Role and gene transfer of Intercellular signal transduction (gap junction)

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10671244
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Thoracic surgery
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: WATANABE Masazumi Tokyo Medical and Dental Univ. Faculty of Medicine, Research Associate, 助手 (10282758)
• Project Period (FY): 1998 – 1999
• Keywords: Gap junction / connexin 43 / gene transfer / adenovirous vector / p16

Research Abstract

1) Gap junctions are composed of narrow membrane channels that serve as conduits for direct cell-to cell transfer of small molecules and ions. Intercellular communication via these gap junctional channels have been proposed to contribute to diverse physiological processes including tissue homeostasis, growth regulation and development. We therefore examined the aged-related changes in the rat hearts.
Methods and Results : Connexin 43, myocardial gap junction protein, was investigated in four groups (6, 30 weeks and 20, 30 months : n=6/group) by quantitative immuno-histochemical localization and electron microscopic study. Quantification of immuno-histochemical localization and westernblotting showed that obvious differences in the number and pattern of connexin 43 bands were noticed between 6, 30 weeks and 20, 30 months.
Conclusion : These age-related changes will need to be included as a potentially important factor for the incidence of arrthythmias and the mechanical, chemical distribu tion in the aged heart.
2) In the present study, we have assessed the efficiency and stability of gene transfer in the rat heart by the novel method.
Methods and Results : The E1E3-deleted adenovirous containing coding sequences of β-galactosidase, (AxCALacZ) were used in this experiment. 10-week-old SD male rats were anesthetized, intubated, and mechanically ventilated. Ascending aorta and pulmonary artery were snared. A 24-gage needle was placed in the ascending aorta through the apex. AxCALacZ (4x10ィイD110ィエD1pfu/ml) in a volume of 100 μL was injected with the clamping the ascending aorta and pulmonary artery for 10 seconds. After 24 hours and 2 weeks of operation, the hearts were resected and stained for LacZ. Western blotting was also done. Gross observation revealed that apparent histochemical expression of β-galactosidase in more than 90% of the area of LV of the heart was detected at 24 hours and 2 weeks after injection.
Conclusion : We have succeeded in establishing an efficient and stable gene transfer method into the heart by adenovirous vectors in rats. This method may provide an effective and useful tool for research and therapy of heart diseases.