)節(jié)軟骨4周,8周及12周后呈現(xiàn)軟骨粗糙,糜爛,裂隙形成,缺損及骨贅形成等早中晚期OA表現(xiàn)。Mankin評(píng)分:對(duì)照組(0.11±0.17)分,4周組(3.89±1.31)分,8周組(8.17±1.67)分,12周組(12.89±1.05)分,各組間相比均有統(tǒng)計(jì)學(xué)差異(p0.01),損傷逐漸加重�；ぴu(píng)分:對(duì)照組:(0.00±0.00)分,1周組:(3.47±0.25)分,2周組:(4.58±0.51)分,4周組:(5.17±0.75)分,8周組:(4.17±0.75)分,12周組:(2.83±0.75)分,各組間有統(tǒng)計(jì)學(xué)意義(p0.01),炎癥逐漸降低。2)免疫組化軟骨細(xì)胞細(xì)胞膜周圍出現(xiàn)環(huán)形MAC沉積,滑膜中沒有MAC。蛋白質(zhì)印跡發(fā)現(xiàn)實(shí)驗(yàn)組有MAC條帶,對(duì)照組沒有。應(yīng)用Elisa法檢測(cè)后,可見OA軟骨加入血清后隨著孵育時(shí)間延長(zhǎng),MAC量逐漸增加,而滑膜與PBS則趨勢(shì)相近。第二部分體外軟骨細(xì)胞培養(yǎng)及補(bǔ)體攻擊實(shí)驗(yàn)1)軟骨細(xì)胞原代培養(yǎng)及鑒定:軟骨細(xì)胞生長(zhǎng)良好,呈三角形、多邊形或長(zhǎng)梭形。甲苯胺藍(lán)染色后可見軟骨細(xì)胞呈多邊形、三角形、長(zhǎng)梭形或類圓形,軟骨細(xì)胞基質(zhì)被染成淺藍(lán)色,細(xì)胞核染成深藍(lán)。Ⅱ型膠原免疫熒光可見細(xì)胞質(zhì)染成紅色。細(xì)胞呈現(xiàn)多邊形或類圓形。2)補(bǔ)體攻擊實(shí)驗(yàn)及免疫熒光檢測(cè)MAC形成:補(bǔ)體攻擊軟骨細(xì)胞后,免疫熒光可見軟骨細(xì)胞失去正常長(zhǎng)梭形、多邊形或三角形的形態(tài),細(xì)胞變圓,細(xì)胞質(zhì)減少,MAC沉積在細(xì)胞膜表面,呈一圈分布。結(jié)論:應(yīng)用膝關(guān)節(jié)行前交叉韌帶切斷加部分內(nèi)側(cè)半月板切除術(shù)可成功制造出早中晚期骨性關(guān)節(jié)炎模型。補(bǔ)體復(fù)合物主要作用于軟骨而非滑膜,OA中滑膜炎是低級(jí)別的,補(bǔ)體系統(tǒng)在OA進(jìn)展中扮演著重要的病理角色。
[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 緇嗚優(yōu),騫惰鍏嶇柅鑽у厜媯，

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