去甲肾上腺素促进内皮祖细胞动员的机制研究
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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%,p<0.05,动员进外周血的EPCs的数量从1.2±0.6%增加到6.2±1.9%,p<0.05,脾脏的EPCs的数量从2.54±0.8%增加到3.1±1.0%,p<0.05。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%,P<0.05,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,P<0.05,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,P>0.05。NE能够显著地促进EPCs的迁移能力(124.1±12.2 VS 71.7±19.6,P<0.05),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,P<0.05和61.3±11.5 VS 124.1±12.2,P<0.05),而美托洛尔无类似作用(112.8±26.0 VS 124.1±12.2,P>0.05)。
(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,P<0.05,而I127能够减轻Erk 1/2和Src的磷酸化,P<0.05,酚妥拉明和美托络尔则没有类似作用,P>0.05。
(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%,P<0.05),而EPCs对NE的敏感性显著地减少(26.6±7.8%VS64.0±13.5%,P<0.05)。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。
Purpose
There are several stages for the endothelial progenitor cells (EPCs) mobilization, such as adhesion, degradation, movement and migration. The number and function of EPCs would change with physiological or pathological conditions in vive. Norepinephrine (NE) is the most important transmitter of human sympathetic nerve system. Up to today, it is not clear whether the EPCs mobilization is regulated by NE. The purpose of this study was to investigate the influence of NE on EPCs mobilization in C57 mouse with limb ischemia, human EPCs proliferation and migration which is cultivated ex vive and the involvement of mitogen-activated protein kinase (MAPK) signal molecular and beta-arrestin 2 in the regulation of EPCs function by NE.
Methods
The regulation of EPCs mobilization by norepinephrine was investigated at molecular level, cellular level and general level.
First, at the general levels limb ischemia mouse were prepared with left femoral artery ligation at the proximal and the distal end. Drugs such as NE 10-8mol, alpha adrenoceptor receptor blocker Phentolamine 10-8mol, beta ladrenoceptor blocker Metoprolol 10-8mol and beta 2 adrenoceptor 1127 10-8mol were intraperitoneal injected. The proportions of EPCs (double positive of CD34 and KDR) in bone marrow, spleen and peripheral circulation were analyzed with flow cytometry at the 7th day after the left femoral artery ligation.
Second at the cellular levels, after 8 days of cultivation of human EPCs, different concentrations of NE (0、0.01、0.1、1、10、100μM) were added into the media, and the proliferation and migration potential were analyzed with MTT and Transwell chamber respectively.
Third, at the molecular levels, the phosphorylation of Mitogen-Activated Protein Kinase (MAPK) signal molecular such as Erk 1/2, JNK and p38 were assay with Western blot.
At the last, PBMCs isolated from 30ml peripheral blood of 4 DM-2 patients and 4 healthy subjects were assayed the expression of beta-arrestin2 with Western blot. The ablations of beta-arrestin 2 were confirmed with Realtime PCR and Western blot. The proliferation and migration of EPCs were assayed after the ablation.
Results
The in vive stydies foud that NE increased mobilization of EPCs from bone marrow to peripheral circulation significantly after limb ischemia in C57 mouse. The proportion of EPCs in bone marrow increased from 2.0±0.4% to 4.7±0.9%, p< 0.05, in the peripheral circulation increased from 1.2±0.6% to 6.2±1.9%, p< 0.05, in spleen increased from 2.5±0.8% to 3.1±1.0%, p< 0.05. The effects of NE could be blocked by Phentolamine and I 127, but could not be blocked by Metoprolol.
The cellular studies found that NE dose dependently increased the proliferation potential of EPCs. With the most effective concentration of NE 10μM, EPCs proliferation increased 103.6±54.6%, P< 0.05. This effect could be blocked by Phentolamine,1127, A6355 and L-NAME, but not by Metoprolol, SP600125,, PD169318, LY294002,, RO106-9920 and SNP, P> 0.05.NE significantly increased the migration potential of EPCs (124.1±12.2 VS 71.7±19.6, P< 0.05). This effect could be blocked by Phentolamine and 1127 (57.2±14.3 vs 124.1±12.2, P< 0.05 and 61.3±11.5 vs 124.1±12.2, P<0.05), but not by Metoprolol (112.8±26.0 VS 124.1±12.2,P>0.05).
The cell signal studies found that NE significantly increased the phosphorylation of Erk 1/2 and signal molecular Src. This effect could be blocked by 1127, P< 0.05, but not by Phentolamine and Metoprolol, P> 0.05.
The cell signal regulation studied found that after the ablation of beta-arrestin 2, EPCs were more sensitive to the addition of VEGF in the media (33.7±6.4% vs 2.1±1.4%, P< 0.05), but less sensitive to the addition of NE (26.6±7.8% vs 64.0±13.5%, P< 0.05). The ablation of beta-arrestin 2 decreased the migration potential and sensitive of EPCs to NE.
Conclusion
NE increases the mobilization of EPCs in mouse with limb ischemia and proliferation, migration potential of EPCs ex vivo. NE increases the phosphorylation of Erk 1/2 and signal molecular Src. Beta-arrestin 2 participates in the regulation of EPCs proliferation and migration.
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%,p<0.05,动员进外周血的EPCs的数量从1.2±0.6%增加到6.2±1.9%,p<0.05,脾脏的EPCs的数量从2.54±0.8%增加到3.1±1.0%,p<0.05。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%,P<0.05,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,P<0.05,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,P>0.05。NE能够显著地促进EPCs的迁移能力(124.1±12.2 VS 71.7±19.6,P<0.05),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,P<0.05和61.3±11.5 VS 124.1±12.2,P<0.05),而美托洛尔无类似作用(112.8±26.0 VS 124.1±12.2,P>0.05)。
(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,P<0.05,而I127能够减轻Erk 1/2和Src的磷酸化,P<0.05,酚妥拉明和美托络尔则没有类似作用,P>0.05。
(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%,P<0.05),而EPCs对NE的敏感性显著地减少(26.6±7.8%VS64.0±13.5%,P<0.05)。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。
Purpose
There are several stages for the endothelial progenitor cells (EPCs) mobilization, such as adhesion, degradation, movement and migration. The number and function of EPCs would change with physiological or pathological conditions in vive. Norepinephrine (NE) is the most important transmitter of human sympathetic nerve system. Up to today, it is not clear whether the EPCs mobilization is regulated by NE. The purpose of this study was to investigate the influence of NE on EPCs mobilization in C57 mouse with limb ischemia, human EPCs proliferation and migration which is cultivated ex vive and the involvement of mitogen-activated protein kinase (MAPK) signal molecular and beta-arrestin 2 in the regulation of EPCs function by NE.
Methods
The regulation of EPCs mobilization by norepinephrine was investigated at molecular level, cellular level and general level.
First, at the general levels limb ischemia mouse were prepared with left femoral artery ligation at the proximal and the distal end. Drugs such as NE 10-8mol, alpha adrenoceptor receptor blocker Phentolamine 10-8mol, beta ladrenoceptor blocker Metoprolol 10-8mol and beta 2 adrenoceptor 1127 10-8mol were intraperitoneal injected. The proportions of EPCs (double positive of CD34 and KDR) in bone marrow, spleen and peripheral circulation were analyzed with flow cytometry at the 7th day after the left femoral artery ligation.
Second at the cellular levels, after 8 days of cultivation of human EPCs, different concentrations of NE (0、0.01、0.1、1、10、100μM) were added into the media, and the proliferation and migration potential were analyzed with MTT and Transwell chamber respectively.
Third, at the molecular levels, the phosphorylation of Mitogen-Activated Protein Kinase (MAPK) signal molecular such as Erk 1/2, JNK and p38 were assay with Western blot.
At the last, PBMCs isolated from 30ml peripheral blood of 4 DM-2 patients and 4 healthy subjects were assayed the expression of beta-arrestin2 with Western blot. The ablations of beta-arrestin 2 were confirmed with Realtime PCR and Western blot. The proliferation and migration of EPCs were assayed after the ablation.
Results
The in vive stydies foud that NE increased mobilization of EPCs from bone marrow to peripheral circulation significantly after limb ischemia in C57 mouse. The proportion of EPCs in bone marrow increased from 2.0±0.4% to 4.7±0.9%, p< 0.05, in the peripheral circulation increased from 1.2±0.6% to 6.2±1.9%, p< 0.05, in spleen increased from 2.5±0.8% to 3.1±1.0%, p< 0.05. The effects of NE could be blocked by Phentolamine and I 127, but could not be blocked by Metoprolol.
The cellular studies found that NE dose dependently increased the proliferation potential of EPCs. With the most effective concentration of NE 10μM, EPCs proliferation increased 103.6±54.6%, P< 0.05. This effect could be blocked by Phentolamine,1127, A6355 and L-NAME, but not by Metoprolol, SP600125,, PD169318, LY294002,, RO106-9920 and SNP, P> 0.05.NE significantly increased the migration potential of EPCs (124.1±12.2 VS 71.7±19.6, P< 0.05). This effect could be blocked by Phentolamine and 1127 (57.2±14.3 vs 124.1±12.2, P< 0.05 and 61.3±11.5 vs 124.1±12.2, P<0.05), but not by Metoprolol (112.8±26.0 VS 124.1±12.2,P>0.05).
The cell signal studies found that NE significantly increased the phosphorylation of Erk 1/2 and signal molecular Src. This effect could be blocked by 1127, P< 0.05, but not by Phentolamine and Metoprolol, P> 0.05.
The cell signal regulation studied found that after the ablation of beta-arrestin 2, EPCs were more sensitive to the addition of VEGF in the media (33.7±6.4% vs 2.1±1.4%, P< 0.05), but less sensitive to the addition of NE (26.6±7.8% vs 64.0±13.5%, P< 0.05). The ablation of beta-arrestin 2 decreased the migration potential and sensitive of EPCs to NE.
Conclusion
NE increases the mobilization of EPCs in mouse with limb ischemia and proliferation, migration potential of EPCs ex vivo. NE increases the phosphorylation of Erk 1/2 and signal molecular Src. Beta-arrestin 2 participates in the regulation of EPCs proliferation and migration.
引文
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50. Rakhit S, Pyne S, Pyne NJ. Nerve growth factor stimulation of p42/p44 mitogen-activated protein kinase in PC 12 cells:role of G(i/o), G protein-coupled receptor kinase 2, beta-arrestin Ⅰ, and endocytic processing. Mol Pharmacol.2001;60(1):63-70.
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