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目的:探讨肝星状细胞条件培养基(hepatic stellate cell conditioned medium,HSC-CM)对人肝癌PLC/PRF/5细胞耐药性的影响及其可能的机制。方法:用无血清RPMI 1640培养肝星状细胞LX-2,使其在缺营养的环境下活化,收集其条件培养上清即为HSC-CM。PLC/PRF/5细胞在HSC-CM条件下培养24 h后,顺铂处理12 h或24 h,采用流式细胞术检测PLC/PRF/5细胞的凋亡情况,MTT法检测PLC/PRF/5细胞的增殖,real-time PCR检测PLC/PRF/5细胞上皮间质转化(epithelialmesenchymal transition,EMT)相关基因的表达水平。结果:顺铂组12和24 h两个时间点PLC/PRF/5细胞的凋亡率为(22.34±1.12)%和(26.78±1.56)%;HSC-CM+顺铂组细胞的凋亡率为(17.22±1.42)%和(21.52±1.76)%,顺铂组细胞凋亡率显著高于HSC-CM+顺铂组(P<0.05)。同在这两个时间点,顺铂组和HSC-CM+顺铂组PLC/PRF/5细胞的增殖活性分别为(68.65±2.56)%和(79.47±1.43)%,(46.54±3.65)%和(62.77±2.89)%,HSC-CM+顺铂组细胞增殖活性均高于顺铂组(P<0.05)。Real-time PCR结果显示,与顺铂组相比较,HSC-CM+顺铂组PLC/PRF/5细胞中上皮标记物钙黏蛋白(E-cadherin)的表达下降(P<0.05),而间质细胞标记物神经黏附素(N-cadherin)、波形蛋白(vimentin)以及EMT相关转录因子Snail和ZEB1的表达显著上调(P<0.01)。结论:HSC-CM可能通过诱导PLC/PRF/5细胞发生EMT,从而增强PLC/PRF/5细胞对顺铂的抵抗作用。
Objective: To investigate the effect of hepatic stellate cell conditioned medium (HSC-CM) on the drug resistance of human hepatoma PLC / PRF / 5 cells and its possible mechanism. Methods: The hepatic stellate cells (LX-2) were cultured in serum-free RPMI 1640 and activated in anaerobic condition. The supernatant of conditioned medium was collected to be HSC-CM. The PLC / PRF / 5 cells were cultured in HSC-CM for 24 h, then treated with cisplatin for 12 h or 24 h. The apoptosis of PLC / PRF / 5 cells was detected by flow cytometry. 5 cells. Real-time PCR was used to detect the expression of related genes of epithelialmesenchymal transition (EMT) in PLC / PRF / 5 cells. Results: The apoptotic rate of PLC / PRF / 5 cells was (22.34 ± 1.12)% and (26.78 ± 1.56)%, respectively at 12 and 24 h after cisplatin treatment. The apoptosis rates of HSC-CM + (17.22 ± 1.42)% and (21.52 ± 1.76)% respectively. The apoptosis rate in cisplatin group was significantly higher than that in HSC-CM + cisplatin group (P <0.05). At the same time point, the proliferation activities of PLC / PRF / 5 cells in cisplatin group and HSC-CM + cisplatin group were (68.65 ± 2.56)% and (79.47 ± 1.43)%, (46.54 ± 3.65)% and (62.77 ± 2.89)% respectively. The cell proliferation activity in HSC-CM + cisplatin group was higher than that in cisplatin group (P <0.05). Real-time PCR results showed that the expression of E-cadherin in PLC / PRF / 5 cells decreased (P <0.05) in HSC-CM + cisplatin group compared with that in cisplatin group The expressions of N-cadherin, vimentin and EMT-related transcription factors Snail and ZEB1 were significantly up-regulated (P <0.01). Conclusion: HSC-CM may enhance the resistance of PLC / PRF / 5 cells to cisplatin by inducing EMT in PLC / PRF / 5 cells.