UBE2L3对牛骨骼肌卫星细胞增殖分化的影响

Effect of UBE2L3 on the proliferation and differentiation of bovine skeletal muscle satellite cells

将分离的牛骨骼肌卫星细胞(BSMSCs)进行体外培养,首先检测泛素结合酶UBE2L3在BSMSCs增殖分化过程中mRNA以及蛋白表达水平的变化。设计UBE2L3的3个干扰RNA(si-UBE2L3-1、si-UBE2L3-2、si-UBE2L3-3),对干扰效果进行筛选。构建UBE2L3过表达质粒载体pcDNA3.1-UBE2L3(pcDNA-UBE2L3),观察3个不同si-RNA中效果最好以及测序正确的过表达质粒转染BSMSCs。进行EdU检测干扰以及过表达UBE2L3后对细胞增殖的影响;诱导BSMSCs进行分化,用显微镜观察细胞在生长过程中的情况,并且观察分化过程中形成肌管数量以及直径大小。利用qRT-PCR以及Western blot技术分别从mRNA和蛋白表达水平检测BSMSCs的分化标志因子MyoG以及MyHC表达情况。综合分析泛素结合酶UBE2L3对BSMSCs增殖分化的影响。结果显示:干扰以及过表达UBE2L3后,EdU染色的阳性细胞数与对照组相比差异不显著;将BSMSCs诱导分化,干扰UBE2L3后,肌管的直径与对照组相比显著升高,2个分化标志因子的蛋白表达水平极显著升高(P<0.01);过表达UBE2L3后,肌管的直径以及数量显著小于对照组,2个分化标志因子的蛋白表达水平显著降低。此试验探明干扰或者过表达UBE2L3蛋白对BSMSCs的增殖没有影响,但对其成肌分化过程具有显著影响。干扰UBE2L3的表达会促进BSMSCs在体外成肌分化过程,过表达UBE2L3会抑制BSMSCs在体外的分化过程。研究结果为进一步探究泛素化途径在牛成肌分化中调控机制研究奠定了基础。

To study the effect of ubiquitin binding enzyme UBE2l3 on the proliferation and differentiation of bovine skeletal muscle satellite cells(BSMSCs),the isolated BSMSCs were cultured in vitro.Firstly, the mRNA and protein expression levels of UBE2l3 during the proliferation and differentiation of BSMSCs were detected.Three interfering RNAs(si-UBE2l3-1,si-UBE2l3-2,si-UBE2l3-3) of UBE2l3 were designed to screen the interference effect.The vector pcDNA3.1-UBE2l3(pcDNA-UBE2l3) was constructed and transfected into BSMSCs.Edu was used to detect the effect of interference and overexpression of UBE2l3 on cell proliferation;BSMSCs were induced to differentiate, and the growth process of cells was observed by microscope, and the number and diameter of myotubes formed during differentiation were observed.The expression of MyoG and MyHC were detected by qRT-PCR and Western blot.The effects of UBE2l3 on the proliferation and differentiation of BSMSCs were analyzed.The results showed that after interference and over expression of UBE2l3,there was no significant difference in the number of positive cells compared with the control group;after inducing the differentiation of BSMSCs and interference with UBE2l3,the diameter of myotubes was significantly increased compared with the control group,and the protein expression levels of two differentiation markers were extremely significantly increased(P<0.01);after overexpression of UBE2l3,the diameter of myotubes was significantly increased compared with the control group(P<0.01).The protein expression levels of the two differentiation markers were significantly decreased,indicating that the interference or overexpression of UBE2l3 had no effect on the proliferation of BSMSCs,but had a significant effect on the myogenic differentiation,interference with the expression of UBE2l3 could promote the myogenic differentiation of bovine skeletal muscle satellite cells in vitro.Overexpression of UBE2l3 could inhibit the myogenic differentiation of BSMSCs invitro.The results laid the foundation for further exploring the regulatory mechanism of ubiquitination pathway in bovine myoblast differentiation.