纤维蛋白支架促进神经干细胞向神经元分化并抑制星形胶质细胞增生

Fibrin scaffold promoted the differentiation of neural stem cells to the neurons and inhibited the proliferation of astrocytes

目的探讨纤维蛋白支架对神经干细胞和星形胶质细胞分化及增殖的影响。方法分别培养胚胎大鼠脊髓来源的神经干细胞和新生鼠脊髓神经胶质细胞,接种于纤维蛋白支架上,同时用多聚赖氨酸修饰的玻片作为对照。于体外培养不同时间后,用神经丝蛋白(NF200)对神经细胞进行免疫荧光染色,测量各复孔(n=4)内NF阳性细胞的突起长度,计算其平均值;用胶质纤维酸性蛋白(GFAP)对胶质细胞进行染色,各复孔(n=4)内统计5个不同视野的胶质细胞总数和GFAP阳性细胞数,计算GFAP阳性细胞相对数量的平均值。比较在纤维蛋白支架和玻片上神经干细胞分化、神经纤维延伸及神经胶质细胞增殖的差异。同时用免疫印迹技术对荧光染色结果进行验证。上述实验各重复3次。结果纤维蛋白支架组的NF阳性纤维明显长于对照组,GFAP阳性星形胶质细胞相对数量明显少于对照组,GFAP的表达水平明显低于对照组。结论纤维蛋白支架可促进神经干细胞向神经细胞分化,并有利于神经纤维的延伸而抑制星形胶质细胞的增殖和成熟。

Objective To investigate the effects of the fibrin scaffold on the differentiation and the proliferation of neural stem cells and astrocytes. Methods Neural stem cells and the gliocytes derived from spinal cord were cultured in vitro respectively. The purified neural stem cells or gliocytes were seeded separately onto the fibrin scaffolds as experimental group and the glass slides modified with poly-L-lysine（PLL）as control group. At different time in culture the neural stem cells were immunofluorescence stained with antibodies against the marker of neurons i. e. neurofilament（NF）. The length of NF-positive neuritis was masured and the average value was calculated in the culture well （n = 4）. The gliocytes were immunofluorescence stained with antibodies against the marker of astrocytes i.e. glial fibrillary acidic protein （ GFAP ）. The total number of the cells and the GFAP-positive cells were counted from 5 different fields of vision in the culture well （n = 4） , then the average ratio of GFAP-positive ceils was calculated. The differentiation of neural stem cells, the extension of neurites and the proliferation of astrocytes on the fibrin scaffolds were compared with those on the slides. The protein of GFAP was detected by Western blotting to analyse the mature degree of astrocytes. All above experiments were repeated 3 times respectively. Results lmmunofluorescence staining showed that the NF-positive neurites in the fibrin scaffold group were longer than those in the control group, whereas GFAP-positive cells were fewer than those in the control group. The expression of GFAP in the cells on the scaffold was lower than that in the control group. Conclusion The fibrin scaffold could promote differentiation of the neural stem cells to neurons and extension of the neurites. Meanwhile, the scaffold could inhibit proliferation and mature of the astrocytes.