脂肪干细胞自体移植促进心肌再生和血管新生

目的探讨体外培养的脂肪干细胞（ADSCs）自体移植后能否在心肌梗死区（MI）分化为心肌细胞,并促进血管新生。方法取新西兰白兔30只,结扎前降支近段制作MI模型。治疗组同时取脂肪组织,体外培养扩增AD-SCs,并给予5-氮胞苷诱导。MI后3周进行细胞自体移植,对照组注射磷酸盐缓冲液,饲养5周后行组织学和心功能检查。结果细胞移植组MI面积显著小于对照组,免疫组织化学检测显示大量ADSCs在MI区分化为心肌细胞,ASDCs在MI边缘较梗死中心分化更为彻底,细胞间互相连接,而MI中心分化的细胞之间连接较少;另有部分ADSCs分化为内皮细胞,整合至毛细血管壁内或者形成血管样结构,治疗组血管密度显著高于对照组。细胞移植前两组动物心功能无明显差别,细胞移植5周后,心功能检查显示:治疗组左心室射血分数和+dp/dtmax明显高于对照组,而心肌功能指数,左室舒张末压和-dp/dtmax明显低于对照组。结论脂肪干细胞移植至MI心肌组织后可以分化为心肌细胞并促进血管新生,进而改善心脏功能。     

心肌内控制释放碱性成纤维细胞生长因子促进血管再生与侧支重构

目的评价心肌内控制释放碱性成纤维细胞生长因子(bFGF)血管再生治疗对急性心肌梗死犬血管再生与侧支重构的影响。方法18只成年健康杂种犬制作急性心肌梗死模型,随机分为单纯心肌梗死组、激光心肌血运重建(TMR)组和bFGF组,每组6只。术后第8周,采用第八因子免疫组织化学染色和计算机形态分析评价血管再生与侧支重构的变化。结果直接血管计数发现,bFGF组镜下总血管计数和直径≥50μm的较大微血管数均高于TMR组和单纯心肌梗死组(P<0.001)。bFGF组的第八因子相关抗原染色显色面积和平均吸光度均高于TMR组与单纯心肌梗死组(P<0.001);在TMR基础上在心肌内控制释放bFGF能使总血管数增加近30%,其中直径≥50μm的微血管数增加近60%。结论采用纤维蛋白胶在心肌内控制释放bFGF的血管再生治疗安全可行,能有效促进缺血心肌内的血管再生和侧支重构。     

Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of myocardial infarction

Transplantation of stem cells may improve regional perfusion and post-infarct ventricular function, but the optimal dose and efficacy of cell delivery via the intravenous route has not been determined. This study tested the hypothesis that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) enhances regional perfusion and improves ventricular function after myocardial infarction. In a closed-chest pig model, the LAD coronary artery was occluded for 75 min by angioplasty balloon inflation followed by 12 weeks of reperfusion. After 15 min of reperfusion, pigs randomly received 1 of 4 treatments: (1) Vehicle (Control, n = 10); (2) 1 × 10⁶ MSCs/kg (1 mill, n = 7); (3) 3 × 10⁶ MSCs/kg (3 mill, n = 8) and (4) 10 × 10⁶ MSCs/kg (10 mill, n = 8). Angiogenesis was demonstrated by immunohistochemical staining, myocardial blood flow (steady state and vasodilator reserve) was measured using 15 μm neutron-activated microspheres, and cardiac function was determined by contrast left ventriculography (ejection fraction) and pressure–volume relationships. After 12 week of reperfusion, von Willebrand Factor-positive vessels and tissue vascular endothelial growth factor (VEGF) expression in the scar zone was significantly greater in all MSCs-treated animals relative to Control. Steady state myocardial blood flow in the scar tissue was comparable among groups. However, adenosine recruited vasodilator reserve in the scar zone induced by intracoronary adenosine was significantly higher in the MSC-treated animals compared to Control. Furthermore, preload-recruitable stroke work and systolic performance were significantly greater compared to Control. In conclusion, these data demonstrate that intravenous delivery of MSCs during early reperfusion augments vasculogenesis, enhances regional perfusion, and improves post-infarct ventricular function. The results suggest that intravenous infusion of MSCs is an effective modality for the treatment of ischemia/reperfusion induced myocardial injury. Returned for 1. Revision: 11 April 2008 1. Revision received: 30 May 2008 Returned for 2. Revision: 11 June 2008 2. Revision received: 7 July 2008 Returned for 3. Revision: 9 July 2008 3. Revision received: 14 July 2008     

急性心肌梗死患者外周血干细胞及干细胞因子的变化

目的观察急性心肌梗死(AMI)患者外周血干细胞和干细胞因子(stem cell factor,SCF)的变化及两者的相关性。方法AMI患者共25例,收集患者入院即刻,第13、、71、4和28天的抗凝血各2 ml,用流式细胞仪检测如下细胞表面标记物:CD34+、血管内皮细胞生长因子受体-2(VEGFR-2+)、CD31+、CD144+、CD133+、CD45+,比较各时间点的差异。同时用酶联免疫吸附法检测血清SCF水平。结果AMI后CD34+细胞逐渐升高,到第7天时达到峰值,而VEGFR-2+、CD31+、CD144+、CD133+细胞则出现下降趋势,并且于第7天时达到谷值(P<0.05)。AMI患者外周血清SCF浓度随时间推移呈下降趋势(P<0.01),但AMI后第1天时SCF浓度与外周血CD34+细胞数呈直线相关(P<0.01)。结论AMI可引起外周血干细胞数量增多以及血清SCF浓度升高,但也许会消耗循环中有内皮功能或向内皮分化的细胞。这些干细胞数量的增多可能受SCF浓度的调节。     

Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue

Success of human myocardial tissue engineering for cardiac repair has been limited by adverse effects of scaffold materials, necrosis at the tissue core, and poor survival after transplantation due to ischemic injury. Here, we report the development of scaffold-free prevascularized human heart tissue that survives in vivo transplantation and integrates with the host coronary circulation. Human embryonic stem cells (hESCs) were differentiated to cardiomyocytes by using activin A and BMP-4 and then placed into suspension on a rotating orbital shaker to create human cardiac tissue patches. Optimization of patch culture medium significantly increased cardiomyocyte viability in patch centers. These patches, composed only of enriched cardiomyocytes, did not survive to form significant grafts after implantation in vivo. To test the hypothesis that ischemic injury after transplantation would be attenuated by accelerated angiogenesis, we created “second-generation,” prevascularized, and entirely human patches from cardiomyocytes, endothelial cells (both human umbilical vein and hESC-derived endothelial cells), and fibroblasts. Functionally, vascularized patches actively contracted, could be electrically paced, and exhibited passive mechanics more similar to myocardium than patches comprising only cardiomyocytes. Implantation of these patches resulted in 10-fold larger cell grafts compared with patches composed only of cardiomyocytes. Moreover, the preformed human microvessels anastomosed with the rat host coronary circulation and delivered blood to the grafts. Thus, inclusion of vascular and stromal elements enhanced the in vitro performance of engineered human myocardium and markedly improved viability after transplantation. These studies demonstrate the importance of including vascular and stromal elements when designing human tissues for regenerative therapies.     

间充质干细胞治疗心肌梗死的现状及展望

间充质干细胞存在于骨髓、脂肪等多种成熟组织,易于体外分离培养。很多临床前及临床研究已经证实其可以通过分化为心肌样细胞、促血管生成、旁分泌等作用参与心肌梗死后组织修复,改善心脏功能。间充质干细胞是理想的心肌再生治疗的细胞来源,现就相关领域研究现状及进展做一综述。     

阵发性房室结折返性室上性心动过速射频消融临床观察

目的: 探讨延迟缺血预处理（delayed ischemic preconditioning,DIPC）对急性心肌梗死(AMI)大鼠远期的心肌保护及其促进小动脉再生的作用。方法: 将54只SD大鼠随机分为4组,即①缺血预处理(IPC)+AMI组:于IPC后24 h,结扎冠状动脉建立AMI大鼠模型(n=24);②AMI组:开胸后,只穿线不进行IPC,于24 h后结扎冠脉建立AMI大鼠模型(n=24);③IPC组:只进行IPC不结扎冠脉(n=3);④假手术组:开胸后,既不进行IPC操作也不结扎冠状动脉(n=3)。前两组于模型建立后3 d、7 d和14 d,用免疫组化染色法检测梗死边缘区毛细血管新生及小动脉再生;后两组分别于IPC后及假手术后24 h,用免疫组化染色法检测梗死边缘区毛细血管新生及血管内皮生长因子（VEGF）、血小板源生长因子-B（PDGF-B）表达。14 d后采用小动物超声检测心功能,Masson’s Trichrome染色法测量心肌梗死(MI)面积。结果: IPC组于IPC后24 h,缺血区的心肌中可见毛细血管新生,VEGF及PDGF-B表达明显增加（与假手术组相比,P<0.05）。与AMI相比,IPC+AMI组于建模后3 d、7 d和14 d,梗死边缘区心肌中毛细血管的密度和小动脉的密度均明显增加（P<0.05）,MI面积减小（P<0.05）,心功能改善（与AMI组相比,左室短轴缩短率增高,P<0.05）。结论: DIPC可减小MI的面积,改善心功能,这一作用与其增加梗死边缘区中的小动脉再生有关;缺血区心肌中VEGF及PDGF-B表达的增加可能是小动脉再生的刺激因素。     

IGF-1预处理对骨髓间充质干细胞心肌再生及抗炎效果的影响

目的:探讨胰岛素样生长因子1(IGF-1)预处理对骨髓间充质干细胞(MSCs)心肌再生及抗炎效果的影响。方法:10μg/L的IGF-1预处理MSCs,流式细胞仪分析趋化因子受体4的表达;建立大鼠心肌梗死模型,尾静脉分别注射不含MSCs的100μl培养液(对照组),含经IGF-1预处理的MSCs(IGF-1-MSCs组)和无预处理的MSCs(MSCs组),每组8只大鼠;MSCs移植4周后观察心肌组织炎性因子(肿瘤坏死因子-α、白细胞介素-1β和白细胞介素-6)在mRNA和蛋白水平的表达,分析移植细胞的归巢率,连接蛋白43(CX43)的表达,心肌特异性蛋白肌钙蛋白T的表达。结果:流式细胞术显示MSCs经IGF-1预处理48h后的趋化因子受体4显著增高。IGF-1-MSCs组MSCs移植4周后:①更多的MSCs归巢到心肌梗死周边区(P<0.05);②显著增加梗死周边区肌钙蛋白T阳性细胞(P<0.05)和CX43密度(P<0.05);③降低了心肌梗死后心肌炎性因子的表达。结论:IGF-1预处理能够显著提高MSCs的心肌再生能力,并提高其在梗死心肌中的抗炎效果。     

Intramyocardial Injection of DNA Encoding Vascular Endothelial Growth Factor in a Myocardial Infarction Model

In a previous study, we observed that one injection of 500g of DNA for the plasmid encoding for vascular endothelial growth factor (ph VEGF₁₆₅) into one site in a rat myocardial infarction model resulted in neovascularization confined to angiomatous structures that did not contribute to regional myocardial blood flow. The purpose of the present study was to determine whether a lower dose (125g DNA), which is the same as that being used in some clinical trials, injected into four separate sites could enhance collateral flow and vascularity to the ischemic bed without inducing angiomas. Rats received injections of 125g DNA of the plasmid encoding phVEGF₁₆₅ or control DNA at four separate sites within the anterior free wall of the left ventricle (LV) supplied by the left coronary artery. The left coronary artery was ligated and hearts analyzed at 4 weeks. In vitro studies confirmed that the phVEGF₁₆₅ used was capable of producing VEGF polypeptide in mammalian cells. The infarct size (percentage of endocardial circumference that infarcted) was similar in controls (42±6%) and treated hearts (39±7%); the LV cavity area did not differ between groups. The number of vascular structures per high-power field within the infarct scar was 10.50±0.68 in controls and 10.00±0.85 in phVEGF₁₆₅-treated rats. Relative regional myocardial blood flow determined by radioactive microspheres and expressed as a ratio of radioactive counts within the scar divided by radioactive counts in the noninfarcted ventricular septum was similar in control (0.74±0.25) and treated hearts (0.88±0.30) (p=not significant). No angiomatous structures were observed. Injections of 125g of DNA of phVEGF₁₆₅ into myocardium to become ischemic had no effect on infarct size or LV cavity size. Unlike higher doses of 500g of DNA, it did not cause gross angiomatous structures; however, it failed to improve neovascularization or regional myocardial blood flow in this rodent model of acute myocardial infarction.     

骨髓间质干细胞移植治疗急性心肌梗死的实验研究

目的 研究同种异体骨髓间质干细胞（bone marrow mesenchymak stem cells MSCs）移植至梗死心肌后对兔的一般情况、心功能、梗死面积和血管新生的影响.方法 开胸结扎冠状动脉左前降支建立急性心肌梗死动物模型,将5-溴脱氧尿核苷（bromodeoxyuridine,BrdU）标记的MSCs注入心肌梗死区.测定各组手术前后体重和心功能并取出心脏作相关检查.结果 与急性心肌梗死组比较,MSCs组术后一般情况好,6周时体重明显增加（P＜0.05）,术后3周、6周左心室射血分数（left ventricular ejection fraction,LVEF）均明显增加（P＜0.05）,梗死面积明显缩小（P＜0.05）,毛细血管数目显著增多（P＜0.01）.MSCs组心肌梗死区可见BrdU阳性细胞,电镜下可见不成熟的心肌样细胞.结论 同种异体MSCs移植治疗急性心肌梗死除提高心功能外,尚可改善兔子的一般情况,增加体重.     

延迟相缺血预处理心肌保护时间窗的临床探讨

目的探讨心肌梗死患者延迟相缺血预处理的心肌保护时间窗。方法行直接冠状动脉介入治疗的急性心肌梗死患者150例,根据梗死前心绞痛发作时间分为三组：无心绞痛组（1组）55例,〈72 h心绞痛组（2组）55例,〉72 h心绞痛组（3组）40例。统计患者一般临床情况、院内预后指标、出院前的左室射血分数,肌酸激酶和肌酸激酶同工酶的曲线下面积反映患者的心肌梗死面积。结果除年龄外,其余各项临床指标在三组间均无统计学差异。2组和3组的心肌梗死面积明显低于1组（P〈0.01）,但2组的充血性心力衰竭/休克、复合终点发生率明显低于1组和3组（P〈0.01或0.05）。梗死前心绞痛的心肌保护作用与年龄、抗心绞痛药物以及侧支循环无关。结论临床缺血预处理的保护时间窗不同于动物实验,可能大于72 h。     

粒细胞集落刺激因子动员骨髓干细胞治疗急性心肌梗死的观察

目的探讨粒细胞集落刺激因子(G-CSF)动员的骨髓来源干细胞对急性心肌梗死(AMI)的治疗作用.方法 27例AMI病人随机分为治疗组和对照组,治疗组连续3 d给予G-CSF 300 μg/d,用201Tl心肌显像比较两组第6天和第30天的梗死面积变化,超声心动图观察3 d内和3个月的心功能变化.结果治疗组第30天的核素梗死缺损区面积明显减少(P＜0.01),核素放射性计数百分比明显增加(P＜0.05),治疗组治疗后各项心功能指标均明显改善(P＜0.01).对照组无明显改变(P＞0.05).结论用G-CSF动员骨髓干细胞可再生成心肌组织,明显缩小AMI的梗死面积及改善心功能.     

应用G-CSF动员骨髓干细胞治疗急性心肌梗塞的研究

目的 研究用粒细胞集落刺激因子(G—CSF)动员骨髓干细胞对急性心肌梗塞进行治疗的可行性。方法 将21例急性心肌梗塞患者随机分成治疗组13例和对照组8例,治疗组连续3大给予G—CSF 300μg/天。用QRS记分系统(ORSs)和SPEGT心肌断层显像方法比较两组第6天和30天的心电图记分和缺损面积变化。结果 治疗组第 30天的心电图记分较第6天明显下降(3.01±0.32vs6.79±0.36,P<0.01),核素梗死缺损区面积明显减少(39.2±7.7 vs 56.9±9.1,P<0.05),核素放射性计数百分比明显增加[(25.1±5.3)％ vs (18.1±6.5)％,P<0.05],对照组无明显改变(P>0.05)。结论 用G—CSF动员骨髓干细胞可迁移心脏内,并再生成心肌组织,明显改善梗死心肌。     

骨髓间质干细胞移植治疗急性心肌梗死研究进展

心肌梗死后导致的心功能障碍和心律失常的细胞学基础是正常心肌细胞的丧失,而代之以纤维瘢痕组织的修复。骨髓间质干细胞具有心肌再生能力,因而显示出其治疗心肌梗死的巨大潜能。现综述骨髓间质干细胞移植治疗急性心肌梗死的兴起、移植途径、作用机制、异体移植和免疫反应、细胞疗法和基因治疗相结合、临床实验的最新研究进展。     

Bone marrow cells adopt the cardiomyogenic fate in vivo

The possibility that adult bone marrow cells (BMCs) retain a remarkable degree of developmental plasticity and acquire the cardiomyocyte lineage after infarction has been challenged, and the notion of BMC transdifferentiation has been questioned. The center of the controversy is the lack of unequivocal evidence in favor of myocardial regeneration by the injection of BMCs in the infarcted heart. Because of the interest in cell-based therapy for heart failure, several approaches including gene reporter assay, genetic tagging, cell genotyping, PCR-based detection of donor genes, and direct immunofluorescence with quantum dots were used to prove or disprove BMC transdifferentiation. Our results indicate that BMCs engraft, survive, and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes. BMCs and myocytes express at their interface connexin 43 and N-cadherin, and this interaction may be critical for BMCs to adopt the cardiomyogenic fate. With time, a large number of myocytes and coronary vessels are generated. Myocytes show a diploid DNA content and carry, at most, two sex chromosomes. Old and new myocytes show synchronicity in calcium transients, providing strong evidence in favor of the functional coupling of these two cell populations. Thus, BMCs transdifferentiate and acquire the cardiomyogenic and vascular phenotypes restoring the infarcted heart. Together, our studies reveal that locally delivered BMCs generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels. This process occurs independently of cell fusion and ameliorates structurally and functionally the outcome of the heart after infarction.     

骨髓间充质干细胞移植治疗心肌梗死的研究进展

溶栓、经皮冠状动脉介入治疗及常规药物治疗可挽救许多急性心肌梗死患者的生命,但不能从根本上恢复心肌细胞数量。骨髓间充质干细胞(BM-MSCS)是骨髓中一种具有多向分化潜能的细胞,可被诱导分化为心肌样细胞,改善梗死区的血液供应及心肌重构,为心肌梗死的治疗带来了新的希望。本文就BM-MSCS移植治疗心肌梗死的方式,移植的时机及临床应用等方面作一综述。     

自体骨髓间充质干细胞移植对犬缺血心肌的修复作用观察

目的 观察自体骨骼间充质干细胞移植对犬缺血心肌的修复作用.方法 通过结扎犬冠状动脉左前降支近端建立心肌梗死(MI)模型.自每只犬的髂后上棘抽取骨髓进行分离、培养、扩增骨髓间充质干细胞(MSCs).2周后用二脒苯基吲哚(DAPI)标记自体MSCs后二次开胸注射到MI瘢痕区及其周围.对照组注射等量无血清培养基.细胞移植前3 d及移植后4周分别行超声心动图和心肌灌注显影检查.结果 细胞移植后4周,两组犬全部存活,超声心动图显示实验组左室射血分数(EF)、每搏量(SV)、左室短轴缩短率(FS)、左室舒张末期内径(LVD)、左室收缩末内径(LVS)分别为56.72%±4.90%、(33.24±8.50)ml、26.16%±3.33%、(3.85±0.36)cm、(2.83±0.30)cm,与移植前的50.40%±6.31%、(25.49±7.67)ml、21.86%±3.04%、(4.24±0.41)cm、(3.14±0.30)cm相比,P均＜0.05;而对照组4周后左室上述指标较移植前差异不显著(P均＞0.05).99mTc-MIBI检查显示细胞移植组原心尖部、前壁缺损区再填充明显,而对照组原心尖部、前壁缺损区再填充不明显.结论 自体MSCs移植能有效地修复损伤的心肌并提高心功能.