PHBV纳米纤维对大鼠骨髓间充质干细胞向神经干细胞分化的形态学影响

Morphological study on inducement and differentiation of nerve cells derived from rat BMSCs on PHBV

目的：探究聚羟基丁酸戊酸共聚酯（PHBV）纳米材料对大鼠间充质细胞向神经干细胞分化的影响,为PHBV纳米材料与骨髓间充质干细胞（BMSCs）联合移植治疗脊髓损伤提供新的依据。方法：采用贴壁分离培养的方法分离纯化大鼠BMSC,传至3-4代后接种至PHBV纳米纤维膜（定向与非定向）上,以接种至玻片上为对照组,倒置显微镜下观察对照组细胞形态,待细胞与PHBV纳米纤维膜融合（24 h）后,以维甲酸（RA）作为增殖及分化诱导因子对BMSCs进行增殖培养、分化诱导,1周后,实验组采用4%多聚甲醛固定,应用电镜观察细胞形态,用Nestin抗体免疫细胞化学染色鉴定神经干细胞。结果：BMSCs与PHBV纳米纤维膜有很好的相容性,加入RA诱导剂72 h后细胞形态开始变化,胞体开始回缩,突起逐渐伸出。电镜观察下定向PHBV组的神经元样细胞被显著拉长,并且细胞沿定向纤维的方向伸展,非定向PHBV组与对照组的神经细胞则无明显的拉长状态。免疫荧光染色验证均有Nestin阳性细胞表达。结论：定向纳米纤维通过引导细胞骨架延伸从而对细胞形貌产生显著影响。与非定向纳米纤维相比,神经元样细胞在定向纳米纤维表面被显著拉长。因此,我们认为定向纳米纤维支架可能在神经组织工程中更有优势。

Objective： To investigate the impact of PHBV nanomaterials on differentiation of bone marrow stromal cells（ BMSCs） into neural stem cells,and to provide a new basis for the treatment of spinal cord injury with combined transplantation containing BMSCs and PHBV. Methods： BMSCs derived from rat bone marrow were used to investigate the effects of aligned（ A-NF） and random—oriented（ NF） PHBV nanomaterials on cell adhesion and differentiation in vitro,SEM was used to observe BMSCs. Results： After six days of adding in the RA medium,we can see cells extend along with the direction of orientation of the fibers. Neural stem cells were detected with immunostaining for Nestin. Conclusion： The A-NF PHBV has a significant effect on cell morphology by guiding the cytoskeleton extending,neural stem cells are significantly elongated on the surface of the A-NF PHBV compared with the NF PHBV.