TLR基因在人肺腺癌细胞和支气管上皮细胞中的表达

Expression of TLRs Genes in Human Pulmonary Adenocarcinoma Cells and Bronchial Epithelial Cells

目的探讨Toll样受体家族(Toll-like receptors,TLRs)基因在人肺腺癌细胞(A549细胞)和人支气管上皮细胞(HBE细胞)中的表达及意义。方法通过GenBank网站查找基因全序列,根据引物设计原则应用软件Primer 5.0设计TLR2、TLR3、TLR9和内参β-actin引物。体外培养A549细胞和HBE细胞,提取细胞总RNA并逆转录得到cDNA,采用实时定量荧光PCR检测TLRs基因mRNA在这2种细胞中的表达。琼脂糖凝胶电泳检测各扩增产物片段大小。采用Western blot检测TLR2、TLR3、TLR9蛋白的表达。结果 TLR2、TLR3、TLR9基因在A549细胞中的表达丰度均比在HBE细胞中高(P<0.01)。琼脂糖凝胶电泳显示扩增产物符合预期片段大小。TLR2、TLR3、TLR9蛋白在A549细胞中的表达相对值分别为(0.472 8±0.010 3)、(0.319 3±0.011 2)和(0.856 9±0.007 9),而在HBE细胞中的表达相对值分别为(0.258 5±0.005 7)、(0.160 7±0.003 0)和(0.699 1±0.010 0),A549细胞中TLR2、TLR3、TLR9表达的相对值均高于HBE细胞(均P<0.01)。结论 TLR2、TLR3、TLR9基因在A549细胞中的表达高于HBE细胞,提示TLRs基因可能参与人肺腺癌的发生发展。

Objective To investigate the expressions of Toll-like receptors（TLRs）genes in human pulmonary adenocarcinoma cells（A549 cells）and human bronchial epithelial cells（HBE cells）and their significance.Methods The primers of human TLR2,TLR3 and TLR9 gene were designed by using the software Primer 5.0 according to the gene sequences available on GenBank.A549 and HBE cells were cultured in vitro.Total RNA was extracted and reverse transcribed to cDNA.Quantitative real-time PCR was employed to detect the mRNA expression of TLRs in A549 and HBE cells.The amplified products were identified by agarose gel electrophoresis.Western blot was used to measure the expression of TLRs protein.Results The expressions of TLR2,TLR3 and TLR9 mRNAs were significantly increased in A549 cells relative to HBE cells（P〈0.01）.The electrophoresis results revealed that the size of the amplified products was consisted with that of expected fragments.The relative expression levels of TLR2,TLR3 and TLR9 proteins were（0.472 8±0.010 3）,（0.319 3±0.011 2）and（0.856 9±0.007 9）in A549 cells,significantly greater than those in HBE cells,which were（0.258 5±0.005 7）,（0.160 7±0.003 0）and（0.699 1± 0.010 0）respectively（P〈0.01 for all）.Conclusion TLR2,TLR3 and TLR9 genes may be involved in the occurrence and progression of human pulmonary adenocarcinoma.