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】：【目的】采用iTRAQ蛋白組學(xué)技術(shù)篩選脊髓損傷(Spinal Cord Injury,SCI)后激活態(tài)雪旺細(xì)胞(Activated Schwann cells,ASCs)聯(lián)合骨髓間充質(zhì)干細(xì)胞(Bone marrow derived mesenchymal stem cells,BMSCs)移植在損傷微環(huán)境中的蛋白表達(dá)水平,并對(duì)差異表達(dá)蛋白進(jìn)行功能注釋,分析其所涉及神經(jīng)修復(fù)相關(guān)的重要功能,初步揭示細(xì)胞移植修復(fù)SCI在基因與分子水平上的調(diào)控機(jī)制,尋找關(guān)鍵調(diào)節(jié)蛋白,分析其作用方式,為進(jìn)一步優(yōu)化細(xì)胞移植修復(fù)脊髓損傷策略和臨床轉(zhuǎn)化應(yīng)用提供理論依據(jù)。【方法】1.細(xì)胞分離培養(yǎng):采用骨髓腔沖洗分離培養(yǎng)大鼠BMSCs,并應(yīng)用流式細(xì)胞術(shù)檢測(cè)胞膜表面分子鑒定BMSCs。應(yīng)用坐骨神經(jīng)預(yù)損傷激活和膠原酶消化分離培養(yǎng)SCs,采用S100免疫熒光染色鑒定SCs。2.實(shí)驗(yàn)分組:54只Wistar成年雌性大鼠隨機(jī)分成6組(每組9只):A組,DMEM空白對(duì)照7天組;B組,DMEM空白對(duì)照14天組;C組,DMEM空白對(duì)照28天組;D組,BMSC聯(lián)合ASC移植7天組;E組,BMSC聯(lián)合ASC移植14天組;F組,BMSC聯(lián)合ASC移植28天組。3.動(dòng)物模型制作與細(xì)胞移植:采用成年雌性Wistar大鼠,利用NYU Impactor II型打擊裝置建立胸10節(jié)段脊髓挫傷模型,打擊重量為10g,高度為2.5cm。在損傷后7天,使用10μl Hamilton微量注射器分別將DMEM,ASCs與BMSCs混合懸液緩慢注射入脊髓損傷區(qū)域,注射劑量為每只15μl,細(xì)胞密度為1×105/μl。分別在移植后7天,14天,28天行心臟灌注,取出損傷區(qū)域0.5cm組織。4.蛋白組學(xué)分析:應(yīng)用裂解液提取法提取脊髓組織蛋白,應(yīng)用2D Quant試劑盒測(cè)定每組蛋白濃度,應(yīng)用Mascot軟件對(duì)二級(jí)質(zhì)譜圖信息進(jìn)行定性定量計(jì)算,采用iTRAQ/TMT質(zhì)譜定量方法鑒定差異蛋白,對(duì)差異蛋白分別進(jìn)行GO功能富集分析與String網(wǎng)絡(luò)分析�！窘Y(jié)果】BMSCs聯(lián)合ASCs移植修復(fù)脊髓損傷后7天,差異表達(dá)蛋白數(shù)為105個(gè),其中,39個(gè)表達(dá)上調(diào),66個(gè)表達(dá)下調(diào);移植后14天,差異表達(dá)蛋白數(shù)為185個(gè),其中,151個(gè)表達(dá)上調(diào),34個(gè)表達(dá)下調(diào);移植后28天,差異表達(dá)蛋白數(shù)為81個(gè),其中,46個(gè)表達(dá)上調(diào),35個(gè)表達(dá)下調(diào)。差異表達(dá)蛋白的GO功能富集分析結(jié)果發(fā)現(xiàn),移植后7天差異蛋白主要富集于炎癥反應(yīng)、免疫應(yīng)答、急性期反應(yīng)等免疫相關(guān)生物學(xué)過程。移植后14天組差異蛋白主要富集于神經(jīng)突觸、神經(jīng)遞質(zhì)運(yùn)輸、金屬離子代謝等生物學(xué)過程。移植后28天組,差異蛋白主要富集于胞外空間重塑、細(xì)胞外基質(zhì)、層黏連蛋白、細(xì)胞粘附、髓鞘化等生物學(xué)過程與細(xì)胞組分。String網(wǎng)絡(luò)互作分析發(fā)現(xiàn)表達(dá)下調(diào)的STAT1蛋白在移植7天后位于蛋白互作網(wǎng)絡(luò)的核心位置�！窘Y(jié)論】1.本研究初步繪制了ASCs聯(lián)合BMSCs移植后脊髓損傷微環(huán)境的蛋白表達(dá)譜,發(fā)現(xiàn)了神經(jīng)絲蛋白,鈣網(wǎng)織蛋白,Lyn蛋白等與脊髓損傷修復(fù)相關(guān)的差異蛋白。2.ASCs聯(lián)合BMSCs移植主要通過抑制炎癥免疫反應(yīng)、抗凋亡、重塑細(xì)胞外基質(zhì)、調(diào)節(jié)損傷局部微環(huán)境等作用機(jī)制促進(jìn)脊髓損傷修復(fù),并提示STAT1蛋白在ASCs聯(lián)合BMSCs移植修復(fù)脊髓損傷中可發(fā)揮免疫調(diào)理和抗凋亡的作用。3.深入探討ASCs聯(lián)合BMSCs聯(lián)合移植促進(jìn)修復(fù)的分子機(jī)制有助于優(yōu)化移植策略的探索研究,為細(xì)胞移植修復(fù)脊髓損傷的臨床治療應(yīng)用奠定基礎(chǔ)。
[Abstract]:[Objective] using the screening of spinal cord injury group (Spinal Cord Injury iTRAQ protein, SCI) after activated Schwann cells (Activated Schwann cells, ASCs) combined with bone marrow mesenchymal stem cells (Bone marrow derived mesenchymal stem cells, BMSCs) transplantation in injury microenvironment in protein expression level, and the differences in protein expression for functional annotation, analysis the important function of the neural repair, preliminarily reveal the mechanism of regulation of cell transplantation in repair of SCI gene and molecular level, to find the key regulatory protein, analyze the effect, provide a theoretical basis for the further optimization strategy of cell transplantation in repair of spinal cord injury and clinical application. [methods] 1. cells isolation: the bone marrow cavity flushing rat BMSCs were isolated and cultured, and flow cytometry was used to detect the cell membrane surface molecular identification of BMSCs. application of sciatic nerve injury pre activation Isolation of SCs and collagenase digestion by S100, immunofluorescence staining of SCs.2. experimental groups: 54 adult female Wistar rats were randomly divided into 6 groups (n = 9): A group, DMEM control group for 7 days; B group, DMEM control group for 14 days; C group, DMEM control group for 28 days; group D, BMSC and ASC 7 days of transplantation group; group E, BMSC and ASC 14 days of transplantation group; group F, BMSC and ASC 28 days of transplantation group.3. animal model and cell transplantation: the adult female Wistar rats, striking device of thoracic spinal cord contusion model in 10 segments by NYU Impactor II, hit the weight is 10g, the height is 2.5cm. in 7 days after injury, using 10 L Hamilton micro syringe respectively DMEM, ASCs and BMSCs mixed suspension was slowly injected into the area of spinal cord injury, the dose per injection is only 15 l, the cell density of 1 * 105/ L. respectively in 7 days after transplantation, 14 days. 28 days after cardiac perfusion, remove the damage zone The domain 0.5cm.4. proteomics analysis: extraction of spinal cord tissue proteins by lysis method, determination of protein concentration in each group using 2D Quant kit, Mascot software is applied to the qualitative and quantitative calculation of the two grade mass spectrum information, the quantitative method for iTRAQ/TMT mass spectrometric identification of differential proteins, the differential proteins were analyzed with String GO enrichment analysis network. [results] BMSCs combined with ASCs transplantation for repair of spinal cord injury after 7 days, the number of differentially expressed protein is 105, among them, 39 upregulated and 66 downregulated; 14 days after transplantation, the differentially expressed protein number 185, among them, 151 upregulated and 34 downregulated; 28 days after transplantation, the differentially expressed proteins in number 81, among them, 46 upregulated and 35 downregulated. Enrichment analysis results show that the protein expression difference of GO function, the 7 day after transplantation proteins mainly enriched in inflammation, immune response A biological reaction in acute stage of immune process. 14 days after transplantation group proteins are mainly enriched in synapses, neurotransmitter transport, metal ion metabolism and other biological processes. 28 days after the transplantation group, the difference of protein enriched in the extracellular space remodeling, extracellular matrix, laminin, cell adhesion, myelin sheath such as biological processes and cellular components of.String network interaction analysis showed that down-regulation of the expression of STAT1 protein in 7 days after transplantation in the protein interaction network core position. [Conclusion] this study draws 1. ASCs combined with BMSCs transplantation after spinal cord injury microenvironment protein expression profile, found neurofilament protein, calreticulin fabric of protein, Lyn protein and repair of spinal cord injury related proteins.2.ASCs combined with BMSCs transplantation mainly by inhibiting the inflammatory immune response, anti apoptosis, extracellular matrix remodeling, regulating the local micro environment damage The mechanism to promote the repair of spinal cord injury, and suggest that STAT1 protein may play immune regulation and anti apoptosis effect of.3. ASCs combined with BMSCs transplantation of promoting the repair mechanism will help to explore the optimization strategy research on ASCs transplantation combined with BMSCs transplantation for repair of spinal cord injury, lay the foundation for clinical application of cell transplantation for repair of spinal cord injury.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 SMITA SAVANT-BHONSALE;MICHAEL CHOPP;;Down-Regulation of Neurocan Expression in Reactive Astrocytes Promotes Axonal Regeneration and Facilitates the Neurorestorative Effects of Bone Marrow Stromal Cells in the Ischemic Rat Brain[J];神經(jīng)損傷與功能重建;2008年06期

2 馮世慶;周先虎;孔曉紅;陳家童;李暉;侯巍;王沛;郭世紱;;自體激活雪旺細(xì)胞移植治療急性脊髓損傷的實(shí)驗(yàn)研究[J];中華骨科雜志;2006年08期

，

本文編號(hào)：1764837