亚甲蓝介导的光动力疗法诱导人表皮鳞状细胞癌A431细胞株凋亡机制研究  

Research on Mechanism of Apoptosis Induced by methylene blue-Based Photodynamic Therapy on A431 cells

作　　者：黎雯 刘成利 曾维花 吴伟伟 Wen Li;Chengli Liu;Weihua Zeng;Weiwei Wu(The Fifth People’s Hospital of Hainan Province,Haikou 570100,China;School of tropical medicine,Hainan Medical University,Haikou 571199,China)

机构地区：[1]海南省第五人民医院,海南海口570100 [2]海南医学院热带医学院,海南海口571199

出　　处：《皮肤科学通报》2023年第3期313-318,共6页Dermatology Bulletin

基　　金：海南省卫生计生行业科研项目(20A200251);海南省临床医学中心建设项目。

摘　　要：目的研究亚甲蓝介导的光动力疗法对人皮肤鳞状细胞癌A431细胞株的杀伤作用以及其诱导凋亡的机制,为亚甲蓝光动力临床应用提供理论依据以及最佳治疗参数。方法采用亚甲蓝与人皮肤鳞状细胞癌A431细胞株共同孵育,经630 nm红光照射。采用CCK-8法检测细胞生长的抑制率,确定培育时间、亚甲蓝浓度对A431细胞株的抑制作用。采用免疫组化法检测A431细胞在亚甲蓝光动力作用前后细胞色素c(Cyt-c)在细胞内的分布情况。最后,采用Western-blot方法检测亚甲蓝光动力治疗后细胞凋亡过程中,线粒体凋亡途径相关蛋白Bcl-2和Bax的表达情况。结果CCK8法检测结果显示亚甲蓝光动力组对A431细胞具有杀伤作用,其杀伤作用与亚甲蓝的药物浓度以及亚甲蓝光动力作用后的孵育时间具有密切相关。Cyt-c免疫荧光结果显示亚甲蓝光光动力作用后,细胞凋亡率空白对照0%,光照对照组8%,亚甲蓝对照组12%,光照后1 h 22%,光照后2 h 26%,光照后4 h 31%,光照后6 h 54%。Western blot的检测结果显示亚甲蓝光动力作用激活A431细胞线粒体凋亡途径,启动Bcl-2家族参与凋亡过程。随着亚甲蓝光动力作用后孵育时间的延长,促凋亡蛋白Bax上调,抗凋亡蛋白Bcl-2下调。结论亚甲蓝光动力能明显抑制A431细胞的增殖,并诱导其发生凋亡。凋亡的发生与启动细胞色素c相关的线粒体凋亡途径相关。Objectives To investigate the killing effect and mechanism of methylene bluemediated photodynamic therapy(MB-PDT)on human epidermoid carcinoma A431 cells.Methods Human epidermoid carcinoma A431 cells were employed in the present study.A431 cells were incubated in different concentration of methylene blue,and illuminated with 630 nm red light.The killing effects of MB-PDT on A431 cells was detected by CCK8 assay.Cytochrome c(Cyt-c)subcellular localization was compared by fluroimmunoassay between pre-MB-PDT and post-MB-PDT.Lastly,the protein expression level of Bcl-2 and Bax were determined by Western blot.Results The results of CCK8 assay showed that MB-PDT presented significant antiproliferation effects on A431 cells in a dose and time dependent manner.Immunofluorescence detection of Cytochrome c found that Cyt-c was released from mitochondrian into the cytosol in apoptosis cells.The apoptosis rates of A431 cells increased to 0,8%,12%,22%,26%,31%,and 54%respectively at blank group,PDT control group,and MB control group,1h,2h,4h,and 6h after MBPDT.Western blot discovered that apoptosis induced by MB-PDT involved upregulation of Bax protein and down-regulation of Bcl-2 protein obviously.Conclusion MB-PDT could significantly inhibit proliferation and induce apoptosis in A431 cells.The apoptosis of A431 cells induced by MB-PDT was related to the Cytochrome c-dependent mitochondrial pathways.

关 键 词：亚甲蓝 光动力疗法 鳞状细胞癌 A431细胞株 凋亡 

分 类 号：R739.5[医药卫生—肿瘤]