光动力疗法联合瘤体输注树突状细胞对小鼠肝癌移植瘤的抑制作用及免疫效应的研究

Anti-tumor and Immunological Effects Induced by the Combination of Photodynamic Therapy and Dendritic Cells on Mouse Hepatoma

目的探讨光动力疗法(photodynamic therapy,PDT)联合瘤体内输注树突状细胞(dendritic cell,DC)的光免疫疗法(photody-namic immuno-therapy,PIT)对小鼠Heps肝癌移植瘤的抑制作用及免疫效应。方法体外培养昆明小鼠骨髓源性DC,4,'6-二脒基-2-苯基吲哚(4,6-diamidino-2-phenylindole,DAPI)荧光染色液标记DC备用。128只昆明小鼠皮下接种Heps肝癌细胞建立肿瘤模型,随机分为对照组、PDT组、DC组和PIT组。对照组小鼠瘤体内注射生理盐水,PDT组单纯PDT治疗,DC组小鼠瘤体内注射DAPI标记的DC,PIT组PDT联合瘤体内注射DAPI标记的DC细胞。治疗后定期测量各组肿瘤体积,记录各组小鼠生存时间,荧光显微镜下计数DC组及PIT组小鼠淋巴结中荧光细胞数目,流式细胞仪测定各组小鼠外周血T细胞亚群,LDH释放法测定各组小鼠脾细胞杀伤活性。结果 (1)与对照组相比,PDT组与PIT组治疗后肿瘤生长明显受抑;(2)PDT组与PIT组小鼠生存时间延长;(3)高倍镜视野下DC组较PIT组荧光细胞数增多(P<0.05);(4)治疗后72 h,PDT及PIT组小鼠外周血CD8+T细胞百分率均明显高于对照组和DC组(P<0.01、P<0.01),其中PIT组较PDT组增高明显(P<0.01),(5)PDT组与PIT组小鼠脾脏细胞杀伤活性较对照组和DC组明显增强(P<0.01,P<0.01)。结论 PDT疗法能够抑制肿瘤生长并激发宿主免疫应答,联合输注DC可增强PDT对小鼠Heps肝癌移植瘤的抑制作用及免疫效应。

Objective To investigate the anti-tumor and immune efficacy of the photodynamic immuno therapy（PIT） combining photodynamic therapy（PDT） and dendritic cells（DC） on murine Heps hepatoma. Methods DCs were obtained through adherent culture and labeled with DAPI in vitro.The hepatoma model was established through the subcutaneous inoculation with Heps cells to 128 mice.They were then divided into four groups at random： the control group,PDT group,DC group and photo immunotherapy（PIT） group.While the mice in the control group being injected with normal saline into the tumor tissue,those in the PDT group were treated only with PDT.For the DC Group,DCs marked by DAPI were injected into the tumor tissue.As to the PIT group,the treatment was performed through the intratumoural injection of the DCs marked by DAPI immediately after PDT exposure.After the therapy,the mice were monitored for tumor growth.Following the treatment,the survival time of the tumor-bearing mice was recorded,and the DAPI-labeled DCs at draining lymph nodes were counted with the fluorescence microscope.At designated times after the treatment,perierpheral blood was collected from the mice in each group,and the T subsets were analyzed with flow cytometric.The cytotoxic activity of splenocytes was tested with standard lactate dehydrogenase（LDH） release assay. Results（1） Compared with the control group,tumor growth was significantly slower in the PDT and PIT groups（P0.01）.（2） In the PDT and PIT groups,the mean survival time was remarkably extended.（3） The number of fluorescent cells at the draining lymph nodes in the DC group was dramatically larger than that of the PIT group.（4） The percentages of CD8＋T cells in peripheral blood in the PDT and PIT groups were much higher than that of the DC and control groups 72 hours after the treatment（P0.01;P0.01）,and the figure of the PIT group was distinctively greater than that of the PDT group.（5） The anti-tumor efficacy of splenocytes in the PDT and PIT groups was impressively greater than that of the DC and control groups（P0.01、P0.01）. Conclusions Photodynamic therapy（PDT） can restrain tumor growth and induce antitumor immune response,and can amplify the restraint on and host immune response against PDT-treated tumor when being used in conjunction with dendritic cell immunotherapy.