5-氮杂胞苷对骨髓间充质干细胞向肌细胞增殖及分化的影响

Influences of 5-azacytidine on mesenchymal stem cells proliferation and differentiation into myoblasts

目的:探讨在不同浓度5-氮杂胞苷(5-Aza)诱导间充质干细胞(MSCs)分化为成肌细胞过程中, 其对干细胞增殖及分化的影响。方法:分离纯化鉴定4周龄Wistar大鼠骨髓MSCs,以不同浓度5-Aza作用,用四唑盐(MTT)法测定细胞生长活力,观察细胞形态变化,通过RT-PCR及电泳测定诱导后大鼠的MSCs中骨骼肌肌动蛋白的表达。结果:0和1 μmol·L-1 5-Aza对细胞增殖无明显影响;随着5-Aza浓度的增高,MSCs 的增殖逐渐减弱;20～30 μmol·L-1 5-Aza对细胞有毒性作用,影响其增殖。3和6 μmol·L-1 5-Aza,MSCs有骨骼肌肌动蛋白的表达;9和12 μmol·L-1 5-Aza,MSCs有骨骼肌肌动蛋白的表达,该浓度作用9 d后有部分细胞体明显增粗,12 d有肌管样细胞出现。结论:5-Aza影响干细胞分化,调控基因的表达及调控MSCs向肌细胞定向分化为肌管样细胞的过程。

Objective To investigate the influences of 5 azacytidine （5-Aza） with different concentrations on mesenchymal stem cells （MSCs） proliferation and differentiation into myoblasts. Methods Bone marrow-derived MSCs of 4 weeks Wistar rats were separated and purified, and then treated with 5-Aza with different concentrations. The growth ability of cell was assayed with methyl thiazolyl tetrazolium （MTT） method. The expression of skeletal muscle actin in MSCs was determined with reverse transcription polymerase chain reaction （RT-PCR） and electrophoresis after MSCs were induced. Results The cell proliferation was not affected by 0 and 1μmol·L^-1 5-Aza . There were expressions of skeletal muscle actin treated with 3-12μmol·L^-1 5-Aza. Some cells were obviously enlarged at the 9th day after induction and myotubu-like cells were found at the 12th day when treated with 9 and 12μmol·L^-1 5-Aza. 20 - 30 μmol·L^-1 5-Aza induced the toxic effect on proliferation of MSCs. With the increase of concentration, the proliferation ability of MSCs was weakened. Conclusion 5-Aza affects the expression of regulatory gene to the stem cells and regulate MSCs to orientationally differentiate into myotube-like cells.