摘要
为了研究改变泛素化蛋白水平对牛骨骼肌卫星细胞体外增殖与成肌分化的影响,试验利用牛骨骼肌卫星细胞体外诱导成肌分化模型,采用1.25μmol/L、2.5μmol/L、5μmol/L、10μmol/L的蛋白酶体抑制剂MG132处理改变牛骨骼肌卫星细胞泛素化蛋白水平,同时设空白对照组和DMSO对照组,通过显微镜观察牛骨骼肌卫星细胞的密度,利用Western-blot技术检测牛骨骼肌卫星细胞蛋白泛素化水平的变化,综合筛选MG132的最佳处理浓度;然后通过EdU增殖检测法检测MG132对牛骨骼肌卫星细胞增殖的影响;最后通过显微镜观察牛骨骼肌卫星细胞分化时期的肌管形成状态,利用Western-blot技术检测牛骨骼肌卫星细胞分化标志因子MyHC的表达情况,综合分析MG132对牛骨骼肌卫星细胞成肌分化的影响。结果表明:牛骨骼肌卫星细胞经不同浓度MG132处理后,空白对照组和DMSO对照组之间细胞密度和蛋白泛素化水平没有明显变化,添加MG132后蛋白泛素化水平明显升高,随着添加浓度的增加对牛骨骼肌卫星细胞生长的抑制作用逐渐增强,综合分析选用1.25μmol/L的MG132进行细胞增殖和分化调控研究;经MG132处理后,牛骨骼肌卫星细胞EdU阳性细胞率显著低于DMSO对照组(P<0.05),诱导分化后的牛骨骼肌卫星细胞形成的肌管数量呈增多趋势,成肌分化标志因子MyHC的表达显著高于DMSO对照组(P<0.05)。说明MG132可以抑制牛骨骼肌卫星细胞的增殖并促进牛骨骼肌卫星细胞的分化。
To investigate the effect of ubiquitinated protein on proliferation and myogenic differentiation of bovine skeletal muscle satellite cells(MSCs) in vitro, the myogenic differentiation model of bovine MSCs in vitro was established, a blank control group and a DMSO control group were set up,and the effects of proteasome inhibitor MG132(1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L) on the level of ubiquitinated protein in bovine MSCs were studied. We observed the density of bovine MSCs by microscope, detected the changes in the protein ubiquitination level of bovine MSCs by Western-blot, and comprehensively screened the best concentration for MG132 treatment. Then, the effect of MG132 on the proliferation of bovine MSCs was detected by EdU proliferation assay. Finally, the myotube formation state during the differentiation period of bovine MSCs was observed by microscope, the expression of differentiation marker factor MyHC in bovine MSCs was detected by Western-blot, and the effect of MG132 on myogenic differentiation of bovine MSCs were analyzed. The results showed that there was no significant change in cell density and protein ubiquitination level between the blank control group and the DMSO control group. After adding MG132, the protein ubiquitination level increased significantly, and with the increase of MG132 concentration, the inhibitory effect on the growth of bovine MSCs was gradually increased. By comprehensive analysis, 1.25 μmol/L MG132 was selected for cell proliferation and differentiation regulation. After treatment with MG132, the rate of EdU positive cells in bovine MSCs was significantly lower than that in DMSO control group(P<0.05), the number of myotubes formed by induced differentiation of bovine MSCs was increased, and the expression of myogenic differentiation marker MyHC was significantly higher than that in the DMSO control group(P<0.05). These results indicated that MG132 could inhibit the proliferation of bovine MSCs and promote the differentiation of bovine MSCs.
作者
朱菲菲
李燕
张林林
李新
郭益文
郭宏
丁向彬
ZHU Feifei;LI Yan;ZHANG Linlin;LI Xin;GUO Yiwen;GUO Hong;DING Xiangbin(College of Animal Science and Veterinary Medicine/Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,Tianjin Agricultural University,Tianjin 300384,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2020年第15期18-22,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31772559)
天津市自然科学基金项目(18JCYBJC29700)
天津市高校“中青年骨干创新人才培养计划”人选项目(J01009030710)。
