干扰HNRNPAB对牛骨骼肌卫星细胞增殖与分化的作用研究

Effects of Interfering HNRNPAB on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells

为了研究核不均一核糖核蛋白AB(heterogeneous nuclear ribonucleoprotein AB,HNRNPAB)对牛骨骼肌卫星细胞增殖与分化的影响。本研究以体外分离培养的鲁西黄牛胎牛原代骨骼肌卫星细胞为试验材料,体外诱导成肌分化,分别收取分化前和分化后第1、2、3天的细胞,提取RNA(每组设置4个重复)或蛋白质(每组设置3个重复),通过qRT-PCR、Western blot检测HNRNPAB在成肌分化前后的表达情况。随后合成HNRNPAB的干扰RNA,并转染牛骨骼肌卫星细胞干扰HNRNPAB的表达,设置阴性对照组;在试验组和对照组中,分别利用EdU试验检测细胞增殖情况,qRT-PCR技术、Western blot技术检测HNRNPAB、增殖标志因子Pax7、Cyclin D1及分化标志因子MyoG、MyHC的mRNA表达水平及蛋白表达水平。结果显示,HNRNPAB在牛骨骼肌卫星细胞成肌分化过程中的mRNA表达呈现先升后降的趋势,在分化第1天表达量最高,分化前后蛋白表达水平也具有显著差异。干扰HNRNPAB后,EdU细胞阳性率极显著上升(P<0.01),增殖标志因子Pax7、Cyclin D1的mRNA水平与对照组相比极显著下降(P<0.01),Pax7蛋白表达水平极显著上升(P<0.01);与对照组相比,分化标志因子MyoG、MyHC的mRNA表达水平极显著升高(P<0.01),蛋白表达水平显著升高(P<0.05)。本研究结果表明,干扰HNRNPAB后降低了牛骨骼肌卫星细胞增殖标志因子的转录水平,提高了Pax7的蛋白表达水平,并促进牛骨骼肌卫星细胞的成肌分化过程。

The study aimed to explore the effect of HNRNPAB(heterogeneous nuclear ribonucleoprotein AB)on the proliferation and differentiation of bovine skeletal muscle satellite cells.In this study,primary skeletal muscle satellite cells of Luxi cattle fetus isolated and cultured in vitro were used as experimental materials.The myogenic differentiation in vitro was induced,and the cells before differentiation and on day 1,2,and 3 after differentiation were obtained.RNA(4 replicates per group)or protein(3 replicates per group)were extracted,qRT-PCR and Western blot were used to detect the expression of HNRNPAB before and after myogenic differentiation.Then the interfering RNA of HNRNPAB was synthesized and transfected into bovine skeletal muscle satellite cells to interfere with the expression of HNRNPAB,and a negative control group was set up;In the experimental group and the control group,EdU was used to detect cell proliferation,qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of HNRNPAB,proliferation marker factor Pax7,Cyclin D1 and differentiation marker factor MyoG and MyHC.The mRNA expression of HNRNPAB in the process of myogenic differentiation of bovine skeletal muscle satellite cells showed a trend of first increase and then decrease,the highest expression was on the first day of differentiation,and the protein expression levels before and after differentiation were also significantly different.After interfering with HNRNPAB,the positive rate of EdU cells increased significantly(P<0.01).The mRNA levels of proliferation marker factor Pax7 and Cyclin D1 significantly decreased compared with the control group(P<0.01).The protein expression level of Pax7 increased significantly(P<0.01).Compared with the control group,the mRNA expression levels of differentiation marker factor MyoG and MyHC were significantly increased(P<0.01),and the protein expression levels of them were also significantly increased(P<0.05).The results of this study showed that interfering with HNRNPAB decreased the transcription level of the proliferation marker factors of bovine skeletal muscle satellite cells,increased the protein expression level of Pax7,and promoted the myogenic differentiation process of bovine skeletal muscle satellite cells.