氨基酮戊酸光动力疗法治疗前后SKH-1小鼠皮肤鳞状细胞癌组织中免疫细胞亚群变化

Changes in immune cell subsets in cutaneous squamous cell carcinoma tissues from SKH-1 mouse models after aminolevulinic acid-based photodynamic therapy

目的研究氨基酮戊酸光动力疗法(ALA-PDT)对小鼠皮肤鳞状细胞癌(cSCC)的免疫效应。方法建立紫外线诱导的SKH-1无毛小鼠cSCC模型,进行ALA-PDT治疗,在治疗前及治疗后1、3、6、12、24 h和3 d、7 d各取5 mm3大小皮肤组织,采用免疫组化及流式细胞仪检测不同时间点小鼠肿瘤组织免疫细胞浸润情况,包括中性粒细胞、巨噬细胞、T细胞、B细胞、自然杀伤细胞和树突细胞。采用SPSS16.0软件进行两样本均数t检验。结果与治疗前相比,小鼠cSCC肿瘤局部中性粒细胞和巨噬细胞数量及比例在ALA-PDT治疗后1 h增高最明显[免疫组化结果(每400倍视野细胞数量):61.22±6.65比22.56±4.13,59.67±4.30比21.89±3.26,均P<0.05;流式细胞仪结果:(35.64±15.33)%比(5.46±2.44)%,(12.15±4.86)%比(1.98±1.49)%,均P<0.05]。同时,免疫组化和流式细胞仪检测均显示肿瘤局部T细胞、B细胞、自然杀伤细胞及树突细胞在治疗后6 h表达显著增高(均P<0.05)。达峰后,肿瘤组织中上述细胞数量和比例下降,但仍高于治疗前,并持续至本研究终点(治疗后第7天)。结论ALA-PDT通过招募免疫细胞发挥抗肿瘤作用,其中以中性粒细胞和巨噬细胞最为明显。

Objective To investigate immune effects of aminolevulinic acid-based photodynamic therapy(ALA-PDT)on mouse models of cutaneous squamous cell carcinoma(cSCC).Methods Ultraviolet-induced SKH-1 hairless mouse models of cSCC were established,and 40 tumor-bearing mice were randomly and equally divided into several groups:control group receiving no treatment,and 7 treatment groups treated with ALA-PDT for 1,3,6,12 and 24 hours,as well as 3 and 7 days respectively.After treatment,these mice were sacrificed at different time points,and skin tissues measuring 5 mm3 in size were resected.Immunohistochemical study and flow cytometry were performed to detect local infiltration of immune cells in cSCC tissues at different time points,including neutrophils,macrophages,T cells,B cells,natural killer cells and dendritic cells.Statistical analysis was done by the two-sample t test using SPSS 16.0 software.Results The number and proportion of local neutrophils and macrophages showed the most significant increase in mouse cSCC tumors 1 hour after ALA-PDT compared with those before treatment(immunohistochemical results[number of cells per 400×field]:61.22±6.65 vs.22.56±4.13,59.67±4.30 vs.21.89±3.26,respectively,both P<0.05;flow cytometry results:35.64%±15.33%vs.5.46%±2.44%,12.15%±4.86%vs.1.98%±1.49%,respectively,both P<0.05).Both immunohistochemical study and flow cytometry showed that the expression of T cells,B cells,natural killer cells and dendritic cells significantly increased in cSCC tissues 6 hours after treatment(all P<0.05).After reaching the peak,the number and proportion of the above-mentioned cells decreased in cSCC tissues,but were still higher than those before treatment,and the increase continued until the end of this study,that is,day 7 after treatment.Conclusion ALA-PDT may exert anti-tumor effects by recruiting immune cells,especially neutrophils and macrophages.