OECs无血清上清液诱导UCB-MSCs向神经细胞分化

发布时间：2018-01-08 13:15

本文关键词：OECs无血清上清液诱导UCB-MSCs向神经细胞分化　出处：《湖南师范大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：探讨大鼠嗅鞘细胞(olfactory ensheathing cells,OECs)无血清上清液体外诱导脐血间充质干细胞(human umbilical cord blood mesenchymal stem cells, HUCB-MSCs)向神经细胞分化的可行性和可能的机制,以及检测诱导分化后神经元细胞的电生理特性。方法：从新生大鼠嗅球分离、提取OECs,采用差速贴壁联合阿糖胞苷抑制法纯化嗅鞘细胞,在细胞最佳状态下无血清培养24h后收集上清液。收集正常足月剖腹产胎儿的脐带血,经肝素抗凝,用密度梯度离心法分离获得脐血的单个核细胞,采用差速贴壁法纯化间充质干细胞,培养、扩增到第3代,记作脐血MSCsP3。实验分为实验组和对照组,DMEM液体中加入OECs无血清上清液培养脐血MSCsP3作为实验组,用DMEM液体培养脐血MSCsP3作为对照组。在一系列时间点(培养后12小时,24小时,72小时,1周,10天和2周)在倒置显微镜下形态学观察生长、分化情况,1周后行免疫组化鉴定,神经元特异性烯醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)和巢蛋白(nestin)分别鉴定神经元、神经胶质细胞和神经干细胞,膜片钳检测神经元电生理特性。结果：用差速贴壁和阿糖胞苷抑制法可获得纯度高达95%的嗅鞘细胞,实验组细胞在培养诱导后相差显微镜下可观察到典型神经元、神经胶质细胞和神经干细胞团,2周后细胞仍维持神经细胞样形态,NSE、GFAP、nestin细胞免疫组化鉴定与形态学结果一致。HUCB-MSCsP3诱导分化后的神经元样细胞检测到快速激活快速失活、能被河豚毒素(TTX)特异性阻断的钠离子电流,能被四乙基氯化铵(TEA)特异性阻断的慢激活慢失活的延迟整流性钾电流,以及缓慢的内向钙离子电流。对照组脐血MSCs培养后呈多形态方向分化。结论：①OECs无血清上清液能诱导脐血MSCs向神经细胞分化;②诱导分化后的神经元具备成熟神经元的电生理特性。
[Abstract]:Objective: to investigate the olfactory ensheathing cells of rat olfactory ensheathing cells. In vitro induction of umbilical cord blood mesenchymal stem cells (OECs) without serum supernatant. Human umbilical cord blood mesenchymal stem cells. The feasibility and possible mechanism of differentiation of HUCB-MSCs into neurons and the electrophysiological characteristics of neuronal cells induced by differentiation were examined. Methods: OECs were isolated from olfactory bulb of newborn rats. OECs were purified by differential adhesion and cytarabine inhibition. The supernatant was collected after 24 hours of serum-free culture in the best condition of the cells. The umbilical cord blood was collected from the normal full-term caesarean section fetus. The mononuclear cells were isolated by density gradient centrifugation after heparin anticoagulant. Mesenchymal stem cells were purified by differential adherent method, cultured and amplified to the third generation, recorded as cord blood MSCsP3. The experiment was divided into experimental group and control group. OECs serum-free supernatant was added to DMEM fluid to culture umbilical cord blood MSCsP3 as experimental group. Umbilical cord blood MSCsP3 was cultured with DMEM liquid as control group. At a series of time points (12 hours, 24 hours, 72 hours, 1 week). After 10 days and 2 weeks, the growth was observed under inverted microscope, and the differentiation was identified by immunohistochemistry after 1 week. The neuron specific enolase (NSE) was identified. Glial fibrillary acidic protein (GFAP) and nestin (nestin) were used to identify neurons, glial cells and neural stem cells respectively. The electrophysiological characteristics of neurons were detected by patch clamp. Results: olfactory ensheathing cells with purity up to 95% could be obtained by differential adhesion and cytarabine inhibition. Typical neurons could be observed under phase contrast microscope in the experimental group. After 2 weeks, the glial cells and the neural stem cells still maintained the neuron-like morphology of NSE-GFAP. The nestin cells were identified by immunohistochemistry. The neuron-like cells induced by HUCB-MSCsP3 showed rapid activation and rapid inactivation. Sodium ion currents specifically blocked by tetrodotoxin TTX, slow activation of slow inactivated delayed rectifier potassium currents specifically blocked by tetraethyl ammonium chloride. And slow inward calcium current. The cord blood MSCs in the control group differentiated in multiple directions after culture. Conclusion the serum free supernatant of 1: 1 OECs can induce the differentiation of cord blood MSCs into neural cells. The neurons after differentiation can possess the electrophysiological characteristics of mature neurons.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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