热休克蛋白90对大鼠骨髓间充质干细胞存活和运动能力的研究
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摘要
第一部分热休克蛋白90对缺氧和缺血清诱导大鼠骨髓间充质干细胞的凋亡保护作用通过PI3K/Akt和ERK1/2通路
实验背景：
随着社会经济的发展以及人们生活方式,饮食习惯和饮食结构的变化,冠心病,中风等心脑血管疾病的发病率和病死率日益增加。同时各种治疗手段的出现,如再血管化介入治疗,冠脉搭桥等虽然在一点程度上起到了积极的治疗效果,但是其高昂的医疗费用一定程度限制了其广泛应用。因此利用自身的骨髓干细胞移植来治疗缺血性疾病是一种低成本,低风险的治疗策略,正日益受到人们的关注。但是绝大部分移植入体内的干细胞在移植后的24小时内,都会经历凋亡过程,最终细胞会死亡。干细胞治疗缺血性心脏病的大型临床循证研究(BOOST研究)提示,经过干细胞移植治疗后的心功能也只提高约5%,因此如何提高移植入体内干细胞的存活和抗凋亡能力一直是摆在科学家面前的难题。
既往研究提示,在心肌梗塞后移植干细胞后,移植细胞的低活力状态限制了这一作用(van der Bogt et al.,2009)。心肌细胞的坏死能诱导并且激活一系列的级联反应,包括氧自由基的激活,促发一些细胞因子的分泌,从而引起移植细胞的凋亡发生(Frangogiannis et al.,2002)。血管丢失和疤痕组织都能减少移植细胞的营养支持,合适的再血管化治疗后能提供给移植细胞合适的生长微环境,增强移植细胞的存活功能。正因为移植细胞的修复心肌功能的短暂性(Daiet al.,2005),人们给予移植细胞缺氧等物理因素和给予细胞因子及药物等预处理方法,来增加移植细胞存活和修复组织的功能。
热休克蛋白(Heat shock protein90, HSP90)是广泛存在于真核生物细胞上,它是一组高度保守的蛋白质,在所有真核细胞中均有表达。HSP90主要作用是结合已获得三级结构的蛋白,参与伴侣蛋白(client protein)的激活与成熟过程。Hsp90作为其他蛋白质的分子伴侣,通过帮助它们折叠并护送它们到达细胞内适当的位置,从而发挥这些蛋白的重要功能。细胞在受到应激刺激后,HSP90表达升高(Calderwood and Ciocca,2008).目前HSP90参与的伴侣蛋白约有100多种,,包括甾体激素受体、螺旋-环-螺旋转录因子、酪氨酸及丝苏氨酸蛋白激酶等,这些多是细胞内信号转导通路的关键蛋白成分,参与细胞正常生理功能,以及细胞的发生和演进作用(Tsutsumi et al.,2009). HSP90具有抗凋亡作用通过细胞内的信号通路(Bishop et al.,2007)。HSP90能拮抗3-hydroxy-kynurenine诱导的神经细胞凋亡作用(Lee et al.,2001). HSP90的心肌保护作用是通过PI3K/Akt通路(Wang et al.,2009).
但是,HSP90对骨髓间充质干细胞的保护作用,目前还不是很明确,因此本研究通过模拟体内缺血的微环境,通过一系列的凋亡检测手段来观察HSP90对由缺氧／缺血清诱导的骨髓间充质干细胞凋亡是否有拮抗作用,并对相关的机制进行初步探讨。
实验目的：
研究HSP90对由缺氧／缺血清诱导的骨髓间充质干细胞凋亡是否有拮抗作用?同时研究那些可能机制参与了HSP90的抗凋亡作用?
实验方法：
本研究通过全骨髓培养的方法获得大鼠骨髓间充质干细胞,经过体外培养以后,取3-5代的骨髓间充质干细胞进行实验。对实验的骨髓间充质干细胞进行细胞表面标志物CD44, CD45,和CD90应用流式细胞仪进行鉴定。对骨髓间充质干细胞给予缺氧/缺血清培养24小时后,获得稳定的凋亡模型。
为了观察HSP90对骨髓间充质干细胞的作用,给予人重组HSP90a(0,0.01,0.1,1和10μmol/L)处理骨髓间充质干细胞24小时,接着再给予常氧(O221%)或缺氧(O21%)/缺血清培养24小时。通过四甲基偶氮唑盐微量酶反应比色法(MTT)对细胞的活力进行检测。对骨髓间充质干细胞的凋亡检测采用Hoechst 33258染色和Annexin V/PI方法。通过以上方法得出HSP90最佳的抗细胞凋亡的浓度。应用实时定量-PCR检测骨髓间充质干细胞细胞膜表达的V-erb-b2 rythroblastic leukemia-viral oncogene homolog 2 (ErbB2)和Toll-like-receptor-4 (TLR-4)的mRNA水平。应用Western blot去观察了凋亡相关蛋白cleaved caspase-3, Bcl-2, Bax, Bcl-xL的水平。以及HSP90抗细胞凋亡的下游信号通路AKT和ERK1／2的磷酸化水平。最后通过比色法检测了细胞上清内的一氧化氮(Nitric oxide, NO)的产量,来反映骨髓间充质干细胞在缺氧／缺血清作用下的一氧化氮的分泌量。
实验结果：
从SD大鼠得到的骨髓间充质干细胞经过培养以后,对其进行细胞鉴定。绝大部份骨髓间充质干细胞的细胞表面的CD44和CD90都是阳性的,而CD45阴性。给予人重组HSP90a(0,0.01,0.1,1和10μmol/L)预处理骨髓间充质干细胞24小时,再给予常氧(O221%)或缺氧(O2 1%)／缺血清培养24小时。实验发现,rhHsp90α在1-10μmol/L浓度范围都能显著的对抗由缺氧／缺血清诱导的干细胞凋亡。Hsp90减少cleaved caspase-3和Bax的表达,增加Bcl-2, Bcl-xL的表达水平。同时,促进NO的分泌,信号通路AKT和ERK1/2参与hsp90的保护作用。
第二部分热休克蛋白90促进骨髓间充质干细胞迁移通过PI3K/Akt和ERK1/2通路
研究背景：
骨髓来源的间充质干细胞(Mesenchymal stem cells, MSCs)是存在于骨髓间质层的一种具有多能潜力的干细胞,它具有向骨骼,软骨,肌肉,等间充质层细胞分化的功能。研究表明,MSCs是治疗心血管疾病细胞疗法的最具有优势的细胞之一。它能够对受损的心肌进行修复,促进损伤局部的血管新生,以及功能恢复。
细胞疗法的机制,主要可能是这些细胞分泌的一些细胞因子,主要包括一些和干细胞迁移,归巢等功能相关的细胞因子,促进干细胞迁移到缺血组织。但是这些细胞因子在损伤局部所起的作用是短暂的,持续时间也不长。因此迁移的细胞也受到很大的限制。
集落刺激因子(G-CSF)及其类似的粒细胞集落刺激因子(GM-CSF),干细胞因子(Stem cell factor SCF),基质细胞衍生因子-1(Stromal derived factor-1 SDF-1), erythropoietin(EPO),其他一些生长因子比如VEGF, angiopoietin-1能对干细胞的迁移和归巢等作用起到调控作用。研究提示,SDF-1及其相应受体CXCR4在干细胞的迁移和归巢中起主要作用,它对干细胞从骨髓动员和促进细胞向缺血组织归巢都起主要作用(Lapidot 2001)。
同时干细胞迁移也需要一些活化的粘附分子作用,如VCAM-1, ICAM-1等。还有细胞外基质降解酶,大部分是降解Ⅳ胶原的基质金属蛋白酶(MMPs),尤其是MMP-2和MMP-9。(Gurjar, Sharma et al.1999)。
热休克蛋白90(Heat shock protein90 Hsp90)是一个高度保守的,在真核生物细胞中表达的分子伴侣蛋白,对维持细胞蛋白的稳态和正常功能,而这些蛋白大部分和细胞正常的增值,分化,生长发育等相关。Hsp90的拮抗剂DMAG-N-oxide能显著减少肿瘤细胞的迁移整合运动以及细胞外基质依赖的细胞骨架的重排(Tsutsumi, Scroggins et al.2008)。
而Hsp90在骨髓间充质干细胞的细胞迁移中扮演什么样的作用,目前还不是很清楚。
实验目的：
研究MSCs在体外经过Hsp90处理后,能否促进骨髓间充质干细胞的细胞迁移活动。并对这些迁移活动的机制进行初步。
实验方法：
经过常规差速贴壁培养得到的骨髓间充质干细胞后,按实验分组分为,正常对照组,转染siRNAHsp90α,17-AAG组,wortmannin组,U0126组。用细胞划痕实验以及Transwell实验观察细胞的迁移能力。用明胶酶谱法检测细胞上清中的MMP-2和MMP-9的活性。用定量-PCR法检测CXCR4和VCAM-1的mRNA表达水平。用Westeen-blot检测了MSCs表达的MMP-2和MMP-9的蛋白水平,以及,phospho-Akt, phospho-ERK1/2的蛋白水平。实验结果：
MSCs经过24小时的RhHsp90 a (10nM)处理后的细胞,和未予RhHsp90 a (10nM)处理组相比,能显著提高MSCs的迁移能力。应用(17-AAG,40 nmol/L,)后,和对照组相比能明显抑制MSC的迁移能力。应用wortmannin (10μm),和U0126 (10μM)后迁移的细胞明显比RhHsp90 a处理组减少。Real-time PCR检测表明,siRNAHsp90 a组和正常对照组相比Hsp90 a的表达下调88.1±3.5%。同时细胞的迁移能力和正常对照组相比也明显下降3倍。明胶酶谱法检测表明,RhHsp90 a处理24小时后,细胞上清中的MMP-2和MMP-9的活力比正常对照组明显增加,细胞用17-AAG (40nM), wortmannin (0.2μM)以及U0126预处理后,细胞的MMP-9活性与RhHsp90 a处理组相比明显被阻断。MSCs给予rhHsp90a(10nM)处理后,MMP-2和MMP-9的蛋白水平与对照组相比明显提高。17-AAG预处理MSCs后,与rhHsp90a组相比,细胞的MMP-9和MMP-2水平显著下降。wortmannin处理后,与rhHsp90a组相比,细胞的MMP-9水平显著下降,而MMP-2的水平没有明显变化。U0126处理后,细胞的MMP-9和MMP-2水平显著下降与RhHsp90a组相比。MSCs经过RhHsp90 a处理后,显著上调VCAM-1和CXCR4的mRNA水平。17-AAG预处理MSCs后,VCAM-1和CXCR4的mRNA水平显著下降。Wortmannin和U0126都能显著降低MSCs的VCAM-1和CXCR4的mRNA表达水平。PI3K/Akt和ERK途径同时参与RhHsp90 a介导的MSCs的迁移作用。
实验结论：
1、RhHsp90 a体外处理能促进骨髓间充质干细胞迁移能力。
2、SiRNAHsp90能干扰Hsp90 a的促进骨髓间充质干细胞迁移能力。
3、MMP-2和MMP-9参与了RhHsp90 a体外处理促进骨髓间充质干细胞迁移能力这一过程。
4、CXCR4和VCAM-1参与了RhHsp90 a体外处理促进骨髓间充质干细胞迁移能力这一过程。
5、PI3K/Akt和ERK途径参与RhHsp90 a介导的骨髓间充质干细胞的迁移作用
ParⅠHeat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathways
Background:
Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability after transplantation limited the reparative capacity of these cells in vivo (der Bogt KE et al.2009). Myocardial necrosis induces complement activation and free radical generation, triggering a cytokine cascade, and then donor cell apoptosis (Frangogiannis et al.2002)。Neovascularization can provide the implanted cells with adequate microenvironment to enhance survival and function, whereas exchange vessels loss and scar formation attenuate the ability to nourish the implanted cells. The donor cell growth appears tenuous and their cardiac reparative benefits are transient (Dai et al. 2005). Preconditioning MSCs with some physical or cytochemical stimuli may improve the therapeutic efficacy of cell therapy, including hypoxia (Hu et al.2008), growth factors (Hahn et al.2008), and some cytokines (Gui et al.2007; Pasha et al.2008), more available for translational application than gene transfection.
Heat shock protein 90 (Hsp90) is deemed as the most active molecular chaperone which plays a critical role in the development and progression of cancer (Tsutsumi et al. 2009). It also acts as a checkpoint, leading to survival or death under stress stimulus.Recently, Hsp90 was found to exert a cardioprotective effect via the PI3K/Akt pathway (1)(Wang et al.2009).
Here, we established an in vitro apoptosis model induced by hypoxia and serum deprivation to investigate the role of exogenous Hsp90 in rat bone marrow MSCs on the apoptosis and signaling molecules involved.
Objective:
To explore the cytopretection of HSp90 against hypoxia and serum deprivation induced apoptosis and the possible mechanisms in rat mesenchymal stem cells.
Methods:
Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was assessed by Hoechest 33258 nuclear staining and flow cytometric analysis with annexin V/PI staining. The gene expression of TLR4 and ErbB2 was detected by real-time PCR. The protein level of cleaved-caspase-3, bcl-2, bcl-xL, bax, total-Erk, phospho-Erk, total-Akt, phospho-Akt, and hsp90 were detected by Western-blot. The production of nitric oxide was measured by spectrophotometric assay.
Results:
Hsp90 improves MSCs viability and protects MSCs against apoptosis induced by serum deprivation and hypoxia. The protective role of Hsp90 not only elevates bcl-2/bax and bcl-xL/bax expression and attenuates cleaved caspase-3 expression via down-regulating membrane TLR-4 and ErbB2 receptors and then activating their downstream PI3K/Akt and ERK1/2 pathways, but also enhances the paracrine effect of MSCs.Hsp90 significantly protected MSCs against apoptosis induced by hypoxia and serum deprivation.
Conclusions:
These findings demonstrated a novel and effective treatment strategy against MSC apoptosis in cell transplantation.
PartⅡHeat shock protein 90 enhances rat mesenchymal stem cells migration via PI3K/Akt and ERK1/2 pathways
Background:
Bone marrow mesenchymal stem cells (MSCs) are pluripotent stem cells that localize in the stromal compartment of the bone marrow, where they support hematopoiesis and differentiate into mesenchymal lineages such as bone, cartilage, muscle tissues. Accumulating evidence suggests that MSCs are attractive candidates for cardiovascular therapy because of their capacity to facilitate myocardial repair and neovascularization in models of cardiac injury. MSCs are able to migrate into the injured myocardium from the circulation and contribute to cardiac repair post myocardial infarction.
Some clinical trial suggest that the benefits of stem cells therapy derive primarily from secretion of paracrine factors, appropriate homing of these cells is essential to enable such factors to function, because their effects are, limited to a short range, requiring relatively high local concentrations. Some chemokine such as granulocyte colony stimulating factor (G-CSF), and the closely related granulocyte/macrophage colony-stimulating factor (GM-CSF), SDF-l,erythropoietin, and stem cell factor (SCF) (the ligand for c-kit) and some growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-1, can regulate the migration of MSCs. The SDF-1/CXCR4 axis is central for stem cell mobilization from the bone marrow and homing to ischemic tissues.
Some clinical trial suggest that the benefits of stem cells therapy derive primarily from secretion of paracrine factors, appropriate homing of these cells is essential to enable such factors to function, because their effects are, limited to a short range, requiring relatively high local concentrations. Some chemokine such as granulocyte colony stimulating factor (G-CSF), and the closely related granulocyte/macrophage colony-stimulating factor (GM-CSF), SDF-l,erythropoietin, and stem cell factor (SCF) (the ligand for c-kit) and some growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-1, can regulate the migration of MSCs. and Adhesive activation such as VCAM-1 and ICAM-1.The SDF-1/CXCR4 axis is central for stem cell mobilization from the bone marrow and homing to ischemic tissues.
Migration of cells requires degradation of basement membrane, which is dependent on the production of matrix-degrading enzymes, especially those capable of degrading type IV collagen, such as MMPs, especially MMP-2 and MMP-9.
Heat shock protein90 (Hsp90) is a highly conserved, constitutively expressed protein in eucaryotic cell, which account for 2 5% of total cellular proteins even in the absence of stress. There are two isoforms:Hsp90 a and Hsp90β, which are essential for the viability of eukaryotic cells.The function of Hsp90 is for maintaining the stability and function of numerous proteins referred to as client proteins.Previous studyshowed that using mAb 4C5, a function-blocking monoclonal antibody against Hsp90 in human breast cancer cells, which altered actin dynamics to inhibit cell invasion.
Whether Hsp90 play an important role in MSCs migration is still unclear.
Objective:
In the present study, we hypothesized that Hsp90 would be involved in the migration of MSCs. Our data may provide new mechanistic insights into the hsp90-mediated migration of MSCs.and, may provide new perspectives in the development of therapeutic strategies targeting MSCs migration and angiogenesis.
Methods:
Rat bone marrow-derived mesenchymal stem cells were harvested from femora and tibia by density gradient centrifugation.The espremiental was divided five groups:control group, Hsp90 a group, siRNAHsp90 a group,17-AAG group, wortmannin group, U0126 group. The ability of MSCs cell migration is determined using the wound healing assay and transwell assay. The activity of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) were estimated by gelatin zymography. The mRNA levels of MMP-2、MMP-9、CXCR4 and VCAM-1 were detected by real-time PCR. The protein expression of MMP-2、MMP-9 and ERK1/2, phospho-ERK1/2, Akt, phospho-Akt was determined by Western-blot.
Results:
Treatment with RhHsp90a significantly enhances MSCs migration. However, sirhsp90 a significantly decreased MSCs migration compared with treatment of hsp90 a. Treatment with RhHsp90 a enhance the MSCs secrete MMP-2 and MMP-9; especially increased the activity of MMP-9.and increased the expression of CXCR4 and VCAM-1. Our data showed that hsp90αmodulate the rat MSCs migration via MMPs and CXCR4. PI3K/Akt and ERK signaling pathways mediates these effects.
Conclusions:
These findings indicated that RhHsp90a promoted MSCs migration, elevated the expression of MMP-2 and MMP-9, and via PI3K/Akt and ERK signaling pathways mediates these effects. Hsp90 is a candidate drug for enhancing MSCs migration.
实验背景：
随着社会经济的发展以及人们生活方式,饮食习惯和饮食结构的变化,冠心病,中风等心脑血管疾病的发病率和病死率日益增加。同时各种治疗手段的出现,如再血管化介入治疗,冠脉搭桥等虽然在一点程度上起到了积极的治疗效果,但是其高昂的医疗费用一定程度限制了其广泛应用。因此利用自身的骨髓干细胞移植来治疗缺血性疾病是一种低成本,低风险的治疗策略,正日益受到人们的关注。但是绝大部分移植入体内的干细胞在移植后的24小时内,都会经历凋亡过程,最终细胞会死亡。干细胞治疗缺血性心脏病的大型临床循证研究(BOOST研究)提示,经过干细胞移植治疗后的心功能也只提高约5%,因此如何提高移植入体内干细胞的存活和抗凋亡能力一直是摆在科学家面前的难题。
既往研究提示,在心肌梗塞后移植干细胞后,移植细胞的低活力状态限制了这一作用(van der Bogt et al.,2009)。心肌细胞的坏死能诱导并且激活一系列的级联反应,包括氧自由基的激活,促发一些细胞因子的分泌,从而引起移植细胞的凋亡发生(Frangogiannis et al.,2002)。血管丢失和疤痕组织都能减少移植细胞的营养支持,合适的再血管化治疗后能提供给移植细胞合适的生长微环境,增强移植细胞的存活功能。正因为移植细胞的修复心肌功能的短暂性(Daiet al.,2005),人们给予移植细胞缺氧等物理因素和给予细胞因子及药物等预处理方法,来增加移植细胞存活和修复组织的功能。
热休克蛋白(Heat shock protein90, HSP90)是广泛存在于真核生物细胞上,它是一组高度保守的蛋白质,在所有真核细胞中均有表达。HSP90主要作用是结合已获得三级结构的蛋白,参与伴侣蛋白(client protein)的激活与成熟过程。Hsp90作为其他蛋白质的分子伴侣,通过帮助它们折叠并护送它们到达细胞内适当的位置,从而发挥这些蛋白的重要功能。细胞在受到应激刺激后,HSP90表达升高(Calderwood and Ciocca,2008).目前HSP90参与的伴侣蛋白约有100多种,,包括甾体激素受体、螺旋-环-螺旋转录因子、酪氨酸及丝苏氨酸蛋白激酶等,这些多是细胞内信号转导通路的关键蛋白成分,参与细胞正常生理功能,以及细胞的发生和演进作用(Tsutsumi et al.,2009). HSP90具有抗凋亡作用通过细胞内的信号通路(Bishop et al.,2007)。HSP90能拮抗3-hydroxy-kynurenine诱导的神经细胞凋亡作用(Lee et al.,2001). HSP90的心肌保护作用是通过PI3K/Akt通路(Wang et al.,2009).
但是,HSP90对骨髓间充质干细胞的保护作用,目前还不是很明确,因此本研究通过模拟体内缺血的微环境,通过一系列的凋亡检测手段来观察HSP90对由缺氧／缺血清诱导的骨髓间充质干细胞凋亡是否有拮抗作用,并对相关的机制进行初步探讨。
实验目的：
研究HSP90对由缺氧／缺血清诱导的骨髓间充质干细胞凋亡是否有拮抗作用?同时研究那些可能机制参与了HSP90的抗凋亡作用?
实验方法：
本研究通过全骨髓培养的方法获得大鼠骨髓间充质干细胞,经过体外培养以后,取3-5代的骨髓间充质干细胞进行实验。对实验的骨髓间充质干细胞进行细胞表面标志物CD44, CD45,和CD90应用流式细胞仪进行鉴定。对骨髓间充质干细胞给予缺氧/缺血清培养24小时后,获得稳定的凋亡模型。
为了观察HSP90对骨髓间充质干细胞的作用,给予人重组HSP90a(0,0.01,0.1,1和10μmol/L)处理骨髓间充质干细胞24小时,接着再给予常氧(O221%)或缺氧(O21%)/缺血清培养24小时。通过四甲基偶氮唑盐微量酶反应比色法(MTT)对细胞的活力进行检测。对骨髓间充质干细胞的凋亡检测采用Hoechst 33258染色和Annexin V/PI方法。通过以上方法得出HSP90最佳的抗细胞凋亡的浓度。应用实时定量-PCR检测骨髓间充质干细胞细胞膜表达的V-erb-b2 rythroblastic leukemia-viral oncogene homolog 2 (ErbB2)和Toll-like-receptor-4 (TLR-4)的mRNA水平。应用Western blot去观察了凋亡相关蛋白cleaved caspase-3, Bcl-2, Bax, Bcl-xL的水平。以及HSP90抗细胞凋亡的下游信号通路AKT和ERK1／2的磷酸化水平。最后通过比色法检测了细胞上清内的一氧化氮(Nitric oxide, NO)的产量,来反映骨髓间充质干细胞在缺氧／缺血清作用下的一氧化氮的分泌量。
实验结果：
从SD大鼠得到的骨髓间充质干细胞经过培养以后,对其进行细胞鉴定。绝大部份骨髓间充质干细胞的细胞表面的CD44和CD90都是阳性的,而CD45阴性。给予人重组HSP90a(0,0.01,0.1,1和10μmol/L)预处理骨髓间充质干细胞24小时,再给予常氧(O221%)或缺氧(O2 1%)／缺血清培养24小时。实验发现,rhHsp90α在1-10μmol/L浓度范围都能显著的对抗由缺氧／缺血清诱导的干细胞凋亡。Hsp90减少cleaved caspase-3和Bax的表达,增加Bcl-2, Bcl-xL的表达水平。同时,促进NO的分泌,信号通路AKT和ERK1/2参与hsp90的保护作用。
第二部分热休克蛋白90促进骨髓间充质干细胞迁移通过PI3K/Akt和ERK1/2通路
研究背景：
骨髓来源的间充质干细胞(Mesenchymal stem cells, MSCs)是存在于骨髓间质层的一种具有多能潜力的干细胞,它具有向骨骼,软骨,肌肉,等间充质层细胞分化的功能。研究表明,MSCs是治疗心血管疾病细胞疗法的最具有优势的细胞之一。它能够对受损的心肌进行修复,促进损伤局部的血管新生,以及功能恢复。
细胞疗法的机制,主要可能是这些细胞分泌的一些细胞因子,主要包括一些和干细胞迁移,归巢等功能相关的细胞因子,促进干细胞迁移到缺血组织。但是这些细胞因子在损伤局部所起的作用是短暂的,持续时间也不长。因此迁移的细胞也受到很大的限制。
集落刺激因子(G-CSF)及其类似的粒细胞集落刺激因子(GM-CSF),干细胞因子(Stem cell factor SCF),基质细胞衍生因子-1(Stromal derived factor-1 SDF-1), erythropoietin(EPO),其他一些生长因子比如VEGF, angiopoietin-1能对干细胞的迁移和归巢等作用起到调控作用。研究提示,SDF-1及其相应受体CXCR4在干细胞的迁移和归巢中起主要作用,它对干细胞从骨髓动员和促进细胞向缺血组织归巢都起主要作用(Lapidot 2001)。
同时干细胞迁移也需要一些活化的粘附分子作用,如VCAM-1, ICAM-1等。还有细胞外基质降解酶,大部分是降解Ⅳ胶原的基质金属蛋白酶(MMPs),尤其是MMP-2和MMP-9。(Gurjar, Sharma et al.1999)。
热休克蛋白90(Heat shock protein90 Hsp90)是一个高度保守的,在真核生物细胞中表达的分子伴侣蛋白,对维持细胞蛋白的稳态和正常功能,而这些蛋白大部分和细胞正常的增值,分化,生长发育等相关。Hsp90的拮抗剂DMAG-N-oxide能显著减少肿瘤细胞的迁移整合运动以及细胞外基质依赖的细胞骨架的重排(Tsutsumi, Scroggins et al.2008)。
而Hsp90在骨髓间充质干细胞的细胞迁移中扮演什么样的作用,目前还不是很清楚。
实验目的：
研究MSCs在体外经过Hsp90处理后,能否促进骨髓间充质干细胞的细胞迁移活动。并对这些迁移活动的机制进行初步。
实验方法：
经过常规差速贴壁培养得到的骨髓间充质干细胞后,按实验分组分为,正常对照组,转染siRNAHsp90α,17-AAG组,wortmannin组,U0126组。用细胞划痕实验以及Transwell实验观察细胞的迁移能力。用明胶酶谱法检测细胞上清中的MMP-2和MMP-9的活性。用定量-PCR法检测CXCR4和VCAM-1的mRNA表达水平。用Westeen-blot检测了MSCs表达的MMP-2和MMP-9的蛋白水平,以及,phospho-Akt, phospho-ERK1/2的蛋白水平。实验结果：
MSCs经过24小时的RhHsp90 a (10nM)处理后的细胞,和未予RhHsp90 a (10nM)处理组相比,能显著提高MSCs的迁移能力。应用(17-AAG,40 nmol/L,)后,和对照组相比能明显抑制MSC的迁移能力。应用wortmannin (10μm),和U0126 (10μM)后迁移的细胞明显比RhHsp90 a处理组减少。Real-time PCR检测表明,siRNAHsp90 a组和正常对照组相比Hsp90 a的表达下调88.1±3.5%。同时细胞的迁移能力和正常对照组相比也明显下降3倍。明胶酶谱法检测表明,RhHsp90 a处理24小时后,细胞上清中的MMP-2和MMP-9的活力比正常对照组明显增加,细胞用17-AAG (40nM), wortmannin (0.2μM)以及U0126预处理后,细胞的MMP-9活性与RhHsp90 a处理组相比明显被阻断。MSCs给予rhHsp90a(10nM)处理后,MMP-2和MMP-9的蛋白水平与对照组相比明显提高。17-AAG预处理MSCs后,与rhHsp90a组相比,细胞的MMP-9和MMP-2水平显著下降。wortmannin处理后,与rhHsp90a组相比,细胞的MMP-9水平显著下降,而MMP-2的水平没有明显变化。U0126处理后,细胞的MMP-9和MMP-2水平显著下降与RhHsp90a组相比。MSCs经过RhHsp90 a处理后,显著上调VCAM-1和CXCR4的mRNA水平。17-AAG预处理MSCs后,VCAM-1和CXCR4的mRNA水平显著下降。Wortmannin和U0126都能显著降低MSCs的VCAM-1和CXCR4的mRNA表达水平。PI3K/Akt和ERK途径同时参与RhHsp90 a介导的MSCs的迁移作用。
实验结论：
1、RhHsp90 a体外处理能促进骨髓间充质干细胞迁移能力。
2、SiRNAHsp90能干扰Hsp90 a的促进骨髓间充质干细胞迁移能力。
3、MMP-2和MMP-9参与了RhHsp90 a体外处理促进骨髓间充质干细胞迁移能力这一过程。
4、CXCR4和VCAM-1参与了RhHsp90 a体外处理促进骨髓间充质干细胞迁移能力这一过程。
5、PI3K/Akt和ERK途径参与RhHsp90 a介导的骨髓间充质干细胞的迁移作用
ParⅠHeat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathways
Background:
Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability after transplantation limited the reparative capacity of these cells in vivo (der Bogt KE et al.2009). Myocardial necrosis induces complement activation and free radical generation, triggering a cytokine cascade, and then donor cell apoptosis (Frangogiannis et al.2002)。Neovascularization can provide the implanted cells with adequate microenvironment to enhance survival and function, whereas exchange vessels loss and scar formation attenuate the ability to nourish the implanted cells. The donor cell growth appears tenuous and their cardiac reparative benefits are transient (Dai et al. 2005). Preconditioning MSCs with some physical or cytochemical stimuli may improve the therapeutic efficacy of cell therapy, including hypoxia (Hu et al.2008), growth factors (Hahn et al.2008), and some cytokines (Gui et al.2007; Pasha et al.2008), more available for translational application than gene transfection.
Heat shock protein 90 (Hsp90) is deemed as the most active molecular chaperone which plays a critical role in the development and progression of cancer (Tsutsumi et al. 2009). It also acts as a checkpoint, leading to survival or death under stress stimulus.Recently, Hsp90 was found to exert a cardioprotective effect via the PI3K/Akt pathway (1)(Wang et al.2009).
Here, we established an in vitro apoptosis model induced by hypoxia and serum deprivation to investigate the role of exogenous Hsp90 in rat bone marrow MSCs on the apoptosis and signaling molecules involved.
Objective:
To explore the cytopretection of HSp90 against hypoxia and serum deprivation induced apoptosis and the possible mechanisms in rat mesenchymal stem cells.
Methods:
Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was assessed by Hoechest 33258 nuclear staining and flow cytometric analysis with annexin V/PI staining. The gene expression of TLR4 and ErbB2 was detected by real-time PCR. The protein level of cleaved-caspase-3, bcl-2, bcl-xL, bax, total-Erk, phospho-Erk, total-Akt, phospho-Akt, and hsp90 were detected by Western-blot. The production of nitric oxide was measured by spectrophotometric assay.
Results:
Hsp90 improves MSCs viability and protects MSCs against apoptosis induced by serum deprivation and hypoxia. The protective role of Hsp90 not only elevates bcl-2/bax and bcl-xL/bax expression and attenuates cleaved caspase-3 expression via down-regulating membrane TLR-4 and ErbB2 receptors and then activating their downstream PI3K/Akt and ERK1/2 pathways, but also enhances the paracrine effect of MSCs.Hsp90 significantly protected MSCs against apoptosis induced by hypoxia and serum deprivation.
Conclusions:
These findings demonstrated a novel and effective treatment strategy against MSC apoptosis in cell transplantation.
PartⅡHeat shock protein 90 enhances rat mesenchymal stem cells migration via PI3K/Akt and ERK1/2 pathways
Background:
Bone marrow mesenchymal stem cells (MSCs) are pluripotent stem cells that localize in the stromal compartment of the bone marrow, where they support hematopoiesis and differentiate into mesenchymal lineages such as bone, cartilage, muscle tissues. Accumulating evidence suggests that MSCs are attractive candidates for cardiovascular therapy because of their capacity to facilitate myocardial repair and neovascularization in models of cardiac injury. MSCs are able to migrate into the injured myocardium from the circulation and contribute to cardiac repair post myocardial infarction.
Some clinical trial suggest that the benefits of stem cells therapy derive primarily from secretion of paracrine factors, appropriate homing of these cells is essential to enable such factors to function, because their effects are, limited to a short range, requiring relatively high local concentrations. Some chemokine such as granulocyte colony stimulating factor (G-CSF), and the closely related granulocyte/macrophage colony-stimulating factor (GM-CSF), SDF-l,erythropoietin, and stem cell factor (SCF) (the ligand for c-kit) and some growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-1, can regulate the migration of MSCs. The SDF-1/CXCR4 axis is central for stem cell mobilization from the bone marrow and homing to ischemic tissues.
Some clinical trial suggest that the benefits of stem cells therapy derive primarily from secretion of paracrine factors, appropriate homing of these cells is essential to enable such factors to function, because their effects are, limited to a short range, requiring relatively high local concentrations. Some chemokine such as granulocyte colony stimulating factor (G-CSF), and the closely related granulocyte/macrophage colony-stimulating factor (GM-CSF), SDF-l,erythropoietin, and stem cell factor (SCF) (the ligand for c-kit) and some growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-1, can regulate the migration of MSCs. and Adhesive activation such as VCAM-1 and ICAM-1.The SDF-1/CXCR4 axis is central for stem cell mobilization from the bone marrow and homing to ischemic tissues.
Migration of cells requires degradation of basement membrane, which is dependent on the production of matrix-degrading enzymes, especially those capable of degrading type IV collagen, such as MMPs, especially MMP-2 and MMP-9.
Heat shock protein90 (Hsp90) is a highly conserved, constitutively expressed protein in eucaryotic cell, which account for 2 5% of total cellular proteins even in the absence of stress. There are two isoforms:Hsp90 a and Hsp90β, which are essential for the viability of eukaryotic cells.The function of Hsp90 is for maintaining the stability and function of numerous proteins referred to as client proteins.Previous studyshowed that using mAb 4C5, a function-blocking monoclonal antibody against Hsp90 in human breast cancer cells, which altered actin dynamics to inhibit cell invasion.
Whether Hsp90 play an important role in MSCs migration is still unclear.
Objective:
In the present study, we hypothesized that Hsp90 would be involved in the migration of MSCs. Our data may provide new mechanistic insights into the hsp90-mediated migration of MSCs.and, may provide new perspectives in the development of therapeutic strategies targeting MSCs migration and angiogenesis.
Methods:
Rat bone marrow-derived mesenchymal stem cells were harvested from femora and tibia by density gradient centrifugation.The espremiental was divided five groups:control group, Hsp90 a group, siRNAHsp90 a group,17-AAG group, wortmannin group, U0126 group. The ability of MSCs cell migration is determined using the wound healing assay and transwell assay. The activity of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) were estimated by gelatin zymography. The mRNA levels of MMP-2、MMP-9、CXCR4 and VCAM-1 were detected by real-time PCR. The protein expression of MMP-2、MMP-9 and ERK1/2, phospho-ERK1/2, Akt, phospho-Akt was determined by Western-blot.
Results:
Treatment with RhHsp90a significantly enhances MSCs migration. However, sirhsp90 a significantly decreased MSCs migration compared with treatment of hsp90 a. Treatment with RhHsp90 a enhance the MSCs secrete MMP-2 and MMP-9; especially increased the activity of MMP-9.and increased the expression of CXCR4 and VCAM-1. Our data showed that hsp90αmodulate the rat MSCs migration via MMPs and CXCR4. PI3K/Akt and ERK signaling pathways mediates these effects.
Conclusions:
These findings indicated that RhHsp90a promoted MSCs migration, elevated the expression of MMP-2 and MMP-9, and via PI3K/Akt and ERK signaling pathways mediates these effects. Hsp90 is a candidate drug for enhancing MSCs migration.
引文
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Wisel, S., M. Khan, et al. (2009). "Pharmacological preconditioning of mesenchymal stem cells with trimetazidine (1-[2,3,4-trimethoxybenzyl]piperazine) protects hypoxic cells against oxidative stress and enhances recovery of myocardial function in infarcted heart through Bcl-2 expression." J Pharmacol Exp Ther 329(2):543-50.
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Bleul, C. C., R. C. Fuhlbrigge, et al. (1996). "A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)." J Exp Med 184(3): 1101-9.
Bowden, R. A., Z. M. Ding, et al. (2002). "Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium." Circ Res 90(5):562-9.
Dias, S., S. V. Shmelkov, et al. (2002). "VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition." Blood 99(7):2532-40.
Gao, F., X. Y. Hu, et al. "Heat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathways." J Zhejiang Univ Sci B 11(8):608-17.
Grenert, J. P., W. P. Sullivan, et al. (1997). "The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation." J Biol Chem 272(38):23843-50.
Guo, Y., G. Hangoc, et al. (2005). "SDF-1/CXCL12 enhances survival and chemotaxis of murine embryonic stem cells and production of primitive and definitive hematopoietic progenitor cells." Stem Cells 23(9):1324-32.
Gurjar, M. V., R. V. Sharma, et al. (1999). "eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity." Arterioscler Thromb Vasc Biol 19(12):2871-7.
Hertlein, E., A. J. Wagner, et al. "17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia:clinical implications of HSP90 inhibition." Blood 116(1):45-53.
Heussen, C. and E. B. Dowdle (1980). "Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates." Anal Biochem 102(1):196-202.
Hyun, Y. M., H. L. Chung, et al. (2009). "Activated integrin VLA-4 localizes to the lamellipodia and mediates T cell migration on VCAM-1."J Immunol 183(1):359-69.
Jo, D. Y, S. Rafii, et al. (2000). "Chemotaxis of primitive hematopoietic cells in response to stromal cell-derived factor-1." J Clin Invest 105(1):101-11.
Lapidot, T. (2001). "Mechanism of human stem cell migration and repopulation of NOD/SCID and B2mnull NOD/SCID mice. The role of SDF-1/CXCR4 interactions." Ann N Y Acad Sci 938:83-95.
Lesko, E., J. Gozdzik, et al. (2007). "HSP90 antagonist, geldanamycin, inhibits proliferation, induces apoptosis and blocks migration of rhabdomyosarcoma cells in vitro and seeding into bone marrow in vivo." Anticancer Drugs 18(10):1173-81.
Lima e Silva, R., J. Shen, et al. (2007). "The SDF-1/CXCR4 ligand/receptor pair is an important contributor to several types of ocular neovascularization." FASEB J 21(12):3219-30.
Loetscher, M., T. Geiser, et al. (1994). "Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes." J Biol Chem 269(1):232-7.
Lu, L., J. Q. Zhang, et al. (2004). "Molecular and cellular events at the site of myocardial infarction:from the perspective of rebuilding myocardial tissue." Biochem Biophys Res Commun 320(3):907-13.
Matsuura, K., A. Honda, et al. (2009). "Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice." J Clin Invest 119(8):2204-17.
Minami, Y, H. Kawasaki, et al. (1991). "Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90." J Biol Chem 266(16):10099-103.
Nemoto, T. and N. Sato (1998). "Oligomeric forms of the 90-kDa heat shock protein.' Biochem J 330 (Pt 2):989-95.
Oberlin, E., A. Amara, et al. (1996). "The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1." Nature 382(6594):833-5.
Oppenheimer-Marks, N., L. S. Davis, et al. (1991). "Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes." J Immunol 147(9):2913-21.
Peled, A., O. Kollet, et al. (2000). "The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells:role in transendothelial/stromal migration and engraftment of NOD/SCID mice." Blood 95(11):3289-96.
Roe, S. M., M. M. Ali, et al. (2004). "The Mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37)." Cell 116(1):87-98.
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