5-氨基乙酰丙酸体外诱导膀胱癌细胞株BIU-87内生性原卟啉IX的动力学研究

Kinetics of 5-ALA induced protoporphyrin IX in bladder cancer cell line BIU-87:an in vitro study

目的 研究5-氨基乙酰丙酸（5-ALA）体外诱导膀胱癌细胞内生性荧光物质原卟啉（PpⅨ）荧光随时间的动力学变化及5-ALA的浓度、pH值对产生PpⅨ的影响。方法 0．5×10^6个／孔BIU-87单细胞悬液接种于6孔板培养24h，加入新鲜培养液和浓度为1mmol／L的5-ALA，于不同时间终止培养，取出用流式细胞仪测各孔细胞内PpⅨ含量。另以不同pH值终浓度为1mmol/L的5-ALA培养液以及不同浓度的5-ALA培养液分别孵育BIU-87细胞2h，同法用流式细胞仪测各孔PpⅨ含量。结果 5-ALA培养液孵育肿瘤细胞2h后终止5-ALA继续作用，肿瘤细胞内PpⅨ含量在第3小时达到最高峰。之后逐渐回落。5-ALA仅作用20min在培养2h后观察PpⅨ含量最低，与40min以后组差异有统计学意义，5-ALA作用40min后PpⅨ逐渐上升，但各观察组间差异无统计学意义。最佳pH值为6、7，而最适宜浓度为1～2mmol／L。结论 5-ALA可体外诱导膀胱癌细胞产生PpⅨ，其含量是-个动态过程，且受5-ALA作用时间、pH值及浓度的影响。实验数据可为临床应用5-ALA诱导荧光的光动力学诊断膀胱肿瘤提供参考。

Objective To investigate the dynamic change of fluorescence of PpⅨ produced by 5- ALA induced bladder cancer cell in vitro and influences of concentration of 5-ALA and pH on PpⅨ production. Methods BIU-87 single cell suspension was inoculated into 6-well plates at a density of 0.5 × 10^6 cells per well for 24 h, and fresh medium and 5-ALA of 1 mmol/L were added. Incubation was terminated after 1,2, 3, 4, 6 and 8 h respectively. The PpⅨ content of each well was determined by flow cytometry. BIU-87 cells were incubated with 1 mol/L 5-ALA at pH 6.0, 6.4, 6.7, 7.0, 8.0 or 5-ALA with different concentrations of 0.2, 0.5, 1,0, 2.0, 5.0 mmol/L. PpⅨ was determined by flow cytometry, Results Stop incubation after 2 hours, PpⅨ content in tumor cells reached peak at third hour, and then fell gradually. Cells incubated for 20 minutes produced the least PpⅨ, significantly less than that of cells incubated for at least 40 minutes, and after 40 minutes, PpⅨ rised slowly, but there were no significant difference between each group. The best pH value is 6.7, and the most fitting concentration is 1 mmol/L- 2 mmol/L. Contusion 5-ALA in vitro induces bladder carcinoma cells to produce PpⅨ whose content is a dynamic process and is easily affected by incubation time, pH value and 5-ALA concentration.