本文選題：創(chuàng)傷性異位骨化 + 大鼠�。� 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：背景與目的:異位骨化是指軟組織中出現(xiàn)成熟骨組織的病理現(xiàn)象。根據其形成原因分為遺傳性和獲得性異位骨化,其中,以創(chuàng)傷性異位骨化最為多見。目前,隨著創(chuàng)傷(交通傷導致的高能量創(chuàng)傷以及人工關節(jié)置換的廣泛應用等等)的不斷增加,異位骨化的發(fā)病率備受關注,一旦發(fā)生,將給患者帶來長期的疼痛,關節(jié)活動受限等癥狀,嚴重的降低了患者的生活質量。目前,臨床上常用的預防異位骨化的方式包括:非甾體類抗炎藥及小劑量放射治療。但因其干擾骨折愈合過程,胃部刺激或潛在的惡性腫瘤危險等副作用,在臨床上使用受限。異位骨化有效治療方法是手術切除異位骨化的骨組織,但會給患者造成二次損傷。為進一步降低異位骨化發(fā)生率,其分子機制及早期預防的研究具有重要意義。本研究基于大鼠跟腱鉗夾、切斷模型誘導創(chuàng)傷性異位骨化,旨在了解創(chuàng)傷性異位骨化早期相關因子的表達情況,探討其在創(chuàng)傷性異位骨化發(fā)病機制中的作用及意義。方法:取8～10周齡雄性SD大鼠68只,體質量(210.1± 10.6)g,隨機分為實驗組及對照組(n=34)。實驗組通過跟腱鉗夾、切斷法制備創(chuàng)傷性異位骨化模型,對照組僅暴露跟腱,跟腱不作任何處理。術后觀察兩組大鼠一般情況,于3、5、8及14天兩組各處死6只大鼠,取跟腱及周圍組織行大體及組織學染色觀察;同時對GEO數(shù)據庫中異位骨化相關因子表達譜及大量文獻進行分析,篩選出25種相關因子。采用實時定量PCR技術檢測不同時間點25種相關因子的基因表達情況,篩選出有統(tǒng)計學及臨床意義的因子,采用免疫組化等技術進行驗證。兩組剩余10只大鼠于術后10周攝X線片,取跟腱及其周圍組織行組織學染色觀察,綜合評價異位骨化形成情況。結果:術后3天實驗組1只大鼠死亡,其余大鼠均存活至實驗完成。大體及組織學染色觀察示:各時間點對照組大鼠跟腱及其周圍組織無明顯變化,呈正常跟腱結構;實驗組大鼠跟腱斷端萎縮,壞死,有炎性細胞浸潤;隨時間延長,其跟腱斷端出現(xiàn)硬結,其硬度不斷增加,有大量不規(guī)則結締組織及軟骨細胞。實時定量PCR數(shù)據顯示:與對照組相比,實驗組BMP-1,TGF-β1,IL-1β,HIF-1α,MMP-2各時間點表達均上調,差異有統(tǒng)計學意義(P0.05)。BMP-4,GDF-8,TNF-α各時間點表達均下調,差異有統(tǒng)計學意義(P0.05)。免疫組化染色證實了BMP-1,TGF-β 1,IL-1 β,HIF-1 α,MMP-2蛋白的存在。結合術后10周X線片及組織學觀察結果,實驗組均發(fā)生異位骨化,對照組無異位骨化發(fā)生。結論:在大鼠跟腱創(chuàng)傷性異位骨化早期,BMP-1,TGF-β 1,IL-1 β,HIF-1 α,MMP-2,BMP-4,GDF-8,TNF-α等因子表達異常,提示其參與了創(chuàng)傷性異位骨化的發(fā)生,并起重要作用。實驗組大鼠早期有大量炎性細胞浸潤以及HIF-1α,IL-1β,TNF-α表達異常,提示局部低氧、炎癥微環(huán)境在創(chuàng)傷性異位骨化發(fā)病機制中起重要作用。這對進一步了解創(chuàng)傷性異位骨化的發(fā)病機制及干預治療提供了新方向。
[Abstract]:Background & objective: ectopic ossification is a pathological phenomenon of mature bone in soft tissue. According to its causes, it can be divided into hereditary and acquired ectopic ossification, among which traumatic ectopic ossification is the most common. At present, with the increasing of trauma (high energy injury caused by traffic injury and extensive application of artificial joint replacement, etc.), the incidence of ectopic ossification has attracted much attention. Once it occurs, it will bring long-term pain to patients. Limited joint movement and other symptoms seriously reduce the quality of life of patients. Currently, nonsteroidal anti-inflammatory drugs and low-dose radiotherapy are commonly used to prevent ectopic ossification. However, it is limited in clinical use because of its side effects such as interfering with fracture healing, gastric irritation or potential malignant tumor risk. The effective treatment of ectopic ossification is surgical excision of ectopic ossified bone tissue, but it can cause secondary injury to patients. In order to further reduce the incidence of ectopic ossification, its molecular mechanism and early prevention are of great significance. The purpose of this study was to investigate the expression of related factors in the early stage of traumatic ectopic ossification and to explore its role and significance in the pathogenesis of traumatic ectopic ossification. Methods: 68 male Sprague-Dawley rats aged 8 weeks and 10 weeks old, weighing 210.1 鹵10.6g, were randomly divided into two groups: the experimental group and the control group. In the experimental group, traumatic ectopic ossification model was established by amputation of Achilles tendon clamp, while the control group only exposed Achilles tendon without any treatment of Achilles tendon. The general condition of the two groups was observed postoperatively, and 6 rats were killed in each group on the 8th and 14th day after operation, and the Achilles tendon and surrounding tissues were taken for gross and histological staining. Meanwhile, the expression profiles of ectopic ossification related factors and a large number of literatures in GEO database were analyzed. 25 related factors were screened out. Real-time quantitative PCR was used to detect the gene expression of 25 related factors at different time points. The factors with statistical and clinical significance were screened and verified by immunohistochemical technique. The remaining 10 rats in the two groups were taken X-ray film 10 weeks after operation. The Achilles tendon and its surrounding tissues were observed by histological staining and the formation of ectopic ossification was evaluated synthetically. Results: one rat died in the experimental group 3 days after operation, and the other rats survived until the experiment was completed. Gross and histological staining showed that the Achilles tendon and its surrounding tissues in the control group had no obvious changes and presented normal Achilles tendon structure at each time point, while in the experimental group, the Achilles tendon had atrophy, necrosis and inflammatory cell infiltration. Its Achilles tendon end appears the hard knot, its hardness unceasingly increases, has a large number of irregular connective tissues and chondrocytes. The real time quantitative PCR data showed that the expression of BMP-1TGF- 尾 1 and IL-1 尾 -HIF-1 偽 was up-regulated at all time points in the experimental group compared with the control group, and the difference was statistically significant (P 0.05). The expression of BMP-4 GDF-8 TNF- 偽 was down-regulated at each time point, and the difference was statistically significant (P0.05). The existence of HIF-1 偽 -MMP-2 protein was confirmed by immunohistochemical staining of BMP-1TGF- 尾 _ 1 and IL-1 尾 -HIF-1 偽. Combined with X ray film and histological observation 10 weeks after operation, ectopic ossification occurred in the experimental group and no heterotopic ossification occurred in the control group. Conclusion: in the early stage of traumatic heterotopic ossification of Achilles tendon, the expression of BMP-1TGF- 尾 1 and IL-1 尾 -HIF-1 偽 may be abnormal, suggesting that BMP-4BMP-4 and GDF-8 TNF- 偽 may participate in the occurrence of traumatic ectopic ossification and play an important role in the development of traumatic ectopic ossification. In the experimental group, a large number of inflammatory cells infiltration and abnormal expression of HIF-1 偽 and IL-1 尾 TNF- 偽 were found in the early stage, suggesting that local hypoxia and inflammatory microenvironment play an important role in the pathogenesis of traumatic ectopic ossification. This provides a new direction for further understanding the pathogenesis and intervention of traumatic ectopic ossification.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R641

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