创伤性异位骨化早期相关因子的研究

发布时间：2018-05-15 13:34

本文选题：创伤性异位骨化 + 大鼠　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景与目的:异位骨化是指软组织中出现成熟骨组织的病理现象。根据其形成原因分为遗传性和获得性异位骨化,其中,以创伤性异位骨化最为多见。目前,随着创伤(交通伤导致的高能量创伤以及人工关节置换的广泛应用等等)的不断增加,异位骨化的发病率备受关注,一旦发生,将给患者带来长期的疼痛,关节活动受限等症状,严重的降低了患者的生活质量。目前,临床上常用的预防异位骨化的方式包括:非甾体类抗炎药及小剂量放射治疗。但因其干扰骨折愈合过程,胃部刺激或潜在的恶性肿瘤危险等副作用,在临床上使用受限。异位骨化有效治疗方法是手术切除异位骨化的骨组织,但会给患者造成二次损伤。为进一步降低异位骨化发生率,其分子机制及早期预防的研究具有重要意义。本研究基于大鼠跟腱钳夹、切断模型诱导创伤性异位骨化,旨在了解创伤性异位骨化早期相关因子的表达情况,探讨其在创伤性异位骨化发病机制中的作用及意义。方法:取8～10周龄雄性SD大鼠68只,体质量(210.1± 10.6)g,随机分为实验组及对照组(n=34)。实验组通过跟腱钳夹、切断法制备创伤性异位骨化模型,对照组仅暴露跟腱,跟腱不作任何处理。术后观察两组大鼠一般情况,于3、5、8及14天两组各处死6只大鼠,取跟腱及周围组织行大体及组织学染色观察;同时对GEO数据库中异位骨化相关因子表达谱及大量文献进行分析,筛选出25种相关因子。采用实时定量PCR技术检测不同时间点25种相关因子的基因表达情况,筛选出有统计学及临床意义的因子,采用免疫组化等技术进行验证。两组剩余10只大鼠于术后10周摄X线片,取跟腱及其周围组织行组织学染色观察,综合评价异位骨化形成情况。结果:术后3天实验组1只大鼠死亡,其余大鼠均存活至实验完成。大体及组织学染色观察示:各时间点对照组大鼠跟腱及其周围组织无明显变化,呈正常跟腱结构;实验组大鼠跟腱断端萎缩,坏死,有炎性细胞浸润;随时间延长,其跟腱断端出现硬结,其硬度不断增加,有大量不规则结缔组织及软骨细胞。实时定量PCR数据显示:与对照组相比,实验组BMP-1,TGF-β1,IL-1β,HIF-1α,MMP-2各时间点表达均上调,差异有统计学意义(P0.05)。BMP-4,GDF-8,TNF-α各时间点表达均下调,差异有统计学意义(P0.05)。免疫组化染色证实了BMP-1,TGF-β 1,IL-1 β,HIF-1 α,MMP-2蛋白的存在。结合术后10周X线片及组织学观察结果,实验组均发生异位骨化,对照组无异位骨化发生。结论:在大鼠跟腱创伤性异位骨化早期,BMP-1,TGF-β 1,IL-1 β,HIF-1 α,MMP-2,BMP-4,GDF-8,TNF-α等因子表达异常,提示其参与了创伤性异位骨化的发生,并起重要作用。实验组大鼠早期有大量炎性细胞浸润以及HIF-1α,IL-1β,TNF-α表达异常,提示局部低氧、炎症微环境在创伤性异位骨化发病机制中起重要作用。这对进一步了解创伤性异位骨化的发病机制及干预治疗提供了新方向。
[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 卤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 伪 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- 伪 was down-regulated at each time point, and the difference was statistically significant (P0.05). The existence of HIF-1 伪 -MMP-2 protein was confirmed by immunohistochemical staining of BMP-1TGF- 尾 _ 1 and IL-1 尾 -HIF-1 伪. 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 伪 may be abnormal, suggesting that BMP-4BMP-4 and GDF-8 TNF- 伪 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 伪 and IL-1 尾 TNF- 伪 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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R641

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