光动力疗法中竹红菌乙素在细胞中含量的检测

Hypocrellin B Concentration Monitoring in vitro in Photodynamic Therapy

目的研究光敏剂孵育时ECV304细胞和培养液中竹红菌乙素(HB)的含量,以及HB在细胞和培养液中的光漂白。方法体外培养ECV304细胞,HB的孵育浓度分别为10、30μg/ml,每个浓度组再分为照光组和未照光组。未照光组HB孵育2 h后,用荧光显微镜观察细胞中HB的荧光分布;然后分别用乙酸乙酯萃取细胞和培养液中的HB后用吸收光谱测定HB的含量。照光组用532 nm激光照射,采用上述方法测量细胞和培养液中HB的含量,并进行HPLC图谱分析。结果HB的孵育浓度为10、30μg/ml,孵育浓度增加细胞中HB浓度并不随之成比例增加,激光照射使细胞和培养液中的HB都发生了光漂白。结论利用HB的浓度吸收光度值的标准曲线,可以估算出细胞中以及光照后HB的含量。

Objective To detect the hypocrellin B （HB） concentration in ECV304 cells and culture medium, and the photobleaching property in vitro. Methods ECV304 ceils were incubated with HB （10ug/ml or 30 ug/ml） for 2 h, at each concentration two groups （ control group without laser irradiation and PDT group） were set. In the control group, after HB incubation, fluorescence image of HB was detected by fluorescence microscopy;HB in cells and RPMI medium 1 640 was extracted by ethyl acetate solution, then to record the absorption spectra and to calculate the concentration from a standard HB concentration curve. In the PDT group, after being exposed to 532 nm laser light, the concentration of HB was measured using the same method as in control group, and HB in cells and RPMI medium 1 640 was analyzed by HPLC. Results When the incubation concentrations were 10 or 30ug/ml, the concentrations of HB in cells were not increased in proportion to incubation concentrations. After PDT, HB in culture medium and cells was bleached. Conclusions We can estimate the concentration of HB in vitro before and after PDT from a standard HB absorption - concentration