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目的:用间断和连续两种诱导方式建立对甲氨蝶呤(MTX)耐药的绒毛膜癌细胞系,并动态检测二氢叶酸还原酶(DHFR)在两种耐药细胞系建立过程中的表达。方法:采用间断和连续诱导法诱导人绒癌细胞系JeG-3,分别建立绒癌MTX耐药细胞系MTXA1(间断诱导浓度为1.1×10-3mol/L)和MTXC2(连续诱导浓度为1.1×10-5mol/L)。细胞计数法(CCK-8)观察耐药细胞的耐药指数;化学发光法和荧光定量PCR技术分别检测不同诱导方法和不同浓度MTX诱导的JeG-3细胞中β人绒毛膜促性腺激素(β-HCG)的分泌量和DHFR mRNA的表达水平。结果:(1)历时1年成功建立了绒癌耐药细胞系MTXA1和MTXC2,其耐药指数分别为21.36和28.84;(2)β-HCG分泌和DHFR mRNA表达水平在间断和连续两种不同诱导方式诱导的绒癌细胞株中呈相同的动态变化趋势:经低浓度MTX诱导后,JeG-3细胞中β-HCG分泌和DHFR mRNA的表达水平上调;但当MTX诱导浓度增加到一定剂量时,两者不再继续上调而呈下降趋势。结论:成功建立了间断和连续两种方式诱导的耐药绒癌细胞系MTXA1和MTXC2。绒癌细胞β-HCG分泌和DHFR mRNA的表达与MTX作用浓度以及耐药性无明显的正相关关系。目前的研究结果表明,DHFR mRNA的表达水平不适宜作为绒癌细胞是否对MTX耐药的指标。
OBJECTIVE: To establish a MTX-resistant choriocarcinoma cell line by intermittent and continuous induction and to detect the expression of dihydrofolate reductase (DHFR) during the establishment of two drug-resistant cell lines expression. Methods: The human choriocarcinoma cell line JeG-3 was induced by intermittent and continuous induction. The MTX-resistant cell line MTXA1 (intermittent induction concentration 1.1 × 10-3 mol / L) and MTXC2 (continuous induction concentration 1.1 × 10-5 mol / L). The drug resistance index of drug-resistant cells was observed by cell counting method (CCK-8). The chemiluminescence and fluorescence quantitative PCR were used to detect the expression of β-human chorionic gonadotropin (β) in JeG-3 cells induced by different concentrations of MTX -HCG) secretion and DHFR mRNA expression levels. Results: (1) MTXA1 and MTXC2 cells were established successfully over one year, and the resistance index was 21.36 and 28.84, respectively. (2) The secretion ofβ-HCG and the expression of DHFR mRNA in two kinds of intermittent and continuous Induced by the induction of choriocarcinoma cells showed the same trend of dynamic changes: induced by low concentrations of MTX, JeG-3 cells in the secretion of β-HCG and DHFR mRNA expression increased; but when MTX induced concentration increased to a certain dose , Both no longer continue to rise but showed a downward trend. Conclusion: The drug-resistant choriocarcinoma cell lines MTXA1 and MTXC2 were successfully established by both intermittent and continuous methods. The expression of β-HCG and DHFR mRNA in choriocarcinoma cells had no significant positive correlation with MTX concentration and drug resistance. The current results show that the expression level of DHFR mRNA is not suitable as an indicator of choriocarcinoma resistance to MTX.