摘要
目的:以耐阿霉素人乳腺癌细胞系MCF-7/ADR为实验对象,验证FOLRa受体基因及其下游基因二氢叶酸还原酶的表达在乳腺癌细胞多药耐药中的作用。
方法:1.细胞形态学观察和计数了解细胞生长情况;2.采用MTT法检测ADR细胞毒性作用;3.RT-PCR检测细胞叶酸受体、二氢叶酸还原酶mRNA的转录水平;4.MTT法检测甲氧苄啶对MCF-7/ADR细胞MDR的逆转作用;5.免疫细胞化学测定细胞P-gp和FOLRa的表达水平。
结果:1.MCF-7/S细胞增殖速度快于MCF-7/ADR细胞;2.MCF-7/ADR细胞对ADR的耐药倍数约为22.5倍;3.MCF-7/S细胞FOLRamRNA转录水平较MCF-7/ADR细胞高,而MCF-7/ADR细胞二氢叶酸还原酶mRNA转录水平较MCF-7/S细胞高;4.24μmol·L~(-1)及以下浓度的甲氧苄啶对MCF-7/ADR细胞有轻度抑制作用(抑制率＜20%),而对MCF-7/ADR细胞的多药耐药无逆转作用;5.免疫细胞化学显示MCF-7/ADR细胞P-gp表达水平较MCF-7/S细胞高,而MCF-7/S细胞FOLRa表达量高于MCF-7/ADR细胞。
结论:1.MCF-7/ADR细胞的FOLRa的mRNA转录水平下调、二氢叶酸还原酶mRNA转录水平升高、细胞P-gp表达增高,说明MCF-7/ADR细胞多药耐药可能与细胞FOLRa的mRNA转录下调、二氢叶酸还原酶mRNA转录升高导致P-gp增加有关;2.甲氧苄啶对MCF-7/ADR细胞多药耐药无逆转作用。
Objective: The adriamycin-resistant human breast cancer cell line MCF-7/ADR were used as the experiment subject to investigate the FOLR1 receptor gene and it's downstream gene dihydrofolate reductase gene expression correlated with the breast cancer cell multidrug-resistance.
Methods: MTT assay were used to investigate the cytotoxicity of ADR and reversal effect of trimethoprim on MCF-7/ADR cells, real time PCR were used to detect transcriptional levels of folate receptor mRNA and dihydrofolate reductase mRNA of MCF-7/S cells and MCF-7/ADR cells,The protein level of P-gp and FOLRa were detected with Immunocytochemistry.
Results: FOLRa receptor mRNA transcriptional levels of MCF-7 cells was higher than MCF-7/ADR cells, dihydrofolate reductase mRNA transcriptional levels of MCF-7/ADR cells was increased; It showed that the The protein level of P-gp of MCF-7/ADR cells was higher than it's of MCF-7/S cells, and The protein level of FOLRa of MCF-7/ADR cells was less than it's of MCF-7/S cells, The concentrations of Trimethoprim of 240μmol·L~(-1) and under it showed mild inhibitory effect on MCF-7/ADR cells (inhibition rate <20%), while non- reversal effect.
Conclusion: The FOLRa receptor transcription level in MCF-7/ ADR cells down-regulated, the transcription level of dihydrofolate reductase in MCF-7/ADR cells up-regulated, the expression of P-gp in MCF-7/ADR cells increased. Those showed the multidrug resistance in MCF-7/ADR cells associated with P-gp increased by The FOLRa receptor transcription level down-regulated and the transcription level of dihydrofolate reductase up- regulated; Trimethoprim had non-reversal effect of multidrug resistance on MCF-7/ADR cells.
引文
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