LPS调节TLR4介导促进心肌细胞存活时间的实验研究
摘要
缺血性心脏病是严重威胁人类生命的疾病之一,其原因是心血管疾病发生、发展过程中大量受损的心肌细胞发生“顿抑”(stunned)、冬眠(hibernation)、坏死以及凋亡,而成熟心肌细胞缺乏分化增殖能力,不能完全代偿受损心肌细胞功能,导致心脏的收缩舒张功能障碍。骨髓间充质干细胞(mesenchymal stem cells, MSCs)具有自我更新、分化增殖和多向分化潜能的特点,还可增加局部的血管、改善血运,保证移植细胞的存活;骨髓间充质干细胞体外培养容易,无免疫排斥、伦理道德等问题,使其成为细胞移植的理想材料。
骨髓干细胞移植到缺血心肌组织中,可以分化为心肌样细胞,并改善心肌梗死和慢性充血性心力衰竭的心功能,促进血管生成和抑制左心室重构。但心肌样细胞在短时间内基本消失凋亡,不能进一步分化为新生的心肌细胞,其机制可能为抑制过程中TLR4-MyD88-NFkB的信号通路被激活,产生大量炎症因子并造成细胞损伤。而利用小剂量TLR4特异性配体Lipopolysaccharide分别预处理接受移植前的个体,刺激兴奋骨髓干细胞和原有心肌细胞TLR4/PI3K/Akt信号途径促进细胞生存以及抑制TLR4-MyD88-NFkB信号途径中NFkB的表达,来减少移植后心肌样细胞在梗死心肌中的凋亡,并促使其进一步向心肌细胞分化。
本论文的主要目的是通过分子生物学手段,利用动物模型来研究抑制心肌梗死区域的炎症反应,减少移植的骨髓干细胞的凋亡,从而有效地提高骨髓干细胞移植治疗缺血性心脏病,并同时为干细胞协同冠脉搭桥手术治疗提供有益的理论与实验依据。
目的:利用小剂量LPS预处理待移植的大鼠,观察移植入大鼠的骨髓间充质干细胞(BMSCs)的在心肌梗塞后心肌微环境内的数量变化、分布情况以及存活时间。
方法:将成年大鼠随即分为对照组、心梗组、LPS(0.1mg/kg)预处理组、LPS(1mg/kg)预处理组、LPS(5mg/kg)预处理组,将经CM-Dil标记后的骨髓来源间充质干细胞分别注射入冠状动脉左前降支中段结扎处周围的5组大鼠左心室壁内,于术后第48h、1、3、4周观察红色荧光标记的MSCs在梗死心肌内存活情况以及数量变化,同时分析移植后的骨髓干细胞的凋亡、存活情况以及相关细胞内信号通路的蛋白表达。
结果:相对小剂量的LPS预处理后,电镜下仅可见少部分线粒体嵴模糊、断裂、减少,凋亡细胞为22.8 %,远远小于心梗组和其他浓度LPS预处理组。心肌梗死后TLR4、NFkB和MyD88 mRNA以及蛋白表达相对于对照组明显上调,小剂量LPS预处理后表达水平均出现下调,1周达到峰值。1周后心梗组和0.1mg/kgLPS预处理组表达红色荧光的细胞明显减少,5mg/kgLPS预处理组荧光细胞的数量和荧光度减少更为明显,而1mg/kgLPS预处理组仍有大量发明亮红色荧光的干细胞,存活的阳性细胞数明显多于心梗组,部分移植心肌干细胞迁移到损伤区的心肌组织,损伤中心已聚有较多的心肌干细胞;到3～4周时,心梗组已很少见到红色荧光,而1mg/kgLPS预处理仍可见散在的荧光,部分已与心肌细胞“融合”。
结论:骨髓间充质干细胞移植后在心肌梗死组织处的存活率极低,其中重要原因就在于心肌梗死后,心肌细胞的缺血、缺氧和炎症等原因造成心肌细胞坏死后微环境改变,并促进了移植干细胞的凋亡。研究发现:小剂量LPS预处理接受移植的个体,可通过减轻局部炎症反应,来减少移植骨髓干细胞在梗死心肌组织中的凋亡,延长其存活时间,改善心功能。?
Ischemic cardiomyopathy is a serious disease that imposes a threat to human. The primary cause of ischemic cardiomyopathy is that the damaged myocardial cells undergo necrosis and apoptosis in the development and progression of cardiovascular disease, while the mature myocardial cells do not have the ability to differentiate or proliferate, and can not fully compensate the affected function of heart muscle cells, leading to cardiac systolic and diastolic dysfunction. Mesenchymal stem cells (MSCs) have the characteristics of self-renewal, differentiation, proliferation and potential of multilineage differentiation. In addition, MSCs are easy to be cultured in vitro, have no immune rejection and ethical issues, thus are ideal for cell transplantation to treat ischemic cardiomyopathy.
When transplanted into ischemic myocardium, bone marrow stem cells could differentiate into cardiomyocyte-like cells to promote angiogenesis and inhibit left ventricular remodeling, thus improving heart function in acute myocardial infarction and chronic congestive heart failure disease. However, these differentiated cardiomyocyte-like cells disappear in a short period and could not be further differentiated into neonatal cardiomyocyte. It has been shown that the activation of TLR4-MyD88-NFkB signaling pathways induces the release of a large number of inflammatory cytokines and thus causes cell damage. The pretreatment of transplant recipients with TLR4-specific ligand lipopolysaccharide(LPS) could stimulate bone marrow stem cells proliferation and activate TLR4/PI3K/Akt signaling pathway in original myocardial, promoting cell survival and inhibiting NFkB expression in TLR4-MyD88-NFkB signaling pathway. Consequently, the apoptosis of cardiomyocyte-like cells after transplantation is reduced and these cells are further induced to differentiate into cardiomyocytes.
The main purpose of this study is to develop novel approach to improve bone marrow stem cell transplantation in the treatment of ischemic heart disease, and a provide useful guidance for coronary artery bypass surgery in combination with stem cells. We employed animal models to investigate whether inhibiting inflammatory response of myocardial infarction region could reduce the apoptosis of transplanted bone marrow stem cell.
Objective: To characterize the change of number, distribution, and survival time in micro-environment after myocardial infarction of implanted rat bone marrow mesenchymal stem cells (BMSCs).
Methods: Adult rats were randomly divided into control group, the myocardial infarction group, LPS (0.1mg/kg) pretreatment group, LPS (1mg/kg) pretreatment group, LPS (5mg/kg) pretreatment group. CM-Dil labeled bone marrow derived mesenchymal stem cells were injected into the left anterior descending artery ligation at left ventricular wall in those rats. 48h, 1, 3, 4 weeks later, the number and survival time of red fluorescent labeled MSCs were recorded in the infarcted myocardium. Simultaneously, the survival, apoptosis and the activation of intracellular signaling pathway were analyzed in the transplanted MSCs.
Results: 22.8%MSCs underwent apoptosis in 0.1mg/kg LPS pretreatment group, much less than the control myocardial infarction group and other dose of LPS pretreatment group. After myocardial infarction TLR4, NFkB, and MyD88 mRNA and protein expression was significantly increased in LPS pretreatment group compared with the control group. A week later red fluorescent cells were decreased significantly in the myocardial infarction group and 0.1mg/kg LPS pretreatment group.
Conclusion: The transplanted MSCs in myocardial infarction have a very low survival rate, perhaps due to the apoptosis induced by the changes in the microenvironment .Low-dose LPS pretreatment could reduce the local inflammatory reaction and the apoptosis of the transplanted bone marrow stem cells in the infarcted myocardium, thu prolonging the survival time of MSCs and improving the heart function.
骨髓干细胞移植到缺血心肌组织中,可以分化为心肌样细胞,并改善心肌梗死和慢性充血性心力衰竭的心功能,促进血管生成和抑制左心室重构。但心肌样细胞在短时间内基本消失凋亡,不能进一步分化为新生的心肌细胞,其机制可能为抑制过程中TLR4-MyD88-NFkB的信号通路被激活,产生大量炎症因子并造成细胞损伤。而利用小剂量TLR4特异性配体Lipopolysaccharide分别预处理接受移植前的个体,刺激兴奋骨髓干细胞和原有心肌细胞TLR4/PI3K/Akt信号途径促进细胞生存以及抑制TLR4-MyD88-NFkB信号途径中NFkB的表达,来减少移植后心肌样细胞在梗死心肌中的凋亡,并促使其进一步向心肌细胞分化。
本论文的主要目的是通过分子生物学手段,利用动物模型来研究抑制心肌梗死区域的炎症反应,减少移植的骨髓干细胞的凋亡,从而有效地提高骨髓干细胞移植治疗缺血性心脏病,并同时为干细胞协同冠脉搭桥手术治疗提供有益的理论与实验依据。
目的:利用小剂量LPS预处理待移植的大鼠,观察移植入大鼠的骨髓间充质干细胞(BMSCs)的在心肌梗塞后心肌微环境内的数量变化、分布情况以及存活时间。
方法:将成年大鼠随即分为对照组、心梗组、LPS(0.1mg/kg)预处理组、LPS(1mg/kg)预处理组、LPS(5mg/kg)预处理组,将经CM-Dil标记后的骨髓来源间充质干细胞分别注射入冠状动脉左前降支中段结扎处周围的5组大鼠左心室壁内,于术后第48h、1、3、4周观察红色荧光标记的MSCs在梗死心肌内存活情况以及数量变化,同时分析移植后的骨髓干细胞的凋亡、存活情况以及相关细胞内信号通路的蛋白表达。
结果:相对小剂量的LPS预处理后,电镜下仅可见少部分线粒体嵴模糊、断裂、减少,凋亡细胞为22.8 %,远远小于心梗组和其他浓度LPS预处理组。心肌梗死后TLR4、NFkB和MyD88 mRNA以及蛋白表达相对于对照组明显上调,小剂量LPS预处理后表达水平均出现下调,1周达到峰值。1周后心梗组和0.1mg/kgLPS预处理组表达红色荧光的细胞明显减少,5mg/kgLPS预处理组荧光细胞的数量和荧光度减少更为明显,而1mg/kgLPS预处理组仍有大量发明亮红色荧光的干细胞,存活的阳性细胞数明显多于心梗组,部分移植心肌干细胞迁移到损伤区的心肌组织,损伤中心已聚有较多的心肌干细胞;到3～4周时,心梗组已很少见到红色荧光,而1mg/kgLPS预处理仍可见散在的荧光,部分已与心肌细胞“融合”。
结论:骨髓间充质干细胞移植后在心肌梗死组织处的存活率极低,其中重要原因就在于心肌梗死后,心肌细胞的缺血、缺氧和炎症等原因造成心肌细胞坏死后微环境改变,并促进了移植干细胞的凋亡。研究发现:小剂量LPS预处理接受移植的个体,可通过减轻局部炎症反应,来减少移植骨髓干细胞在梗死心肌组织中的凋亡,延长其存活时间,改善心功能。?
Ischemic cardiomyopathy is a serious disease that imposes a threat to human. The primary cause of ischemic cardiomyopathy is that the damaged myocardial cells undergo necrosis and apoptosis in the development and progression of cardiovascular disease, while the mature myocardial cells do not have the ability to differentiate or proliferate, and can not fully compensate the affected function of heart muscle cells, leading to cardiac systolic and diastolic dysfunction. Mesenchymal stem cells (MSCs) have the characteristics of self-renewal, differentiation, proliferation and potential of multilineage differentiation. In addition, MSCs are easy to be cultured in vitro, have no immune rejection and ethical issues, thus are ideal for cell transplantation to treat ischemic cardiomyopathy.
When transplanted into ischemic myocardium, bone marrow stem cells could differentiate into cardiomyocyte-like cells to promote angiogenesis and inhibit left ventricular remodeling, thus improving heart function in acute myocardial infarction and chronic congestive heart failure disease. However, these differentiated cardiomyocyte-like cells disappear in a short period and could not be further differentiated into neonatal cardiomyocyte. It has been shown that the activation of TLR4-MyD88-NFkB signaling pathways induces the release of a large number of inflammatory cytokines and thus causes cell damage. The pretreatment of transplant recipients with TLR4-specific ligand lipopolysaccharide(LPS) could stimulate bone marrow stem cells proliferation and activate TLR4/PI3K/Akt signaling pathway in original myocardial, promoting cell survival and inhibiting NFkB expression in TLR4-MyD88-NFkB signaling pathway. Consequently, the apoptosis of cardiomyocyte-like cells after transplantation is reduced and these cells are further induced to differentiate into cardiomyocytes.
The main purpose of this study is to develop novel approach to improve bone marrow stem cell transplantation in the treatment of ischemic heart disease, and a provide useful guidance for coronary artery bypass surgery in combination with stem cells. We employed animal models to investigate whether inhibiting inflammatory response of myocardial infarction region could reduce the apoptosis of transplanted bone marrow stem cell.
Objective: To characterize the change of number, distribution, and survival time in micro-environment after myocardial infarction of implanted rat bone marrow mesenchymal stem cells (BMSCs).
Methods: Adult rats were randomly divided into control group, the myocardial infarction group, LPS (0.1mg/kg) pretreatment group, LPS (1mg/kg) pretreatment group, LPS (5mg/kg) pretreatment group. CM-Dil labeled bone marrow derived mesenchymal stem cells were injected into the left anterior descending artery ligation at left ventricular wall in those rats. 48h, 1, 3, 4 weeks later, the number and survival time of red fluorescent labeled MSCs were recorded in the infarcted myocardium. Simultaneously, the survival, apoptosis and the activation of intracellular signaling pathway were analyzed in the transplanted MSCs.
Results: 22.8%MSCs underwent apoptosis in 0.1mg/kg LPS pretreatment group, much less than the control myocardial infarction group and other dose of LPS pretreatment group. After myocardial infarction TLR4, NFkB, and MyD88 mRNA and protein expression was significantly increased in LPS pretreatment group compared with the control group. A week later red fluorescent cells were decreased significantly in the myocardial infarction group and 0.1mg/kg LPS pretreatment group.
Conclusion: The transplanted MSCs in myocardial infarction have a very low survival rate, perhaps due to the apoptosis induced by the changes in the microenvironment .Low-dose LPS pretreatment could reduce the local inflammatory reaction and the apoptosis of the transplanted bone marrow stem cells in the infarcted myocardium, thu prolonging the survival time of MSCs and improving the heart function.
引文
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[51]Li W,Ma N,Nesselmann C,Klopsch C,Ladilov Y et al.Bcl-2 engineered MSCs inhibited apoptosis and improved heart function.Stem Cells.2007 Aug;25(8):2118-27.
[52]Fallach R, Shainberg A, Avlas O, Fainblut M, Chepurko Y, Porat E, Hochhauser E.Cardiomyocyte Toll-like receptor 4 is involved in heart dysfunction following septic shock or myocardial ischemia.J Mol Cell Cardiol. 2010 Jun;48(6):1236-44
[53]Jun Muto, Kenshi Yamasaki, Kristen R. Taylor and Richard L. Gallo Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS. Molecular Immunology, Volume 47, Issues 2-3, December 2009, Pages 449-456
[54]Takeishi Y, Kubota I.Role of Toll-like receptor mediated signaling pathway in ischemic heart.Front Biosci. 2009 Jan 1;14:2553-8.
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[77]Deuse T, Peter C, Fedak PW, Doyle T, Reichenspurner H, Zimmermann WH, Eschenhagen T, Stein W, Wu JC, Robbins RC, Schrepfer S.Hepatocyte growth factor or vascular endothelial growth factor gene transfer maximizes mesenchymal stem cell-based myocardial salvage after acute myocardial infarction.Circulation. 2009 Sep 15;120(11 Suppl):S247-54.
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[51]Li W,Ma N,Nesselmann C,Klopsch C,Ladilov Y et al.Bcl-2 engineered MSCs inhibited apoptosis and improved heart function.Stem Cells.2007 Aug;25(8):2118-27.
[52]Fallach R, Shainberg A, Avlas O, Fainblut M, Chepurko Y, Porat E, Hochhauser E.Cardiomyocyte Toll-like receptor 4 is involved in heart dysfunction following septic shock or myocardial ischemia.J Mol Cell Cardiol. 2010 Jun;48(6):1236-44
[53]Jun Muto, Kenshi Yamasaki, Kristen R. Taylor and Richard L. Gallo Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS. Molecular Immunology, Volume 47, Issues 2-3, December 2009, Pages 449-456
[54]Takeishi Y, Kubota I.Role of Toll-like receptor mediated signaling pathway in ischemic heart.Front Biosci. 2009 Jan 1;14:2553-8.
[55]Chao W, Shen Y, Zhu X, Zhao H, Novikov M, Schmidt U, Rosenzweig A. Lipopolysaccharide improves cardiomyocyte survival and function after serum deprivation.Biol Chem. 2005 Jun 10;280(23):21997-2005. Epub 2005 Mar 26.
[56]Godement P, Vanselow J, Thanos S, Bonhoeffer F. A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue.Development. 1987 Dec;101(4):697-713.
[57] van Gaalen SM, Kruyt MC, Geuze RE, de Bruijn JD, Alblas J, Dhert WJ.Use of fluorochrome labels in in vivo bone tissue engineering research.Tissue Eng Part B Rev. 2010 Apr;16(2):209-17.
[58]Teupser D, Thiery J, Walli AK, Seidel D. Determination of LDL-and scavenger -receptor act ivity in adherent and non-adherent cultured cells with a new single-step fluorometric assay. Biochim Biophys Acta. 1996 Oct 18;1303(3):193-8.
[59]Janssens S, Dubois C, Bogaert J, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlledtrial. Lancet 2006;367:113-21.
[60]Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004;364:141-8.
[61]Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells,2007. 25:2739–49
[62]Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res,2007,327:449–62
[63]Majumdar MK, Thiede MA, Mosca JD, Moorman M, Gerson SL. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs)and stromal cells. J. Cell Physiol. 1998,176:57–66
[64]Horwitz E, Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy,2005,7:393–95
[65]Gnecchi M,He H,Liang O D,et al. Paracrine action accounts for marked protection of ischemic heart by Akt modified mesenchymal stem cells.Nat Med, 2005, 11 (4): 367-368.
[66]Ripa RS,Haack-SorensenM,Wang Y, Jorgensen E, Mortensen S, et al. Bone marrow derived mesenchymal cell mobilization by granulocyte-colony stimulating factor after acute myocardial infarction: results from the Stem Cells in Myocardial Infarction (STEMMI) trial. Circulation, 2007, 116:I24–30
[67]Lazarus H, Haynesworth S, Gerson S, Rosenthal N, Caplan A. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells):implications for therapeutic use. Bone Marrow Transplant. 1995.16:557–64
[68]Nagaya N, Fujii T, Iwase T, Ohgushi H, Itoh T, et al. Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis. Am. J. Physiol. Heart Circ. Physiol,2004, 287: H2670–76
[69]Phinney D, Prockop D. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair-current views. Stem Cells 2007.25:2896–902
[70]Block G, Ohkouchi S, Fung F, Frenkel J, Gregory C, et al. Multipotent stromal cells are activated to reduce apoptosis in part by upregulation and secretion of stanniocalcin-1. Stem Cells,2009,27:670–81
[71]Klyushnenkova E, Mosca JD, Zernetkina V, Majumdar MK, Beggs KJ, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J. Biomed. Sci. 2005,12:47–57
[72]Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ. Expansion of human adult stem cells from bone marrow stroma:conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells, 2002,20:530–41
[73]Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat. Med. 2006,12:459–65
[74]Sheng X G,Feng J Z,Wu S,et al.Differentiation of rabbit bone marrowmesenchymal stem cells into myogenic cells in vitro and expression of vascular endothelial growth factor gene after transfection.Exp Hematol.2004 Mar;24(3)290-4
[75]Tang Y, Zhao Q, Zhang Y, Cheng L, Liu M, et al. Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium. Regul. Pept,2004,117:3–10
[76]Esposito C, Pertile E, Grosjean F, Castoldi F, Diliberto R, Serpieri N, Arra M, Villa L, Mangione F, Esposito V, Migotto C, Valentino R, Dal Canton A.The improvement of ischemia/reperfusion injury by erythropoetin is not mediated through bone marrow cell recruitment in rats.Transplant Proc. 2009 May;41(4):1113-5.
[77]Deuse T, Peter C, Fedak PW, Doyle T, Reichenspurner H, Zimmermann WH, Eschenhagen T, Stein W, Wu JC, Robbins RC, Schrepfer S.Hepatocyte growth factor or vascular endothelial growth factor gene transfer maximizes mesenchymal stem cell-based myocardial salvage after acute myocardial infarction.Circulation. 2009 Sep 15;120(11 Suppl):S247-54.
[78]Imanishi Y, Saito A, Komoda H, Kitagawa-Sakakida S, Miyagawa S, Kondoh H, Ichikawa H, Sawa Y.Allogenic mesenchymal stem cell transplantation has a therapeutic effect in acute myocardial infarction in rats.J Mol Cell Cardiol. 2008 Apr;44(4):662-71.
[79]Song JQ, Teng X, Cai Y, Tang CS, Qi YF.Activation of Akt/GSK-3beta signaling pathway is involved in intermedin(1-53) protection against myocardial apoptosis induced by ischemia/reperfusion.Apoptosis. 2009 Nov;14(11):1299-307
[80]Afzal MR, Haider HKh, Idris NM, Jiang S, Ahmed RP, Ashraf M.Preconditioning promotes survival and angiomyogenic potential of mesenchymal stem cells in the infarcted heart via NF-kappaB signaling.Antioxid Redox Signal. 2010 Mar 15;12 (6): 693-702.
[81]ChanK.L,Wong,K.F. Luk,J.M.Role of LPS/CD14/TLR4-mediated inflammation in necrotizing enterocolitis: pathogenesis and therapeutic implications.World J. Gastroenterol. 15 (38), 4745-4752 (2009)
[82]Yong-Chen Lu, Wen-Chen Yeh and Pamela S. LPS/TLR4 signal transduction pathway .Cytokine, Volume 42, Issue 2, May 2008, Pages 145-151