组织特异性细胞外基质对体外培养hPDLSCs干细胞特性的维持及体内分化的研究

发布时间：2018-04-15 09:25

本文选题：细胞外基质 + 牙周膜干细胞　；参考：《第四军医大学》2014年硕士论文

【摘要】：牙周炎是人类古老而普遍存在的疾病之一，牙周炎的基本病理变化是由细菌感染引起的牙周组织局部的炎症，随着炎症的进展，牙周组织逐渐丧失附着，牙槽骨进行性吸收，造成牙齿松动。现有的治疗方法都有各自的局限性，无法实现理想的牙周再生。近年来，干细胞和再生医学的蓬勃发展为牙周炎的治疗带来了曙光，一般认为，牙周组织再生的基础是来自于牙周膜干细胞的扩增和多向分化能力，而干细胞的增殖、分化受到多种因素的影响，在炎症状态下其增殖和多向分化能力均受到显著抑制，，因此，改善牙周膜干细胞的炎症微环境，使得牙周膜干细胞大量扩增并多向分化是获得牙周再生的关键。细胞外基质（ECM）是蛋白和多糖连接而成的网状支架，通过多条途径控制着干细胞的命运，且有研究表明脱细胞的骨髓细胞ECM对骨髓间充质干细胞体外扩增的干性保持以及多向分化具有重要的促进作用，而ECM对牙周干细胞的影响研究甚少。本课题采用脱细胞技术获得牙周膜细胞和骨髓细胞两种ECM，模拟牙周膜干细胞的体内微环境，以实现在体外多次扩增传代的条件下牙周膜干细胞依然能够保持良好的干细胞特性，获得大量高质量的牙周膜干细胞用于基础研究；通过组织特异性ECM对牙周膜干细胞的培养，与骨粉结合植入裸鼠体内，观察牙周再生情况，为临床应用提供一定的理论依据。研究内容：（1）组织特异性ECM（hPDLCs-ECM和hBMCs-ECM）的制备。（2）研究组织特异性ECM（hPDLCs-ECM和hBMCs-ECM）对体外培养人牙周膜干细胞（hPDLSCs）的生物学特性的影响。（3）研究组织特异性ECM（hPDLCs-ECM和hBMCs-ECM）培养人牙周膜干细胞（hPDLSCs）后，观察其在裸鼠体内的生长与分化。研究方法：（1）利用有限稀释克隆法从牙周膜细胞（hPDLCs）中分离纯化人牙周膜干细胞（hPDLSCs），通过检测克隆形成、表面分子标志以及多向诱导分化实验鉴定hPDLSCs。脱细胞处理人牙周膜细胞（hPDLCs）和人骨髓细胞（hBMCs）以制备组织特异性ECM。（2）利用克隆形成实验检测两种组织特异性ECM对于体外培养hPDLSCs克隆形成的影响，利用成骨成脂诱导实验检测两种组织特异性ECM对体外培养hPDLSCs成骨与成脂的影响，以反映ECM对hPDLSCs干细胞特性的影响。（3）将hPDLSCs与骨髓细胞ECM和牙周膜细胞ECM共培养，结合无机牛骨粉植入裸鼠皮下，8周后取材进行HE染色、Masson三色染色、苦味酸-天狼猩红以及免疫组织化学染色观察新生成的组织。研究结果：（1）通过有限稀释法获得的hPDLSCs，具有克隆形成能力，表达间充质干细胞标志，并具有多向分化能力。光学显微镜下，脱细胞处理之后，无细胞结构存在，ECM呈网状膜片。（2）接种在两种组织特异性ECM上的hPDLSCs的克隆形成率均明显高于对照组，经过多次传代之后，ECM中培养的hPDLSCs仍保持较高的克隆形成能力。接种在两种组织特异性ECM上的hPDLSCs多次传代之后的成骨成脂分化能力也明显高于对照组。（3）hPDLSCs和hBMCs-ECM、hPDLCs-ECM共培养后与无机牛骨粉混合，植入裸鼠皮下，在hBMCs-ECM组生成了类似骨质和牙周膜纤维样结构，hPDLCs-ECM组生成牙周膜样纤维，含大量的Ⅰ型胶原和Ⅲ型胶原，对照组仅有疏松的纤维结缔组织生成，主要为Ⅰ型胶原。结论: （1）通过有效的脱细胞处理方法可以制得结构完整和具有良好生物学功能的ECM（hPDLCs-ECM和hBMCs-ECM）。（2）组织特异性ECM（hPDLCs-ECM和hBMCs-ECM）对体外培养人牙周膜干细胞（hPDLSCs）维持干细胞特性有显著的促进作用，hPDLSCs经体外多次传代扩增后仍保持较高的自我更新和多向分化的能力。（3）组织特异性ECM能够促使hPDLSCs在体内生成类骨质和牙周膜样纤维结构，体现出良好的牙周组织再生能力。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.42

【参考文献】