MPC30-DEA70-loaded transforming growth factor beta1 antisense oligonucleotide for transfection of cardiomyocytes
10.3969/j.issn.2095-4344.2015.33.017
- VernacularTitle:磷酸胆碱聚合物MPC30-DEA70负载转化生长因子β1AS-ODN转染心肌细胞
- Author:
Yu YANG
;
Min ZHANG
;
Jianrong XU
;
Xuefeng LIN
;
Xia ZHAO
;
Zhirong WANG
;
Xichuan CAO
;
Zhuoqi ZHANG
- Publication Type:Journal Article
- Keywords:
Transforming Growth Factor beta1;
Oligonucleotides,Antisense;
Transfection;
Myocytes,Cardiac;
Genes
- From:
Chinese Journal of Tissue Engineering Research
2015;(33):5332-5337
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Currently, antisense oligonucleotides (AS-ODN) have a good prospect in gene therapy, but AS-ODN with smal molecular weight cannot easily enter into the cels, which is susceptible to nuclease degradation. Therefore, there is stil 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 chemicaly synthesized transforming growth factor-β1 (TGF-β1) AS-ODN into myocardial cels. 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.
