【作者】 赵慧英；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2004， 博士

【摘要】 成体干细胞的发现及其诱导分化为神经细胞的研究为人类及动物神经系统各种疾病的治疗和损伤的修复开辟了一条新途径。为了寻求一种自体取材方便、安全有效的可用于诱导分化为神经细胞的成体干细胞，为神经系统疾病的替代治疗提供理想的移植材料。本实验在建立了胚胎小鼠神经干细胞和山羊皮肤干细胞体外分离、培养体系的基础上，分别对神经干细胞定向诱导分化多巴胺能神经元和皮肤干细胞诱导分化神经细胞的条件进行了研究。1．分离培养了小鼠胚胎大脑皮层神经干细胞。原代细胞在培养的第2～3 d即可见到分裂形成的细胞球，5～7 d形成许多大的神经球；生长曲线显示神经干细胞的生长呈持续增长趋势；单细胞克隆试验显示单个细胞可以自我分裂增殖形成神经球，构成的神经球的细胞继续传代培养，同样具有增殖和分化能力，其克隆率可达到93%；所培养的神经球经免疫细胞化学反应呈nestin阳性着色。2．分离神经干细胞时采用先机械吹打、过滤、静置，取上清，再离心的方法，换液时采取只加入不吸弃的方法，有利于获得纯化、健康的神经干细胞。采用只添加bFGF的干细胞培养液培养，可保持神经干细胞长期增殖并传至12代。培养液中撤除bFGF，并添加2%～5%的FBS后，神经干细胞所分化的细胞分别呈NF、GAFP和Galc-C阳性表达，表明神经干细胞可分化为神经元、星形胶质细胞和少突胶质细胞，三者分别占分化细胞的21.1%、59.7%、19.2%。3．将NSCs重悬于含10% BSA和8% DMSO的NSCs冻存液中，采取4℃放置30 min→-20℃放置2 hr→放置液氮管口，气相中下降3次，每次30 min→最后置于液氮中的步骤冻存细胞；按常规方法对冻存细胞用1%～2% BSA的神经干细胞复苏液进行复苏，观察细胞复苏后的存活率。结果证明冻存时间长短对细胞的存活率的影响差异不显著，各代冻存细胞之间复苏后的存活率无显著性差异。 4．成功地用含2% B27、100 pg/mL白细胞介素-1、0.05 g/L维生素C、5 u/mL红细胞生成素的DMEM/F12组成的诱导剂将大脑皮质和中脑来源的神经干细胞诱导分化为多巴胺能神经元。所诱导细胞具有典型的神经细胞的形态特征，进行TH抗体免疫细胞化学染色鉴定，细胞的胞质呈棕黄色阳性着色，胞核不着色呈空泡状。TH表达的阳性细胞率，随着诱导时间的增加而增加；中脑来源的神经干细胞的诱导细胞的TH阳性率明显高于大脑皮层来源的神经干细胞的诱导细胞的TH阳性率，数据统计分析显示二者差异极显著；中脑源和大脑源的神经干细胞诱导的TH细胞的阳性率分别达到22.49%、10.65%。 <WP=7>5．用纹状体提取液与DMEM/F12按1∶1的比例组成的诱导液将中脑来源的14.49%的神经干细胞诱导为多巴胺能神经元，低于诱导剂组诱导分化的多巴胺能神经元。6．山羊皮肤干细胞在体外培养开始呈圆形或椭圆型、核大而明显，随后成集落样生长，并可维持14～20天。在角质细胞无血清条件培养液和自制的干细胞普通培养液均生长良好，两种培养液对皮肤干细胞克隆形成率的影响不显著,但对克隆持续时间的影响差异显著,且条件培养液中的皮肤干细胞克隆持续时间显著地高于普通培养液的干细胞克隆持续时间。皮肤干细胞在胚胎小鼠成纤维细胞饲养层和山羊成纤维细胞饲养层上均生长良好，两种饲养层细胞对皮肤干细胞克隆开始形成的时间的影响差异不显著，但对皮肤干细胞克隆持续时间的影响差异显著（P=0.049<0.05），胚胎小鼠成纤维细胞饲养层组显著地大于山羊成纤维细胞组。7．皮肤干细胞及干细胞克隆均表达整合素-β1，大小较一致呈圆形的细胞，其胞质被染成棕黄色，中央的细胞核不着色，如圆环，克隆样集落则整个被染成棕黄色。8．由山羊神经干细胞培养液上清1份与山羊成纤维细胞培养液上清1份混合，再添加10 ng/mL IGF-1和0.05 g/L Vc组成的诱导液可有效地将12％的山羊皮肤干细胞诱导为神经元样细胞。在培养的第5天可见3%的皮肤干细胞形状变长，有突起样的结构从细胞发出并呈神经细胞样结构；随诱导时间延长,突起更加明显,到第8～10天可以看到形状典型的神经细胞，皮肤干细胞变为神经元样的细胞。经免疫细胞化学染色，神经元样细胞呈NF染色阳性，证明为神经细胞。更多还原

【Abstract】 The found of adult stem cells and the study of induced them into neurons have opened up a new way for curing nerve system diseases and damages of human and animal. In order to explore a kind of adult stem cell, which can be used to induce into neuron expediently、safely and effectively，to become a ideal substitute material for transplantaton，the present study has established the culture system of fetal mouse neural stem cell and epidermal stem cell in vitro, and probed the condition of directed differentiated into dopaminergic neuron from neural stem cell and epidermal stem cell into neurons. 1．Neural stem cells were dissociated and cultured from fetal mouse cerebral. The growth curve revealed that neural stem cell grew continually when cultured in vitro. Single cell could proliferate to become neurosphere，and the cells derived from the obtained neurosphere had proliferation and differentiation capacity when passaged, the clone form rate was 93%. Immunocytochemistry study indicated that the neurosphere cells were nestin positive.2．It was found that the following methods was favorite for obtaining pure and normal neural stem cells, pipetted firstly, then filtrated, and after stay for a while collected supernatant liquid to centrifuge. When culture media were refreshed by just adding new media and no discarding. The neural stem cells could sustain proliferation status and until to 12 passages when cultured with stem cell culture media supplement bFGF. Removed bFGF from culture media, replaced with 2-5% FBS, neural stem cells could differentiate to three kinds cells，which express positive staining of NF、GAFP、 Galc-C respectively, they are neurons、oligodendrocytes、astrocytes, their percentage were 21.1%, 59.7%, 19.2% respectively.3．NSCs were cryopreserved using 10% BSA and 8%DMSO NSCs cryopreservation media by following protocol, 30 min at 4℃→2 hr at -20℃→3 falls in nitrogen steam up liquid nitrogen, each 30min→put into liquid nitrogen. Thawed NSCs using standard method and cultured, the results showed that there was no significant effect of cryopreservation duration on NSCs viability, and the viability were not different significantly within passages.Cerebral NSCs and diencephalons NSCs were successfully induced to be <WP=9>dopaminergic neuron by DMEM/F12 supplement 2% B27+100pg/mL Il-1 + 0.05g/L Vc + 5 u/mL Erythropoietin (inducer group).Induced cells had typical neural cell morphology character, immunocytochemistry check with TH antibody revealed that plasma was positive stain to brown-yellow, nucleus was not stained. The percentage of TH positive cells increased along with the increase of induction times. TH positive cell rate derived from diencephalons (22.49%) was significantly higher than that of cerebral derivation (10.65%). When corpus striatum extract + DMEM/F12 (1:1) used as induction media (corpus striatum group), 14.49% diencephalons NSCs was differentiated into dopaminergic neuron, significantly lower than inducer group.Goat epidermal stem cells were round or elliptical at beginning of in vitro culture with big obvious nucleus, then became colony-like, could grow 14～20 d. They grew well in Ca2+ -free keratinocyte conditioned media and self-made stem cell culture media. The two culture media had no significant effect on epidermal stem cell clone form rate, but had significant effect on clone sustaining time. The clone sustaining time in conditioned media was higher significantly than that in self-made stem cell culture media. Epidermal stem cells could grow on mouse fetal fibroblast feeder layer or goat fibroblasts feeder layer. The two feeder layer had no significant effect on the beginning time of epidermal stem cell clone form, but had significant effect on clone sustaining period（P=0.049<0.05）. The clone sustaining time on mouse fetal fibroblast feeder layer was higher significantly than that on goat fibroblasts feeder layer. Epidermal stem cells and stem cell clone all express integrin-β1. The plasma of those cells that had round shape and same size, stained to be brow更多还原