P2X7受体在ECM促进牙周膜干细胞成骨分化中作用的研究

发布时间：2018-05-10 13:45

  本文选题：细胞外基质 + 牙周膜干细胞　； 参考：《第四军医大学》2016年硕士论文

【摘要】：牙周炎是一种发生在牙周组织的常见感染性疾病,主要症状为牙龈萎缩,附着丧失,牙骨质、牙周膜的破坏。炎症的不断进展最终将引起牙槽骨的吸收并最终导致牙齿脱落。目前临床通用的牙周炎治疗方法虽然可以很大程度上去除病因阻止牙周炎恶化,但受损的牙周组织却因有限的自我修复能力而无法复原,这是目前牙周炎诊疗中面临的难题。因此,如何有效实现完整的牙周组织再生是现在牙周组织工程学研究的重要方向。牙周组织工程学的研究对象主要包括种子细胞,生物载体支架以及微环境,其中,种子细胞——牙周膜干细胞(periodontal ligament stem cells,PDLSCs)的增殖和分化是牙周组织再生的生物学基,而细胞外基质(extracellular matrix,ECM)微环境是维持干细胞干性的重要因素。因此,不同组织特异性ECM势必会影响PDLSCs的成骨分化,而这方面的研究尚不明确。另一方面,近期的许多研究发现,P2X7受体在多种组织来源的间充质干细胞向成骨细胞分化过程发挥重要作用,这提示P2X7很可能参与ECM微环境对PDLSCs成骨分化的影响。因此,本课题通过体外制取人牙周膜细胞和骨髓细胞的ECM,模拟不同的h PDLSCs成骨分化微环境;观察组织特异性ECM对h PDLSCs生物学特性的影响;随后对h PDLSCs成骨分化与其细胞表面P2X7受体表达进行检测,并明确该受体在h PDLSCs成骨分化中发挥的作用;最后在骨髓ECM微环境中对P2X7受体和h PDLSCs的成骨分化关系进行深入分析,希望能够进一步明确P2X7受体在ECM促进h PDLSCs成骨分化的作用。研究内容:1.组织特异性ECM在牙周膜干细胞成骨分化中作用的研究。2.P2X7受体在h PDLSCs成骨分化中作用的研究。3.P2X7受体在ECM促进牙周膜干细胞成骨分化过程中作用的研究。研究方法:1.观察不同组织来源的ECM对h PDLSCs成骨分化的影响。体外脱细胞处理人颌骨骨髓细胞和牙周膜细胞,制备组织特异性ECM。分离培养鉴定h PDLSCs,之后将h PDLSCs分别接种在h PDLCs-ECM,h BMCs-ECM,Fibronectin以及普通培养板(TCP)上进行成骨诱导,7d后利用RT-PCR检测成骨相关基因RUNX2和OCN的表达,成骨诱导21天后,茜素红染色等方法检测四组成骨差异。2.研究P2X7受体与h PDLSCs成骨分化的关系。分离培养h PDLSCs,分为4组,α-MEM组细胞用α-MEM普通培养基培养,成骨诱导液组细胞用成骨诱导液培养,激动剂组用100 nmol/L三磷酸腺苷+成骨诱导液培养,拮抗剂组用100nmol/L KN-62+成骨诱导液培养,于成骨诱导7d时,RT-PCR检测成骨相关基因OCN和Runx2的m RNA表达,同时用RT-PCR以及Western blot检测P2X7受体在基因和蛋白水平的表达;在成骨诱导21d后利用茜素红染色检测成骨效果。3.P2X7受体在ECM促进h PDLSCs成骨分化中的作用。体外实验:将h PDLSCs分别接种在h BMCs-ECM与普通培养板(TCP)上,成骨诱导液诱导7d后,免疫荧光观察P2X7受体的表达;细胞接种到ECM和TCP表面,并在ECM表面增加一组抑制剂KN-62组,在TCP表面增加一组激动剂ATP组,成骨诱导7d后进行Western blot检测P2X7受体表达,采用Real-time PCR检测OCN、Runx2的表达;诱导21天后茜素红染色观察成骨结果;体内实验:将h PDLSCs分别接种于TCP和ECM上制备细胞膜片,包裹40mg羟基磷灰石HA颗粒,移植于裸鼠背部皮下,于SPF级环境饲养。60天后,处死裸鼠,取出移植物,固定液固定。10%EDTA溶液脱钙10天,脱水、包埋、切片并染色,观察两组新骨形成差异。研究结果:1.通过有限稀释法获得的h PDLSCs,表面标记物符合间充质干细胞的特性,具有克隆形成能力和多向分化潜能。分离培养的细胞在四组中生长状况良好,其中h BMCs-ECM组和h PDLCs-ECM组在成骨诱导后,成骨相关基因表达显著高于其余两组,其中h BMCs-ECM组成骨效果高于h PDLCs-ECM组,有统计学差异。2.外源性三磷酸腺苷可在h PDLSCs成骨诱导过程中明显促进h PDLSCs的成骨分化。三磷酸腺苷可以激活h PDLSCs中P2X7受体表达,且P2X7受体的表达与h PDLSCs的成骨效果正相关。3.体外实验中,细胞生长在h BMCs-ECM表面诱导7d后,P2X7受体表达显著高于TCP表面;Western blot结果显示h BMCs-ECM表面P2X7受体表达显著高于TCP组,而在ECM表面加入拮抗剂后显著降低了P2X7表达,在TCP表面添加了激动剂后提高了P2X7表达;成骨诱导21天后茜素红染色结果显示,h BMCs-ECM组钙化结节显著高于TCP组,ECM表面加入拮抗剂后显著降低了矿化形成,而TCP表面加入激动剂后显著提高了矿化形成能力;体内实验,HE染色镜下观察,在h BMCs-ECM表面形成的牙周膜干细胞膜片在体内新生骨量明显多于在TCP表面。研究结论:1.组织特异性ECM能有效促进了h PDLSCs成骨分化的潜能,其中h BMCs-ECM的效果好于h PDLCs-ECM。2.P2X7受体与h PDLSCs成骨分化的过程密切相关。3.P2X7受体在组织特异性ECM促进h PDLSCs成骨分化中,发挥着重要作用。
[Abstract]:Periodontitis is a common infectious disease occurring in the periodontium. The main symptoms are gingival atrophy, loss of attachment, cementum, periodontal membrane destruction. The continuous progress of inflammation will eventually lead to the absorption of alveolar bone and eventually lead to tooth loss. The current clinical periodontitis treatment method can greatly remove the cause of the disease. It is a difficult problem to prevent periodontitis from deteriorating, but the damaged periodontal tissue is unable to recover because of the limited self repair ability. This is a difficult problem in the diagnosis and treatment of periodontitis. Therefore, how to effectively realize the complete periodontal tissue regeneration is an important direction in the study of periodontal tissue engineering. The research object of periodontal tissue engineering mainly includes seeds. Cell, biological carrier scaffold and microenvironment, in which the proliferation and differentiation of periodontal ligament stem cells (PDLSCs) is the biological basis of periodontal tissue regeneration, and the extracellular matrix (extracellular matrix, ECM) microenvironment is an important factor in the maintenance of stem cell stem. Therefore, different tissues are specific. Sexual ECM is bound to affect the osteogenic differentiation of PDLSCs, and the research in this area is not clear. On the other hand, many recent studies have found that P2X7 receptors play an important role in the differentiation of osteoblasts from a variety of tissue derived mesenchymal stem cells. This suggests that P2X7 may be involved in the effect of ECM microenvironment on the osteogenesis of PDLSCs. The ECM of human periodontal membrane cells and bone marrow cells in vitro was used to simulate different h PDLSCs osteogenesis microenvironment, and the effect of tissue specific ECM on the biological characteristics of H PDLSCs was observed. Then, the osteogenic differentiation of H PDLSCs and the expression of P2X7 receptor on the surface of the cell surface were detected, and the receptor played in the H PDLSCs osteogenesis differentiation. Effect; finally the osteogenic differentiation of P2X7 receptor and H PDLSCs in bone marrow ECM microenvironment is deeply analyzed. Hope to further clarify the role of P2X7 receptor in ECM promoting the osteogenesis of H PDLSCs. Research contents: 1. research on the role of tissue specific ECM in the osteogenesis of periodontal ligament stem cells;.2.P2X7 receptor in H PDLSCs osteogenesis Research on the role of.3.P2X7 receptor in the process of ECM promoting osteogenic differentiation of periodontal ligament stem cells. Study methods: 1. the effect of ECM on the osteogenesis of H PDLSCs by different tissue sources. In vitro decellular decellular treatment of human bone marrow cells and periodontal ligament cells, the preparation of group woven specific ECM. isolation and identification of H PDLSCs, after the preparation of H PDLSCs H PDLSCs was inoculated on H PDLCs-ECM, H BMCs-ECM, Fibronectin, and common culture plate (TCP) to induce osteogenesis, and RT-PCR was used to detect the expression of RUNX2 and OCN in bone related genes after 7d, and 21 days after induction of osteogenesis, and the relationship between the osteogenesis differentiation of four bone differentiation receptors was detected by alizarin red staining and other methods. H PDLSCs was cultured in 4 groups. The cells in group a -MEM were cultured with alpha -MEM medium. The cells in the osteogenic induction group were cultured with osteogenic induction solution. The agonist group was cultured with 100 nmol/L adenosine triphosphate + osteogenic induction solution, and the antagonist group was cultured with 100nmol/L KN-62+ osteogenic induction solution. When osteogenesis induced 7d, RT-PCR detected OCN and Runx2 of osteogenesis related genes. The expression of M RNA, simultaneously using RT-PCR and Western blot to detect the expression of P2X7 receptor at the gene and protein level, and the effect of.3.P2X7 receptor on ECM promoting h PDLSCs osteogenesis by using alizarin red staining after osteogenesis induced 21d. After induction of 7D, the expression of P2X7 receptor was observed by immunofluorescence; the cells were inoculated to the surface of ECM and TCP, and a group of inhibitor KN-62 groups were added to the surface of ECM, and a group of activator ATP groups on the TCP surface was added to the TCP surface. The expression of P2X7 receptors was detected by Western blot after the induction of 7D, and the expression was detected with the induction of Alizarin 21 days later. The results of osteogenesis were observed by red staining. In vivo, H PDLSCs was inoculated on TCP and ECM to prepare cell diaphragms, 40mg hydroxyapatite HA particles were coated on the back of nude mice. After.60 days in the SPF environment, nude mice were killed and removed, and the fixed solution was immobilized on.10% EDTA solution for 10 days. Dehydration, embedding, sectioning and staining were observed. The difference between the two groups of new bone formation. 1. the H PDLSCs obtained by the finite dilution method, the surface markers conformed to the characteristics of mesenchymal stem cells, and had the ability of clonogenesis and multidirectional differentiation. The cultured cells grew well in the four groups, in which the H BMCs-ECM group and the H PDLCs-ECM group were induced by the osteogenesis, and the osteogenic related genes were induced. The expression of H BMCs-ECM was higher than that of the other two groups, in which the bone effect was higher than that of the H PDLCs-ECM group. There was a statistically significant difference between.2. and PDLCs-ECM in the osteogenesis of H PDLSCs. Adenosine triphosphate could activate the P2X7 receptor in H PDLSCs and the expression of the receptor and the osteogenesis effect of H. In positive correlation.3., the expression of P2X7 receptor was significantly higher than that on the surface of TCP after the cell growth was induced on the surface of H BMCs-ECM. The Western blot results showed that the expression of P2X7 receptor on the surface of H BMCs-ECM was significantly higher than that of the TCP group, and the expression was significantly reduced after the addition of antagonists on the surface. The results of alizarin red staining after 21 days of osteogenesis showed that the calcified nodule of H BMCs-ECM group was significantly higher than that in group TCP, and the mineralization was significantly reduced after the ECM surface was added to the antagonist, and the mineralization ability was significantly increased after the TCP surface added to the agonist; in vivo experiments, under the HE staining microscope, the membrane of the periodontal membrane stem cells formed on the surface of H BMCs-ECM. The new bone mass in the body is more than that on the TCP surface. Conclusion: 1. the tissue specific ECM can effectively promote the potential of H PDLSCs osteogenesis, and the effect of H BMCs-ECM is better than that of H PDLCs-ECM.2.P2X7 receptor and H PDLSCs, which is closely related to the differentiation of the.3.P2X7 receptor in the tissue specific ECM. Important role.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781.4
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本文编号：1869547