AKT 修饰的自体冠状动脉间充质干细胞预防猪梗死心肌的重构和修复。

AKT-modified autologous intracoronary mesenchymal stem cells prevent remodeling and repair in swine infarcted myocardium.

机构信息

Department of Cardiovascular Surgery, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China.

出版信息

Chin Med J (Engl). 2010 Jul;123(13):1702-8.

PMID:20819633

Abstract

BACKGROUND

Transplantation of adult bone marrow-derived mesenchymal stem cells (MSCs) has been proposed as a strategy for cardiac repair following myocardial damage. However cell transplantation strategies to replace lost myocardium are limited by the inability to deliver large numbers of cells that resist peritransplantation graft cell death. Accordingly, we set out to isolate and expand adult swine bone marrow-derived MSCs, and to engineer these cells to overexpress AKT1 (protein kinase B), to test the hypothesis that AKT1-engineered MSCs are more resistant to apoptosis and can enhance cardiac repair after transplantation into the ischemic swine heart.

METHODS

The CDS (regulation domain of AKT1) AKT1-cDNA fragment was amplified, and MSCs were transfected following synthesis with a pCDH1-AKT1 shuttling plasmid. Western blotting analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed. Myocardial infarction (MI) models were constructed in Meishan pigs, and cardiac function was evaluated by magnetic resonance imaging (MRI) measurements and echocardiography 4 weeks later. All pigs were assigned to four groups: control (A), DMEM (B), MSC (C), and AKT-transfected (D). MSCs were transfected with the AKT1 gene, and autologous BrdU-labeled stem cells (1 x 10(7)/5 ml) were injected into left anterior descending coronary atery (LAD) of the infarct heart in groups C and D. In group B, DMEM was injected using the same approach. In group A, there was no injection following LAD occlusion. After 4 weeks, cardiac function and regional perfusion measurements were repeated by MRI and echocardiography, and histological characteristics of the hearts were assessed. Connecxin-43 (CX-43), BrdU, and von Willebrand factor (VWF) immunoreactivity was tested using enzyme linked immunosorbent assay (ELISA). Vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1) were analyzed at the same time.

RESULTS

AKT1-cDNA was cloned into pCDH1-MCS1-EF1-copGFP and the sequence was confirmed. AKT mRNA expression was detected at 24 hours after transfection. AKT1 expression in MSCs remained strong after 2 weeks, according to real-time RT-PCR and Western blotting. Prior to cell implantation, end-diastolic left ventricular dimension (EDLVd) increased and stroke volume (SV) decreased in the MI hearts. MRI scans revealed significantly improved cardiac function following implantation, and implanted MSCs prevented thinning and expanding in the infarct region, as well as improved contraction and increased perfusion in all groups compared to control hearts. The left ventricular chamber size was smaller in cell-transplanted hearts than in control hearts. Moreover, group D exhibited significant improvement. The expression of CX-43, BrdU, and VWF could be found in the immunohistochemical pathological sections of group C and group D. The level of VEGF reached a high level 1 week after implanting the MSCs, but the level of TGF-beta1 decreased gradually.

CONCLUSIONS

The AKT1-expressing lentiviral vector resulted in stable over-expression of AKT1 in MSCs. MSC engraftment in host myocardium improved cardiac function by attenuating contractile dysfunction and pathological thinning of the infracted left ventricular wall, which likely resulted from myocardial regeneration and angiogenesis.

摘要

背景

移植成体骨髓间充质干细胞（MSCs）已被提议作为心肌损伤后心脏修复的一种策略。然而，用于替代失活心肌的细胞移植策略受到能够递送大量抵抗移植后细胞死亡的细胞的能力的限制。因此，我们着手分离和扩增成年猪骨髓来源的 MSCs，并对这些细胞进行工程改造以过表达 AKT1（蛋白激酶 B），以检验 AKT1 工程化 MSCs 对细胞凋亡的抵抗能力更强，并能增强移植到缺血性猪心脏后的心脏修复的假设。

方法

扩增 CDS（AKT1 的调节域）AKT1-cDNA 片段，并在合成 pCDH1-AKT1 穿梭质粒后转染 MSCs。通过 Western blot 分析和实时逆转录-聚合酶链反应（RT-PCR）进行分析。在梅山猪中构建心肌梗死（MI）模型，并在 4 周后通过磁共振成像（MRI）测量和超声心动图评估心功能。所有猪均分为 4 组：对照组（A）、DMEM（B）、MSC（C）和 AKT 转染组（D）。将 AKT1 基因转染到 MSCs 中，将自体 BrdU 标记的干细胞（1x107/5ml）注入 C 和 D 组的梗死心脏的左前降支（LAD）。在 B 组中，采用相同的方法注入 DMEM。在 A 组中，LAD 闭塞后无注射。4 周后，通过 MRI 和超声心动图重复进行心功能和局部灌注测量，并评估心脏的组织学特征。使用酶联免疫吸附测定（ELISA）检测连接蛋白 43（CX-43）、BrdU 和血管性血友病因子（VWF）的免疫反应性。同时分析血管内皮生长因子（VEGF）和转化生长因子-β1（TGF-β1）。

结果

AKT1-cDNA 被克隆到 pCDH1-MCS1-EF1-copGFP 中，并确认了序列。转染后 24 小时检测到 AKT mRNA 表达。根据实时 RT-PCR 和 Western blot，MSC 中的 AKT1 表达在转染后 2 周仍保持较强。在细胞植入前，MI 心脏的左心室舒张末期直径（EDLVd）增加，心搏量（SV）减少。MRI 扫描显示，植入细胞后心功能明显改善，与对照组心脏相比，植入的 MSCs 可防止梗死区变薄和扩张，并改善所有组的收缩和增加灌注。与对照组心脏相比，细胞移植心脏的左心室腔更小。此外，D 组表现出显著改善。在 C 组和 D 组的免疫组织化学病理切片中可以发现 CX-43、BrdU 和 VWF 的表达。MSC 植入后 1 周 VEGF 水平达到较高水平，但 TGF-β1 水平逐渐下降。