血管内皮祖细胞分泌内皮素1增强共培养骨髓间充质干细胞成骨效应的机制研究

Bone regeneration is required to repair mandible defects arising from trauma, inflammation, or tumor. Bone tissue engineering is becoming a primary approach in bone reconstruct. Moreover, this approach can eliminate the donor-site pain, limited vailability of autologous grafts and immunogenicity of allogenic grafts, and reduce the surgery time. Bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells having the potential to differentiate into bone, cartilage. the osteogenic stem cells with osteogenic factors were less effective in treating large bone defects due to inefficient blood supply in the entire bone graft. In order to gain an insight into the vascularization process, the co-culture methods with angiogenic cells have been established. One potential approach to achieve such vascularization was the application of endothelial progenitor cells (EPCs). Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial progenitor cells and osteoblast-like cells during osteogenesis. Study showed that BMSCs behave differently in terms of proliferation and differentiation when cultured in association with endothelial cells in various coculture models than when they are cultured alone. In our previous experiment, an increase in alkaline phosphatase activity (ALP) was observed when BMSCs were cocultured with EPCs,the up regulation of gene related to osteogenesis and angiogenesis such as COL1, OCN, BMP2, VEGF, cdh5, vWF were also detected. By using the BMSCs/EPCs/scaffold construct in rat critical size bone defect model, we found the defect was almost complete healed in BMSCs/EPCs group after 7 weeks. We conclude there is reciprocal regulation bewteen BMSCs and EPCs. Endothlin 1(ET-1) might be a important factor. Therefore, the objective of this study is to developed co-culture system of BMSCs /EPCs, to investigate the mechanism of ET-1 synthesis in EPCs. qRT-PCR and western blot were used to investigate the mechanism of ET-1 enhanced BMSCs osteogenesis via MAPK signaling pathway. the result is confirm by silence and overexpression the key gene in MAPK path way, and the osteogenic effect of the silenced/overexpressed BMSCs/EPCs is evaluted in vitro by using qRT-PCR, western blot, ALP assay and von kossa staining. We can figure out the reciprocal regulation and functional relationship between EPCs and BMSCs by analysing the result, and find a better way in bone tissue engineering, which has the potential to provide reconstructive surgeons with new and advanced treatment modalities for wounded patients by promoting vascularized bone regeneration.

骨修复过程中的血管化是骨组织工程的关键，近期研究表明成骨细胞及其前体细胞与血管内皮祖细胞（EPCs）间可能通过相互交流耦合机制及旁分泌信号等方式进行功能联系，调控成骨与血管新生。在前期工作中我们发现体外共同培养的BMSCs/EPCs中成骨成血管相关基因的表达上调，矿化结节形成增加，大鼠颅骨缺损修复实验表明缺损区新骨及新血管生成有明显增加。这可能是由于EPCs合成的内皮素（ET-1）增加，促进BMSCs成骨分化引起的。因此我们拟利用体外BMSCs/EPCs共培养体系，对两种细胞间相互作用机制进行深入研究。我们采用生物学相关技术研究BMSCs通过PI3K/HIF1a通路促进EPCs合成ET-1的机制，同时分析ET-1通过MAPK通路对BMSCs成骨分化的调控机制，并通过基因沉默/过表达信号通路中的关键基因进行验证，阐明EPCs调控BMSCs增殖，分化等生物学行为的分子机制。

研究表明成骨细胞及其前体细胞与血管内皮祖细胞（EPCs）间可能通过相互交流耦合机制及旁分泌信号等方式进行功能联系，调控成骨与血管新生。在前期工作中我们发现体外共同培养的BMSCs/EPCs能够增强成骨成血管相关基因的表达，并且矿化结节形成增加，大鼠颅骨缺损修复实验表明缺损区新骨及新血管生成有明显增加。因此我们拟利用体外BMSCs/EPCs共培养体系，对两种细胞间相互作用机制进行深入研究。本课题的主要内容是：（1）建立BMSCs/EPCs共培养体系并鉴定（2）明确与EPCs刺激BMSCs成骨增强有关的通路。（3）通过体外实验检测BMSCs成骨相关基因表达情况、蛋白合成情况及矿化结节形成情况，验证共培养体系中BMSCs成骨增强与该通路有关。.结果发现：（1）EPCs与BMSCs共培养后，上清中VEGF与ET-1的浓度增加。（2）共培养系统中，过表达/阻断PKC信号通路影响上清中VEGF，ET-1浓度，BMSCs通过激活PKC引起细胞外调节性激酶ERK磷酸化，进而影响EPCs的VEGF，ET-1合成分泌，而HIF1a通路对常规氧浓度下VEGF，ET-1合成没有影响。（3）在BMSCs/EPCs共培养后，BMSCs中的P38MAPK及p42/44MAPK通路被激活，成骨相关基因表达上调，蛋白合成增加，矿化结节形成增加。促进BMSCs增殖及向成骨方向分化。（4）在BMSCs/EPCs中沉默MKK后BMSCs成骨能力明显下降，p42/44MAPK沉默后BMSCs成骨体外矿化影响较小，BMSCs/EPCs共培养后，EPCs可能主要通过P38MAPK影响BMSCs向成骨方向分化。.本研究对 BMSCs与EPCs非接触培养下的相互作用机制进行了研究，结果表明BMSCs可以通过PKC引起EPCs分泌VEGF，ET-1，而EPCs可以通过p38MAPK，P42/44MAPK信号通路促进BMSCs增殖及向成骨方向分化。本课题的研究结果提示我们可以利用不同细胞间旁分泌信号交联耦合机制调控成骨与血管新生，寻找BMSCs向成骨转化的最佳条件，通过研究多种调控因素间的相互作用，为临床上治疗骨缺损提供新的细胞构成设想及新的策略。