h-MSCs的体外培养及向神经元样细胞分化的研究

发布时间：2018-02-26 17:10

  本文关键词： 骨髓间充质干细胞 细胞培养技术 神经元 睫状神经节神经营养因子 碱性成纤维细胞生长因子　出处：《南华大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的 采用体外分离、培养人骨髓间充质干细胞(human Mesenchymal stem cells, h-MSCs),以及用碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和不同浓度的睫状神经节神经营养因子(ciliary neurotrophic factor,CNTF)对其进行预诱导及诱导,通过观察细胞形态学变化,及免疫细胞化学染色CD34、CD44、CD54、NSE和Nestin的表达,探讨建立一种较简便有效的体外扩增培养h-MSCs的体系,以及CNTF对h-MSCs分化为神经元样细胞的诱导作用和适宜的诱导浓度。 方法 骨髓来源于南华大学附属第二医院和第一医院血液内科行骨髓穿刺检查患者(经患者同意),已排除肿瘤、传染病及血液系统疾病,年龄18～55岁,性别不限,男39例,女29例,共68例。采用密度梯度离心法和全骨髓贴壁法相结合的方法,分离获得h-MSCs,利用含血清的DMEM/F12培养基体外扩增,以及细胞冻存、复苏。相差倒置显微镜下观察细胞形态学变化,并绘制第1、3、6代h-MSCs的生长曲线。取传至第3代的h-MSCs,分为4组进行诱导分化:实验组、单纯预诱导组、单纯诱导组和空白对照组。实验组以bFGF预诱导24h后,再以5ng/mL、10ng/mL和20ng/mL浓度梯度的CNTF诱导12h;单纯预诱导组仅行bFGF预诱导而无CNTF诱导;单纯诱导组不经bFGF预诱导仅行CNTF诱导,诱导液中的CNTF亦分为3个浓度梯度(5ng/mL、10ng/mL和20ng/mL);空白对照组为无处理组,既无bFGF预诱导亦无CNTF诱导。应用免疫细胞化学技术对培养的h-MSCs进行CD34、CD44和CD54鉴定及对诱导分化后的细胞进行NSE和Nestin鉴定。 结果 1.h-MSCs在体外培养条件下,呈长梭形,类似成纤维细胞,可稳定增值传代并可冻存复苏,复苏后细胞生长特性与冻存前的细胞相似; 2.第1、3、6代h-MSCs生长曲线呈“S”形。第1代与第3代的细胞浓度比较差异无统计学意义(P=0.704),第1代与第6代比较差异无统计学意义(P=0.132),第3代与第6代比较差异亦无统计学意义(P=0.256); 3.应用免疫细胞化学技术检测培养的h-MSCs,显示CD44, CD54均呈阳性表达,CD34表达呈阴性; 4.h-MSCs在bFGF预诱导24h后,细胞形态基本无改变。CNTF诱导5h后部分细胞体积变小,以细胞核为中心收缩,逐渐呈锥形,形成突起。CNTF诱导12h后,实验组中10ng/mLCNTF组和20ng/mLCNTF组细胞形态改变明显,有2个或多个细长突起,类似神经元;5ng/mLCNTF亚组仅有少部分细胞形态稍发生变化。空白对照组、单纯预诱导组和单纯诱导组的细胞形态基本无改变,仍为宽大扁平的梭形。 5.免疫细胞化学检测显示诱导后的细胞神经元特异性烯醇化酶(neuron specific enolase, NSE)和巢蛋白(Nestin)均呈阳性表达。各组阳性细胞率间比较,差异均有统计学意义(F=98.822,F=105.395,均P0.01);实验组与空白对照组、单纯预诱导组及单纯诱导组比较,差异均有统计学意义(均P0.01),但后三组之间比较差异无统计学意义(均P0.01);实验组内两两之间比较显示,20ng/mL组与10ng/mL组阳性细胞率差异无统计学意义(P=0.413,P=0.496),但均高于5ng/mL组(均P0.01)。10ng/mLCNTF实验组NSE阳性细胞率(46.38±4.99)%,Nestin阳性细胞率(45.76±4.46)%,为最多。 结论 1.梯度离心法和全骨髓贴壁法相结合的方法,是一个较简便有效的体外h-MSCs培养、纯化体系; 2. h-MSCs可在体外培养扩增,并可冻存、复苏,复苏后的细胞生长特性与冻存前的细胞相似; 3.在本实验条件下,CNTF可诱导h-MSCs定向分化为神经元样细胞;且10ng/mLCNTF可能为较适宜的诱导浓度。
[Abstract]:Purpose The expression of CD34 , CD44 , CD54 , NSE and Nestin in human bone marrow mesenchymal stem cells ( hMSCs ) and the expression of CD34 , CD44 , CD54 , NSE and Nestin in vitro were investigated by using basic fibroblast growth factor ( bFGF ) and different concentrations of ciliary neurotrophic factor ( CNTF ) . method Bone marrow derived from bone marrow puncture examination patients ( consent of patients ) from the Second Affiliated Hospital of South China University and the First Affiliated Hospital of the First Affiliated Hospital of South China University . The growth curve of h - MSCs was observed by means of density gradient centrifugation and full bone marrow adherent method . The cells were divided into 4 groups : experimental group , pre - induction group , simple induction group and blank control group . Results 1 . h - MSCs can be cultured in vitro under the conditions of long shuttle shape and similar fibroblast , and can be stably added and recovered , and the growth characteristics of the cells after resuscitation are similar to those of the cells before freezing ; 2 . The growth curve of the 1st , 3rd , 6th generation h - MSCs was " S " . There was no significant difference between the 1st and 3rd generation cell concentrations ( P = 0.704 ) . There was no significant difference between the 1st and 6th generations ( P = 0.132 ) . There was no significant difference between the 3rd and 6th generations ( P = 0.256 ) . 3 . The expression of CD44 and CD54 was positive and CD34 was negative in cultured h - MSCs . After 12 hours of induction of CNTF , the morphological changes of the cells in the 10 ng / mLCNTF group and 20ng / mLCNTF group were significantly changed in the experimental group . 5 . The positive rates of NSE and Nestin in the cells were significantly higher than those in the control group ( F = 98.822 , F = 105.395 , P0.01 ) . There was no significant difference between the two groups ( P = 0.413 , P = 0.496 ) . The positive cell rates of NSE in the experimental group were 46.38 卤 4.99 % and 45.76 卤 4.46 % , respectively . Conclusion 1 . The method of combination of gradient centrifugation and full - bone marrow adherent method is a simple and effective method for culturing and purifying h - MSCs in vitro . 2 . h - MSCs can be cultured in vitro and can be frozen , recovered , and the cell growth after resuscitation is similar to that before freezing . 3 . Under the condition of this experiment , CNTF can induce the differentiation of h - MSCs into neuron - like cells , and 10 ng / mL CNTF may be a suitable induction concentration .

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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