摘要
目的:目前国内外研究介绍的嗅鞘细胞培养方法存在繁杂或重复性差的问题,不利于实际应用。为此实验应用酶消化法体外分离培养大鼠嗅鞘细胞,观察其形态学及表型特征。方法:实验于2005-11/2006-03在上海中医药大学附属龙华医院脊柱病研究所完成。①选取新生1周龄雄性SD大鼠1只,浸入体积分数为0.75的乙醇中3min,碘伏消毒。打开前颅,暴露嗅球,完整取下两个嗅球,放入4℃预冷的DMEM抗菌液中清洗2遍,洗掉嗅球表面的血液等杂质,剥下嗅球被膜,将嗅球用眼科剪剪碎,加入质量浓度为2.5g/L的胰酶,在37℃孵育箱磁力搅拌下孵育15min,DMEM终止消化。细胞液过100μm细胞筛,离心去除上清液,用DMEM稀释细胞浓度至1×109L-1,种植于无包被处理的6孔细胞培养板内常规培养。按差速贴壁法在培养18～20h后吸出细胞悬液,重新种植于包被神经生长因子低亲和力受体p75的6孔细胞培养板内。观察细胞生长情况,每2～3d换液1次,培养14d。②将培养不同时间的嗅球成鞘细胞于倒置显微镜、透射电镜下观察其形态变化,并行苏木精-伊红染色。使用免疫组化法检测嗅鞘细胞的特征性标志神经生长因子低亲和力受体p75的表达情况,阳性表达为棕黄色,无着色为阴性。结果:①倒置显微镜形态学观察:差速贴壁培养第2天可见有较多嗅鞘细胞贴壁生长,细胞突起较短,胞体较大,透亮度一般,成团或散在生长,较难辨认细胞的形态。5～6d可见较典型的嗅鞘细胞形状,主要以梭形和多突起细胞为主,细胞立体感强、透亮、杂质细胞较少、本底清楚,亦可见有少量成纤维细胞开始生长。9d时嗅鞘细胞数量明显增加,其生长方向呈较一致的条索状。至14d无论在细胞数量及质量上都呈明显的降低趋势。②苏木精-伊红染色结果:细胞多呈梭形,还可见扁平细胞,核为圆形或椭圆形,有时见双核,胞浆充实无空泡。③透射电
AIM： At present, the domestic and overseas cultural methods of olfactory ensheathing cells comprise some problems, such as complications or poor reproducibility. So in this experiment applied enzyme digestion was performed to culture the olfactory ensheathing cells from a rat in vitro. In addition, the morphology and phenotypes of this cell population was monitored. METHODS： The experiment was conducted from November 2005 to March 2006 at the Spinal Institute of the Shanghai University of Traditional Chinese Medicine. （1）A neonatal .male SD rat with an age of 1 week was selected and immersed in alcohol of 0.75 volume fraction for 3 minutes and degermed with iodophors. Subsequently, proso-cranium was unfolded to expose the olfactory bulbs. In this way, two integrity olfactory bulbs were obtained, put into DMEM culture fluid at 4 ℃ and cleaned twice to wash out superficial blood. Olfactory bulbs＇ envelopes were stripped and the olfactory bulbs were sheared off with eye scissors. 2.5 g/L diastase vera were added before the olfactory bulb was incubated with magnetic agitating in a incubation box at 37 ℃ for 15 minutes. This process was stopped with DMEM. Enchylema was sieved using a 100 μm meshwork. Supematant fluid was removed after centrifugation. The cells were attenuated to 1×10^9 L^-1 by DMEM, and then cultured routinely in 6-well culture plate without coating. Cell suspension was drawn off after culturing for 18-20 hours by differential adhesion method. The cells were cultured in 6-well cell culture plate coated with the special nerve growth factor receptor p75. Cell growth was monitored and the culture medium was changed every 2-3 days, Cells were successively cultured for 14 days. （2）The morphology of the olfactory ensheathing cells from the olfactory bulbs was examined by inverted and transmission electron microscope. Cells were colorized with haematoxylin-eosine （HE）. The special nerve growth factor receptor p75 was determined by immunohistochemical analysis. It was colored as positi
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第20期3892-3895,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
上海市教委科研基金资助(05cz12)~~
