本文選題：細(xì)胞外基質(zhì) + 牙周膜干細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2016年碩士論文

【摘要】：牙周炎是一種發(fā)生在牙周組織的常見感染性疾病,主要癥狀為牙齦萎縮,附著喪失,牙骨質(zhì)、牙周膜的破壞。炎癥的不斷進(jìn)展最終將引起牙槽骨的吸收并最終導(dǎo)致牙齒脫落。目前臨床通用的牙周炎治療方法雖然可以很大程度上去除病因阻止牙周炎惡化,但受損的牙周組織卻因有限的自我修復(fù)能力而無法復(fù)原,這是目前牙周炎診療中面臨的難題。因此,如何有效實(shí)現(xiàn)完整的牙周組織再生是現(xiàn)在牙周組織工程學(xué)研究的重要方向。牙周組織工程學(xué)的研究對(duì)象主要包括種子細(xì)胞,生物載體支架以及微環(huán)境,其中,種子細(xì)胞——牙周膜干細(xì)胞(periodontal ligament stem cells,PDLSCs)的增殖和分化是牙周組織再生的生物學(xué)基,而細(xì)胞外基質(zhì)(extracellular matrix,ECM)微環(huán)境是維持干細(xì)胞干性的重要因素。因此,不同組織特異性ECM勢(shì)必會(huì)影響PDLSCs的成骨分化,而這方面的研究尚不明確。另一方面,近期的許多研究發(fā)現(xiàn),P2X7受體在多種組織來源的間充質(zhì)干細(xì)胞向成骨細(xì)胞分化過程發(fā)揮重要作用,這提示P2X7很可能參與ECM微環(huán)境對(duì)PDLSCs成骨分化的影響。因此,本課題通過體外制取人牙周膜細(xì)胞和骨髓細(xì)胞的ECM,模擬不同的h PDLSCs成骨分化微環(huán)境;觀察組織特異性ECM對(duì)h PDLSCs生物學(xué)特性的影響;隨后對(duì)h PDLSCs成骨分化與其細(xì)胞表面P2X7受體表達(dá)進(jìn)行檢測(cè),并明確該受體在h PDLSCs成骨分化中發(fā)揮的作用;最后在骨髓ECM微環(huán)境中對(duì)P2X7受體和h PDLSCs的成骨分化關(guān)系進(jìn)行深入分析,希望能夠進(jìn)一步明確P2X7受體在ECM促進(jìn)h PDLSCs成骨分化的作用。研究?jī)?nèi)容:1.組織特異性ECM在牙周膜干細(xì)胞成骨分化中作用的研究。2.P2X7受體在h PDLSCs成骨分化中作用的研究。3.P2X7受體在ECM促進(jìn)牙周膜干細(xì)胞成骨分化過程中作用的研究。研究方法:1.觀察不同組織來源的ECM對(duì)h PDLSCs成骨分化的影響。體外脫細(xì)胞處理人頜骨骨髓細(xì)胞和牙周膜細(xì)胞,制備組織特異性ECM。分離培養(yǎng)鑒定h PDLSCs,之后將h PDLSCs分別接種在h PDLCs-ECM,h BMCs-ECM,Fibronectin以及普通培養(yǎng)板(TCP)上進(jìn)行成骨誘導(dǎo),7d后利用RT-PCR檢測(cè)成骨相關(guān)基因RUNX2和OCN的表達(dá),成骨誘導(dǎo)21天后,茜素紅染色等方法檢測(cè)四組成骨差異。2.研究P2X7受體與h PDLSCs成骨分化的關(guān)系。分離培養(yǎng)h PDLSCs,分為4組,α-MEM組細(xì)胞用α-MEM普通培養(yǎng)基培養(yǎng),成骨誘導(dǎo)液組細(xì)胞用成骨誘導(dǎo)液培養(yǎng),激動(dòng)劑組用100 nmol/L三磷酸腺苷+成骨誘導(dǎo)液培養(yǎng),拮抗劑組用100nmol/L KN-62+成骨誘導(dǎo)液培養(yǎng),于成骨誘導(dǎo)7d時(shí),RT-PCR檢測(cè)成骨相關(guān)基因OCN和Runx2的m RNA表達(dá),同時(shí)用RT-PCR以及Western blot檢測(cè)P2X7受體在基因和蛋白水平的表達(dá);在成骨誘導(dǎo)21d后利用茜素紅染色檢測(cè)成骨效果。3.P2X7受體在ECM促進(jìn)h PDLSCs成骨分化中的作用。體外實(shí)驗(yàn):將h PDLSCs分別接種在h BMCs-ECM與普通培養(yǎng)板(TCP)上,成骨誘導(dǎo)液誘導(dǎo)7d后,免疫熒光觀察P2X7受體的表達(dá);細(xì)胞接種到ECM和TCP表面,并在ECM表面增加一組抑制劑KN-62組,在TCP表面增加一組激動(dòng)劑ATP組,成骨誘導(dǎo)7d后進(jìn)行Western blot檢測(cè)P2X7受體表達(dá),采用Real-time PCR檢測(cè)OCN、Runx2的表達(dá);誘導(dǎo)21天后茜素紅染色觀察成骨結(jié)果;體內(nèi)實(shí)驗(yàn):將h PDLSCs分別接種于TCP和ECM上制備細(xì)胞膜片,包裹40mg羥基磷灰石HA顆粒,移植于裸鼠背部皮下,于SPF級(jí)環(huán)境飼養(yǎng)。60天后,處死裸鼠,取出移植物,固定液固定。10%EDTA溶液脫鈣10天,脫水、包埋、切片并染色,觀察兩組新骨形成差異。研究結(jié)果:1.通過有限稀釋法獲得的h PDLSCs,表面標(biāo)記物符合間充質(zhì)干細(xì)胞的特性,具有克隆形成能力和多向分化潛能。分離培養(yǎng)的細(xì)胞在四組中生長狀況良好,其中h BMCs-ECM組和h PDLCs-ECM組在成骨誘導(dǎo)后,成骨相關(guān)基因表達(dá)顯著高于其余兩組,其中h BMCs-ECM組成骨效果高于h PDLCs-ECM組,有統(tǒng)計(jì)學(xué)差異。2.外源性三磷酸腺苷可在h PDLSCs成骨誘導(dǎo)過程中明顯促進(jìn)h PDLSCs的成骨分化。三磷酸腺苷可以激活h PDLSCs中P2X7受體表達(dá),且P2X7受體的表達(dá)與h PDLSCs的成骨效果正相關(guān)。3.體外實(shí)驗(yàn)中,細(xì)胞生長在h BMCs-ECM表面誘導(dǎo)7d后,P2X7受體表達(dá)顯著高于TCP表面;Western blot結(jié)果顯示h BMCs-ECM表面P2X7受體表達(dá)顯著高于TCP組,而在ECM表面加入拮抗劑后顯著降低了P2X7表達(dá),在TCP表面添加了激動(dòng)劑后提高了P2X7表達(dá);成骨誘導(dǎo)21天后茜素紅染色結(jié)果顯示,h BMCs-ECM組鈣化結(jié)節(jié)顯著高于TCP組,ECM表面加入拮抗劑后顯著降低了礦化形成,而TCP表面加入激動(dòng)劑后顯著提高了礦化形成能力;體內(nèi)實(shí)驗(yàn),HE染色鏡下觀察,在h BMCs-ECM表面形成的牙周膜干細(xì)胞膜片在體內(nèi)新生骨量明顯多于在TCP表面。研究結(jié)論:1.組織特異性ECM能有效促進(jìn)了h PDLSCs成骨分化的潛能,其中h BMCs-ECM的效果好于h PDLCs-ECM。2.P2X7受體與h PDLSCs成骨分化的過程密切相關(guān)。3.P2X7受體在組織特異性ECM促進(jìn)h PDLSCs成骨分化中,發(fā)揮著重要作用。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R781.4
，

本文編號(hào)：1869547