胰酶限时消化在嗅鞘细胞培养中的运用

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、24h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、24h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18h、36h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±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、24h两次差速贴壁+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。

Application of pancreatic enzyme with limited digestion time for olfactory ensheathing cell culturing

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 o...