Background and Objectives; Thymosin beta 4 (T棺4), a small acidic peptide isolated originally from calf thymus, was postulated to be a thymic hormone at first. Recent studies showed that T棺4 repairs damaged myocardium via promotion of cardiomyocyte (CMC) migration, survival and reduction of apoptosis, inflammation. But there are very limited data the relation between survival signal and T棺4 in the heart. Therefore, we investigated effects of T棺4 gene therapy in the aspect of survival signal in rat CMCs in hypoxic condition.
Methods; The effects of Lentiviral-T棺4 on hypoxia injury were examined using rat neonatal CMCs (rCMCs). We ligated T棺4 with Lentiviral vector using multi-cloning site, and produced by Lenti-virus for gene transfer using calcium phosphate method at 293T cell. Primary cultures of rCMCs were prepared from the ventricles of 1-day-old Sprague-Dawley rats. rCMCs (3��10⁵ cells) were infected by Lenti-T棺4 (1.6��10⁶ IU) or Lenti-GFP as control and then left in hypoxia using GasPak for 12 hours. Apoptosis severity was evaluated by FACS analysis with Annexin-���-phycoerythrin staining. And also, proteins (e.g. T棺4, p-ERK/ERK, p-Akt/Akt, Bcl-2/Bax, survivin) related with cell survival signal were measured by Western blot.
Result; More than 90% of rCMCs expressed GFP in confocal laser microscope. FACS analysis showed that 17.62% decrease of apoptosis in rCMCs/Lenti-T棺4 group compared with control group (Lenti-GFP; 57.34%, Lenti-T棺4; 47.24%) (Table). Also, the expressions of T棺4, p-ERK/ERK, p-Akt/Akt, Bcl-2/Bax, survivin were significantly enhanced at rCMCs/Lenti-T棺4 group in Western blot (Figure).
Conclusion;With there results, T棺4 has considerable influence on CMC survival in hypoxic damage via cell survival signals. Animal implication of T棺4 in ischemic injured myocardium is on the way.