携带bFGF_2基因的腺病毒转染内皮祖细胞用于缺血心肌移植的实验研究
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摘要
缺血性心脏病是目前发病率和死亡率较高的疾病之一。其传统治疗方法无论是PCI还是CABG,效果都受到很多因素的制约。弥漫性、钙化病变或血管末梢病变,均无法进行完全的血运重建。据报道完全的血运重建在CABG患者所占比例小于37%。然而与未完全血运重建相比,一旦进行了完全的血运重建,患者的5年生存率即会显著提高、心绞痛发生率显著降低。因此,PCI或CABG术后,未达到完全血运重建的患者,仍需要附加其它的治疗措施。近年来分子生物学的迅猛发展使心血管疾病的基因治疗成为可能。缺血性心脏病基因治疗的含义是把生长因子基因导入缺血冠脉或心肌中,使之转录、表达有特定生物功能的生长因子蛋白质,从而促心肌血管新生,改善心肌缺血症状,发挥治疗作用。
心肌缺血时,心肌能逐渐建立自身的侧支循环,靠代偿性血管新生,适应局部的缺血缺氧。这种血管新生是由多种生长因子参与的。成纤维细胞生长因子(FGF)是一种作用很强的血管生长因子,它通过促进新生血管的形成,改善局部缺血组织的血运微环境,为受损组织的修复和再生创造条件。FGF分bFGF(碱性)和aFGF(酸性)两种,它们通过相同的受体起作用,有相同的生物学效应。但是,bFGF的促血管新生作用是aFGF的30～100倍,而成为极具临床治疗价值的细胞生长因子。大量动物实验和临床研究证明,FGF可增加缺血性心脏病患者的侧枝循环,因此,通过人为给予外源性FGF(重组蛋白或基因),增加缺血心肌局部生长因子的浓度,可以刺激血管再生,缓解组织缺血,改善心脏功能。但在某些情况下,如衰老、糖尿病、高胆固醇血症等,不仅内源性FGF合成减少,而且血管内皮细胞功能障碍,对FGF的反应能力也下降。在上述情况下,仅仅给以促血管生长因子,不能有效地改善组织缺血。因此,国外很多学者考虑在给予外源性促血管生长因子的情况下,还应提供大量功能良好的内皮细胞,来补充那些因血管再生而增生、迁移、重构的血管壁内皮细胞。
近来研究发现,在人的骨髓、外周血和脐血中存在有内皮细胞的前体细胞-内皮祖细胞(endothelial progenitor cells,EPCs)。EPCs能特异性归巢于缺血部位并分化为成熟血管内皮细胞参与血管新生,通过补充高增殖活性的EPCs促进旁路血管新生来代偿缺血部位的血供以达到治疗的目的,称为“supply side”策略。这一发现为缺血性疾病治疗提供了新思路。可将内皮祖细胞作为血管基因转移的自身载体,转染促血管生长基因的内皮祖细胞,通过血管新生与血管发生双重机制,增强心肌缺血部位的血管生长,改善心功能。但是,以内皮祖细胞为载体表达bFGF基因是否能够增强移植细胞功能并进一步促进缺血心肌再血管化尚未见报道。
本课题将对携带bFGF_2基因的腺病毒载体Ad.bFGF_2进行扩增及鉴定,同时诱导培养大鼠骨髓内皮祖细胞,用Ad.bFGF_2在体外转染骨髓内皮祖细胞并检测其转染效率、蛋白表达及对内皮祖细胞的促增殖及分化作用,在体外研究的基础上,将转染Ad.bFGF_2后的内皮祖细胞移植到大鼠缺血心肌,研究移植细胞的存活,及其对于促进血管生长、增加血流灌注、改善心功能的协同治疗作用。
一、碱性成纤维细胞生长因子基因重组腺病毒载体(Ad.bFGF_2)的扩增及鉴定(实验一)
本研究以携带bFGF_2基因的重组腺病毒载体Ad.bFGF_2及Ad-GFP为原料,在293细胞中进行四代扩增,对重组腺病毒DNA进行PCR检测及接种COS-7细胞行RT-PCR鉴定,将鉴定好的病毒应用氯化铯梯度离心纯化和TCID_(50)方法测定滴度,并对其使用安全性进行鉴定。
研究中筛选扩增完成含有目的基因的重组腺病毒Ad.bFGF_2,PCR法证明重组腺病毒中稳定地整合有bFGF_2的基因,RT-PCR方法检测出重组腺病毒感染细胞中的bFGF_2特异性mRNA,表明这些基因可被有效地转录。在293细胞中筛选、扩增出的第4代高滴度重组腺病毒,TCID_(50)方法测定滴度,得到Ad.bFGF_2滴度为1.1×10~9pfu/ml。经氯化铯密度梯度离心后的滴度达到2.0×10~(12)pfu/ml。制备的重组腺病毒Ad.bFGF_2以50的MOI值感染Hela细胞后,观察7天,细胞未出现病态反应,证明其具有良好的使用安全性。
二、体外分离大鼠骨髓单核细胞并诱导培养内皮祖细胞(实验二)
EPCs在骨髓中的含量很少,因此要获得足量的EPCs,体外细胞培养条件下进行分离、纯化和扩增是十分必要的。本研究通过Ficoll密度梯度离心分离大鼠骨髓单核细胞,差速贴壁后置于纤维连接素包被的培养皿中加入10%FCS的DMEM选择性培养基进行诱导培养内皮祖细胞。用倒置相差显微镜观察培养细胞的生长状态,用CD31、CD34、VRGFR-2、Ⅷ因子免疫组化和BS1-Lectin结合能力鉴定培养细胞类型,流式细胞仪检测培养细胞的纯度,透射电镜观察细胞超微结构和内皮细胞特征性细胞器。
结果显示:原代培养7d时形成由内皮祖细胞组成的细胞集落;10d时形成明显克隆,两周左右,细胞生长至80%～90%融合。传代培养后,细胞形态及密度均匀,前3天为相对抑制期,此后呈指数形式快速增殖,10天后达到80%-90%融合,可进行传代。细胞表面抗原免疫组化检测结果表明,CD34、VEGFR-2、Ⅷ因子、CD31相关抗原表达均为阳性,分别为90%,92%,95%和65%,85%的细胞可结合BS1-Lectin。流式细胞仪检测结果显示,BS1-Lectin阳性的细胞占85.5%。透射电镜观察可见内皮细胞最具特征性的细胞器Weibel-Palade小体。培养液NO含量检测,与主动脉内皮细胞相比无明显差异。这表明诱导培养出的梭形贴壁细胞就是分化中的内皮祖细胞
三、Ad.bFGF_2转染EPCs后bFGF_2的表达、分泌及促EPCs增殖及分化作用的研究(实验三)
为了解重组腺病毒载体对EPCs的最佳转染倍数,本课题以不同MOI值的腺病毒载体Ad-GFP体外转染EPCs,检测转染效率。然后以最佳转染倍数进行EPCs的体外转染研究。Ad.bFGF_2以MOI=50转染EPCs后,RT-PCR检测细胞中bFGF_2 mRNA的转录水平,以免疫组化染色、Western-blot检测转染Ad.bFGF_2后,目的蛋白在细胞内的表达及细胞外的分泌情况:硝酸还原酶法检测细胞培养液中NO水平。用MTT法检测转染Ad.bFGF_2后对EPCs的促增殖作用;将转染后的EPCs种植于细胞外基质Matrigel上,观察细胞分化情况。
结果显示:随着MOI值逐渐增大,Ad-GFP体外转染EPCs的转染阳性率也逐渐增高。MOI=50时阳性率可达90%以上,MOI再增大,阳性率增高不明显。RT-PCR、免疫组化、Western-blot等方法检测表明:MOI=50时Ad.bFGF_2可成功将bFGF_2基因转入EPCs中,并有目的蛋白表达与分泌。与对照组相比,Ad.bFGF_2转染组的EPCs培养液中NO含量更高。MTT检测显示Ad.bFGF_2转染后对EPCs有明显的促增殖作用;转染后的EPCs在细胞外基质(Matrigel Matrix)上生长时,出现分化,细胞相互连接成毛细血管网络样结构,说明Ad.bFGF_2转染能在体外条件下诱导EPCs的分化,刺激血管发生。
四、转染bFGF_2基因的EPCs用于缺血心肌移植的实验研究(实验四)
在上述体外研究的基础上,这一部分研究是将转染Ad.bFGF_2的EPCs进行体内实验,对细胞移植与促血管生长因子联合治疗的效果进行探讨。
取Wistar大鼠60只,随机分为4组,每组15只,结扎左冠状动脉前降支建立急性心肌梗死模型,10分钟后进行细胞移植,实验前移植细胞先以PKH-26和BrdU进行双标记:组Ⅰ移植转染Ad.bFGF_2的EPCs;组Ⅱ单纯移植EPCs;组Ⅲ注射AD.bFGF_2;组Ⅳ注射PBS。手术后7天荧光显微镜观察移植细胞的存活率,免疫组化染色及western-blot检测bFGF蛋白的表达,TUNEL染色观察缺血区心肌细胞凋亡的情况;术后4周抗BrdU及抗Ⅷ因子免疫组化染色评价移植细胞分化、心肌血管再生状况,心脏超声检测评价心脏的大小及收缩、舒张功能
研究表明:移植的EPCs在移植区可存活,与其它组比较,组Ⅰ经Ad.bFGF_2转染的移植细胞存活数量较多;免疫组化及western-blot检测移植区有明显bFGF蛋白的表达;另TUNEL染色显示缺血区心肌细胞凋亡明显减少。术后4周的BrdU及抗Ⅷ因子免疫组化染色结果显示:移植细胞出现分化,形成血管内皮;转染Ad.bFGF_2后的EPCs促血管形成作用明显优于其他各组;心功能得到显著改善。
研究结论:
本课题研究结果显示:将Ad.bFGF_2转染的EPCs移植于心脏缺血梗死区后,移植细胞能长期存活,如同时有bFGF_2基因的有效表达,二者可发挥协同作用,显著增加移植细胞成活率和新生血管数目,减少缺血区心肌细胞凋亡,改善左室舒张末容积和收缩、舒张功能。当然,目前无论是基因治疗还是细胞治疗都还有许多缺陷,还存在许多未知的、尚待解决的问题,有待我们更深入探讨。
Background:
Ischemic heart disease (IHD) is the primary cause of death in the industrialized world. Traditionally, patients with IHD were treated with medicine, percutaneous transluminal ballon angioplasty, or coronary artery bypass graft. Despite the fact that advances in modern medical, surgical and interventional treatment of the disease have produced significantly improved results, the efficacy of all these methods is limited in patients with diffuse distal coronary artery disease. According to reports, complete revascularization was found in only about 37 percent of patients who had coronary artery bypass graft, however, once the myocardium was revascularized completely, the incidents of angina would be decreased and the incidence of 5 year survive was improved significantly. Therefore, additional therapeutic measures were needed in those patients. Recent advances in vascular biology suggest the possibility of a novel therapeutic approach to treatment of advanced coronary artery disease. The definition of gene therapy in ischemic heart desease is to transfect special angiogenic growth factor genes and enhances the expressing of growth factors into myocardium. This approach seeks to augment normal collateral development by exposing the heart to growth factors capable of stimulating the growth of new blood vessels.
During myocardial ischemia, compensatory angiogenesis occurred to adapt to ischemic and anoxaemia. In this procedure, growth factors were essential. Fibroblast growth factor (FGF) is the major regulators of vascular growth. It stimulates proliferation of new vessels, improve the microenvironment of ischemic tissue and benefit for tissular reparation or anagenesis. Fibroblast growth factor was divided into two kinds (aFGF and bFGF). They have the same receptors and biological effect .The effect of bFGF is 10 to 30 times power than aFGF in regulators of vascular proliferation. Experimental and clinical studies have shown that FGF may stimulate vessel growth to the ischemic region. Recently, using FGF treatment in patients with severe ischemic heart disease has been implemented as a medical therapy for coronary artery disease. However, under some circumstances, such as in old and feeble, diabetes or hypercholesteremia patients, it exists not only synthesis decreasing of FGF , but also functional impairment of endothelial cells (ECs) that makes its reaction to FGF decrease. Under this circumstance, only using FGF doesn't improve blood supply of ischemic tissue efficiently, so it was considered that ECs should be provided under using FGF by many oversea scholars.
It was found in recently research that there were endothelial progenitor cells (EPCs), the precursor cell of ECs which existed in bone marrow, peripheral blood and cord blood. EPCs may be distributed to ischemic position specially, differentiate to mature ECs and take part in angiogenesis. This approach is called "supply side" strategy that collateral development is augmented by supplying active EPCs. The method of alleviating myocardial remodeling by implanting cells which are transfected with special angiogenic growth factor genes to enhance the angiogenesis may present a new approach to the treatment of ischemic heart disease. However, whether transfecting with special angiogenic growth factor genes reinforce the function of implanting cells or not isn't reported in previous documents.
In our study, Firstly, recombinant adenovirus (Ad) vector carrying the basic fibroblast growth factor 2 (bFGF_2) was amplifed, identified, and rat bone marrow endothelial progenitor cells were induced. Then EPCs with Ad. bFGF_2 was transfected in vitro and the efficiency of transfection and protein expression were tested. The proliferation and differentiation effect of Ad. bFGF_2 on EPCs were certified. Finally, the EPCs transfected by Ad. bFGF_2 were transplanted into rat ischemic myocardium and the survival of the transplanted cells was found and the ability to increase the growth of neovascularization in ischemic zone which enhance local blood perfusion and subsequently improve myocardial function was identified.
Part 1. Propagation, identification of recombinant adenovirus carrying thebasic fibroblast growth factor 2 gene.
In this study, human embroynic kidney 293 cells were transfected with Ad.bFGF_2 and Ad-GFP. Virus clones were isolated and propagated for restriction analysis. After amplified in 293 cells, the obtained adenovirus DNA was detected by PCR. Meanwhile, recombinant adenovirus was further identified by RT-PCR in COS-7 cells. The desired Ad vectors were purified by density gradient ultracentrifuge and titrated, and the safety of using was tested.
Results: Recombinant adenoviral bFGF_2 was propagated successfully, in which the expressing of bFGF_2 was confirmed by PCR and RT-PCR. The Ad. bFGF_2 were amplified in 293 cells for 4 times. The viral particle tite of Ad. bFGF_2 were determined with the TCID50. The viral particle tite of Ad. bFGF_2 was 1.1×10~9pfu/ml, and viral particle tite of Ad. bFGF_2 was 2.0×10~(12)pfu/ml after purified by density gradient ultracentrifuge. Hela cells which were infected with Ad. bFGF_2 for 7 days didn't show up pathosis reaction, so the safety of using was proved.
Part 2. Isolation, Culture and identification of endothelial progenitor cells.
The number of EPCs is estimated to be too few in bone marrow, so it is important to expand and purify the EPCs for the following experiments. Bone marrow cells suspension of adult rat was prepared by discontinuous gradient centrifugation on Ficoll, mononeuclear cells in the middle layer was collected, cultured for 24 hours, then; suspending cells were put into selective culture medium and cultured in the 10% fetal calf serum coated plate. EPCs were isolated, cultured and expanded. The cells were identified by transmission electron microscope, flow cytometer, immunohistochemical staining with anti-factorⅧ, CD31, CD34, VRGFR-2 antigen, the ability of secreting nitrous oxide and combining BS1-Lectin.
Results: Colonies of EPCs formed at 7th day at primary culture. At about 14th day, EPCs got together about 80～90%, and serial subcultivation was made. The passage EPCs proliferated fast, and could be subcultured about every 10 days. EPCs were identified by flow cytometer and immunohistochemical staining (anti-factorⅧ, CD31, CD34, VRGFR-2 antigen) with high purity. Under transmission electron microscope, the cells displayed
Weibel-Palade bodies which are characteristic of endothelial cells. The cells could also combine BS-1 lectin and secrete nitrous oxide. So, the cells induced and cultured in vitro have the same characters as endothelial cells.
Part 3. Transfection of Ad.bFGF_2 to EPCs and its effects of proliferationand differentiation on EPCs in vitro.
To determine the best MOI, transfection efficiency of adenovirus vector to EPCs were identified by infection with various titrations of Ad-GFP. Ad. bFGF_2 was transfected to EPCs with MOI 50:1. After Ad. bFGF_2 transfection, bFGF_2 mRNA transcription was detected by RT-PCR. bFGF_2 protein expression in EPCs and its secretion in culture medium were measured by immunohistochemical staining, Western-blot respectively. The level of NO in culture medium was also measured. The ability of proliferation of Ad. bFGF_2 on EPCs were measured by MTT assay. Finally, the differentiatial ability of Ad. bFGF_2 on EPCs was examined
Results: The transfection efficiency of Ad-GFP to EPCs was increased with MOI. When MOI reached 50, the efficiency was increased to more than 90%. From then on, the transfection efficiency increased limitedly. So 50:1 was choosed as the most suitable MOI. The RT-PCR, immunohistochemical staining and Western-blot showed that bFGF_2 gene could be correctly transfected into EPCs and bFGF_2 protein could express in EPCs. Its secretion in culture medium was detected. After the transfection, the level of NO in the culture medium was also higher than control group. EPCs transfected with Ad. bFGF_2 were revealed a significant increase of cell number which showed by MTT assay. Furthermore, when seeded on Matrigel Matrix, EPCs transfected by Ad. bFGF_2 were induced into capillary-like structures.
Part 4.Studies of transplantation of endothelial progenitor cells transfected by bFGF_2 gene into ischemic myocardium.
Transplantation of EPCs transfected by Ad. bFGF_2 was carried out in vivo following the above studies. Sixty Wistar rats in which acute myocardial infarction was generated by ligating the left anterior descending coronary artery were divided into four groups (GroupⅠ, transplanted with EPCs transfected by Ad. bFGF_2; GroupⅡ, transplanted with EPCs alone; GroupⅢ, injected with Ad. bFGF_2 alone; GroupⅣ, injected with PBS). EPCs were labeled with PKH-26 and BrdU before transplantation. Ten minutes after myocardial infarction the animals were used for transplantation. Seven days after transplantation, survival of transplanted cells, expression of bFGF_2 and apoptosis of ischemic myocardium were measured by immunohistochemical staining, Western-blot, tunnel staining respectively. Four weeks later, the cardiac function of all alive animals was assessed by echocardiography. Finaly, the hearts were harvested for the pathological examination.
Results: Seven days after transplantation, the transplanted EPCs can survive in the zone of ischemic myocardium. There were more cells survive in group I than that in the other groups. More bFGF protein was expressed and less apoptosis of ischemic myocardium. Four weeks later, the histological study showed differentiation of the transplanted EPCs which formed vascular endothelium. Angiogenesis was more remarkable than that in the other Groups. Echocardiography showed that the systolic and diastolic function in Group I were significantly improved compared with the other groups.
Conclusions:
The transplantation of EPCs transfected with Ad. bFGF_2 could improve the survival ability of transplanted cells, inhibit apoptosis of ischemic myocardium. With bFGF_2 protein expressed, The transfected EPCs have strong potency to protect cardiac function by stimulating angiogenesis. Such a strategy might be useful in the treatment of patients with ischemic heart disease. This dissertation is just the beginning of the reseach of angiogenesis by gene or cell therapy. There are more unknown questions to be studied and reasearched for us.
心肌缺血时,心肌能逐渐建立自身的侧支循环,靠代偿性血管新生,适应局部的缺血缺氧。这种血管新生是由多种生长因子参与的。成纤维细胞生长因子(FGF)是一种作用很强的血管生长因子,它通过促进新生血管的形成,改善局部缺血组织的血运微环境,为受损组织的修复和再生创造条件。FGF分bFGF(碱性)和aFGF(酸性)两种,它们通过相同的受体起作用,有相同的生物学效应。但是,bFGF的促血管新生作用是aFGF的30～100倍,而成为极具临床治疗价值的细胞生长因子。大量动物实验和临床研究证明,FGF可增加缺血性心脏病患者的侧枝循环,因此,通过人为给予外源性FGF(重组蛋白或基因),增加缺血心肌局部生长因子的浓度,可以刺激血管再生,缓解组织缺血,改善心脏功能。但在某些情况下,如衰老、糖尿病、高胆固醇血症等,不仅内源性FGF合成减少,而且血管内皮细胞功能障碍,对FGF的反应能力也下降。在上述情况下,仅仅给以促血管生长因子,不能有效地改善组织缺血。因此,国外很多学者考虑在给予外源性促血管生长因子的情况下,还应提供大量功能良好的内皮细胞,来补充那些因血管再生而增生、迁移、重构的血管壁内皮细胞。
近来研究发现,在人的骨髓、外周血和脐血中存在有内皮细胞的前体细胞-内皮祖细胞(endothelial progenitor cells,EPCs)。EPCs能特异性归巢于缺血部位并分化为成熟血管内皮细胞参与血管新生,通过补充高增殖活性的EPCs促进旁路血管新生来代偿缺血部位的血供以达到治疗的目的,称为“supply side”策略。这一发现为缺血性疾病治疗提供了新思路。可将内皮祖细胞作为血管基因转移的自身载体,转染促血管生长基因的内皮祖细胞,通过血管新生与血管发生双重机制,增强心肌缺血部位的血管生长,改善心功能。但是,以内皮祖细胞为载体表达bFGF基因是否能够增强移植细胞功能并进一步促进缺血心肌再血管化尚未见报道。
本课题将对携带bFGF_2基因的腺病毒载体Ad.bFGF_2进行扩增及鉴定,同时诱导培养大鼠骨髓内皮祖细胞,用Ad.bFGF_2在体外转染骨髓内皮祖细胞并检测其转染效率、蛋白表达及对内皮祖细胞的促增殖及分化作用,在体外研究的基础上,将转染Ad.bFGF_2后的内皮祖细胞移植到大鼠缺血心肌,研究移植细胞的存活,及其对于促进血管生长、增加血流灌注、改善心功能的协同治疗作用。
一、碱性成纤维细胞生长因子基因重组腺病毒载体(Ad.bFGF_2)的扩增及鉴定(实验一)
本研究以携带bFGF_2基因的重组腺病毒载体Ad.bFGF_2及Ad-GFP为原料,在293细胞中进行四代扩增,对重组腺病毒DNA进行PCR检测及接种COS-7细胞行RT-PCR鉴定,将鉴定好的病毒应用氯化铯梯度离心纯化和TCID_(50)方法测定滴度,并对其使用安全性进行鉴定。
研究中筛选扩增完成含有目的基因的重组腺病毒Ad.bFGF_2,PCR法证明重组腺病毒中稳定地整合有bFGF_2的基因,RT-PCR方法检测出重组腺病毒感染细胞中的bFGF_2特异性mRNA,表明这些基因可被有效地转录。在293细胞中筛选、扩增出的第4代高滴度重组腺病毒,TCID_(50)方法测定滴度,得到Ad.bFGF_2滴度为1.1×10~9pfu/ml。经氯化铯密度梯度离心后的滴度达到2.0×10~(12)pfu/ml。制备的重组腺病毒Ad.bFGF_2以50的MOI值感染Hela细胞后,观察7天,细胞未出现病态反应,证明其具有良好的使用安全性。
二、体外分离大鼠骨髓单核细胞并诱导培养内皮祖细胞(实验二)
EPCs在骨髓中的含量很少,因此要获得足量的EPCs,体外细胞培养条件下进行分离、纯化和扩增是十分必要的。本研究通过Ficoll密度梯度离心分离大鼠骨髓单核细胞,差速贴壁后置于纤维连接素包被的培养皿中加入10%FCS的DMEM选择性培养基进行诱导培养内皮祖细胞。用倒置相差显微镜观察培养细胞的生长状态,用CD31、CD34、VRGFR-2、Ⅷ因子免疫组化和BS1-Lectin结合能力鉴定培养细胞类型,流式细胞仪检测培养细胞的纯度,透射电镜观察细胞超微结构和内皮细胞特征性细胞器。
结果显示:原代培养7d时形成由内皮祖细胞组成的细胞集落;10d时形成明显克隆,两周左右,细胞生长至80%～90%融合。传代培养后,细胞形态及密度均匀,前3天为相对抑制期,此后呈指数形式快速增殖,10天后达到80%-90%融合,可进行传代。细胞表面抗原免疫组化检测结果表明,CD34、VEGFR-2、Ⅷ因子、CD31相关抗原表达均为阳性,分别为90%,92%,95%和65%,85%的细胞可结合BS1-Lectin。流式细胞仪检测结果显示,BS1-Lectin阳性的细胞占85.5%。透射电镜观察可见内皮细胞最具特征性的细胞器Weibel-Palade小体。培养液NO含量检测,与主动脉内皮细胞相比无明显差异。这表明诱导培养出的梭形贴壁细胞就是分化中的内皮祖细胞
三、Ad.bFGF_2转染EPCs后bFGF_2的表达、分泌及促EPCs增殖及分化作用的研究(实验三)
为了解重组腺病毒载体对EPCs的最佳转染倍数,本课题以不同MOI值的腺病毒载体Ad-GFP体外转染EPCs,检测转染效率。然后以最佳转染倍数进行EPCs的体外转染研究。Ad.bFGF_2以MOI=50转染EPCs后,RT-PCR检测细胞中bFGF_2 mRNA的转录水平,以免疫组化染色、Western-blot检测转染Ad.bFGF_2后,目的蛋白在细胞内的表达及细胞外的分泌情况:硝酸还原酶法检测细胞培养液中NO水平。用MTT法检测转染Ad.bFGF_2后对EPCs的促增殖作用;将转染后的EPCs种植于细胞外基质Matrigel上,观察细胞分化情况。
结果显示:随着MOI值逐渐增大,Ad-GFP体外转染EPCs的转染阳性率也逐渐增高。MOI=50时阳性率可达90%以上,MOI再增大,阳性率增高不明显。RT-PCR、免疫组化、Western-blot等方法检测表明:MOI=50时Ad.bFGF_2可成功将bFGF_2基因转入EPCs中,并有目的蛋白表达与分泌。与对照组相比,Ad.bFGF_2转染组的EPCs培养液中NO含量更高。MTT检测显示Ad.bFGF_2转染后对EPCs有明显的促增殖作用;转染后的EPCs在细胞外基质(Matrigel Matrix)上生长时,出现分化,细胞相互连接成毛细血管网络样结构,说明Ad.bFGF_2转染能在体外条件下诱导EPCs的分化,刺激血管发生。
四、转染bFGF_2基因的EPCs用于缺血心肌移植的实验研究(实验四)
在上述体外研究的基础上,这一部分研究是将转染Ad.bFGF_2的EPCs进行体内实验,对细胞移植与促血管生长因子联合治疗的效果进行探讨。
取Wistar大鼠60只,随机分为4组,每组15只,结扎左冠状动脉前降支建立急性心肌梗死模型,10分钟后进行细胞移植,实验前移植细胞先以PKH-26和BrdU进行双标记:组Ⅰ移植转染Ad.bFGF_2的EPCs;组Ⅱ单纯移植EPCs;组Ⅲ注射AD.bFGF_2;组Ⅳ注射PBS。手术后7天荧光显微镜观察移植细胞的存活率,免疫组化染色及western-blot检测bFGF蛋白的表达,TUNEL染色观察缺血区心肌细胞凋亡的情况;术后4周抗BrdU及抗Ⅷ因子免疫组化染色评价移植细胞分化、心肌血管再生状况,心脏超声检测评价心脏的大小及收缩、舒张功能
研究表明:移植的EPCs在移植区可存活,与其它组比较,组Ⅰ经Ad.bFGF_2转染的移植细胞存活数量较多;免疫组化及western-blot检测移植区有明显bFGF蛋白的表达;另TUNEL染色显示缺血区心肌细胞凋亡明显减少。术后4周的BrdU及抗Ⅷ因子免疫组化染色结果显示:移植细胞出现分化,形成血管内皮;转染Ad.bFGF_2后的EPCs促血管形成作用明显优于其他各组;心功能得到显著改善。
研究结论:
本课题研究结果显示:将Ad.bFGF_2转染的EPCs移植于心脏缺血梗死区后,移植细胞能长期存活,如同时有bFGF_2基因的有效表达,二者可发挥协同作用,显著增加移植细胞成活率和新生血管数目,减少缺血区心肌细胞凋亡,改善左室舒张末容积和收缩、舒张功能。当然,目前无论是基因治疗还是细胞治疗都还有许多缺陷,还存在许多未知的、尚待解决的问题,有待我们更深入探讨。
Background:
Ischemic heart disease (IHD) is the primary cause of death in the industrialized world. Traditionally, patients with IHD were treated with medicine, percutaneous transluminal ballon angioplasty, or coronary artery bypass graft. Despite the fact that advances in modern medical, surgical and interventional treatment of the disease have produced significantly improved results, the efficacy of all these methods is limited in patients with diffuse distal coronary artery disease. According to reports, complete revascularization was found in only about 37 percent of patients who had coronary artery bypass graft, however, once the myocardium was revascularized completely, the incidents of angina would be decreased and the incidence of 5 year survive was improved significantly. Therefore, additional therapeutic measures were needed in those patients. Recent advances in vascular biology suggest the possibility of a novel therapeutic approach to treatment of advanced coronary artery disease. The definition of gene therapy in ischemic heart desease is to transfect special angiogenic growth factor genes and enhances the expressing of growth factors into myocardium. This approach seeks to augment normal collateral development by exposing the heart to growth factors capable of stimulating the growth of new blood vessels.
During myocardial ischemia, compensatory angiogenesis occurred to adapt to ischemic and anoxaemia. In this procedure, growth factors were essential. Fibroblast growth factor (FGF) is the major regulators of vascular growth. It stimulates proliferation of new vessels, improve the microenvironment of ischemic tissue and benefit for tissular reparation or anagenesis. Fibroblast growth factor was divided into two kinds (aFGF and bFGF). They have the same receptors and biological effect .The effect of bFGF is 10 to 30 times power than aFGF in regulators of vascular proliferation. Experimental and clinical studies have shown that FGF may stimulate vessel growth to the ischemic region. Recently, using FGF treatment in patients with severe ischemic heart disease has been implemented as a medical therapy for coronary artery disease. However, under some circumstances, such as in old and feeble, diabetes or hypercholesteremia patients, it exists not only synthesis decreasing of FGF , but also functional impairment of endothelial cells (ECs) that makes its reaction to FGF decrease. Under this circumstance, only using FGF doesn't improve blood supply of ischemic tissue efficiently, so it was considered that ECs should be provided under using FGF by many oversea scholars.
It was found in recently research that there were endothelial progenitor cells (EPCs), the precursor cell of ECs which existed in bone marrow, peripheral blood and cord blood. EPCs may be distributed to ischemic position specially, differentiate to mature ECs and take part in angiogenesis. This approach is called "supply side" strategy that collateral development is augmented by supplying active EPCs. The method of alleviating myocardial remodeling by implanting cells which are transfected with special angiogenic growth factor genes to enhance the angiogenesis may present a new approach to the treatment of ischemic heart disease. However, whether transfecting with special angiogenic growth factor genes reinforce the function of implanting cells or not isn't reported in previous documents.
In our study, Firstly, recombinant adenovirus (Ad) vector carrying the basic fibroblast growth factor 2 (bFGF_2) was amplifed, identified, and rat bone marrow endothelial progenitor cells were induced. Then EPCs with Ad. bFGF_2 was transfected in vitro and the efficiency of transfection and protein expression were tested. The proliferation and differentiation effect of Ad. bFGF_2 on EPCs were certified. Finally, the EPCs transfected by Ad. bFGF_2 were transplanted into rat ischemic myocardium and the survival of the transplanted cells was found and the ability to increase the growth of neovascularization in ischemic zone which enhance local blood perfusion and subsequently improve myocardial function was identified.
Part 1. Propagation, identification of recombinant adenovirus carrying thebasic fibroblast growth factor 2 gene.
In this study, human embroynic kidney 293 cells were transfected with Ad.bFGF_2 and Ad-GFP. Virus clones were isolated and propagated for restriction analysis. After amplified in 293 cells, the obtained adenovirus DNA was detected by PCR. Meanwhile, recombinant adenovirus was further identified by RT-PCR in COS-7 cells. The desired Ad vectors were purified by density gradient ultracentrifuge and titrated, and the safety of using was tested.
Results: Recombinant adenoviral bFGF_2 was propagated successfully, in which the expressing of bFGF_2 was confirmed by PCR and RT-PCR. The Ad. bFGF_2 were amplified in 293 cells for 4 times. The viral particle tite of Ad. bFGF_2 were determined with the TCID50. The viral particle tite of Ad. bFGF_2 was 1.1×10~9pfu/ml, and viral particle tite of Ad. bFGF_2 was 2.0×10~(12)pfu/ml after purified by density gradient ultracentrifuge. Hela cells which were infected with Ad. bFGF_2 for 7 days didn't show up pathosis reaction, so the safety of using was proved.
Part 2. Isolation, Culture and identification of endothelial progenitor cells.
The number of EPCs is estimated to be too few in bone marrow, so it is important to expand and purify the EPCs for the following experiments. Bone marrow cells suspension of adult rat was prepared by discontinuous gradient centrifugation on Ficoll, mononeuclear cells in the middle layer was collected, cultured for 24 hours, then; suspending cells were put into selective culture medium and cultured in the 10% fetal calf serum coated plate. EPCs were isolated, cultured and expanded. The cells were identified by transmission electron microscope, flow cytometer, immunohistochemical staining with anti-factorⅧ, CD31, CD34, VRGFR-2 antigen, the ability of secreting nitrous oxide and combining BS1-Lectin.
Results: Colonies of EPCs formed at 7th day at primary culture. At about 14th day, EPCs got together about 80～90%, and serial subcultivation was made. The passage EPCs proliferated fast, and could be subcultured about every 10 days. EPCs were identified by flow cytometer and immunohistochemical staining (anti-factorⅧ, CD31, CD34, VRGFR-2 antigen) with high purity. Under transmission electron microscope, the cells displayed
Weibel-Palade bodies which are characteristic of endothelial cells. The cells could also combine BS-1 lectin and secrete nitrous oxide. So, the cells induced and cultured in vitro have the same characters as endothelial cells.
Part 3. Transfection of Ad.bFGF_2 to EPCs and its effects of proliferationand differentiation on EPCs in vitro.
To determine the best MOI, transfection efficiency of adenovirus vector to EPCs were identified by infection with various titrations of Ad-GFP. Ad. bFGF_2 was transfected to EPCs with MOI 50:1. After Ad. bFGF_2 transfection, bFGF_2 mRNA transcription was detected by RT-PCR. bFGF_2 protein expression in EPCs and its secretion in culture medium were measured by immunohistochemical staining, Western-blot respectively. The level of NO in culture medium was also measured. The ability of proliferation of Ad. bFGF_2 on EPCs were measured by MTT assay. Finally, the differentiatial ability of Ad. bFGF_2 on EPCs was examined
Results: The transfection efficiency of Ad-GFP to EPCs was increased with MOI. When MOI reached 50, the efficiency was increased to more than 90%. From then on, the transfection efficiency increased limitedly. So 50:1 was choosed as the most suitable MOI. The RT-PCR, immunohistochemical staining and Western-blot showed that bFGF_2 gene could be correctly transfected into EPCs and bFGF_2 protein could express in EPCs. Its secretion in culture medium was detected. After the transfection, the level of NO in the culture medium was also higher than control group. EPCs transfected with Ad. bFGF_2 were revealed a significant increase of cell number which showed by MTT assay. Furthermore, when seeded on Matrigel Matrix, EPCs transfected by Ad. bFGF_2 were induced into capillary-like structures.
Part 4.Studies of transplantation of endothelial progenitor cells transfected by bFGF_2 gene into ischemic myocardium.
Transplantation of EPCs transfected by Ad. bFGF_2 was carried out in vivo following the above studies. Sixty Wistar rats in which acute myocardial infarction was generated by ligating the left anterior descending coronary artery were divided into four groups (GroupⅠ, transplanted with EPCs transfected by Ad. bFGF_2; GroupⅡ, transplanted with EPCs alone; GroupⅢ, injected with Ad. bFGF_2 alone; GroupⅣ, injected with PBS). EPCs were labeled with PKH-26 and BrdU before transplantation. Ten minutes after myocardial infarction the animals were used for transplantation. Seven days after transplantation, survival of transplanted cells, expression of bFGF_2 and apoptosis of ischemic myocardium were measured by immunohistochemical staining, Western-blot, tunnel staining respectively. Four weeks later, the cardiac function of all alive animals was assessed by echocardiography. Finaly, the hearts were harvested for the pathological examination.
Results: Seven days after transplantation, the transplanted EPCs can survive in the zone of ischemic myocardium. There were more cells survive in group I than that in the other groups. More bFGF protein was expressed and less apoptosis of ischemic myocardium. Four weeks later, the histological study showed differentiation of the transplanted EPCs which formed vascular endothelium. Angiogenesis was more remarkable than that in the other Groups. Echocardiography showed that the systolic and diastolic function in Group I were significantly improved compared with the other groups.
Conclusions:
The transplantation of EPCs transfected with Ad. bFGF_2 could improve the survival ability of transplanted cells, inhibit apoptosis of ischemic myocardium. With bFGF_2 protein expressed, The transfected EPCs have strong potency to protect cardiac function by stimulating angiogenesis. Such a strategy might be useful in the treatment of patients with ischemic heart disease. This dissertation is just the beginning of the reseach of angiogenesis by gene or cell therapy. There are more unknown questions to be studied and reasearched for us.
引文
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22. Gehling UM, Ergun S, Schumacher U, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood. 2000;95:3106-3112.
23. Quinci Nadia, Soligo D, Caneva L, et al. Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells. British Journal of Haematology. 2001;115:186-192
24. Shi Q, Rafii S, Wu MH, et al. Evidence FOR circulating bone marrow-derived endothelial cells. Blood. 1998; 92:362-367
25. Yamaguchi TP, Dumont DJ, Conlon RA, et al. Fik-1, and fit-related receptor tyrosine kinase is an early maker for endothelial cell precursors. Development. 1993; 118:489-498
26. Krause DS, Fackler MJ, Civin CI, et al. CD34:structure,biology and clinical utility. Blood. 1996;87:1-13
27. Khakoo AY, Finkel T. Endothelial progenitor cells. Annu Rev Med, 2005,56:79-101.
28. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000, 105:1527-1536
29. Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res. 2003,93:17-24.
30. Griese DP, Ehsan A, Melo LG, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy. Circulation, 2003, 108: 2710-2715.
31. Kong D, Melo Lq Gnecchi M, et al. Cytokine-Induced Mobilization of Circulating Endothelial Progenitor Cells Enhances Repair of Injured Arteries. Circulation, 2004,110:203 9-2046.
1.Hockel M,Schlenger K,Doctroro S,et al.Therapeutic angiogenesis.Arch Surg.1993,128:423-429.
2.Scott R J,Morrow LA.Growth factors and angiogenesis.Cardiovasc Res.1993,27(7)1155-1161.
3.Sato K,Laham RJ,Pearlman JD,et al.Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia.Ann Thorac Surg.2000,70(6):2113-8.
4.Lazarous D F,Shou M,Scheinowitz M,et al.Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury.Circulation.1996,94:1074-1082.
5.Rajanayagam MA,Shou M,Thirumurti V,et al.Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs.J Am Coll Cardiol.2000,35:519-526.
6.Moore MAS.Putting the neo into neoangiogenesis.J Clin Invest.2002,109:313-315.
7.Takahashi JC,Saiki M,Miyatake SI,et al.Adenovirus-meditated gene transfer of basic fibroblast growth factor induces in vitro angiogenesis.Atherosclerosis.1997,132:199-205.
8.Kubota Y,Kleinman HK.Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structure J Cell Biol.1988,107:1589-1598.
9.Hockel M,Schlenger K,Doctroro S,et al.Therapeutic angiogenesis.Arch Sung.1993,128:423-429.
10..Kaplittm G,Leone P,Samulski R,et al.Lone term gene exprssion and phenotypic correction using adno-associated virus vectors in the mammalian brain. Nat Genet. 1994, 8(2):148-154.
11. Hong SS, Karayan L, Tournier J, et al. Adenovirus type 5 fiber knob binds to MHC class I α 2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J. 1997,16:2291-2306.
12. Greber UF,Willetts M, Webster P, et al. Stepwise dismantling of adenovirus 2 during enter into cells.Cell. 1993,75:477-486.
13. Alyson KE, Erik FP, Mauricio A, et al. Quantitative determination of Adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci USA. 1993,90:11498-11502.
14. Yeh P, Perricaudet M. Advances in adenoviral vectors: From genetic engineering to their biology. FASEB J. 1997,11:615-623.
15. Frey M, Hackett NR, Bergelson JM, et al. High-Efficiency Gene Transfer Into Ex Vivo Expanded Human Hematopoietic Progenitors and Precursor Cells by Adenovirus Vectors. Blood. 1998; 91:2781-2792
16. Jueren L, Fang X, Kurt M, et al. Gene therapy: Adenovirus-mediated himan bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cells proliferation and differentiation in vitro and bone formation in vivo. Journal of orthopaedic research. 1999, 17:43-50.
17. Tsutomu W, Charles K, Kazuhiko I, et al. Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors. Blood. 1996, 87:5032-5039
18. Nielsen LL, Gurnani M, Syed J, et al. Recombinant E1-deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and liver histology in tumor xenograft models. Hum GeneTher. 1998, 9(5):681-694.
19. Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med Bull. 1995, 51(1):31-44.
20. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Thr. 2001, 8: 837-845.
21. Yau TM, Fung K, Weisel RD, et al. Enhenced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001, 104:I218-I222.
22. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Tnr. 2001, 8: 837-845.
23. Jikui S, Neil T, Linda D, et al. Ex vivo adenovirus mediated gene transfer of human conjunctival epithelium. Br J Ophthalmol. 2001,85:861-867.
24. Leor J, Quinones MJ, Patterson M, et al. Adenovirus-mediated gene transfer into myocardium: feasibility, timing, and location of expression. J Mol Cell Cardiol. 1996, 28:2057-2067.
25. Gospodanvicz D. Purrification of fibroblast growth factor from bovine pituitary. J Biol Chem.1975, 250:2515-2519.
26. Yang HT, Deschenes MR, Olilive RW et al. Basic fibroblast growth factor increase collateral blood flow in rat with femoral arteral ligation. CirRes. 1996, 79(1):62-69.
27. Fernandez B, Alexadra B, Swen W, et al. Transgenic myocardial overexpression of FGF-I increases coronary artery density and branching. Cir Res. 2000, 87(3):207-213.
28. Kubota Y, Kleinman HK. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structure. J Cell Biol. 1988, 107:1589-1598.
1.Scott RJ.Morrow LA.Growth factors and angiogenesis.Cardiovasc Res. 1993,27(7)1155-1161.
2. Lazarous D F, Shou M, Scheinowitz M, et al. Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury. Circulation. 1996, 94:1074-1082.
3. Sato K, Laham RJ, Pearlman JD, et al. Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia. Ann Thorac Surg. 2000, 70(6):2113-8.
4. Rajanayagam MA, Shou M, Thirumurti V, et al. Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs. J Am Coll Cardiol. 2000, 35:519-526.
5. Padua RR, Sethi R, Dlialla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem.1995,143:129-135.
6. Hristov M, Weber C. Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med, 2004,8(4):498-508.
7. Weinsaft JW, Edelberg JM. Aging-associated changes in vascular activity:a potential link to geriatric cardiovascular disease. Am J Geriatr Cardiol. 2001,10:348-354
8. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Ihvest. 2001,107:1395-1402
9. Laham R J, Sellke FW, Edelman ER, et al. Local perivascular delivery of basic growth factor in patients undergoing coronary bypass surgery. Results of a phase I randamiled Double-blind, placebo-controlled trial. Circulation. 1999,(100):1865-1871.
10. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Sung .1993, 128:423-429.
11. Kawamoto A, Gwon HC, Lwaguro H, et al. Therapeutic potential of ex-vivo expanded endothelial progenitor cell for myocardial ischemia. Circulation. 2001,103(5): 634-637.
12. Aoki J, Serruys PW, van Beusekom H, et al. Endothelial progenitor cell capture by stents coated with an tibody against CD_(34): the HEALINGFIM(Healthy Endothelial Accelerated lining Inhibits Neointimal Growth-First In Man)Registry. J Am Coil Csrdiol. 2005,45(10): 1574-1579.
13. Kawamoto A, Murayama T, Kusano K, et al. Synergistic dc effect of bone marrow mobilization and vascular endothelial growthfactor-2 gene therapy in myocardial ischemia. Circulatio, 2004, 110(11):1398-1405.
14. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Ihvest. 2001;107:1395-1402
15. Isner JM, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair. Ann NYAS. 2001,953:75-8
16. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis,reduces remodeling and improves cardiac function. Nat Med. 2001,7:430-436
17. Fuchs S, Baffour R, Zhou YF, et al. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol. 2001,37:1726-1732
18. Hu MC, Wang YP, Qiu WR. Human fibroblast growth factor-18 stimulates fibro-blast cell proliferation and is mapped to chromosome 14p11.Oncogene.1999,18: 2635-2642.
19. Miyake A, Konishi M, Martin FH, et al. Structure and expression of a novel member, FGF-16, on the fibroblast grow th factor family. [J]Biochem Biophys Res Comon. 1998, 243:148-152.
20. Coulier F, Pontarotti P, Rollbin R, et al. Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families. J Mol Evol. 1997, 44(1):43-56.
21. Anvensa P, Oliretti G, Capass JM. Cellar basis of ventricular remodeling. Am J Cardiol. 1991,68:7-14
22. Ikenaga S, Hamano K, Nishida M, et al. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res. 2001,96: 277-283.
23. Scorsin M, Hagege AA, Marotte F, et al. Does transplantation of cardiomyocytes improve function of infarcted myocardium? Circulation. 1997, 96:11188-193
24. Tavlor DA. Atkins BZ. Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transptantation. Nat Med.1998,4:929-933
25. Scorsin M, Hagege AA, Dolizy I, et al. Can cellular transplantation improve function in doxorubicin-induced heart failure? Circulation. 1998,98:II 151 -156
26. Yan TM, Fung K, Weisel RD, et al. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001,104:1218-222.
27. Ken S, Bari M, Ryszard TS, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation. 2001,104 [suppl I]:I207-212
28. Ziche M, Morbidelli L, Choudhuri R, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997,99: 2625-2634.
29. Takeshita S, Isshiki T, Tanaka E, et al. Use of synchrotron radiation microangiography to assess development of small collateral arteries in rat model of hindlimb ischemia. Circulation.1997,95:805.
30. Anvensa P, Oliretti G, Capass JM. Cellar basis of ventricular remodeling. Am J Cardiol. 1991,68:7-14
31. Miyatake K, yamagishi M, et al. New method for evaluating left ventricular wall motion by color-coded tissue Doppler imaging:in vitro and in vivo studies. J Am Coll Cardiol. 1995,25:717-724
1. Scott RJ, Morrow LA. Growth factors and angiogenesis. Cardiovasc Res. 1993,27(7)1155-1161.
2. Lazarous D F, Shou M, Scheinowitz M, et al. Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury. Circulation. 1996, 94:1074-1082.
3. Sato K, Laham RJ, Pearlman JD, et al. Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia. Ann Thorac Surg. 2000, 70(6):2113-8.
4. Rajanayagam MA, Shou M, Thirumurti V, et al. Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs. J Am Coll Cardiol. 2000, 35:519-526.
5. Padua RR, Sethi R, Dlialla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem. 1995; 143:129-135.
6. Hristov M, Weber C. Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med, 2004,8(4):498-508.
7. Weinsaft JW, Edelberg JM. Aging-associated changes in vascular activity:a potential link to geriatric cardiovascular disease. Am J Geriatr Cardiol. 2001 ;10:348-354
8. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Ihvest. 2001;107:1395-1402
9. Laham R J, Sellke FW, Edelman ER, et al. Local perivascular delivery of basic growth factor in patients undergoing coronary bypass surgery. Results of a phase I randamiled Double-blind, placebo-controlled trial. Circulation. 1999(100): 1865-1871.
10. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Sung .1993; 128:423-429.
11. Kawamoto A, Gwon HC, Lwaguro H, et al. Therapeutic potential of ex-vivo expanded endothelial progenitor cell for myocardial ischemia. Circulation. 2001,103(5): 634-637.
12. Aoki J, Serruys PW, van Beusekom H, et al. Endothelial progenitor cell capture by stents coated with an tibody against CD34: the HEALINGFIM(Healthy Endothelial Accelerated lining Inhibits Neointimal Growth-First In Man)Registry. J Am Coil Csrdiol. 2005, 45(10): 1574-1579.
13. Kawamoto A, Murayama T, Kusano K, et al. Synergistic dc effect of bone marrow mobilization and vascular endothelial growthfactor-2 gene therapy in myocardial ischemia. Circulatio, 2004,110(11):1398-1405.
14. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Divest. 2001 ;107:1395-1402
15. Isner JM, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair. Ann NYAS. 2001;953:75-8
16. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis,reduces remodeling and improves cardiac function. Nat Med. 2001;7:430-436
17. Fuchs S, Baffour R, Zhou YF, et al. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol. 2001;37:1726-1732
18. Hu MC, Wang YP, Qiu WR. Human fibroblast growth factor-18 stimulates fibro-blast cell proliferation and is mapped to chromosome 14 p11.Oncogene. 1999,18: 2635-2642.
19. Miyake A, Konishi M, Martin FH, et al. Structure and expression of a novel member, FGF-16, on the fibroblast grow th factor family. [J]Biochem Biophys Res Comon. 1998, 243:148-152.
20. Coulier F, Pontarotti P, Rollbin R, et al. Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families. J Mol Evol. 1997, 44(1):43-56.
21. Anvensa P, Oliretti G, Capass JM. Cellar basis of ventricular remodeling. Am J Cardiol. 1991;68:7-14
22. Ikenaga S, Hamano K, Nishida M, et al. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res. 2001 ;96: 277-283.
23. Scorsin M, Hagege AA, Marotte F, et al. Does transplantation of cardiomyocytes improve function of infarcted myocardium? Circulation. 1997; 96:11188-193
24. Tavlor DA. Atkins BZ. Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transptantation. Nat Med.1998;4:929-933
25. Scorsin M, Hagege AA, Dolizy I, et al. Can cellular transplantation improve function in doxorubicin-induced heart failure? Circulation. 1998;98:II151-156
26. Yan TM, Fung K, Weisel RD, et al. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001; 104:1218-222.
27. Ken S, Bari M, Ryszard TS, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation. 2001; 104 [suppl I]:I207-212
28. Ziche M, Morbidelli L, Choudhuri R, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99: 2625-2634.
29. Takeshita S, Isshiki T, Tanaka E, et al. Use of synchrotron radiation microangiography to assess development of small collateral arteries in rat model of hindlimb ischemia. Circulation.1997;95:805.
1. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Surg. 1993,128:423-429.
2. Scott RJ, Morrow LA. Growth factors and angiogenesis. Cardiovasc Res. 1993,27(7)1155-1161.
3. Sato K, Laham RJ, Pearlman JD, et al. Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia. Ann Thorac Surg. 2000,70(6):2113-8.
4. Lazarous D F, Shou M, Scheinowitz M, et al. Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury. Circulation. 1996, 94:1074-1082.
5. Rajanayagam MA, Shou M, Thirumurti V, et al. Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs. J Am Coll Cardiol. 2000, 35:519-526.
6. Moore MAS. Putting the neo into neoangiogenesis. J Clin Invest. 2002,109:313-315.
7. Takahashi JC, Saiki M, Miyatake SI, et al. Adenovirus-meditated gene transfer of basic fibroblast growth factor induces in vitro angiogenesis. Atherosclerosis. 1997, 132:199-205.
8. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Sung. 1993,128:423-429.
9. .Kaplittm G, Leone P, Samulski R, et al. Lone term gene exprssion and phenotypic correction using adno-associated virus vectors in the mammalian brain. Nat Genet.1994,8(2):148-154.
10. Hong SS, Karayan L, Tournier J, et al. Adenovirus type 5 fiber knob binds to MHC class I α_2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J. 1997,16: 2291-2306.
11. Greber UF,Willetts M,Webster P, et al. Stepwise dismantling of adenovirus 2 during enter into cells.Cell. 1993,75:477-486.
12. Alyson KE, Erik FP, Mauricio A, et al. Quantitative determination of Adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci USA. 1993,90:11498 -11502.
13. Yeh P, Perricaudet M. Advances in adenoviral vectors: From genetic engineering to their biology. FASEB J. 1997,11:615-623.
14. Frey M, Hackett NR, Bergelson JM, et al. High-Efficiency Gene Transfer Into Ex Vivo Expanded Human Hematopoietic Progenitors and Precursor Cells by Adenovirus Vectors. Blood. 1998; 91:2781-2792
15. Jueren L, Fang X, Kurt M, et al. Gene therapy: Adenovirus-mediated himan bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cells proliferation and differentiation in vitro and bone formation in vivo. Journal of orthopaedic research. 1999,17:43-50.
16. Tsutomu W, Charles K, Kazuhiko I, et al. Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors. Blood. 1996, 87:5032-5039
17. Nielsen LL, Gurnani M, Syed J, et al. Recombinant E1-deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and liver histology in tumor xenograft models. Hum GeneTher. 1998, 9(5):681-694.
18. Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med Bull. 1995, 51(1):31-44.
19. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Tnr. 2001, 8: 837-845.
20. Yau TM, Fung K, Weisel RD, et al. Enhenced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001,104:1218-1222.
21. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Thr. 2001, 8: 837-845.
22. Jikui S, Neil T, Linda D, et al. Ex vivo adenovirus mediated gene transfer of human conjunctival epithelium. Br J Ophthalmol. 2001,85:861-867.
23. Leor J, Quinones MJ, Patterson M, et al. Adenovirus-mediated gene transfer into myocardium: feasibility, timing, and location of expression. J Mol Cell Cardiol. 1996, 28:2057-2067.
24. Yang HT, Deschenes MR, Olilive RW et al. Basic fibroblast growth factor increase collateral blood flow in rat with femoral arteral ligation. CirRes. 1996, 79(1):62-69.
25. Fernandez B, Alexadra B, Swen W, et al. Transgenic myocardial overexpression of FGF-I increases coronary artery density and branching. Cir Res. 2000, 87(3):207-213.
2.Schumacher B,Pecher P.Induction of neoangigenesis inischemic myocardium by human growth factor[J].Circulation.1998,97:645.
3.Fernande BZ,Buehler AK,Wolfman SC,et al.Transgenic myocardial over expression of FGF in creases coronary artery density and branching[J].Circ Res.2000,5(2):87207-87213.
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7.Kremer EJ,Perricaudet M.Adenovirus and adeno-associated virus mediated gene transfer.Br Med Bull.1995,51(1):31-44.
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21. Peichev M, Naiyer AJ, Pereira D, et al. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood. 2000;95: 952-958
22. Gehling UM, Ergun S, Schumacher U, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood. 2000;95:3106-3112.
23. Quinci Nadia, Soligo D, Caneva L, et al. Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells. British Journal of Haematology. 2001;115:186-192
24. Shi Q, Rafii S, Wu MH, et al. Evidence FOR circulating bone marrow-derived endothelial cells. Blood. 1998; 92:362-367
25. Yamaguchi TP, Dumont DJ, Conlon RA, et al. Fik-1, and fit-related receptor tyrosine kinase is an early maker for endothelial cell precursors. Development. 1993; 118:489-498
26. Krause DS, Fackler MJ, Civin CI, et al. CD34:structure,biology and clinical utility. Blood. 1996;87:1-13
27. Khakoo AY, Finkel T. Endothelial progenitor cells. Annu Rev Med, 2005,56:79-101.
28. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000, 105:1527-1536
29. Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res. 2003,93:17-24.
30. Griese DP, Ehsan A, Melo LG, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy. Circulation, 2003, 108: 2710-2715.
31. Kong D, Melo Lq Gnecchi M, et al. Cytokine-Induced Mobilization of Circulating Endothelial Progenitor Cells Enhances Repair of Injured Arteries. Circulation, 2004,110:203 9-2046.
1.Hockel M,Schlenger K,Doctroro S,et al.Therapeutic angiogenesis.Arch Surg.1993,128:423-429.
2.Scott R J,Morrow LA.Growth factors and angiogenesis.Cardiovasc Res.1993,27(7)1155-1161.
3.Sato K,Laham RJ,Pearlman JD,et al.Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia.Ann Thorac Surg.2000,70(6):2113-8.
4.Lazarous D F,Shou M,Scheinowitz M,et al.Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury.Circulation.1996,94:1074-1082.
5.Rajanayagam MA,Shou M,Thirumurti V,et al.Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs.J Am Coll Cardiol.2000,35:519-526.
6.Moore MAS.Putting the neo into neoangiogenesis.J Clin Invest.2002,109:313-315.
7.Takahashi JC,Saiki M,Miyatake SI,et al.Adenovirus-meditated gene transfer of basic fibroblast growth factor induces in vitro angiogenesis.Atherosclerosis.1997,132:199-205.
8.Kubota Y,Kleinman HK.Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structure J Cell Biol.1988,107:1589-1598.
9.Hockel M,Schlenger K,Doctroro S,et al.Therapeutic angiogenesis.Arch Sung.1993,128:423-429.
10..Kaplittm G,Leone P,Samulski R,et al.Lone term gene exprssion and phenotypic correction using adno-associated virus vectors in the mammalian brain. Nat Genet. 1994, 8(2):148-154.
11. Hong SS, Karayan L, Tournier J, et al. Adenovirus type 5 fiber knob binds to MHC class I α 2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J. 1997,16:2291-2306.
12. Greber UF,Willetts M, Webster P, et al. Stepwise dismantling of adenovirus 2 during enter into cells.Cell. 1993,75:477-486.
13. Alyson KE, Erik FP, Mauricio A, et al. Quantitative determination of Adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci USA. 1993,90:11498-11502.
14. Yeh P, Perricaudet M. Advances in adenoviral vectors: From genetic engineering to their biology. FASEB J. 1997,11:615-623.
15. Frey M, Hackett NR, Bergelson JM, et al. High-Efficiency Gene Transfer Into Ex Vivo Expanded Human Hematopoietic Progenitors and Precursor Cells by Adenovirus Vectors. Blood. 1998; 91:2781-2792
16. Jueren L, Fang X, Kurt M, et al. Gene therapy: Adenovirus-mediated himan bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cells proliferation and differentiation in vitro and bone formation in vivo. Journal of orthopaedic research. 1999, 17:43-50.
17. Tsutomu W, Charles K, Kazuhiko I, et al. Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors. Blood. 1996, 87:5032-5039
18. Nielsen LL, Gurnani M, Syed J, et al. Recombinant E1-deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and liver histology in tumor xenograft models. Hum GeneTher. 1998, 9(5):681-694.
19. Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med Bull. 1995, 51(1):31-44.
20. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Thr. 2001, 8: 837-845.
21. Yau TM, Fung K, Weisel RD, et al. Enhenced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001, 104:I218-I222.
22. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Tnr. 2001, 8: 837-845.
23. Jikui S, Neil T, Linda D, et al. Ex vivo adenovirus mediated gene transfer of human conjunctival epithelium. Br J Ophthalmol. 2001,85:861-867.
24. Leor J, Quinones MJ, Patterson M, et al. Adenovirus-mediated gene transfer into myocardium: feasibility, timing, and location of expression. J Mol Cell Cardiol. 1996, 28:2057-2067.
25. Gospodanvicz D. Purrification of fibroblast growth factor from bovine pituitary. J Biol Chem.1975, 250:2515-2519.
26. Yang HT, Deschenes MR, Olilive RW et al. Basic fibroblast growth factor increase collateral blood flow in rat with femoral arteral ligation. CirRes. 1996, 79(1):62-69.
27. Fernandez B, Alexadra B, Swen W, et al. Transgenic myocardial overexpression of FGF-I increases coronary artery density and branching. Cir Res. 2000, 87(3):207-213.
28. Kubota Y, Kleinman HK. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structure. J Cell Biol. 1988, 107:1589-1598.
1.Scott RJ.Morrow LA.Growth factors and angiogenesis.Cardiovasc Res. 1993,27(7)1155-1161.
2. Lazarous D F, Shou M, Scheinowitz M, et al. Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury. Circulation. 1996, 94:1074-1082.
3. Sato K, Laham RJ, Pearlman JD, et al. Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia. Ann Thorac Surg. 2000, 70(6):2113-8.
4. Rajanayagam MA, Shou M, Thirumurti V, et al. Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs. J Am Coll Cardiol. 2000, 35:519-526.
5. Padua RR, Sethi R, Dlialla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem.1995,143:129-135.
6. Hristov M, Weber C. Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med, 2004,8(4):498-508.
7. Weinsaft JW, Edelberg JM. Aging-associated changes in vascular activity:a potential link to geriatric cardiovascular disease. Am J Geriatr Cardiol. 2001,10:348-354
8. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Ihvest. 2001,107:1395-1402
9. Laham R J, Sellke FW, Edelman ER, et al. Local perivascular delivery of basic growth factor in patients undergoing coronary bypass surgery. Results of a phase I randamiled Double-blind, placebo-controlled trial. Circulation. 1999,(100):1865-1871.
10. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Sung .1993, 128:423-429.
11. Kawamoto A, Gwon HC, Lwaguro H, et al. Therapeutic potential of ex-vivo expanded endothelial progenitor cell for myocardial ischemia. Circulation. 2001,103(5): 634-637.
12. Aoki J, Serruys PW, van Beusekom H, et al. Endothelial progenitor cell capture by stents coated with an tibody against CD_(34): the HEALINGFIM(Healthy Endothelial Accelerated lining Inhibits Neointimal Growth-First In Man)Registry. J Am Coil Csrdiol. 2005,45(10): 1574-1579.
13. Kawamoto A, Murayama T, Kusano K, et al. Synergistic dc effect of bone marrow mobilization and vascular endothelial growthfactor-2 gene therapy in myocardial ischemia. Circulatio, 2004, 110(11):1398-1405.
14. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Ihvest. 2001;107:1395-1402
15. Isner JM, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair. Ann NYAS. 2001,953:75-8
16. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis,reduces remodeling and improves cardiac function. Nat Med. 2001,7:430-436
17. Fuchs S, Baffour R, Zhou YF, et al. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol. 2001,37:1726-1732
18. Hu MC, Wang YP, Qiu WR. Human fibroblast growth factor-18 stimulates fibro-blast cell proliferation and is mapped to chromosome 14p11.Oncogene.1999,18: 2635-2642.
19. Miyake A, Konishi M, Martin FH, et al. Structure and expression of a novel member, FGF-16, on the fibroblast grow th factor family. [J]Biochem Biophys Res Comon. 1998, 243:148-152.
20. Coulier F, Pontarotti P, Rollbin R, et al. Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families. J Mol Evol. 1997, 44(1):43-56.
21. Anvensa P, Oliretti G, Capass JM. Cellar basis of ventricular remodeling. Am J Cardiol. 1991,68:7-14
22. Ikenaga S, Hamano K, Nishida M, et al. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res. 2001,96: 277-283.
23. Scorsin M, Hagege AA, Marotte F, et al. Does transplantation of cardiomyocytes improve function of infarcted myocardium? Circulation. 1997, 96:11188-193
24. Tavlor DA. Atkins BZ. Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transptantation. Nat Med.1998,4:929-933
25. Scorsin M, Hagege AA, Dolizy I, et al. Can cellular transplantation improve function in doxorubicin-induced heart failure? Circulation. 1998,98:II 151 -156
26. Yan TM, Fung K, Weisel RD, et al. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001,104:1218-222.
27. Ken S, Bari M, Ryszard TS, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation. 2001,104 [suppl I]:I207-212
28. Ziche M, Morbidelli L, Choudhuri R, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997,99: 2625-2634.
29. Takeshita S, Isshiki T, Tanaka E, et al. Use of synchrotron radiation microangiography to assess development of small collateral arteries in rat model of hindlimb ischemia. Circulation.1997,95:805.
30. Anvensa P, Oliretti G, Capass JM. Cellar basis of ventricular remodeling. Am J Cardiol. 1991,68:7-14
31. Miyatake K, yamagishi M, et al. New method for evaluating left ventricular wall motion by color-coded tissue Doppler imaging:in vitro and in vivo studies. J Am Coll Cardiol. 1995,25:717-724
1. Scott RJ, Morrow LA. Growth factors and angiogenesis. Cardiovasc Res. 1993,27(7)1155-1161.
2. Lazarous D F, Shou M, Scheinowitz M, et al. Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury. Circulation. 1996, 94:1074-1082.
3. Sato K, Laham RJ, Pearlman JD, et al. Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia. Ann Thorac Surg. 2000, 70(6):2113-8.
4. Rajanayagam MA, Shou M, Thirumurti V, et al. Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs. J Am Coll Cardiol. 2000, 35:519-526.
5. Padua RR, Sethi R, Dlialla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem. 1995; 143:129-135.
6. Hristov M, Weber C. Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med, 2004,8(4):498-508.
7. Weinsaft JW, Edelberg JM. Aging-associated changes in vascular activity:a potential link to geriatric cardiovascular disease. Am J Geriatr Cardiol. 2001 ;10:348-354
8. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Ihvest. 2001;107:1395-1402
9. Laham R J, Sellke FW, Edelman ER, et al. Local perivascular delivery of basic growth factor in patients undergoing coronary bypass surgery. Results of a phase I randamiled Double-blind, placebo-controlled trial. Circulation. 1999(100): 1865-1871.
10. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Sung .1993; 128:423-429.
11. Kawamoto A, Gwon HC, Lwaguro H, et al. Therapeutic potential of ex-vivo expanded endothelial progenitor cell for myocardial ischemia. Circulation. 2001,103(5): 634-637.
12. Aoki J, Serruys PW, van Beusekom H, et al. Endothelial progenitor cell capture by stents coated with an tibody against CD34: the HEALINGFIM(Healthy Endothelial Accelerated lining Inhibits Neointimal Growth-First In Man)Registry. J Am Coil Csrdiol. 2005, 45(10): 1574-1579.
13. Kawamoto A, Murayama T, Kusano K, et al. Synergistic dc effect of bone marrow mobilization and vascular endothelial growthfactor-2 gene therapy in myocardial ischemia. Circulatio, 2004,110(11):1398-1405.
14. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Divest. 2001 ;107:1395-1402
15. Isner JM, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair. Ann NYAS. 2001;953:75-8
16. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis,reduces remodeling and improves cardiac function. Nat Med. 2001;7:430-436
17. Fuchs S, Baffour R, Zhou YF, et al. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol. 2001;37:1726-1732
18. Hu MC, Wang YP, Qiu WR. Human fibroblast growth factor-18 stimulates fibro-blast cell proliferation and is mapped to chromosome 14 p11.Oncogene. 1999,18: 2635-2642.
19. Miyake A, Konishi M, Martin FH, et al. Structure and expression of a novel member, FGF-16, on the fibroblast grow th factor family. [J]Biochem Biophys Res Comon. 1998, 243:148-152.
20. Coulier F, Pontarotti P, Rollbin R, et al. Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families. J Mol Evol. 1997, 44(1):43-56.
21. Anvensa P, Oliretti G, Capass JM. Cellar basis of ventricular remodeling. Am J Cardiol. 1991;68:7-14
22. Ikenaga S, Hamano K, Nishida M, et al. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res. 2001 ;96: 277-283.
23. Scorsin M, Hagege AA, Marotte F, et al. Does transplantation of cardiomyocytes improve function of infarcted myocardium? Circulation. 1997; 96:11188-193
24. Tavlor DA. Atkins BZ. Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transptantation. Nat Med.1998;4:929-933
25. Scorsin M, Hagege AA, Dolizy I, et al. Can cellular transplantation improve function in doxorubicin-induced heart failure? Circulation. 1998;98:II151-156
26. Yan TM, Fung K, Weisel RD, et al. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001; 104:1218-222.
27. Ken S, Bari M, Ryszard TS, et al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation. 2001; 104 [suppl I]:I207-212
28. Ziche M, Morbidelli L, Choudhuri R, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99: 2625-2634.
29. Takeshita S, Isshiki T, Tanaka E, et al. Use of synchrotron radiation microangiography to assess development of small collateral arteries in rat model of hindlimb ischemia. Circulation.1997;95:805.
1. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Surg. 1993,128:423-429.
2. Scott RJ, Morrow LA. Growth factors and angiogenesis. Cardiovasc Res. 1993,27(7)1155-1161.
3. Sato K, Laham RJ, Pearlman JD, et al. Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischemia. Ann Thorac Surg. 2000,70(6):2113-8.
4. Lazarous D F, Shou M, Scheinowitz M, et al. Comparative effects of Basic Fibroblast Growth factor and Vascular Endothelial Growth Factor on coronary collateral Development and the Arteial response to injury. Circulation. 1996, 94:1074-1082.
5. Rajanayagam MA, Shou M, Thirumurti V, et al. Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs. J Am Coll Cardiol. 2000, 35:519-526.
6. Moore MAS. Putting the neo into neoangiogenesis. J Clin Invest. 2002,109:313-315.
7. Takahashi JC, Saiki M, Miyatake SI, et al. Adenovirus-meditated gene transfer of basic fibroblast growth factor induces in vitro angiogenesis. Atherosclerosis. 1997, 132:199-205.
8. Hockel M, Schlenger K, Doctroro S, et al. Therapeutic angiogenesis. Arch Sung. 1993,128:423-429.
9. .Kaplittm G, Leone P, Samulski R, et al. Lone term gene exprssion and phenotypic correction using adno-associated virus vectors in the mammalian brain. Nat Genet.1994,8(2):148-154.
10. Hong SS, Karayan L, Tournier J, et al. Adenovirus type 5 fiber knob binds to MHC class I α_2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J. 1997,16: 2291-2306.
11. Greber UF,Willetts M,Webster P, et al. Stepwise dismantling of adenovirus 2 during enter into cells.Cell. 1993,75:477-486.
12. Alyson KE, Erik FP, Mauricio A, et al. Quantitative determination of Adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci USA. 1993,90:11498 -11502.
13. Yeh P, Perricaudet M. Advances in adenoviral vectors: From genetic engineering to their biology. FASEB J. 1997,11:615-623.
14. Frey M, Hackett NR, Bergelson JM, et al. High-Efficiency Gene Transfer Into Ex Vivo Expanded Human Hematopoietic Progenitors and Precursor Cells by Adenovirus Vectors. Blood. 1998; 91:2781-2792
15. Jueren L, Fang X, Kurt M, et al. Gene therapy: Adenovirus-mediated himan bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cells proliferation and differentiation in vitro and bone formation in vivo. Journal of orthopaedic research. 1999,17:43-50.
16. Tsutomu W, Charles K, Kazuhiko I, et al. Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors. Blood. 1996, 87:5032-5039
17. Nielsen LL, Gurnani M, Syed J, et al. Recombinant E1-deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and liver histology in tumor xenograft models. Hum GeneTher. 1998, 9(5):681-694.
18. Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med Bull. 1995, 51(1):31-44.
19. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Tnr. 2001, 8: 837-845.
20. Yau TM, Fung K, Weisel RD, et al. Enhenced myocardial angiogenesis by gene transfer with transplanted cells. Circulation. 2001,104:1218-1222.
21. Ohara N, Koyama H, Miyata T, et al. Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia. Gene Thr. 2001, 8: 837-845.
22. Jikui S, Neil T, Linda D, et al. Ex vivo adenovirus mediated gene transfer of human conjunctival epithelium. Br J Ophthalmol. 2001,85:861-867.
23. Leor J, Quinones MJ, Patterson M, et al. Adenovirus-mediated gene transfer into myocardium: feasibility, timing, and location of expression. J Mol Cell Cardiol. 1996, 28:2057-2067.
24. Yang HT, Deschenes MR, Olilive RW et al. Basic fibroblast growth factor increase collateral blood flow in rat with femoral arteral ligation. CirRes. 1996, 79(1):62-69.
25. Fernandez B, Alexadra B, Swen W, et al. Transgenic myocardial overexpression of FGF-I increases coronary artery density and branching. Cir Res. 2000, 87(3):207-213.