新型二氢卟吩类光敏剂KAE介导的光动力疗法对HeLa细胞的杀伤作用  

Photodynamic Therapeutic Effect of a Novel Chlorin-type Photosensitizer-KAE on HeLa Cells

作　　者：刘浩林 邱海霞 曾晶 赵洪友 顾瑛 LIU Haolin;QIU Haixia;ZENG Jing;ZHAO Hongyou;GU Ying(Medical School of Chinese PLA,Beijing 100853,China;Department of Laser Medicine,the First Medical Center of Chinese PLA General Hospital;Institute of Engineering Medicine,Beijing Institute of Technology;Precision Laser Medical Diagnosis and Treatment Innovation Unit,Chinese Academy of Medical Sciences)

机构地区：[1]解放军医学院,北京市100853 [2]中国人民解放军总医院第一医学中心激光医学科 [3]北京理工大学医工融合研究院 [4]中国医学科学院精准激光医学诊疗创新单元

出　　处：《中国激光医学杂志》2021年第6期301-305,356,共6页Chinese Journal of Laser Medicine & Surgery

基　　金：国家自然基金资助(61635014);北京理工大学青年教师学术启动计划(1870012222103);中国医学科学院医学与健康科技创新工程项目资助(2019-I2M-5-061)。

摘　　要：目的观察新型二氢卟吩类光敏剂N-二氢卟吩e6三葡甲氨盐(N-chlorin e6 triglumine,简称KAE)介导的光动力疗法(photodynamic therapy,PDT)对宫颈癌HeLa细胞的体外杀伤效应,并与临床常用光敏剂血卟啉衍生物(Hematoporphyrin derivatives,HpD)介导的光动力疗法(HpD-PDT)的效应进行比较。方法(1)采用紫外可见分光光度计检测光敏剂KAE和HpD分别在磷酸缓冲盐溶液(phosphate buffer saline,PBS)和含10%胎牛血清的DMEM中的吸收光谱;(2)采用荧光分光光度计检测KAE与HeLa细胞孵育不同时间后的荧光强度,将荧光最高峰的数值作为衡量HeLa细胞内KAE含量的指标;(3)根据不同浓度KAE或HpD介导的光动力疗法处理后HeLa细胞的存活率拟合曲线方程并计算半数抑制浓度(50%inhibitory concentration,IC50),用以评估KAE-PDT和HpD-PDT对HeLa细胞的杀伤效率。结果(1)吸收光谱结果:KAE在可见光范围内主要有两个吸收峰,其中主吸收峰在400 nm附近,次吸收峰位于红光区内660nm附近。在PBS和含有10%胎牛血清的DMEM溶液的峰值波长分别为654.5 nm、657.5 nm。HpD在可见光范围内的主吸收峰在400 nm左右,在红光区内的次吸收峰较弱。(2)HeLa细胞对KAE吸收特点检测结果,HeLa细胞对KAE的吸收量在孵育的前4 h内迅速增加,其后呈缓慢增加趋势。(3)细胞杀伤效果,在本实验所选浓度范围内,KAE-PDT和HpD-PDT对HeLa细胞的杀伤作用均随着光敏剂孵育浓度升高而增强,KAE-PDT杀伤HeLa细胞的IC50值为1.3μg/m L;HpD-PDT杀伤HeLa细胞的IC50值为2.85μg/m L。结论新型二氢卟吩类光敏剂KAE在红光区的吸收峰(约660 nm)与临床常用光敏剂HpD(约630 nm)相比红移较为明显,KAE-PDT对HeLa细胞的体外杀伤效力强于HpD-PDT。Objective To observe the in vitro effect of photodynamic therapy(PDT)mediated by a new type of chlorin photosensitizer-KAE(Nchlorin e6 triglumine)on HeLa cells,and compare it with the effect of hematoporphyrin derivatives(HpD)-mediated photodynamic therapy(HpD-PDT).Methods(1)UV-Vis spectrophotometer was used to detect the absorption spectra of photosensitizers KAE and HpD in PBS and DMEM containing 10%fetal calf serum.(2)Fluorescence spectrophotometer was used to detect the fluorescence of KAE in cells incubated with KAE for different lengths of time.The fluorescence intensity in HeLa cells was set as an indicator of the KAE level in HeLa cells.(3)The 50%inhibitory concentration(IC50)was calculated according to the survival rates of HeLa cells treated with PDT mediated by different concentrations of KAE or HpD.IC50 was used to evaluate and compare the efficacy of KAE-PDT and HpDPDT on HeLa cells.Results KAE showed two absorption peaks in the rang of visible light.The main absorption peak was around 400 nm,and the secondary peak in the red region was around 660 nm.In PBS,the peak was at 654.5 nm(absorbance is 0.3457).In DMEM solution containing 10%FBS,it shifted to 657.5 nm(absorbance:0.3591).the main absorption peak of HpD in the visible light range was around 400 nm,and there was a weak secondary absorption peak in the red region.The uptake of KAE by HeLa cells increased rapidly in the first 4 h after incubation.After 4 h,it showed a slow increase trend.Within the concentration range of KAE and HpD selected in this experiment,the cell killing effect of KAE-PDT and HpD-PDT on HeLa cells increased with the increase of the incubation concentrations,and the IC50 of KAE-PDT and HpD-PDT was 1.3μg/ml and 2.85μg/ml respectively.Conclusions Compared with that of the clinical photosensitizer HpD,the absorption peak(about 660 nm)of the new chlorin-type photosensitizer KAE in the red light region is stronger and obviously red-shifted.The in vitro efficacy of PDT mediated by KAE is higher than that of PDT mediated by HpD.

关 键 词：KAE 血卟啉衍生物 光动力疗法 HELA细胞 

分 类 号：R454.2[医药卫生—治疗学] R737.33[医药卫生—临床医学]