摘要
目的：探讨人脐血单个核细胞(HUMNCs)静脉多次移植对急性心肌梗死(AMI)后心肌胶原网络重构的影响。
方法：自健康、足月分娩孕妇取脐血,分离并培养脐血单个核细胞(HUMNCs),60只健康中国家兔随机分为4组：(1)假手术组(n=15),冠脉前降支(LAD)过线不结扎,术后7d,9d,11d,13d,15d经耳缘静脉注入生理盐水500uL；(2)心肌梗死对照组(n=15),结扎冠脉前降支,术后7d,9d,11d,13d,15d同途径注入等量生理盐水；(3)HUMNCs单次静脉移植组(n=15),结扎冠脉前降支,术后7d,同途径注入含有3x106HUMNCs生理盐水500uL,9d,11d,13d,15d同途径注入等量生理盐水；(4)HUMNCs多次静脉移植组(n=15),结扎冠脉前降支,术后7d,9d,11d,13d,15d经耳缘静脉注入含有3×106 HUMNCs生理盐水500uL。术后1周,3周和5周行超声心动图检测左室舒张末内径(LVEDd)和左室射血分数(LVEF)；免疫组化检测心肌BrdU阳性细胞,HE染色和改良Masson's三色染色观测心肌胶原病理变化。
结果：(1)与假手术组比较,术后1周,3周,5周两细胞移植组和对照组超声LVEDd显著增加,LVEF显著降低,均有显著统计学意义(P<0.01)；与对照组比较,术后3周和5周两细胞移植组LVEDd减低,LVEF增加,有统计学意义(P<0.05)；与单次细胞移植组比较,多次细胞移植组术后3周LVEDd,LVEF改善,但无统计学意义(P>0.05),但术后5周LVEF显著改善,并有统计学意义(P<0.05)；
(2)免疫组化示经静脉单次与多次移植组的梗死周边区均存在BrdU阳性细胞,且多次移植组BrdU阳性细胞计数多于单次移植组；改良Masson's三色染色结果显示,术后3周及5周假手术组心肌细胞间有少量蓝色的、呈条索状与心肌纤维平行排列的胶原纤维；与假手术组比较,术后3周对照组及两细胞移植组梗死区及非梗死区胶原密度显著增加,梗死区域胶原纤维部分融合,排列较紊乱,心肌基本组织结构破坏；术后5周两细胞移植组梗死周边区域心肌细胞间胶原含量较对照组明显减少,梗死区域胶原纤维融合较少,排列较有序,且与单次细胞移植组比较,多次细胞移植组进一步改善。
结论：(1)多次静脉移植人脐血单个核细胞迁移并存活至急性心肌梗死梗死周边区域,数量多于单次移植,伴心功能明显改善。
(2)经静脉单次与多次移植人脐血单个核细胞均降低心肌梗死区域内胶原纤维的沉积和融合,促进心肌胶原网络有序排列,且多次移植优于单次移植。
(3)多次静脉移植脐血单个核细胞改善急性心肌梗死后心脏功能、阻抑心肌胶原纤维重构,其疗效优于单次静脉移植。
Objective:To investigate the influence of multiple transplantation intravenously on the remodeling of cardiac collagen network after myocardial infarction by human umbilical cord blood mononuclear cells (HUMNCs)
Methods:HUMNCs were harvested, cultured and expanded from umbilical cord blood of healthy full-term pregnant females. 60 Chinese rabbits were divided into four groups randomly. The sham operation group (n=15) was intravenously injected with normal saline 7d,9d, 11d,13d,15d after the non-ligation of the LAD. The control group (n=15), which had undergone ligation of the left anterior coronary artery (LAD), was intravenously injected with normal saline 7d,9d, 11d,13d,15d after operation. The single transplantation group (n=15) were intravenously injected with human umbilical cord blood mononuclear cells (HUMNCs) labeled with bromodexyuridine (BrdU) 7d after operation, and normal saline 9d, 11d,13d,15d after the ligation of the LAD. The multiple transplantation group (n=15) were intravenously injected with human umbilical cord blood mononuclear cells (HUMNCs) labeled with BrdU 7d,9d,11d, 13d,15d after the ligation of the LAD. Echocardiography examination was performed to measure left ventricular end-diastolic dimension(LVEDd) and left ventricular ejection fraction (LVEF) at 1 week,3 weeks and 5 weeks after operation. The rabbits were sacrificed for immunohistochemistry staining to identify the survival of transplanted cells (anti-BrdU monoclonal antibody), and for HE staining and Masson's trichrome staining to observe the structure of the cardiac collagen network.
Results:(1) Transthoracic echocardiography at 1 week,3 weeks and 5 weeks after the operation showed a significant enlargement of left ventricular end-diastolic dimension (LVEDd) and left ventricular ejection fraction (LVEF) in the cell groups and the control group, compared to the sham operation group(P<0.01). At 3 weeks and 5 weeks after the operation, the both cell transplatation groups showed a less enlargement of left ventricular end-diastolic dimension (LVEDd) and less reduction of and left ventricular ejection fraction (LVEF), compared to the control group (P<0.05), and while the heart function of the multiple transplantation group was improved compared with the single transplantation group at 5 weeks after the operation (P<0.05).
(2) The BrdU positive cells were found in the peri-myocardial infarction area in the both transplantation groups, and while the number of the BrdU positive cells in the multiple transplantation group are more than that in the single transplantation group. Masson's trichrome staining showed that collagen fibers were arranged regularly in the myocardial tissue of the sham operation group's rabbits at 3 and 5 weeks after operation. Compared to the sham operation group, the counts of the collagen fiber in the control group and the cell transplatation groups were significantly increased at 3 weeks after the operation, and the collagen fibers were fused partially, and arranged irregularly, the basic structure of myocardium was destroyed. The collagen fibers in the multiple cell transplantation group were arranged more regularly, compared to in the single group.
Conclusion:(1) The human umbilical cord blood mononuclear cells (HUMNCs) delivered by intravenous injection could migrate to the infarct region and survive in the peri-myocardial infarction area, and the number of the transplanted cells in the myocardium in the multiple transplantation group was increased, associated with more improvement of the heart function, compared to the single transplantation group.
(2) The single and multiple intravenous transplantation of human cord blood mononuclear cells could reduce the collagen deposition and integration of the collagen fuse, and promote orderly arrangement of myocardial collagen network in the myocardial infarction region. Compared with the single-cell transplantation group, the multiple cell transplantation group further improved.
(3) The multiple intravenously transplanted cord blood mononuclear cell transplantation for myocardial infarction was superior to improve cardiac function and inhibit myocardial collagen remodeling, compared to single intravenously transplanted human cord blood mononuclear cells.
引文
[1]Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med, 2001,344(23):1750-1757
[2]Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell,2003,114(6):763-776
[3]Javed MJ, Mead LE, Prater D, et al. Endothelial colony forming cells and mesenchymal stem cells are enriched at different gestational ages in human umbilical cord blood. Pediatr Res,2008,64(1):68-73
[4]李聪,漆洪波.脐血采集和保存的实用问题.实用妇产科杂志,2009,(04)：207-209
[5]Ma N, Stamm C, Kaminski A, et al. Human cord blood cells induce angiogenesis following myocardial infarction in NOD/scid-mice. Cardiovasc Res, 2005,66(1):45-54
[6]赵林,王雷,贾三庆,等.人脐血干细胞移植促进心肌梗死大鼠血管新生的机制研究.临床心血管病杂志,2008,24(7)：540-544
[7]Xing YL, Shen LH, Li HW, et al. Optimal time for human umbilical cord blood cell transplantation in rats with myocardial infarction. Chin Med J (Engl), 2009,122(23):2833-2839
[8]Yu G, Borlongan CV, Stahl CE, et al. Transplantation of human umbilical cord blood cells for the repair of myocardial infarction. Med Sci Monit, 2008,14(10):RA163-172
[9]Jiang CY, Gui C, He AN, et al. Optimal time for mesenchymal stem cell transplantation in rats with myocardial infarction. J Zhejiang Univ Sci B, 2008,9(8):630-637
[10]徐叔云,卞如濂,陈修.药理学实验方法学.第第3版版：北京：人民卫生出版社,2002.229-230
[11]Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol,2000,109(1):235-242
[12]刘素芳,鄢文海,韩雪飞,等.人脐血间充质细胞体外分离培养方法及培养基特性.中国临床康复,2005,9(46)：34-35
[13]Lee OK, Kuo TK, Chen WM, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood,2004,103(5):1669-1675
[14]Stute N, Holtz K, Bubenheim M, et al. Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use. Exp Hematol, 2004,32(12):1212-1225
[15]张勇刚,王黎明,杨明理,等.自体血清培养人脐血间充质干细胞的实验研究.生物医学工程学杂志,2008,25(5)：1155-1160
[16]金玮,杨安怀,邢怡桥.人脐血间充质干细胞的体外培养及生物学特性.武汉大学学报：医学版,2007,28(4)：488-491
[17]Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science,1997,275(5302):964-967
[18]Dudek AZ, Bodempudi V, Welsh BW, et al. Systemic inhibition of tumour angiogenesis by endothelial cell-based gene therapy. Br J Cancer, 2007,97(4):513-522
[19]Yin AH, Miraglia S, Zanjani ED, et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood,1997,90(12):5002-5012
[20]Davani S, Deschaseaux F, Chalmers D, et al. Can stem cells mend a broken heart. Cardiovasc Res,2005,65(2):305-316
[21]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. Am J Physiol Heart Circ Physiol, 2004,287(6):H2670-2676
[22]赵林,王雷,贾三庆,等.人+细胞静脉移植对急性心肌梗死大鼠血流动力学指标和超声心动图指标的影响.中国组织工程研究与临床康复,2007,11(20)：3948-3951
[23]Kraitchman DL, Tatsumi M, Gilson WD, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation, 2005,112(10):1451-1461
[24]Guo J, Lin G, Bao C, et al. Insulin-like growth factor 1 improves the efficacy of mesenchymal stem cells transplantation in a rat model of myocardial infarction. J Biomed Sci,2008,15(1):89-97
[25]Abbott JD, Huang Y, Liu D, et al. Stromal cell-derived factor-lalpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation, 2004,110(21):3300-3305
[26]Tiyyagura SR, Pinney SP. Left ventricular remodeling after myocardial infarction: past, present, and future. Mt Sinai J Med,2006,73(6):840-851
[27]郭豫涛[1],李小鹰[1],吴迪[2],等.骨髓干细胞移植通过基质金属蛋白酶及其抑制剂降低大鼠缺血性心力衰竭心室重构.中华心血管病杂志,2006,34(9)：784-788
[28]Wang JA, Luo RH, Zhang X, et al. Bone marrow mesenchymal stem cell transplantation combined with perindopril treatment attenuates infarction remodelling in a rat model of acute myocardial infarction. J Zhejiang Univ Sci B, 2006,7(8):641-647
[29]钟竑[1],赵宏光[2],马南[1],等.脐血内皮祖细胞移植对犬急性梗死心肌胶原网络重构的影响.江西医学院学报,2007,47(5)：18-21,F0003
[30]Liew A, Barry F, O'Brien T. Endothelial progenitor cells:diagnostic and therapeutic considerations. Bioessays,2006,28(3):261-270
[31]Rabelink TJ, de Boer HC, de Koning EJ, et al. Endothelial progenitor cells:more than an inflammatory response. Arterioscler Thromb Vasc Biol, 2004,24(5):834-838
[32]Ishizawa K, Kubo H, Yamada M, et al. Hepatocyte growth factor induces angiogenesis in injured lungs through mobilizing endothelial progenitor cells. Biochem Biophys Res Commun,2004,324(1):276-280
[33]Ishizawa K, Kubo H, Yamada M, et al. Bone marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema. FEBS Lett, 2004,556(1-3):249-252
[34]Ishizawa K, Kubo H, Yamada M, et al. Bone marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema. FEBS Lett, 2004,556(1-3):249-252
[35]Palange P, Testa U, Huertas A, et al. Circulating haemopoietic and endothelial progenitor cells are decreased in COPD. Eur Respir J,2006,27(3):529-541
[36]Henning RJ, Shariff M, Eadula U, et al. Human cord blood mononuclear cells decrease cytokines and inflammatory cells in acute myocardial infarction. Stem Cells Dev,2008,17(6):1207-1219
[37]Xu X, Xu Z, Xu Y, et al. Effects of mesenchymal stem cell transplantation on extracellular matrix after myocardial infarction in rats. Coron Artery Dis, 2005,16(4):245-255
[38]Xu X, Xu Z, Xu Y, et al. Selective down-regulation of extracellular matrix gene expression by bone marrow derived stem cell transplantation into infarcted myocardium. Circ J,2005,69(10):1275-1283
[39]Misao Y, Takemura G, Arai M, et al. Bone marrow-derived myocyte-like cells and regulation of repair-related cytokines after bone marrow cell transplantation. Cardiovasc Res,2006,69(2):476-490
[40]Sun Y, Zhang JQ, Zhang J, et al. Angiotensin Ⅱ, transforming growth factor-betal and repair in the infarcted heart. J Mol Cell Cardiol, 1998,30(8):1559-1569
[41]Zuscik MJ, Pateder DB, Puzas JE, et al. Lead alters parathyroid hormone-related peptide and transforming growth factor-betal effects and AP-1 and NF-kappaB signaling in chondrocytes. J Orthop Res,2002,20(4):811-818
[1]Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for cardiomyocyte renewal in humans. Science,2009,324(5923):98-102
[2]Reffelmann T, Konemann S, Kloner RA. Promise of blood- and bone marrow-derived stem cell transplantation for functional cardiac repair:putting it in perspective with existing therapy. J Am Coll Cardiol,2009,53(4):305-308
[3]Chachques JC, Acar C, Herreros J, et al. Cellular cardiomyoplasty:clinical application. Ann Thorac Surg,2004,77(3):1121-1130
[4]Bartunek J, Dimmeler S, Drexler H, et al. The consensus of the task force of the European Society of Cardiology concerning the clinical investigation of the use of autologous adult stem cells for repair of the heart. Eur Heart J, 2006,27(11):1338-1340
[5]Lipinski MJ, Biondi-Zoccai GG, Abbate A, et al. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: a collaborative systematic review and meta-analysis of controlled clinical trials. J Am Coll Cardiol,2007,50(18):1761-1767
[6]Tendera M, Wojakowski W, Ruzyllo W, et al. Intracoronary infusion of bone marrow-derived selected CD34+CXCR4+ cells and non-selected mononuclear cells in patients with acute STEMI and reduced left ventricular ejection fraction: results of randomized, multicentre Myocardial Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) Trial. Eur Heart J,2009,30(11):1313-1321
[7]Stagg MA, Coppen SR, Suzuki K, et al. Evaluation of frequency, type, and function of gap junctions between skeletal myoblasts overexpressing connexin43 and cardiomyocytes:relevance to cell transplantation. FASEB J, 2006,20(6):744-746
[8]Guan K, Wagner S, Unsold B, et al. Generation of functional cardiomyocytes from adult mouse spermatogonial stem cells. Circ Res,2007,100(11):1615-1625
[9]Bartunek J, Sherman W, Vanderheyden M, et al. Delivery of biologics in cardiovascular regenerative medicine. Clin Pharmacol Ther,2009,85(5):548-552
[10]Fernandez-Aviles F, San RJA, Garcia-Frade J, et al. Experimental and clinical regenerative capability of human bone marrow cells after myocardial infarction. Circ Res,2004,95 (7):742-748
[11]Chachques JC, Salanson-Lajos C, Lajos P, et al. Cellular cardiomyoplasty for myocardial regeneration. Asian Cardiovasc Thorac Ann,2005,13(3):287-296
[12]Gnecchi M, Zhang Z, Ni A, et al. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res,2008,103(11):1204-1219
[13]Fedak PW, Szmitko PE, Weisel RD, et al. Cell transplantation preserves matrix homeostasis:a novel paracrine mechanism. J Thorac Cardiovasc Surg, 2005,130(5):1430-1439
[14]余国龙[2],蒋路平[1],谢秀梅[1],等.自体骨髓干细胞移植治疗心力衰竭的研究进展.基础医学与临床,2004,24(6)：696-700
[15]Yu G, Borlongan CV, Stahl CE, et al. Transplantation of human umbilical cord blood cells for the repair of myocardial infarction. Med Sci Monit, 2008,14(10):RA163-172
[16]Fernandes S, Amirault JC, Lande G, et al. Autologous myoblast transplantation after myocardial infarction increases the inducibility of ventricular arrhythmias. Cardiovasc Res,2006,69(2):348-358
[17]Villa A, Sanchez PL, Fernandez-Aviles F. Ventricular arrhythmias following intracoronary bone marrow stem cell transplantation. Europace, 2007,9(12):1222-1223
[18]Krupnick AS, Kreisel D, Riha M, et al. Myocardial tissue engineering and regeneration as a therapeutic alternative to transplantation. Curr Top Microbiol Immunol,2004,280:139-164
[19]Memon IA, Sawa Y, Miyagawa S, et al. Combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells in canine hearts for ischemic cardiomyopathy. J Thorac Cardiovasc Surg,2005,130(3):646-653
[20]Miyagawa S, Matsumiya G, Funatsu T, et al. Combined autologous cellular cardiomyoplasty using skeletal myoblasts and bone marrow cells for human ischemic cardiomyopathy with left ventricular assist system implantation:report of a case. Surg Today,2009,39(2):133-136
[21]杨水祥,徐静,徐桂玉,等.自体骨髓干细胞移植治疗心肌梗死6例4年随访.中国组织工程研究与临床康复,2008,(34)：6657-6660
[22]Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction:the BOOST randomised controlled clinical trial. Lancet,2004,364(9429):141-148
[23]石宗华,高传玉,马许辉,等.经冠状动脉自体骨髓造血干细胞移植对急性心肌梗死的疗效.岭南心血管病杂志,2007,(06)：398-402
[24]聂颖,郭艳红,郭丽君,等.自体骨髓干细胞移植改善心肌梗死后左心功能不全患者心脏功能.北京大学学报(医学版),2007,(06)：634-637
[25]Erbs S, Linke A, Schachinger V, et al. Restoration of microvascular function in the infarct-related artery by intracoronary transplantation of bone marrow progenitor cells in patients with acute myocardial infarction:the Doppler Substudy of the Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial. Circulation, 2007,116(4):366-374
[26]Monnet E, Chachques JC. Animal models of heart failure:what is new. Ann Thorac Surg,2005,79(4):1445-1453
[27]王建安,谢小洁,何红,等.骨髓间质干细胞移植治疗原发性扩张型心肌病的疗效与安全性.中华心血管病杂志,2006,34(2)：107-110
[28]Huang RC, Yao K, Li YL, et al. [Transplantation of autologous bone marrow mononuclear cells on patients with idiopathic dilated cardiomyopathy:early results on effect and security]. Zhonghua Xin Xue Guan Bing Za Zhi, 2006,34(2):111-113
[29]张彤,刘超,杨俊,等.外周血干细胞移植治疗扩张型心肌病42例3个月随访.中国组织工程研究与临床康复,2008,12(16)：3045-3048
[30]Vilas-Boas F, Feitosa GS, Soares MB, et al. Bone marrow cell transplantation to the myocardium of a patient with heart failure due to Chagas' disease. Arq Bras Cardiol,2004,82(2):185-187,181-184
[31]Vilas-Boas F, Feitosa GS, Soares MB, et al. [Early results of bone marrow cell transplantation to the myocardium of patients with heart failure due to Chagas disease]. Arq Bras Cardiol,2006,87(2):159-166
[32]Pillekamp F, Khalil M, Emmel M, et al. Stem cells in pediatric heart failure. Minerva Cardioangiol,2008,56(3):335-348
[33]Olgunturk R, Kula S, Sucak GT, et al. Peripheric stem cell transplantation in children with dilated cardiomyopathy:Preliminary report of first two cases. Pediatr Transplant,2009,
[34]Rupp S, Bauer J, Tonn T, et al. Intracoronary administration of autologous bone marrow-derived progenitor cells in a critically ill two-yr-old child with dilated cardiomyopathy. Pediatr Transplant,2009,13(5):620-623
[35]Coletta JE, Rosenthal N, Costa MA. Cardiac mapping and stem cell delivery for the damaged myocardium. Expert Rev Cardiovasc Ther,2008,6(9):1181-1190
[36]王梦洪,付勇南,郑泽琪,等.自体骨髓间充质干细胞移植治疗心肌梗死的时机选择.临床心血管病杂志,2009,(04)：263-265
[37]Al KA, Ge Y, Shum-Tim D, et al. Cellular cardiomyoplasty:routes of cell delivery and retention. Front Biosci,2008,13:2421-2434
[38]McCue JD, Swingen C, Feldberg T, et al. The real estate of myoblast cardiac transplantation:negative remodeling is associated with location. J Heart Lung Transplant,2008,27(1):116-123
[39]Thompson RB, Parsa CJ, den Bos EJ v, et al. Video-assisted thoracoscopic transplantation of myoblasts into the heart. Ann Thorac Surg, 2004,78(1):303-307
[40]Chachques JC, Herreros J, Trainini J, et al. Autologous human serum for cell culture avoids the implantation of cardioverter-defibrillators in cellular cardiomyoplasty. Int J Cardiol,2004,95 Suppl 1:S29-33
[41]Shafy A, Lavergne T, Latremouille C, et al. Association of electrostimulation with cell transplantation in ischemic heart disease. J Thorac Cardiovasc Surg, 2009,138(4):994-1001
[42]Akhyari P, Kamiya H, Haverich A, et al. Myocardial tissue engineering:the extracellular matrix. Eur J Cardiothorac Surg,2008,34(2):229-241
[43]Genovese JA, Spadaccio C, Chachques E, et al. Cardiac pre-differentiation of human mesenchymal stem cells by electrostimulation. Front Biosci, 2009,14:2996-3002
[44]Prasad SM, Czepiel M, Cetinkaya C, et al. Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation. Cell Prolif, 2009,42(1):63-74
[45]Chachques JC, Duarte F, Cattadori B, et al. Angiogenic growth factors and/or cellular therapy for myocardial regeneration:a comparative study. J Thorac Cardiovasc Surg,2004,128(2):245-253