【作者】 李巧；

【导师】 王志刚；

【作者基本信息】 重庆医科大学 ， 影像医学与核医学， 2010， 博士

【摘要】 冠心病是影响人类生命健康的主要疾病之一,虽然传统的药物治疗、冠状动脉介入治疗以及冠脉旁路移植等技术的广泛应用,使冠心病患者的病死率呈下降趋势,但相当一部分冠心病患者仍不适宜上述治疗方法。心肌血管新生是当前缺血性心脏病研究的重点和难点。近年来,国内外学者利用干细胞移植,通过血管及其心肌细胞的再生来治疗缺血性心脏病(Ischemic heart disease,IHD),目前研究较多的是单个核细胞(Mononuclear cells,MNCs)和间充质干细胞(Mesenchymal stem cells,MSCs),但是由于移植成分较复杂,随着临床应用例数的增多,不良反应的报告也日趋增多。内皮祖细胞(Endothelial progenitor cells,EPCs)是一类能循环、增殖并分化为血管内皮细胞(Endothelial cells,ECs)的前体细胞,但尚未表达成熟血管ECs表型,也未形成血管。它包括从血液母细胞到成熟ECs之间多个阶段的细胞。EPCs有促进组织器官,尤其是缺血组织器官ECs修复,建立侧支循环和恢复血供的作用,它不仅参与胚胎时期的血管形成,而且在出生后成体的血管生成中也起重要作用。干细胞移植主要有经冠状动脉内注射、经心内膜下注射、开胸心内注射及经静脉移植等几种方式。研究表明,采用上述方式进行细胞移植均可不同程度的改善缺血性心脏病患者的心功能,但是需要高浓度的干细胞,而且前三种移植方式必须在介入操作情况下才能完成;经静脉干细胞移植途径接近于无创,但大量细胞可随血流迁移至其他组织、器官,引起不必要的血管新生。因此,很有必要寻找一种既能通过静脉途径输入又能在靶组织内定向转移的细胞移植方法。近年来研究发现,超声微泡作为增强组织灰阶显像的造影剂,也可用于药物或基因传递。将特定的药物或基因与微泡结合在一起,通过外周血管注射,经体表超声定位辐照破坏微泡,可使药物或基因在特定组织内定位释放。微泡破裂后产生的生物学效应可使靶组织微环境改变,如毛细血管内皮间隙增宽,细胞膜通透性增高,从而使药物或基因进入特定的组织内。另外,超声辐照微泡可以引起局部组织的炎症反应,引起炎细胞聚集,炎性因子的释放,从而产生一些潜在的生物学效应。有研究证实,超声破坏微泡可增加局部组织血管内皮细胞粘附分子1 (Vascular cell adhere molecular-1,VCAM-1)的表达,从而促进骨髓MNCs粘附于内皮上,增加MNCs的归巢数量。在诸多用于治疗IHD的基因选择上,缺氧诱导因子1α(Hy- poxia inducible factor-1α,HIF-1α)作为机体氧稳态调节的重要转录因子日渐引起人们的关注, HIF广泛存在于人和哺乳动物的体内,可以上调多达150种细胞因子的表达,是组织、细胞在缺氧应答反应中最重要的调节因子。此外有研究证实,HIF-1α可以促进基质细胞衍生因子(Str- omal cell-derived factor-1,SDF-1)的表达,而SDF-1作为一种趋化因子,在干细胞的流动及迁移中起非常重要的作用,可增加干细胞对缺血组织粘附及归巢。基于上述理论基础,超声破坏微泡能否作为一种新型的促进EPCs定向移植的有效方法有待进一步探讨,因此本课题利用超声破坏微泡联合载HIF-1α及增强型绿色荧光蛋白(Enhanced green flucorescent protein,EGFP)的重组腺病毒Ad-EGFP/HIF-1α介导EPCs移植,观察其对大鼠心肌梗死(Myocardial infarction,MI)的治疗效果,为IHD的治疗提供一种新的途径和方法。综上,本?课题研究主要包括以下三个部分:第一部分大鼠骨髓源性EPCs的分离、培养及鉴定目的研究大鼠骨髓来源的EPCs的分离、培养、鉴定方法以及向ECs分化的诱导条件。方法Percoll密度梯度离心法分离SD大鼠股骨及胫骨骨髓MNCs,经差速贴壁后取2次贴壁细胞置于纤维连接蛋白铺被的培养板中,血管内皮细胞生长因子(Vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(Basic fibroblast growthfactor,bFGF)及表皮细胞生长因子(Epidermal growth factor,EGF)诱导下培养2w,观察其形态学改变,对其进行免疫细胞化学染色以及Dil标记的乙酰化低密度脂蛋白(Acetyl low density lipoprotein,acLDL)、FITC标记的荆豆凝集素1 (Ulex europaeus agglutinin,UEA-1)双荧光染色对其进行鉴定。结果普通光学显微镜观察:贴壁细胞呈铺路石、团簇样生长,梭形、线样排列等特殊形态;免疫细胞化学染色:贴壁细胞CD133、CD34、Flk-1(VEGFR)、vWF (Ⅷ因子)在不同时段呈阳性表达;激光共聚焦显微镜观察:EPCs吞噬Dil-acLDL及结合FITC-UEA-1呈双染色。结论采用Percoll密度梯度离心法,在VEGF、bFGF及EGF培养体系下,可以获得较高纯度的EPCs,该细胞具有ECs的特性,经体外诱导可以分化为ECs。第二部分Ad-EGFP/HIF-1α感染大鼠EPCs的实验目的研究含HIF-1α及EGFP的Ad-EGFP/HIF-1α感染大鼠EPCs的可行性。方法采用Percoll密度梯度离心法分离SD大鼠骨髓MNCs,VEGF、bFGF及EGF诱导培养,观察其形态学变化,对其进行免疫学及功能学鉴定;Ad-EGFP/HIF-1α在HNK293细胞中进行扩增,之后感染体外培养的EPCs,分别于24,48,72,96 h采用倒置荧光显微镜观察细胞表达EGFP的情况;MTT法检测细胞生长活性;流式细胞仪检测转染率;RT-PCR法测定HIF-1α在EPCs中的表达。结果倒置荧光显微镜下可观察到EPCs内EGFP的表达,随时间延长,表达EGFP细胞数逐渐增多;MTT检测EPCs在24,48,72,96 h存活率分别为99.83%,99.70%,99.32%,99.57%;流式细胞仪计数显示,EPCs在24,48,72,96 h的转染率分别为23.05%,45.94%,78.91%,85.64%;通过RT-PCR法检测到EPCs内HIF-1α的表达。结论重组腺病毒Ad-EGFP/HIF-1α能够有效地感染EPCs,感染后在mRNA水平可检测到HIF-1α的表达,并对EPCs活性无明显影响,为进一步研究转基因的EPCs移植促进血管新生奠定了基础。第三部分超声微泡联合Ad-EGFP/HIF-1α介导EPCs移植治疗大鼠心肌梗死的实验研究第一节超声微泡联合Ad-EGFP/HIF-1α介导EPCs归巢大鼠缺血心肌目的研究超声破坏微泡联合Ad-EGFP/HIF-1α介导EPCs移植归巢大鼠缺血心肌的可行性及有效性。方法采用Percoll密度梯度离心法分离SD大鼠骨髓MNCs,VEGF、bFGF及EGF诱导培养,观察其形态学改变,对其进行免疫学及功能学鉴定;Ad-EGFP/HIF-1α在HNK293细胞中进行扩增,之后感染体外培养的EPCs。将30只SD大鼠建立MI模型后随机分为5组:①空白对照组(C),②超声+微泡组(US+MB),③内皮祖细胞组(EPCs),④超声+内皮祖细胞组(US+EPCs),⑤超声+微泡+内皮祖细胞组(US+MB+EPCs)。建模成功后第3天,经尾静脉注入超声微泡,同时超声基因转染治疗仪辐照大鼠心前区,辐照条件:300KHz、2W/cm2,辐照10s,间隔5s,共5min。建模成功后第5天、第7天行同样处理,之后通过尾静脉注射感染Ad-EGFP/HIF-1α的EPCs。EPCs移植后48h将大鼠处死,激光共聚焦显微镜观察大鼠心肌组织内EPCs的分布; Western blot检测HIF-1α的表达。结果US+MB+EPCs组绿色荧光强度高于其他各组,Western blot显示,US+MB+EPCs组HIF-1α蛋白表达高于其他组(P<0.05)。结论超声破坏微泡联合Ad-EGFP/HIF-1α介导EPCs移植可有效促进EPCs归巢,为干细胞移植治疗IHD提供了一种新的途径。第二节超声微泡联合Ad-EGFP/HIF-1α介导EPCs移植治疗大鼠心肌梗死的研究目的研究超声破坏微泡联合Ad-EGFP/HIF-1α介导EPCs移植治疗大鼠MI的可行性。方法采用Percoll密度梯度离心法分离SD大鼠骨髓MNCs,VEGF、bFGF及EGF诱导培养,观察其形态学改变,对其进行免疫学及功能学鉴定;Ad-EGFP/HIF-1α在HNK293细胞中进行扩增,之后感染体外培养的EPCs。将30只SD大鼠建立MI模型后随机分为5组:①空白对照组(C),②超声+微泡组(US+MB),③内皮祖细胞组(EPCs),④超声+内皮祖细胞组(US+EPCs),⑤超声+微泡+内皮祖细胞组(US+MB+EPCs)。建模成功后第3天,经尾静脉注入超声微泡,同时超声基因转染治疗仪辐照大鼠心前区,辐照条件:300KHz、2W/cm2,辐照10s,间隔5s,共5min。建模成功后第5天、第7天行同样处理,之后通过尾静脉注射感染Ad-EGFP/HIF-1α的EPCs。EPCs移植后4w,超声心动图检测大鼠左室收缩功能,HE染色检测心肌梗死边缘区新生血管情况,免疫组织化学(Immunohis-tochemistry,IHC)染色法检测CD34的表达及微血管密度(Microvessel density,MVD);Western blot检测VEGF蛋白的表达。结果超声心动图检测左室收缩功能显示,US+MB+EPCs组射血分数(Ejection fraction,EF)、短轴缩短率(Fractional shortening,FS)较其他各组测值高,差异有统计学意义(P<0.05);HE染色显示,US+MB+EPCs组有较多的新生血管;IHC染色显示,新生血管被染成棕黄色,US+MB+EPCs组表达最多,MVD计数与其他各组相比,差异有统计学意义(P<0.05);Western blot显示,US+MB+EPCs组的VEGF蛋白表达高于其他各组(P<0.05)。结论超声破坏微泡联合Ad-EGFP/ HIF-1α介导EPCs移植可通过促进VEGF的高效表达、刺激MI区血管生成等途径改善心功能,为IHD的治疗提供了一种新的途径和方法。更多还原

【Abstract】 Ischemic heart disease (IHD) is one of the major serious global health problems. Although coronary heart disease (CHD) mortality has decreased with the extensive application of drugs,percutaneous coronary intervention and coronary artery bypass graft, a number of patients are still not suitable for those treatments. Myocardial angiogenesis is the focal and difficult point of the current studies of ischemic heart disease. In recent years, researchers treated ischemic heart disease by using stem cells transplantation, which become a hot spot direction. The current studies most focus on mononuclear cells (MNCs) and mesenchymal stem cells (MSCs). But with the increased number of cases in clinical application, reports of adverse reaction are also increased, especially arrhythmias, etc.Endothelial progenitor cells (EPCs) can proliferate and differentiate into vascular endothelial cells (ECs) without expressing phenotype of mature ECs and forming vascular precursors. It includes multiple stages of cells from the mother cells to mature blood vessel ECs. EPCs can promote repairment of ECs, establishment of collateral circulation and restoration of blood supply particularly in ischemic tissues and organs. It can not only involved blood vessel formation in embryonic, but also play an important role in angiogenesis after birth.The main ways of stem cells transplantation are focus on coronary artery injection, endometrial injection, open-chest cardiac injection and autologous transplantation, et al. Studies have shown that adopted those approaches above could improve cardiac function in patients with ischemic heart disease, but those approaches need a high concentration of cells and also involved interventional operation. Transplantation from intravenous injection is a noninvasive way, but a large number of blood cells may migrate to other tissues and organs which may cause unexpected angiogenesis. Therefore, it is necessary to find a way to transplant stem cells by intravenous injection and the cells can shift into targeted tissue.Recent studies have found that as an ultrasound contrast agent, micro- bubbles were not only enhance the gray-scale ultrasound imaging, but also can be used as a new drug or gene delivery vehicles. After ultrasound Irridiation, cavitation and mechanical effects produced by microbubbles destruction could increase permeability of cells membrane, make rupture of microvessel, broaden the endothelial cell gap, so that the target gene could reach the tissue and cells from the broken capillaries and endothelial cell gap, which enhanced the targeted therapy and efficacy of gene. Meanwhile, ultrasound targeted microbubbles distruction (UTMD) can cause local tissue inflammation and accumulation of inflammatory cells, release of inflammatory factors, which can induced some potential biological effects. Researches has shown that ultrasound microbubbles destruction could increase the expression of local vascular cell adhere molecular 1 (VECM- 1), there by, promot MNCs adhere on the endothelium.HIF-1αas an important regulator of oxygen homeostasis transcription factor caused more concern. HIF exist widly in human and mammals, it can promote the expression of more than150 cytokines. It is the most important regulator in the hypoxic response reactions.Based on the theories above, whether Ultrasound microbubbles destruction could be used as a new effective way of stem cells transplantion need to be further explored. Therefore, this subject was to transplant EPCs used ultrasound microbubbles combined with recombinant adenovirus (Ad- EGFP/HIF-1α), then observed the effect of the therapeutic of myocardial infarction, so as to offer a new approach and methods to treat ischemic heart disease.Overall,The study was composed of the following three parts.PARTⅠIsolation, Cultivation and Identification of Endothelial Progenitor Cells from Rat Bone MarrowObjective To investigate the methods of isolating and culturing endothelial progenitor cells (EPCs) from rat bone marrow,as well as their differentiating into endothelial cells. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation, then plated on dishes coated with fibronectin and induced with VEGF, bFGF and EGF for two weeks. The expression of cell markers was assessed by immunocytochemistry, and the attached cells were stained with Dil-ac-LDL and FITC-UEA-1. Results The attached EPCs were able to line up in the typical cord-like structure and formed clusters and cobblestone. Adherent cells showed CD34,CD133,Flk-1 andⅧ/vWF positive in different time and took up Dil-acLDL,bound FITC-UEA-1 double positive fluorescence by laser confocal microscopy. Conclusions The mononuclear cells can be isolated from rat bone marrow using percoll density gradient centrifugation,and after induction using VEGF, bFGF and EGF, we can get high purified EPCs with the same characteristics as the endothelial cells,and EPCs can be differentiated into endothelial cells in vitro.PARTⅡInfection of Recombinant Adenovirus Ad-EGFP/ HIF-1αto Rat Endothelial Progenitor Cells in VitroObjective To investigate the feasibility of infection of recombinant adenovirus Ad-EGFP/HIF-1αto rat EPCs. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation and induced with VEGF, bFGF and EGF. The expression of cell markers was assessed by immunocytochemistry. Ad-EGFP/HIF-1αwas amplified in HNK293 cells, and then transfected EPCs in vitro. Expression of EGFP in infected EPCs was observed by fluorescence microscope, cell growth activity was detected by MTT, infection efficiency was detected by flow cytometry, and expression of HIF-1αin EPCs was verified by RT-PCR analysis. Results After EPCs were infected with recombinant adenovirus, green fluorescence was found and increased gradually with the time in EPCs.The survival rates of EPCs had no significant change after infection, and the infection rate increased gradually with the time. HIF-1αwas detected in infected cells by RT-PCR. Conclnsions The recombinant adenovirus Ad-EGFP/HIF-1αcan infect EPCs effectively, without significant effect on activity of EPCs.PART III Combination of Ultrasound Microbubbles and Recombinant Adenovirus Ad-EGFP/ HIF-1αMediate EPCs to Rat Myocadial Infarction SECTIONⅠStudy on Myocardium-Targeted Homing ofIntravenously Implantated EPCs by Ultrasound Microbubbles and Ad-EGFP/ HIF-1αObjective To explore the feasibility of Myocardium-targeted homing of intravenously implantated EPCs by Ultrasound Microbubbles and Ad-EGFP/ HIF-1α. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation and induced with VEGF, bFGF and EGF. The expression of cell markers was assessed by immunocytochemistry. Ad-EGFP/HIF-1αwas amplified in HNK293 cells, and then transfected EPCs in vitro. Thrity Sprague Dawley rats were randomly divided into 5 groups after the models of myocardial infarction were prepared.①Blank control group(C),②Ultrasound+Microbubbles group(US+MB),③Endothelial progenitor cells group (EPCs),④Ultra- sound+Endothelial progenitor cells group (US+EPCs) and⑤Ultrasound+ Microbubbles+Endothelial progenitor cells group (US+MB+EPCs). All the rats were sacrificed 48h after EPCs implantation. The intensity of EGFP in the rat myocardium was observed using laser confocal microscopy. The expression of HIF-1αwas respectively detected by Western blot. Results The EGFP intensity in US+MB+EPCs group was the highest among all the groups,and its expression of HIF-1αwas higher than those of other groups. Conclusions Intravenously implantated EPCs by ultrasound microbubbles and Ad-EGFP/ HIF-1αcould augment the homing ability of EPCs to the infracted myocardium, which providing a novel strategy for EPCs implantation of ischemic heart disease.SECTIONⅡCombination of Ultrasound Microbubbles and Recombinant Adenovirus Ad-EGFP/ HIF-1αMediate EPCs to Rat Myocadial Infarction Objective To explore the feasibility of mediated EPCs treating acute myocardial infarction by ultrasound microbubbles and Ad-EGFP/ HIF-1α. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation and induced with VEGF, bFGF and EGF. The expression of cell markers was assessed by immunocyto- chemistry. Ad-EGFP/HIF-1αwas amplified in HNK293 cells, and then transfected EPCs in vitro. Thrity Sprague-Dawley rats were randomly divided into 5 groups after the models of myocardial infarction were prepared.①Blank control group(C),②Ultrasound+Microbubbles group (US+MB),③Endothelial progenitor cells group (EPCs),④Ultrasound+ Endothelial progenitor cells group (US+EPCs) and⑤Ultrasound+Micro- bubbles+Endothelial progenitor cells group (US+MB+EPCs). All the rats were sacrificed 4w after EPCs implantation. The CD34 expression was detected by IHC, and Microvessel density (MVD) was deternined. The expression of VEGF was respectively detected by Western blot. Results IHC showed that CD of US+MB+EPCs group was the highest among all the groups (84.24±3.80/HP vs 17.50±2.28/HP, 27.69±6.42/HP, 54.42±3.54/HP, 67.80±5.72/HP). The expression of VEGF of US+MB+EPCs group was higher than those of other groups. Conclusions Combination of ultrasound microbubbles and recombinant adenovirus Ad-EGFP/ HIF-1αcould mediate EPCs effectively and noninvasively to the ischemic myocardium, and promote the expression of VEGF and angiogenesis, which providing a novel strategy for gene therapy of ischemic heart disease.更多还原