HMME-PDT对原代培养瘢痕成纤维细胞中Cyc表达的影响  

The Investigation of Cytochrome C in HSF after HMME-PDT

作　　者：蔡宏[1] 顾瑛[2] 曾晶[2] 刘玮[1] 

机构地区：[1]解放军空军总医院皮肤科,北京市100141 [2]解放军总医院激光医学科

出　　处：《中国激光医学杂志》2012年第5期273-277,357,共6页Chinese Journal of Laser Medicine & Surgery

摘　　要：目的观察血卟啉单甲醚(hematoporphyrin monomethyl ether,HMME)-光动力学疗法(photodynamic therapy,PDT)对增生性瘢痕成纤维细胞(hypertrophic scar fibroblast,HSF)中细胞色素C(cytochrome C,Cyc)表达的影响。方法采用组织块法原代培养瘢痕组织来源的成纤维细胞,建立HSF的传代细胞系,实验用第4～6代细胞。取对数生长期的HSF细胞,分为对照组和HMME-PDT组,其中HMME-PDT组的处理方法为培养的HSF与4μg/ml的HMME避光孵育3h,PDT治疗采用波长为630nm的激光,功率密度10mW/cm2,能量密度2.5J/cm2,两组细胞爬片细胞经固定、打孔后加入Cyc(1∶50)抗体以及FITC标记的二抗(1∶100),荧光显微镜下观察Cyc的荧光强度。用PI单染后经FCM检测细胞凋亡率。每标本计数105个细胞,实验重复5次。结果对照组HSF的细胞浆中Cyc-FITC荧光微弱,PDT组荧光显著增强;PI染色分析表明对照组凋亡率低(2.45±0.22%),PDT组的凋亡率显著升高(30.54±3.78%),二者相比差异有显著意义(P<0.05)。结论光动力治疗后导致的线粒体功能障碍可能在瘢痕成纤维细胞凋亡中发挥了重要作用。Objective To observe the effect of HMME-PDT on the expression of Cytochrome C in human hypertrophic scar fibroblast （HSF）. Methods Fibroblasts were cultured from untreated burn hypertrophic scars, and the cells of the 4th-6th generations were used in the experiment. Having grown to the level of 80% confluence, the fibroblasts were divided into 2 groups： the control （untreated） group and HMME-PDT group. For the HMME-PDT group, the medium was removed, serum-free medium with 4 μg/ml HMME was added, and the cells were given HMME for4 h. The cells were then irradiated with 630 nm light produced by a diode laser （630 nm） at a pewer of 10 mW/cm^2 （ fluence ： 2. 5 J/em^2 ）. Immunofluorescence staining method was applied for the observation of Cyc expression in HSF with fluorescence microscope. The apoptotic rate of the cells was detected with FCM after pepodium iodide （PI） staining. Results Being almost negative in the control group, the Cyc-FITC staining result was highly enhanced in the HMME-PDT group. The detection results of FCM indicated that the apoptotic rate grew significantly after the HMME-PDT （30. 54 ＋ 3. 78% VS 2. 45 ~ 0.-22%, P〈0.05）.Conclusions The mitochondria dysfunction caused by PDT may play a major role in leading the apoptosis of hypertrophic scar cells.

关 键 词：血卟啉单甲醚 光动力学疗法 瘢痕成纤维细胞 细胞色素C 

分 类 号：R318.08[医药卫生—生物医学工程]