核干细胞因子增强间充质干细胞心肌保护作用

赵九洲; 林晨; 贺媛; 于卫华; 郭永珍; 陈有虎; 李聪叶; 陶凌; 郭艳杰; 刘瑞; 闫文俊

doi:10.12125/j.chj.201908036

核干细胞因子增强间充质干细胞心肌保护作用

Nucleostemin enhances therapeutic efficacy of mesenchymal stromal cells in treatment of acute myocardial infarction

摘要

摘要:
目的研究核干细胞因子（nucleostemin，NS）过表达对脂肪来源间充质干细胞（ADSC）移植后的生存和心肌保护作用的影响。
方法分离培养小鼠ADSC，分别转染NS腺病毒（ADSC-NS）和对照腺病毒（ADSC-Con），CM-DiI标记ADSC。制作小鼠心肌梗死（MI）模型，随机分为以下4组：①Sham组（与MI相同操作但不结扎冠脉），②MI+vehicle组（冠脉结扎后，在左心室前壁、侧壁和后壁立即心肌点注射总量20 μl PBS），③MI + ADSC-Con组（心肌点注射20 μl含2 × 10⁵新鲜ADSC-Con的悬液），④MI + ADSC-NS组（心肌点注射20 μl含2 × 10⁵的新鲜ADSC-NS的悬液）。MI 3 d后，免疫荧光和流式检测ADSC存活率，TUNEL法检测心肌细胞凋亡。MI 4周后，超声心动图检测心脏结构和功能，MASSON-三色染色测定MI面积，实时定量PCR检测心房钠尿肽（ANP）和脑钠尿肽（BNP）表达水平。在细胞水平将ADSC分为以下各组：①对照腺病毒（ADSC-Con）组，②NS腺病毒（ADSC-NS）组，③对照腺病毒 + 对照（ADSC-Con + Con）组，④NS腺病毒+对照（ADSC-NS + Con）组，⑤对照腺病毒+双氧水（ADSC-Con + H₂O₂）组，⑥ NS腺病毒+双氧水（ADSC-NS + H₂O₂）组。检测NS过表达对ADSC增殖、迁移和抗凋亡作用的影响。
结果 MI 3 d后，与ADSC-Con相比，ADSC-NS显著增加ADSC移植后在心肌组织中的存活率（P < 0.05），显著降低心肌细胞凋亡率（P < 0.05）。MI 4周后，ADSC-NS移植显著增加左心室射血分数，降低左心室内径，降低MI面积和ANP、BNP的mRNA表达。在细胞水平，NS过表达促进ADSC增殖和抗凋亡作用，对ADSC的迁移无显著影响。
结论 NS过表达通过增加ADSC的增殖和抗凋亡能力，增强ADSC移植对心脏的保护作用。

Abstract:
AIM To investigate the effects of nucleostemin (NS) on the survival and cardioprotective effects of adipose tissue-derived mesenchymal stromal cells (ADSC).
METHODS ADSC obtained from C57BL/6J mice were transfected with adenovirus harboring NS (ADSC-NS) or control (ADSC-Con). Mice were subjected to myocardial infarction (MI) or sham operations. Immediately after coronary artery occlusion, MI mice were intramyocardially injected with ADSC-NS (2 × 10⁵ fresh ADSC-NS per mouse) or ADSC-Con (2 × 10⁵ fresh ADSC-Con per mouse), both of which were stained with CM-DiI dye before transplantation. The survival of ADSC and cardiomyocyte apoptosis were determined 3 days after MI. Cardiac functions, fibrosis, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA expressions were evaluated 4 weeks after MI. In the in vitro studies, cultured ADSC were subjected to the following six groups: (1) ADSC-Con, (2) ADSC-NS, (3) ADSC-Con + Control, (4) ADSC-NS+Control, (5) ADSC-Con + H₂O₂, (6) ADSC-NS + H₂O₂. ADSC proliferation, migration and anti-apoptosis were investigated.
RESULTS Both immunostaining and flow cytometric analyses showed that ADSC-NS significantly increased (P < 0.05) ADSC survival in myocardial peri-infarcted area 3 days after injection as compared to ADSC-Con. In addition, ADSC-NS treatment significantly (P < 0.05) reduced cardiomyocyte apoptosis. Echocardiographic studies showed that ADSC-NS, not ADSC-Con, significantly improved the left ventricular ejection fraction (LVEF) 4 weeks after MI. ADSC-NS transplantation significantly mitigated MI-induced fibrosis. In vitro studies showed that NS over-expression significantly increased ADSC growth and reduced H₂O₂-induced ADSC apoptosis.
CONCLUSION NS over-expression increases ADSC survival and cardioprotection.

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