摘要
目的:干细胞治疗是新兴的治疗心血管疾病的方法,为心肌病的治疗提供了新的策略。内皮祖细胞(EPCs)和间充质干细胞(MSCs)是骨髓干细胞最重要的组成成分,是干细胞研究和组织工程学研究的重要种子细胞。目前国内外绝大部分基础和临床研究均选用单一的内皮祖细胞或间充质干细胞,在治疗缺血性心肌病或扩张型心肌病的研究中,取得了令人鼓舞的结果。EPCs和MSCs具有不同的生物学行为:实验证实前者在体外培养或移植到体内可分化为内皮细胞,可以修复受损的血管内皮并参与血管新生;而后者具有多向分化潜能,与心肌细胞共同培养及用5氮杂胞苷诱导在体外可以分化为心肌细胞,移植到体内4周就可以表达心肌细胞的特异性标志,并可以和原有的心肌细胞形成连接,将其移植到心肌内可以弥补丢失的心肌细胞。大剂量的儿茶酚胺类药物具有心肌细胞毒性,可引起心肌细胞的坏死并进而发展为心肌病。异丙肾上腺素(ISO)皮下注射诱导的心肌病是经典的儿茶酚胺类药物中毒性心肌病模型,我们研究的目的是将EPCs和MSCs联合移植于ISO损伤的大鼠心肌,观察其对心功能、血流动力学指标和心肌局部血流的影响,并探讨其对儿茶酚胺类药物中毒性心肌病的治疗效果和可能的相关机制,为心肌病的治疗寻找新的思路,并为EPCs和MSCs联合移植治疗的临床应用提供线索和依据。
方法:骨髓细胞悬液取自SD大鼠的股骨和胫股,用Ficoll分离液和差速贴壁法分离雄性SD大鼠的骨髓单个核细胞,分别用EGM-2和DMEM培养液培养EPCs和MSCs,用于细胞移植的干细胞为原代培养的EPCs和第三代MSCs。90只雌性SD大鼠,通过腹股沟皮下注射ISO(250mg/kg)制作儿茶酚胺类药物中毒性心肌病模型。将65只模型成功的大鼠随机分为五组,假手术组(开胸手术并注射生理盐水)、对照组(只注射EBM-2培养液)、MSCs组(注射2×106个MSCs)、EPCs组(注射2×106个EPCs)、MSCs+EPCs组(注射1×106个EPCs和1×106个MSCs),在直视下分5点将干细胞直接注射到大鼠的心肌内。在注射ISO前、注射药物四周和细胞移植三个月后,分别做心脏超声测量其左室舒张末期内径(LVEDD)、左室收缩期末期内径(LVESD)、室间隔厚度(IVST)、左室前壁厚度(LVAWT)、后壁厚度(LVPWT),计算左室收缩末期容积(LVESV)、左室舒张末期容积(LVEDV)、左室射血分数(EF)、短轴缩短率(FS)。在细胞移植后三个月,处死动物之前,通过颈动脉插管到左心室,测量左室收缩期压力上升和下降的最大速度(±dp/dt),左室舒张期末压力(LVEDP);应用彩色微球技术评估心肌局部血流(RMBF);Y染色体原位杂交和免疫荧光技术确定雌性大鼠心肌标本中移植的雄性干细胞的定位和分化;应用免疫组化技术通过vWF染色半定量分析心肌组织内毛细血管密度;Massion’s染色分析心肌组织纤维化程度;Tunel染色评价心肌细胞凋亡的比率。同时测量心肌组织内血管内皮生长因子(VEGF),碱性成纤维生长因子(b-FGF)及血管生成素2(Ang-2)的浓度及其mRNA的表达情况。
结果:90只雌性大鼠注射ISO四周后有71存活,其中有6只因EF下降未超过20%被排除,其余65只大鼠被随机分为五组(每组13只),手术及干细胞移植后各组均有大鼠死亡,终点时每组存活的大鼠:假手术组11只、对照组11只、MSCs组10只、EPCs组9只、MSCs+EPCs组11只。药物注射后4周,与基线比较大鼠的EF、FS明显下降(P<0.05);LVEDD、LVESV、LVEDV明显增加(P<0.05)。细胞移植3月后,接受细胞治疗的三组(MSCs组、EPCs组和EPCs+MSCs组)EF、FS较细胞移植前明显改善(P<0.05);LVEDD、LVESV、LVEDV较移植前有明显的缩小(P<0.05);细胞移植组与对照组和假手术组比较也有明显差异(P<0.05);而假手术组和对照组与手术前比较,各项指标都没有明显变化,两组间比较也没有明显差异(P<0.05)。细胞移植后三个月,接受细胞移植三组动物的血流动力学指标:±dp/dt、LVEDP较假手术组和对照组有明显的改善,表现为±dp/dt加快, LVEDP降低(P<0.05)。更重要的是EPCs+MSCs组与MSCs组、EPCs组比较,除LVEDD外的各项心功能指标及血流动力学指标改善得更为明显(P<0.05);但是MSCs组和EPCs组比较没有明显差别(P>0.05)。LVEDD在接受细胞治疗的三组中比较没有明显差异(P>0.05)。彩色微球技术测定RMBF是评价心肌血流灌注的金指标,我们的实验发现EPCs+MSCs组和EPCs组的RMBF在五组中是最高的(P<0.05);MSCs组也高于对照组和假手术组(P<0.05);但EPCs+MSCs组与EPCs组比较没有显著性差异(P>0.05)。心肌内毛细血管密度检测结果解释了RMBF在各组间出现差异的原因,EPCs+MSCs组和EPCs组心肌内毛细血管密度明显高于MSCs组、对照组和假手术组(P<0.05);EPCs+MSCs组和EPCs组比较、对照组和假手术组比较均没有达到统计学差异(P>0.05)。Y染色体原位杂交能检测到雌性大鼠心肌内移植的雄性干细胞,结果显示:EPCs+MSCs组和MSCs组心肌细胞、血管和间质组织内可以观察到Sry阳性细胞;而EPCs组只能在血管壁内看到Sry阳性细胞。假手术组和对照组没有发现阳性细胞。为了明确移植细胞的分化方向,我们应用免疫荧光技术,结果发现EPCs组移植的细胞表达内皮细胞表面标志(vWF),而MSCs组一些移植的细胞表达心肌细胞表面标志(troponin T)。其次无论是单独移植还是两种细胞联合移植都减轻了心肌纤维化程度和心肌细胞的调亡比率(P<0.05),而EPCs+MSCs组减轻的程度明显大于其它四组(P<0.05)。我们的研究还证实心功能的改善、心肌血流灌注和心肌内血管密度的增加与心肌内血管生长因子:VEGF、b-FGF、Ang-2分泌增多有关,而它们的分泌与其mRNA转录水平增高是一致的。MSCs组心肌组织内VEGF,b-FGF及Ang-2蛋白的浓度与假手组和对照组相比明显升高(P<0.05),但比EPCs+MSCs组和EPCs组低(P<0.05),对照组与假手术之间比较没有统计学差异(P>0.05)。值得提到的是:EPCs组的b-FGF水平较EPCs+MSCs组更高,P<0.05。QRT-PCR分析显示EPCs+MSCs组和EPCs组VEGF、b-FGF、Ang-2mRNA的转录高于MSCs组、假手术组和对照组(P<0.05)。但b-FGFmRNA的表达在EPCs组和EPCs+MSCs组没有明显差别(P>0.05),假手术组和对照组三种细胞因子mRNA的比较均没有达到统计学差异(P>0.05)。
结论:EPCs和MSCs联合移植治疗ISO损伤性心肌病,3个月后,移植的细胞可以分化为心肌细胞和内皮细胞或整合于心肌细胞和内皮细胞内,明显改善了心功能和血流动力学指标,增加心肌的血流灌注,其机制与EPCs联合MSCs移植诱导心肌内血管新生、减少心肌细胞的纤维化程度和心肌细胞的凋亡数量及移植细胞所引起的自分泌和旁分泌多种生长因子有关。EPCs和MSCs联合移植为儿茶酚胺类药物中毒性心肌病的治疗提供了一种的新方法和实验依据。
Objective Stem cell transplantation is emerging as a potential therapeutic approach to treating cardiac diseases, and providing a new method for various cardiomyopathies. Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) are important components of stem cells derived from bone marrow. Some studies showed they had the capacities to either repair damaged endothelium and generate new blood vessels, or regenerate new cardiocytes in the ischemic area of myocardium. While EPCs and MSCs have different biologic properties, their potential synergetic role in treating degenerative disease or damage myocardium might have better results. In this study, we aimed to investigate whether simultaneous implantation of MSCs and EPCs into the myocardium in rats with isoproterenol injured cardiomyopathy could enhance regional myocardium blood flow (RMBF) and improve cardiac function.
Methods Bone marrow mononuclear cells (BMNCs) of 50 male rats were separated by Ficoll density gradient centrifugation, and EPCs and MSCs were cultured in EGM-2 and DMEM, respectively. The primary passage of EPCs and the third passage of MSCs were used for cells transplantation. 90 female rats (150-220g) received inguinal subcutaneous injections of ISO (250mg/kg) for two consecutive days. Before injection of ISO and 4 weeks after injection of ISO, heart function was assessed by transthoracic echocardiography to confirm the successful models (EF decreasing by 20% with the dilation of left ventricular). These successful models were then randomly divided into five groups: sham group, control group, MSCs group, EPCs group and EPCs+MSCs group. 2×106 EPCs, 2×106 MSCs and mixture of 1×106 EPCs and 1×106 MSCs resolved in 200μL EBM-2 were given evenly to left ventricular free wall for EPCs group, MSCs group and EPCs+MSCs group, respectively. Another vehicle group only received 200μL EBM-2 to test the effect of EBM-2 medium. And the sham control group was given saline. Echocardiography examination was repeated at 3 months after cell therapy for left ventricular end diastolic diameter (LVEDD), left ventricular anterior wall thickness (LVAWT), left ventricular posterior wall thickness (LVPWT) and interventricular septum thickness (IVST). Left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), ejection factors (EF), fractional shortening ratio (FS) were calculated by an independent investigator. The left ventricular end-diastolic pressure (LVEDP), the maximal rate of pressure rise (+dp/dtmax), the maximal rate of pressure fall (-dp/dtmax) were analyzed a right carotid artery catheter. RMBF was measured by colored micro spheres (CM) on the last day of the study. Fluorescence in situ hybridization (FISH) was performed to detect male cells in the female rat hearts. Immunofluorescence staining was performed with monoclonal mouse anticardiac troponin T, and polyclonal rabbit anti–von Willebrand factor (vWF) was used to assay the differentiation of transplanted cells. To detect fibrosis in myocardium, Masson’s trichrome staining was performed on the left ventricle tissue. To detect capillary density in the myocardium, immunohistochemical staining of endothelial cells was performed using an antibody against vWF. The ratio of apoptosis cell in myocardium was analyzed by Tunel staining. The protein expression of vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (b-FGF) and angiopoietin 2 (Ang-2) were measured by ELISA assay. Quantitative real time polymerase chain reaction(QRT-PCR), using SYBR? Green I as a dye, was performed to analyze the mRNA expression of VEGF, b-FGF and Ang-2.
Results 71 of 90 female rats survived from the ISO injection intervention. Whereas, the 6 female rats didn’t manifest the significant decrease of cardiac function, and were excluded from the study. These 65 rats were then randomly divided into five groups (13 rats in each group). At the completion of 3-month cell therapy treatment, there were 11 rats in sham group, 11 rats in control group, 10 rats in MSCs group, 9 rats in EPCs group and 11 rats in MSCs+EPCs group to survive. 4 weeks after ISO injection, compared with baseline, the cardiac function showed decrease in EF and FS (P<0.05). However, LVEDD, LVESV and LVEDV were increased significantly (P<0.05). 3 months after cell transplantation, MSCs group, EPCs group and EPCs+MSCs group were improved in EF, FS, LVESV, LVEDV, +dp/dt, -dp/dt and LVEDP (P<0.05). There were no difference between sham group and the control group (P>0.05). However, there showed no statistical difference in LVEDD between EPCs+MSCs group and single stem cells treatment groups (P>0.05). Besides LVEDD, EPCs+MSCs group demonstrated even better cardiac function than either MSCs group or EPCs group (P<0.05). The combination of MSCs and EPCs induced greater RMBF comparing as the other three groups (sham group, control group and MSCs group, P<0.05), but was not significantly greater than EPCs group (P>0.05). Total capillary density in the myocardial tissue had the similar results with RMBF. The Sry positive cells detected in cardiac tissue in EPCs+MSCs group and MSCs group, however, in EPCs group they only were found in blood vessels. No Sry positive cell was presented in the sham group and control group. Immunofluorescence staining demonstrated that some of Sry positive MSCs were positive for the cardiac markers (cardiac troponin T) and some of EPCs were positive for vWF. EPCs+MSCs group had lest collagen deposition compared with the other four groups (P<0.05). There are less the ratio of apoptosis cell in EPCs+MSCs group than the other four groups (P<0.05). VEGF, b-FGF and Ang-2 protein levels slightly increased in MSCs group compared with sham group and control group (P<0.05). Both EPCs+MSCs group and EPCs group demonstrated further increase in protein expression level than MSCs group (P<0.05). Interestingly, EPCs group had higher protein expressions of b-FGF than the EPCs+MSCs groups (P<0.05). QRT-PCR analysis demonstrated that higher mRNA expressions of were found in the EPCs+MSCs group and EPCs group than either MSCs group or sham group or control group (P<0.05). But, mRNA expression of b-FGF was not different between the EPCs group and the EPCs+MSCs group (P>0.05).
Conclusion The transplantation of EPCs combined with MSCs can significantly improve the cardiac function and myocardium flow for the rats with isoproterenol injured cardiomyopathy, as comparing with MSCs or EPCs transplantation. The effects are related to increase neovascularization, decrease fibrosis and apoptosis cardiocytes in cardiac tissue. The transplanted EPCs and MSCs can differentiate into cardiocytes and endothelial cells, and promote the expression of various growth factors such as VEGF, b-FGF and Ang-2. This study provided a novel treatment method for isoproterenol injured cardiomyopathy and presented experimental data for the application of combined transplantation of two kinds of stem cell.
引文
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