Transfecting hyperpolarization-activated cyclic nucleotide-gated channel 2 gene into porcine bone marrow mesenchymal stem cells
10.3969/j.issn.1673-8225.2009.49.013
- VernacularTitle:超极化激活环化核苷酸门控通道基因转染猪骨髓间充质干细胞
- Author:
Yinglong YAO
;
Hao ZHANG
;
Dejun GONG
;
Zhigang SONG
;
Zhiyun XU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(49):9673-9676
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Hyperpolarization-activated cyclic nucleotide-gated (HCN) current plays an important role in regulating heart spontaneous pulsation.OBJECTIVE: To observe target gene expression and electrophysiological characteristics of pig bone marrow mesenchymal stem cells (BMSCs) transfected with hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) gene recombinant adenovirus.DESIGN, TIME AND SETTING: Cell-gene in vitro study was performed at the Laboratory of Thoracic and Cardiovascular Surgery,Second Military Medical University of Chinese PLA from July 2007 to March 2008.MATERIALS: Yorkshire pig was supplied by Animal Institute, Second Military Medical University of Chinese PLA. HCN2 plasmid was presented by Professor Dario DiFrancesco from Italy. Recombinant adenovirus Ad.HCN2 was constructed and stored using Ad5 in this laboratory.METHODS: Pig BMSCs were isolated with combination of gradient centrifugation of Percoll and adherent treatment in vitro.Ad.HCN2 was transfected at multiplicity of infection=50. We also set non-transfection and transfected Ad.Null groups.MAIN OUTCOME MEASURES: Expression of HCN2 mRNA was detected with RT-PCR, and expression of HCN2 channel protein was examined with immunofluorescent staining. Electrophysiology of HCN2 channel protein was measured with whole-cell patch clamp.RESULTS: No amplified fragments were found in the non-transfection and transfected Ad.Null groups, but amplified fragments were determined at 250-500 bp following Ad.HCN2 amplification, which was the same as plasmid carrying HCN2 gene. Staining strength of cell nuclei following transfection was significantly weaken compared with cell membrane and plasma, which showed identical distribution as HCN2 protein. No HCN2 protein was detected in the non-transfection and transfected Ad.Null groups.Pacemaker current could be recorded with a whole-cell patch clamp. It was fully activated around -140 mV with an activation threshold of -60 mV, presenting voltage dependence. CsCI (4 mmol/L) reversibly blocked the inward currents. No pacemaker current was detected in the non-transfection and transfected Ad.Null groups.CONCLUSION: The HCN2 recombinant adenovirus carrier was transferred into serial subcultivation porcine bone marrow mesenchymal stem cells. HCN2 channel protein has been expressing. Pacemaker current could be recorded with a whole-cell patch clamp.
