姜黄素介导光动力治疗对宫颈癌H8细胞的作用研究

Photodynamic effects of curcumin on human cervical cancer H8 cell

目的 探讨姜黄素介导光动力疗法（PDT）治疗老年宫颈癌抗肿瘤的作用机制. 方法 噻唑蓝（MTT）法评价姜黄素介导PDT在不同药物浓度、不同光照剂量下对宫颈癌H8细胞增殖的影响,并筛选出最佳作用参数.观察细胞形态学变化,流式细胞术检测细胞凋亡率.实时荧光转录聚合酶链反应（RT-PCR）法分析H8细胞在姜黄素PDT治疗前后对凋亡抑制基因Bcl-2、P53、Survivin表达的影响.选择人宫颈癌H8细胞系建立裸鼠宫颈癌移植瘤模型,分为对照组、单纯激光照射组、单纯姜黄素组、姜黄素PDT治疗组（每组10只）.通过苏木精-伊红常规染色（HE）观察姜黄素-PDT体内杀伤效应,免疫组化法检测PDT治疗前后Bcl 2蛋白表达的影响. 结果 姜黄素5μmol/L和蓝光照射剂量100 J/cm2时,姜黄素-PDT对H8细胞增殖具有明显抑制作用,并可诱导凋亡,且呈浓度和光剂量依赖效应.在光学显微镜下可见典型凋亡形态改变,核染色质凝集、核固缩、核碎裂、凋亡小体形成.PDT组总凋亡率为（47.21±4.11）％,高于对照组（1.71±0.16）％,差异有统计学意义（P＜0.01）.姜黄素-PDT可抑制Bcl-2、P53、Survivin基因的mRNA表达.裸鼠移植瘤PDT组HE染色可观察到受激光照射的皮下肿瘤组织区域坏死.姜黄素-PDT可抑制移植瘤组织的Bcl 2的蛋白表达（P＜0.01）. 结论 姜黄素-PDT在体内、体外对宫颈癌H8细胞均具有杀伤效应,此作用与促进肿瘤细胞凋亡、抑制Bcl-2基因表达有关.

Objective To study the effects of curcumin mediated photodynamic therapy （PDT）on the growth and proliferation in human cervical cancer cell line H8 in vitro and in vivo,and to investigate its antitumor mechanisms.Methods The effects of curcumin mediated PDT on proliferation of human cervical cancer H8 cell by MTT assay was used to screen the optimal parameter.Changes in cell morphology were observed by May-Gr ＆#252;nwald-Farbstoff Giemsa staining.The apoptosis rate was estimated by flow cytometry.The effect of PDT by curcumin on the expressions of Bcl-2,P53 and survivin in H8 cells was detected by fluorescence real-time reverse transcription-polymerase chain reaction （RT-PCR）.Forty BALB/C nude mice underwent subcutaneous injection of H8 cell line so as to establish animal models,and then were randomly divided into four equal groups：control group,irradiation alone group,curcumin alone group,curcumin PDT group.HE staining and pathological examination were performed.Immunohistochemical study was conducted to detect the protein expression of the apoptosis inhibiting genes of Bcl-2.Results The proliferation inhibition of H8 cells was obvious after PDT when curcumin 5μmol/L with irradiation 100 J/cm2,and with dose dependent manner.Typical morphologic features of apoptosis appeared characterizedly by marked chromatin condensation,nuclear pyknosis and fragmentation,and the appearance of apoptotic bodies.The total apoptosis rate was higher in PDT group [（47.21 ± 4.11）%]than in control group（1.71 ±0.16） % （P〈0.01）.The mRNA expression of Bcl-2,P53 and survivin in H8 cells were suppressed significantly.HE staining showed remarkable subcutaneous necrosis in the PDT group.Immunohistochemistry showed remarkable down-regulation of protein expression of Bcl-2（P〈0.01）.Conclusions Curcumin-mediated photodynamic therapy has a significant killing effect on H8 cells in vivo and in vitro.Its antitumor effect might be related to induction of Tumor cell apoptosis and suppression of Bcl-2 mRNA and protein expression.