【作者】 李升；

【导师】 曾水林；

【作者基本信息】 东南大学 ， 人体解剖与组织胚胎学， 2006， 硕士

【摘要】 目的探讨嗅鞘细胞(OECs)联合神经干细胞(NSCs)脑内移植对NSCs增殖、分化及对PD大鼠的治疗作用。方法1、NSCs培养:取E14.5 d SD大鼠腹侧中脑组织制备成单细胞悬液,接种到含有表皮生长因子(Epidermal growth factor,EGF ),碱性成纤维细胞生长因子(Fibroblast growth factor-basic,bFGF、)B27(B27 suplement)的DMEM/F12(1:1)的无血清培养液中培养,待形成原代克隆球后,用有限稀释法进行单细胞克隆和传代扩增,以获得大量来源相同的NSCs克隆球,并进行Brdu和Nestin免疫细胞化学检测。2、OECs培养:取E14.5 d SD大鼠两侧嗅球组织制备成细胞悬液,离心后去上清,将细胞浓度调整到1×106个/ ml,移至含20%胎牛血清OECs培养液培养。3、将培养传至第三代的OECs与NSCs共培养。用P75免疫组化法,P75/Nestin, P75 ,Brdu/Nestin ,GFAP,NF,NF/TH免疫荧光法分别鉴定OECs和NSCs的增殖与分化。4、立体定位下,于左侧黑质致密部(SNC)和腹侧被盖(VTA)注射6-OHDA制备PD大鼠模型。将成功PD大鼠模型随机分成5组,即假手术对照组(注入生理盐水);NSCs移植组;OECs移植组;OECs+NSCs移植组和模型对照组。5、将上述培养传代至第3代的NSCs、OECs和OECs+NSCs,浓度为1.0x106细胞/μl,分别移植到PD大鼠吻侧纹状体内,于移植后7 d、14 d、30 d、60 d检测PD大鼠旋转行为变化后,深麻下处死,灌流固定取脑做冰冻切片,行HE染色和Brdu,TH,P75, Brdu/TH, Brdu/P75免疫组化和免疫荧光染色。结果1、OECs在培养7 d时,绝大多数呈梭形且发出2至3个突起,少量呈扁平椭圆形,免疫组化呈P75阳性反应。2、NSCs培养7 d时,显示典型的神经球悬浮生长,呈Brdu/Nestin免疫荧光双标阳性;10 d后细胞活性下降,球体积不再增大,球中心透亮度明显下降;14 d后偶见神经球贴壁分化,GFAP和NF免疫荧光检测显示,大部分为红色荧光标记的GFAP+细胞,少数为绿色荧光标记的NF+细胞。3、OECs+NSCs共培养组在5 d时形成神经球;10 d时球体积不断增大,球中心透亮度良好;12 d后神经球的体积不再增大,开始贴附在OECs上生长,可见神经球向四周伸出突起并开始分化;14 d时紧贴OECs生长的NSCs,同OECs广泛交织在一起,P75/Nestin免疫荧光染色可见红色荧光Nestin标记的NSCs,绿色荧光P75标记的OECs; GFAP和NF免疫荧光染色显示,大部分为绿色荧光标记的NF+细胞,小部分为红色荧光标记的GFAP+细胞。OECs+NSCs共培养组和NSCs单独培养组NF阳性细胞率分别为69.5%,47.2%,前者明显高于后者(P < 0.05);其中向TH分化的比例,共培养组是36.7%,而NSCs单独培养组仅6.34%(P < 0.05)。4、细胞移植后第1周,各组大鼠的旋转行为无明显变化;第30天和60天时,与假手术组和模型对照组比较,旋转行为有较为明显的改善(P < 0.05),其中OECs+NSCs共移植组的旋转行为改善较单纯移植NSCs组和单纯移植OECs更为明显(P < 0.05)。5、HE染色显示,移植后一天,移植细胞呈簇状或串状沿针道分布,细胞数量较多。6、Brdu免疫荧光染色显示,移植区Brdu阳性细胞为FITC绿色荧光标记,大多数细胞较小,无突起,呈圆形或椭圆形,少数呈多边形或不规则形态。随着时间的推移,Brdu阳性细胞数量越来越少。7、TH免疫荧光显示,从第7天起,在移植区可见胞浆呈罗丹明红色荧光标记的TH阳性神经元(15-27个/视野),呈圆形或椭圆形,无明显突起,分布在针道与纹状体实质的交界处;在第30天,在针道的两侧TH阳性神经元增多(28-34个/视野),,有的可见少许突起。NSCs移植组和OECs移植组在移植区也可见少数罗丹明红色荧光标记的TH阳性神经元。经过图像处理和数据统计,OECs+NSCs移植组TH神经元阳性率高于NSCs、OECs单独移植组(P<0.05)结论1、从E14.5胚胎大鼠腹侧中脑组织可以分离得到具有不断增殖和自我更新能力的NSCs。2、在体外培养条件下,OECs有促进NSCs增殖和诱导其分化的作用,其中分化成神经元的比例高于星形胶质细胞,并有部分可以分化为TH阳性神经元。3、NSCs、OECs分别单独移植或OECs+NSCs联合移植入PD大鼠的纹状体内,对于PD大鼠的旋转行为均有改善,但联合移植优于单独移植。4、在体移植情况下,OECs能够诱导胚鼠中脑来源的NSCs向TH阳性神经元分化。5、OECs可供选择用于中枢神经系统损伤修复和再生的理想替代种子细胞,与NSCs联合移植效果更好。更多还原

【Abstract】 In vitro:To investigate the effects of olfactory ensheathing cells(OECs) on proliferation and differentiation of neural stem cells(NSCs). NSCs and OECs were separated from ventral midbrain and olfactory bulb of embryonic rats aged 14.5 days. Divide the cells into two groups: group A :NSCs co-cultured with OECs group ;group B :NSCs cultured solely. Observe the proliferation and differentiation of NSCs and OECs by P75 stained immunochemistry and P75, Brdu/Nestin,GFAP,NF,P75/Nestin single or double-stained immunofluorescence respectively . Most of OECs on the 7th day tested by P75 stained immunochemistry appears to be positive. NSCs formed typical floaty neurospheres on the 7th day, the results of immunofluorescence double stained by Brdu/Nestin were positive .On the 14th day, some neurospheres in group B were proned to differentiation. Most of the cells were GFAP positive cells marked with red fluorescein . Few of them were NF positive cells marked by green fluorescein .The neurospheres in group A grew accreting on OECs, stretched its tubcles began differentiation from the 12th day. On the 14th day , both kinds of cells clustered together intensively. P75/Nestin immunofluorescence showed Nestin positive NSCs marked by red fluorescein and P75 positive OECs marked by green fluorescein . GFAP and NF stain showed the majority are NF positive cells marked by green fluorescein , the minority are GFAP positive cells marked by red fluorescein. Not only percentage of the NF positive cell was 69.5% more than 47.2% of control group but also percentage of the NF/TH positive cell was 36.7% more than 6.34% of control group.In vivo:To observe the effect of neural stem cells and olfactory ensheathing cells co-graft in the rat model of Parkinson disease. The singel cell suspensions derived from E14.5 rat embryonic mesencephlon were prepared and plated into the serum-free medium containing DMEM/F12(1:1),EGF,bFGF,and B27supplement.After the primary neurosphere were formed,the single cell suspension of primary neurosphere were diluted by limiting dilution method,and single cell clone sphere of neural stem cell were obtained with successive proliferation. The neurospheres were labled by using immunofluorescence detection of Brdu and Nestin.The rat models of PD were established with injecting 6-OHDA into the SNC and VTA of the rat brains and divided into three groups:NS(0.9%saline)tranplatation group,NSCs tranplatation group,OECs tranplatation group and NSCs+OECs tranplatation group.The concentration of cells for tranplatation was1.0x105cells/μl.The change of rotation behavior was measured compared with three groups at different time point(7d,14d,30d,60d) after tranplatation.The survived cells were detected by HE and Brdu immunocytochemical stain;the differentiated cells were marked by TH immunocytochemical stain. 1、Single cell neuropheres consisting lots of cells were isolated from E14.5 rat embryonic mesencephlon after two weeks culturing.Large amounts of single cell clone spheres derived from one NSCs were obtained by successive passage.The neuropheres exhibited both Brdu and Nestin positive(90%).2、Compared with control groups,there was no obvious changes in the first three weeks after transplantation.But at 30d and 60d time point,the rotation of rat groups of NSCs ,groups of OECs and groups of NSCs + OECs transplatation were distinctly improved( P<0.05). 3、The brain tissue were stained HE one day later after transplantation.The cells were gathered around the transplatation channel. 4、Brdu immunocytochemistry:Brdu+ cells body were small and karyons were snuff color .The cells shape were roundness,oblong,polygon.A large amount of cells were found in the core of the transplantation.And the Brdu+ cells were decreased quickly as time went through.5、TH immunocytochemistry: The emergence of TH+ cells was at 14d after transplantation.TH+ cells shape were roundness or oblong and there were no perimeters on the cells.The positive cells were filemot.TH+ cells were gradually increased at 30d after transplantetion.We also found small perimeters on these cells.Compare with NSCs group,there are more TH+ cells in the NSCs+ OECs group. 1、Our cell line belong to NSCs and is multipotent, which has the ability to undergo self-renew. So there are the stem cells of the central nervous system. 2、The results indicated OECs can promote the proliferation and differentiation of NSCs. 3、The transplantation of NSCs has a little efficiency on the treatment of PD.4、OECs may promote the NSCs derived from E14.5 rat embryonic mesencephlon to turn into dopaminergic neurons.The rotation behavior were obvious meliorated in the PD rats by using NSCs+ OECs.更多还原