【作者】 陈景；

【导师】 陈龙邦；

【作者基本信息】 南京大学 ， 肿瘤学， 2012， 硕士

【摘要】 研究背景与目的：食管癌是来源于食管粘膜上皮的肿瘤,包括鳞状细胞癌和腺癌,在亚洲以食管鳞癌为主,我国是食管癌的高发区,尤以河南林县为重。食管癌的发病率在所有肿瘤中为第8位,死亡率在所有肿瘤中为第6位,90%的患者确诊时已为Ⅲ期或Ⅳ期。放疗、化疗仍为晚期食管癌患者的主要治疗手段,但晚期食管癌患者对化疗的反应率仅20～40%,中位生存期为8～10个月,如何提高食管癌患者对放化疗的敏感性仍然是一个研究热点。分子靶向治疗是近年研究的热点,其中靶向于表皮生长因子受体(epidermal growth factor receptor, EGFR)通路的药物研究最为深入。EGFR是表皮生长因子受体,在许多肿瘤细胞中表达,而食管癌组织多可检测到EGFR的过表达,其中以食管鳞癌多见。针对EGFR的靶向药物包括酪氨酸激酶抑制剂(Tyrosine kinase inhibitors,TKI)和抗EGFR抗体。尼妥珠单抗是人源化EGFR局粘附性单克隆抗体,能够特异性识别EGFR胞外Ⅲ区结构域,阻断EGFR下游信号通路,具有选择性强、人源化程度高、半衰期长、毒副反应较小等特点。体内外试验证实尼妥珠单抗具有促进肿瘤细胞凋亡、抑制肿瘤细胞增殖、抗肿瘤血管生成等作用。尼妥珠单抗能够提高头颈部恶性肿瘤的放、化疗敏感性,我国已批准尼妥珠单抗用于头颈部恶性肿瘤的治疗。目前尼妥珠单抗在头颈部恶性肿瘤中的研究相对较多,而在食管癌中的研究较少,本实验研究尼妥珠单抗对食管鳞癌Eca109、TE-1细胞化、放疗敏感性的影响,并初步探讨其可能的机制。方法：1.流式细胞仪检测食管鳞癌Eca109、TE-1细胞EGFR表达的平均荧光强度,免疫组化法检测TE-1、Eca109细胞EGFR的表达水平。2.MTT法检测尼妥珠单抗、紫杉醇、顺铂、5-Fu单药以及尼妥珠单抗分别联合紫杉醇、顺铂、5-Fu对Eca109、TE-1增殖的影响。3.细胞克隆形成实验检测尼妥珠单抗对Eca109、TE-1细胞放疗敏感性的影响。4.流式细胞仪分析尼妥珠单抗对Eca109、TE-1细胞周期分布的影响。5.流式细胞仪分析尼妥珠单抗分别与紫杉醇、顺铂、5-Fu联合对Eca109、TE-1细胞凋亡的影响。6.透射电镜观察尼妥珠单抗对食管癌Eca109、TE-1细胞自噬的影响。7. RT-PCR、Western blotting方法检测尼妥珠单抗对Eca109、TE-1细胞becline-1mRNA及蛋白水平的影响。结果：1.Eca109细胞EGFR表达平均荧光强度(MFI)为66.08,TE-1细胞平均荧光强度为123.17,免疫组化Eca109细胞EGFR表达为+++,TE-1细胞+。2.尼妥珠单抗增强紫杉醇、顺铂对Eca109细胞的杀伤效应,但不能增强紫杉醇、顺铂对TE-1细胞的杀伤效应,尼妥珠单抗未增强5-Fu对Eca109、TE-1细胞的杀伤效应。3.尼妥珠单抗增强Eca109细胞的放疗敏感性,但对TE-1细胞的放疗敏感性无明显影响。4.尼妥珠单抗明显增强紫杉醇、顺铂诱导的Eca109细胞凋亡,但未增强5-Fu诱导的Eca109细胞凋亡;而在TE-1细胞中,尼妥珠单抗对三种化疗药物诱导的凋亡无明显增强作用。5.尼妥珠单抗诱导Eca109细胞G0/G1期细胞周期阻滞,S期细胞减少；但对TE-1细胞的周期分布无明显影响。6.尼妥珠单抗诱导Eca109细胞自噬增强,对TE-1细胞自噬无明显影响。7.尼妥珠单抗诱导Eca109细胞Becline-1mRNA和蛋白水平表达上调,对TE-1细胞Becline-1的表达无影响。结论：1.尼妥珠单抗增强EGFR高表达的食管鳞癌Eca109细胞的化、放疗敏感性,而对EGFR低表达的食管鳞癌TE-1细胞的化、放疗敏感性无明显影响。2.尼妥珠单抗增强食管鳞癌Eca109细胞的化、放疗敏感性的机制可能与诱导细胞凋亡增加、细胞周期G0/G1期阻滞、自噬增强等机制有关。更多还原

【Abstract】 Background and objective:Esophageal cancer which derived from the mucous membrane of esophagus includes squamous cell carcinoma and adenocarcinoma.The main histological typle of esophageal cancer is squamous cell carcimoma in our country. Esophageal cancer is the eighth most common cancer worldwide. Most patients are in III/IV stage when diagnosed, especially in developing countries, which also results in a poor prognosis. Chemoradiotherapy are still reserved as a significant approach to patients suffering from advanced carcinoma, however, the effectiveness was limited, only20to40%of patients were observed to be responsive to it. Thus, how to improve the therapeutic benefit remains to be a research hotspot. Nimotuzumab is a humanized monoclonal antibody which can bind to domain III of the extracellular region of the EGFR with a moderate affinity and inhibits its downstream signal pathway. Compared to other annti-EGFR monoclonal antibodies, it carries a slight side effect. Nimotuzumab revealed a remarkable anti-proliferative, pro-apoptotic and anti-angiogenic effect in vitro as well as in vivo studies and enhanced the radiotherapy sensitivity of malignant tumors, such as glioma and nasopharyngeal carcinoma. Due to its modulation effect on radiochemotherapy sensitivity, it has been combined with chemotherapeutic drugs and radiotherapy. Moreover, it has been approved in the treatment of different tumors in different countries. In our contry, it has been recommended for the treatment of head and neck cancer patients. In this study, we observed the effect of nimotuzumab on the radiochemotherapy sensitivity of esophageal squamous cancer cells and explored the mechanism underlying the combinational scheme.Methods:1.The mean fluorescence intensity of EGFR expression on TE-1and Eca109esophageal cancer cells were analyzed by Flow cytomy, while, the degree of EGFR expression was detected by immunohistochemistry.2. MTT assay was applied to evaluate the effect of the combination of nimotuzumab and paclitaxel(PTX)/cis-platinum(DDP)/5-florouracil(5-Fu)on the proliferation of Eca109and TE-1esophageal cancer cells.3. The effect of h-R3on radiotherapy sensitivity of Eca109and TE-1esophageal cancer cells was observed by Cell cloning experiments.4. Flow cytometry was used to analyse the changes of apoptosis rate of Eca109and TE-1cells induced by the the combination scheme.5. The changes of cell cycle distribution underlying the Eca109and TE-1cells were also detected by flow cytometry.6. Transmission electron microscope reserved as a significant method to observe the effect of nimotuzumab on the autophagy of Eca109and TE-1cells.7. The mRNA expression level of Becline-1expressed on Eca109and TE-1cells were analyzed by RT-PCR and the protein expression level of becline-1was measured by western blotting.Results:1. Eca109cells highly expressed EGFR, but TE-1cells carried a low expression level of EGFR.2. Nimotuzumab could enhanced the response of PTX/DDP/5-Fu to Eca109cells, but no enhancement was observed on TE-1cells.3. Nimotuzumab could enhanced radiotherapy sensitivity of Eca109cells, however, it have no obvious effect on that of TE-1cells.4. The combination of nimotuzumab and PTX/DDP induced a higher apoptosis rate underlying the Eca109cells, but had no obvious effect on apoptosis rate of TE-1cells.5. The retardant of G0/G1phase of the cell cycle and the decrease of S phase of the cell cycle induced by nimotuzumab were observed in Eca109cell line but not in TE-1cell line.6. Nimotuzumab induced an enhancement of autophagy existing in Eca109cells, but had no apparent effect on that of TE-1cells.7. Nimotuzumab could up-regulate Becline-1expression on Eca109cells but exerted no effect on expression level of Becline-1in TE-1cells Conclusion:1. Nimotuzumab could enhance the chemosensitivity and radiosensitivity of Eca109esophageal cancer cells which possess of a high level of EGFR expression, but the TE-1cells with a low level of EGFR expression revealed no obvious response to nimotuzumab.2. The superiority of the combinational project was achieved by a series of possible mechanisms, such as the the retardant of G0/G1phase of the cell cycle and the enhancement of apoptosis and autophagy.更多还原