嗅鞘细胞培养、鉴定及耳蜗移植的初步研究

发布时间：2019-05-28 08:15

【摘要】：目的建立嗅鞘细胞(olfactory ensheathing cells,OECs)的培养、鉴定及示踪方法,并初步探讨OECs耳蜗移植的可行性。方法 1、细胞培养及纯化:对新生3天Sprague-Dawley大鼠嗅球来源的OECs进行原代培养,然后用差速贴壁法+抗有丝分裂法纯化培养。在不同时间点于倒置显微镜下观察细胞的形态特点及生长情况,利用低亲和力神经生长因子受体(NGFR-p75)行免疫组织化学染色观察,并做细胞纯度的鉴定。 2、动物实验:将纯化培养7天的OECs与5-溴-2'-脱氧尿嘧啶核苷(5-bromo-2'-deoxyuridine,BrdUrd)孵育48小时,以标记OECs。作成年豚鼠耳后切口,暴露、打开听泡,在耳蜗底周近镫骨动脉处钻孔,注入含OECs的悬液。细胞移植1天后,取豚鼠耳蜗作冰冻切片,免疫荧光染色后,在激光共聚焦显微镜下观察移植细胞的分布情况。结果 1、纯化培养后的OECs迅速增殖,于第9天细胞数量最多,纯度最高可达85%。 2、BrdUrd与OECs共同孵育48小时后,其标记率可达到90%以上。 3、移植后1天OECs主要分布于豚鼠耳蜗的鼓阶中。结论 1、采用差速贴壁法+抗有丝分裂法可获得高纯度的OECs。 2、BrdUrd可成功标记OECs,是安全、有效、简单、标记率高的示踪方法。 3、OECs耳蜗移植后短期内可在鼓阶存活,长期转归有待进一步观察。
[Abstract]:Objective to establish a method for culture, identification and tracer of olfactory ensheathing cells (olfactory ensheathing cells,OECs), and to explore the feasibility of cochlear transplantation with OECs. Methods 1. Cell culture and purification: the primary culture of OECs from olfactory bulb of neonatal Sprague-Dawley rats was carried out, and then purified by differential adherent method against mitosis. The morphological characteristics and growth of the cells were observed under inverted microscope at different time points. The low affinity nerve growth factor receptor (NGFR-p75) was used for histochemical staining and the purity of the cells was identified. 2. Animal experiment: OECs purified for 7 days was incubated with 5 bromo 2 deoxyuridine (BrdUrd) for 48 hours to label OECs.. The posterior ear incision of adult guinea pigs was exposed, the auditory vesicle was opened, the cochlea was drilled near the stapes artery around the cochlear floor, and the suspension containing OECs was injected. One day after cell transplantation, the cochlea of guinea pig was taken for frozen section. after immunofluorescence staining, the distribution of the transplanted cells was observed under laser confocal microscope. Results 1. The purified OECs proliferated rapidly, the number of cells was the highest on the 9th day, and the highest purity was 85%. 2. After incubated with OECs for 48 hours, the labeling rate of BrdUrd was more than 90%. 3. 1 day after transplantation, OECs was mainly distributed in the tympanic stage of guinea pig cochlea. Conclusion 1. High purity OECs. can be obtained by differential adherent method against mitosis. 2. 2, BrdUrd can successfully mark OECs, is a safe, effective, simple and high labeling rate tracer method. 3, OECs cochlear transplantation can survive in the tympanic stage in a short period of time, and the long-term outcome needs to be further observed.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

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