骨髓间充质干细胞过表达MIR126移植通过AKT/ERK相关途径促进缺血心肌血管新生
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摘要
研究背景
目前,缺血性心肌病是人类死亡和致残的主要疾病之一。由于冠脉的狭窄和闭塞导致组织缺血和缺氧,伴随心绞痛、心梗、心律失常及心衰等症状,当心功能恶化到Ⅳ级时,1年死亡率可达60%。在过去几十年里,药物治疗、经皮冠状动脉腔内成形+支架术、冠状动脉搭桥术及心脏移植等已经显著改善了缺血性心肌病患者的预后。尽管这些治疗措施已经取得了很大的进步,但仍存在许多限制,因此,很多学者致力于干细胞移植的研究。
心肌梗死的修复过程中常看到冠脉侧支循环的形成,侧支循环能够改善缺血心肌灌注,减少心肌坏死,恢复“冬眠”心肌细胞的活性,从而减少心功能障碍的发生。然而机体自身生成的新生血管往往不足以代偿冠脉闭塞诱发的心肌缺血。因此,刺激缺血区小血管生长,促进侧支循环的形成,完成缺血区血管的自我搭桥便成了十分诱人且似乎直观合理的治疗方法。近年来,随着对血管新生和血管生长因子研究的不断深入,治疗性血管新生已成为治疗缺血性心脏病的新方法。
目前有关MSCs移植治疗心肌梗死的动物实验表明,MSCs植入损伤部位后可长期存活,并分化为具有心肌细胞特征的类心肌细胞,促进“血管新生”,改善血液动力学。但仍然存在着大量有待解决的问题。例如如何增加移植后的干细胞存活率；改善梗死区的微环境,增加迁移或归巢到梗死区干细胞的数量等。近来,对于miRNAs调控干细胞的发生发展、自我更新、分化、抗凋亡及炎症和血管新生等一系列生物过程知识的积累,有学者认为miRNAs可以作为干细胞移植治疗缺血性心肌病研究的一个新的切入点。
MiRNA126是血管内皮上的特殊的MiRNA,它在心梗后的血管发生和维持血管的完整性起着重要作用。MiRNA126可通过细胞分裂素活化蛋白酶发信号给VEGF与FGF来起作用。Spred-1和PIK3R2/p85-b是对Ras/细胞分裂素活化蛋白酶的抑制因子,可被MiRNA126抑制,而Spred-1和PIK3R2/p85-b可能抑制ERK\AKT途径从而抑制血管生成。因此,上调MiRNA126可减轻Spred-1和PIK3R2/p85-b对VEGF及FGF的抑制作用,有利于血管发生。我们推测MiRNA126通过对干细胞的调节可能为干细胞移植治疗缺血性心脏病开辟一条新途径。
第一部分小鼠骨髓间充质干细胞的体外培养、诱导及鉴定
研究目的
建立小鼠骨髓间充质干细胞(MSCs)的分离、培养方法,研究其在体外生长增殖的生物学特性及表型特征.
研究方法
应用60%的Percoll细胞分离液分离骨髓间质干细胞,接着进行培养和传代。应用MTT方法测定培养细胞的生长曲线,应用流式细胞仪测定细胞周期和鉴定培养细胞的纯度,同时成骨诱导分化及成脂诱导分化鉴定骨髓间质干细胞
实验结果
原代培养的BMSCs24小时内贴壁并伸展成多角形、梭形,前3天为相对抑制期,其后细胞增殖速度逐渐加快,呈对数形式,7天左右至平台期,细胞覆盖率达90%左右,约在2周内传至第3代时可到106个数量级细胞,能够满足细胞移植数量的需要,应用流式细胞仪检测BMSCs的标志,显示CD29, CD44阳性,而CD34, CD45阴性,符合BMSCs特征。
实验结论
本课题应用60%Percoll细胞分离液成功地分离和培养出较为均一的骨髓间质干细胞。骨髓间质干细胞体外培养生长稳定,传代后细胞生长较快,适应能力较强,细胞大多数处于静止期,增殖潜力巨大。
第二部分MiR126转染小鼠骨髓间充质干细胞
研究目的：
1构建miR-126的慢病毒表达载体,包装生产重组慢病毒,并进行慢病毒滴度、活性测定。
2包装生产重组慢病毒,慢病毒滴度、活性测定
研究方法
我们利用Gateway clone构建pDown-miR126及pLV.EX3d.P/puro-TRE> miRl26>IRES/eGFP。制备编码了慢病毒颗粒的重组病毒质粒及其pLV/helper-SL3, pLV/helper-SL4, pLV/helper-SL5辅助包装原件载体质粒,三种质粒载体分别进行高纯度无内毒素抽提,重组质粒DNA进行去内毒素质粒提取后,与包装系统质粒混合物按Invitrogen公司Lipofectamine2000使用说明进行共转染293TN细胞,然后进行慢病毒的包装,包装完成之后进行重组慢病毒滴度检测。
实验结果：
(1)成功构建miR-126的慢病毒表达载体。
(2)成功进行了LV-miR-126重组病毒的生产和滴度测定。
(3)慢病毒感染293TN细胞后,能够大大增强目的基因的表达。
实验结论：
通过慢病毒途径可以有效增强miR-126在宿主细胞中的表达
第三部分观察MiRNA126对ERK\AKT调控作用
研究目的：
在MSCs中过表达miRNA126以观察其对ERK\AKT的影响及体外诱导骨髓间充质干细胞分化为血管内皮细胞。
研究方法：
总RNA提取是根据All-in-One miRNA qRT-PCR Detection Kit生产厂家的说明进行,miR126的表达行quantitative real-time RT-PCR检测,测试miRNA以U6作为单拷贝基因对照。阴性对照组及控制组相比在过表达miRNA126的MSCs中ERK\AKT的表达行Westernblot检测。在第3代,过表达miR-126骨髓间充质干细胞与骨髓间充质干细胞分别按1×109/L密度接种,加入含血管内皮细胞生长因子、碱性成纤维细胞因子的DMEM培养基向血管内皮细胞诱导分化,ECMatrixTM凝胶体外血管新生实验观察血管新生情况。
实验结果：
在过表达miRNA126的MSCs中ERK\AKT表达明显增加及过表达miRNA126的MSCs中毛细血管密度明显增加。
实验结论：
在MSCs中过表达MiRNA126对ERK\AKT具有调控作用,能促进干细胞分化为血管内皮细胞。
第四部分骨髓间充质干细胞过表达MIR126移植通过AKT/ERK途径促血管新生及改善心脏功能
研究目的：
比较骨髓间充质干细胞过表达MIR126移植与单纯BM-MSCs移植在小鼠心梗模型中的促血管新生作用及其机制。
研究方法：
通过结扎C57小鼠冠状动脉前降支制备心肌梗死模型,C57小鼠随机分为3组：MIR126-MSC组(即过表达MIR126的MSCs移植,n=15),MSC组(单纯的MSCs移植,n=15)以及PBS对照组(注射PBS, n=15)。在术后14天,进行细胞移植。实验组每只C57小鼠接受总计5×106(0.1m1)个过表达MIR126骨髓间充质干细胞或MSCs,分成6个点注入C57小鼠缺血心肌；对照组注入同等体积的无菌PBS。6周后行组织学及超声检查。
实验结果：
细胞移植6周后,MSC组和MIR126-MSC组缺血肌肉组织的血管新生均明显增强,以MIR126-MSC组的毛细血管密度最高(P<0.01)。Western-blot结果显示,MSC组和MIR126-MSC组缺血肌肉组织中ERK1, AKT蛋白表达均明显高于PBS对照组,以MIR126-MSC组的表达量最高(P<0.05)。细胞移植6周后,组MiRNA126-MSCs组、BMSCs组心功能都较移植前有明显改善(p<0.05),而其中MiRNA126-MSCs细胞移植组EF、FS改善程度要明显好于单纯细胞移植组(P<0.05)。
实验结论：
与单纯的MSC细胞移植疗法相比,过表达MIR126骨髓间充质干细胞移植具有更强的促进血管新生和侧枝血管形成的能力。我们推测过表达MIR126骨髓间充质干细胞移植通过ERK/AKT途径改善心功能,MIR126可能为干细胞移植治疗缺血性心脏病开辟一条新途径。
Backgroud
Ischemic heart disease which triggers dysfunction and the death of cardiomyocytes is the most common cause of death throughout the world despite continued advances in the prevention and treatment of coronary artery disease. The symptoms of ischemic heart disease are angina, myocardial infarction, arrhythmia and heart failure, which are the consequence of decreased patency of atherosclerotic vessels. The mortality of heart failure can reach60%within one year for patients in New York Heart Association functional class IV.Over the past decades,improvements in medical therapy, percutaneous transluminal coronary angioplasty(PTCA), coronary artery bypass surgery(CABG) and heart transplantation, have dramatically improved the prognosis of ischemic heart disease. However, all the methods including drugs, thrombolysis and revascularization are not able to influence the dead myocardium. Many researchers devote to studies in the field of myocardium regeneration and repairing insulted myocardium with stem cells.
During AMI repairs, coronary collateral formed which could improve myocardial perfusion, circulation(CC)often decrease myocardial necrosis, restore hibernating myocardial viability and reduce left ventricular function disorder. As self neovascularisation is insufficient for compensation myocardial ischemia induced from CHD, stimulating small vessels to develop in ischemic region and promoting to form CC become attractive and reasonable strategy. As study of the angiogenesis and VGF development, theraputic angiogenesis has become a new approach to treat IHD. This strategy is designed to promote the development of supplemental collateral blood vessels that will constitute endogenous bypass conduits around occluded native arteries, a strategy termed "therapeutic angiogenesis."
Recentely,a few studys on MSCs to treat MI in animal experiment have proven that MSCs have the ability of differentiating into cardiac myocytes, living long time, promoting angiogenesis, and improving haemodynamics. However, some problems, for example, how to further augment the implanted cells survival and other functions to overcome the limitations for their clinical applications, are still to be explored. Since the knowledge of a variety of biological processes, including developmental timing, signal transduction, tissue differentiation and maintenance, Antiapoptosis, Anti-inflammation and neoangiogenesis under miRNAs regulate, new therapeutic targets for ischemic heart disease may identified in these non-coding RNAs.
MiR-126is an endothelial cell-specific miRNA that plays an essential role in neoangiogenesis following MI and in maintenance of vascular integrity [94]. MiR-126functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1and phosphoinositol-3kinase regulatory subunit2(PIK3R2/p85-beta). MiR-126represses the expression of spredl and PIK.3R2, which negatively regulate VEGF signaling via the ERK and AKT pathways, respectively. Thus, in the absence of miR-126, Spred-1/PI3k expression is elevated, resulting in repression of angiogenic signaling. Conversely, miR-126overexpression relieves the repressive influence of Spred-1/PI3k on the signaling pathways activated by VEGF and FGF, favoring angiogenesis. It is reasonable to speculate that miRNA126, a novel but practical regulator of stem cell implantation for ischemic heart disease, introduce a promising strategy for the treatment of myocardial infarction.
Part Ⅰ. MSCs isolation, culture and identification
Objective
This study was to establish the experimental method for isolating, culturing, proliferating and inducing mice bone marrow mesenchymal stem cells in vitro.
Methods
We isolated MSCs from animal bone marrow and separated MSCs by Percoll Centrifugation.MSCs were cultured and subcultured in low-glucose Dulbecco's modified Eagle's medium (DMEM) with10%selected fetal calf serum. Cell growth patterns were evaluated by MTT test. Cell cycle and surface antigenic features were analyzed by flow cytometry technique.
Results:
To isolate MSCs from bone marrow aspiration, the samples were fractionated with Percoll by density gradient centrifugation for mononuclear cell isolation. Through replacing culture medium, the non-adherent cells were removed and the remaining tightly adhered cells were MSCs. The morphology of cultured MSCs was fibroblast-like form. Primary cultured BMSCs adhered to and stretched into polygon or spindle within24hours. The first3days was the relative inhibition period, and then the cell proliferation accelerated gradually in a pattern of logarithm and reached the period of plateau by Day7or so, with90% cell coverage. The number of P3BMSCs can reach1062weeks later, and that can satisfy the required number of cell transplantation. The signs of BMSCs with positive CD29, CD44, CD90and negative CD34, CD45were detected by the flow cytometry, and that is consisted with the characteristics of BMSCs.
Conclusions:
In this study, we believed the adherent cells in cultured medium were mainly MSCs obtained by Percoll fractionation. Numerous molecular markers of MSCs were characterized, but unique marker of MSCs and any marker of blood cells and endothelial cells were not identified. MSCs showed stable growth in vitro, easy survival in subculture and rapid proliferation in culture medium.
Part II Transfection of miR-126in mesenchymal stem cellsand its expression
Object:
Construct Lentivirus vector to express miR-126, produce pseudoviral particles and determine its titer.
Methods:
Lentiviral MicroRNAs expression vector was constructed with Gateway system. Mature miR126, TRE promoter and eGFP sequences were inserted into plasmids to produce pUp-TRE, pDown-miR126and pTail-IRES/eGFP; scramble sequence was set as negative control pLV.EX3d.P/puro-TRE> miR126>IRES/eGFP was obtained with incubation of donors and accepter vectors catalyzed by LR clonase. Plasmid was then sequenced and purified for lentivirus envelope.
Envelope helper plasmids:pLV/helper-SL3, pLV/helper-SL4, pLV/helper-SL5, with pLV.EX3d.P/puro-TRE-miR126-IRES/eGFP or pLVrtTA/neo which contains the imperative elements for virus packaging, were co-transfected into293T cells with lipofectamine2000, according to the manufacture's instructions for the generation of Lenti-miR126-eGFP/puro or Lenti-rtTA/neo respectively.
To perform lentiviral infections, the mice MSCs cells were plated at40%-50%confluence and incubated overnight. Cells were firstly treated by Lenti-rtTA/neo, Selection was terminated when control cells were completely dead and antibiotic free medium were used for propagation. Neomycin resistant cells were then infected by Lenti-miR126-eGFP/puro and grown with2μg/ml Puromycin. Double resistance cells were ultimately obtained, and2μg/ml doxycycline was added to medium and intrigue expression of miR126.
Results:
(1) MiR-126genomic sequence was amplified and lentiviral expression vector were constructed.
(2) LV-miR-126pseudovirus was packaged and the titer was determined:
(3)293cell infected by LV-miR-126can over-express miR-126.
Conclusion:
LV-miR-126infection can enhance the expression level of miR-126in293cell lines.
Part III MiR126promote the expression of ERK and AKT in MSCs
Objective:
To evaluate the role of miR-126in ERKI and AKT signal transduction pathways.
Methods:
Total RNA was extracted from cells with All-in-One miRNA qRT-PCR Detection Kit according to the manufacturer's instructions. Expression of miR126was detected by quantitative real-time RT-PCR using the All-in-OneTM miRNA qPCR Primer. The relative levels of miR126transcripts were normalized to the control U6mRNA. Relative gene expression was quantified using the GraphPad Prism4.0software and expressed as%of the control.
The miR126transfected and nontransfected MSCs were collected separately for ERK1and PERK1、AKT and pAKT Western blot assay. Chemiluminent detection was performed with the ECL kit. Bands on Western blots were quantified by densitometry using Quantity One software.
Results:
Results uncovered that under higher expression of miR126, ERK1and pERK1、 AKT、pAKT and capillarynetwork density were dramatically increased (P＜0.01).
Conclusions:
MiR-126represses the expression of spredl and PIK3R2, which negatively regulate VEGF signaling via the ERK and AKT pathways, respectively.
Part IV Transplantation of mesenchymal stem cells overexpressing MiR126Enhances Ischemic angiogenesis via AKT/ERK-related pathway
Objective:
To whether a investigate new strategy that combines MSCs transplantation and ex vivo miR-126transferring with lentiviral vectors was more therapeutically efficient than MSCs cell therapy alone in a C57mice Myocardial infarction model
Methods:
Mice models of myocardial infarction were established by ligation of coronary artery. The ligated animals were randomly divided into three groups (15in each) and, after2weeks, were intramyocardial injected at the heart infarct zone with miR126-transfected MSCs (miR126-MSCs Group) MSCs (MSCs group), or medium (PBS group).Six weeks later, histological study and Echocardiographic assessment were performed.
Results:
Capillary density of the infarcted region was significantly improved in the miR126-MSCs group compared with the MSC group and the PBS group (both P＜0.01). Western blot showed that ERK1,pERK1, AKT and pAKT gene were dramatically enhanced in the miR126-MSC group compared with the MSC group and the PBS group (both P<0.05) and. Echocardi ography showed MiR126led to sustained improvement in cardiac function, as assessed by left ventricle ejection fraction (LVEF) and fraction of shortening(FS).
Conclusions:
The present study shows that Mesenchymal stem cells overexpressing of MiR126transplantation therapy induced more potent angiogenesis and collateral vessel formation than MSC cell therapy alone. We put forward the hypothesis that transplantation of MSCs transfected with mi R126can improve angiogenesis and cardiac function in infarcted area of mice heart, which may be due to stimulation of AKT/ERK-related pathway.
目前,缺血性心肌病是人类死亡和致残的主要疾病之一。由于冠脉的狭窄和闭塞导致组织缺血和缺氧,伴随心绞痛、心梗、心律失常及心衰等症状,当心功能恶化到Ⅳ级时,1年死亡率可达60%。在过去几十年里,药物治疗、经皮冠状动脉腔内成形+支架术、冠状动脉搭桥术及心脏移植等已经显著改善了缺血性心肌病患者的预后。尽管这些治疗措施已经取得了很大的进步,但仍存在许多限制,因此,很多学者致力于干细胞移植的研究。
心肌梗死的修复过程中常看到冠脉侧支循环的形成,侧支循环能够改善缺血心肌灌注,减少心肌坏死,恢复“冬眠”心肌细胞的活性,从而减少心功能障碍的发生。然而机体自身生成的新生血管往往不足以代偿冠脉闭塞诱发的心肌缺血。因此,刺激缺血区小血管生长,促进侧支循环的形成,完成缺血区血管的自我搭桥便成了十分诱人且似乎直观合理的治疗方法。近年来,随着对血管新生和血管生长因子研究的不断深入,治疗性血管新生已成为治疗缺血性心脏病的新方法。
目前有关MSCs移植治疗心肌梗死的动物实验表明,MSCs植入损伤部位后可长期存活,并分化为具有心肌细胞特征的类心肌细胞,促进“血管新生”,改善血液动力学。但仍然存在着大量有待解决的问题。例如如何增加移植后的干细胞存活率；改善梗死区的微环境,增加迁移或归巢到梗死区干细胞的数量等。近来,对于miRNAs调控干细胞的发生发展、自我更新、分化、抗凋亡及炎症和血管新生等一系列生物过程知识的积累,有学者认为miRNAs可以作为干细胞移植治疗缺血性心肌病研究的一个新的切入点。
MiRNA126是血管内皮上的特殊的MiRNA,它在心梗后的血管发生和维持血管的完整性起着重要作用。MiRNA126可通过细胞分裂素活化蛋白酶发信号给VEGF与FGF来起作用。Spred-1和PIK3R2/p85-b是对Ras/细胞分裂素活化蛋白酶的抑制因子,可被MiRNA126抑制,而Spred-1和PIK3R2/p85-b可能抑制ERK\AKT途径从而抑制血管生成。因此,上调MiRNA126可减轻Spred-1和PIK3R2/p85-b对VEGF及FGF的抑制作用,有利于血管发生。我们推测MiRNA126通过对干细胞的调节可能为干细胞移植治疗缺血性心脏病开辟一条新途径。
第一部分小鼠骨髓间充质干细胞的体外培养、诱导及鉴定
研究目的
建立小鼠骨髓间充质干细胞(MSCs)的分离、培养方法,研究其在体外生长增殖的生物学特性及表型特征.
研究方法
应用60%的Percoll细胞分离液分离骨髓间质干细胞,接着进行培养和传代。应用MTT方法测定培养细胞的生长曲线,应用流式细胞仪测定细胞周期和鉴定培养细胞的纯度,同时成骨诱导分化及成脂诱导分化鉴定骨髓间质干细胞
实验结果
原代培养的BMSCs24小时内贴壁并伸展成多角形、梭形,前3天为相对抑制期,其后细胞增殖速度逐渐加快,呈对数形式,7天左右至平台期,细胞覆盖率达90%左右,约在2周内传至第3代时可到106个数量级细胞,能够满足细胞移植数量的需要,应用流式细胞仪检测BMSCs的标志,显示CD29, CD44阳性,而CD34, CD45阴性,符合BMSCs特征。
实验结论
本课题应用60%Percoll细胞分离液成功地分离和培养出较为均一的骨髓间质干细胞。骨髓间质干细胞体外培养生长稳定,传代后细胞生长较快,适应能力较强,细胞大多数处于静止期,增殖潜力巨大。
第二部分MiR126转染小鼠骨髓间充质干细胞
研究目的：
1构建miR-126的慢病毒表达载体,包装生产重组慢病毒,并进行慢病毒滴度、活性测定。
2包装生产重组慢病毒,慢病毒滴度、活性测定
研究方法
我们利用Gateway clone构建pDown-miR126及pLV.EX3d.P/puro-TRE> miRl26>IRES/eGFP。制备编码了慢病毒颗粒的重组病毒质粒及其pLV/helper-SL3, pLV/helper-SL4, pLV/helper-SL5辅助包装原件载体质粒,三种质粒载体分别进行高纯度无内毒素抽提,重组质粒DNA进行去内毒素质粒提取后,与包装系统质粒混合物按Invitrogen公司Lipofectamine2000使用说明进行共转染293TN细胞,然后进行慢病毒的包装,包装完成之后进行重组慢病毒滴度检测。
实验结果：
(1)成功构建miR-126的慢病毒表达载体。
(2)成功进行了LV-miR-126重组病毒的生产和滴度测定。
(3)慢病毒感染293TN细胞后,能够大大增强目的基因的表达。
实验结论：
通过慢病毒途径可以有效增强miR-126在宿主细胞中的表达
第三部分观察MiRNA126对ERK\AKT调控作用
研究目的：
在MSCs中过表达miRNA126以观察其对ERK\AKT的影响及体外诱导骨髓间充质干细胞分化为血管内皮细胞。
研究方法：
总RNA提取是根据All-in-One miRNA qRT-PCR Detection Kit生产厂家的说明进行,miR126的表达行quantitative real-time RT-PCR检测,测试miRNA以U6作为单拷贝基因对照。阴性对照组及控制组相比在过表达miRNA126的MSCs中ERK\AKT的表达行Westernblot检测。在第3代,过表达miR-126骨髓间充质干细胞与骨髓间充质干细胞分别按1×109/L密度接种,加入含血管内皮细胞生长因子、碱性成纤维细胞因子的DMEM培养基向血管内皮细胞诱导分化,ECMatrixTM凝胶体外血管新生实验观察血管新生情况。
实验结果：
在过表达miRNA126的MSCs中ERK\AKT表达明显增加及过表达miRNA126的MSCs中毛细血管密度明显增加。
实验结论：
在MSCs中过表达MiRNA126对ERK\AKT具有调控作用,能促进干细胞分化为血管内皮细胞。
第四部分骨髓间充质干细胞过表达MIR126移植通过AKT/ERK途径促血管新生及改善心脏功能
研究目的：
比较骨髓间充质干细胞过表达MIR126移植与单纯BM-MSCs移植在小鼠心梗模型中的促血管新生作用及其机制。
研究方法：
通过结扎C57小鼠冠状动脉前降支制备心肌梗死模型,C57小鼠随机分为3组：MIR126-MSC组(即过表达MIR126的MSCs移植,n=15),MSC组(单纯的MSCs移植,n=15)以及PBS对照组(注射PBS, n=15)。在术后14天,进行细胞移植。实验组每只C57小鼠接受总计5×106(0.1m1)个过表达MIR126骨髓间充质干细胞或MSCs,分成6个点注入C57小鼠缺血心肌；对照组注入同等体积的无菌PBS。6周后行组织学及超声检查。
实验结果：
细胞移植6周后,MSC组和MIR126-MSC组缺血肌肉组织的血管新生均明显增强,以MIR126-MSC组的毛细血管密度最高(P<0.01)。Western-blot结果显示,MSC组和MIR126-MSC组缺血肌肉组织中ERK1, AKT蛋白表达均明显高于PBS对照组,以MIR126-MSC组的表达量最高(P<0.05)。细胞移植6周后,组MiRNA126-MSCs组、BMSCs组心功能都较移植前有明显改善(p<0.05),而其中MiRNA126-MSCs细胞移植组EF、FS改善程度要明显好于单纯细胞移植组(P<0.05)。
实验结论：
与单纯的MSC细胞移植疗法相比,过表达MIR126骨髓间充质干细胞移植具有更强的促进血管新生和侧枝血管形成的能力。我们推测过表达MIR126骨髓间充质干细胞移植通过ERK/AKT途径改善心功能,MIR126可能为干细胞移植治疗缺血性心脏病开辟一条新途径。
Backgroud
Ischemic heart disease which triggers dysfunction and the death of cardiomyocytes is the most common cause of death throughout the world despite continued advances in the prevention and treatment of coronary artery disease. The symptoms of ischemic heart disease are angina, myocardial infarction, arrhythmia and heart failure, which are the consequence of decreased patency of atherosclerotic vessels. The mortality of heart failure can reach60%within one year for patients in New York Heart Association functional class IV.Over the past decades,improvements in medical therapy, percutaneous transluminal coronary angioplasty(PTCA), coronary artery bypass surgery(CABG) and heart transplantation, have dramatically improved the prognosis of ischemic heart disease. However, all the methods including drugs, thrombolysis and revascularization are not able to influence the dead myocardium. Many researchers devote to studies in the field of myocardium regeneration and repairing insulted myocardium with stem cells.
During AMI repairs, coronary collateral formed which could improve myocardial perfusion, circulation(CC)often decrease myocardial necrosis, restore hibernating myocardial viability and reduce left ventricular function disorder. As self neovascularisation is insufficient for compensation myocardial ischemia induced from CHD, stimulating small vessels to develop in ischemic region and promoting to form CC become attractive and reasonable strategy. As study of the angiogenesis and VGF development, theraputic angiogenesis has become a new approach to treat IHD. This strategy is designed to promote the development of supplemental collateral blood vessels that will constitute endogenous bypass conduits around occluded native arteries, a strategy termed "therapeutic angiogenesis."
Recentely,a few studys on MSCs to treat MI in animal experiment have proven that MSCs have the ability of differentiating into cardiac myocytes, living long time, promoting angiogenesis, and improving haemodynamics. However, some problems, for example, how to further augment the implanted cells survival and other functions to overcome the limitations for their clinical applications, are still to be explored. Since the knowledge of a variety of biological processes, including developmental timing, signal transduction, tissue differentiation and maintenance, Antiapoptosis, Anti-inflammation and neoangiogenesis under miRNAs regulate, new therapeutic targets for ischemic heart disease may identified in these non-coding RNAs.
MiR-126is an endothelial cell-specific miRNA that plays an essential role in neoangiogenesis following MI and in maintenance of vascular integrity [94]. MiR-126functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1and phosphoinositol-3kinase regulatory subunit2(PIK3R2/p85-beta). MiR-126represses the expression of spredl and PIK.3R2, which negatively regulate VEGF signaling via the ERK and AKT pathways, respectively. Thus, in the absence of miR-126, Spred-1/PI3k expression is elevated, resulting in repression of angiogenic signaling. Conversely, miR-126overexpression relieves the repressive influence of Spred-1/PI3k on the signaling pathways activated by VEGF and FGF, favoring angiogenesis. It is reasonable to speculate that miRNA126, a novel but practical regulator of stem cell implantation for ischemic heart disease, introduce a promising strategy for the treatment of myocardial infarction.
Part Ⅰ. MSCs isolation, culture and identification
Objective
This study was to establish the experimental method for isolating, culturing, proliferating and inducing mice bone marrow mesenchymal stem cells in vitro.
Methods
We isolated MSCs from animal bone marrow and separated MSCs by Percoll Centrifugation.MSCs were cultured and subcultured in low-glucose Dulbecco's modified Eagle's medium (DMEM) with10%selected fetal calf serum. Cell growth patterns were evaluated by MTT test. Cell cycle and surface antigenic features were analyzed by flow cytometry technique.
Results:
To isolate MSCs from bone marrow aspiration, the samples were fractionated with Percoll by density gradient centrifugation for mononuclear cell isolation. Through replacing culture medium, the non-adherent cells were removed and the remaining tightly adhered cells were MSCs. The morphology of cultured MSCs was fibroblast-like form. Primary cultured BMSCs adhered to and stretched into polygon or spindle within24hours. The first3days was the relative inhibition period, and then the cell proliferation accelerated gradually in a pattern of logarithm and reached the period of plateau by Day7or so, with90% cell coverage. The number of P3BMSCs can reach1062weeks later, and that can satisfy the required number of cell transplantation. The signs of BMSCs with positive CD29, CD44, CD90and negative CD34, CD45were detected by the flow cytometry, and that is consisted with the characteristics of BMSCs.
Conclusions:
In this study, we believed the adherent cells in cultured medium were mainly MSCs obtained by Percoll fractionation. Numerous molecular markers of MSCs were characterized, but unique marker of MSCs and any marker of blood cells and endothelial cells were not identified. MSCs showed stable growth in vitro, easy survival in subculture and rapid proliferation in culture medium.
Part II Transfection of miR-126in mesenchymal stem cellsand its expression
Object:
Construct Lentivirus vector to express miR-126, produce pseudoviral particles and determine its titer.
Methods:
Lentiviral MicroRNAs expression vector was constructed with Gateway system. Mature miR126, TRE promoter and eGFP sequences were inserted into plasmids to produce pUp-TRE, pDown-miR126and pTail-IRES/eGFP; scramble sequence was set as negative control pLV.EX3d.P/puro-TRE> miR126>IRES/eGFP was obtained with incubation of donors and accepter vectors catalyzed by LR clonase. Plasmid was then sequenced and purified for lentivirus envelope.
Envelope helper plasmids:pLV/helper-SL3, pLV/helper-SL4, pLV/helper-SL5, with pLV.EX3d.P/puro-TRE-miR126-IRES/eGFP or pLVrtTA/neo which contains the imperative elements for virus packaging, were co-transfected into293T cells with lipofectamine2000, according to the manufacture's instructions for the generation of Lenti-miR126-eGFP/puro or Lenti-rtTA/neo respectively.
To perform lentiviral infections, the mice MSCs cells were plated at40%-50%confluence and incubated overnight. Cells were firstly treated by Lenti-rtTA/neo, Selection was terminated when control cells were completely dead and antibiotic free medium were used for propagation. Neomycin resistant cells were then infected by Lenti-miR126-eGFP/puro and grown with2μg/ml Puromycin. Double resistance cells were ultimately obtained, and2μg/ml doxycycline was added to medium and intrigue expression of miR126.
Results:
(1) MiR-126genomic sequence was amplified and lentiviral expression vector were constructed.
(2) LV-miR-126pseudovirus was packaged and the titer was determined:
(3)293cell infected by LV-miR-126can over-express miR-126.
Conclusion:
LV-miR-126infection can enhance the expression level of miR-126in293cell lines.
Part III MiR126promote the expression of ERK and AKT in MSCs
Objective:
To evaluate the role of miR-126in ERKI and AKT signal transduction pathways.
Methods:
Total RNA was extracted from cells with All-in-One miRNA qRT-PCR Detection Kit according to the manufacturer's instructions. Expression of miR126was detected by quantitative real-time RT-PCR using the All-in-OneTM miRNA qPCR Primer. The relative levels of miR126transcripts were normalized to the control U6mRNA. Relative gene expression was quantified using the GraphPad Prism4.0software and expressed as%of the control.
The miR126transfected and nontransfected MSCs were collected separately for ERK1and PERK1、AKT and pAKT Western blot assay. Chemiluminent detection was performed with the ECL kit. Bands on Western blots were quantified by densitometry using Quantity One software.
Results:
Results uncovered that under higher expression of miR126, ERK1and pERK1、 AKT、pAKT and capillarynetwork density were dramatically increased (P＜0.01).
Conclusions:
MiR-126represses the expression of spredl and PIK3R2, which negatively regulate VEGF signaling via the ERK and AKT pathways, respectively.
Part IV Transplantation of mesenchymal stem cells overexpressing MiR126Enhances Ischemic angiogenesis via AKT/ERK-related pathway
Objective:
To whether a investigate new strategy that combines MSCs transplantation and ex vivo miR-126transferring with lentiviral vectors was more therapeutically efficient than MSCs cell therapy alone in a C57mice Myocardial infarction model
Methods:
Mice models of myocardial infarction were established by ligation of coronary artery. The ligated animals were randomly divided into three groups (15in each) and, after2weeks, were intramyocardial injected at the heart infarct zone with miR126-transfected MSCs (miR126-MSCs Group) MSCs (MSCs group), or medium (PBS group).Six weeks later, histological study and Echocardiographic assessment were performed.
Results:
Capillary density of the infarcted region was significantly improved in the miR126-MSCs group compared with the MSC group and the PBS group (both P＜0.01). Western blot showed that ERK1,pERK1, AKT and pAKT gene were dramatically enhanced in the miR126-MSC group compared with the MSC group and the PBS group (both P<0.05) and. Echocardi ography showed MiR126led to sustained improvement in cardiac function, as assessed by left ventricle ejection fraction (LVEF) and fraction of shortening(FS).
Conclusions:
The present study shows that Mesenchymal stem cells overexpressing of MiR126transplantation therapy induced more potent angiogenesis and collateral vessel formation than MSC cell therapy alone. We put forward the hypothesis that transplantation of MSCs transfected with mi R126can improve angiogenesis and cardiac function in infarcted area of mice heart, which may be due to stimulation of AKT/ERK-related pathway.
引文
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