心脉隆注射液诱导大鼠骨髓间充质干细胞分化为神经元样细胞的研究

发布时间：2018-05-17 17:44

本文选题：心脉隆注射液 + 骨髓间充质干细胞　；参考：《山东大学》2012年硕士论文

【摘要】：研究目的近年来,通过细胞工程技术体外分离和培养所需的靶细胞,已成为神经退行性疾病细胞替代治疗的研究热点。其中骨髓间充质干细胞(Bone marrow-derived mesenchvmal stem cells, BMSCs)由于来源丰富、取材简便,易于体外分离、培养、纯化,具有多向分化潜能等优点,已成为一种良好的替代治疗的靶细胞,在神经再生领域有着广泛的应用价值。心脉隆注射液是由美洲大蠊干品经过浸渍、减压浓缩、分离得到心脉隆浸膏再溶解后制得的,主要有扩血管、利尿、改善微循环、纠正神经内分泌失衡等作用。本研究以大鼠骨髓间充质干细胞为研究对象,心脉隆注射液为诱导剂,探讨心脉隆注射液体外诱导大鼠骨髓间充质干细胞(BMSCs)向神经元样细胞分化的可行性。研究方法 1.贴壁筛选法培养大鼠骨髓间充质干细胞(BMSCs),倒置显微镜下逐日观察原代、传代细胞的生长情况及形态特征。 2.流式细胞学检测细胞表面标志物CD29、CD90、CD34、CD45。 3.以心脉隆注射液为诱导剂,定向诱导第3代大鼠骨髓间充质干细胞(BMSCs)向神经元样细胞转化,每隔1h倒置相差显微镜下观察细胞形态变化。 4.免疫细胞荧光法鉴定诱导4h、12h、36h后巢蛋白(Nestin)、神经元特异性烯醇化酶(Neuron specific enolase, NSE)、微管相关蛋白2(Microtubule associated protein-2, MAP2)、胶质纤维酸性蛋白(Glial fibrillary acidic protein, GFAP)的表达情况。 5. RT-PCR生检测诱导36h后神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)mRNA的表达。结果 1.原代接种的细胞散在分布,折光性强,混杂其他细胞,1d后可见少量单核圆形细胞贴壁生长,3d后细胞大部分贴壁,有聚集生长倾向；8-10d后细胞呈集簇状生长,每个集簇细胞中心呈放射状或螺旋状,细胞铺满皿底并融合。传代后细胞增殖速度加快,3-5代后已纯化为全梭形细胞,呈鱼群样排列,6代后细胞生长减慢,部分开始变得宽大扁平。 2.流式细胞学检测示CD29、CD90高表达(阳性细胞百分比分别为99.06%、99.61%),CD34、CD45低表达(阳性细胞百分比分别为2.03%、2.00%)。 3.诱导2h后实验组细胞形态即开始改变,部分长梭形细胞的胞体变小,变为不规则形或圆形。随着诱导时间延长,突起不断增长,呈双极、多极形,36h诱导达到高峰,细胞形态明显变化,每个细胞有两个或多个突起并形成网状结构,呈典型神经元样细胞。对照组细胞形态无明显变化。 4.诱导4h、12h后,实验组Nestin阳性表达率分别为(81.0±1.6)%、(22.5±1.9)%,36h后,Nestin|阴性。诱导4h后,实验组NSE、MAP2阴性,12h、36h后,NSE阳性表达率分别为(73.5±2.2)%、(94.3±1.8)%,MAP2阳性表达率分别为(80.0±2.2)%、(96.4±2.8)%。诱导4h、12h、36h后实验组GFAP阴性,对照组Nestin、NSE、MAP2、GFAP均为阴性。 5.RT-PCR结果显示,诱导36h后,实验组细胞表达NSE mRNA,不表达GFAP mRNA。对照组细胞NSE、GFAP mRNA均不表达。结论 1.贴壁筛选法是一种简单、快速分离及纯化大鼠骨髓间充质干细胞的方法。 2.心脉隆注射液可在体外快速、高效地诱导大鼠骨髓间充质干细胞向神经元样细胞分化。 3.心脉隆注射液在体外诱导大鼠骨髓间充质干细胞分化为神经元样细胞前,经历了短暂的神经前体细胞阶段。
[Abstract]:Purpose of study

In recent years , it has become a hot spot for the replacement therapy of neurodegeneration diseases by means of cell engineering technique in vitro isolation and culture . Bone marrow - derived mesenchymal stem cells ( MSCs ) are widely used in the field of nerve regeneration .

Research Methods

1 . The rat bone marrow mesenchymal stem cells were cultured with adherent screening method . The growth and morphological characteristics of primary and passaged cells were observed on a day - by - day basis under an inverted microscope .

2 . Flow cytometry was used to detect CD29 , CD90 , CD34 , CD45 in cell surface markers .

3 . In the third generation rat bone marrow mesenchymal stem cells were transformed into neuron - like cells , and the morphological changes of the cells were observed at every 1h inverted phase contrast microscope .

4 . The expression of Nestin , Neuron specific protein ( NSE ) , microtubules associated protein - 2 ( MAP2 ) and Glial glial acidic protein ( GFAP ) were identified by immunofluorescence .

5 . The expression of NSE and GFAP mRNA were detected by RT - PCR in 36 hours after induction .

Results

1 . The cells of primary inoculated cells were distributed , the refractive index was strong , and other cells were mixed . After 1d , a small number of single - core round cells were observed . After 3 days , most of the cells were adherent , and there was a tendency of aggregation and growth .
After 8 - 1days , the cells were clustered , and the centers of each cluster were radial or spiral , the cells were plated with the bottom of the dish and fused . After passage , the cell proliferation was accelerated . After 3 - 5 generations , the cells were purified into full spindle cells .

2 . Flow cytometry showed that CD29 and CD90 were highly expressed ( 99 . 06 % , 99 . 61 % , respectively ) .

3 . After 2 hours of induction , the cell morphology of the experimental group began to change , and the cells of some long spindle cells became irregular or round . As the induction time was prolonged , the growth of the cells became irregular or round . As the induction time was prolonged , the number of the cells increased , and the shape of the cells changed obviously . The cells had two or more protrusions and formed a net structure , showing the typical neuron - like cells .

4 . After 4 h , 12 h , the expression rates of Nestin positive in experimental group were ( 81.0 卤 1.6 ) % , ( 22.5 卤 1.9 ) % , 36h , Nestin immunoreactive expression rates were ( 73.5 卤 2.2 ) % , ( 94.3 卤 1.8 ) % , and MAP2 positive expression rates were ( 80.0 卤 2.2 ) % , ( 96.4 卤 2.8 ) % , respectively .

5 . RT - PCR showed that NSE mRNA and GFAP mRNA were not expressed in the experimental group after 36 hours of induction . The NSE and GFAP mRNA in the control group were not expressed .

Conclusion

1 . The adherent screening method is a simple and rapid method for the isolation and purification of rat bone marrow mesenchymal stem cells .

2 . Xinmailong injection can induce the differentiation of rat bone marrow mesenchymal stem cells into neuron - like cells rapidly and efficiently in vitro .

3 . After inducing the differentiation of bone marrow mesenchymal stem cells into neuron - like cells in vitro , Xinmailong injection has experienced a transient neural progenitor cell stage .
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】