Notch信号通路在姜黄素介导光动力治疗宫颈癌的作用研究

Cervical cancer is the 2nd most frequently occurred tumor in female reproductive system in the world. And its incidence rate is high and rapidly increasing among young women. Notch signaling pathway closely correlates with carcinogenesis and development of cervical cancer, is a potential control-target to improve the effectiveness of PDT. Traditional treatment, like surgery and radio/chemo therapy, could cause serious damage to ovarian and vaginal functions. While photodynamic therapy (PDT), as its high possibilities to retain patients' organ and reproductive function, is a new and effective noninvasive therapy to cancers with bright prospect. Our preliminary studies demonstrated that, PDT is an effective measure for cervical cancer treatment. ..Curcumin, as a high efficiency and low toxicity anti-cancer drug and photosensitiser, has co-activation and complemention effects with PDT. It has been proven in treatment of some malignant tumor, such as nasopharyngeal carcinoma and basal cell carcinoma...Our study is aiming to find, on both cellular and molecular levels, the optimal interaction parameters of curcumin mediated PDT on cervical cancer cell, Me180; analyze the role of Notch and downstream genes, such as NF-KB, COX-2, VEGF-A, VEGF-C, and VEGFR-3, before and after PDT; based on which, to further observe the influences of PDT on animal model of cervical cancer; identify the possible targets in PDT on cervical cancer through blocking Notch receptor. We hope our studies could provide new ideas and experimental evidences for clinical PDT on cervical cancer.

宫颈癌是当前妇女第二大恶性肿瘤，发病率高且趋于年轻化。Notch信号通路与其发生发展密切相关。手术创伤、放化疗对患者卵巢和阴道功能造成严重毁损。而光动力治疗（PDT）是无创、有效的肿瘤新疗法之一，因能保留器官和生育功能而极具诱人前景。我们前期研究表明，光动力治疗宫颈癌是有效手段。姜黄素作为一种高效、低毒的抗癌新药和光敏剂，与PDT具有协同和互补作用，已应用于鼻咽癌、基底细胞癌等其它恶性肿瘤治疗。本课题拟以姜黄素为光敏剂，通过从细胞、分子水平观察姜黄素介导PDT对宫颈癌Me180细胞的最佳作用参数、分析治疗前后Notch及下游NF-κB、COX-2、VEGF-A、-C、VEGFR-3各环节发生的作用，在此基础上进一步从整体水平观察PDT对宫颈癌动物模型的影响，同时通过阻断剂阻断Notch受体，以明确Notch在PDT治疗宫颈癌中的可能作用靶点，为临床光动力治疗宫颈癌提供新思路和实验依据。

目的：通过从细胞、分子水平观察姜黄素介导PDT对宫颈癌Me180细胞的最佳作用参数、在此基础上进一步观察PDT对宫颈癌动物模型的影响，分析治疗前后Notch及下游NF-κB、COX-2、VEGF-A、-C、VEGFR-3各环节发生的作用，同时通过阻断剂阻断Notch受体，以明确Notch信号通路在PDT治疗宫颈癌中的可能作用靶点。.方法：MTT 法检测Me180细胞存活率、正交试验设计优选最佳作用范围。分析细胞凋亡、细胞周期变化，明确PDT体外杀伤作用。选择宫颈癌Me180细胞系建立裸鼠宫颈癌移植瘤模型，通过体内抑瘤实验、苏木精-伊红常规染色明确体内杀伤效应。采用Real-time PCR、Western Blot、免疫组化等方法综合分析PDT治疗前后和加入阻断剂DAPT的PDT治疗前后Notch1和下游NF-КB、COX2、VEGF-A、VEGF-C、VEGFR-3的基因、蛋白表达的变化。.结果：最佳参数组合为：姜黄素10 μM、激光剂量200 J/cm2、照射360 s、照射后孵育24 h。Hoechst荧光染色和流式检测均表明姜黄素-PDT和1 μM DAPT均可诱导Me180凋亡，加DAPT后有协同交互作用，统计学差异显著。细胞周期分析显示治疗组显著的G0-G1期阻滞，S 期明显减少。裸鼠移植瘤治疗组肿瘤体积在治疗后1-7天保持不变或略减小，与模型组相比有统计学差异。HE染色见各治疗组镜下肿瘤组织区域坏死明显，其中以姜黄素+PDT+DAPT组最为明显。在体内和体外实验中，PT-PCR结果均显示姜黄素-PDT可以抑制Notch1的mRNA表达，但对Notch2抑制不明显。加入DAPT后，在抑制Notch1方面有增效作用。Western blot和免疫组化结果表明：对VEGFR-3和COX-2，各组无明显差异；姜黄素-PDT组对Notch1、NF-κB、VEGF-A、VEGF-C的蛋白表达有明显抑制作用。加入DAPT后，抑制作用更加明显。.结论：姜黄素-PDT光动力疗法在体内、体外对宫颈癌Me180细胞均具显著杀伤效应，对Notch-1以及其下游蛋白NF-κB、VEGF的表达均有显著抑制作用。姜黄素-PDT光动力疗法在Notch受体被DAPT阻断的情况下，还能与DAPT发挥联合增效的抗肿瘤效果，说明Notch信号通路并不是姜黄素-PDT光动力疗法的唯一靶点。