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在肿瘤中,黏蛋白O-糖基化有着重要的生物学功能.控制O-糖基化起始合成的是多肽∶N-乙酰氨基半乳糖转移酶家族,研究该酶家族对阐明O-糖基化在肿瘤中的作用机制有重要的意义.探讨了靶向干扰ppGalNAc-T2基因表达对白血病Jurkat细胞株增殖及迁移的影响.首先合成ppGalNAc-T2特异shRNA干扰及对照序列,将其连接至慢病毒干扰载体YH1;重组载体经双酶切、测序鉴定正确后与包装质粒共转染293T细胞,获得的病毒颗粒经过滤纯化后感染Jurkat细胞,流式细胞分选仪进行细胞分选以获得ppGalNAc-T2基因稳定干扰表达的Jurkat细胞,然后使用RT-PCR和Western blot方法对各组别细胞中ppGalNAc-T2基因表达情况进行分析,以确定ppGalNAc-T2基因表达被有效干扰;进一步利用MTT实验和Transwell实验分析ppGalNAc-T2基因干扰表达对Jurkat细胞增殖及迁移的影响.结果表明,成功构建了靶向干扰ppGalNAc-T2基因表达的慢病毒载体,感染Jurkat细胞后能稳定干扰ppGalNAc-T2基因表达.MTT和Transwell实验研究发现,下调ppGalNAc-T2基因表达对Jurkat细胞增殖和迁移有抑制作用.
In tumors, mucin O-glycosylation has important biological functions.Studies on the initiation of O-glycosylation are the polypeptide: N-acetylgalactosaminyltransferase family, the enzyme family to elucidate the O-sugar The mechanism of the role of the gene in tumorigenesis is of great significance.To investigate the effect of targeted interference with the gene expression of ppGalNAc-T2 on the proliferation and migration of leukemia Jurkat cells.Firstly, ppGalNAc-T2 specific shRNA interference and control sequences were synthesized and ligated to Lentiviral vector YH1; Recombinant vector was double digested and sequenced to identify the correct 293T cells were co-transfected with the packaging plasmid, the resulting virus particles were purified by infection Jurkat cells, flow cytometry to obtain cells sorted The expression of ppGalNAc-T2 gene in Jurkat cells stably interfered with ppGalNAc-T2 gene was analyzed by RT-PCR and Western blot. The results showed that ppGalNAc-T2 gene expression was effectively inhibited by MTT assay And Transwell assay were used to analyze the effect of ppGalNAc-T2 gene expression on the proliferation and migration of Jurkat cells.The results showed that the gene targeting ppGalNAc-T2 gene Viral vector, stably infected Jurkat cells and interfere with gene expression .MTT Transwell experimental study found ppGalNAc-T2, ppGalNAc-T2 gene expression down-regulated inhibition of Jurkat cell proliferation and migration.