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目的:运用基因重组技术构建人MAL-AT-1基因种属同源序列(Species homologous frag-ments/shf)重组真核荧光表达载体,并探讨其在MALAT-1高表达的人鼻咽癌细胞株C666-1中的表达和对其侵袭转移的影响。方法:应用RT-touchdown PCR的方法,从鼻咽癌患者正常切缘组织中扩增出MALAT-1基因的种属同源序列,将所得的cDNA序列定向克隆至真核表达载体pEGFP-C1质粒中,构建出重组真核荧光表达载体;采用脂质体Lipofectamine 2000介导重组质粒pEGFP-C1-MALAT-1/shf瞬时转染MALAT-1高表达的人鼻咽癌细胞株C666-1;通过实时荧光定量PCR方法检测MALAT-1基因种属同源序列转染组和空载体转染组细胞中MALAT-1/shf的表达水平;细胞基质胶侵袭实验研究转染组细胞侵袭能力的变化。结果:经酶切鉴定及测序分析,证实成功构建人MALAT-1/shf重组真核荧光表达载体。pEGFP-C1-MALAT-1/shf(6 918~8 441 bp)转染组平均侵袭细胞数(51.20±5.933)显著高于MALAT-1/mock组(13.40±3.847),差异具有统计学意义(P=0.000)。这说明转染pEGFP-C1-MALAT-1/shf(6 918~8 441 bp)重组质粒后,细胞的侵袭能力明显增强。结论:成功构建了人MALAT-1基因种属同源序列真核荧光表达载体pEGFP-C1-MALAT-1/shf,并成功在MALAT-1高表达的人鼻咽癌细胞株C666-1中表达,MALAT-1/shf(6 918~8 441 bp)与人鼻咽癌C666-1细胞株的侵袭转移密切相关。
OBJECTIVE: To construct a recombinant eukaryotic expression vector of human MAL-AT-1 gene homologous sequences (shy) by using gene recombination technique and to explore its expression in human nasopharyngeal carcinoma cells with high MALAT-1 expression Expression of strain C666-1 and its effect on invasion and metastasis. Methods: The species homologous sequences of MALAT-1 gene were amplified from the normal marginal tissues of patients with nasopharyngeal carcinoma by RT-touchdown PCR. The obtained cDNA sequence was cloned into the eukaryotic expression vector pEGFP-C1 The recombinant plasmid pEGFP-C1-MALAT-1 / shf was transfected into human nasopharyngeal carcinoma cell line C666-1 with high expression of MALAT-1 by Lipofectamine 2000. The expression of MALAT-1 / shf in MALAT-1 gene-transfected and empty vector transfected cells was detected by real-time fluorescent quantitative polymerase chain reaction (MALAT-1) Results: The recombinant plasmid of human MALAT-1 / shf eukaryotic expression vector was confirmed by restriction enzyme analysis and sequencing analysis. The average number of invasive cells (51.20 ± 5.933) in pEGFP-C1-MALAT-1 / shf (6 918-8 441 bp) transfection group was significantly higher than that in MALAT-1 / mock group (13.40 ± 3.847), the difference was statistically significant P = 0.000). This shows that the transfected pEGFP-C1-MALAT-1 / shf (6 918 ~ 8 441 bp) recombinant plasmid, the cell invasion was significantly enhanced. CONCLUSION: The eukaryotic expression vector pEGFP-C1-MALAT-1 / shf of human MALAT-1 gene has been successfully constructed and successfully expressed in human nasopharyngeal carcinoma cell line C666-1 with high expression of MALAT-1 , MALAT-1 / shf (6918 ~ 8441 bp) were closely related to the invasion and metastasis of human nasopharyngeal carcinoma cell line C666-1.