胰酶限时消化在嗅鞘细胞培养中的运用

摘要

目的 探索出一套高效、实用的大鼠嗅球嗅鞘细胞(OECs)的培养和纯化方案.方法 分离SD大鼠嗅球的外两层组织,剪切消化成细胞悬液进行接种.采用3种方法进行OECs的培养和纯化:(1)A组分别经6h、24 h两次差速贴壁去除杂质细胞,培养至12d左右消化重悬细胞进行爬片分析.(2)B组分别经6h、24 h两次差速贴壁去除杂质细胞,培养至12d左右经胰酶限时消化,留成纤维细胞于瓶壁,重悬的细胞进行爬片分析.(3)C组采用经典的Nash法(分别经18 h、36 h两次差速贴壁去除杂质细胞),培养至11d左右消化重悬细胞进行爬片分析.采用NGFRP75与碘化丙啶(PI)双染的方法进行OECs的鉴定和纯度分析.结果 在共聚焦显微镜下呈双染阳性的细胞为OECs,OECs多数突起细长,呈双极或三极,少量呈单极或多极.A、B、C组所纯化的OECs纯度分别为(67.3±6.2)％、(83.7±7.7)％和(74.6士9.5)％,3组间比较有统计学差异(F=13.633,P＜0.01),B组所得OECs纯度均较另两组高(P＜0.05).结论 经6h、24 h两次差速贴壁十胰酶限时消化可以获得较高纯度的OECs,能够满足动物实验的需要.%Objective To explore an efficient and practical method in culturing and purifing procedures of rat olfactory ensheathing cells (OECs). Methods The outer two layers of SD rat olfactory bulb were peel off, cut and digested into monoplast suspension for seeding. Three kinds of purification methods were taken for comparison: (1) In group A, the monoplast suspension was incubated twice for 6 h + 24 h subsequently according to different adherence, the purified OECs were cultured for about 12 days and then were analyzed on coverslip. (2) In group B, the monoplast suspension was incubated as group A and then pancreatic enzyme with limited digestion time was adopted to remain fibroblasts on the sidewall, the suspension was drawn off for analysis as group A. (3) In group C, the monoplast suspension was incubated by Nash' s method (incubate twice for 18 h + 36 h subsequently according to differential adherence), the purified OECs were cultured for about 11 days and then the cells were analyzed on coverslip. The immunofluorescence staining of NGFRp75 and PI were adopted to identify and analyze the purity of the OECs. Results OECs were positive for both FITC-NGFRp" and PI under the laser scanning confocal microscope (LSCM). OECs presented thin and long processes, the major of them were bipolar or tripolar, only a few were unipolar or multipolar. The purity of OECs was (67. 3±6. 2)% in group A, (83. 7±7. 7)% in group B, (74. 6±9. 5)% in group C, respectively. There was significant difference among the three groups (F= 13.633, P < 0. 01), LSD test showed that there was significant difference between group B and group A or group C (P<0. 05). Conclusions The purification procedure of OECs through twice incubation for 6 h and 24 h subsequently according to differential adherence, and digestion time limiting is effective, the high purity of OECs can meet the creteria for animal experiments.