成年犬自体骨骼肌成肌细胞移植于急性心肌梗死区的实验研究

目的观察心肌内和冠状动脉内注射法移植自体骨骼肌成肌细胞于急性心肌梗死区的生长分化特点。方法以改良成年犬骨骼肌成肌细胞培养方法进行细胞分离及扩增。结扎冠状动脉前降支中段,建立急性心肌梗死模型。分为直接注射对照、移植,冠状动脉内注射对照、移植4组,每组5只。不开通冠状动脉,分别向梗死心肌内和梗死相关冠状动脉内注射自体骨骼肌成肌细胞(1·0~1·4×108个)或等量生理盐水。移植4周后通过HE染色、PTH染色、骨骼肌特异性慢肌球蛋白抗体免疫组化染色和透射电镜寻找梗死区内存在新生肌组织的证据并观察其生长特点。结果经心肌内直接注射和冠状动脉内注射移植自体骨骼肌成肌细胞4周后,透射电镜及HE染色下均可在梗死区内找到新生幼稚肌原性细胞存在,PTH染色证实有新生的横纹肌组织形成,骨骼肌特异性慢肌球蛋白抗体免疫组化染色发现有骨骼肌原性的成熟肌组织存在;成肌细胞直接注射组内新生的肌组织排列较为密集,而冠状动脉内成肌细胞注射组内的新生肌组织排列较分散。结论通过心肌内直接注射和经梗死相关冠状动脉注射将自体骨骼肌成肌细胞移植到急性心肌梗死区后均能形成成熟的肌组织,为以骨骼肌成肌细胞进行急性心肌梗死的细胞心肌成形治疗提供了组织学依据。     

经冠脉注射法移植成年犬自体骨骼肌成肌细胞治疗急性心肌梗死的疗效研究

目的观察经冠状动脉内注射法移植自体骨骼肌成肌细胞治疗急性心肌梗死的疗效。方法以改良成年犬骨骼肌成肌细胞培养方法进行细胞分离及扩增；结扎冠状动脉前降支中段，建立急性心肌梗死模型；分为冠脉内注射对照及冠脉内成肌细胞注射组，每组8只；冠状动脉结扎前、后及4周时测定左室功能指标、4周时行超声心动图检查。结果自体骨骼肌成肌细胞经冠脉注射移植组术后4周CO比对照组显著增加（P〈0．05）且比冠脉结扎后显著升高（P〈0．05）；术后4周时LVEF和SV比对照组上升21．1％～31．4％（P均〉0．05）。结论经冠脉内注射移植自体骨骼肌成肌细胞可能提高急性心肌梗死后收缩功能。     

经冠状动脉注射法自体骨骼肌成肌细胞移植治疗兔急性心肌梗死

目的 观察经冠状动脉注射自体骨骼肌成肌细胞（SM）移植到兔急性心肌梗死（AMI）区后的生长分化特点和疗效。方法 取日本大耳白兔45只,随机分为经冠脉注射SM移植组、对照组和假手术组,各15只。SM移植组取臀肌分离、纯化SM并体外扩增。结扎兔左冠脉前降支（LAD）,建立AMI模型。再灌注后,SM移植组经冠脉注射自体SM悬液1 ml(5×106个细胞)。对照组以相同的方法建立AMI模型,注入等量的无血清培养液。假手术组除不结扎左前降支外,其余操作均同对照组。4周后,通过HE 染色和抗5-溴脱氧尿核苷（BrDU）抗体、抗骨骼肌特异性慢β-肌凝蛋白重链（slow-MHC）抗体的免疫组化染色评价移植细胞的转归。术后24 h和4周,以超声心动图仪测量3组的左室射血分数（LVEF） 和左室短轴缩短率（FS）。结果 SM移植组在术后4周,HE染色后可在梗死区内找到新生的多核肌样细胞。抗BrdU 抗体和抗Slow-MHC抗体的免疫组化染色呈阳性。术后24 h,假手术组的LVEF 和FS 明显高于对照组和SM移植组（P<0.01）。而术后4周,与对照组比较,假手术组和SM移植组的LVEF和FS明显改善（P<0.01）。结论 经冠脉注射法移植自体SM可在AMI区存活,修复受损心肌并提高心脏的功能。     

骨骼肌成肌细胞移植对慢性心力衰竭犬左心室功能的影响

目的:观察自体骨骼肌成肌细胞移植对慢性心力衰竭犬的左心室重塑及左心室功能的影响.方法:19只慢性心力衰竭犬模型建立后随机分为实验组(n=9):采用开胸直接心肌内注射自体骨骼肌成肌细胞;对照组(n=10):开胸心肌内注射等量生理盐水.移植前及移植后5周行超声心动图检查,病理组织标本行Desmin和Brd-U免疫荧光双重染色.结果:①移植后较移植前,实验组收缩末期心室腔面积(ESA)、舒张末期心室腔面积(EDA)均明显下降(P<0.05),射血分数显著升高(P<0.05),差异有统计学意义;而对照组ESA和EDA在移植前、移植后差异无统计学意义(P>0.05);②实验组心肌注射部位可见存活细胞,经Desmin和Brd-U免疫荧光双重染色证实来自于移植的骨骼肌成肌细胞.结论:自体骨骼肌成肌细胞对慢性心力衰竭犬的左心室重塑及功能有明显改善作用.     

成年犬自体骨髓基质干细胞经冠状动脉移植在心梗后心肌组织内分化

目的　探讨经冠状动脉移植成年犬自体骨髓基质干细胞在心梗后心肌组织内的存活、分化情况。方法　结扎犬冠状动脉前降支制备心肌梗死模型 ;细胞移植组将体外培养、诱导的自体骨髓基质干细胞标记后在心肌梗死后 4周经冠状动脉植入心脏 ;对照组植入等量培养基 ;细胞移植 4周后取犬心脏 ,行组织学检查、免疫组织化学检查及电镜检查。结果　细胞移植组植入骨髓基质干细胞在心肌组织内存活 ;免疫组织化学检查结蛋白、肌钙蛋白I染色阳性 ;电镜示心肌疤痕组织中可见胞浆丰富的异源性细胞 ,圆形或椭圆形 ,细胞体积较大 ,核糖体丰富 ,线粒体较丰富 ,有肌丝样结构 ,单核。对照组心脏标本中可见疤痕组织 ;心肌细胞结蛋白染色阴性 ,肌钙蛋白I染色阳性 ;电镜未发现异源性细胞。结论　成年犬骨髓基质干细胞经冠状动脉移植后在心肌组织内存活并分化为肌源性细胞。     

自体成肌细胞和骨髓间充质干细胞移植对缺血坏死心肌的修复作用

目的 观察自体骨骼肌成肌细胞(SMs)和骨髓间充质干细胞(BMSCs)经冠脉移植至缺血坏死心肌后的分化情况及对缺血坏死心肌的修复作用.方法 取日本大耳白兔45只,随机分为SMs、BMSCs移植组和对照组,每组15只.移植组分别取臀肌和骨髓体外培养SMs和BMSCs.各组均结扎左前降支建立缺血坏死心肌模型.分别于再灌注后1 h经左前降支注射5-溴脱氧尿核苷(BrdU)标记的SMs、BMSCs悬液1 ml(5×1012个/L)或等量DMEM培养液.移植后24 h和4周用超声心动仪检查心脏功能变化.移植后4周以病理组织学检查HE染色及BrdU抗体、结蛋白(Desmin)抗体和骨骼肌特异性慢肌球蛋白重链(Slow-MHC)单克隆抗体,免疫组化染色评价移植细胞的分化情况.结果 移植后4周,SMs和BMSCs组在左室射血分数[(61.9±3.6)%、(62.4±2.6)%]和左室短轴收缩率[(31.6±2.3)%、(30.8±1.9)%]较对照组[(53.2 ±2.3)%、(23.8±0.9)%]均有明显改善(P＜0.05);SMs与BMSCs组比较差异无统计学意义(P＞0.05);HE染色显示移植组心肌纤维排列有序,其中SMs组可见新生的多核肌样细胞.移植SMs和BMSCs BrdU抗体免疫组化染色阳性.SMs组SMsDesmin抗体和Slow-MHC单克隆抗体免疫组化染色阳性.结论 经冠脉自体移植的SMs和BMSCs可在缺血坏死心肌内存活.对心肌损伤具有修复作用,并同等程度地提高了心脏功能.     

自体骨骼肌成肌细胞移植对心肌梗死兔心室重构和血管新生的影响

目的观察经冠状动脉途径移植自体骨骼肌成肌细胞(SMs)对急性心肌梗死兔心室重构和血管新生的影响。方法取日本大耳白兔45只,随机分为3组:经冠状动脉注射SMs移植组(移植组)、对照组和假手术组,每组15只。术后4周,检测各组免左心室质量及左心室质量指数,并以免疫组织化学法和ELISA法分别检测各组兔新生血管数目和血管内皮生长因子(VEGF)浓度。结果术后4周,与假手术组比较,移植组和对照组左心室质量及左心室质量指数明显增加(P0.01);与对照组比较,移植组左心室质量及左心室质量指数明显降低(P0.01)。与假手术组比较,对照组和移植组新生血管数目、梗死心肌组织中VEGF浓度明显增多,而移植组较对照组增多更明显(P0.05,P0.01)。结论经冠状动脉途径自体SMs移植可增加VEGF表达,诱导血管新生,改善心肌梗死后心室重构。     

骨骼肌成肌细胞移植治疗急性心肌梗死的研究进展

骨骼肌成肌细胞具有来源广泛、易于获得,自体移植不受供体来源限制,增殖能力及抗缺血、缺氧能力强大,易在梗死区内存活等多种优点,是干细胞治疗的重要来源。该文主要阐述骨骼肌成肌细胞移植治疗急性心肌梗死的研究进展。     

经冠脉移植骨髓单个核细胞治疗心肌梗死的实验研究

目的观察经皮冠状动脉内移植自体骨髓单个核细胞于急性心肌梗死区域后的定植、生长、分化特点和疗效。方法中华小型猪冠状动脉前降支结扎90 m in再灌注制备心肌梗死模型,分为移植组和对照组,于心肌梗死后1周分别经皮冠状动脉内移植PKH26标记的骨髓单个核细胞和等体积培养基。心肌梗死后6周通过血流动力学和超声心动图指标检测心功能变化并进行病理检查。结果在移植了骨髓细胞的梗死区域内可找到发出红色荧光的移植细胞,其VIII因子和Desm in免疫组化染色均为阳性;HE染色、PTH染色及透射电镜下可观察到幼稚的心肌细胞,未见细胞融合现象。移植组的血流动力学指标显示心肌梗死6周时,较结扎后左室舒张末压显著改善（P<0.05）,较同期对照组左室压力最大上升速度显著升高（P<0.05）;心肌梗死6周时的超声心动图指标和对照组相比,各项指标均无显著差异,术后-术前射血分数的差值显著高于对照组（P<0.05）;其梗死区的小血管数量显著高于对照组（P<0.01）。结论经冠状动脉移植的骨髓单个核细胞可定植于心肌梗死区,并向心肌细胞和血管内皮细胞方向分化,同时能显著地促进小血管再生,有改善心功能的潜能。     

成熟心耳肌细胞自体移植的实验研究

目的将急性分离的成年兔自体左心耳心肌细胞移植到自体梗死区心肌后,研究其存活情况,以及对心律的影响。方法结扎成年兔的冠状动脉前降支,建立心肌梗死模型,4周后获取自体左心耳组织,急性消化分离为单细胞,经DAPI标记后,分别将细胞悬液和培养基注射到移植组和对照组梗死区内。4周后行心电图检查并取移植区组织进行组织学观察。结果4周后移植组与对照组兔全部存活。心电图检查,移植组心率略高于对照组（P<0.05）,未见异位心律。心肌组织切片见对照组梗死区内为典型心肌梗死后改变,移植组梗死区内有“细胞岛”形成,荧光检测证明移植的心耳肌细胞在移植区存活。结论急性分离的自体左心耳心肌细胞移植到梗死区心肌内可以存活,并不产生异位心律。     

Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation

OBJECTIVES: We report histological analysis of hearts from patients with end-stage heart disease who were transplanted with autologous skeletal myoblasts concurrent with left ventricular assist device (LVAD) implantation. BACKGROUND: Autologous skeletal myoblast transplantation is under investigation as a means to repair infarcted myocardium. To date, there is only indirect evidence to suggest survival of skeletal muscle in humans. METHODS: Five patients (all male; median age 60 years) with ischemic cardiomyopathy, refractory heart failure, and listed for heart transplantation underwent muscle biopsy from the quadriceps muscle. The muscle specimen was shipped to a cell isolation facility where myoblasts were isolated and grown. Patients received a transplant of 300 million cells concomitant with LVAD implantation. Four patients underwent LVAD explant after 68, 91, 141, and 191 days of LVAD support (three transplant, one LVAD death), respectively. One patient remains alive on LVAD support awaiting heart transplantation. RESULTS: Skeletal muscle cell survival and differentiation into mature myofibers were directly demonstrated in scarred myocardium from three of the four explanted hearts using an antibody against skeletal muscle-specific myosin heavy chain. An increase in small vessel formation was observed in one of three patients at the site of surviving myotubes, but not in adjacent tissue devoid of engrafted cells. CONCLUSIONS: These findings represent demonstration of autologous myoblast cell survival in human heart. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results establish the feasibility of myoblast transplants for myocardial repair in humans.     

经冠状动脉自体骨髓单个核细胞移植治疗急性心肌梗死的安全性观察

目的采用随机对照研究方法观察经冠状动脉自体骨髓单个核细胞(BM-MNCs)移植治疗急性心肌梗死(AMI)的安全性。方法入选184例AMI患者,分为细胞移植组92例和对照组92例,两组患者在药物治疗基础上,分别在介入治疗同时,经微导管于梗死相关动脉内支架远端注入自体BM-MNCs悬液或等量的肝素生理盐水(对照组)。记录骨髓收集及分离过程中可能出现的迷走反射(如面色苍白、晕厥、恶心、低血压甚至休克)、心绞痛发作、心力衰竭加重等,细胞悬液输注中可能出现的心律失常(窦性心动过缓、窦性停搏或三度房室传导阻滞、心室颤动)、低血压、栓塞等不良反应。监测术后1周内体温、心率、血压、心肌酶谱变化,并根据预先设定的时间点行24小时动态心电图、血常规、肝肾功能、血糖、血脂、血尿酸、心肌酶谱和高敏C反应蛋白(hs-CRP)检测,定期复查冠状动脉造影和经胸超声心动图。计划随访2年,记录主要心脏事件发生率、支架内再狭窄以及肿瘤等发生情况。结果在骨髓收集和分离过程中患者未出现心绞痛发作、心力衰竭加重等并发症,迷走反射相关不良反应均为一过性。两组患者平均随访(14．5±8．1)个月。与对照组相比,移植组围手术期及随访期间不良事件发生率差异无统计学意义:围手术期(≤1周)4．3％比5．7％(P>0．05);术后3个月为3．5％比2．4％(P>0．05);术后6个月至30个月为11．7％比14．3％(P>0．05)。未发现与移植相关的恶性心律失常、感染、心肌缺血加重、支架内再狭窄增加等。随访期间未发生死亡、新发肿瘤、栓塞等事件。同时,与对照组相比,BM-MNCs移植可以在介入治疗的基础上进一步提高左室射血分数。结论经冠状动脉自体BM-MNCs移植治疗AMI安全可行,且能改善心功能。     

Lack of additional benefit of intracoronary transplantation of autologous peripheral blood stem cell in patients with acute myocardial infarction.

BACKGROUND: Recently the potential of myocardial repair by transplantation of autologous bone marrow stem cells has been suggested. Whether the additional intracoronary transplantation of autologous peripheral blood stem cells (PBSC), which were mobilized by granulocyte-colony-stimulating factor (G-CSF), could safely improve myocardial function in patients with acute myocardial infarction (AMI) was investigated. METHODS AND RESULTS: Seventy-three patients with AMI who had successfully undergone percutaneous coronary intervention (PCI) were enrolled in the present prospective nonrandomized open-labeled study. Ten patients with elective PCI received G-CSF for 4 days followed by intracoronary PBSC transplantation. Thirty-two patients with primary PCI and 31 patients with recent AMI and elective PCI served as controls. The left ventricular (LV) function was evaluated using echocardiography and magnetic resonance imaging. G-CSF and intracoronary transplantation of PBSC did not incur any periprocedural myocardial damage. After 6 months, the LV ejection fraction was significantly improved in the cell therapy group. For 2 years of the follow-up period, there was no adverse clinical events, except one asymptomatic in-stent restenosis. However, comparable improvement of the LV ejection fraction was also identified in the primary PCI and elective PCI control groups. CONCLUSIONS: In the present study, additional intracoronary infusion of PBSC was safe and feasible for the patients with AMI who had undergone PCI, but did not lead to a significant improvement in LV function compared to standard reperfusion treatment.     

骨骼肌干细胞移植对犬缺血心肌结构的影响

目的　观察自体骨骼肌干细胞移植于缺血心肌后对心肌结构变化的影响。方法　取 12只成年犬臀大肌 ,分离卫星细胞、培养、传代、用 4’ ,6 二乙酰基 2 苯基吲哚 (DAPI)标记卫星细胞 ;在已建立的急性心肌梗死动物模型基础上 ,将DAPI标记的卫星细胞 ,自左冠状动脉前降支灌入缺血心肌中。分别于 2、4、8周后取出心脏 ,对缺血心肌的纤维化程度及植入的卫星细胞进行观察。结果　在 2 4例标本中卫星细胞分化成为带有横纹德肌纤维 ;卫星细胞在缺血心肌中可分化成心肌细胞样细胞 ;在卫星细胞移植区域 ,原有心肌细胞由于得到保护而不发生玻璃样变性 ,且排列有序 ;对照组缺血心肌发生玻璃样变性 ,心肌细胞基本结构紊乱。结论　自体骨骼肌卫星细胞在缺血心肌中可分化成心肌细胞样细胞 ,并可抑制缺血心肌的纤维化 ,心肌的基本结构得到保护 ;干细胞移植有望为心肌损伤提供一条新的治疗途径     

Transcatheter transplantation of autologous skeletal myoblasts in postinfarction patients with severe left ventricular dysfunction.

PURPOSE: To report a case-controlled safety and feasibility study of transcatheter transplantation of autologous skeletal myoblasts as a stand-alone procedure in patients with ischemic heart failure. METHODS: Six men (mean age 66.2+/-7.2 years) were eligible for transcatheter transplantation of autologous skeletal myoblasts cultured from quadriceps muscle biopsies. Six other men (mean age 65.7+/-6.3 years) were selected as matched controls (no muscle biopsies). A specially designed injection catheter was advanced through a femoral sheath into the left ventricle cavity, where myoblasts in solution (0.2 mL/injection) were injected into the myocardium via a 25-G needle. At baseline and in follow-up, both groups underwent Holter monitoring, a 6-minute walk test, New York Heart Association (NYHA) class determination, and echocardiography with dobutamine challenge. RESULTS: Skeletal myoblast transplantation was technically successful in all 6 patients with no complications; 19+/-10 injections were performed per patient (210 x 10(6)+/-150 x 10(6) cells implanted per patient). Left ventricular ejection fraction (LVEF) rose from 24.3%+/-6.7% at baseline to 32.2%+/-10.2% at 12 months after myoblast implantation (p=0.02 versus baseline and p<0.05 versus controls); in matched controls, LVEF decreased from 24.7%+/-4.6% to 21.0%+/-4.0% (p=NS). Walking distance and NYHA functional class were significantly improved at 1 year (p=0.02 and p=0.001 versus baseline, respectively), whereas matched controls were unchanged. CONCLUSIONS: Transcatheter transplantation of autologous skeletal myoblasts for severe left ventricular dysfunction in postinfarction patients is feasible, safe, and promising. Scrutiny with randomized, double-blinded, multicenter trials appears warranted.     

Autologous skeletal myoblast transplantation for the treatment of postinfarction myocardial injury: phase I clinical study with 12 months of follow-up

Background

Experimental studies have shown that skeletal myoblast transplantation into an area of postinfarction left ventricular injury results in an increase of segmental contractile performance that could be related to transplanted myoblasts. Initial experience with autologous skeletal myoblast transplantation in patients with postinfarction myocardial injury has also been obtained.

Methods

Patients who survived an acute myocardial infarction and were scheduled to undergo coronary artery bypass grafting were screened by means of dobutamine stress echocardiography and included into the study when no contractility changes within akinetic/dyskinetic segments were observed. Ten patients who gave informed consent were enrolled, and autologous myoblasts (satellite cells) were isolated from the skeletal muscle biopsy. Myoblast injections into the akinetic/dyskinetic area were performed after constriction of the anastomoses during the coronary artery bypass grafting procedure.

Results

Myoblast transplantations were performed after 3 weeks of in vitro culture in all patients. One patient died of a recent infarction at day 7 postoperatively because of a recent infarction in a remote area of the left ventricle. The left ventricular ejection fraction increased from 25% to 40% (mean, 35.2%) before the procedure to 29% to 47% (mean, 42.0%) during the 4-month visit (P <.05), and the effect was maintained throughout 12 months of follow-up. Sustained ventricular tachycardia was observed in 2 patients in the early postoperative period and in the other 2 patients after 2 weeks of follow-up. Prophylactic amiodarone infusion was used in the remaining 8 patients and prevented sustained ventricular tachycardia episodes.

Conclusions

Autologous skeletal myoblast transplantation for the treatment of postinfarction heart failure is feasible. Our initial observations justify further research to validate this method in a clinical practice.     

Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: clinical experience with six-month follow-up

OBJECTIVES: We report on the procedural and six-month results of the first percutaneous and stand-alone study on myocardial repair with autologous skeletal myoblasts. BACKGROUND: Preclinical studies have shown that skeletal myoblast transplantation to injured myocardium can partially restore left ventricular (LV) function. METHODS: In a pilot safety and feasibility study of five patients with symptomatic heart failure (HF) after an anterior wall infarction, autologous skeletal myoblasts were obtained from the quadriceps muscle and cultured in vitro for cell expansion. After a culturing process, 296 +/- 199 million cells were harvested (positive desmin staining 55 +/- 30%). With a NOGA-guided catheter system (Biosense-Webster, Waterloo, Belgium), 196 +/- 105 million cells were transendocardially injected into the infarcted area. Electrocardiographic and LV function assessment was done by Holter monitoring, LV angiography, nuclear radiography, dobutamine stress echocardiography, and magnetic resonance imaging (MRI). RESULTS: All cell transplantation procedures were uneventful, and no serious adverse events occurred during follow-up. One patient received an implantable cardioverter-defibrillator after transplantation because of asymptomatic runs of nonsustained ventricular tachycardia. Compared with baseline, the LV ejection fraction increased from 36 +/- 11% to 41 +/- 9% (3 months, p = 0.009) and 45 +/- 8% (6 months, p = 0.23). Regional wall analysis by MRI showed significantly increased wall thickening at the target areas and less wall thickening in remote areas (wall thickening at target areas vs. 3 months follow-up: 0.9 +/- 2.3 mm vs. 1.8 +/- 2.4 mm, p = 0.008). CONCLUSIONS: This pilot study is the first to demonstrate the potential and feasibility of percutaneous skeletal myoblast delivery as a stand-alone procedure for myocardial repair in patients with post-infarction HF. More data are needed to confirm its safety.