周敏　胡鸣　何松明

[摘要] 目的 通過比较两种不同的新生大鼠施万细胞（SCs）原代培养的方法（双酶消化法与单酶消化法），探索出一种细胞纯度更高、获取时间更短的SCs细胞培养方法。方法 取4～5 d SD乳鼠双侧坐骨神经，分别采用单酶和双酶消化法，联合运用半植块法、差速消化法、差速贴壁法以及抗有丝分裂法原代培养纯化SCs。培养SCs 48 h和6 d后倒置显微镜观察两组细胞形态，7 d后分别进行SCs消化与传代，细胞计数板计数两组的SCs总数，传代48 h后用免疫荧光标记S-100蛋白对两组细胞进行鉴定，CCK-8测定细胞生长曲线。结果 用等量的神经组织，与单酶消化法比较，双酶消化法培养的SCs所获得SCs数目远高于单酶消化法所获得细胞数目（2倍以上）。免疫荧光结果显示，双酶消化法纯度[（95.29±1.71）%]高于单酶消化法[（72.41±2.16）%]（P < 0.05），两种方法P3代细胞生长曲线差异无统计学意义（P > 0.05）。 结论 在短时间内，用双酶消化法联合半植块法、差速消化法、差速贴壁法以及抗有丝分裂法可获得足量和高纯度的SCs。

[关键词] 施万细胞；原代培养；双酶消化法；单酶消化法

[中图分类号] R329.21 [文献标识码] A [文章编号] 1673-7210（2018）05（a）-0014-04

Experimental study on culturing Schwann cells of neonatal rats in vitro

ZHOU Min HU Ming HE Songming HUANG Guotao CHEN Qian HONG Li

Department of Gynecology and Obstetrics， People's Hospital of Wuhan University， Hubei Province， Wuhan 430060， China

[Abstract] Objective To explore a culture of SCs method with higher cell purity and shorter time by comparing the two methods including the double-enzyme digestion and single-enzyme digestion of primary culture of Schwann cells （SCs） using the neonatal rats. Methods Bilaterally sciatic nerves were dissected from 4 to 5-day-old neonatal SD rats and double-enzyme digestion method and single-enzyme digestion method which both combined with hemi-explants-culture method， differential digestion method， differential adhesion method， and cytosine arabinoside were used to culture and purify SCs， respectively. The morphology of the SCs was observed under the inverted microscope at 48 hours and 6 days of culture. Then the SCs were digested and passaged after 7 days in culture and counted under the cell counter. The purity of SCs was identified by S-100 immunofluorescence staining. CCK-8 was used to measure the P3 cells growth curve， respectively. Results With the same number of neonatal rats， double-enzyme digestion method acquired SCs at least twice than single-enzyme digestion method. （95.29±1.71）% of S-100 positive SCs cultured were shown by immunofluorescence staining in the double-enzyme digestion method group， and （72.41±2.16）% in the single-enzyme digestion method group （P < 0.05）. There was no difference between the two cultures in the growth curve of P3 cells （P > 0.05）. Conclusion The double-enzyme digestion combined with hemi-explants-culture method， differential digestion method， differential adhesion method， and cytosine arabinoside may obtain sufficient amount of high-purity SCs in a short time and be applied in the further cytological experimental research on peripheral nerve regeneration.

[Key words] Schwann cell； Primary culture； Double-enzyme digestion method； Single-enzyme digestion method

施万细胞（Schwann cells，SCs）是一类特殊的神经胶质细胞，其包裹着外周神经轴突形成有髓神经纤维的髓鞘。当周围神经受损后，SCs与神经元之间的信号转导对神经轴突的生长和维持有重要作用[1-2]。SCs主要通过调节突触前神经轴突末梢的活动、产生神经营养因子，以及清除轴突、髓鞘碎片，在基底膜形成有效的基板，形成再生的神经元轴突生长的通道，从而促进轴突再生[3-6]。因此，研究周围神经再生就要求能够在短时间内高效率提供足量高纯度的SCs[7]。……