Dlx-5和Msx-1在双膦酸盐相关性颌骨坏死致病机制中的作用

发布时间：2018-05-01 22:37

本文选题：远中缺失同源盒基因5 + 肌节同源盒基因1　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:1.颅面骨和髂骨胚胎来源不同,它们之间存在不同的基因表达模式和骨代谢水平。远中缺失同源盒基因(Distal-less homeobox,Dlx)和肌节同源盒基因(Msh homeobox,Msx)在骨代谢方面发挥重大作用,尤其是Dlx-5和Msx-1。本研究对象为长期接受唑来膦酸药物的大鼠,通过检测其Dlx-5和Msx-1蛋白及基因表达水平的变化,从而探究唑来膦酸对颅面骨和髂骨的影响是否存在差异。2.建立双膦酸盐相关性颌骨坏死(Bisphosphonate-related osteonecrosis of the jaw,BRONJ)动物模型,观察BRONJ的临床表现、影像学改变及组织病理学特点,探究Dlx-5和Msx-1在BRONJ致病机制中发挥的作用。方法:1.36只雌性白化封闭群大鼠(Sprague-Dawley,SD)随机分成三组,每组12只。唑来膦酸组腹腔注射唑来膦酸(0.2 mg/kg),每周3次持续12周。生理盐水组腹腔注射等量生理盐水持续12周,另外一组为对照组,不做任何处理。利用免疫组织化学染色、蛋白质印迹法(Western blot)和实时定量-聚合酶链反应(Real time-polymerase chain reaction,RT-PCR)检测颅面骨(包括上颌骨、下颌骨和顶骨)和髂骨中Dlx-5和Msx-1的表达水平。2.24只雌性SD大鼠随机分为两组,每组12只。唑来膦酸组腹腔注射唑来膦酸(0.2 mg/kg),每周3次持续12周。对照组腹腔注射等量生理盐水,每周3次持续12周。12周后所有大鼠麻醉下拔除左下第一磨牙,临床观察拔牙创愈合情况。拔牙后8周所有动物被处死。对大鼠左下颌骨进行X线片检查和Micro-CT检查,探究其影像学改变。对下颌骨软硬组织进行苏木精-伊红(Hematoxylin-eosin,HE)染色和Masson染色,探究其组织病理学特点,进一步确定BRONJ动物模型。通过Western blot、RT-PCR技术检测Dlx-5和Msx-1在大鼠下颌骨拔牙创周围硬组织中的表达水平。结果:1.分别与生理盐水组和对照组比较,唑来膦酸组上、下颌骨中Dlx-5的蛋白和基因表达水平升高(P0.05),上、下颌骨中Msx-1的蛋白和基因水平降低(P0.01)。在蛋白和基因水平上,唑来膦酸组髂骨中的Dlx-5和Msx-1的表达水平均降低(P0.05)。唑来膦酸组与其他两组比较,顶骨中Dlx-5和Msx-1的蛋白和基因表达水平均无显著变化(P0.05),它们之间的差异无统计学意义。2.拔牙后8周,临床观察发现唑来膦酸组大鼠拔牙创不愈合,并且实验动物口内可见死骨暴露。影像学检查及组织病理学检查均证实BRONJ的发生。Masson染色结果显示唑来膦酸组拔牙创周围牙龈胶原纤维纤细。Western blot、RT-PCR检测结果显示唑来膦酸组拔牙创周围颌骨中Dlx-5蛋白和基因表达水平均升高(P0.01),同时Msx-1表达水平降低(P0.01)。结论:1.唑来膦酸作用下颅面骨和髂骨中Dlx-5和Msx-1表达水平的变化趋势存在差异性,这在一定程度上表明了唑来膦酸对大鼠骨骼的影响存在位置差异性。与顶骨和髂骨相比,颌骨对唑来膦酸的应用最为敏感。2.腹腔注射唑来膦酸联合拔牙的方法能够成功诱导大鼠发生BRONJ样病变。在BRONJ动物模型中Dlx-5和Msx-1表达水平的变化趋势能够促进骨形成,进一步破坏骨代谢平衡,导致拔牙创局部骨硬化,其在BRONJ的发生、发展过程中发挥重要作用。
[Abstract]:Objective: 1. craniofacial bone and iliac bone have different embryonic sources, and there are different gene expression patterns and bone metabolism levels between them. Distal-less homeobox (Dlx) and Msh homeobox (Msx) genes (Msh homeobox, Msx) play a major role in bone metabolism, especially in Dlx-5 and Msx-1. based subjects for long-term acceptance of azole By detecting changes in the levels of Dlx-5 and Msx-1 protein and gene expression in the rat, the difference in the effects of zoledronic acid on the craniofacial bone and iliac bone was detected by.2., and the establishment of the bisphosphonate associated osteonecrosis (Bisphosphonate-related osteonecrosis of the jaw, BRONJ) animal model and the clinical manifestations of BRONJ were observed. The role of Dlx-5 and Msx-1 in the pathogenesis of BRONJ was investigated by imaging changes and histopathological features. Methods: 1.36 female albino closed group rats (Sprague-Dawley, SD) were randomly divided into three groups, 12 in each group. Zoledronic acid group was intraperitoneally injected with zoledronic acid (0.2 mg/kg) and lasted 3 times a week for 12 weeks. The normal saline lasted for 12 weeks, and the other group was a control group without any treatment. The expression level of Dlx-5 and Msx-1 in the cranial bone (including the maxilla, mandible and parietal bone) and the iliac bone was detected by immunohistochemical staining, Western blot and real-time quantitative polymerase chain reaction (RT-PCR),.2. .24 female SD rats were randomly divided into two groups, each group was 12. Zoledronic acid group was intraperitoneally injected with zoledronic acid (0.2 mg/kg) and lasted 3 times a week for 12 weeks. The control group was injected with equal amount of saline, 3 times a week after 12 weeks of.12 weeks, all rats were extracted to remove the left inferior molars and observe the healing of tooth extraction at 8 weeks after extraction. The X-ray examination and Micro-CT examination of the left mandible of the rat were carried out to explore the imaging changes. The histopathological features of the soft and hard tissue of the mandible were studied by Hematoxylin-eosin and HE staining and Masson staining, and the model of BRONJ animal was further determined. Dlx-5 and Msx-1 were detected by Western blot and RT-PCR technology. Results: 1. compared with the normal saline group and the control group, 1. the levels of protein and gene expression in the mandible increased (P0.05) in the zoledronic acid group and in the zoledronic acid group (P0.05), and the protein and gene level of Msx-1 in the mandible decreased (P0.01). The expression level of Dlx-5 and Msx-1 in the bone decreased (P0.05). There was no significant change in the protein and gene expression levels of Dlx-5 and Msx-1 in the parietal bone compared with the other two groups (P0.05). There was no significant difference between them, and there was no significant difference between them in.2. after the extraction of teeth 8 weeks after the extraction of the azolidonic acid group. The imaging examination and histopathological examination showed that the.Masson staining results of BRONJ showed that the gingival collagen fibrils were fine.Western blot in the azolidonic acid group, and the RT-PCR detection results showed that the level of Dlx-5 protein and gene expression in the azolidonic acid group was elevated (P0.01) in the surrounding jaw of the azolidonic acid group. The expression level of Msx-1 was reduced (P0.01). Conclusion: there is a difference in the expression of Dlx-5 and Msx-1 in the cranial bone and iliac bone under the action of 1. zoledronic acid, which, to a certain extent, indicates that zoledronic acid has a position difference in the effect of zoledronic acid on the skeleton of rats. Compared with the parietal bone and iliac bone, the maxillofacial application is the most sensitive.2. to the zoledronic acid. Intraperitoneal injection of zoledronic acid combined with tooth extraction can successfully induce BRONJ like lesions in rats. In the BRONJ animal model, the changes in the expression of Dlx-5 and Msx-1 can promote bone formation, further destroy the balance of bone metabolism, and lead to local osseosclerosis, which plays an important role in the occurrence and development of BRONJ.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782

【参考文献】