MyoD-EGFP融合蛋白発現遺伝子導入による線維芽細胞の筋細胞再分化過程の光学的定量分析

Fibroblast is the mesenchymal cell that develops connective tissues of many organs. It can be cultured easily, but it scarcely redifferentiate. As it is known that fibroblast C3H/10T1/2 redifferentiates into muscle cell though in low efficiency, we studied the redifferentiation of various fibroblasts Balb/3T3, C3H/10T1/2, Balb/3T12-3, or C3H/MCA cell line by using MyoD-EGFP chimeric protein expression with transfection of the cDNA. And we also analyzed the qualitative characters of redifferentiation by the expressed MyoD-EGFP chimeric protein with that by MyoD protein derived from a transfected 2-cystronic vector that expressed both MyoD and EGFP respectively. Myogenesis of these mouse embryonic fibroblasts was analyzed by myosine immunochemical staining or L-type Ca2+ channel recording. And we further studied whether 5-azacytidine had some effect on the redifferentiation into myogenic cells. These transfected fibroblasts first fused together and then differentiated into myoblasts. The cell line, that is known to be more tumorigenic such as Balb/3T12-3 or C3H/MCA, fused more effectively than that of Balb/3T3 or C3H/10T1/2. The fused cell, whose original cell line was more tumorigenic, was also differentiated more effectively. It was also observed that DNA demethylation in fibroblast Balb/3T12-3 by 5-azacytidine effectively facilitated the redifferentiation into myotube. The molecular mechanism, that explains the facts that the effective redifferentiation relies on high tumorigenicity and gene demethylation of the fibroblast, must be studied further. [Jpn J Physiol 54 Suppl:S80 (2004)]