本文選題：細胞外基質(zhì) + 牙周膜干細胞�。� 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：牙周炎是人類古老而普遍存在的疾病之一，牙周炎的基本病理變化是由細菌感染引起的牙周組織局部的炎癥，隨著炎癥的進展，牙周組織逐漸喪失附著，牙槽骨進行性吸收，造成牙齒松動。現(xiàn)有的治療方法都有各自的局限性，無法實現(xiàn)理想的牙周再生。近年來，干細胞和再生醫(yī)學(xué)的蓬勃發(fā)展為牙周炎的治療帶來了曙光，一般認為，牙周組織再生的基礎(chǔ)是來自于牙周膜干細胞的擴增和多向分化能力，而干細胞的增殖、分化受到多種因素的影響，在炎癥狀態(tài)下其增殖和多向分化能力均受到顯著抑制，，因此，改善牙周膜干細胞的炎癥微環(huán)境，使得牙周膜干細胞大量擴增并多向分化是獲得牙周再生的關(guān)鍵。細胞外基質(zhì)（ECM）是蛋白和多糖連接而成的網(wǎng)狀支架，通過多條途徑控制著干細胞的命運，且有研究表明脫細胞的骨髓細胞ECM對骨髓間充質(zhì)干細胞體外擴增的干性保持以及多向分化具有重要的促進作用，而ECM對牙周干細胞的影響研究甚少。本課題采用脫細胞技術(shù)獲得牙周膜細胞和骨髓細胞兩種ECM，模擬牙周膜干細胞的體內(nèi)微環(huán)境，以實現(xiàn)在體外多次擴增傳代的條件下牙周膜干細胞依然能夠保持良好的干細胞特性，獲得大量高質(zhì)量的牙周膜干細胞用于基礎(chǔ)研究；通過組織特異性ECM對牙周膜干細胞的培養(yǎng)，與骨粉結(jié)合植入裸鼠體內(nèi)，觀察牙周再生情況，為臨床應(yīng)用提供一定的理論依據(jù)。 研究內(nèi)容： （1）組織特異性ECM（hPDLCs-ECM和hBMCs-ECM）的制備。 （2）研究組織特異性ECM（hPDLCs-ECM和hBMCs-ECM）對體外培養(yǎng)人牙周膜干細胞（hPDLSCs）的生物學(xué)特性的影響。 （3）研究組織特異性ECM（hPDLCs-ECM和hBMCs-ECM）培養(yǎng)人牙周膜干細胞（hPDLSCs）后，觀察其在裸鼠體內(nèi)的生長與分化。 研究方法： （1）利用有限稀釋克隆法從牙周膜細胞（hPDLCs）中分離純化人牙周膜干細胞（hPDLSCs），通過檢測克隆形成、表面分子標(biāo)志以及多向誘導(dǎo)分化實驗鑒定hPDLSCs。脫細胞處理人牙周膜細胞（hPDLCs）和人骨髓細胞（hBMCs）以制備組織特異性ECM。 （2）利用克隆形成實驗檢測兩種組織特異性ECM對于體外培養(yǎng)hPDLSCs克隆形成的影響，利用成骨成脂誘導(dǎo)實驗檢測兩種組織特異性ECM對體外培養(yǎng)hPDLSCs成骨與成脂的影響，以反映ECM對hPDLSCs干細胞特性的影響。 （3）將hPDLSCs與骨髓細胞ECM和牙周膜細胞ECM共培養(yǎng)，結(jié)合無機牛骨粉植入裸鼠皮下，8周后取材進行HE染色、Masson三色染色、苦味酸-天狼猩紅以及免疫組織化學(xué)染色觀察新生成的組織。 研究結(jié)果： （1）通過有限稀釋法獲得的hPDLSCs，具有克隆形成能力，表達間充質(zhì)干細胞標(biāo)志，并具有多向分化能力。光學(xué)顯微鏡下，脫細胞處理之后，無細胞結(jié)構(gòu)存在，ECM呈網(wǎng)狀膜片。 （2）接種在兩種組織特異性ECM上的hPDLSCs的克隆形成率均明顯高于對照組，經(jīng)過多次傳代之后，ECM中培養(yǎng)的hPDLSCs仍保持較高的克隆形成能力。接種在兩種組織特異性ECM上的hPDLSCs多次傳代之后的成骨成脂分化能力也明顯高于對照組。 （3）hPDLSCs和hBMCs-ECM、hPDLCs-ECM共培養(yǎng)后與無機牛骨粉混合，植入裸鼠皮下，在hBMCs-ECM組生成了類似骨質(zhì)和牙周膜纖維樣結(jié)構(gòu)，hPDLCs-ECM組生成牙周膜樣纖維，含大量的Ⅰ型膠原和Ⅲ型膠原，對照組僅有疏松的纖維結(jié)締組織生成，主要為Ⅰ型膠原。 結(jié)論: （1）通過有效的脫細胞處理方法可以制得結(jié)構(gòu)完整和具有良好生物學(xué)功能的ECM（hPDLCs-ECM和hBMCs-ECM）。 （2）組織特異性ECM（hPDLCs-ECM和hBMCs-ECM）對體外培養(yǎng)人牙周膜干細胞（hPDLSCs）維持干細胞特性有顯著的促進作用，hPDLSCs經(jīng)體外多次傳代擴增后仍保持較高的自我更新和多向分化的能力。 （3）組織特異性ECM能夠促使hPDLSCs在體內(nèi)生成類骨質(zhì)和牙周膜樣纖維結(jié)構(gòu)，體現(xiàn)出良好的牙周組織再生能力。
[Abstract]:Periodontitis is one of the ancient and widespread disease, the basic pathological changes of periodontitis is caused by bacterial infection of periodontal tissue local inflammation, along with the development of inflammation, periodontal attachment loss gradually, alveolar bone absorption caused by loose teeth. Current treatment methods have their own limitations, can not be achieved the ideal periodontal regeneration. In recent years, rapid development of stem cell and regenerative medicine brought the dawn, for the treatment of periodontitis is generally believed that the basis of periodontal tissue regeneration from periodontal ligament stem cell expansion and differentiation ability, and stem cell proliferation and differentiation is affected by many factors, in under the condition of inflammation proliferation and differentiation capacity were inhibited significantly, therefore, improve the periodontal ligament stem cells in the inflammatory microenvironment, the periodontal ligament stem cells proliferation and differentiation is periodontal again The key. The extracellular matrix (ECM) protein and polysaccharide is stent connected, through multiple pathways that control the fate of stem cells, and studies have shown that acellular bone marrow cells of ECM on bone marrow mesenchymal stem cells in vitro and keep dry differentiation has an important role in promoting ECM, on the periodontal stem cells studied very little. This subject was obtained by periodontal ligament cells and bone marrow cells of two ECM acellular technology, simulation of periodontal ligament stem cells in vivo microenvironment, periodontal membrane to achieve in vitro passage under the condition of stem cells can still maintain stem cell characteristics the access to a large number of high quality of periodontal ligament stem cells for basic research; through the organization of specific ECM on periodontal ligament stem cells implanted in nude mice, combined with bone, observe the periodontal regeneration, and clinical application The theoretical basis.
Research content:
(1) preparation of tissue specific ECM (hPDLCs-ECM and hBMCs-ECM).
(2) the study of the effects of tissue specific ECM (hPDLCs-ECM and hBMCs-ECM) on the biological characteristics of human periodontal ligament stem cells (hPDLSCs) in vitro.
(3) to investigate the growth and differentiation of human periodontal ligament stem cells (hPDLSCs) in nude mice after the study of tissue specific ECM (hPDLCs-ECM and hBMCs-ECM).
Research methods:
(1) using limited dilution cloning cells from periodontal ligament (hPDLCs) in the separation and purification of human periodontal ligament stem cells (hPDLSCs), formed by detecting clone, surface markers and multi-directional differentiation experiments identified hPDLSCs. acellular human periodontal ligament cells (hPDLCs) and human bone marrow cells (hBMCs) by the organization specific to ECM.
(2) using clone formation assay for detection of two kinds of tissue specific ECM effects on hPDLSCs colony formation in vitro by osteogenic and adipogenic induction experiments to detect two kinds of tissue specific ECM hPDLSCs osteogenic and adipogenic effects of in vitro culture, to reflect the ECM stem cell character of hPDLSCs.
(3) the hPDLSCs and ECM of bone marrow cells and periodontal ligament cells ECM were cultured with bovine bone mineral powder and implanted subcutaneously in nude mice after 8 weeks of HE staining, Masson staining and picrosirius red staining and immunohistochemistry of newly formed tissue.
The results of the study:
(1) the hPDLSCs obtained by the limited dilution method has the ability of clone forming, expressing mesenchymal stem cell markers, and has the ability of multidirectional differentiation. Under the microscope, after acellular treatment, there is no cell structure, and ECM is reticular membrane.
(2) cloning hPDLSCs vaccination in two kinds of tissue specificity of the ECM formation rate were significantly higher than the control group, after several passages after ECM cultured in hPDLSCs still maintain high clonality. After hPDLSCs inoculation in two kinds of tissue specific ECM on several passages of osteogenic and adipogenic differentiation ability significantly higher than the control group.
(3) hPDLSCs and hBMCs-ECM, hPDLCs-ECM co culture mixed with the inorganic bone powder, and implanted subcutaneously in nude mice in the hBMCs-ECM group generated similar bone and periodontal membrane fiber like structure, hPDLCs-ECM group generated periodontal ligament fibers containing type I collagen and type III collagen a fibrous connective tissue formation, the control group only loose the mainly type I collagen.
Conclusion:
(1) ECM (hPDLCs-ECM and hBMCs-ECM) with good structure and good biological function can be obtained by effective cell removal.
(2) tissue-specific ECM (hPDLCs-ECM and hBMCs-ECM) can significantly promote the growth of human periodontal ligament stem cells (hPDLSCs) and maintain the characteristics of stem cells. HPDLSCs still maintains a high ability of self-renewal and multidifferentiation after repeated passage in vitro.
(3) tissue specific ECM can induce hPDLSCs to produce bone like and periodontal ligament structure in the body, which reflects the good regeneration ability of periodontal tissue.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R781.42

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