HGF基因转染脂肪来源干细胞移植改善兔急性心肌梗死后心功能的研究

目的探讨脂肪来源干细胞(adipose derived stem cells,ADSC)移植和肝细胞生长因子(hepatocyte growthfactor,HGF)基因联合治疗兔急性心肌梗死(acute myocardial infarction,AMI)的疗效及其机制。方法家兔30只,复制急性心肌梗死模型。随机分为AMI/ADSC+hHGF组、AMI/ADSC组、AMI未治疗组,每组10只。另外随机选取10只兔作为假手术组。AMI术后3 d,取兔腿部脂肪,分离ADSC培养,流式细胞术鉴定其表面标记。构建pcDNA3.1-hHGF重组表达载体,移植前用脂质体包裹表达载体转染ADSCs,孵育24 h。AMI术后14 d,自体细胞移植,AMI未治疗组接受等量的无血清培养基注射,假手术组只开胸,不注射。细胞移植后28 d,超声心动图检测左室功能指标;取心肌组织,酶联免疫吸附法测定心肌组织中HGF的含量;计算心肌血管密度和纤维化面积。结果 AMI/ADSC+hHGF组各项参数明显优于AMI/ADSC组;AMI/ADSC+hHGF组HGF含量高于AMI/ADSC组以及AMI组。AMI/ADSC+hHGF组梗死区血管的数量及心肌纤维化面积等参数明显优于AMI/ADSC组。结论转染HGF基因的脂肪来源干细胞移植可改善兔心肌梗死后的心功能,其疗效明显优于单纯干细胞移植。可能通过增加干细胞的存活、改善梗死区的血供及抗纤维化效应起作用。     

棕色脂肪干细胞与白色脂肪干细胞移植对心肌梗死大鼠心功能的影响

目的对比研究棕色脂肪干细胞（BADSCs）与白色脂肪干细胞（ADSCs）治疗急性心肌梗死大鼠的效果。方法采用酶消化法分别分离大鼠腹股沟脂肪组织及肩胛骨下脂肪组织,获得ADSCs与BADSCs,并进行流式分析与多谱系分化鉴定。30只雄性SD大鼠随机分为PBS对照组、ADSCs移植组及BADSCs移植组,建立急性心肌梗死模型,将各组对应细胞直接注入大鼠心肌梗死边缘区。移植后4周进行心功能检测并利用组织学检测移植细胞的体内分化与血管化作用。结果术后4周,与PBS对照组相比,ADSCs以及BADSCs移植组大鼠心功能均显著提高（P〈0.05）,且纤维化程度显著减弱（P〈0.05）。免疫荧光结果表明,移植的BADSCs成心肌分化数量显著高于ADSCs。免疫组化结果显示,ADSCs移植组以及BADSCs移植组大鼠心肌梗死区血管密度均较PBS对照组显著增加（P〈0.05）,ADSCs移植组血管密度显著高于BADSCs移植组（P〈0.05）。结论不同来源脂肪干细胞移植均具有改善心肌梗死患者心功能的作用,虽然BADSCs体内促血管化作用不如ADSCs,但其具有明显的心肌分化能力,可有效改善心脏功能。     

脂肪来源干细胞生长行为特征及自体移植大鼠心肌梗死的生物行为影响

目的探讨脂肪来源的干细胞生长行为特征及自体移植大鼠心肌梗死的生物行为影响。方法将200～250g的雄性Wistar大鼠随机分为正常心脏移植组和心肌梗死移植组。取附睾处脂肪,分离获得脂肪来源的干细胞,DAPI标记第3代细胞,通过细胞计数和免疫组化检测细胞心肌生物学行为变化。结果细胞一般传2代可以获得较纯的脂肪来源的干细胞。脂肪来源的干细胞植入后第4周,心肌特异性肌钙蛋白T(TnT)免疫组化检查表达阳性率为(16.24±2.31)%,其呈向心肌梗死周边扩散和迁移的趋势,而对照组植入的标记细胞TnT表达阴性,并堆积。结论从脂肪组织中可获得具有多分化潜能的脂肪来源的干细胞并能在体外稳定增殖、传代。移植入大鼠心肌梗死模型ADSCs可以向心肌分化,其机理可能是受体内微循环影响。     

转染hVEGF165基因的骨髓间充质干细胞移植治疗兔心肌梗死

1 材料和方法 1.1 材料 健康纯种新西兰大白兔48只,雌雄不拘,体质量2.0～2.5 kg,兔龄4～5 mo,由岭南科研动物中心提供.低糖DMEM(Gibco产品);Percoll分离液(Pharmacia产品);VIII因子多克隆抗体及免疫组化检测试剂盒(武汉博士德生物工程有限公司);BrdU mAb BrdU检测试剂盒(Roche产品);AdTrackCMV-VEGF165真核表达质粒(由广东省人民医院中心实验室惠赠);LipofectamineTM 2000 reagent(GIBCOL).     

脂肪间充质干细胞移植对兔心肌梗死后微血管生成及心功能的影响

目的 探讨脂肪间充质干细胞(adipose tissue-derived mesenchymal stem cells,ADMSCs)移植对兔心肌梗死后微血管生成的可能机制及心功能的变化情况.方法 30只健康日本大耳白兔,完全随机分为假手术组、心肌梗死对照组和ADMSCs移植组(ADMSCs组).结扎兔前室间支,建立急性心肌梗死(acute myocardial infarction, AMI)动物模型,AMI 1 h 内将4',6-二脒-2-苯基吲哚(4',6-diamidio-2-phenylindole,DAPI)标记的第3代ADMSCs植入ADMSCs组梗死心肌,对照组及假手术组注射等量磷酸盐缓冲溶液(phosphate buffered solution, PBS).术前及术后4周分别做超声心动图检查其心功能变化.处死动物,取其心脏,采用免疫组织化学法检测梗死区新生血管微血管密度,RT-PCR 检测血管内皮生长因子(vascular endothelial growth factor, VEGF)和碱性成纤维生长因子(basic fibroblast growth factor, bFGF)的表达.结果 ADMSCs组荧光显微镜下可以观察到DAPI标记细胞存在,部分分化为心肌样细胞;超声心动图证实移植后4周ADMSCs组心功能较心肌梗死对照组改善(P<0.05);ADMSCs组较心肌梗死对照组梗死区微血管密度明显增高[(20.00±2.65)vs(7.75±2.12),P<0.05];ADMSCs组bFGF和VEGF的表达水平均较心肌梗死对照组明显增高[(0.590±0.028)vs(0.569±0.021),(0.913±0.030)vs(0.886±0.049),P<0.05].结论 ADMSCs移植促进心肌梗死区微血管的生成,并改善心脏功能,其机制可能与促进bFGF、VEGF的表达有关.     

脂肪来源干细胞(ADSC)在脂肪移植中的应用

自体脂肪移植是整形外科一个常见的手术,临床应用广泛。然而,移植脂肪的存活率具有很大的不确定性。脂肪来源干细胞(adipose-derived stem cell, ADSC)是近年来从脂肪组织中发现的一类具有多功能分化能力的干细胞。ADSC能够在自体脂肪移植中起到辅助作用,增加脂肪存活率,这一技术又叫做细胞辅助脂肪移植术(cell-assisted lipotransfer, CAL)。本文对CAL的现状进行综述,为ADSC在脂肪移植中的临床应用和基础研究提供参考。     

脂肪来源的间充质干细胞对大鼠心肌梗死后心功能的保护作用

目的探讨脂肪来源的间充质干细胞(ADSCs)对大鼠急性心肌梗死(AMI)后心功能的影响。方法结扎SD大鼠左冠状动脉前降支制备心肌梗死模型。同种异体ADSCs体外分离、培养、纯化、扩增,在大鼠AMI区域周围进行心外膜下移植。将24只大鼠随机分为3组,每组8只:A组为AMI模型组,只给予前降支结扎;B组为细胞培养基(DMEM)移植组,结扎后心外膜注射DMEM;C组为ADSCs治疗组,结扎前降支后接受ADSCs移植治疗。术后7 d、28 d各组大鼠行心脏超声检测左室射血分数(LVEF)、左室短轴缩短率(FS),28 d超声检测后行血流动力学测量左室收缩压、左室舒张末压、左心室压力最大变化速率,评价ADSCs移植对AMI后大鼠心功能的影响;心脏组织行TTC染色,观察AMI面积。结果与AMI模型组比较,ADSCs治疗组的LVEF、FS及血流动力学指标明显提高(P0.01)。TTC染色观察心肌梗死面积,ADSCs治疗组心肌梗死面积明显减小(P0.01)。结论 ADSCs可减少AMI的面积,改善心功能。     

人胎盘来源间质干细胞移植治疗改善心肌梗死大鼠的心功能

目的 从人胎盘中分离培养间充质干细胞,评价其生物学特性并观察其改善大鼠心梗模型心功能的能力.方法 用酶消化法自人胎盘组织中分离培养,并以流式细胞术对其进行鉴定.利用Percoll密度梯度离心方法从骨髓中分选出人骨髓来源间充质干细胞.将两种来源细胞进行大鼠心梗模型移植,4周后超声心动图检测大鼠心脏功能.结果 人胎盘组织中可分离培养出间充质干细胞,并且胎盘来源间充质干细胞与骨髓来源间充质干细胞具有相似的改善心梗后心功能的能力.结论 胎盘来源间充质干细胞是心肌组织工程良好前景的种子细胞来源之一.     

HGF基因转染关节软骨细胞及其生物学特性影响

目的观察携带有绿色荧光蛋白（gree fluorescent protein，GFP）标记的肝细胞生长因子（hepatocyte growth factor，HGF）cDNA转染兔软骨细胞后的主要生物学特性。方法取兔膝关节软骨细胞体外培养至第二代，HGFcDNA转染软骨细胞，空白质粒组为对照组；噻唑蓝（MTT）检测软骨细胞的增殖能力，SABC免疫组化及原位杂交法测其Ⅱ型胶原在转染前后的变化与mRNA的表达。结果软骨细胞在转染后可较长时间的保持其软骨细胞的生物学特性，并可促进软骨细胞的增殖，SABC免疫组化测第VI代软骨细胞的Ⅱ型胶原表达仍呈强阳性，而对照组软骨细胞则明显减弱，说明HGF基因转染软骨细胞后可表达其生物学功能。基因瞬间转染率大约为31．23名。结论GFP标记的HGF基因以脂质体为载体转染兔膝关节软骨细胞后可发挥其生物学功能，加速细胞的生长，在荧光显微镜下可直观的计算软骨细胞的瞬间转染率。     

血管内皮生长因子转染骨髓间充质干细胞心肌移植对心肌梗死后大鼠心功能及血管新生的作用

目的建立重组人血管内皮生长因子(VEGF)165基因转染大鼠骨髓间充质干细胞(MSC)的方法,探讨该细胞心肌移植对缺血性心脏病心功能及血管新生的影响,并比较联合治疗与单独基因或细胞治疗的疗效差异。方法采用密度梯度离心-贴壁培养法获取Wistar近交系大鼠MSC,用脂质体将pcDNA3.1-hVEGF165转入该细胞,通过ELISA、RT—PCR和Western印迹检测后者VEGF的表达情况;以Wistar近交系大鼠建立心肌缺血模型,随机分成4组(每组12只),心肌梗死模型建立2周后,联合组在心肌梗死区移植转染VEGF165基因的MSC,细胞组移植等量的MSC,基因组注射脂质体-pcDNA3.1-VEGF165DNA复合物,对照组注射等容积培养液;另取12只未结扎冠脉的大鼠为假手术组。细胞移植4周后,用Buxco系统检测心功能;然后处死动物,取心肌标本,测量心肌梗死面积;用5-溴脱氧尿嘧啶(Brdu)、肌钙蛋白T免疫组化双染法确定移植细胞的存活与分化;用Ⅷ因子染色法检测血管新生,RT-PCR法检测VEGF165基因的体内表达情况。结果(1)pcDNA3.1-hVEGF165基因通过脂质体转染大鼠MSC后获稳定表达;(2)移植4周后,联合组心肌梗死面积(27．8％±3．0％)明显低于细胞组(37．0％±10．1％)与基因组(37．1％±5．2％,均P<0．05),心功能改善优于细胞组与基因组;(3)联合组心肌梗死区毛细血管密度(每视野40．2个±5．5个)高于细胞组(27．2个±6．3个,P<0．01)和对照组(18．5个±5．8个,P<0．01),较基因组(35．8个±7．7个)亦有增加的趋势(P=0．189);(4)Brdu、肌钙蛋白T双染示各治疗组心肌梗死区心肌细胞数量不同程度的多于对照组;(5)联合组VEGF基因的体内表达(hVEGFmRNA相对含量0．18±0．04)高于基因组(0．10±0．03,P<0．01)。结论转染VEGF基因的MSC移植可使鼠冠脉结扎造成的心肌梗死面积缩小、心功能明显改善,其疗效优于单独应用基因或细胞治疗,为缺血性心脏病的细胞基因联合治疗提供了理论依据。     

Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction

Background Adipose-derived stem cells （ADSCs） are capable of differentiating into cardiomyogenic and endothelial cells in vitro. We tested the hypothesis that transplantation of ADSCs into myocardial scar may regenerate infracted myocardium and restore cardiac function. Methods ADSCs were isolated from the fatty tissue of New Zealand white rabbits and cultured in Iscove＇s modified dulbecco＇s medium. Three weeks after ligation of left anterior descending coronary artery of rabbits, either a graft of untreated ADSCs （UASCs, n=14）, 5-azacytidine-pretreated ADSCs （AASCs, n=13）, or phosphate buffer saline （n=13） were injected into the infarct region. Transmural scar size, cardiac function, and immunohistochemistry were performed 5 weeks after cell transplantation. Results ADSCs in culture demonstrated a fibroblast-like appearance and expressed CD29, CD44 and CD105. Five weeks after cell transplantation, transmural scar size in AASC-implanted hearts was smaller than that of the other hearts. Many ADSCs were differentiated into cardiomyocytes. The AASCs in the prescar appeared more myotube-like. AASCs in the middle of the scar and UASCs, in contrast, were poorly differentiated. Some ADSCs were differentiated into endothelial cells and participate in vessel-like structures formation. All the ADSC-implanted hearts had a greater capillary density in the infarct region than did the control hearts. Statistical analyses revealed significant improvement in left ventricular ejection fraction, myocardial performance index, end-diastolic pressure, and peak ＋dP/dt, in two groups of ADSC-implanted hearts relative to the control hearts. AASC-implanted hearts had higher peak -dP/dt values than did control, higher ejection fraction and peak ＋dP/dt values than did UASC-implanted hearts. Conclusions ADSCs transplanted into the myocardial scar tissue formed cardiac islands and vessel-like structures, induced angiogenesis and improved cardiac function. 5-Azacytidine pretreatment before implantation is desirable for augmenting myogenesis. Transplantation of 5-azacytidine-treated ADSCs into the myocardial scar was more efficient than that of untreated ADSCs in preservation of cardiac function.     

转染VEGF165基因的自体成肌细胞治疗心肌梗死的实验研究

目的:将体外培养、纯化并转染VEGF165基因的新西兰兔自体骨骼肌成肌细胞植入心肌梗死区,观测转染VEGF165基因的成肌细胞移植对改善梗死区心肌血运重建和心功能的影响。方法:采用pcDNA3.1-VEGF165和pcDNA3.1质粒转染经体外培养及纯化的新西兰兔自体骨骼肌成肌细胞。结扎兔冠状动脉致局部心肌梗死后2周,分别于心肌梗死区移植转染VEGF165基因成肌细胞(实验组,n=8)和空载体成肌细胞(对照组,n=8),4周后观察心肌梗死边缘区毛细血管密度、植入细胞的形态鉴定和心功能改善情况。结果:成功将pcDNA3.1-VEGF165和pcDNA3.1质粒转染体外培养的兔自体骨骼肌成肌细胞,成肌细胞移植后能在梗死区种植成活、分化。实验组细胞移植区域的毛细血管密度较对照组高(P<0.05)。经Buxco系统有创心功能测定显示:实验组较对照组的左心室等容收缩期室内压最大上升速率[+dp/dtmax,(1607.23±102.67)mmHg/s vs(1217.77±89.91)mmHg/s]和左心室等容舒张期室内压最大下降速率[-dp/dtmax,(1535.09±81.34)mmHg/s vs(1174.58±91.50)mmHg/s]均有所改善。结论:转染VEGF165的成肌细胞能改善心肌梗死区域的血液供应,增加心肌收缩力,改善心功能。     

Granulocyte-macrophage colony stimulating factor improves cardiac function in rabbits following myocardial infarction

To investigate the therapeutic potency of recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) in a rabbit myocardial infarction model. Methods: A myocardial infarction was created by the ligation of the major ventricular branch of the left coronary artery in rabbits. After myocardial infarction, the ani-mals were randomly assigned to GM-CSF treatment group, untreated groups and sham-operated group. The rabbits of the treated group were injected into GM-CSF by subcutaneous administration, 10μg/kg/day, once a day for 5 days.The untreated and sham-operated group received a equal saline in the same manner as treated group. Six weeks later echocardiography and haemodynamic assessment were undertaken to assesse cardiac function. The size of the infarct re-gion of the heart were also studied. Restflts: The untreated group exhibited significant higher left ventricle end-diastolic pressure, higher central venous pressure, and with significant lower mean blood pressure, lower peak first derivative of left ventricle pressure (dP/dt) than the sham group. Also, Rabbits in untreated group display significant systolic dys-function shown by the decreased ejection fraction, diastolic dysfunction shown by increasing in the ratio of E wave to A wave (E/A), and display left ventricle enlargement. However, GS-CSF singnificanfly prevented heart dysfunction, left ventricle enlargement, and reduced infarct size in treatment group. Conclusion: Administration GM-CSF after cardiac infarction can improve heart function. These findings indicate the technique may be a novel and simple therapeutic method for ischemic mvocardium.     

Transplantation of magnetically labeled mesenchymal stem cells improves cardiac function in a swine myocardial infarction model

Background Mesenchymal stem cells （MSCs） transplantation provides a new approach for myocardial repair. However, many important fundamental questions about MSCs transplantation remain unanswered. There is an urgent need to identify MSCs from the beating heart and analyze the efficacy of this new approach. This study aimed to localize the magnetically labeled MSCs （MR-MSCs） and monitor the restorative effects of MR-MSCs with magnetic resonance （MR） imaging. Methods Acute myocardial infarction （AMI） was created in swine by a balloon occlusion of the left anterior descending coronary artery. Cells were delivered via intracoronary infusion after myocardial infarction. Infarct size change and cardiac function were assessed with 3.0T MR scanner. The results were then confirmed by histological and western blot analysis. All statistical procedures were performed with Systat （SPSS version 12.01）. Results A total of 26 swine were divided into four groups （sham-operated group, n=6; AMI group with PBS transplantation, n=6; labeled MSCs group, n=7; unlabeled MSCs group, n=7）. MSCs, MR-MSCs （10~cells） or PBS were delivered by intracoronary injection after MI and serial cardiac MR imaging studies were performed at 0, 4 and 8 weeks after transplantation. MR imaging demonstrated MI size decreased after MSCs transplantation in labeled and unlabeled groups, however, increases were seen in the AMI group at 8 weeks after MI. The left ventricular ejection fraction （LVEF） was slightly increased in the AMI group （（41.87~2.45）% vs （39.04~2.80）%, P 〉0.05）, but significantly improved in the MR-MSCs group （（56.85~1.29）% vs （40.67~2.00）%, P 〈0.05） and unlabeled group （（55.38~1.07）% vs （41.78~2.08）%, P 〈0.05） at 8 weeks after treatment. MR-MSCs were further confirmed by Prussian blue and immunofluorescent staining. Western blot analysis demonstrated that there was an increased expression of cardiomyocyte markers such as myosin heavy chain and troponin T in the MSCs trea     

自体脂肪干细胞治疗心肌梗死的实验研究

目的探讨自体脂肪干细胞治疗心肌梗死的作用机制。方法用9μmol/L的5-氮胞苷(5-aza)培养基诱导自体脂肪干细胞24h,2、3周后免疫细胞化学法染色鉴定心肌特异肌钙蛋白T(cTnT)和肌纤蛋白(desmin)的表达。冷冻法制作大鼠心肌梗死模型。1周后,经尾静脉移植自体脂肪干细胞。TTC染色测梗死体积,插管测左心室血流动力学变化。结果移植自体脂肪干细胞后,梗死体积减小(P<0.01),移植3w后,实验组左心室收缩压及左心室等容收缩期/舒张期内压最大上升或下降速率较对照组升高(P<0.05～0.01),左心室舒张末压较对照组减小(P<0.05)。结论经5-aza诱导的大鼠自体脂肪干细胞能减小心肌梗死体积及改善大鼠心肌梗死后的心脏功能。     

自体成肌细胞移植改善兔心肌梗死后心功能的实验研究

目的:将体外培养并纯化的自体骨骼肌成肌细胞植入心肌梗死区,观察其心脏功能有无改善及改善程度,以探讨成肌细胞移植治疗的机制.方法:采用反复差速贴壁法体外培养、纯化及鉴定新西兰兔自体成肌细胞.结扎冠状动脉致局部心肌梗死后2周,分别将成肌细胞和对照的细胞培养液注入心肌梗死区,4周后观察心功能改善情况,鉴定心肌梗死边缘区毛细血...     

Vascular endothelial growth factor expressing mesenchymal stem cells improves cardiac function in chronic myocardial infarction in pigs

Transplantation of mesenchymal stem cells （MSCs） for myocardial reconstruction has shown promise in both animal models and human phase 1 clinical studies. Vascular endothelial growth factor （VEGF） is a strong therapeutic agent for treating ischaemia by inducing angiogenesis. The feasibility of ex vivo MSCs mediated gene transfer is documented. Matsumoto and colleagues have recently reported genetically engineered MSCs carrying VEGF165 delivery for revascularization in a model of acute myocardial infarction （MI）. The promising data from our laboratory in both angiogenesis and MSCs transplantation in cunicular heart model of acute MI have prompted us to attempt the combined and simultaneous application of the two strategies.