P38/AKT通路调控骨髓间充质干细胞在不同空间结构纳米纤维环支架中的定向分化

文题释义： 拓扑结构：泛指组织工程支架的一系列细微的结构差异，涵盖多方面因素，如支架质地、硬度等材料特性相关的属性；支架表面的光滑程度、支架内部的孔隙率等结构特性等；实验主要指静电纺丝纤维环支架中纤维排列方式。 差异分化：实验中用于描述骨髓间充质干细胞在不同条件下(主要指支架拓扑结构的不同)，接受外界信息后发生一系列变化，通过细胞通路等机制的调节分化为不同细胞的现象，如实验中骨髓间充质干细胞在不同支架中分别向成纤维细胞或成软骨细胞分化。对差异分化的机制进行研究，有助于今后通过改变条件诱导细胞向所需方向分化、发挥组织工程技术的优势更好修复组织的目的。 背景：以往研究表明多种材料可用于组织工程支架的构建，支架表面的拓扑结构特征对干细胞增殖、分化等生物学行为有调节作用，但其具体机制尚不明确。 目的：研究骨髓间充质干细胞在纳米结构支架中定向分化过程中P38、AKT通路的作用。 方法：构建3种结构不同的纳米纤维支架，即取向纳米纤维支架AFS、取向纳米纱支架AYS、三维多孔纳米纤维支架3-DPS；将大鼠骨髓间充质干细胞分别接种于3种纳米纤维支架表面，成软骨诱导培养后，分别进行细胞形态、黏附、增殖检测，利用qRT-PCR检测细胞关键表型分子(Ⅱ型胶原α1、Ⅰ型胶原α1、蛋白聚糖、Sox-9)mRNA的表达水平，Western blot测定细胞内P38、AKT、ERK1/2、JNK的蛋白表达水平。 结果与结论：①3-DPS支架组培养4，8 h的细胞黏附率高于其余两组支架(P < 0.05)，培养7 d的细胞增殖快于其余两组支架(P < 0.05)；②骨髓间充质干细胞在3种材料表面均贴附牢固，生长状态良好，其中在AFS、AYS支架上呈类成纤维细胞样生长，在3-DPS支架上呈类软骨细胞样生长；③成软骨诱导3周后，3-DPS支架组Ⅱ型胶原α1、蛋白聚糖、Sox9 mRNA表达高于其余两组支架(P < 0.05)，Ⅰ型胶原α1 mRNA表达低于其余两组支架(P < 0.05)；AFS支架组、AYS支架组中Ⅱ型胶原α1、蛋白聚糖、Sox9 mRNA表达无明显差异(P > 0.05)；④成软骨诱导3周后，3-DPS支架组p-AKT、p-P38蛋白相对表达量高于其余两组支架(P < 0.05)，3组间AKT总蛋白表达量及ERK1/2、JNK、P38、p-ERK1/2、p-JNK、p-P38蛋白表达量比较均无显著性意义；⑤结果表明，不同结构支架形貌可通过Integrin-FAK信号通路下游的P38、AKT通路诱导骨髓间充质干细胞的的定向分化。 ORCID: 0000-0003-3571-8840(王爽) 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

P38/Akt pathway regulates the oriented differentiation of bone marrow mesenchymal stem cells in nanofiber annulus fibrosus scaffolds with different spatial structures

BACKGROUND:Previous studies have shown that a variety of materials can be used for the construction of tissue engineering scaffolds.The topological structure of the scaffold surface has a regulatory effect on the biological behaviors such as stem cell proliferation and differentiation,but the specific mechanism is still unclear.OBJECTIVE:To investigate the role of P38 and Akt pathways in the oriented differentiation of bone marrow mesenchymal stem cells in nanofiber scaffolds.METHODS:Three kinds of nanofiber scaffolds(AFS,AYS,3-DPS)with different structures were constructed.Rat bone marrow mesenchymal stem cells were inoculated on the surface of three kinds of nanofiber scaffolds.After osteogenic induction,cell morphology,adhesion and proliferation were detected.mRNA expression levels of key phenotype molecules(COLIα1,COLIIα1,Aggrecan,Sox-9)were measured using qRT-PCR.Intracellular P38,AKT,ERK1/2 and JNK expression was detected by western blot assay.RESULTS AND CONCLUSION:After 4 and 8 hours of culture,cell adhesion rate of the 13-DPS scaffold group was higher than that of the AFS and AYS scaffold groups(P<0.05).After 7 days of culture,cells of the 13-DPS scaffold group proliferated faster than those of AFS and AYS scaffold groups(P<0.05).Bone marrow mesenchymal stem cells adhered firmly and grew well on three kinds of scaffolds.Fibroblast-like growth was observed on the AFS and AYS scaffolds and chondrocyte-like growth was observed on the 3-DPS scaffold.After 3 weeks of cartilage induction,mRNA expression of COLIIα1,Aggrecan and Sox-9 was higher,and the mRNA expression of COLIα1 was lower,in the 3-DPS scaffold group compared with the other two groups(both P<0.05).After 3 weeks of cartilage induction,relative expression level of p-AKT and p-P38 in the 3-DPS scaffold group was significantly higher than that in the other two groups(both P<0.05).There were no significant differences in AKT total protein and ERK1/2,JNK,P38,p-ERK1/2,p-JNK and p-P38 protein expression levels among three groups.These findings suggest that nanofiber annulus fibrosus scaffolds with different spatial structures can induce the oriented differentiation of bone marrow mesenchymal stem cells through the P38 and AKT pathway,which were the downstream of the Integrin-FAK signaling pathway.