Bcl-2和cMyc基因调控牙髓干细胞与骨髓来源细胞竞争及促进牙髓再生的实验研究

Background: Recently, multiple studies have shown that continued root development and ingrowth of new vital tissue into the canal space can be achieved after disinfection, evoked bleeding into the canal for an immature tooth with infected pulp necrosis. In further animal study, we found that the ingrowth of new tissue had little similarity to normal pulp tissue, consisting of cementum, periodontal ligament and bone. Key issues: It was hypothesized that remaining vital pulp and/or apical papilla may reconstitute pulp/dentin after disinfection. However, the cells might be derived from several sources, including bone marrow-derived cells, bone and periodontal ligament. The type of tissue healing might be determined upon the stem cells capacity recruited into the given location. A competition between different tissue compartment cells whereby a disinfected pulp space might be occupied by PDL cells and bone cells. Thus, the source of stem cells responsible to tissue metaplasia is fundamental issue in understanding the revascularization mechanism and therapy for this disease. Aims: To determine whether bone marrow-derived cells contribute to the pulp tissue revascularization; whether implantation of dental pulp stem cells (DPSCs) can compete with the recruited bone marrow-derived cells to influence their growth and contribution to pulp/dentin tissue regeneration; whether coexpression of cMyc and Bcl-2 in DPSCs can regulate DPSCs proliferation and apoptosis, enhance pulp-like tissue regeneration.Research plan: First, the chimeric model mice are created: isolated whole bone marrow-derived cells from GFP-trangenic mice are injected into the tail veins of recipient wild-type C57BL/6 mice that have been irradiated with a lethal dose. Next, the necrotic immature teeth are induced in mandibular first molars followed by canal disinfection, blood clot formation and coronal sealing. In another group, after canal disinfection, dental pulp stem cell microspheres are injected into the canals and then sealed. The jaws will be dissected for radiographic, histological and immunohistochemical analyses. The type of tissue regenerated, GFP+ cells distribution and dual immunofluorescence will be evaluated. Secondly, DPSCs will be transfected with c-Myc and Bcl-2. Overexpression of c-Myc and Bcl-2 on cell proliferation and apoptosis will be assessed by detection of Ki-67 and TUNEL method. Then gene-modified DPSCs will be cultured in 3-dimensional microsphere, and injected into mice model of molar apical periodontitis. Cell survival, histological and immunohistological analysis will be performed. Significance: The demonstration of host bone marrow-derived cell origin of the newly generated tissue and the results of DPSCs with or without expression of cMyc and Bcl-2 competing with the recruited bone marrow-derived cells to influence tissue regeneration lays the groundwork for future targeting on regulation of both implanted DPSCs and the recruited bone marrow-derived cells.

牙髓坏死的年轻恒牙经过牙髓再血管化处理，牙根能继续发育，并有新生组织长入根管。动物实验证实，根管内的新生组织含有类牙骨质和骨样组织。但牙根继续发育的机理和新生组织的细胞来源仍不清楚。干细胞可能有多个来源，包括骨髓、牙周膜和牙髓组织。再生组织的类型取决于募集干细胞的来源，干细胞之间的竞争。因此，探索骨髓来源细胞是否参与牙髓再血管化、研究牙髓干细胞和骨髓来源细胞的竞争机制以及对新生组织的影响，能为牙髓再生新方法提供生物学基础，也有助于丰富细胞竞争理论在组织再生中的应用研究。本课题拟建立骨髓嵌合体小鼠磨牙根尖周炎模型，进行牙髓再血管化，通过组织学、免疫组化研究新生组织结构、骨髓来源细胞的分布和分化；Bcl-2和cMyc基因转染牙髓干细胞，促进其增殖，抑制凋亡，通过动物实验，探索Bcl-2和cMyc基因过表达能否提高植入根管内的牙髓干细胞的竞争力，诱导募集的宿主干细胞凋亡，改善牙髓新生组织的结构。

本研究首先证明了骨髓来源细胞能够迁移到牙髓损伤区域, 参与牙髓再血管化的过程。骨髓来源细胞能分化为nestin+/GFP+, a-SMA/GFP+, and NeuN/GFP+ cells牙髓组织特异性的细胞。通过转染Bcl-2和cMyc基因，能显著促进牙髓干细胞的增殖，减少细胞的凋亡和死亡，进一步提高体内牙髓组织再生能力。通过将牙髓干细胞植入根管内进行牙髓再生，并分析新生组织的结构。结果表明根管内新生组织更近似牙周组织，而不是牙髓样组织。Bcl-2转染细胞不仅能促进细胞的存活，而且在缺氧条件下，其促进细胞分泌VEGF水平，促进血管网形成。动物体内研究表明，转染Bcl-2的牙髓干细胞有助于促进新生血管组织和牙髓组织的再生。