Chinese Journal of Tissue Engineering Research ›› 2015, Vol. 19 ›› Issue (33): 5332-5337.doi: 10.3969/j.issn.2095-4344.2015.33.017

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MPC30-DEA70-loaded transforming growth factor beta1 antisense oligonucleotide for transfection of cardiomyocytes 

Yang Yu1, Zhang Min1, Xu Jian-rong1, Lin Xue-feng1, Zhao Xia1, Wang Zhi-rong1, Cao Xi-chuan2, Zhang Zhuo-qi1   

  1. 1Department of Cardiology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221006, Jiangsu Province, China; 2School of Materials Science and Engineering, China University of Mining and Technology, Xuzhou 221116, Jiangsu Province, China
  • Online:2015-08-13 Published:2015-08-13
  • Contact: Zhang Zhuo-qi, Department of Cardiology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221006, Jiangsu Province, China
  • About author:Yang Yu, Master, Associate chief physician, Department of Cardiology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221006, Jiangsu Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 30800219, 30670557

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Abstract:

BACKGROUND: Currently, antisense oligonucleotides (AS-ODN) have a good prospect in gene therapy, but AS-ODN with small molecular weight cannot easily enter into the cells, which is susceptible to nuclease degradation. Therefore, there is still a lack of fundamental understanding about how to improve their transfection efficiency, and target-based transferring.

OBJECTIVE: To investigate whether a weak cationic and phosphorylcholine-containing diblock copolymer (MPC30-DEA70) can act as a carrier system to deliver a chemically synthesized transforming growth factor-β1 (TGF-β1) AS-ODN into myocardial cells.

METHODS: MPC30-DEA70 was compounded with TGF-β1 AS-ODN at various N/P ratios and the MPC30-DEA70/TGF-β1 AS-ODN complexes were characterized by DNA electrophoresis. MTT assay was used to observe the biocompatibility. Confocal laser scanning microscope was used to observe the distribution and location of MPC30- DEA70/TGF-β1 AS-ODN in cells. Flow cytometry was used to detect the transfection efficiency and fluorescence intensity of MPC30-DEA70/TGF-β1 AS-ODN in cells. Western blot and RT-PCR methods were employed to measure the expression of TGF-β1 in cells.

RESULTS AND CONCLUSION: Cell growth inhibition showed that the MPC30-DEA70 had low cytotoxicity to myocardial cells within the effective transfection dosage range (< 20 mg/L). Data from the flow cytometry test indicated a clear trend of increasing transfection efficiency with the increasing of N/P ratios. At high N/P ratios, the expression levels of TGF-β1 mRNA and protein in myocardial cells were significantly lower. This study shows that MPC30-DEA70 can work as an effective transgenic vector in myocardial cells. TGF-β1 AS-ODN can silence the expression of TGF-β1 gene efficiently and specially, and may antagonize TGF-β1-mediated biological function.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Transforming Growth Factor beta1, Oligonucleotides, Antisense, Transfection, Myocytes, Cardiac, Genes

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