白细胞介素-12基因缺陷促进内皮祖细胞浸润改善缺血再灌注后心肌损伤的研究
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摘要
目的:探讨白细胞介素-12(IL-12)调控小鼠心肌缺血再灌注损伤(IRI)的作用机制。方法:复制小鼠心肌IRI模型,分成四组:野生型(WT)假手术组;白细胞介素-12p35亚基(IL-12p35)基因缺陷假手术组;WT手术组;IL-12p35基因缺陷手术组。超声检测心脏结构及功能;采用Masson三色特殊染色法检测组织内胶原沉积;TUNEL检测心肌细胞凋亡;免疫组化染色及qRT-PCR检测内皮祖细胞(EPC)招募相关的趋化因子粒细胞-巨噬细胞集落刺激因子(GM-CSF)表达以及EPC细胞表面趋化因子受体(CXCR4)和血管内皮细胞生长因子受体(VEGFR)表达;流式细胞术检测IRI后梗死部位EPC数量。结果:缺血再灌注术后14d,与对照组相比,IL-12p35基因缺陷小鼠的心脏射血分数明显升高[WT vs.IL-12p35基因缺陷:(48.7±5.9)vs.(55.2±6.7)%,P<0.05],心功能显著改善;心肌纤维化程度明显减轻(P<0.05);术后1d,IL-12p35缺陷小鼠心肌细胞凋亡数量较对照组,差异无统计学意义;术后7d,IL-12p35基因缺陷小鼠心脏中趋化因子GM-CSF、EPC细胞表面受体CXCR4和VEGFR表达较对照组显著增加(P<0.05),干扰素-γ(IFN-γ)表达较对照组减少(P<0.05);且与对照组相比,IRI后梗死部位EPC数量明显升高(P<0.05)。结论:IL-12基因缺陷表现出明显的抗心肌IRI的作用,其机制是GM-CSF表达增加促进内皮祖细胞(EPC)浸润,从而促进了血管生成和抑制心力衰竭。
Objective: To investigate the mechanism of interleukin-12( IL-12) in regulating myocardial ischemia-reperfusion injury( IRI) in mice. Methods: The mice model of ischemia-reperfusion was duplicated and divided into 4 groups: WT sham operation group,IL-12p35 KO sham operation group,WT operation group and IL-12p35 KO operation group. Ultrasound was used to detect the structure and function of the heart.Masson trichrome staining was used to detect the collagen deposition in the tissue. TUNEL was used to detect the apoptosis of cardiomyocytes. The expression of chemotactic factor( GM-CSF) related to EPC recruitment was detected by immunohistochemistry and qRT-PCR. The expression of CXCR4 and VEGFR on EPCs cells was detected by flow cytometry. Results: At 14 days after ischemia/reperfusion,the ejection fraction of IL-12p35 KO mice were significantly increased( P < 0. 05),the cardiac function was significantly improved,and the degree of myocardial fibrosis was significantly reduced compared with the control group( P < 0. 05). At 1 day after operation,the number of apoptotic cardiomyocytes in IL-12p35 KO mice was not significantly different from that in the control group. At 7 days after operation,the chemokines GM-CSF,Compared with the control group,the expression of CXCR4 and VEGFR on the cell surface was significantly increased( P < 0. 05) and the expression of IFN-γ was decreased( P < 0. 05). Compared with the control group,the number of EPCs at infarction was significantly increased( P < 0. 05). Conclusion: IL-12 deficiency shows obvious anti-myocardial ischemia-reperfusion injury. The mechanism is that the increased expression of GM-CSF can promote the infiltration of endothelial progenitor cells( EPCs),thus promoting angiogenesis and inhibiting heart failure.
Objective: To investigate the mechanism of interleukin-12( IL-12) in regulating myocardial ischemia-reperfusion injury( IRI) in mice. Methods: The mice model of ischemia-reperfusion was duplicated and divided into 4 groups: WT sham operation group,IL-12p35 KO sham operation group,WT operation group and IL-12p35 KO operation group. Ultrasound was used to detect the structure and function of the heart.Masson trichrome staining was used to detect the collagen deposition in the tissue. TUNEL was used to detect the apoptosis of cardiomyocytes. The expression of chemotactic factor( GM-CSF) related to EPC recruitment was detected by immunohistochemistry and qRT-PCR. The expression of CXCR4 and VEGFR on EPCs cells was detected by flow cytometry. Results: At 14 days after ischemia/reperfusion,the ejection fraction of IL-12p35 KO mice were significantly increased( P < 0. 05),the cardiac function was significantly improved,and the degree of myocardial fibrosis was significantly reduced compared with the control group( P < 0. 05). At 1 day after operation,the number of apoptotic cardiomyocytes in IL-12p35 KO mice was not significantly different from that in the control group. At 7 days after operation,the chemokines GM-CSF,Compared with the control group,the expression of CXCR4 and VEGFR on the cell surface was significantly increased( P < 0. 05) and the expression of IFN-γ was decreased( P < 0. 05). Compared with the control group,the number of EPCs at infarction was significantly increased( P < 0. 05). Conclusion: IL-12 deficiency shows obvious anti-myocardial ischemia-reperfusion injury. The mechanism is that the increased expression of GM-CSF can promote the infiltration of endothelial progenitor cells( EPCs),thus promoting angiogenesis and inhibiting heart failure.
引文
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[15]张晶,李涛涛,马友才,等.心肌梗死小鼠趋化因子MIP1α和MIP1β对巨噬细胞招募作用的研究.心肺血管病杂志,2015,34:580-584.
[16]Codarri L,Gyülvészi G,Tosevski V,et al.RORγt drives production of the cytokine GM-CSF in helper T cells,which is essential for the effector phase of autoimmune neuroinflammation.Nat Immunol,2011,12(6):560.
[2]Heusch G,Gersh BJ.The pathophysiology of acute myocardial infarction and strategies of protection beyond reperfusion:a continual challenge.Eur Heart J,2017,38(11):774-784.
[3]Sebastian Z,Neurath MF.Interleukin-12:Functional activities and implications for disease.Cytokine Growth Factor Rev,2015,26(5):559-568.
[4]Li Y,Zhang C,Wu Y,et al.Interleukin-12p35 deletion promotes CD4 T-cell-dependent macrophage differentiation and enhances angiotensin II-Induced cardiac fibrosis.Arterioscler Thromb Vasc Biol,2012,32(7):1662-1674.
[5]Kan X,Wu Y,Ma Y,et al.Deficiency of IL-12p35 improves cardiac repair after myocardial infarction by promoting angiogenesis.Cardiovasc Res,2016,109(2):249.
[6]崔巍,李玉琳,王吉静,等.细胞组学技术在心肌细胞凋亡研究中的应用.心肺血管病杂志,2013,32:768-772.
[7]Thierfelder WE,van Deursen JM,Yamamoto K,et al.Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells.Nature,1996,382(6587):171-174.
[8]Ali M,Mali V,Haddox S,et al.Essential role of IL-12 in angiogenesis in type 2 diabetes.Am J Pathol,2017,187(11):2590-2601.
[9]Zykov MV,Barbarash OL,Kashtalap VV,et al.Interleukin-12serum level has prognostic value in patients with ST-segment elevation myocardial infarction.Heart Lung,2016,45(4):336-340.
[10]Hansson GK,Libby P.The immune response in atherosclerosis:a double-edged sword.Nature Reviews Immunology,2006,6(7):508.
[11]Elizabeth J,Harvey,Dipak P.Ramji.Interferon-γand atherosclerosis:Pro-or anti-atherogenic?.Cardiovasc Res,2005,67(1):11-20.
[12]Dimmeler S,Burchfield J,Zeiher AM.Cell-based therapy of myocardial infarction.Arterioscler Thromb Vasc Biol,2008,28(2):208.
[13]Shantsila E,Watson T,Lip GY.Endothelial progenitor cells in cardiovascular disorders.J Am Coll Cardiol,2007,49(7):741.
[14]Takahashi T,Kalka C,Masuda H,et al.Ischemia-and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med,1999,5(4):434-438.
[15]张晶,李涛涛,马友才,等.心肌梗死小鼠趋化因子MIP1α和MIP1β对巨噬细胞招募作用的研究.心肺血管病杂志,2015,34:580-584.
[16]Codarri L,Gyülvészi G,Tosevski V,et al.RORγt drives production of the cytokine GM-CSF in helper T cells,which is essential for the effector phase of autoimmune neuroinflammation.Nat Immunol,2011,12(6):560.