关键词: pGCSIL/GFP载体, 二氢叶酸还原酶, 高效液相色谱法, 凋亡, 透射电子显微术

Abstract: Background and purpose: Dihydrofolate reductase (DHFR) is expressed highly in platinum-resistant ovarian cancer. This study aimed to explore the relationship between the silence of DHFR gene and platinum drug resistance in ovarian cancer, and lay the foundation for the treatment of platinum-resistant ovarian cancer. Methods: To design targeting hairpin siRNA of DHFR gene, the optimal siRNA silent sequence was selected, and lentiviral vector carrying DHFR gene was constructed successfully, named DHFR-pGCSIL-SKOV3 cell. Flow cytometry was used to detect the cell apoptosis of DHFR-pGCSIL-SKOV3 cells, pGCSIL-SKOV3 cells and SKOV3 cells incubated in various concentrations of cisplatin (2.5, 5.0, 10.0 and 20.0 μg/mL) at different time points (24, 48 and 72 h), and cell cycle changes of these cells at IC₅₀ cisplatin concentration (4.4 μg/mL). High performance liquid chromatography was used to test intracellular concentration of cisplatin at different induction concentration of cisplatin (2.5, 5.0 and 7.5 μg/mL) and various time points (24 and 48 h). Ultrastructural changes of these cells at concentration of cisplatin IC₅₀ (4.4 μg/mL) were observed by transmission electron microscope. Results: After annealing double-strand nucleotide was connected to pGCSIL/GFP vector, sequencing result was correct. SKOV3 cell were transfected with virus particles followed by Western blot detection of interference effect. Flow cytometry was used to detect apoptosis in three groups of cells, and increased apoptosis rate was found at the raised cisplatin concentration (2.5, 5.0, 10.0 and 20.0 μg/mL) at 24, 48 and 72 h in DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3 and SKOV3 cells. The apoptosis rate in DHFR-pGCSIL-SKOV3 was significantly higher than that in pGCSIL-SKOV3 and SKOV3 cells at 24 and 48 h (P<0.05). Flow cytometry was adopted to test cells cycle of 3 groups at different time period under IC50 cisplatin concentration (4.4 μg/mL), the results indicated that G₀/G₁ phase cell rate of DHFR-pGCSIL-SKOV3 was much more than the others, of which G₂/M and S phase cell rates were on the contrary. While at 72 h, 3 groups were mainly G₂/M and S phase cell rates, DHFR-pGCSIL-SKOV3 was lower than the others. High performance liquid chromatography method was used to detect intracellular cisplatin concentration at 24 and 48 h after the cells were incubated at various concentrations of cisplatin (2.5 and 5.0 μg/mL). The results showed the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was significantly higher than that of pGCSIL-SKOV3 and SKOV3 cells. However, after incubation at cisplatin concentration of 7.5 μg/mL, the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was significantly lower than that of pGCSIL-SKOV3 and SKOV3 cells at 24 h, while higher than pGCSIL-SKOV3 and SKOV3 cells at 48 h (P=0.034, P=0.014). We observed ultrastructural changes of three different cell lines induced by IC₅₀ cisplatin concentration(4.4 μg/mL) at different time points by the electron microscope. We found that the microfilaments were increased and gathered together and mitochondrial structure was also changed obviously without the drug. However, there was rare microfilament in three groups of cells at 24 and 48 h, while at 72 h, obviously increased inordinate microfilaments were observed. Conclusion: We successfully constructed pGCSIL lentivirus interference carrier carrying DHFR gene. The research indicates that down-regulation of DHFR gene is related to cisplatin drug resistance in ovarian cancer. The results laid the foundation for us to investigate the molecular mechanisms of multidrug-resistance in tumor.

Key words: pGCSIL/GFP vector, Dihydrofolate reductase, High performance liquid chromatography, Apoptosis, Transmission electron microscopy