ERK1/2信号通路在3种骨肉瘤细胞中的作用和表达水平的研究

Explore the role and expression of ERK1/2 signaling pathway in three kinds of osteosarcoma

目的观察细胞外调节蛋白激酶(ERK1/2)激动剂表皮生长因子(EGF)和抑制剂PD98059对3种骨肉瘤细胞株U2OS、MG63、UMR106的增殖影响以及ERK1/2基因和蛋白在3种细胞株中的表达水平,筛选出ERK1/2基因和蛋白表达最高的骨肉瘤细胞株用于后续实验研究。方法采用细胞计数和CCK8法分别测定U2OS、MG63、UMR106细胞的生长情况,同时使用EGF和PD98059分别干预3种细胞株,观察其对3种骨肉瘤细胞增殖的影响;采用实时荧光定量PCR(RT-qPCR)检测3种细胞株中ERK1/2基因的表达;采用Western-blot检测3种细胞株中ERK1/2蛋白、p-ERK1/2蛋白的表达情况。结果细胞计数和CCK8结果均显示,U2OS和UMR106在正常情况下培养48 h可达最佳生长状态,而MG63细胞的最佳生长状态时间为24 h;EGF在干预细胞24 h和48 h均能使U2OS、MG63、UMR106细胞增殖,而PD98059在干预细胞24 h和48 h均能使3种细胞的增殖受到抑制(P<0.05)。RT-qPCR结果显示在培养48 h后,U2OS细胞中的ERK1/2基因表达水平最高。Western-blot结果显示在培养48 h后,U2OS细胞中ERK1/2和p-ERK1/2的蛋白表达水平最高,UMR106细胞次之,而MG63细胞中ERK1/2和p-ERK1/2的蛋白表达水平最低(P<0.05)。结论U2OS细胞株生长期长,增殖活力强,同时ERK1/2基因、蛋白以及p-ERK1/2蛋白的表达水平高,适合用于骨肉瘤ERK1/2信号通路的研究。

Objective To select osteosarcoma cell lines with the highest expression of ERK1/2 gene and protein for sub-sequent experimental study,by observing the proliferation effects of ERK1/2 agonist(EGF)and inhibitor PD98059 on three 08-teosarcoma cell lines U2OS,MG63 and UMR106 and the expression of ERK1/2 gene and protein in the three cell lines.Methods The growth of U2OS,MG63 and UMR106 cells was measured by cell count and CCK8 method,and the effects of EGF and PD98059 on the proliferation of the three cells were observed.The gene expression of ERK1/2 in three cell lines was detected by RT-qPCR.The protein expression of ERK1/2 and p-ERK1/2 in three cell lines were detected by Western blot.Results Cell count and CCK8 showed that the optimal growth state of U2OS and UMR106 under normal conditions could reach was 48 h,while MG63 cells was 24 h.EGF could induce the proliferation of all three kinds of cells at 24 h and 48 h,while PD98059 could inhibit the proliferation of them at 24 h and 48 h(P<0.05)。RT-qPCR showed that the gene expression level of ERK1/2 in U2OS cells was the highest after 48 h culture.Western:blot showed that the protein expression levels of ERK1/2 and P ERK1/2 in U2OS cells were the highest after 48 h culture,followed by UMR106 cells,and MG63 cells(P<0.05).Conclusion U2OS cell has long growth cycle,strong proliferation activity,and high expression of ERK1/2 gene,protein and pr ERK1/2 protein,suggesting that U2OS cells could be used as the research object in subsequent experiments.