【作者】 程鑫；

【导师】 李凯； 于津浦；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:近年来,肺癌的发病率和死亡率持续上升,已居各种恶性肿瘤的首位:其中非小细胞肺癌（non-small cell lung cancer,NSCLC）占80%左右,而确诊时约50%的患者为不可手术的晚期患者。化疗在NSCLC的综合治疗中占有重要地位,但化疗效果差,容易发生耐药。1971年Folkman教授首次提出肿瘤血管生成依赖学说,提出血管生成不仅在肿瘤的生长过程中必需,而且在肿瘤的侵袭和转移阶段也发挥作用,抗血管生成疗法成为肿瘤治疗领域的一线曙光。血管内皮抑素（Endostatin）为内源性血管生成抑制剂,能特异性地作用于血管内皮细胞,抑制其生长和迁移、诱导其凋亡,从而抑制血管形成和肿瘤生长。近年来也有研究表明,它还可直接抑制肿瘤细胞生长、诱导肿瘤细胞凋亡,但关于其具体作用机制及信号传导通路尚不十分清楚。恩度^TM（Endostar）是由我国自主研发的新型重组人血管内皮抑素,临床试验结果显示其具有良好的临床应用前景,且与化疗有增效作用,然对其增效机制、作用特点及最佳用法方面的研究尚不十分清楚。我们选用Calu-6人肺腺癌细胞系,观察:1、血管内皮抑素（下简称内皮抑素）对肺癌细胞Calu-6是否具有生长抑制作用、内皮抑素是否能够诱导肺癌细胞凋亡及其相关机制。2、内皮抑素与顺铂（DDP）的联合是否具有协同作用,如果有,则进一步探讨最佳联合用药的时间、剂量关系及可能的相关机制,以期指导临床联合用药。3、两种内皮抑素（Endostatin、Endostar）的作用效果有无差异,以期了解各种血管内皮抑素对肿瘤细胞的作用是否具相同机制、指导制定各血管内皮抑素的共同用药原则。方法:1、MTT法测定各药物对肺癌细胞Calu-6的生长抑制率。2、流式细胞术检测细胞凋亡及坏死率。3、ELISA检测细胞凋亡相关蛋白（Bcl-2/Bax、sFas/sFasL）的表达。结果:内皮抑素具有抑制Calu-6细胞增殖的作用（P＜0.05）并呈现时间依赖性、与剂量无明显依赖、但存在最佳作用浓度;其还可诱导Calu-6细胞凋亡、下调Bcl—2表达、但对Bax的表达无明显影响、也未检测到sFas/sFasL的表达。在内皮抑素与DDP联合用药方案中,同时给药较单药DDP诱导凋亡及下调Bcl-2表达明显,序贯给药与单药DDP间无明显差异。恩度、内皮抑素的作用效果差异无统计学意义。结论:1、恩度、内皮抑素与DDP对Calu-6细胞均具有生长抑制作用,DDP呈现明显的时间和剂量依赖性,恩度和内皮抑素在一定范围内具有明显时间依赖性、而剂量依赖关系不明显。但恩度与内皮抑素存在一最佳作用浓度,未达到或超过该最佳作用浓度时的抗肿瘤作用可能均不能得到最大发挥。2、恩度、内皮抑素与DDP均能诱导Calu-6细胞凋亡、下调Bal-2的表达,对Bax的表达无明显影响。恩度与内皮抑素的单独作用效果弱于DDP单药;各联合给药方案中,序贯给药较DDP单药作用无明显差异,以同时给药诱导凋亡及下调Bcl-2的表达最明显。实验各组均未检测到对Fas/FasL的表达,关于其是否通过Fas/FasL途径诱导凋亡尚不能明确,有待进一步的深入研究。3、恩度与内皮抑素在抑制Calu-6细胞生长、诱导其凋亡、下调BCl-2的表达等方面作用效果无明显统计学差异,说明两种内皮抑素的作用机制相似;这提示我们可能制定各血管内皮抑素的共同用药原则、指导临床治疗。更多还原

【Abstract】 Objective: Recently, the incidence and mortality of lung cancer continue to increase and get highest in all of malignancy. About 80% of them are non- small cell lung cancer and 50% of patients given the diagnosis of NSCLC belong to advanced disease. Chemotherapy plays a key role in treatment of advanced NSCLC. But, efficacy of chemotherapy is very limited and the phenomenon of drug resistance in NSCLC is very common. 1971 the professor Folkman firstly proposed the theory of tumor depending on angiogenesis. He proposed that the angiogenesis was essential not only in tumor growth, but also in tumor invasion and metastasis. Antiangiogenesis become the first light of morning in tumor therapy. Endostatin is an endogenous inhibitor of angiogenesis, specifically acting on endothelial cells, inhibiting it’s migration, inducing it’s apoptosis, thus inhibiting the forming of new vessels and tumor growth. Recently some research indicated that Endostatin can directly inhibit tumor cell growth, but the mechanism of action and signal conduction pathway is not very clear. Endostar is the new recombinated human endostatin. Clinical trial results indicated that it had a good perspective on clinical application and synergetic effect with chemotherapy. But it is not clear about the synergetic mechanism, action characteristic and best regimen. We choosing Calu-6, the human lung adenocarcinoma cell, to observe the effects of Endostatin and Endostar on human lung cancer cells (Calu-6) proliferation and the apoptosis- induced effects and apoptosis associated mechanism. To observe the synergistic inhibiting effects of Endostatin, Endostar and Cisplatin on Calu-6 cells proliferation. If synergistic effect was defined, we further investigate the optimistic time or dose of the combination regimen and the possible mechanism. To assess whether or not the effects of two Endostatins (Endostar, Endostatin) are different.Methods:The inhibiting effects of Endostatin and Cisplatin on Calu-6 cells proliferation were studied by MTT assay. The cell apoptosis was measured by cell flow cytometry (FCM).The expression of apoptosis associated protein (Bcl-2/Bax、sFas/sFasL) was analyzed by enzyme linked immunosorbent assay (ELISA).Results: Endostatin can inhibit Calu-6 cell proliferation (P<0.05) on time-dependence; no any evidence for its effect on dosage-dependant has been proved, however, an optimistic dosage revealed. Also it can induce apoptosis by down-regulating the expression of Bcl-2 without obvious effect on Bax. The expression of sFas/sFasL hasn’t been found. Among combinative protocol, simultaneous administration of Endostatin and Cisplatin represented more obvious effect on inducing apoptosis and down-regulating Bcl-2 than Cisplatin alone. Successive administration represented no obvious effect than Cisplatin. There was no significant difference on effect between Endostar and Endostatin.Conclusions:Cisplatin inhibit growth of Calu-6 cell on time-dependence and dose-dependence. Endostatin inhibit growth of Calu-6 cell on time-dependence and have an optimistic dosage .Both of them can induce cell apoptosis with the mechanism concerned with the down-regulation of Bcl-2 without obvious effect on Bax. The expression of sFas/sFasL hasn’t been found. It still need more efforts to investigate. Among combinative protocol, simultaneous administration of Endostatin and Cisplatin represented more obvious effect on inducing apoptosis and down-regulating Bcl-2 than Cisplatin alone. Successive administration represented no obvious effect than Cisplatin. The mechanisms of Endostatins’ effect are similar. It can point out to draw up the common principle of endostatin to guide clinical action.更多还原