干细胞修复心肌损伤的实验研究

【作者】 郭静；

【导师】 李新立；

【作者基本信息】 南京医科大学 ， 内科学（心血管病学）， 2014， 博士

【摘要】 课题一 单核趋化因子-1促进间充质干细胞在扩张型心肌病中的归巢背景：扩张型心肌病(Dilated Cardiomyopathy, DCM)是导致心力衰竭最常见的非缺血性心肌病。间充质干细胞(mesenchymal stem cells, MSCs)移植作为治疗DCM的极具潜力的方法是目前研究的热点。然而关于静脉移植MSCs对DCM的治疗作用报道相对较少,DCM中MSCs向心脏组织归巢的机制也尚不清楚。方法：从C57/BL6小鼠骨髓分离培养出小鼠MSCs并在体外进行扩增培养。通过腹腔注射盐酸阿霉素构建DCM模型。MSCs或生理盐水经颈内静脉注射到小鼠体内。移植后4周超声心动图及血流动力学评估心脏功能包括缩短分数,左室舒张末内径,左室收缩末压及左心室压最大上升速率。Masson三色染色检测心肌纤维化水平。应用RT-PCR检测心肌组织中单核趋化蛋白-1 (monocyte chemotactic protein-1, MCP-1)、基质细胞衍生因子-1 (stromal cell-derived factor-1, SDF-1)、巨噬细胞炎症蛋白-1α(macrophage inflammatory protein-1α (MIP-1α)及单核趋化蛋白-3 (monocyte chemotactic protein-3,MCP-3) mRNA表达水平。Western blot验证心肌中MCP-1蛋白表达水平。Western blot及流式细胞仪测定MCP-1的受体C-C chemokine receptor type 2 (CCR2)在MSCs表达情况。利用transwell小室方法检测MCP-1对MSCs体外的趋化作用,体内实验通过观察抑制了MSCs的CCR2表达后对其向心肌趋化的影响进一步证实MCP-1/CCR2信号通路在MSCs向扩张的心肌归巢中的作用。结果：静脉移植MSCs可明显改善DCM小鼠的心功能并减轻其心肌纤维化。DCM心肌中MCP-1基因及蛋白水平都较正常对照组显著升高,而SDF-1,MIP-1α及MCP-3则无明显变化。MSCs表面表达MCP-1受体CCR2。MCP-1在体外可促进MSCs的迁移,抑制了MSCs的CCR2的表达后可减少MSCs向扩张的心肌的归巢量。结论：本研究显示静脉移植MSCs能够改善DCM的心脏功能,并发现这种过程是MCP-1/CCR2信号通路参与了MSCs在DCM中的归巢所致,为MSCs治疗DCM提出了新的治疗靶点。课题二 人心脏来源的iPS细胞系的建立及在小鼠心肌梗死模型中的应用背景：可诱导的多能干细胞(Induced pluripotent stem cells, iPSCs)具有保持其来源组织表观遗传特性的记忆功能。这种表观遗传记忆可以影响iPSCs的分化及其分化的细胞相应的功能。因此我们设想心脏来源的iPSCs在治疗心肌损伤方面可能会有更为出色的表现。方法：从心力衰竭病人心肌组织中分离培养出心脏成纤维细胞,以Oct3/4、Sox2,.Klf4和cMyc作为编程基因,通过仙台病毒感染的方式将其重编程为人的iPSCs (hciPSCs)。hciPSC建立成功并经生物学鉴定后,利用细胞因子Activin A、BMP4和bFGF在体外诱导其向心肌细胞分化。结扎小鼠左冠状动脉前降支建立心肌梗死模型。将分化的心肌细胞形成的细胞膜片移植到心肌梗死的小鼠心脏上,4周后通过人特异性抗体Human specific nuclei antibody (HNA)及 Human specific cardiac troponinT (hcTNT)免疫荧光染色方法评估移植细胞的存活率及成熟情况,超声心动图测定心脏功能,TUNEL染色观察小鼠心肌细胞凋亡情况,内皮细胞特异性抗体CD31染色检测心肌组织血管密度。结果：我们成功地将人的心脏成纤维细胞重编程为hciPSCs,对其生物学鉴定显示hciPSCs保持正常的细胞核型,表达多能分化的标志Tral-60、SSEA4和Oct4,多能基因Oct3/4、Sox2和Nanog表达量与成熟的iPS细胞系相似,在免疫缺陷的小鼠体内可形成具有三个胚层结构的畸胎瘤。hciPSCs可在体外高效地分化为心肌细胞(效率可达92%以上)。分化的心肌细胞形成的细胞膜片移植到心肌梗死小鼠心脏4周后通过计算心肌组织中HNA和hcTNT阳性细胞数量得到移植细胞存活率可达30%以上。与移植后1天相比,移植的心肌细胞显示出更为清晰的细胞骨架,排列更加规律。移植后4周细胞移植组左室射血分数(left-ventricular ejection fraction,EF%)及缩短分数(fractional shortening, FS%)都较心肌梗死组明显升高(p<0.05)。TUNEL染色可见细胞移植组小鼠心肌中TUNEL阳性细胞数明显少于心肌梗死组(p<0.05),并且CD31染色提示在心肌梗死边缘区,细胞移植组的血管密度明显高于心肌梗死组(p<0.05)。结论：将人心脏成纤维细胞重编程的iPSCs分化的心肌细胞移植到心肌梗死小鼠心脏表面,4周后发现大量心肌细胞在小鼠体内存活并发育成熟,并且明显改善小鼠的心脏功能,减少了缺血区域心肌的凋亡并促进心肌梗死边缘区的血管生成。更多还原

【Abstract】 Section I Monocyte chemotactic protein-1 promotes the myocardial homing of mesenchymal stem cells in dilated cardiomyopathyBackground:Dilated cardiomyopathy (DCM) is the most common form of non-ischemic cardiomyopathy that leads to heart failure. Mesenchymal stem cells (MSCs) are under active investigation currently as a potential therapy for DCM. However, little information is available about the therapeutic potential of intravenous administration of MSCs for DCM. Moreover, how MSCs home to the myocardium in DCM is still unclear.Methods:MSCs were isolated from bone marrow of C57/BL6 mice and expanded in vitro. DCM was induced by intraperitoneally administering Doxorubicin and MSCs or vehicles were infused through the internal jugular vein. Cardiac functions including the percentage of fractional shortening, left ventricular diastolic dimension, left ventricular end-diastolic pressure, and left ventricular maximum dp/dt were evaluated by echocardiographic and hemodynamic studies. Fibrosis was determined by Masson’s trichrome staining. The mRNA expression levels of monocyte chemotactic protein-1 (MCP-1), stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein-la (MIP-lα), and monocyte chemotactic protein-3 (MCP-3) were determined using real time polymerase chain reactions and the protein expression level of MCP-1 was detected with Western blot. The MSCs expression of C-C chemokine receptor type 2 (CCR2), a MCP-1 receptor, is confirmed by Western blot and flow cytometry analysis. The chemotactic effects of MCP-1/CCR2 were checked by assessing the migration in vitro and in vivo.Results:MSCs transplantation improved the cardiac function and decreased the myocardial fibrosis of mice with DCM. MCP-1 was up-regulated in dilated myocardial tissue both at the mRNA and protein level while SDF-1, MIP-1α and MCP-3 remain unchanged. CCR2 was present in MSCs. MCP-1 promoted MSCs migration in vitro while CCR2 inhibition decreased the migration of MSCs to the dilated heart.Conclusions:This study shows that peripheral intravenous infusion of MSCs can support the functional recovery of DCM, and this effort is caused by the myocardial homing of MSCs involving of MCP-1/CCR2 pathway. Modulation of MCP-1/CCR2 signaling system could be as a novel therapeutic target for DCM.Section Ⅱ Generation of human induced pluripotent stem cells from adult cardiac fibroblast and the application in the mouse model of myocardium infarctionBackground:Induced pluripotent stem cells (iPSCs) retain epigenetic characteristics from their tissues of origin. This epigenetic memory can influence iPSC differentiation and the subsequent function of iPSC-derived cells. Thus, we assume that iPSC-derived cell therapy may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells.Methods:Cardiac fibroblasts from patient with heart failure were used to generate human iPSC lines (hciPSC) by Sendai virus transduction using Oct3/4, Sox2, Klf4 and cMyc as programming gene. After the establishment and biology identification, hciPSC were induced to differentiate into cardiomyocyte (hciPSC-CMs) via cytokine including Activin A, BMP4 and bFGF in vitro. Myocardial infarction (MI) was created by occluding the left anterior descending artery (LAD) in immunodeficient mice. Four weeks after transplantation of hciPSC-CM sheets, immunistaining of human specific nuclei antibody (HNA) and human specific cardiac troponinT (hcTNT) were performed to evaluate the engraftment rate and cell maturation, the heart function was detected by echo with the measurements of left-ventricular ejection fraction (EF%) and fractional shortening (FS%). TUNEL assay was used for the assessment of cell apoptosis and vascular density in the heart tissue was measured by CD31 staining.Results:We showed that human cardiac iPSCs can be generated from adult cardiac fibroblasts, and the biology identification illuminated that iPSCs displayed a normal human chromosomal structure, expressed the pluripotency markers Tral-60, SSEA4, and Oct4 and the mRNA levels of pluripotency gene such as Oct3/4, Sox2, and Nanog were similar to the levels observed in a control line of iPSCs. Pluripotency of the hciPSCs was confirmed via the formation of teratoma which contained cells of all three developmental germ layers. HciPSC can be differentiated into cardiomyocytes (CMs) with greater than 92% CM purity. Four weeks after hciPSC-CMs transplantation into the mice with myocardium infarction, more than 30% of the original cells survived in the mouse heart based on the number of hcTNT+ and HNA+ cells and the cell sheets had become structurally organized, with aligned myocytes. The EF% and FS% were improved in the cell transplantation group compared with MI group, p<0.05. TUNEL assay showed that the apoptotic cells were 45% less in myocardium underneath of the cell sheet in the hciPSC-CM group than myocardium of the MI group, p<0.05. The myocardial vascular density was significantly higher in the peri-infarct region of hciPSC-CM hearts than in the corresponding regions of hearts from MI animals, p<0.05.Conclusions:Human cardiac iPSCs can be generated from adult cardiac fibroblasts successfully and differentiate into cardiomyocytes with high efficiency. HciPSC-CMs can survive and mature in the mice of myocardium infarction on the 4 weeks after transplantation and improve the heart function which is likely secondary to a combination of the reduction of apoptosis and increased vascular density.更多还原