CAJ | 학술논문

目的探讨氨基酮戊酸光动力疗法(ALA-PDT)对小鼠皮肤鳞状细胞癌(SCC)的作用机制.方法先建立SKH-1无毛小鼠SCC模型,ALA-PDT治疗前即对照组3只,ALA-PDT治疗后1、3、6、12、24h组各3只.以透射电镜及TUNEL法观察ALA-PDT治疗后1～24h SCC组织肿瘤细胞死亡方式(坏死、凋亡、自噬)；以免疫组化技术检测治疗后7 d SCC组织细胞自噬标记物LC3B、肿瘤局部微血管CD34、肿瘤局部免疫细胞浸润包括树突细胞CD1a、CD4+T淋巴细胞和CD8+T淋巴细胞表达的变化.采用SPSS17.0软件进行统计学分析,两样本均数比较采用t检验.结果透射电镜观察发现,ALA-PDT治疗后24 h,肿瘤细胞逐渐发生坏死和凋亡,且以细胞坏死为主,巨噬细胞中可见自噬小体.TUNEL检测显示,肿瘤细胞凋亡显著增加(ALA-PDT治疗前及治疗后24 h分别为2.00±0.69和7.30±2.18,P＜0.05).免疫组化显示,与治疗前比较,ALA-PDT治疗后7d,CD34表达显著下降(分别为19.00±2.66和1.33±0.58,P＜0.01),肿瘤局部CD1a表达明显增高(分别为10.33±1.88和23.01±2.04,P＜0.05),CD4+T细胞数(分别为12.40±2.27和28.67±1.76,P＜0.05)和CD8+T细胞数(分别为11.67±1.45和25.79±2.37,P＜0.05)明显增多,同时肿瘤间质中LC3B表达明显增多(分别为21.44±4.3和30.6±3.21,P＜0.05).结论 ALA-PDT可通过细胞坏死和凋亡两种方式直接杀伤SCC细胞,未发现肿瘤细胞自噬性死亡；ALA-PDT可损伤SCC组织微血管内皮细胞,诱导局部树突细胞以及CD4+、CD8+T细胞聚集.
목적 탐토안기동무산광동력요법(ALA-PDT)대소서피부린상세포암(SCC)적작용궤제.방법 선건립SKH-1무모소서SCC모형,ALA-PDT치료전즉대조조3지,ALA-PDT치료후1、3、6、12、24h조각3지.이투사전경급TUNEL법관찰ALA-PDT치료후1～24h SCC조직종류세포사망방식(배사、조망、자서)；이면역조화기술검측치료후7 d SCC조직세포자서표기물LC3B、종류국부미혈관CD34、종류국부면역세포침윤포괄수돌세포CD1a、CD4+T림파세포화CD8+T림파세포표체적변화.채용SPSS17.0연건진행통계학분석,량양본균수비교채용t검험.결과 투사전경관찰발현,ALA-PDT치료후24 h,종류세포축점발생배사화조망,차이세포배사위주,거서세포중가견자서소체.TUNEL검측현시,종류세포조망현저증가(ALA-PDT치료전급치료후24 h분별위2.00±0.69화7.30±2.18,P＜0.05).면역조화현시,여치료전비교,ALA-PDT치료후7d,CD34표체현저하강(분별위19.00±2.66화1.33±0.58,P＜0.01),종류국부CD1a표체명현증고(분별위10.33±1.88화23.01±2.04,P＜0.05),CD4+T세포수(분별위12.40±2.27화28.67±1.76,P＜0.05)화CD8+T세포수(분별위11.67±1.45화25.79±2.37,P＜0.05)명현증다,동시종류간질중LC3B표체명현증다(분별위21.44±4.3화30.6±3.21,P＜0.05).결론 ALA-PDT가통과세포배사화조망량충방식직접살상SCC세포,미발현종류세포자서성사망；ALA-PDT가손상SCC조직미혈관내피세포,유도국부수돌세포이급CD4+、CD8+T세포취집.
Objective To investigate the mechanisms underlying the effect of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on cutaneous squamous cell carcinoma (SCC) in mice.Methods A model of cutaneous SCC was established in 21 SKH-1 hairless mice,which were treated with topical ALA 8％ cream followed by single irradiation with He-Ne laser at a total dose of 30 J/cm2 (ALA-PDT).Three mice were sacrificed before and at 1,3,6,12,24 hours and 7 days after the irradiation,separately,and SCC tissue was taken from the mice.Transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were performed to determine the pattern of tumor cell death(necrosis,apoptosis and autophagy) during 1-24 hours after ALA-PDT,and immunohistochemical techniques were used to estimate the expressions of LC3B and CD34 on SCC cells,as well as the quantity of CD1a+ cells,CD4+ T and CD8+ T lymphocytes in SCC tissue 7 days after the irradiation.Statistical analysis was done by two-sample t test using SPSS 17.0 software.Results TEM showed gradual necrosis and apoptosis (especially necrosis) of tumor cells and formation of autophagosomes in macrophages within 24 hours after ALA-PDT.The number of apoptotic cells per high power field (× 400) in SCC tissue significantly increased at 24 hours compared with that before ALA-PDT (7.30 ± 2.18 vs.2.00 ± 0.69,P ＜ 0.05).As immunohistochemistry revealed,there was a significant decrease in the number of CD34+ cells (1.33 ± 0.58 vs.19.00 ± 2.66,P＜ 0.01),but a marked increase in that of CD1a+ ce1ls (23.01 ± 2.04 vs.10.33 ± 1.88,P＜ 0.05),CD4+ T cells (28.67 ± 1.76 vs.12.40 ± 2.27,P＜ 0.05),CD8+ T cells (25.79 ± 2.37 vs.11.67 ± 1.45,P ＜ 0.05) and LC3B+ interstitial cells (30.6 ± 3.21 vs.21.44 ± 4.3,P ＜ 0.05) per high power field (× 400) in SCC tissue on day 7 compared with that before ALA-PDT.Conclusions ALA-PDT may directly kill SCC cells by inducing cell necrosis and apoptosis rather than autophagy.Additionally,ALA-PDT can injure microvascular endothelial cells and cause the aggregation of dendritic cells,CD4+ T cells and CD8+ T cells in SCC tissue.