吗啡对人肝癌细胞增殖和迁移能力的影响：离体实验

Effect of morphine on proliferation and migration of human hepatocellular carcinoma cells： an m vitro experiment

目的评价吗啡对人肝癌细胞增殖和迁移能力的影响。方法实验1人肝癌细胞以2×10^5个／孔接种于6孔板中（2ml／孔），采用随机数字表法，将细胞分为6组（n=12）：对照组（C组）、10ng／ml吗啡组（M1组）、25ng／ml吗啡组（M2组）、50ng／ml组（M3组）、100ng／ml（M4组）和200ng／ml（M5组），M1组-M5组加入相应终浓度的吗啡，C组使用等量PBS处理，培养或孵育48h。采用Real—timePCR法检测μ阿片受体（MOR1）mRNA的表达。C组和M1组采用Westernblot法测定MOR1的表达。实验Ⅱ人肝癌细胞接种于96孔培养板（1×10^3个/孔）或Transwell小室（200μl）。采用随机数字表法分为2组（n=9）：对照组（c组）和吗啡组（M组）。M组加入吗啡，终浓度100ng／ml，C组加入等量PBS，终体积为100μl／孔。于孵育或培养第1～7天时采用MTT法测定细胞增殖能力。于孵育或培养30h时采用Transwell法检测细胞迁移能力。结果实验I与C组比较，M1组M5组MOR1mRNA表达上调，M4组MOR1表达上调（P〈0．01）。与M4组比较，M1组-M3组、M5组MOR1mRNA表达下调（P〈0．01）。实验Ⅱ与C组比较，M组孵育第4～7天时细胞增殖能力增强，穿过Transwell小室的细胞计数升高（P〈0．05或0．01）。M组孵育第4～7天细胞增殖能力逐渐增强（P〈0．05）。结论吗啡可促进人肝癌细胞增殖和迁移，机制与上调MOR1表达有关。

Objective To evaluate the effect of morphine on the proliferation and migration of hu- man hepatocellular carcinoma （HCC） cells in an in vitro experiment. Methods The experiment was performed in 2 parts. Experiment I Human HCC cells were inoculated in 6-well plates at a density of 2× 10^5 cells/well （2 ml/well） and divided into 6 groups （n = 12 each） using a random number table： control group （C group） and 10, 25, 50, 100 and 200 ng/ml morphine groups （M1-M5 groups）. Morphine at the final concentration of 10, 25, 50, 100 and 200 ng/ml was added in M1-M5 groups, respectively. The equal volume of phosphate buffer solution was added in group C. The cells were cultured or incubated for 48 h. The expression of μl-opioid receptor （ MOR1 ） mRNA was measured by real-time polymerase chain reaction. The expression of MOR1 was detected by Western blot in C and M4 groups. Experiment Ⅱ Human HCC cells were inoculated in 96-well plates （1 ×10^3 cells/well） or in Transwell chambers （200 pA） and divided into 2 groups （n = 9 each） using a random number table： control group （C group） and morphine group （M group）. Morphine was added at the final concentration of 100 ng/ml in group M, and the equal volume of phosphate buffer solution （final volume 100 Ixl/well） was added in group C. The cells were cultured or in- cubated for 7 days. The cell proliferation was detected by methyl thiazolyl tetrazolium assay at 1-7 days of incubation or culture. The cell migration was determined by Transwell chamber assay at 30 h of incubation or culture. Results Experiment I Compared with group C, the expression of MOR1 mRNA was signifi- cantly up-regulated in M1-M5 groups, and the expression of MOR1 was significantly up-regulated in group M4 （P〈0. 01 ）. Compared with group M4 , the expression of MOR1 mRNA was significantly down-regulated in Mt-M3 and Ms groups （P〈0.01）. Experiment Ⅱ Compared with group C, the cell proliferation was significantly enhanced on 4th-7th days of incubation, and the number of cells passing through Transwell chambers was increased in group M （P〈0.05 or 0.01）. The cell proliferation was gradually enhanced on 4th-7th days of incubation in group M （P〈0. 05）. Conclusion Morphine can promote the proliferation and migration of human HCC ceils, and the mechanism is related to up-regulation of MOR1 expression in an in vitro experiment.