阳离子卟啉对宫颈癌细胞株Caski的光动力学杀伤效应

摘要

Objective To investigate the photodynamic effect of meso-5,10,15,20-Tetrakis-(N-methyl-4-pyridyl) (TMPyP4) on cervical cancer cell line Caski and possible mechanisms, and provide a foundation theory of PDT in both animal model and clinic. Methods The cervical cancer cell line Caski were divided into 4 groups as follows:normal control group,simple PDT group,simple TMPyP4 group and TMPyP4-PDT group. The in vitro suppressive effect of TMPyP4-PDT on the proliferation of Caski cells was measured by MTT colorimetry, and the morphology of the cells was examined under light microscope. The cell apoptosis was measured by flow cytometry (FCM) with An-nexin V/PI staining. The expression levels of Ki-67,MCM2,P16,CA-Ⅸ and NF-κB mRNA were detected by RT-PCR. Results The sup-pressive effect of simple TMPyP4 group and TMPyP4-PDT group gradually increased with the increase of the dosage (P < 0.01). The sup-pressive effect of simple PDT group was not significant (P > 0.05). More adherent cells without nuclear pyknosis and nuclear fragmentation of simple PDT group were observed under the light microscope; cytoplasmic vacuolization, cell shrinkage, nuclear fragmentation and apoptotic body formation of simple TMPyP4 group and TMPyP4-PDT group were observed under the light microscope. FCM analysis showed that TMPyP4-PDT could induce the apoptosis of Caski cells in a dose dependent manner. RT-PCR analysis showed that, with the increase of the dosage,the expressions of Ki-67,MCM2, CA-Ⅸ and NF-κB mRNA were significantly decreased and the expressions of P16 mRNA were sig-nificantly increased (all P < 0.01,compared with normal control group). Conclusion TMPyP4-PDT has distinctive inhibition effects on cervical cancer cell line Caski and the mechanism may relate to increased activity of P16 and reduced expressions of Ki-67,MCM2,CA-Ⅸ and NF-κB mRNA.%目的 探讨阳离子卟啉(TMPyP4)对宫颈癌细胞株Caski的光动力学杀伤效果和可能作用机制,为光动力学治疗(PDT)在宫颈癌动物荷瘤模型以及临床应用中提供理论依据.方法 以宫颈癌细胞株Caski为研究对象,以TMPyP4为光敏剂,按照不同处理因素分为正常对照组、单纯PDT组、单纯TMPyP4组和TMPyP4-PDT组.采用四甲基偶氮唑蓝法检测TMPyP4-PDT对宫颈癌细胞株Caski体外生长的抑制作用,光镜下观察细胞形态变化.采用Annexin V-PI双标记法检测细胞凋亡率.采用实时荧光定量PCR检测TMPyP4-PDT对Caski细胞Ki-67、MCM2、P16、CA-Ⅸ和NF-κB mRNA表达水平的影响.结果 单纯TMPyP4组和TMPyP4-PDT组对细胞增殖有明显的抑制作用(P＜0.01),且呈剂量依赖性；单纯PDT组对细胞增殖的抑制作用不明显(P＞0.05).光镜下可观察到单纯PDT组贴壁细胞多,无核固缩及核碎裂样改变；单纯TMPyP4组及TMPyP4-PDT组细胞出现胞质空泡化、细胞体积缩小、细胞核碎裂及凋亡小体形成等.流式细胞术检测结果显示,TMPyP4-PDT可诱导Caski细胞凋亡,且随着药物浓度的增加,细胞凋亡百分比增加.实时荧光定量PCR检测发现,随着药物浓度的增加,Caski细胞Ki-67、MCM2、CA-Ⅸ和NF-κB mRNA表达水平明显降低,P16 mRNA表达水平升高,与正常对照组比较差异均有统计学意义(P＜0.01).结论 TMPyP4-PDT对宫颈癌细胞株Caski有明显的杀伤作用,其机制可能与正性调节P16以及负性调节Ki-67、MCM2、CA-Ⅸ和NF-κB mRNA表达量有关.