去甲肾上腺素促进内皮祖细胞动员的机制研究

发布时间：2018-03-14 14:21

  本文选题：肢体缺血　切入点：内皮祖细胞　出处：《第二军医大学》2010年博士论文　论文类型：学位论文

【摘要】： 研究目的 EPCs的动员机制存在多个环节,如黏附、降解、运动、迁移等。在机体某些生理或病理状态下,外周血中EPCs的数量和功能也会发生变化。去甲肾上腺素是人体交感神经的主要的神经递质之一。其对于EPCs动员的影响仍不是十分清楚。本研究的目的是：探讨NE对肢体缺血C57小鼠EPCs动员的影响,以及对体外培养的人EPCs的增殖、迁移能力的影响；探讨MAPK信号通路介导NE的促进EPCs的作用以及beta-arrestin 2在EPCs功能调节中的作用。 研究方法 本课题从在体实验水平、细胞实验水平和分子调控机制等多个层面逐层深入研究NE调节EPCs动员的机制。 (1)在体实验：采用结扎股动脉的方法制备左下肢缺血的小鼠模型,于术后的第l至5天每天给予药物腹腔注射,具体药物以下：NE 10"8mol、α阻滞剂酚妥拉明10"8mol、beta1受体阻滞剂美托洛尔10-8mol和beta2受体阻滞剂10-8mol。于术后第7天取骨髓单个核细胞、脾脏细胞和小鼠外周血单个核细胞,流式检测CD34+KDR+的细胞所占的比率。 (2)细胞功能学研究：在体外培养人外周血EPCs的培养基中加入不同浓度的NE(0、0.01、0.1、1、10、100μM)干预72小时, MTT评价EPCs的增殖能力,Transwell小室实验评价EPCs的迁移能力。 (3)细胞信号研究：Western-blot检测检测丝裂原激活蛋白激酶(Mitogen Activated Protein Kinase, MAPK),即细胞外信号调节激酶Erk1/2、c-Jun氨基端激酶Jnk和p38 MAPK的磷酸化水平。 (4)细胞信号通路的蛋白调控机制研究：随机选取4名对照者和4名糖尿病患者的外周血30ml,分离PBMCs, Western-blot检测beta-arrestin 2的表达。基因沉默的方法干扰EPCs beta-arrestin 2的表达,Realtime PCR和Western-blot的方法评价沉默的效率,采用MTT何Transwell小室实验的方法研究beta-arrestin2在EPCs增殖能力和迁移能力的调控中的作用。 结果 (1)在体实验发现：NE能够显著地促进肢体缺血C57小鼠骨髓EPCs的增殖以及其动员进外周血的能力。注射了NE组的C57小鼠骨髓的EPCs的数量从2.0±0.4%增加到4.7±0.9%,p0.05,动员进外周血的EPCs的数量从1.2±0.6%增加到6.2±1.9%,p0.05,脾脏的EPCs的数量从2.54±0.8%增加到3.1±1.0%,p0.05。而NE的这种作用能够被Q肾上腺素受体阻断剂酚妥拉明和beta2肾上腺受体阻断剂I127阻断,但是不能被beta1肾上腺素受体阻断剂阻断。 (2)细胞功能学实验发现：NE能够浓度依赖性地促进体外培养的EPCs的增殖,其中以NE 10μM的作用最强,其增殖率为103.6±54.6%,P0.05,NE的促进EPCs增殖作用能够被酚妥拉明和I127阻断,但是不能被美托络尔阻断。另外ERK 1/2抑制剂A6355和eNOS抑制剂L-NAME能够阻断也能够阻断NE的这种作用,P0.05,但是JNK抑制剂SP600125、p38抑制剂PD169318、PI3K抑制剂LY294002 1μM、、p65抑制剂R0106-9920和NO供体SNP均无类似作用,P0.05。NE能够显著地促进EPCs的迁移能力(124.1±12.2 VS 71.7±19.6,P0.05),酚妥拉明和I127能够阻断NE的促迁移能力(57.2±14.3 VS 124.1±12.2,P0.05和61.3±11.5 VS 124.1±12.2,P0.05),而美托洛尔无类似作用(112.8±26.0 VS 124.1±12.2,P0.05)。 (3)细胞信号通路的研究发现：NE能够浓度依赖性地增加EPCs的Erk 1/2和信号分子Src的的磷酸化,P0.05,而I127能够减轻Erk 1/2和Src的磷酸化,P0.05,酚妥拉明和美托络尔则没有类似作用,P0.05。 (4)信号调控机制研究发现：基因沉默beta-arrestin 2后,EPCs对VEGF的敏感性显著地增加(33.7±6.4%VS 2.1±1.4%,P0.05),而EPCs对NE的敏感性显著地减少(26.6±7.8%VS64.0±13.5%,P0.05)。基因沉默beta-arrestin 2后,EPCs的迁移能力下降,对NE的敏感性下降。 结论 NE促进肢体缺血时骨髓EPCs的动员以及增殖、迁移能力。NE能够激活EPCs Src-MAPK信号通路,而beta-arrestin 2参与EPCs的增殖和迁移能力的调节。
[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.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R341

【共引文献】

相关期刊论文 前3条

1 毕凌云;杨达胜;梁斌;张瑞霞;白海涛;;动员自体骨髓干细胞对大鼠急性肾小管坏死的治疗[J];中国中西医结合肾病杂志;2012年08期

2 梁斌;杨达胜;;动员自体骨髓干细胞对大鼠急性肾小管坏死的治疗作用[J];中国社区医师(医学专业);2010年20期

3 毕凌云;郭金岗;张瑞霞;赵静丽;梁斌;赵德安;杨达胜;;干细胞因子和粒细胞集落刺激因子动员自身骨髓干细胞治疗缺血再灌注肾损伤[J];中国组织工程研究;2012年41期

相关硕士学位论文 前1条

1 郑莉萍;大鼠外周血内皮祖细胞培养及鉴定的实验研究[D];郑州大学;2010年

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