大鼠坐骨神经雪旺细胞体外培养纯化的实验研究

Experimental study of purifying rat sciatic nerve Schwann cells in vitro

目的:探索大鼠坐骨神经雪旺细胞体外培养纯化的方法,以获取大量的雪旺细胞应用于周围神经组织工程研究。方法:取预变性坐骨神经,去神经外膜,剪碎块后,用胰酶及胶原酶组成的混合酶消化15min,加入含10%胎牛血清的DMEM吹打成悬液进行培养。24h后将培养液全部吸出,组织块中的细胞游出铺满皿底后,将组织块移入新的培养皿中,反复3次后将得到雪旺细胞扩大培养,加入b-FGF(20ng/mL),待细胞铺满瓶底的80%～90%时,即可传代。纯化后细胞行S-100免疫细胞化学鉴定。结果:双酶消化法结合差速贴壁法可去除较多杂细胞,应用b-FGF刺激雪旺细胞增殖,可获得大量高纯度的雪旺细胞,经S-100蛋白免疫细胞化学鉴定,雪旺细胞纯度达96%以上。结论:双酶消化法、差速贴壁法结合b-FGF刺激增殖的方法可从坐骨神经中分离纯化大量雪旺细胞。

Objective：To explore the method of the rat sciatic nerve Schwann cells purification for obtaining a large number of Schwann cells to be used in peripheral nerve tissue engineering.Methods：To adopt predegenerated sciatic nerve,remore epineurium,shear fragments,digest for 15 min with the mixed enzyme consisted of trypsin and collagenast,then add with 10% FBS DMEM for wind and percussion into a suspension culture.24 h later,sucking out of the culture solution,removing the tissue pieces to a new culture dish after the cell moving out from the tissue pieces and covering the dish bottom.This process was repeated for 3 times.Cultured expansion of Schwann cells was abtained.Adding b-FGF （20 ng/mL）,when cells covered 80%~90% of the bottom,the passage could be carried out.Purified cell line was performed by S-100 immunocytochemical identification.Results：Dual-enzyme digestion method and differential adherent method could remove more miscellaneous cells.b-FGF could stimulate proliferation of Schwann cells,a large number of highpurity Schwann cells could be abtained,by S -100 protein immunocytochemistry identification Schwann cell purity was above 96% .Conclusion：Dual-enzyme digestion,differential adherence method and b-FGF can stimulate the proliferation.This method can purify a large number of Schwann cells from sciatic nerve.