本文選題：經(jīng)誘導(dǎo)性多能干細(xì)胞　切入點(diǎn)：神經(jīng)干細(xì)胞　出處：《濟(jì)南大學(xué)》2015年碩士論文

【摘要】：目的比較抑制劑濃度及誘導(dǎo)初始密度的不同對(duì)尿液細(xì)胞重編程所得誘導(dǎo)性多能干細(xì)胞(urine cells derived induced pluripotent stem cells,UC-iPSC)向神經(jīng)干細(xì)胞分化效率及神經(jīng)干細(xì)胞(neural stem cells,NSC)生存狀態(tài)的影響,從而確定一種較穩(wěn)定、高效的誘導(dǎo)分化的方法;檢測(cè)UC-iPSC源性神經(jīng)干細(xì)胞在新生大鼠缺血缺氧性腦病(hypoxic-ischemic encephalopathy,HIE)動(dòng)物模型腦內(nèi)的生存、遷移及分化能力。方法比較不同誘導(dǎo)體系誘導(dǎo)UC-iPSC向神經(jīng)干細(xì)胞分化的效率及神經(jīng)干細(xì)胞生存狀態(tài)的影響:A體系:SB431542和Drosomophorin的濃度均為5 um/ml,誘導(dǎo)初始密度為100%,B體系:SB431542的濃度為5 um/ml,Drosomophorin的濃度為1 um/ml,誘導(dǎo)初始密度為30-50%。通過觀察誘導(dǎo)獲得的神經(jīng)干細(xì)胞的形態(tài)學(xué)變化;q-PCR比較神經(jīng)干細(xì)胞相關(guān)基因Pax6,Nestin,Sox1,Sox2的表達(dá)量及流式細(xì)胞術(shù)比較誘導(dǎo)第16天Pax6的陽(yáng)性率等方法比較兩種體系的誘導(dǎo)效率。建立HIE模型:取出生7天的SD大鼠,行左側(cè)頸總動(dòng)脈結(jié)扎、離斷手術(shù),繼而在8%O2,92%N2 38℃條件下缺氧120 mins,建立HIE的疾病動(dòng)物模型。造模后第7天,TTC染色觀察大腦損傷灶。神經(jīng)干細(xì)胞移植:動(dòng)物模型建立后第3天,取已鑒定的UC-iPSC源性神經(jīng)干細(xì)胞3μL、1×105移植入疾病模型鼠側(cè)腦室內(nèi)。分別于第7、28天,心臟灌流取腦、固定、切片、免疫熒光染色,激光共聚焦顯微鏡觀察分析外源性神經(jīng)干細(xì)胞在模型動(dòng)物腦內(nèi)的生存、遷移及分化的情況。結(jié)果A體系獲得的第1代神經(jīng)干細(xì)胞,呈明亮的圓球形,聚集緊密;B體系獲得的第1代神經(jīng)干細(xì)胞,則呈灰暗的不規(guī)則形,聚集松散。q-PCR顯示A體系誘導(dǎo)獲得的細(xì)胞神經(jīng)干相關(guān)基因的表達(dá)量高于B體系。流式細(xì)胞術(shù)分析誘導(dǎo)第3代神經(jīng)干細(xì)胞高表達(dá)Pax6,Nestin,Sox1,Sox2等神經(jīng)干細(xì)胞相關(guān)基因。自發(fā)分化第20天,形成明顯的神經(jīng)纖維束,免疫熒光證明該細(xì)胞高表達(dá)TUJ-1,MAP2等神經(jīng)元的特異性標(biāo)志物及部分細(xì)胞表達(dá)星形膠質(zhì)細(xì)胞的特異性標(biāo)志物GFAP。TTC染色顯示建模后第7天在腦皮質(zhì)、紋狀體及海馬部位存在明顯的白色損傷區(qū)域。UC-iPSC源性神經(jīng)干細(xì)胞移植后第7天,免疫熒光染色可在左側(cè)腦室內(nèi)及室管壁檢測(cè)到Nestin與Stem121雙陽(yáng)性細(xì)胞,即移植的神經(jīng)干細(xì)胞,成球形聚集,在側(cè)腦室及損傷灶之間檢測(cè)到呈遷移狀Nestin陰性的移植細(xì)胞,第28天損傷灶周圍檢測(cè)到TUJ-1與Stem121雙陽(yáng)性及GFAP與Stem121雙陽(yáng)性的細(xì)胞。結(jié)論在體外誘導(dǎo)UC-iPSC向神經(jīng)干細(xì)胞分化的A,B兩種體系中,A體系優(yōu)于B體系,且A體系獲得神經(jīng)干細(xì)胞高表達(dá)神經(jīng)干細(xì)胞相關(guān)基因,具有正常的自發(fā)分化能力。移植的神經(jīng)干細(xì)胞在HIE模型鼠腦內(nèi)可生存、遷移及向神經(jīng)元和膠質(zhì)細(xì)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，

本文編號(hào)：1712417