本文選題：肢體缺血　切入點(diǎn)：內(nèi)皮祖細(xì)胞　出處：《第二軍醫(yī)大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 研究目的 EPCs的動員機(jī)制存在多個環(huán)節(jié),如黏附、降解、運(yùn)動、遷移等。在機(jī)體某些生理或病理狀態(tài)下,外周血中EPCs的數(shù)量和功能也會發(fā)生變化。去甲腎上腺素是人體交感神經(jīng)的主要的神經(jīng)遞質(zhì)之一。其對于EPCs動員的影響仍不是十分清楚。本研究的目的是：探討NE對肢體缺血C57小鼠EPCs動員的影響,以及對體外培養(yǎng)的人EPCs的增殖、遷移能力的影響；探討MAPK信號通路介導(dǎo)NE的促進(jìn)EPCs的作用以及beta-arrestin 2在EPCs功能調(diào)節(jié)中的作用。 研究方法 本課題從在體實(shí)驗(yàn)水平、細(xì)胞實(shí)驗(yàn)水平和分子調(diào)控機(jī)制等多個層面逐層深入研究NE調(diào)節(jié)EPCs動員的機(jī)制。 (1)在體實(shí)驗(yàn)：采用結(jié)扎股動脈的方法制備左下肢缺血的小鼠模型,于術(shù)后的第l至5天每天給予藥物腹腔注射,具體藥物以下：NE 10"8mol、α阻滯劑酚妥拉明10"8mol、beta1受體阻滯劑美托洛爾10-8mol和beta2受體阻滯劑10-8mol。于術(shù)后第7天取骨髓單個核細(xì)胞、脾臟細(xì)胞和小鼠外周血單個核細(xì)胞,流式檢測CD34+KDR+的細(xì)胞所占的比率。 (2)細(xì)胞功能學(xué)研究：在體外培養(yǎng)人外周血EPCs的培養(yǎng)基中加入不同濃度的NE(0、0.01、0.1、1、10、100μM)干預(yù)72小時, MTT評價EPCs的增殖能力,Transwell小室實(shí)驗(yàn)評價EPCs的遷移能力。 (3)細(xì)胞信號研究：Western-blot檢測檢測絲裂原激活蛋白激酶(Mitogen Activated Protein Kinase, MAPK),即細(xì)胞外信號調(diào)節(jié)激酶Erk1/2、c-Jun氨基端激酶Jnk和p38 MAPK的磷酸化水平。 (4)細(xì)胞信號通路的蛋白調(diào)控機(jī)制研究：隨機(jī)選取4名對照者和4名糖尿病患者的外周血30ml,分離PBMCs, Western-blot檢測beta-arrestin 2的表達(dá)�；虺聊姆椒ǜ蓴_EPCs beta-arrestin 2的表達(dá),Realtime PCR和Western-blot的方法評價沉默的效率,采用MTT何Transwell小室實(shí)驗(yàn)的方法研究beta-arrestin2在EPCs增殖能力和遷移能力的調(diào)控中的作用。 結(jié)果 (1)在體實(shí)驗(yàn)發(fā)現(xiàn)：NE能夠顯著地促進(jìn)肢體缺血C57小鼠骨髓EPCs的增殖以及其動員進(jìn)外周血的能力。注射了NE組的C57小鼠骨髓的EPCs的數(shù)量從2.0±0.4%增加到4.7±0.9%,p0.05,動員進(jìn)外周血的EPCs的數(shù)量從1.2±0.6%增加到6.2±1.9%,p0.05,脾臟的EPCs的數(shù)量從2.54±0.8%增加到3.1±1.0%,p0.05。而NE的這種作用能夠被Q腎上腺素受體阻斷劑酚妥拉明和beta2腎上腺受體阻斷劑I127阻斷,但是不能被beta1腎上腺素受體阻斷劑阻斷。 (2)細(xì)胞功能學(xué)實(shí)驗(yàn)發(fā)現(xiàn)：NE能夠濃度依賴性地促進(jìn)體外培養(yǎng)的EPCs的增殖,其中以NE 10μM的作用最強(qiáng),其增殖率為103.6±54.6%,P0.05,NE的促進(jìn)EPCs增殖作用能夠被酚妥拉明和I127阻斷,但是不能被美托絡(luò)爾阻斷。另外ERK 1/2抑制劑A6355和eNOS抑制劑L-NAME能夠阻斷也能夠阻斷NE的這種作用,P0.05,但是JNK抑制劑SP600125、p38抑制劑PD169318、PI3K抑制劑LY294002 1μM、、p65抑制劑R0106-9920和NO供體SNP均無類似作用,P0.05。NE能夠顯著地促進(jìn)EPCs的遷移能力(124.1±12.2 VS 71.7±19.6,P0.05),酚妥拉明和I127能夠阻斷NE的促遷移能力(57.2±14.3 VS 124.1±12.2,P0.05和61.3±11.5 VS 124.1±12.2,P0.05),而美托洛爾無類似作用(112.8±26.0 VS 124.1±12.2,P0.05)。 (3)細(xì)胞信號通路的研究發(fā)現(xiàn)：NE能夠濃度依賴性地增加EPCs的Erk 1/2和信號分子Src的的磷酸化,P0.05,而I127能夠減輕Erk 1/2和Src的磷酸化,P0.05,酚妥拉明和美托絡(luò)爾則沒有類似作用,P0.05。 (4)信號調(diào)控機(jī)制研究發(fā)現(xiàn)：基因沉默beta-arrestin 2后,EPCs對VEGF的敏感性顯著地增加(33.7±6.4%VS 2.1±1.4%,P0.05),而EPCs對NE的敏感性顯著地減少(26.6±7.8%VS64.0±13.5%,P0.05)�；虺聊琤eta-arrestin 2后,EPCs的遷移能力下降,對NE的敏感性下降。 結(jié)論 NE促進(jìn)肢體缺血時骨髓EPCs的動員以及增殖、遷移能力。NE能夠激活EPCs Src-MAPK信號通路,而beta-arrestin 2參與EPCs的增殖和遷移能力的調(diào)節(jié)。
[Abstract]:research objective
The mobilization mechanism of EPCs have many aspects, such as adhesion, degradation, movement and migration. In the physiological or pathological conditions, the number and function of EPCs in peripheral blood will change. Norepinephrine is one of the main neurotransmitter of vagus nerve. The effects of EPCs mobilization is still not very clear. The purpose of this study is: To investigate the effect of NE on EPCs mobilization of limb ischemia in C57 mice, and the proliferation of cultured human EPCs, affect the migration ability of MAPK; signaling pathway mediated by NE to promote the role of EPCs and beta-arrestin 2 in regulating EPCs function.
research method
In this study, the mechanism of EPCs mobilization by NE is studied in depth from the level of body experiment, the level of cell experiment and the mechanism of molecular regulation and control.
(1) in vivo: mice model was induced by occlusion of the femoral artery by the left lower limb ischemia, the day after L to 5 days given intraperitoneal injection of drugs, drug specific as follows: NE 10 "8mol, alpha blocker phentolamine 10" 8mol, beta1 10-8mol and beta2 receptor blocker metoprolol blocker 10-8mol. on the seventh day after transplantation of bone marrow mononuclear cells, spleen cells and mouse peripheral blood mononuclear cells, the ratio of flow cytometry CD34+KDR+ cells.
(2) cell function research: in vitro culture of human peripheral blood EPCs medium, adding different concentrations of NE (0,0.01,0.1,1,10100 M) for 72 hours, MTT to evaluate EPCs proliferation ability, Transwell chamber experiment to evaluate EPCs migration ability.
(3) cellular signal research: Western-blot detection and detection of Mitogen Activated Protein Kinase (MAPK), namely extracellular signal regulated kinase Erk1/2, c-Jun amino terminal kinase Jnk and p38 MAPK phosphorylation level.
(4) to study the protein regulation mechanism of cell signaling pathways: randomly selected peripheral blood 30ml, 4 control subjects and 4 diabetic patients with isolated PBMCs, detect the expression of Western-blot beta-arrestin 2. The expression of gene silencing method of interference of EPCs beta-arrestin 2, Realtime PCR and Western-blot efficiency evaluation method of silence, by what MTT Transwell assay method of beta-arrestin2 in the regulation of proliferation and migration of EPCs in vitro.
Result
(1) in vivo: NE can significantly promote the limb ischemia C57 mouse bone marrow EPCs proliferation and mobilization of peripheral blood injection ability. The number of C57 in bone marrow of mice NE group EPCs increased from 2 + 0.4% to 4.7 + 0.9%, P0.05, the number of mobilization of peripheral blood EPCs increased from 1.2 + 0.6% to 6.2 + 1.9%, P0.05, the number of spleen EPCs increased from 2.54 + 0.8% to 3.1 + 1% p0.05., and the effect of NE can be Q adrenoceptor antagonist phentolamine and beta2 adrenergic receptor blockers I127, but not by beta1 adrenergic receptor blocking agent blocking.
(2) cell function experiments showed that NE concentration dependently promote the proliferation of EPCs, which had the strongest effect of NE 10 M, the proliferation rate was 103.6 + 54.6%, P0.05, NE in promoting the proliferation of EPCs can be blocked by phentolamine and I127, but not by beauty care network Seoul. In addition ERK 1/2 blocking inhibitor A6355 and eNOS inhibitor L-NAME could block can block this effect, the NE P0.05, but JNK inhibitor SP600125, p38 inhibitor PD169318, PI3K inhibitor LY294002 1 M, p65, R0106-9920 and NO inhibitor SNP had no similar donor for P0.05.NE can significantly promote the migration of EPCs (124.1 + 12.2 VS 71.7 + 19.6, P0.05), phentolamine and I127 could inhibit the pro migratory capacity of NE (57.2 + 14.3 VS 124.1 + 12.2, P0.05 + 11.5 and 61.3 + 12.2 VS 124.1, P0.05), but no similar effects of metoprolol (112.8 + 26 VS 124.1 + 12.2, P0 .05).
(3) the study of cell signaling pathway showed that NE can increase the phosphorylation of EPCs Erk 1/2 and signal molecule Src, P0.05, while I127 can reduce the phosphorylation of Erk 1/2 and Src, P0.05, phentolamine and mesolol have no similar effect.
(4) study on the regulatory mechanism of beta-arrestin gene silencing signal found: 2, the sensitivity of EPCs to VEGF significantly increased (33.7 + 2.1 + 1.4% 6.4%VS, P0.05), and the sensitivity of EPCs to NE significantly decreased (26.6 + 7.8%VS64.0 + 13.5%, P0.05). Beta-arrestin gene silencing after 2, decreased the migration capacity of EPCs, decreased sensitivity to NE.
conclusion
NE promotes the mobilization and proliferation of bone marrow EPCs during limb ischemia..NE can activate EPCs Src-MAPK signaling pathway, while beta-arrestin 2 is involved in the regulation of EPCs proliferation and migration.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R341

【共引文獻(xiàn)】

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3 畢凌云;郭金崗;張瑞霞;趙靜麗;梁斌;趙德安;楊達(dá)勝;;干細(xì)胞因子和粒細(xì)胞集落刺激因子動員自身骨髓干細(xì)胞治療缺血再灌注腎損傷[J];中國組織工程研究;2012年41期

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1 鄭莉萍;大鼠外周血內(nèi)皮祖細(xì)胞培養(yǎng)及鑒定的實(shí)驗(yàn)研究[D];鄭州大學(xué);2010年

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本文編號：1611587