新生SD大鼠海马神经干细胞体外培养、冷冻复苏及诱导分化后钾通道的变化

发布时间：2018-06-13 13:44

本文选题：NSCs + 新生SD大鼠　；参考：《河北大学》2012年硕士论文

【摘要】：神经干细胞(neural stem cells, NSCs)的发现是在研究造血发生和神经发育的基础上开始的。研究结果显示，胚胎和成人脑组织中均存在NSCs。它强大的增殖能力使得NSCs易于在体外大量繁殖，在体内移植时常常具有低免疫原性，因此在临床治疗中具有广阔的应用前景。本文选择的实验材料为新生SD (sprague-dawley)大鼠，首先探讨了NSCs的体外分离、培养及鉴定的方法。无菌条件下分离出大鼠的海马组织，采用无血清培养法进行培养。研究结果表明，以5×10~5个/mL的细胞密度接种到培养瓶中，传代后的细胞生长稳定，经免疫荧光鉴定均为Nestin阳性。建立了一种快速获得NSCs的体外培养及鉴定系统。在此基础上，，以DMEM/F12培养基、二甲基亚砜DMSO、胎牛血清FBS作为主要成分，设计了不同配比的冻存保护液，对NSCs冷冻保存进行了研究。分别冻存1周、2周及1个月后台盼蓝染色检测其复苏率，结果显示D10组(70%DMEM/F12+20%FBS+10%DMSO)作为冻存保护液，复苏率分别为(54.00±1.73)%，(59.00±1.16)%，(58.00±2.08)%，显著优于其余各组，且复苏后不影响其原有的生物学特性。此外，本文还对NSCs诱导分化后不同天数的钾通道的变化进行了研究。将培养的第3代NSCs进行诱导分化，对NSCs以及诱导分化后的神经元和星形胶质细胞进行免疫荧光鉴定，并用全细胞膜片钳记录其分化5d,10d,15d,20d钾电流的变化。研究结果表明，去除生长因子后在培养液中添加10%FBS将NSCs诱导分化为神经元和星形胶质细胞，经NSE和GFAP检测均为阳性；膜片钳记录到的钾电流随着分化天数的延长而不断增高，说明诱导后的神经元在电生理功能上逐渐发育成熟。
[Abstract]:The discovery of neural stem cells (NSCs) is based on the study of hematopoiesis and neural development. The results showed that NSCs were present in both embryonic and adult brain tissues. Because of its strong proliferative ability NSCs are easy to proliferate in vitro and have low immunogenicity when transplanted in vivo. Therefore NSCs have a broad application prospect in clinical treatment. The experimental materials selected in this paper were neonatal SD sprague-dawley rats. The methods of isolation, culture and identification of NSCs in vitro were studied. Rat hippocampal tissue was isolated under aseptic condition and cultured by serum-free culture. The results showed that the cells inoculated with 5 脳 10 ~ 5 / mL cell density in the culture flask had stable growth and were identified as nestin positive by immunofluorescence. An in vitro culture and identification system of NSCs was established. On this basis, DMEM / F12 medium, DMSO-dimethyl sulfoxide and FBS of fetal bovine serum were used as the main components to design different ratio of frozen preservation solution, and the cryopreservation of NSCs was studied. The rate of resuscitation was detected by backstage trypan blue staining for 1 week, 2 weeks and 1 month respectively. The results showed that the recovery rate of D10 group was 54.00 卤1.73 + 59.00 卤1.16 + 58.00 卤2.080.The recovery rate of D10 group was 54.00 卤1.73 + 1.16%, and the original biological characteristics were not affected after resuscitation. In addition, the changes of potassium channels in different days after induction and differentiation of NSCs were studied. The third generation of cultured NSCs were induced to differentiate. The NSCs and the differentiated neurons and astrocytes were identified by immunofluorescence, and the changes of potassium currents were recorded by whole-cell patch clamp for 5 days, 10 days, 15 days and 20 days after differentiation. The results showed that NSCs were induced to differentiate into neurons and astrocytes by adding 10s to the culture medium after the removal of growth factors. Both NSE and GFAP were positive. The potassium currents recorded by patch clamp increased with the prolongation of differentiation days, indicating that the induced neurons developed and matured in electrophysiological function.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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