摘要
研究背景
心肌梗死(Myocardial infarction, MI)是由于冠状动脉循环改变引起冠状血流和心肌需求之间不平衡而导致的心肌损害,是临床上一种严重的缺血性心脏病(Ischemic heart disease, IHD)。目前,治疗冠心病的常用方法诸如药物、经皮冠状动脉腔内成形术及冠状动脉旁路移植等虽然能改善心肌缺血,却不能使梗死区的血管再生。新近国内外兴起的一些干细胞移植实验治疗缺血性心脏病,通过再生血管,为治疗这类疾病提供了新思路。
干细胞移植治疗心肌梗死在大量动物实验及临床中的应用已取得了令人鼓舞的疗效,越来越多的证据表明,骨髓来源的干细胞能够参与血管新生,改善心肌梗死后血流灌注及心脏功能。被用于移植的细胞种类很多,其中骨髓间充质干细胞(Mesenchymal stem cells, MSCs)目前被公认最适合用于细胞移植治疗,其易于分离提取,具有高度的扩增潜能,有良好的基因稳定性、组织相容性好、不涉及伦理道德问题等优点,成为研究的热点。
但干细胞移植治疗心肌缺血目前还存在一些问题,包括干细胞移植途径的有创性、细胞移植效率低下及干细胞的定向归巢能力欠佳等。如何寻找一种无创、移植效率更高及定向能力好的移植途径与方法呢?研究显示,脉冲式治疗超声联合白蛋白微泡Optison能有效将经静脉移植的骨髓单个核细胞靶向运送至心肌病动物模型,心肌毛细血管密度、缺血心肌的血流灌注,VCAM-1,ICAM-1等黏附分子表达均得到了显著提高,心脏功能也得到明显改善,表明超声介导的微泡破坏能提高干细胞的靶向归巢能力进而更有效地实现干细胞移植治疗心脏病的效果。
研究目的
1.探讨诊断超声联合微泡促进静脉移植骨髓MSCs归巢兔缺血心肌的可行性及其机制;
2.探讨诊断超声联合微泡静脉移植MSCs改善梗死后心肌血流灌注和心功能的有效性;
3.初步探讨诊断超声联合微泡作用下无创移植MSCs改善心肌梗死后心功能的可能机制。
研究方法
采用密度梯度离心联合贴壁培养法进行兔骨髓MSCs的分离、纯化及培养,检测其生物学特性。流式细胞仪检测细胞表面标记分子,成脂、成骨诱导分化进行细胞鉴定。
冠状动脉左前降支结扎法建立兔心肌梗死模型,采用血清学、影像学及病理学方法评价模型的建立。
以荧光标记物DAPI标记MSCs,移植48h观察标记MSCs在静脉移植MSCs组与超声+微泡+MSCs组心肌、肺等脏器的荧光分布并进行阳性细胞计数和统计学比较。MSCs移植后4周透射电镜观察各组(PBS组、超声+微泡组、静脉移植MSCs组与超声+微泡+MSCs组)心肌缺血区血管超微结构。免疫组织化学检测心肌缺血区VCAM-1、SDF-1表达并行定量分析。
MSCs移植后4周HE染色检测心肌缺血区的血管密度(Capillary density,CD)、免疫组织化学检测CD34表达、western blot检测血管内皮生长因子(Vascular endothelial growth factor,VEGF)水平,心肌声学造影(Myocardial contrast echocardiography, MCE)评价心肌血流灌注。Masson染色检测心肌胶原纤维形成,并计算各组胶原面积。免疫组织化学检测各组心肌梗死区Bax蛋白的表达情况。通过M型超声与双平面Simpson法检测左室收缩功能。
结果
培养的MSCs贴壁生长,呈梭形为主;细胞表面高表达CD44,几乎不表达CD45,不表达CD34。成脂诱导培养后油红O染色胞浆脂滴形成,成骨诱导后碱性磷酸酶染色见钙颗粒,茜素红S染色钙结节形成。
冠状动脉结扎法建立MI模型后,MCE显示左室前壁、前间隔呈灌注缺损,Masson染色左室前壁梗死区呈蓝色染色。心肌梗死后兔左室收缩功能明显降低。
荧光显微镜下计数并比较静脉移植MSCs组与超声+微泡+MSCs组心肌梗死及周围区DAPI阳性细胞数,分别为(147±19)个,(213±27)个,差异有统计学意义(P<0.01)。在正常供血区心肌内未见阳性细胞。透射电镜显示超声+微泡+MSCs组与超声+微泡组心肌缺血区血管一侧内皮细胞间隔增大,血管通透性增加。免疫组织化学检测SDF-1在各组中的心肌细胞胞膜与胞浆均有棕黄色阳性表达,超声+微泡+MSCs组的灰度(188.45±2.83)与静脉移植MSCs组(183.94±7.29)间差异无统计学意义(P>0.05),高于超声+微泡组(175.46±8.13)和PBS组(170.52±6.04)(两者P<0.05)。超声+微泡+MSCs组的VCAM-1阳性细胞数量(77±44)个显著高于静脉移植MSCs组(34±18)个,超声+微泡组(12±3)个及PBS组(8±5)个(三者P<0.01)。
HE染色检测超声+微泡+MSCs组平均CD为(44±23)个,高于PBS组(19±10)个、超声+微泡组(22±5)个及MSCs组(26±7)(三者P<0.01)。Masson染色检测心肌胶原面积分别为PBS组(16.88±5.84)%、超声+微泡组(14.77±2.95)%、MSCs组(12.05±4.83)%和超声+微泡+MSCs组(8.97±3.5)%,超声+微泡+MSCs组与前三组比较,胶原面积分别缩小约25.6%、40.1%及46.8%。免疫组织化学检测超声+微泡+MSCs组CD34阳性血管最多,分布密集,MSCs组较丰富,超声+微泡组次之,PBS组的阳性血管最少。Bax蛋白阳性表达在PBS组最高,超声+微泡组次之,MSCs组进一步减少,超声+微泡+MSCs组最少。M型超声检测超声+微泡+MSCs组的FS(%)测值显著高于PBS组和超声+微泡组比较, P<0.01,高于静脉移植MSCs组,P<0.05。EF(%)比较,超声+微泡+MSCs组较PBS组及超声+微泡组显著增高(P<0.01),与静脉移植MSCs组比较,差异有统计学意义(P<0.05)。双平面Simpson法EF值超声+微泡+MSCs组高于PBS组(P<0.01)、超声+微泡组(P<0.01)及静脉移植MSCs组(P<0.05)。
结论
1.成功实现兔骨髓源性MSCs的分离、纯化及培养,密度梯度离心联合贴壁培养法是获取、纯化、扩增MSCs简便、易行的方法。细胞生长曲线、细胞周期、透射电镜检测提示培养的MSCs呈低分化状态,有较强的自我更新能力。流式细胞仪检测培养细胞高表达CD44,CD45呈阴性表达,免疫细胞化学检测CD34阴性,体外成脂、成骨诱导分化成功,判定所获细胞为MSCs。
2.冠状动脉左前降支结扎法可建立稳定的兔MI模型,血清学、影像学及病理学检测可有效评价模型是否成功建立。
3. DAPI作为一种荧光剂,可有效示踪MSCs在体内各脏器、各部位的分布,其方法简单、操作简便,可应用于干细胞的标记。
4.诊断超声联合脂膜微泡通过声孔效应可增加心肌梗死兔心肌血管通透性,为经静脉移植的MSCs归巢到达缺血心肌提供了更多的通道与机会。微泡介导的超声空化效应可刺激超声辐照心肌缺血区小血管破裂及炎症发生,促进局部VCAM-1等黏附分子的表达,进而增强移植的MSCs向心肌缺血区聚集与归巢;同时,诊断超声介导的微泡破坏效应通过促进VEGF生成与表达、刺激心肌缺血区血管生成等途径能更加有效改善心肌梗死后血流灌注。
5. MSCs移植后通过旁分泌效应促进血管生成因子VEGF、归巢相关因子SDF-1、黏附分子VCAM-1的表达及抑制凋亡相关因子Bax表达,进而有效增强MSCs的趋化、黏附、聚集及归巢能力,通过促进血管新生及抑制凋亡等机制来改善心肌梗死后心功能。
6.静脉移植MSCs在超声联合微泡介导下通过抑制心肌梗死后胶原纤维形成,抑制心脏重构来改善心功能。
7.本研究方法以无创、有效促进MSCs定向归巢及改善心肌梗死后心功能在干细胞治疗心肌梗死中显示出较好的应用前景,有望为干细胞移植治疗冠心病提供一种新方法。
Backgrounds
Myocardial ischemia associated with coronary artery disease is a leading cause of morbidity and mortality in clinical practice. Transplantation of mesenchymal cells has been clinically applied for the patients with ischemic heart diseases via medicines,Percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass graft surgery (CABG) procedures which are effective for revascularization and might improve the myocardial perfusion, but are hard to regenerate the myocytes. Lately, stem cell transplantation is applied in the treatment of coronary artery disease (CHD) and developed rapidly,which can not only generate new vessels,but also regenerate necrotic myocardium.
Currently one of the most popular cell types used by cardiomyoplasty investigators is bone marrow derived mesenchymal stem cells(BMSCs). BMSCs transplantation has shown promise for cardiac regeneration not only in animal models of myocardial infarction, but also in patients with CHD. Several studies have indicated that BMSCs offer a novel therapeutic option in the treatment of heart diseases. BMSCs transplantation leads to a significant improvement in the myocardial perfusion and post-infarction left ventricular function through angiogenesis. MSCs are pluripotent cells capable of high proliferation and differentiation into multiple cell types. What has made MSCs particularly appealing to investigators in cellular cardiomyoplasty is that they are easily obtained from bone marrow,can be expanded in culture, have good gene stability and histocompatibility,and are not involved in ethic problems. MSCs have high proliferative and selfrenewal capability. They exhibit multilineage differentiation capacity being capable to give rise to diverse cells like osteoblasts,chondrocytes,adipocytes,myocytes,tenocytes and possibly neural cells.
Although the implantation of MSCs showed some favorable effects on cardiac remodeling and function,the efficiency of cell delivery was still limited by some factors,such as that the cell therapies were invasive and the targeted homing ability of implanted cells need to be improved. How to solve these existing problems is critial and pressing in the cell therapy in coronary artery diseases. Zen et al reported that therapeutic ultrasound-mediated albumin microbubbles destruction combined with the intravenous transfusion of BM-MNCs could greatly enhance the neovascularization by an increase in the endothelial attachment of BM-MNCs, leading to an improvement in blood perfusion and cardiac function of BIOTO2 cardiomyopathy, which resulted in an improvement of cardiac function via the inhibition of myocyte apoptosis and interstitial fibrosis. Their results suggest that US + Microbubble + MNC treatment maybe a feasible, efficient and non-invasive system for targeted cell delivery to the myocardium.
Objectives
1. To study the feasibility of the improvement of targeted homing of bone marrow derived mesenchymal stem cells by diagnostic ultrasound-mediated microbubbles destruction to the ischemic heart and its mechanism.
2. To study the efficacy of intravenous MSCs implantation by diagnostic ultrasound-mediated mirobubbles destruction on the improvement of myocardial perfusion and cardiac function after rabbit myocardial infarction.
3. To study the preliminary mechanisms of the improvement of the cardiac function by MSCs transplantation by diagnostic ultrasound-mediated microbubbles destruction after myocardial infarction.
Methods
Density gradient centrifugation and adherent culture method were used in the isolation and cultivation of MSCs. The morphological and other characterics were detected. Flow cytometry (FCM) and immune cells chemistry were applied to detect the expression of CD44 and CD45 on the stem cells. Osteogenic and adipogenesis induction of MSCs were performed.
Myocardial infarction (MI) model was established by ligation of the left anterior descending coronary artery (LAD). The successful building of MI was assessed with serology, ultasonography and pathology.
Sixteen models were equally devided into DAPI-labeled MSCs infusion group and DAPI MSCs + diagnostic US + microbubbles group. After 48 hours of cell transplantation,the survival of implanted cells was identified by the number of DAPI-positive cells in frozen sections (8μm in thickness) made from hearts of MI and lung under fluorescent microscope. Permeability of microvessel in MI area was assessed in sections of the anterior wall (right under the irradiation of diagnostic ultrasound) under Transmission electronic microscope. Expressions of VCAM-1(Vascular cell adhesion molecule 1) and SDF-1(Stromal cell-derived factor-1) in MI area were assessed with IHC after cell therapy.
After 4 weeks of treatment, capillary density was assessed with HE staining, CD34 expression with IHC, VEGF level with western blot and myocardial perfusion with myocardial contrast echocardiography (MCE), which would prove the mechanism of angiogenesis. Myocardial collagen fiber area was analyzed with Q-Win image software with Masson staining to give evidence of inhibition in fibrosis by this implantation method. Protein Bax was detected with IHC in 4 groups. Echocardiography was performed blindly to assess the cardiac function. Fractional shortening (FS) and ejection fraction (EF) were detected with M mode echocardiography. Apical two and four chamber views were obtained and EF with biplane Simpson’s method was examined, too.
Results
Most of the cultured MSCs were spindle shaped and became more uniform after several passages. These expanded MSCs were uniformly positive for CD44 (96.02%) and negative for CD45 (2.47%). The cultured cells were uniformly negative for CD34 with immunocytochemistry. Adipogenic and osteocyte differentiation was successfully induced in MSCs.
MI models were builded by ligation of LAD. Perfusion defect was observed in the anterior and anteroseptal wall of LV with MCE,a blue dye was detected in the MI area with Masson staining,and LV systolic function was attenuated greatly after MI.
The DAPI positive cells were located in infarcted and border area,while there was nearly none in the normal myocardium. The number of DAPI positive cells in MI area of US+Buble+MSCs group (214±27) was much more than that of MSCs infusion group(147±19) (P < 0.01). There were some endothelial cells in part of vessel wall impaired in ischemic myocardium under ultrasonic irradiation, the intercellular spaces increased in UMC(US+Microbubble+MSCs) group and UM (US+Bubble)group. The number of VCAM-1 positive cells of group UMC (77±44) was markedly increased compared with MSCs infusion (MSC) group(34±18), UM group (12±3) and PBS group (8±5), all the P<0.01. The scale of SDF-1 analyzed with Q-Win was increased in both MSCs treated groups, especially in UMC group.
The number of capillaries stained by HE in UMC group (47±23) was much greater than that of the MSC group (26±7), the UM group (22±5) and the PBS group (19±10) under light microscope. Western blotting results showed that the level of VEGF in infarcted zone was higher in the UMC group (236.59±47.13) than that of the MSC group (151.48±25.07), the UM group (133.47±20.16) and the PBS group (89.43±20.43), with all P < 0.01. CD34 assessed with IHC reached the highest in the UMC group, followed by the MSCs group, the UM group and the PBS group. The collagen percentage area(%) of UMC group (9±4) was decreased by 25.6% compared with MSC group (13±5), decreased by 40.1% with UM group (15±3) and 46.8% with PBS group(17±6). There were significant differences of gray scale (analyzed with histogram of Photoshop) in anterior wall between the PBS group (52±12) and UM group (59±10), MSC group(64±16) and UMC group(71±13), with P = 0.091, 0.032 and 0.000,respectively. There was significant difference of FS between the UMC group (31±4)% and the MSC group [ (27±3)%, P<0.05], the UM group [ (22±3)%,P<0.01] and the PBS group [(20±3)%, P<0.01], respectively. So was the difference of EF between the UMC group [(62±6)%] and the MSC group [(54±5)%, P<0.05], the UM group [(47±4)%, P<0.01] and the PBS group [(43±5.0)%, P<0.01], respectively. And so was the EF(%) of biplane Simpson rule between the UMC group [(49±4)%] and the PBS group [(35±5)%, P<0.01], the UM group [(35±6)%, P<0.01] and the MSC group [(42±5)%, P<0.05], respectively.
Conclusions
1. Purified MSCs can be obtained via the density gradient centrifugation and adhere culture methods using morphological observation, surface marker identification and inducing differentiation. It is a simple available method for the isolation and culture of MSCs.
2. DAPI fluorescent agent is a good stem cell label molecule, which could efficiently track the distribution of DAPI labeled MSCs in heart, lung and other organs.
3. The intercellular space of vessel wall was increased with diagnostic ultrasound-mediated microbubble destruction, which facilitated the homing and gathering of MSCs to the MI area. Higher expression of VCAM-1 in the US + Microbubble +MSCs group may be induced by both the US + Bubble-mediated response and the paracrine effect of the implanted MSCs, which caused the enhanced attachment of transfused BM-MSCs onto the targeted endothelial layer.
4. Intravenous infusion of rabbits BM-MSCs combined with US+Bubbles can stimulate neovessel formation by supplying angiogenic factors (VEGF) and inducing angiogenesis in ischemic heart and enhance the cardiac perfusion. The upregulation of VEGF, SDF-1, VCAM-1 and inhibition of Bax protein were induced by Paracrine effect of the implanted MSCs, which augmented the adhension, homging and gathering ability to the infarcted myocardium, and resulted in the improvement of cardiac function through the neogenesis and the inhibition of apoptosis.
5. Intravenous infusion of BM-MSCs combined with diagnostic US + Bubble may assist in the inhibition of fibrosis and LV remodeling, which causes the improvement in cardiac function.
6. Intravenous implantation of BM-MSCs combined with diagnostic US + Bubble can enable targeted delivery of BM-MSCs to the infarcted myocardium and induce the regional angiogenic response, resulting in an improvement of LV perfusion and cardiac function of ischemic heart associated with the inhibition of cardiac fibrosis and remodeling.
7. Myocardium-targeted homing of intravenously implantated mesenchymal stem cells by diagnostic ultrasound-mediated microbubble destruction may be feasible as a non-invasive and efficient angiogenic cell therapy to the ischemic heart disease.
引文
1. Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature 2004, 428: 664-668.
2. Chen SL, Fang WW, Qian J, et al. Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction. Chin Med J (Engl), 2004, 117: 1443-1448.
3. Li Y, Chen Y, Tian J, et al. Screening of differentially expressed genes in rats with cardiomyopathy after bone marrow mesenchymal stem cell transplantation. Zhonghua Er Ke Za Zhi, 2006, 44: 787-791.
4. Toma C, Pittenger MF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation, 2005, 105: 93-98.
5. Siminiak T, Czepcczynski R, Grygieska B, et al. Evidence for extravasation of intacoronary administered bone-marrow derived CD34+ stem cells in patients with acute myocardial infarction.Circulation, 2004, 110:Ⅲ-51.
6. Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infracted myocardium:feasibility, cell migration, and body distribution. Circulation, 2003: 108: 863-868.
7. Mangi AA, Noiscus N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infracted hearts. Nat Med, 2003, 9: 195-201.
8.杨春,马爱群,蔡萍.大鼠急性心肌梗死后骨髓干细胞归巢于缺血心肌的时间窗研究.中国病理生理杂志.2005, 21: 1539–1543.
9. Price RJ, Kaul S. Contrast ultrasound targeted drug and gene delivery: an update on a new therapeutic modality. J Cardiovasc Pharmacol Ther, 2002, 7: 171-180.
10. Teupe C, Richter S, Fisslthaler B, et al. Vascular gene transfer of phosphomimetic endothelial nitric oxide synthase (S1177D) using ultrasound-enhanced destruction of plasmid local microbubbles improves vasoreactivity. Circulation, 2002, 105: 1104–1109.
11. Taniyama Y, Tachibana K, Hiraoka K, et al. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation. 2002, 105:1233–1239.
12. Zen K, Okigaki M, Hosokawa Y, et al. Myocardium-targeted delivery of endothelial progenitor cells by ultrasound-mediated microbubble destruction improves cardiac function via an angiogenic response. J Mol Cell Cardiol. 2006, 40: 799-809.
13. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med, 2001, 226 :507-520.
14. Lee HS, Huang GT, Chiang H, et al. Multipotential mesenchymal stem cells from femoral bone marrow near the site of osteonecrosis. Stem Cells, 2003, 21: 190 - 199 .
15. Pountos I, Corscadden D, Emery P, et al. Mesenchymal stem cell tissue engineering: techniques for isolation, expansion and application. Injury, 2007, 38: S23 - 33.
16. PittengerMF, Martin BJ. Mesenchymal stem cells and their potential cardiac therapeutics. Circ Res, 2004, 95: 9 - 20.
17. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284: 143-147.
18.侯炳波,王挹青.骨髓间充质干细胞通过旁分泌作用治疗心肌缺血研究进展.中国分子心脏病学杂志, 2007, 7: 117-120.
19. Trotha R, Hanck T, Konig W, et a1. Rapid ribesequencing-an effective diagnostic tool for detecting microbial infection. Infect, 2001, 29: 12- 16.
20. Lisignoli G, Remiddi G, Cattini L. et al.An elevated number of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a gradient isolation procedure. Connect Tissue Res, 2001, 42: 49- 57.
21. Conget P A, Minguell J J. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells .J Cell Physiol, 1999, 181: 67- 73.
22.刘侠,乐以伦.碱性磷酸酶与钙化.中国生物医学工程学报, 1997, 16: 71 - 82.
23. Alborzi A, Mac K, Glackin CA, et al. Endochondral and in tramembranous fetal bone development osteoblastic cell proliferation, and expression of alkaline phosphatase, twist, and histone H4 . J Craniofac Genet Dev Biol (Denmark), 1996, 16: 94-106.
24.夏冰,王捷,郭立达,等.不同种属骨髓间充质干细胞体外培养特性的比较研究.中国微侵袭神经外科杂志(CMINSJ), 2006, 11: 555-557.
25.苏立,陈运贞.骨髓间充质干细胞的生物学特性.医学综述, 2004, 10: 306-309.
26. Saito T, Kuang JQ, Bittira B, et al . Xenotransplant cardiac chimera: immune tolerance of adult stem cells. Ann Thorac Surg, 2002, 74: 19-24.
27. Wang JS, Shum-Tim D, Calipeau J, et al. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg, 2000, 120: 999—1006.
28.金旭红,杨柳.骨髓间充质干细胞标记及活体示踪技术研究进展,中华创伤杂志, 2007, 23: 311-313.
29. Schuster MD, Kocher AA, Seki T, et al. Myocardial neovascularization by bone marrow angioblasts results in cardiomyocyte regeneration. Am J Physiol Heart Circ Physiol, 2004, 287: H525-532.
30. Dobaczewski M, Akrivakis S, Nasser K, et al .Vascular mural cells in healing canine myocardial infarcts. J Histochem Cytochem, 2004, 52: 1019 - 1029.
31. Pilla JJ, Blom AS, Brockman DJ, et al .Ventricular constraint using the acorn cardiac support device reduces myocardial akinetic area in an ovine model of acute infarction. Circulation, 2002, 106: 1207-1211.
32. Crottogini AJ, Lascano EC, Barra JG, et al. Effect of long termoral nisoldipine on infarct size and collateral development in pigs undergoing gradual occlusion of the left circumflex artery. J Cardiovasc Pharmacol, 1990, 15: 644–654.
33. Vracko R, Thorning D, Fredericks RG, et al. Myocyte reactions at the borders of injured and healing rat myocardium. Lab I nvest, 1988, 59: 104-114.
34. Lee BH, Kim WH, Choi MJ, et al . Chronic heart failure model in rabbits based on the concept of the bifurcation trifurcation coronary artery branching pattern. Artif Organs, 2002, 26: 360 - 365.
35. Degabriele NM, Griesenbach U, Sato K, et al . Critical appraisal ofthe mouse model of myocardial infarction. Exp Physiol, 2004, 89: 497 - 505.
36.赵洪云,管慧红,钟雪云,等.开胸冠状动脉结扎法制作大鼠心肌梗死模型.国外医学内科学分册, 2005, 32: 540-542.
37.胡晓军,孙有刚,郭瑞强.等.存活心肌的超声心动图评价.临床超声医学杂志, 2001, 3: 170-172.
38. Villanueva FS. The use of myocardial contrast echocardiography in clinical evaluationafter myocardial infarction. Coronary Artery Disease, 2000, 11: 235 - 242.
39.周俐红,王琦,申彪,等.兔心脏冠状动脉的大体解剖.新乡医学院学报, 2003, 20: 96-97.
40. Salto Tellez M, Yung Lim S, Oakley RM, et al. Myocardial infarction in the mouse: a quantifiable and highly reproducible experimental model. Cardiovasc Pathol, 2004, 13(2): 91 - 97.
41.李国营,田素民,于连发,等.兔心肌缺血及缺血再灌注模型的制备.解剖学研究, 2002, 24: 237-238.
42.王太重,崔敏,王文武,等.心肌梗死的实验室诊断进展:心肌损伤标志物.中国介入心脏病学杂志, 2000, 8: 163.
43.程芮,王士雯,张友荣,等.3种不同途径移植自体骨髓间充质干细胞治疗急性心肌梗死效果比较.中华实验外科杂志.2005, 22: 1504-1506.
44. Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation, 2002, 106: 1913-1918.
45.曹丰,裴雪涛,李艳华,等.移植时机对骨髓间充质干细胞修复梗死心肌的影响.第四军医大学学报, 2005, 26: 22- 27.
46.余其贵,吴继雄.骨髓间充质干细胞移植治疗缺血性心脏病研究进展.中国心血管病研究杂志, 2007, 5: 232-234
47. Strauer BE, Brehm M, Zeus T, et a1. Intracoronary human autologous stem cells transplantation for myocardial regeneration following myocardial infarction. Dtsch Med Wochenscher, 2001, 126: 932- 938.
48. Kocher AA, MD Schuster, MJ Szaboles et al. Neovascularization of ischemic myocardium by human bonemarrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med, 2001, 7: 430-436.
49. Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesencymal stem cells to the infracted myocardium: feasibility, cell migration, and body distribution. Circulation, 2003, 108: 863-868.
50. Vulliet PR, Greeley M, Halloran SM, et al. Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet, 2004, 363: 783-784.
51. Mangi AA, Noiscus N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infracted hearts. Nat Med, 2003, 9: 195-201.
52. Wollert KC, Drexler H. Clinical application of stem cells for the heart. Circ Res, 2005, 96: 151-63.
53. Orlic D, Kajstura J, Chimenti S, et al . Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci, 2001, 98: 10344-10349.
54. Li P, Cao L Q, Dou C Y, et al, Impact of myocardial contrast echocardiography on vascular permeability: an in vivo dose response study of delivery mode, pressure amplitude and contrast dose. Ultrasound in Med Biol, 2003, 29: 1341-1349.
55.王雅婕,刘徳英,刘政,等.超声造影在心脏负荷实验中对心肌微血管的损伤效应.中国超声医学杂志, 2008, 24: 106-108.
56. Imada T, Tatsumi T,Mori Y, et al. Targeted delivery of bone marrow mononu-clear cells by ultrasound destruction of microbobbles induces both angiogenesis and arteriogenesis response. Arterioscler Thromb Vasc Biol, 2005, 25: 2128-2134 .
57.马军,葛均波.骨髓干细胞归巢损伤脏器机制的研究概况.中国临床医学, 2004, 11: 453-454.
58. Kraitchman DL, M Tatsumi and WD Gilson. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation, 2005, 112: 1454–1464.
59. Noort WA, AB Kruisselbrink, PS Anker, et al. Mesenchymal stem cells remote engraftment of human umbilical cord blood-derived CD34+cells in NOD/SCID mice. Exp Hematol, 2002, 30: 870–878.
60. Krause U, Harter C, Seckinger A, et al. Intravenous delivery of autologous mesenchymal stem cells limits infarct size and improves left ventricular function in the infarcted porcine heart. Stem Cells Dev, 2007, 16: 31-37.
61. Tang YL, Zhao Q, Qin X,et al. Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. Ann Thorac Surg. 2005, 80: 229-237.
62. Peled A, Petit I , Kollet O, et al. Dependence of human stem cell engraftment and repopulation of NOD /SCID mice on CXCR4. Science, 1999, 283: 845 - 848.
63. Ratajczak MZ, Majka M, Kucia M, et al. Expression of functional CXCR4 by muscle satellite cells and secretion of SDF-1 by muscle derived fibroblasts is associated with the presence of both muscle progenitors in bone marrow and hematopoietic stem /progenitor cells in muscles. Stem Cells, 2003, 21: 363 - 371.
64. Reca R, Mastell osD, MajkaM, et al . Functional receptor for C3a anaphylatoxin is expressed by normal hemato-poietic stem /progenitor cells, and C3a enhances their homing related responses to SDF-1. Blood, 2003, 101: 3784 - 3793.
65. KorblingM, Estrov Z. Adult stem cells for tissue repair: a new therapeutic concept. N Engl J Med, 2003, 349: 570 - 582 .
66. Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation, 2003, 107: 1322 - 1328 .
67. Peled A, Kollet O, Ponomaryov T, et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34 (+) cells: role in transendothelial/stromal migration and engraftment of NOD /SCI D mice. Blood, 2000, 95 (11): 3289 - 3296.
68. Kudo M, Wang YG, Wani MA, et al. Implantation of bone marrow stem cells reduces the infarction and fibrosis in ischemic mouse heart. J Mol Cell Cardiol, 2003,35: 1113 - 1119.
69. Shintani S, Murohara T, Ikeda H, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 2001, 103: 897-903
70. Tang YL, Zhao Q, Zhang YC, et al. Autologous mesenchymal stem cell transplantation induces VEGF and neovascularization in ischemic myocardium. Regul Pept, 2004, 117: 3-10
71. Kinnaird T, Stabile E, Burnett M S, et al. Local delivery of marrow derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation, 2004, 109: 1543-1549.
72. Shake J G, Gruber P J, Baumgartner W A , et al. Mesenchymal stem cell implantation in a swine myocardial infarct model:engraftment and functional effects. Ann Thorac Surg, 2002 ,73: 1919 - 1925.
73.郝崇伟,杨云华.骨髓间充质干细胞移植改善心功能机制的研究进展.中国心血管病研究. 2007, 11: 845-847.
74. Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation, 2001,104: 1046.
75.韩瑜,陈曦,陈静海,等.骨髓间充质干细胞条件培养液对心脏成纤维细胞胶原合成的影响.中国分子心脏病学杂志, 2003, 83: 778-781.
76. Fucher S, Baffour R, Zhou YE, 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
77. Tang J, Xie Q, Pan G, et al. Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion. Eur J Cardiothorac Surg, 2006, 30:353-361.
78. Epstein SE, Kornowski R, Fuchs S, et al. Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects. Circulation, 2001, 104: 115-119.
79.徐亚丽,高云华,方针强,等.诊断超声联合微泡增强骨髓间充质干细胞归巢兔缺血心肌的研究.中华超声影像学杂志, 2008, 17: 899-902
80. Song J, Qi M, Kaul S, Price RJ. Stimulation of arteriogenesis in skeletal muscle by microbubble destruction with ultrasound. Circulation, 2002, 106: 1550–1555.
81.张群霞,王志刚,冉海涛,等.超声破坏微泡促进骨骼肌血管新生的实验研究.中国超声医学杂志, 2005, 21: 172-174.
82. Pons Jr, Huang Y, Arakawa-Hoyt J, et al.VEGF improves survival of mesenchymal stem cells in infarcted hearts. Biochem Biophysi Res Commu, 2008, 376: 419–422.
83.高云华,谭开彬,刘平,等.一种新型长循环超声造影剂增强心脏显像的实验研究.中国超声医学杂志, 2004, 20: 81-84.
84. Gnecchi M, He H, Liang O D, et al. Paracrine action accounts for marked protect ion of ischemic heart by Akt modified mesenchymal stem cells. Nat Med, 2005, 11: 367-368.
85.王彤,黄子通,符岳,等.心肌梗死后MSCs治疗改进心功能及心肺复苏结果的实验研究.中山大学学报(医学科学版), 2007, 28 (3) : 9– 11.
86. Kinnaird T, Stabile E, BurnettMS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivoarteriogenesis through paracrine mechanisms. Circ Res, 2004, 94: 678 - 685.
87. Yoon Young-sup, Andrea Wecker, Lindeay Heyd, et al. Clonally expanded novel multipotnet stem cells from human bone marrow regenerate myocardium after myocardial infarction. J Clin Invest, 2005, 115: 326-338.
88. Song J, Cottler PS, Klibanov AL, et al. Microvascular remodeling and accelerated hyperemia blood flow restoration in arterially occluded skeletal muscle exposed to ultrasonic microbubble destruction. Am J Physiol. 2004, 287: H2754–H2761.
89.刘德英,刘政,李秋颖,等.超声造影显像模式对实验兔心肌血管通透性的影响.临床超声医学杂志, 2006, 8: 577-580.
1. Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infracted myocardium:feasibility, cell migration, and body distribution[J ], Circulation, 2003, 108(7): 863-868.
2.张静,谢明星,王新房.超声心动图评价心肌细胞移植对Wistar大鼠心肌梗死后左心室重构及心功能的影响[J ].中华超声影像学杂志, 2006, 15(3): 216-219.
3. Orlic D, Kajstura J, Chimenti S, el al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival[J ]. PNAS, 200l, 98: 10344-10349.
4. Perin EC , Dohmann HF ,Borojevic R et al. Transendocardial autologous bone marrow cell transplantation for severe, chronic ischemic heart failure[J]. Circulation, 2003, 13, 107: 2294-2302.
5.崔继福,赵学,吴宗贵,经冠状动脉移植自体骨髓基质干细胞改善成年心肌梗死犬的心功能[J ].中国临床康复, 2005, 9(22): 49-52.
6. Wang JS, Shum-Tim D, Chedrawy E, et al. The coronary delivery of marrow stem cells for myocardial regeneration: pathophysiologic and therapeutic implications[J]. J Thorac Cardiovasc Surg, 2001, 122(4): 699-705.
7. Strauer BE, Brehn M, Zeus T, et al. Repair of infracted myocardium by autologous intraconronary mononuclear bone marrow cell transplan-tation in humans[J ]. Circulation, 2002, 106 :1913-1918.
8. Saito T , Kuang JQ , Lin CC ,et al. Transcoronary implantation of bone marrow stromal cells ameliorates cardiac function after myocardial infarction[J]. J Thorac Cardiovasc Surg, 2003, 126: 114-123.
9. Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal cells derived factor 1 on stem cell homing and tissue regeneration in ischaemic cardiomyopathy[J]. Lancet, 2003, 362: 697-703.
10.孟玲,刘国树,曹丰,等.经静脉途径移植骨髓间充质干细胞修复梗死心肌的可行性与安全性研究[J ].中华老年心脑血管病杂志, 2005, 7(3): 184-187.
11. Kocher AA, Schuster MD, Szabolcs MJ, et al.Neovascularization of ischemic myocardium by human bone marrow-derived angioblasta prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function[J]. Nat Med, 2001, 7:430-436.
12.余其贵,吴继雄.骨髓间充质干细胞移植治疗缺血性心脏病研究进展[J].中国心血管病研究杂志, 2007, 5(3): 232-234.
13. Toma C, Pittenger MF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart[J].Circulation.2005, 105:93-98.
14. Siminiak T, Czepcczynski R, Grygieska B, et al. Evidence for extravasation of intacoronary administered bone-marrow derived CD34+ stem cells in patients with acute myocardial infarction[J ]. Circulation, 2004, 110:Ⅲ-51.
15. Mangi AA, Noiscus N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore perormance of infracted hearts[J], Nat Med, 2003, 9(9): 195-201.
16.杨春,马爱群,蔡萍.大鼠急性心肌梗死后骨髓干细胞归巢于缺血心肌的时间窗研究[J ].中国病理生理杂志, 2005, 21(8):1539–1543.
17. Zen K, Okigaki M, Hosokawa Y, et al. Myocardium-targeted delivery of endothelial progenitor cells by ultrasound-mediated microbubble destruction improves cardiac function via an angiogenic response[J]. J Mol Cell Cardiol. 2006, 40(6): 799-809.
1. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999, 284 (5411) : 143-147.
2. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues [J] Science, 1997, 276( 5309): 71- 74.
3. Minguell JJ , Erices A , Conget P.Mesenchymal stem cells[J] . Exp Biol Med, 2001, 226 (6): 507-520.
4. Trotha R, Hanck T, Konig W, et a1. Rapid ribesequencing-an effective diagnostic tool for detecting microbial infection[J]. Infect, 2001, 29: 12-16.
5.王彤,符岳,方向韶,等.体外诱导MSCs向心肌细胞的分化和鉴定[ J ].中山大学学报(医学科学版), 2007, 28 (3): 9 - 11.
6. Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics [J]. Circ Res, 2004, 95 (1): 9 - 20.
7. Mangi AA, Noiseux N, Kong D, et al. Mesenchymal stem cells modified with Akt pevent remodeling and restore performance of infarcted hearts [J]. Nat Med, 2003, 9 (9): 1195 - 1201.
8. Djouad F, Hence P, Bony C, et a1. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals[J]. Blood, 2003, 102: 3837- 3844.
9.曹丰,裴雪涛,李艳华,等.移植时机对骨髓间充质干细胞修复梗死心肌的影响[J ].第四军医大学学报, 2005, 26: 22- 27.
10. StrauerBE, B rehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans [J]. Circulation, 2002, 106 (11): 1913-1918.
11. Nagaya N, Fujii T, Iwase T, et al. Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis[J]. Am J Physiol Heart Circ Physiol, 2004, 287(6): H2670-H2676
12. Tang J, Xie Q, Pan G, et al. Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion[J]. Eur J Cardiothorac Surg. 2006, 30(2): 353-361
13. Krause U, Harter C, Seckinger A, et al. Intravenous delivery of autologous mesenchymal stem cells limits infarct size and improves left ventricular function in the infarcted porcine heart[J]. Stem Cells Dev. 2007, 16(1): 31-37.
14. Zen K, Okigaki M, Hosokawa Y, et al. Myocardium-targeted delivery of endothelial progenitor cells by ultrasound-mediated microbubble destruction improves cardiac function via an angiogenic response[J]. J Mol Cell Cardiol. 2006, 40(6): 799-809.
15. Strauer BE, Brehm M, Zeus T, et a1. Intracoronary human autologous stem cells transplantation for myocardial regeneration following myocardial infarction[J]. DtschMed Wochenscher, 2001, 126: 932- 938.
16. Assmus B, Schachiger V, Teupe C, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction[J]. Circulation, 2002, 106: 3009-3017.
17. Chen SL, FangWW, Ye F, et al. Effect on left ventricular function of intracoro-nary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction [J]. Am J Cardiol, 2004, 94 (1): 92-95.
18. Tang Y L, Zhao Q, Qin X, et al. Paracrine act ion enhances theeffects of auto logous mesenchymal stem cell t ransp lantat ion on vascular regenerat ion in rat model of myocardial infarction[J]. A nn Tho rac Surg, 2005, 80(1): 229-236.
19. Pak H N, Qayyum M, Kim D T, et al. Mesenchymal stem cell injection induces cardiac nerve sprouting and increased tenascin expression in a swine model of myocardial infarct ion[J]. J Cardiovasc Elect rophysiol, 2003, 14(8): 841-848.
20. Xu X, Xu Z, Xu Y, et al . Effects of mesenchymal stem cell transplantation on extracellular matrix after myocardial infarction in rats[J]. Coron Artery Dis, 2005, 9: 124-129.
21. Hakuno D, Fukuda K, Makino S, et al. Bone marrow-derived regenerated cardimyocytes(CMG Cells) express functional adrenergic and muscarinic receptors[J]. Circulation, 2002, 105: 380- 386.
22. Kocher AA, Shuster MD, Szabolcs M, et al. Neovascularization of ischemic myocardium by human bone - marrow- derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function[J]. Nat Med, 2001, 7: 430- 436.
23. Krampera M, Glennie S, Dyson J, et a1.Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen specific T cells to their cognate peptide[J]. Blood, 2001, 101: 3722- 3729.
24. Toma C, PittengerMF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adultmurine heart[J]. Circulation, 2002, 105(8): 93-98.