[Study of myoblast culture and myoblast transfer therapy in dystrophic mice].

Abstract

In this report, we study the suitable conditions for myoblast cultures through analysis of myoblast growth and differentiation, and then try to develop a mouse model for myoblast transfer therapy (MTT). Recently, some research has indicated that Muscular Dystrophy Murine Mice (MDX) have an X-linked recessive dystrophin deficiency which is caused by dystrophin gene point mutation at the X chromosome. Therefore, MDX mice are usually used for MTT models of muscular dystrophy disease. Control mice, C57BL10/SCSN (B-10) were chosen as a source of normal myoblasts. Myoblasts isolated from the hindlimb muscle tissues of two- to three-day-old neonatal B-10 mice were cultured in vitro for one to seven days. Through our modifyied techniques of isolation and culturing conditions, a myoblast purity of 70% could be achieved, with fibroblast the only contaminating cell type. The proliferative capacity and the doubling time of myoblasts were counted from analysis of growth kinetics. While differentiative capacity was analyzed morphologically, we found the fusion of myoblasts was time-dependent. Immunostaining myoblasts of different stages with anti-dystrophin antibody showed that purified myoblasts with the capacity of fusion can express dystrophin and can be utilized as a donating source in MTT. In the MTT experiment, eight young MDX mice were injected with normal myoblasts at a concentration of 1 x 10(6) cells. All transplated mice received daily cyclosporine A injection for immunosuppression. Two to three months later, dystrophin was found in the myoblast-transferred muscles while staining immunocytochemically. The result suggests that we successfully transferred the normal dystrophin gene from the normal myoblasts into the MDX mice since their myoblast-injected muscle could express dystrophin.