本文選題：脊髓損傷 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的:脊髓損傷(spinal cord injuries,SCI)是中樞神經(jīng)系統(tǒng)創(chuàng)傷性疾病,具有高發(fā)病率,高致殘率,目前尚無有效治愈的方法。本課題組前期研究發(fā)現(xiàn)自體激活雪旺細(xì)胞(SCs)不僅能分泌神經(jīng)營養(yǎng)因子,抑制神經(jīng)元凋亡,還可以誘導(dǎo)骨髓間充質(zhì)干細(xì)胞(BMSCs)向神經(jīng)元方向分化,促進(jìn)軸突再生和功能恢復(fù),但具體的激活及誘導(dǎo)機(jī)制尚不清楚。本課題應(yīng)用iTRAQ(Isobaric Tag for Relative and Absolute Quantitation)技術(shù)深入研究BMSCs與SCs共培養(yǎng)后的蛋白質(zhì)組學(xué)變化,建立其差異蛋白譜,尋找參與細(xì)胞移植干預(yù)修復(fù)的潛在細(xì)胞信號轉(zhuǎn)導(dǎo)通路,確立細(xì)胞信號通路中關(guān)鍵調(diào)節(jié)位點(diǎn)的改變和調(diào)控方式,進(jìn)一步明確BMSCs與SCs共移植的修復(fù)SCI機(jī)理。研究內(nèi)容與方法:本課題應(yīng)用采用胰酶消化組織塊和機(jī)械分離法培養(yǎng)雪旺細(xì)胞,同時應(yīng)用胰酶快速消化法和雙30分鐘差速貼壁法純化SCs。用改進(jìn)的全骨髓貼壁法,分離培養(yǎng)大鼠BMSCs。應(yīng)用S100免疫熒光染色鑒定SCs,應(yīng)用流式細(xì)胞儀和三系分化鑒定BMSCs。本實(shí)驗(yàn)共分為3組,單純BMSCs組(SC0),BMSCs與SCs共培養(yǎng)3天組(SC3),BMSCs與SCs共培養(yǎng)7天組(SC7)。本實(shí)驗(yàn)采用半定量換液的方式進(jìn)行共培養(yǎng)。應(yīng)用裂解液提取法提取各組蛋白,應(yīng)用2D Quant試劑盒測定每組蛋白濃度,采用iTRAQ/TMT質(zhì)譜定量方法鑒定差異蛋白。應(yīng)用Mascot軟件對二級譜圖信息進(jìn)行定性定量計算。對差異蛋白分別進(jìn)行GO、KEGG、Interpro功能的注釋與功能分析,對差異蛋白進(jìn)行顯著性富集分析,應(yīng)用STRING數(shù)據(jù)庫對差異蛋白進(jìn)行網(wǎng)絡(luò)互作關(guān)系分析。研究結(jié)果:本實(shí)驗(yàn)以表達(dá)上調(diào)或下調(diào)1.3倍以上作為差異表達(dá)蛋白。SC3d組與SC0d組相比,上調(diào)蛋白29個,下調(diào)蛋白45個;SC7d組與SC0d組相比,上調(diào)蛋白43個,下調(diào)蛋白32個;SC7d組與SC3d組相比,上調(diào)蛋白83個,下調(diào)蛋白24個。對差異表達(dá)蛋白進(jìn)行GO注釋和亞細(xì)胞定位分析顯示:差異表達(dá)蛋白共涉及13種生物過程,9種細(xì)胞組份,9種分子功能;主要位于細(xì)胞核、細(xì)胞質(zhì)、胞外蛋白,質(zhì)膜等亞細(xì)胞結(jié)構(gòu)中。對鑒定的差異表達(dá)蛋白進(jìn)行蛋白功能(GO/level2)、功能結(jié)構(gòu)域、KEGG Pathway富集分析顯示,發(fā)現(xiàn)SC3d vs.SC0d比較組的差異表達(dá)蛋白顯著富集的功能term相對較少,SC7d vs.SC0d組差異蛋白主要富集在脂類代謝、糖類代謝,生物降解等生物過程,顯著富集在溶酶體、細(xì)胞骨架和細(xì)胞外基質(zhì)等細(xì)胞組份中,顯著富集在水解酶活性,糖胺聚糖結(jié)合和糖類結(jié)合等分子功能中,3個比較組差異蛋白顯著富集的代謝通路為鞘糖脂生物合成代謝,神經(jīng)鞘脂類代謝,溶酶體代謝途徑,顯著富集的功能結(jié)構(gòu)域?yàn)樘擒真I鍵水解酶催化結(jié)構(gòu)域,鈣調(diào)蛋白同源結(jié)構(gòu)域,糖基水解酶家族13�；趯Ω鹘M差異蛋白GO(三個本體)、KEGG和Interpro共五項(xiàng)功能富集分析的結(jié)果cluster分析顯示,總共54個生物過程term得以聚類,38個細(xì)胞組份功能term得以聚類,38個分子功能term得以聚類。KEEG聚類分析主要集中在SC7 vs.SC3比較組,主要為神經(jīng)節(jié)鞘磷脂生物合成代謝、溶酶體代謝途徑、鞘磷脂代謝途徑等,功能結(jié)構(gòu)域聚類分析同樣主要集中在SC7 vs.SC3比較組,主要為硫酸酯酶、糖苷水解酶催化結(jié)構(gòu)域、糖苷水解酶超家族等結(jié)構(gòu)域。結(jié)論:本研究應(yīng)用定量蛋白組學(xué)技術(shù),繪制了BMSCs與SCs共培養(yǎng)后的差異蛋白表達(dá)譜,為揭示BMSCs與SCs共移植到脊髓損傷部位發(fā)揮作用的深層機(jī)制奠定了一定的基礎(chǔ),對于這些問題的研究將有助于深化和擴(kuò)大干細(xì)胞臨床治療應(yīng)用。
[Abstract]:Objective: spinal cord injuries (SCI) is a traumatic disease of the central nervous system, with high incidence and high disability rate. There is no effective cure at present. In our previous study, it was found that autoactivated Schwann cells (SCs) can not only secrete a god management factor, inhibit neuronal apoptosis, but also induce bone marrow mesenchymal cells. Stem cells (BMSCs) differentiate into neuron direction, promote axonal regeneration and function recovery, but the specific activation and induction mechanism is not clear. This subject uses iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) technology to study the changes in egg white substance after BMSCs and SCs co culture, and establish its differential protein spectrum and seek participation. Cell transplantation intervention to repair the potential cell signal transduction pathway, establish the change and regulation of key regulatory sites in cell signaling pathway, and further clarify the mechanism of BMSCs and SCs transplantation for the repair of SCI. Enzyme rapid digestion method and double 30 minute differential adherence method were used to purify SCs. with improved full bone marrow adherence method. The isolated and cultured rat BMSCs. was identified by S100 immunofluorescence staining, and SCs was identified by S100 immunofluorescence staining. The use of flow cytometry and three line differentiation identification were divided into 3 groups, pure BMSCs group (SC0), BMSCs and SCs co culture 3 days group (SC3), BMSCs and SCs co culture for 7 days Group (SC7). In this experiment, a semi quantitative solution was used to co culture. The protein was extracted by the lysate extraction method, the concentration of each protein was determined by 2D Quant kit and the differential protein was identified by iTRAQ/TMT mass spectrometry. Mascot software was used to make a qualitative and quantitative calculation of the two level spectrum information. The difference protein was G respectively. O, KEGG, Interpro function annotation and functional analysis, the difference protein was significantly enriched and analyzed. STRING database was used to analyze the network interaction of differential proteins. The results were as follows: in this experiment, the expression of protein.SC3d in the.SC3d group was up to 1.3 times up or down. Compared with the SC0d group, the protein was up regulated and down regulated by 45 proteins. Group SC7d was up to 43 and 32 down-regulation of protein compared with group SC0d, and 83 up and 24 down regulated proteins in group SC7d and SC3d group. The GO annotation and subcellular localization analysis of differentially expressed proteins showed that the differentially expressed proteins involved 13 biological processes, 9 cell components and 9 molecular functions, mainly located in the nucleus and cytoplasm. The protein function (GO/level2), functional domain and KEGG Pathway enrichment analysis showed that the differentially expressed proteins in the SC3d vs.SC0d group showed significant enrichment of the function term relatively less, and the SC7d vs.SC0d group difference protein was mainly enriched in lipid metabolism and saccharide generation. The biological processes such as biodegradation, such as biodegradation, were significantly enriched in the lysosome, cytoskeleton and extracellular matrix, and were significantly enriched in the function of hydrolase, glycosaminoglycan binding and saccharide binding. The metabolic pathways of the 3 comparative groups were the glycolipid biosynthesis metabolism, neurinosin metabolism, and dissolution. Enzyme metabolism pathway, the significant functional domain is glycoside bond key hydrolase domain, calmodulin homologous domain, sugar based hydrolase family 13. based on the analysis of five functions of different proteins GO (three bodies), KEGG and Interpro, cluster analysis showed that term was clustered in a total of 54 biological processes, 38 Cell component function term was clustered, and 38 molecular functional term clustering analysis was mainly concentrated in SC7 vs.SC3 comparison group, mainly ganglion sphingomyelin biosynthesis metabolism, lysosome metabolic pathway and sphingomyelin metabolic pathway, and functional domain clustering analysis was mainly concentrated in SC7 vs.SC3 comparison group, mainly sulfuric acid. Esterase, glycoside hydrolase catalyzed domain and glycoside hydrolase superfamily. Conclusion: This study used quantitative proteomics technology to draw the differential protein expression profiles of BMSCs and SCs co culture, which laid a certain foundation for revealing the deep mechanism of BMSCs and SCs transplantation to the spinal cord injury. The research will help to deepen and expand the clinical application of stem cells.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.2

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相關(guān)期刊論文 前8條

1 Zhiyuan Li;Zhanxiu Zhang;Lili Zhao;Hui Li;Suxia Wang;Yong Shen;;Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury[J];Neural Regeneration Research;2014年08期

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本文編號：2064886