UC-iPSC向NSC分化体系的比较及NSC在HIE模型中的初步研究

发布时间：2018-04-05 00:30

  本文选题：经诱导性多能干细胞　切入点：神经干细胞　出处：《济南大学》2015年硕士论文

【摘要】：目的比较抑制剂浓度及诱导初始密度的不同对尿液细胞重编程所得诱导性多能干细胞(urine cells derived induced pluripotent stem cells,UC-iPSC)向神经干细胞分化效率及神经干细胞(neural stem cells,NSC)生存状态的影响,从而确定一种较稳定、高效的诱导分化的方法;检测UC-iPSC源性神经干细胞在新生大鼠缺血缺氧性脑病(hypoxic-ischemic encephalopathy,HIE)动物模型脑内的生存、迁移及分化能力。方法比较不同诱导体系诱导UC-iPSC向神经干细胞分化的效率及神经干细胞生存状态的影响:A体系:SB431542和Drosomophorin的浓度均为5 um/ml,诱导初始密度为100%,B体系:SB431542的浓度为5 um/ml,Drosomophorin的浓度为1 um/ml,诱导初始密度为30-50%。通过观察诱导获得的神经干细胞的形态学变化;q-PCR比较神经干细胞相关基因Pax6,Nestin,Sox1,Sox2的表达量及流式细胞术比较诱导第16天Pax6的阳性率等方法比较两种体系的诱导效率。建立HIE模型:取出生7天的SD大鼠,行左侧颈总动脉结扎、离断手术,继而在8%O2,92%N2 38℃条件下缺氧120 mins,建立HIE的疾病动物模型。造模后第7天,TTC染色观察大脑损伤灶。神经干细胞移植:动物模型建立后第3天,取已鉴定的UC-iPSC源性神经干细胞3μL、1×105移植入疾病模型鼠侧脑室内。分别于第7、28天,心脏灌流取脑、固定、切片、免疫荧光染色,激光共聚焦显微镜观察分析外源性神经干细胞在模型动物脑内的生存、迁移及分化的情况。结果A体系获得的第1代神经干细胞,呈明亮的圆球形,聚集紧密;B体系获得的第1代神经干细胞,则呈灰暗的不规则形,聚集松散。q-PCR显示A体系诱导获得的细胞神经干相关基因的表达量高于B体系。流式细胞术分析诱导第3代神经干细胞高表达Pax6,Nestin,Sox1,Sox2等神经干细胞相关基因。自发分化第20天,形成明显的神经纤维束,免疫荧光证明该细胞高表达TUJ-1,MAP2等神经元的特异性标志物及部分细胞表达星形胶质细胞的特异性标志物GFAP。TTC染色显示建模后第7天在脑皮质、纹状体及海马部位存在明显的白色损伤区域。UC-iPSC源性神经干细胞移植后第7天,免疫荧光染色可在左侧脑室内及室管壁检测到Nestin与Stem121双阳性细胞,即移植的神经干细胞,成球形聚集,在侧脑室及损伤灶之间检测到呈迁移状Nestin阴性的移植细胞,第28天损伤灶周围检测到TUJ-1与Stem121双阳性及GFAP与Stem121双阳性的细胞。结论在体外诱导UC-iPSC向神经干细胞分化的A,B两种体系中,A体系优于B体系,且A体系获得神经干细胞高表达神经干细胞相关基因,具有正常的自发分化能力。移植的神经干细胞在HIE模型鼠脑内可生存、迁移及向神经元和胶质细胞分化。
[Abstract]:Objective to compare the effects of different concentrations of inhibitors and initial density of induction on the differentiation efficiency of induced pluripotent stem cell urine (cells derived induced pluripotent stem cells) into neural stem cells (NSCs) and the survival state of neural stem cells (NSCs) obtained by urine cell reprogramming.Thus, a stable and efficient method for inducing differentiation was established, and the ability of survival, migration and differentiation of UC-iPSC derived neural stem cells in the brain of neonatal rats with hypoxic-ischemic encephalopathy was detected.Methods the effects of different induction systems on the differentiation of UC-iPSC into neural stem cells and the survival status of neural stem cells were compared. The concentration of 1: a system: SB431542 and Drosomophorin were both 5 渭 m / ml, and the initial induction density was 100 渭 m / ml. The initial density of inducing UC-iPSC was 5 ump / ml / ml Drosomophorin.It is 1 um / ml, and the initial density of induction is 30-50.The morphological changes of induced neural stem cells were observed. The expression of Pax6Nestinsil Sox2 and the positive rate of Pax6 on the 16th day after induction were compared by q-PCR and flow cytometry, respectively. The induction efficiency of the two systems was compared by flow cytometry.HIE model was established: SD rats born 7 days old were taken out, left common carotid artery was ligated, left carotid artery was amputated, then the animal model of HIE was established by hypoxia at 88 鈩，

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