成年大鼠嗅球嗅鞘细胞的纯化实验(英文)

Purifying olfactory ensheathing cells from the olfactory bulb of adult rats

背景:嗅鞘细胞的纯化方法以及嗅鞘细胞纯度的不同被认为与嗅鞘细胞移植后的效果有关。因此,开发高效、便于统一的纯化方法对嗅鞘细胞移植研究的标准化非常重要。目的:为嗅鞘细胞移植研究的标准化提供一个高效、便于统一的纯化方法。设计:随机对照实验。单位:东南大学临床医学院附属中大医院骨科,东南大学临床医学院中心实验室,东南大学临床医学院实验动物中心。材料:实验于2006-02/08在东南大学临床医学院中心实验室完成。选用28只成年雌性SD大鼠,体质量200￣250g;DMEM/F-12(GIBCO);2.5g/L胰蛋白酶(GIBCO);左旋多聚赖氨酸(SIGMA);牛垂体萃取液(SIGMA);胎牛血清(杭州四季青生物试剂公司);兔抗p75抗体(SIGMA);生物素化羊抗兔IgG(武汉博士德生物技术公司);MTT试剂盒(SIGMA)。方法:将SD大鼠嗅球中分离出嗅鞘细胞的原代培养物,体外培养8d,将培养物分为4组:差速贴壁组、免疫吸附组、改良方法组、对照组。①改良方法组细胞悬液接种到未经包被的培养瓶在37℃、体积分数为0.05CO2的环境中孵育1h,将上清液接种到培养瓶中(底面用1mg/L的兔抗p75抗体润湿后在37℃下烘干,再用DMEM/F-12洗涤1次)。上清液在这种已包被兔抗p75抗体的培养瓶中37℃、体积分数为0.05CO2下孵育45min,DMEM/F-12洗涤5遍以清除未贴壁的细胞,用细胞刮刀收获贴壁细胞、离心、重新悬浮在含20mg/L牛垂体萃取液和10万U/L青链霉素的D/F-10S中;Nash差速贴壁组细胞按Nash的方法进行操作;抗p75抗体免疫吸附组细胞按照Ramo’n-Cueto的方法进行操作;上述纯化方法获得的3组细胞悬液分别接种到包被左旋多聚赖氨酸的24孔细胞培养板中,在37℃、体积分数为0.05CO2的环境中培养14d。对照组未经纯化的细胞悬液同样重新悬浮在含20mg/L牛垂体萃取液和10万U/L青链霉素的D/F-10S中并接种到包被左旋多聚赖氨酸的24孔细胞培养板中,培养条件同其他组。②在各组处理结束后2,5,8,10,12,14d进行嗅鞘细胞纯度比较,每组每个时间点都随机选择15个视野计算嗅鞘细胞比例,这15个数值的平均值代表嗅鞘细胞纯度,从而评估这种改良方法的纯化效率。③分别用MTT法检测处理后第14天各组细胞活力。主要观察指标:各组嗅鞘细胞纯度、处理后14d细胞活力检测结果。结果:①改良方法组每个时间点的嗅鞘细胞纯度都高于其他3组(P<0.05～0.01),各组嗅鞘细胞纯度都随培养时间延长而降低,但改良方法组的纯度变化最小,改良方法组最后1个时间点的纯度仍然很高(92.1±1.2)%,而其他组最高只有(85.2±2.2)%。②处理结束后第14天,改良方法组嗅鞘细胞细胞活力与其他组细胞活力差异无显著性(P=0.895)。结论:本组纯化成年大鼠嗅球嗅鞘细胞的改良方法是高效的,而且对嗅鞘细胞的活力无额外损害,将有益于嗅鞘细胞研究的标准化。

BACKGROUND：The diversity of purification procedures resulting in various pudties of olfactory ensheathing cells （OECs） used for grafting is considered to be relevant in the effectiveness of OECs transplant. It is important to develop a well-defined method which produces OECs of great purity and is easy to unify for the future standardization of research involving OECs. OBJECTIVE： To establish a method being easy to unify for purifying OECs to acquire highly and uniformly enriched population of OECs for standardized studies on cell transplantation. DESIGN： Randomized and controlled experiment SETTING： Department of Orthopaedics, Affiliated Zhongda Hospital of Southeast University School of Clinical Medicine; Central Laboratory of Southeast University School of Clinical Medicine; Experimental Animal Center of Southeast University School of Clinical Medicine. MATERIALS： This experiment was carded out in the Central Laboratory of Southeast University School of Clinical Medicine from February to August 2006. Twenty-eight adult female SD rats weighing 200-250 g were selected in this study. The main reagents were detailed as follows： DMEM/F-12 （GIBCO）; 2.5 g/L trypsin （GIBCO）; poly-L-lysine （SIGMA）; bovine pituitary extract （BPE, SIGMA）; fetal bovine serum （FBS, Sijiqing Biological Agent Co., Ltd., Hangzhou）; rabbit anti-low-affinity nerve growth factor receptor （anti-P^75, SIGMA）; biotinylated goat anti-rabbit IgG （Boster Bioengineering Co., Ltd,, Wuhan）; methyl thiazolyl tetrazolium （MTT） kit （SIGMA）. METHODS： Pdmary cultures of OECs were separated from adult SD rats olfactory bulbs. At day 8 in vitro, the primary cultures were divided randomly into 4 groups, namely differential adhesion method group, immunoadsorption method group, the modified method group, and control group. ① The cell suspension in the modified method group was seeded into uncoated flasks and incubated at 37 ℃ in 0.05 volume fraction of CO2 for 1 hours. The supernatants were seeded into flasks that had been prepared as follows. The bottoms of these flasks were moistened with anti-P^75 （1 mg/L） and were made to dry at 37 %, and then they were washed one time with DMEM/F-12. The supernatants were incubated on the anti-P ^75-treated flasks for 45 minutes at 37 %, 0.05 volume fraction of CO2. For removing unbound cells, the flasks were washed five times with DMEM/F-12. The bound cells were detached from the flasks with a cell scraper, centrifuged, and resuspended in D/F-10S with 10^5 U/L penicillin/streptomycin and 20 mg/L BPE. The cell suspension in differential anchoring method group or immunoadsorptlon method group was purified as previously described by Nash or Ramo＇n-Cueto respectively. Three groups of cell suspensions resulted from the above three methods were seeded respectively onto poly-L-lysine-coated 24-well cell culture chambers and incubated for 14 days at 37 ℃ in 0.05 volume fraction of CO2. Without purification, the cell suspension in control group was also resuspended in D/F-10S with 105 U/L penicillin/streptomycin and 20 mg/L BPE and seeded onto poly-L-lysine-coated 24-well cell culture chambers and incubated under the same culture condition as the other groups. ② Pudty comparisons for the four groups were made at 2, 5, 8, 10, 12 and 14 days after the end of their respective manipulation to evaluate the effectiveness of the modified method. At per time point in each of the four groups, fifteen visual fields of cultures were selected randomly to count the proportion of OECs and the mean which was determined by averaging the 15 values represented the purity of OECs. ③ At the day of 14, viabilities of OECs in the four groups were assessed by MTT assays. MAIN OUTCOME MEASURES ： OECs purities at per time point and viabilities of OECs at the day of 14 in each of the four groups after the end of their respective manipulation. RESULTS： ① The purities of OECs in the modified method group at each time point were greater （P 〈 0.05-0.01） than counterparts in the other three groups. OECs purities decreased with culture prolongation in all groups, but the changes of purities over the whole period of observation in the modified method group were the least. The last purities of OECs yielded from the modified method were still extremely great （92.1 ±1.2）%, whereas the parallels in the others were no more than （85.2±2.2）%. ② There was no significant difference in viabilities of OECs between the modified method group and any of the others at the day of 14 （P =0.895）. CONCLUSION ： The modified method for purifying OECs from the adult rat olfactory bulb is highly effective without extra impairment on the viability of OECs and will be beneficial to the future standardization of research involving OECs.