实时荧光定量PCR法检测人骨肉瘤耐MTX细胞系中RFC、DHFR、GST-π的mRNA表达_临床医学论文.docVIP

实时荧光定量PCR法检测人骨肉瘤耐MTX细胞系中RFC、DHFR、GST-π的mRNA表达_临床医学论文 实时荧光定量PCR法检测人骨肉瘤耐MTX细胞系中RFC、DHFR、GST-π的mRNA表达_临床医学论文 【摘要】 ［目的］研究RFC(还原叶酸载体)、GST-π（谷胱甘肽S转移酶π）、DHFR（二氢叶酸还原酶） mRNA在人骨肉瘤U2-OS细胞系及人骨肉瘤耐药细胞系U2-OS/R1-R3中的表达差异，并探讨其在人骨肉瘤MTX化疗耐药中的意义。［方法］采用冲击并逐步增加诱导药物浓度诱导的方法，诱导人骨肉瘤细胞系U2-OS，并建立耐MTX细胞系（U2-OS/R1-R3），利用荧光定量PCR技术在mRNA 水平检测RFC、GST-π、DHFR在U2-OS及（U2-OS/R1-R3）中的表达。［结果］本试验成功建立三株人骨肉瘤耐药细胞系U2-OS/R1-R3中，荧光定量PCR结果显示MTX耐药与DHFRmRNA表达增加有关，与RFCmRNA的表达降低有关，与 GST-πmRNA的表达增加有关。［结论］本试验在基因水平探讨人骨肉瘤细胞MTX化疗耐药机制， DHFR、GST-π基因表达的增加及RFC基因表达的降低参与了人骨肉瘤细胞的MTX耐药，为临床探索骨肉瘤患者的MTX耐药机制提供了重要的依据，为临床筛选MTX化疗不敏感患者提供重要的依据。 【关键词】 实时荧光定量PCR； 甲氨喋呤（MTX） 还原叶酸载体（RFC）； 二氢叶酸还原酶（DHFR）； 谷胱甘肽S转移酶（GST-π） Abstract: ［Objective］To study the difference in expression of RFC, GST-π, DHFRmRNA between human osteosarcoma U2-OS cell line and the MTX-resisitant variants U2-OS/R1-R3, and to investigate the significance in MTX resistance for human osteosarcoma. ［Methods］Three resistant MTX human osteosarcoma cell lines were established by pulse exposure parental cell line(U2-OS) in gradually increased dose of MTX . The expression of RFC，GST-π，DHFRmRNA were assayed by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).［Results］Three MTX-resistant variants(U2-OS/R1-R3) were successfully established , the results of the FQ-PCR revealed that the MTX resistance was associated with the decreased expression of the RFC mRNA and increased expression of DHFR mRNA and GST-π mRNA.［Conclusion］The author investigated the MTX resistant mechanism of human osteosarcoma cell line at a gene level. The decreased expression of RFC mRNA and the increased expression of DHFR mRNA and GST-π mRNA participate in the MTX resistance in human osteosarcoma cell lines U-2 OS. This provides the evidence for exploring the MTX resistance mechanism in clinical osteosarcoma patients ,and helps to screen the patients who are insensitive to MTX chemotherapy. Key words：real-time fluorescence quantitative polymerase chain reaction(FQ-PCR); methotrexate(MTX); re