摘要
目的探讨移植用成年大鼠嗅球嗅鞘细胞(OECs)的培养和纯化方法。方法取成年SD大鼠嗅球,将分离消化所得细胞悬液进行接种。然后分别于接种后2 h、6 h和18 h 3个时间点进行单步骤差速贴壁法去除成纤维细胞,纯化嗅鞘细胞,纯化培养约11 d时采用神经生长因子受体P^(75)(NGFRP^(75))和碘化丙啶(PI)双染OECs并对其纯度及细胞数量进行免疫荧光鉴定分析。结果纯化培养11 d后在共聚焦显微镜下呈双染阳性的细胞为OECs,数量级达10~5/mL,形态以梭形、双极或三极突起为主,亦有少量多极或单极细胞。2 h、6 h和18 h组嗅鞘细胞纯度分别为(53±5)%、(73±8)%和(69±13)%,组间差异有统计学意义(F=11.766,P<0.001)。结论分别经2、6、18 h单步骤差速贴壁法纯化的OECs在纯度和数量级上均可满足移植用细胞的要求,且方法简单、经济、快捷。
Objective To explore a method to culture and purify olfactory bulb-derived olfactory ensheathing ceils （OB-OECs） from adult rats for transplantation. Methods The olfactory bulbs were harvested from adult SD rats, and cut into pieces. Through enzyme digestion, the monoplast suspension was made and seeded in the flasks. After being incubated 2 hours （group A）, 6 hours （group B） or 18 hours （group C） separately, the supernatant was obtained to seed in the new culture flasks and incubated for about 11 days. Olfactory ensheathing cells（OECs） were purified through above mothods which named modified single step Nash＇s differential attachment technique. The purified OECs were identified by the immunofluorescenee staining of neural growth factor receptor p^75 （NGFR p^75） and Propidium Iodide （PI）. The purity and quantity of OECs were calculated at last. Results After being cultured for 11 days, OECs which were stained by both FITC-NGFR p^25 and PI could be observed with the help of laser scanning confocal microscope （LSCM）. The OECs＇ quantity was about 10^6/mL. And there were many bipolar, tripolar cells and a few multipolar cells which were all confirmed as the OECs. By using our modified single step Nash＇s technique, the purity of the OECs in group A was （53±5）%, group B was （73±8）%, group C was （69±13）% respectively. And there was significant difference among the three groups （F= 11. 766, P〈0. 001）. Conclusions The purity and quantity of the OECs that were obtained with our 2 h, 6 h and 18 h modified single step Nash＇s differential attachment technique could meet the requirement of transplantation, and the technique was inexpensive and simple.
出处
《中国神经免疫学和神经病学杂志》
CAS
2009年第4期289-292,共4页
Chinese Journal of Neuroimmunology and Neurology
关键词
嗅鞘细胞
细胞纯化
差速贴壁法
细胞移植
嗅球
olfactory ensheathing cells
purification of cells
Nash＇s differential attachment
transplantation
olfactory bulbs
