体外原代培养纯化新生鼠嗅鞘细胞的实验方法:差速贴壁+阿糖胞苷+胰酶法

Primary culture and purification of neonatal rat olfactory ensheasing cells in vitro with Nash's method, cytarabine and trypsinization

目的:通过细胞形态学观察及生物学特性鉴定,建立一种经济实用的体外原代培养纯化新生鼠嗅鞘细胞的实验方法。方法:实验于2006-06在武汉理工大学完成。①阿糖胞苷(Sigma,批号w10562);胎牛血清(杭州四季青产品);胰蛋白酶(Amresco);多聚赖氨酸(Sigma);胶质纤维酸性蛋白,神经生长因子受体蛋白抗体(博士德)。②选取出生3d内的Wistar大鼠5只,乙醇浸泡后断头处死,取嗅球的最外两层,通过1.25g/L胰蛋白酶消化分离嗅鞘细胞,体外原代培养。③采用Nash差速贴壁+阿糖胞苷+胰酶法纯化嗅鞘细胞。培养18h后将未贴壁的细胞悬液转种于另一未涂层的器皿中,再培养36h,重复上述步骤移入0.1g/L多聚赖氨酸包被的塑料培养瓶中进行培养,纯化后的细胞在24h内陆续贴壁,常规培养2d,加入终浓度为2mg/L的阿糖胞苷,作用48h后,换上新的培养基,继续培养1d,弃去培养基,用无钙镁的Hanks液清洗2次,然后用1.25g/L的胰酶消化10min,待细胞突起回缩、胞体变圆时,立刻加入纯血清终止,其血清终浓度为20%,制备单细胞悬液。④倒置显微镜下观察其形态变化,苏木精-伊红染色,同时行神经生长因子受体蛋白和胶质纤维酸性蛋白免疫组化染色鉴定嗅鞘细胞,并计算嗅鞘细胞阳性百分率。结果:①活细胞形态观察:分离培养的嗅鞘细胞具有双极或多极突起,且突起细长,相互交织。②嗅鞘细胞的苏木精-伊红染色鉴定:细胞呈三角形或梭形,有长的突起,胞浆被染成粉红色,胞核呈蓝紫色,胞核内深染的为核仁,有1～3个核仁。③嗅鞘细胞的胶质纤维酸性蛋白免疫组化染色鉴定:细胞呈现棕黄色,胞核淡染,阳性细胞成网状连接,胞体多为三角形,有细长突起,阳性率91.5%。④嗅鞘细胞的P75免疫组化染色鉴定:阳性细胞呈绿色,多数细胞染色阳性,阳性率89%。结论:实验所采用的Nash差速贴壁+阿糖胞苷+胰酶法分离纯化培养嗅鞘细胞切实可行,为神经诱导修复材料的研究提供种子细胞。

AIM： To set up an economic and useful method to culture and purify the olfactory ensheathing calls （OECs） of neonatal rats in vitro through observation and identification of morphological features. METHODS： The experiment was conducted in Wuhan University of Technology on June 2006. （1） Materials： Cytarabine （Sigma with the batch number of w10562）, fetal bovine serum （produced by Hangzhou Sijiqing）, trypsin （Ameresco）, polylysine （Sigma）, glial flbrillary acidic protein （GFAP）, and neural growth factor of protein receptors （NGFR-p75）. （2） Five Wister rats of 3 days old were selected and executed after being dipped into grain alcohol to obtain two outer layers from the olfactory bulbs, which were then digested into 1.25 g/L trypsinization solution to separate the OECs for primary culture in vitro. （3） Cells were purified with the method of Nash＇s differential attachment, cytarabine and trypsinization. Eighteen hours later, the suspending liquid of non-attachment calls was diverted to another culture vessels and cultured for 36 hours. Then all of attachment calls were diverted to 0.1 g/L pelylysine solution and cultured for 2 days. After cultured for 48 hours in the 2 mg/L cytarabine solution, and the calls were updated with fresh culture medium. After 1 day, the calls were cleaned twice with the Hanks solution, and then digested for 10 minutes with 1.25 g/L trypsinization solution; Pure fetal bovine serum with the concentration of 20% was added immediately while the call process condensed and the call body tumed into round, so as to prepare for single call suspending liquid. （4） The hematoxylin and eosin staining, immunohistochemical staining of neural growth factor of protein receptors and glial fibrill aryacidic protein were performed respectively to observe and identify the morphological features of OECs under the electron inversion microscope. And the proportion of masculine rate of OECs was calculated. RESULTS： （1） Observation on morphology of live OECs： Cultured OECs appeared with bipolar or multipelar slender and interlacing processes, which ware slim and interlaced with each other. （2） The identification of OECs stained by HE： Cells were in triangle and fusiform with long process, red cytoplasm, and blue karyons. There were 1-3 blue-black nucleoli in the karyons. （3） The identification of OECs stained by GFAP immunocytochemistry： Cells with bipolar or multipelar slender and interlacing processes were brown and the nucleoluses were hazel. The positive rate was 91.5%. （3） The identification of OECs stained by p75 immunocytochemistry： Most calls were positive and appeared green. The positive rate was 89%. CONCLUSION： The method of culturing OECs with Nash＇s differential attachment, cytarabine and trypsinization is available and provide seed calls for the study of nerve guiding recovery materials.