Xu Hongxin, Zhu Gangyan, Tian Yihao
Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
Department of Geriatrics, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
J Huazhong Univ Sci Technolog Med Sci. 2012 Feb;32(1):36-41. doi: 10.1007/s11596-012-0006-x. Epub 2012 Jan 27.
Bone marrow mesenchymal stem cells (MSCs) have shown potential for cardiac repair following myocardial injury, but this approach is limited by their poor viability after transplantation. The present study was to investigate whether trimetazidine (TMZ) could improve survival of MSCs in an ex vitro model of hypoxia, as well as survival, differentiation, and subsequent activities of transplanted MSCs in rat hearts with acute myocardial infarction (AMI). MSCs at passage 3 were examined for their viability and apoptosis under a transmission electron microscope, and by using flow cytometry following culture in serum-free medium and exposure to hypoxia (5% CO(2), 95% N(2)) for 12 h with or without TMZ. Thirty Wistar rats were divided into 3 groups (n=10 each group), including group I (AMI control), group II (MSCs transplantation alone), and group III (TMZ+MSCs). Rat MSCs (4×10(7)) were injected into peri-infarct myocardium (MSCs group and TMZ+MSCs group) 30 min after coronary artery ligation. The rats in TMZ+MSCs group were additionally fed on TMZ (2.08 mg·kg(-1)·day(-1)) from day 3 before AMI to day 28 after AMI. Cardiac structure and function were assessed by echocardiography at 28th day after transplantation. Blood samples were collected before the start of TMZ therapy (baseline), and 24 and 48 h after AMI, and inflammatory cytokines (CRP, TNF-α) were measured. Then the survival and differentiation of transplanted cells in vivo were detected by immunofluorescent staining. The cellular apoptosis in the peri-infarct region was detected by using TUNEL assay. Furthermore, apoptosis-related proteins (Bcl-2, Bax) within the post-infarcted myocardium were detected by using Western blotting. In hypoxic culture, the TMZ-treated MSCs displayed a two-fold decrease in apoptosis under serum-free medium and hypoxia environment. In vivo, cardiac infarct size was significantly reduced, and cardiac function significantly improved in MSCs and TMZ+MSCs groups as compared with those in the AMI control group. Combined treatment of TMZ with MSCs implantation demonstrated further decreased MSCs apoptosis, further increased MSCs viability, further decreased infarct size, and further improved cardiac function as compared with MSCs alone. The baseline levels of inflammatory cytokines (CRP, TNF-α) had no significant difference among the groups. In contrast, all parameters at 24 h were lower in TMZ+MSCs group than those in MSCs group. Furthermore, Western blotting indicated that the expression of anti-apoptotic protein Bcl-2 was up-regulated, while the pro-apoptotic protein Bax was down-regulated in the TMZ+MSCs group, compared with that in the MSCs group. It is suggested that implantation of MSCs combined with TMZ treatment is superior to MSCs monotherapy for MSCs viability and cardiac function recovery.
骨髓间充质干细胞（MSCs）已显示出在心肌损伤后心脏修复的潜力，但这种方法受到移植后其生存能力差的限制。本研究旨在探讨曲美他嗪（TMZ）是否能在体外缺氧模型中提高MSCs的存活率，以及在急性心肌梗死（AMI）大鼠心脏中移植的MSCs的存活、分化及后续活性。对第3代MSCs在无血清培养基中培养并暴露于缺氧环境（5% CO₂，95% N₂）12 h，添加或不添加TMZ，然后通过透射电子显微镜及流式细胞术检测其活力和凋亡情况。30只Wistar大鼠分为3组（每组n = 10），包括I组（AMI对照组）、II组（单纯MSCs移植组）和III组（TMZ + MSCs组）。冠状动脉结扎30分钟后，将大鼠MSCs（4×10⁷）注射到梗死周边心肌（MSCs组和TMZ + MSCs组）。TMZ + MSCs组的大鼠从AMI前3天至AMI后28天额外给予TMZ（2.08 mg·kg⁻¹·天⁻¹）。移植后第28天通过超声心动图评估心脏结构和功能。在TMZ治疗开始前（基线）、AMI后24小时和48小时采集血样，检测炎症细胞因子（CRP、TNF-α）。然后通过免疫荧光染色检测体内移植细胞的存活和分化情况。使用TUNEL法检测梗死周边区域的细胞凋亡。此外，通过蛋白质免疫印迹法检测梗死心肌内凋亡相关蛋白（Bcl-2、Bax）。在缺氧培养中，TMZ处理的MSCs在无血清培养基和缺氧环境下凋亡减少了两倍。在体内，与AMI对照组相比，MSCs组和TMZ + MSCs组的心脏梗死面积显著减小，心脏功能显著改善。与单纯MSCs相比，TMZ与MSCs植入联合治疗进一步降低了MSCs凋亡，进一步提高了MSCs活力，进一步减小了梗死面积，并进一步改善了心脏功能。各组炎症细胞因子（CRP、TNF-α）的基线水平无显著差异。相比之下，TMZ + MSCs组在24小时时的所有参数均低于MSCs组。此外，蛋白质免疫印迹法表明，与MSCs组相比，TMZ + MSCs组抗凋亡蛋白Bcl-2的表达上调，而促凋亡蛋白Bax的表达下调。提示MSCs植入联合TMZ治疗在MSCs活力和心脏功能恢复方面优于MSCs单一疗法。
