胰岛素诱导牛成肌细胞成脂分化的研究

Insulin induced adipogenic differentiation of bovine myoblasts

【目的】建立稳定成熟的牛成肌细胞胰岛素诱导成脂分化体系,为肉牛肉脂品质的改良提供参考。【方法】取1日龄荷斯坦公牛犊后腿肌肉,用Ⅳ型胶原酶消化法分离成肌细胞,用胰岛素诱导成肌细胞成脂分化,以未添加胰岛素的培养基为对照组,检测成肌细胞成脂分化过程中细胞形态的变化,并分析与成肌细胞和脂肪细胞分化相关基因(C/EBPα、C/EBPβ、C/EBPδ、PPARγ、Myf5、MyoD)分别诱导第3,6,9,12,15天相对表达量的变化,分析牛成肌细胞的诱导分化情况。【结果】成功分离培养了牛原代成肌细胞。胰岛素处理组细胞中脂滴的油红O染色明显,C/EBPβ、C/EBPδ、PPARγ和MyoD相对表达量明显增高,C/EBPα和Myf5基因相对表达量则下降;用DMEM高糖培养基+体积分数10%胎牛血清(FBS)培养时,C/EBPs基因相对表达量总体高于DMEM高糖培养基+体积分数20%FBS+体积分数10%马血清(HS)。【结论】成功分离并鉴定了牛成肌细胞,建立了适于牛成肌细胞的体外培养体系,用胰岛素诱导成肌细胞成脂分化效果好。

[Objective] A stable mature bovine myoblasts insulin induced adipogenic differentiation sys- tem was established to improve beef quality. [Method] Hind leg muscles were isolated from 1-day-old calf, and digested with collagenase to obtain the myoblasts, and then adipogenic differentiation was induced by insulin. The medium without adding insulin was taken as the control group. Cell morphology and expres- sion of differentiation associated genes of myoblasts and adipocytes （C/EBPa, C/EBPfl, C/EBPS, PPARγ, My f5 and MyoD） at 3,6,9,12 ,and 15 days after induction were measured and bovine myoblasts trans-dif- ferentiation was analyzed. [Result] We have successfully isolated and cultured the primary bovine myo- blasts. Relative expressions of C/EBP, C/EBPS, PPAR and MyoD in groups treated with insulin were higher than that of control,while relative expressions of C/EBPa and My f5 were lower. Oil red O staining of treated groups was obvious. C/EBPs expression in DMEM-volume fraction 10% FBS group was higher than that in DMEMvolume fraction 20% FBS^volume fraction 10% HS group. [Conclusion] The pres ent study successfully isolated myoblasts from calf and established an optimal in vitro culture system. Adi- pogenic differentiation of myoblasts was successfully conducted as well.