The overexpression of miR-378 promotes the therapeutic effects of bone marrow mesenchymal stem cell transplantation on myocardial infarction

doi:10.3969/j.issn.2095-4344.2017.09.015

Abstract

Abstract:

BACKGROUND: Recently, miR-378 has been shown to modulate the anti-hypoxia capacity of bone marrow mesenchymal stem cells (BMSCs) and reduce cell apoptosis under hypoxic conditions.

OBJECTIVE: To investigate the benefits of miR-378-upregulated BMSC transplantation in a rat model of acute myocardial infarction.

METHODS:Primary rat BMSCs were cultured in vitro. Until passage 3, the cells were infected with the lentivirus carrying synthetic miR-378 gene fragments. A rat model for acute myocardial infarction was constructed by ligating the left anterior descending artery. Thereafter, the animals were randomly assigned to three groups: control group (n=10), BMSC^null group (n=16) and BMSC^miR-378 group (n=16). In the latter two groups, 50 μL of normal saline containing 1×10⁷ empty virus-transfected or miR-378-transfected BMSCs was injected into the region of myocardial infarction, respectively. Only 50 μL of normal saline was injected in the control group. Twenty-four hours later, the apoptosis of transplanted BMSCs was evaluated with TUNEL, and expression level of vascular endothelial growth factor and transforming growth factor-β was detected using western blot assay. Four weeks after treatment, the left ventricular function of rats was assessed by echocardiography, and then histological and molecular biology analyses were performed.

RESULTS AND CONCLUSION: At 24 hours postoperatively, there were less apoptotic BMSCs and higher expression levels of vascular endothelial growth factor and transforming growth factor-β in the BMSC^miR-378 group than in the BMSC^null group (n=6, P < 0.001). Four weeks later, there were more transplanted BMSCs and BMSCs-derived cardiomyocytes in the BMSC^miR-378 group than the BMSC^null group (n=10, P < 0.001). Moreover, increased new vessel density (P < 0.001), decreased infarcted area (P < 0.001), preserved left ventricular ejection fraction (P < 0.05), reduced left ventricular end-diastolic volume (P < 0.05) were found in the BMSC^miR-378 group, compared with the other two groups. The above parameters were better in the BMSC^null group than the control group (P < 0.05). Overall, the upregulation of miR-378 could enhance the capability of BMSCs against hypoxia, and consequently promote myocardial repair after implantation, providing a new strategy for cell therapy of myocardial infarction.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Myocardial Infarction, Bone Marrow, Mesenchymal Stem Cell Transplantation, MicroRNAs, Tissue Engineering

CLC Number:

R394.2

Zhang Xiu-min, Yu Bo, Li Xue-yuan. The overexpression of miR-378 promotes the therapeutic effects of bone marrow mesenchymal stem cell transplantation on myocardial infarction[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1390-1396.

Figures/Tables 2

2.2 移植后细胞凋亡率及细胞因子表达移植24 h后，TUNEL染色结果显示(图2A，B)BMSCnull组移植骨髓间充质干细胞凋亡比率为(88.5±6.7)%，而BMSCmiR-378组的细胞凋亡比率明显减低[(74.9±5.4)%，n=6，P < 0.001]，提示miR-378转染明显抑制移植骨髓间充质干细胞的凋亡。Western blotting结果显示(图2C和D)，BMSCmiR-378组血管内皮生长因子和转化生长因子β1的蛋白表达水平明显高于BMSCnull组(n=6，P < 0.001)，提示miR-378转染明显提高移植骨髓间充质干细胞的旁分泌功能。 2.3 移植骨髓间充质干细胞存活与心肌细胞分化移植4周后，免疫荧光染色结果显示(图3A-C)，BMSCmiR-378组中GFP+细胞数量明显高于BMSCnull组[(23.4±3.1)/高倍视野，(7.6±1.7)/高倍视野，n=10，P < 0.001]，提示miR-378转染增加骨髓间充质干细胞存活数量。另外，BMSCmiR-378组中GFP+/cTNT+细胞数量亦明显高于BMSCnull组[(20.6±2.9)/高倍视野，(5.2±1.3)/高倍视野，n=10，P < 0.001]，提示miR-378转染促进骨髓间充质干细胞向心肌细胞分化。 2.4 血管再生与心肌梗死面积评价移植4周后，抗vWF染色结果显示(图4A，B)BMSCmiR-378组新生血管密度明显高于BMSCnull组和对照组[(5 839±353)像素/高倍视野， (4 148±296)像素/高倍视野，(2 314±262)像素/高倍视野，n=10，P均< 0.001]。Masson染色提示(图4A，C)，BMSCmiR-378组心肌梗死面积明显小于BMSCnull组和对照组[(33 716±2 851)像素/高倍视野，(64 288±3 590)像素/高倍视野，(91 626±3 817)像素/高倍视野，n=10，P < 0.001]。与对照组比较，BMSCnull组新生血管密度和心肌梗死面积差异也有显著性意义(P < 0.001)。 2.5 心功能与心室重构术后4周，BMSCmiR-378组左室射血分数[(58±3)%]明显优于BMSCnull组[(50±3)%，n=10，P=0.016]和对照组[(41±2)%，n=10，P < 0.001]；左室舒张末容积[(1.82±0.16) mL]较BMSCnull组[(2.21± 0.19) mL，n=10，P=0.037]和对照组[(2.64±0.22) mL，n=10，P=0.003]亦有明显改善。BMSCnull组与对照组比较也有改善(P < 0.05)，证实了未处理骨髓间充质干细胞的治疗效果。"

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