外周血来源间充质干细胞向成牙骨质细胞定向诱导分化及其机制研究

The use of peripheral blood derived mesenchymal stem cells (PB-MSCs) has become a new focus in regenerative medicine due to their advantages such as their abundant resources, accessibility and facility for cell isolation. It is reasonable to predict that PB-MSCs may have the potential to become a new cell source for periodontal tissue regeneration. In this proposal, PB-MSCs are isolated from human peripheral blood using modified and optimized density gradient centrifugation and adherence screening methods. The cells are characterized by colony-forming ability, proliferative capacity, migratory potential, the cell surface antigens, differentiation ability, alkaline phosphatase activity and relative gene expression. Then, we established and optimized an artificial microenvironment that mimics the embryonic cementum development for the induction of PB-MSCs to differentiate into cementoblasts, where the conditioned medium from developing apical tooth germ cells (APTG-CM) and enamel matrix proteins (EMPs) isolated from pig developing enamel matrix are used, alone or in combination. Western Blot and iRNA are adopted to study the signal transduction during the differentiation process and the key molecular mechanisms are identified. Finally, the cementum regenerative potential of PB-MSCs was assessed by an in vivo ectopic transplantation model, where the PB-MSCs cell sheets containing the optimized culture medium (APTG-CM/EMPs) were implanted via a human dentin carrier. This research will provide the first experimental evidence that use PB-MSCs for regeneration of tooth cementum, a very important tissue but its formation is extremely difficult. The induction of PB-MSCs differentiation into cementoblasts may offers new insights into the establishment of extracellular microenvironment mimicking tissue development for periodontal tissue engineeirng. The elucidation of the cellular and molecular mechanisms during the differentiating process and the in vitro and in vivo data obtained from this proposal may open the door to a more wide use of PB-MSCs in periodontal tissue engineering and tooth/root regeneration.

外周血间充质干细胞（PB-MSCs）来源充足、取材简便，成为再生医学领域关注的新焦点，有望成为牙周组织再生新的种子细胞来源。本项目拟采用密度梯度离心法结合贴壁筛选法高效分离培养人PB-MSCs，对获得的干细胞进行分子生物学鉴定；应用根端牙胚细胞的条件培养液（APTG-CM）或/和来自猪牙胚上皮根鞘的釉基质蛋白（EMPs）模拟胚胎期牙骨质发育的微环境，探索体外诱导PB-MSCs向成牙骨质细胞定向分化的方法，建立优化的条件诱导模式；初步通过Western Blot和RNA干扰等方法研究其分化过程中的信号转导机制，寻求定向分化关键靶标；最后采用细胞膜片技术，将定向诱导后的PB-MSCs与牙本质载体复合，植入免疫缺陷鼠体内观察牙骨质再生情况。研究结果可望为PB-MSCs用于牙周组织再生提供理论基础，为仿生微环境条件下成牙骨质细胞定向诱导分化提供新思路，为牙周组织工程、牙根及牙再生开辟新的研究空间。

种子细胞来源受限是牙周组织工程研究的瓶颈问题。外周血来源间充质干细胞（PB-MSCs）来源充足、取材简便，有望成为牙周组织再生新的种子细胞来源。牙周组织功能重建的关键和难点是牙骨质的再生，探索PB-MSCs诱导分化为成牙骨质细胞是其应用于牙周治疗领域、成为牙周组织工程种子细胞的关键。.本项目通过采用percoll密度梯度离心法和贴壁筛选法联合应用、加入抗坏血酸等方法成功建立了一套有效分离扩增PB-MSCs的技术和方法，并证实PB-MSCs具有与BM-MSCs类似的生物学特征。还通过将牙胚根部细胞条件培养液（ATGC-CM）和釉基质蛋白（EMPs）联合培养基和PB-MSCs共同培养成功建立了共培养体系，证实此培养体系可有效促进PB-MSCs向成牙骨质细胞分化，PB-MSCs增殖活性降低，碱性磷酸酶ALP活性明显增高，矿化结节形成显著增多，矿化相关基因和蛋白表达明显增强，牙骨质特异性蛋白CAP表达阳性。在此培养体系中加入ERK1/2激酶抑制剂后，ALP活性升高、矿化能力增强等标志成牙骨质方向分化的活动均受到抑制，并且PB-MSCs经定向诱导培养后检测到ERK1/2 磷酸化蛋白表达水平明显增高，而应用ERK1/2激酶抑制剂可抑制PB-MSCs定向分化相关基因表达，提示PB-MSCs的成牙骨质向分化与ERK1/2 信号通路密切相关。除此以外，还通过体内实验证实经诱导培养的PB-MSCs细胞膜片复合牙本质载体植入动物体内可成功重建牙骨质。以上实验和研究首次证实人PB-MSCs可在一定的诱导条件下向成牙骨质细胞定向分化，其细胞膜片植入体内可成功重建牙骨质，PB-MSCs有望成为牙周组织工程理想的种子细胞。本研究为牙周组织工程提供了新的理想种子细胞来源，为解决牙周组织再生种子细胞来源受限等瓶颈问题提供了研究基础，为牙周组织工程开辟了新的研究空间，有望加速牙周组织再生临床转化的进程，也为PB-MSCs在再生医学研究领域的推广应用提供了理论基础和实验依据。