PDGF-BB激活NR4A1/SP1复合体募集内源性MSCs促进组织工程骨修复的机制研究

Classic tissue engineered-bone (TEB) brings the hope for the repair of large bone defect, however, it is restricted due to the long amplification cycle of seed cells and Ethics. Selective cell retention (SCR) technology can construct TEB rapidly for its integration of components including of the seed cells, scaffolds, and growth factors and so on. The recruitment and homing of endogenous MSCs play an important role for damaged tissue, however, the key of research is, how they are recruited to TEB? Our preliminary study found that the high relevance of exogenous PDGF-BB among PKC, nuclear receptor NR4A1, transcription factor SP1 for MSCs migration. According to the study of the professional fields, we propose that PDGF-BB could bind to receptors β in the membrane surface of MSCs, increasing the Ca2+ concentration of cytosolic, and activation of PKC regulate the cytoskeletal F-actin of MSCs through NR4A1/SP1 complex, and mobilize the migration of MSCs to the anchor area for ossification. In this way, we will identify the key bit of signal pathway by the activation and inhibition of target molecule, and validate the direct regulation of NR4A1/SP1 complex for migration of MSCs by siRNA technology, and confirm the expression of key molecules in the signal pathway in vivo by Western blot and other methods, and further observe its efficacy on post-osteogenic. This research will provide experimental basis for the rapid construction of TEB by the signal pathways of PDGF-BB activated migration for endogenous MSCs.

经典的组织工程骨（TEB）为大段骨缺损修复带来希望，但受种子细胞扩增周期长、伦理等限制；选择性细胞滞留（SCR）技术可将种子细胞、支架、生长因子等关键成份快速整合，实现TEB快速构建。内源性MSC募集和归巢是受损组织修复的重要机制，而其如何被募集到TEB是研究的关键。前期研究发现，PDGF-BB与MSC的PKC、核受体NR4A1、转录因子SP1的表达高度相关。提出，PDGF-BB可能结合MSC膜β受体后，致胞浆Ca2+ 升高，激活的PKC通过NR4A1/SP1调控细胞骨架 F-actin，调动MSC向锚着点迁移，促进骨修复。藉此，可望通过靶分子激活/阻断等明确通路的关键环节；拟采用siRNA技术验证复合体对MSC迁移的直接作用；在体内用免疫印迹等印证通路关键分子的表达，并观察对成骨效果的影响。本项目通过阐明外源性PDGF-BB激活内源性MSC迁移的信号通路，为TEB的快速构建提供实验基础。

组织工程骨的快速构建策略（STEB）是将成骨关键成份包括：干细胞、生长因子和支架材料快速整合，以达到自体骨同等的修复效果。但STEB与TEB相比，干细胞和生长因子的量相差1-2个数量级，而二者的成骨效果相当。研究表明，内源性间充质干细胞（MSCs）的募集和归巢是重要的成骨机制之一，结合前期基础，我们提出：PDGF-BB激活NR4A1/SP1复合体募集内源性MSCs促进组织工程骨修复的成骨机制。. 本研究明确了25ng/ml的PDGF-BB是诱导MSCs迁移的最佳浓度，其机制为：PDGF-BB结合MSCs膜表面的β受体，激活蛋白激酶PKC，抑制NR4A1磷酸化（p-NR4A1），间接促进NR4A1与SP1结合，NR4A1/SP1复合体调节细胞迁移。.主要研究内容，包括：①体外研究了PDGF-BB激活NR4A1/SP1复合体对MSCs迁移的影响；②研究信号通路中的信号传导关系；③验证PDGF-BB在动物体内募集内源性MSCs并促进骨形成的作用。. 本研究取得了一定的重要结果及关键数据，主要包括：①筛选并验证了PDGF-BB诱导MSCs迁移的最佳浓度，发现PDGF-BB受体（PDGFR-β）抑制剂CP-673451能明显抑制BMSCs的迁移。②通过激活/阻断研究，发现PKC是通路的关键分子，RNA沉默分别及同时沉默NR4A1、SP1对MSCs的迁移率具有显著的统计学差异（P<0.05）。③明确了NR4A1/SP1复合体的形成及影响因素，即与PDGF-BB浓度、p-NR4A1磷酸化程度相关。④阐明了NR4A1/SP1复合体对MSCs的迁移起决定性作用。⑤体内验证了PDGF-BB与PKC、NR4A1、SP1及NR4A1/SP1复合体的关系并分析了成骨效果，发现PDGFR-β抑制剂组的成骨效果显著低于正常PDGF-BB组，二者之间的差异有统计学意义（P <0.05），微观结构下观察抑制剂组的新生骨体积及成熟度明显低于正常组。. 本研究的科学意义在于：①证实了"PDGF-BB激活NR4A1/SP1复合体调节内源性MSCs迁移" 的科学假说，将进一步拓宽MSCs生物学信号传导通路；②NR4A1/SP1复合体调节细胞迁移，促进骨形成。这为组织工程骨的快速构建策略提供理论基础，是组织工程骨临床应用研究的一个创新。