TNF对离体培养肌管蛋白降解率的影响及其机制的研究

EFFECTS OF TUMOR NECROSIS FACTOR ON DEGRADATION OF LONG-LIVED PROTEIN IN CULTURED MYOTUBES

目的 研究肿瘤坏死因子α(TNF α)对体外培养的骨骼肌肌管内长寿命蛋白降解的影响及其可能的作用机制。方法 通过无菌分离Wistar大鼠(2日龄)下肢肌肉 ,组织块培养法增殖、传代纯化成肌细胞 ,融合形成肌管 ,使用L [3,5 3 H] 酪氨酸标记肌管蛋白后 ,随机分成四组 :对照组和TNF组分别应用不含TNF和含有TNF α(2 0 0 0U/mL)的培养液培养肌管 12、2 4、36和 4 8h。MG132组和TNF +MG132组则分别使用含蛋白酶体抑制剂MG132 (5 0 μmol/L)或含有MG132 (5 0 μmol/L) +TNF α(2 0 0 0U/mL)的培养液培养肌管2 4h。应用液体闪烁计数仪测定培养液和细胞内L [3,5 3 H] 酪氨酸的含量 ,计算肌管长寿命蛋白的降解率。结果 长寿命蛋白降解率TNF组各时间点均较对照组显著增加(P <0 0 1) ,MG132组较对照组显著降低(P <0 0 1) ,TNF +MG132组显著低于TNF组 ,差异有显著性意义 (P <0 0 1)。结论 泛素 蛋白酶体途径是骨骼肌肌管长寿命蛋白的重要降解途径之一 ;TNF α能作用于骨骼肌肌管 ,增强蛋白降解率 ;泛素蛋白酶途径是TNF

Objective To study the effects of tumor necrosis factord (TNF α)on degradation of long lived protein in cultured myotubes, myoblasts were proliferated in tissue block culture and fused into myotubes. Methods Then the protein in myotubes was radiolabelled with L [3,5 3 H] tyrosine. Myotubes were either cultured with TNF α 2000U/mL or without TNF α, and 12h, 24h, 36h, 48h later, the amounts of L [3,5 3 H] tyrosine in culture medium and cells were determined, and the degradation rates of long lived protein were calculated. Other myotubes were cultured either with 50μmol/mL proteasome inhibitor MG132 or 50μmol/mL MG132 and TNF α 2000U/mL, and long lived proteolytic rates were calculated by the same method after 24h culture. Results The long lived proteolytic rates in myotubes cultured with TNF α were increased significantly at all time points compared with control group ( P <0 01). The long lived proteolytic rates in myotubes cultured with MG132 was decreased significantly compared with control group ( P <0 01). The long lived proteolytic rates in myotubes cultured with MG132 and TNF α were notably lower than that in TNF group ( P <0 01). Conclusions The ubiquitin proteasome system was one of the important pathways for degradation of long lived protein in cultured myotubes. TNF α conld directly act on myotubes and increase the proteolytic rates. The ubiquitin proteasome pathway was one of the important pathways that mediated the high proteolytic rate of myotubes induced by TNF α