肝细胞生长因子及骨髓间充质干细胞联合移植治疗慢性缺血性心脏病的实验研究

目的研究经肝细胞生长因子(HGF)及骨髓间充质干细胞(MSCs)联合移植治疗慢性缺血性心脏病的疗效。方法采集香猪髂骨骨髓,用密度梯度法和贴壁分离法相结合的方法分离、培养MSCs,通过细胞表面标记(CD34、CD44、CD71、Ⅷ因子和desmin)成份鉴定;HGF基因的重组腺病毒(AdHGF)+MSCs共培养并检验HGF对MSCs生物学特征的影响。经左胸冠状动脉回旋支放置Ameroid环建立慢性心肌缺血香猪模型,将40只小型香猪采用随机对照分为5组(n=8),于缺血心肌处分别注射5×106/ml MSCs+4×109pfu 200μl AdHGF(MSCs+AdHGF组)、4×109pfu 200μl AdHGF(AdHGF组)、5×106/ml MSCs 200μl(MSCs组),4×109pfu 200μl AdNull(AdNull组)和生理盐水1 ml(对照组)。治疗4周后行心脏超声心动图,数字减影动脉造影(DSA),单光子发射计算机体层摄影(SPECT)心肌灌注检查及心肌凋亡细胞等检测。结果流式细胞仪检测MSCs表面标记CD44、CD71阳性,CD34、Ⅷ因子、desmin阴性;增殖细胞抗原(PCNA)蛋白表达显示HGF具有较强刺激MSCs增殖分化作用;彩色超声心动图检查提示:经治疗后MSCs+AdHGF组左心室舒张期末容积(LVEDV)、左心室射血分数(LVEF)、短轴缩短率(FS)明显改善,差异有统计学意义(P<0.05);DSA检测缺血区新生血管数MSCs+AdHGF组较AdHGF组、MSCs组明显增多,差异有统计学意义(P<0.05);SPECT显示MSCs+AdHGF组左室心肌增厚、灌注明显改善,运动增强,差异有统计学意义(P<0.05);心肌HE染色血管密度,MSCs+AdHGF组明显高于AdHGF组和MSCs组(39.4±1.2)个/HPF vs.(36.5±1.4)个/HPF、(34.5±1.7)个/HPF,P<0.05];心肌TUNEL染色法检测,MSCs+AdHGF组、AdHGF组细胞凋亡率明显低于MSCs组(P<0.05)。结论 MSCs+AdHGF联合具有促进慢性缺血心肌新血管生成、抑制细胞凋亡、改善心功能等作用;MSCs+AdHGF联合治疗缺血性心脏病作用较单纯HGF或MSCs移植更明显。

Combination Transplantation of Bone Marrow Mesenchymal Stem Cells and Hepatocyte Growth Factor Gene in the Treatment of Chronic Ischemic Heart Disease in Pigs

Objective To investigate the effects of hepatocyte growth factor(HGF) gene transfected bone marrow mesenchymal stem cells(MSCs) transplantation in pigs with chronic ischemic heart disease.Methods MSCs were isolated from pig bone marrow by density gradient centrifugation and adherent cell culture,purified,and determined by cell surface antigens(CD34,CD44,CD71,Ⅷ factor and desmin).MSCs were transfected by adenovirus expressing hepatocyte growth factor(AdHGF),and the influence of HGF on the biological characteristics of MSCs was tested.The pig model of chronic myocardial ischemia was established by placing Ameroid ring inside the left circumflex coronary artery via left thoracotomy.A total of 40 pigs were randomly divided into 5 groups(n=8) and were injected 5×106/ml MSCs+4×109 pfu 200 μl AdHGF(MSCs+AdHGF group),4×109 pfu 200 μl AdHGF(AdHGF group),5×106/ml MSCs 200 μl(MSCs group),4×109 pfu 200 μl AdNull(AdNull group)and 1 ml saline(control group) into the ischemic myocardium respectively.Echocardiogram,digital subtraction angiography(DSA) of coronary artery,single photon emission computed tomography(SPECT) myocardial perfusion imaging and cardiomyocyte apoptosis were examined after 4 weeks.Results Positive CD44 and CD71 and negative CD34,Ⅷ factorand desmin were detected in MSCs by flow cytometer.HGF had a strong influence on stimulating the proliferation and differentiation of MSCs.Echocardiogram examination showed that left ventricular end-diastolic volume(LVEDV),left ventricular ejection fraction(LVEF),fractional shortening(FS) of MSCs+AdHGF group were significantly increased after treatment(P<0.05).DSA detection showed that ischemic neovascularization of MSCs+AdHGF group was significantly higher than those of AdHGF group and MSCs group(P<0.05).SPECT showed that the left ventricular myocardium of MSCs+AdHGF group appeared thickened,myocardial perfusion was significantly improved and the myocardial motion was significantly increased(P<0.05).Vascular density of MSCs+AdHGF group was significantly higher than those of AdHGF group and MSCs group by HE stain of myocardium (39.4±1.2) / HPF vs.(36.5±1.4) / HPF and(34.5±1.7) / HPF,P<0.05].Cardiomyocyte apoptosis rate of MSCs+AdHGF group was significantly lower than those of AdHGF group and MSCs group by TUNEL stain(P<0.05).Conclusion Combination transplantation can promote the angiogenesis of chronic ischemic myocardium,inhibit cardiomyocyte apoptosis and improve heart function in pigs with chronic ischemic heart disease.The effect of HGF gene transfected MSCs transplantation is better than that of MSCs or HGF transplantation alone.