本文選題：骨髓間充質(zhì)干細胞 + 神經(jīng)生長因子��； 參考：《山東醫(yī)藥》2016年39期

【摘要】：目的觀察神經(jīng)生長因子(NGF)誘導大鼠骨髓間充質(zhì)干細胞(BMSCs)向神經(jīng)細胞分化的效果,并分析相關(guān)蛋白網(wǎng)絡(luò)。方法將4周齡健康SD大鼠4只,脫臼處死取股骨分離BMSCs并傳代。取第5代BMSCs制備細胞爬片,分為觀察組和對照組兩組,觀察組加入3%DMSO、60 ng/m L NGF、DMEM/F12預誘導液預誘導2 h,預誘導完成后加入100 ng/m L NGF、DMEM/F12誘導液誘導48 h。對照組不加任何藥物。誘導完成后倒置顯微鏡觀察兩組細胞形態(tài)變化,用免疫組化法檢測兩組BMSCs中的神經(jīng)元特異性烯醇酶(NSE)蛋白,用實時熒光定量PCR法檢測BMSCs中的NSE mRNA。采用生物信息學軟件STRING10.0分析NGF在誘導BMSCs向神經(jīng)細胞分化中參與的蛋白網(wǎng)絡(luò),并對網(wǎng)絡(luò)系統(tǒng)中編碼蛋白的基因進行功能富集分析。結(jié)果鏡下可見觀察組細胞體積增大,細胞核固縮,核質(zhì)比降低,細長突起明顯,部分細胞間以網(wǎng)絡(luò)狀連接。對照組細胞密度較均勻,形態(tài)以長梭形為主。誘導完成第1、2、4天,觀察組NSE mRNA相對表達量分別為1.34±0.06、2.23±0.23、1.56±0.09,兩組比較,P均0.05。觀察組誘導完成第1、2天時NSE蛋白陽性細胞數(shù)分別為(35±8)、(133±6)個,對照組未檢測到NSE蛋白陽性細胞,兩組比較,P均0.05。以NGF為種子節(jié)點得到與NGF直接發(fā)生作用的105個蛋白節(jié)點,共3個中心節(jié)點,分別為NGF、神經(jīng)生長因子受體和泛素C。對網(wǎng)絡(luò)中的各個節(jié)點進行GO功能富集分析,結(jié)果富集在神經(jīng)分化、神經(jīng)發(fā)育及調(diào)控神經(jīng)凋亡等生物學過程。NGF與Ras蛋白特定鳥嘌呤核苷酸釋放因子1、腦衍生神經(jīng)營養(yǎng)因子、生長抑制蛋白、NGF、NGFR、神經(jīng)營養(yǎng)受體酪氨酸激酶1、神經(jīng)營養(yǎng)受體酪氨酸激酶2、神經(jīng)營養(yǎng)因子3、極微小蛋白激酶C、GTP結(jié)合蛋白RAC、核糖體蛋白S27A、信號傳導子及轉(zhuǎn)錄激活子3、酪氨酸羥化酶、泛素A-52、泛素B和UBC等蛋白相互作用調(diào)控神經(jīng)分化過程。模塊分析結(jié)果顯示中心節(jié)點NGF、NGFR和UBC在模塊1中發(fā)揮核心的作用。結(jié)果 NGF可誘導大鼠BMSCs向神經(jīng)細胞分化。以NGF為種子節(jié)點,篩選到105個與NGF直接發(fā)生互作的蛋白節(jié)點。NGF、NGFR和UBC在NGF誘導大鼠BMSCs向神經(jīng)細胞分化的蛋白網(wǎng)絡(luò)中發(fā)揮核心作用。
[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 鹵0.066 鹵0.23 鹵0.23 鹵0.23 鹵1.56 鹵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 鹵8), (鹵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.
【作者單位】： 河北北方學院基礎(chǔ)醫(yī)學院;
【基金】：河北北方學院重大項目(120177) 河北省高等學�？茖W技術(shù)研究指導項目(Z2015047)
【分類號】：R329.2

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