Tolerance of rat bone marrow mesenchymal stem cells overexpressing human heme oxygenase 1 to ischemia/hypoxia injury

doi:10.3969/j.issn.2095-4344.2017.29.005

Abstract

Abstract:

BACKGROUND: Under ischemia/hypoxia microenvironment, very low survival rate of transplanted bone marrow mesenchymal stem cells (BMSCs) in the host limits its efficacy in the treatment of acute myocardial infarction.
OBJECTIVE: To explore the tolerance of human heme oxygenase 1 (hHO-1) gene modified rat BMSCs to ischemia/hypoxia injury.
METHODS: The rat BMSCs were transfected with hHO-1 recombinant adenovirus. Western blot assay was used to determinate the optimal time of hHO-1 protein expression. hHO-1 modified rat BMSCs were cultured in hypoxia and serum-free conditions that simulated ischemia/hypoxia microenvironment in vivo. Cell counting kit-8 and trypan blue staining were performed to detect the survival rate of BMSCs at 12, 24, 48, 72 hours after culture under the ischemia/hypoxia microenvironment. Flow cytometry was used to detect apoptosis in BMSCs at 24 hours after culture under the ischemia/hypoxia microenvironment.
RESULTS AND CONCLUSION: The expression of hHO-1 protein was highest at 4 days after transfection. Under the ischemia/hypoxia microenvironment for 12, 24, 48, 72 hours, the survival rates of transfected BMSCs were significantly higher as compared with the untransfected cells (P < 0.05), shown by the cell counting kit-8 and trypan blue staining. In addition, the results from flow cytometry showed that there was a higher survival rate of transfected BMSCs than untransfected cells at 24 hours of culture under the ischemia/hypoxia microenvironment (P < 0.05). To conclude, hHO-1 modified rat BMSCs have stronger tolerance to the ischemia/hypoxia microenvironment.

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: Bone Marrow, Mesenchymal Stem Cells, Heme Oxygenase-1, Adenoviridae Infections, Tissue Engineering

CLC Number:

R394.2

Deng Ning-bo, Han Teng-long, Zeng Yuan-qing, Jiang Zhi-xin. Tolerance of rat bone marrow mesenchymal stem cells overexpressing human heme oxygenase 1 to ischemia/hypoxia injury[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(29): 4617-4622.

Figures/Tables 1

2.1 骨髓间充质干细胞鉴定结果经流式细胞仪检测，结果显示第3代骨髓间充质干细胞的特异性表面标志物CD45阴性，阳性率为0.5%(图1A2)；CD29阳性，阳性率为98.0%(图1A3)；CD90阳性，阳性率为99.2%(图1A4)。成脂诱导3周后，骨髓间充质干细胞有脂滴形成，油红O染色呈红色(图1B2)，成骨诱导3周后，骨髓间充质干细胞有钙结节形成，茜素红染色呈红色(图1C2)。 2.2 hHO-1蛋白表达空白组(未转染腺病毒)及对照组(转染空载质粒腺病毒)未见明显条带，HO-1组(转染hHO-1重组腺病毒)随着时间延长目的蛋白的表达量增加，在第4天达到最大表达量(图2)。 2.3 锥虫蓝染色法检测细胞存活率正常培养条件下的对照组和HO-1组的生存率分别为(98.02±0.78)%，(98.33± 0.77)%(n=30，t=-1.535，P=0.130)，差异无显著性意义。缺血缺氧培养12，24，48，72 h的对照组和HO-1组的生存率分别为(70.37±2.28)% vs. (82.32±1.61)%(n=30，t=-23.358，P=0.000)，(49.41±3.27)% vs. (60.90±2.58)% (n=30，t=-15.107，P=0.000)，(20.93±2.67)% vs. (25.17± 2.61)%(n=30，t=-6.182，P=0.000)，(13.60±2.21)% vs. (15.20±1.74)%(n=30，t=-3.121，P=0.003)，差异有显著性意义(图3)。 2.4 CCK-8法检测细胞存活率对照组和HO-1组缺血缺氧12 h生存率分别为(79.32±1.26)%，(89.31±2.06)% (n=60，t=-32.030，P=0.000)，缺氧24 h生存率分别为(55.95±2.27)%，(67.73±2.38)%(n=60，t=-27.734，P=0.000)，缺血缺氧48 h生存率分别为(22.69±1.54)%，(27.24±1.80)%(n=60，t=-14.858，P=0.000)，缺血缺氧72 h生存率分别(13.15±1.30)%，(18.06±1.89)% (n=60，t=-16.554，P=0.000)，差异均有显著性意义(图4)。 2.5 流式细胞术检测细胞凋亡阴性对照生存率为(97.39±1.15)%(图5A)，缺血缺氧24 h后对照组及HO-1组生存率分别为(52.72±4.66)%，(62.15±3.88)%(n=27，t= -8.076，P=0.000)(图5B，C)，差异有显著性意义。"

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