神经生长因子诱导大鼠骨髓间充质干细胞向神经细胞分化的效果观察及相关蛋白网络分析

发布时间：2018-07-08 11:09

本文选题：骨髓间充质干细胞 + 神经生长因子　；参考：《山东医药》2016年39期

【摘要】：目的观察神经生长因子(NGF)诱导大鼠骨髓间充质干细胞(BMSCs)向神经细胞分化的效果,并分析相关蛋白网络。方法将4周龄健康SD大鼠4只,脱臼处死取股骨分离BMSCs并传代。取第5代BMSCs制备细胞爬片,分为观察组和对照组两组,观察组加入3%DMSO、60 ng/m L NGF、DMEM/F12预诱导液预诱导2 h,预诱导完成后加入100 ng/m L NGF、DMEM/F12诱导液诱导48 h。对照组不加任何药物。诱导完成后倒置显微镜观察两组细胞形态变化,用免疫组化法检测两组BMSCs中的神经元特异性烯醇酶(NSE)蛋白,用实时荧光定量PCR法检测BMSCs中的NSE mRNA。采用生物信息学软件STRING10.0分析NGF在诱导BMSCs向神经细胞分化中参与的蛋白网络,并对网络系统中编码蛋白的基因进行功能富集分析。结果镜下可见观察组细胞体积增大,细胞核固缩,核质比降低,细长突起明显,部分细胞间以网络状连接。对照组细胞密度较均匀,形态以长梭形为主。诱导完成第1、2、4天,观察组NSE mRNA相对表达量分别为1.34±0.06、2.23±0.23、1.56±0.09,两组比较,P均0.05。观察组诱导完成第1、2天时NSE蛋白阳性细胞数分别为(35±8)、(133±6)个,对照组未检测到NSE蛋白阳性细胞,两组比较,P均0.05。以NGF为种子节点得到与NGF直接发生作用的105个蛋白节点,共3个中心节点,分别为NGF、神经生长因子受体和泛素C。对网络中的各个节点进行GO功能富集分析,结果富集在神经分化、神经发育及调控神经凋亡等生物学过程。NGF与Ras蛋白特定鸟嘌呤核苷酸释放因子1、脑衍生神经营养因子、生长抑制蛋白、NGF、NGFR、神经营养受体酪氨酸激酶1、神经营养受体酪氨酸激酶2、神经营养因子3、极微小蛋白激酶C、GTP结合蛋白RAC、核糖体蛋白S27A、信号传导子及转录激活子3、酪氨酸羟化酶、泛素A-52、泛素B和UBC等蛋白相互作用调控神经分化过程。模块分析结果显示中心节点NGF、NGFR和UBC在模块1中发挥核心的作用。结果 NGF可诱导大鼠BMSCs向神经细胞分化。以NGF为种子节点,筛选到105个与NGF直接发生互作的蛋白节点。NGF、NGFR和UBC在NGF诱导大鼠BMSCs向神经细胞分化的蛋白网络中发挥核心作用。
[Abstract]:Objective to observe the effect of nerve growth factor (NGF) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into neural cells and to analyze the related protein networks. Methods BMSCs were isolated from femur of 4 weeks old healthy SD rats. The fifth generation of BMSCs were divided into two groups: the observation group and the control group. The observation group was induced by 3DMSO-60 ng/m / L NGFMEM / F12 pre-induction solution for 2 h, and after the preinduction was completed, 100 ng/m L NGFN DMEM / F12 induction solution was added for 48 h. The control group was not treated with any drugs. The morphologic changes of the two groups were observed by inverted microscope after induction. The neuron-specific enolase (NSE) protein in BMSCs was detected by immunohistochemistry and the NSE mRNA in BMSCs was detected by real-time fluorescence quantitative PCR. Bioinformatics software STRING10.0 was used to analyze the protein networks involved in NGF inducing BMSCs to differentiate into neural cells, and the functional enrichment of the genes encoding proteins in the network system was analyzed. Results in the observation group, the cell volume increased, the nucleus became pyknosis, the ratio of nucleus to cytoplasm decreased, the slender process was obvious, and some of the cells were connected with each other in the form of network. The density of cells in the control group was uniform, and the shape of the cells was mainly fusiform. The relative expression of NSE mRNA in the observation group was 1.34 卤0.066 卤0.23 卤0.23 卤0.23 卤1.56 卤0.09 on the 4th day of induction, respectively, compared with 0.05 in the two groups. The number of NSE positive cells in the observation group was (35 卤8), (卤6) at the first day of induction, but no positive cells were detected in the control group (P < 0.05). 105 protein nodes directly interacting with NGF were obtained by using NGF as seed nodes. The three central nodes were NGF, NGF receptor and ubiquitin C. The results of go function enrichment analysis showed that NGF and Ras protein specific guanine nucleotide releasing factor 1, brain derived neurotrophic factor, and so on, were enriched in the biological processes of nerve differentiation, nerve development and regulation of neuronal apoptosis. Growth suppressor protein NGFN NGFR, neurotrophic receptor tyrosine kinase 1, neurotrophic receptor tyrosine kinase 2, neurotrophic factor 3, very small protein kinase Con GTP binding protein RAC, ribosomal protein S27A, signal transduction and transcriptional activator 3, tyrosine hydroxylase, The interaction of ubiquitin A-52, ubiquitin B and UBC regulates the process of neural differentiation. The results of the module analysis show that the central nodes NGF, NGFR and UBC play a central role in module 1. Results NGF could induce BMSCs to differentiate into neural cells. Using NGF as seed node, 105 protein nodes interacting directly with NGF. NGF NGFR and UBC played a central role in the protein network of NGF induced BMSCs to differentiate into neural cells.
【作者单位】：河北北方学院基础医学院;
【基金】：河北北方学院重大项目(120177) 河北省高等学校科学技术研究指导项目(Z2015047)
【分类号】：R329.2

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