【作者】 吴欣；

【导师】 王杰军；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2008， 博士

【摘要】 背景胃癌是全世界最常见的恶性肿瘤之一,其发病率位于恶性肿瘤第四位,居癌症死因的第二位。手术切除是早期胃癌的标准治疗,可是,80%至90%患者确诊时已是晚期,不能手术根治,全身化疗是晚期胃癌患者的主要治疗手段。虽然随机临床研究已证实化疗可以为晚期胃癌患者带来生存益处,但标准化疗常有严重的不良反应,从而降低患者的生活质量,也严重限制了化疗的使用。为了避免传统化疗引起的问题,研究者开始寻找新的用药方式,旨在提高抗肿瘤和/或抗转移效果,降低毒性。血管生成(angiogenesis)对维持原发肿瘤的生长和转移至关重要。自1971年Folkman首次提出针对肿瘤血管生成的治疗以来,血管生成已成为癌症治疗一个靶点。研究发现,某些细胞毒性药物的确能抑制血管生成,尤其是以小剂量、高频率给药时。与最大耐受剂量(maximum tolerable dose,MTD)化疗相比,小剂量、高频率或持续给药对肿瘤血管内皮的靶向作用更强。这种化疗方案称为“节拍性化疗”(metronomic chemotherapy)或“抗血管生成化疗”(antiangiogenicchemotherapy)。由于节拍性化疗以肿瘤血管内基因稳定、活化的内皮细胞为治疗靶点,降低了耐药性的发生,提高了治疗效果,又因其剂量较低,还能降低化疗的毒副作用。为了探讨胃癌治疗治疗新方法,本课题以MKN-45荷瘤裸鼠为模型,研究节拍性化疗的抗肿瘤血管生成作用。研究证实,血管生成并不是肿瘤获得微循环的唯一机制。肿瘤细胞在行为上与胚胎干细胞类似。肿瘤细胞可模拟其他类型细胞,对微环境变化作出适应性应答。肿瘤细胞表达多种细胞表型基因的能力,称为肿瘤细胞可塑性。肿瘤细胞表达内皮细胞相关基因,形成输送液体的管道结构,即称为血管生成拟态(vasculogenic mimicry,VM),其是肿瘤可塑性的一种表现。研究证实,人胃癌细胞系可表达一系列与上皮细胞生理相关的基因,如Von Willebrand因子、CD31、VE-钙粘蛋白(VE-cadherin),表明胃癌细胞具有肿瘤细胞可塑性,提示胃癌中可能存在血管生成拟态。基于以上研究的启示,本课题拟在荷瘤裸鼠中研究MKN-45肿瘤细胞是否具有形成VM的能力,以及节拍性化疗对VM是否具有抑制作用。目的确定优福定(UFT)节拍性化疗的最佳生物学剂量(optimum biologic dose,OBD),以及在MKN-45荷瘤裸鼠中检测UFT节拍性化疗抗血管生成效应和抑制血管生成拟态作用,并比较UFT节拍性化疗与MTD UFT和CTX节拍性化疗的疗效。材料与方法1.MKN-45、SGC-7901人胃癌细胞系、Huh-7人肝癌细胞系和U87人神经胶质瘤细胞系接种至裸鼠皮下形成皮下移植瘤。移植后3周处死裸鼠,三色流式细胞术检测MKN-45、SGC-7901、Huh-7和U87荷瘤裸鼠外周血内皮祖细胞(circulatingendothelial progenitor cells,CEPs)。使用小鼠内皮VEGF受体2(flk-1)、Sca-1和CD117来定义CEP。以0、5、10、15、20、25mg/kg/d UFT治疗MKN-45荷瘤裸鼠,治疗3周处死,流式细胞术检测CEPs。2.将未荷瘤裸鼠作为空白组,MKN-45荷瘤裸鼠用生理盐水组、UFT节拍性化疗(20mg/kg/d),MTD UFT(50mg/kg,5天连续使用)和CTX节拍性化疗(20mg/kg/d)。治疗1周、2周和3周时,每组处死5只,流式细胞术检测CEPs。治疗3周后免疫组化检测肿瘤微血管密度(MVD)。3.MKN-45荷瘤裸鼠分组为:生理盐水组(A)、UFT LMD(20mg/kg/d)组(B),UFTMTD(50mg/kg,5天连续使用)组(C)。UFT治疗1周、2周和3周时,每组处死5只。H&E和PAS染色肿瘤切片,显微镜观察,计数血管生成拟态(VM)。免疫组化检测CD31表达。采集第3周裸鼠血浆,ELISA法检测人血管内皮生长因子(VEGF)。结果1.SGC-7901、MKN-45、Huh-7和U87等动物模型中CEP水平升高。U87荷瘤裸鼠中CEPs数最高,而Huh-7荷瘤裸鼠中CEPs数最低。CEPs数与癌细胞系具有一致性。2.使用CEPs作为标记物,确定UFT节拍性化疗的OBD为20mg/kg。3.UFT节拍性化疗可抑制MKN-45荷瘤裸鼠中CEP和MVD,但CTX节拍性化疗却均未抑制CEP流动和MVD。4.MKN-45肿瘤细胞在活体内可形成VM。UFT节拍性化疗抑制VM形成,而MTD UFT却没有。结论1.不同荷瘤裸鼠中外周血CEP水平是不同的。2.外周血CEP检测确定UFT的OBD为20mg/kg。3.UFT可通过抑制血管生成,对MKN-45胃癌产生抗肿瘤效应。4.UFT节拍性化疗可抑制MKN-45胃癌的VM。更多还原

【Abstract】 BackgroudGastric carcinoma remains a common disease worldwide.It represents the fourth most frequent malignancy and second leading cause of cancer-related death worldwide.Surgical resection is the standard treatment for patients with early-stage gastric cancer.Howerver, 80 to 90%of all patients are either diagnosed at an advanced stage when the tumour is inoperable.Patients with advanced disease are mainly treated with chemotherapy. Although several randomised trials have provided evidence that chemotherapy improves survival in these patients,the standard chemotherapeutic regimens often seriously impair the quality of life and cause serious side effects,which pose serious constraints on the use of chemotherapy.In order to avoid the problems caused by traditional chemotherapeutic treatments, several researchers began to search for new modalities of drug administration oriented towards a more efficient and non-toxic antitumoral and/or antimetastic therapy. Angogenesis is necessary to sustain the growth of both the primary tumor and the development of metastases.Therapeutic targeting of tumor angiogenesis was first proposed by Folkman in 1971.From then on,the concept of angiogenesis as a therapeutic target in cancer has gained considerable momentum.Experiments had shown that some cytoxic drugs inhibited angiogenesis effectively,especially when administered more frequently and at lower dosage.Compared to maximum tolerable dose(MTD) chemotherapy,drugs being given at lower doses more frequently have stronger target effects on tumor vessel endothelial cells.This type of regimen is called metronomic chemotherapy or antiangiogenic chemotherapy.The metronomic chemotherapy affects the actively dividing and genetically stable endothelial cells,thereby reducing the chances of drug resistance to minimum.Because of its lower dosage,and more frequent schedule,it has lower toxicity. To develop a new approach for the treatment of gastric cancer,we want to explore whether antiangiogenic effect will be seen in nude mice bearing gastic cancer cells with this therapeutic approaches.Recent evidences suggest that angiogenesis may not be the only mechanism by which tumors acquire a microcirculation.Tumour cells are able to behave like stem cells. Therefore,cancer cells can mimic other cell types and can adapt to microenvironmental changes.Tumour cell plasticity refers to the ability of tumour cells to express genes associated with multiple cellular phenotypes.Vasculogenic mimicry,in which tumour cells express genes associated with endothelial cells and form fluid-conducting channels,is an example of this plasticity.Some evidences suggested that gastric carcinoma cell lines expressed a set of genes,many of which had been implicated in epithelial cell biology, such as Von Willebrand factor,CD31,VE-cadherin.That means gastric carcinoma cells may be have tumor cell plasticity,and form VM in vivo.Based on these evidences,we want to explore wethere MKN-45 tumor cells can form VM in vivo and the effect of metronomic chemotherapy on VM.ObjectTo assess endothelial progenitor cells(EPCs) in the peripheral blood of SGC 7901, MKN45,Huh-7 and U87 xenograft in BALB/c nude mice,to determined the optimum biologic dose(OBD) of metronomic LIFT,and to assess antiangiogenic effect and its effect on vasculogenic mimicry of metronomic UFT in human MKN-45 gastric cancer,compare its efficacy with MTD chemotherapy and metronomic CTX.Materials and Methods1.MKN-45 and SGC-7901 human gastric carcinoma cell lines,Huh-7 human hepatoma cell lines and U87 human glioblastoma cell lines were inoculated subcutaneously in nude mice to develop s.c.growing tumors.Nude mice were sacrificed at the 3th week after tumor implantation,EPCs in the peripheral blood of SGC-7901, MKN-45,Huh-7 and U87 xenograft in BALB/c nude mice were measured by 3-color flow cytometry.CEP(EPC in the peripheral blood) subset were depicted using the endothelial murine markers VEGF receptor 2 fetal liver kinase 1(flk-1),Sca-1,and CD117.After nude mice bearing MKN-45 human gastric cancer cell lines were treated with 0,5,10,15,20, 25mg/kg/d UFT,mice were sacrificed at 3th week,evaluation of CEPs was carried out on blood by flow cytometry.2.Except for blank group,which was tumor-free nude mice,xenografts of MKN-45 gastric cancer cell lines were subjected to either continuous treatment with normal saline, low doses of UFT(20mg/kg/d),maximum tolerable doses of UFT(50mg/kg,5 consecutive days) and low doses of cyclophosphamide(CTX,20mg/kg/d).At the 1th,2th,3th week after the treatment,5 in each group were sacrificed,and evaluation of CEPs was carried out on blood by flow cytometry.Tumor microvessel density was evaluated by immunohistochemistry at the 3th week after the treatment.3.Nude mice bearing MKN-45 human gastric carcinoma cell lines were deviding into three group:control group(A),UFT LMD(20mg/kg/d) group(B),UFT MTD (50mg/kg,5 consecutive days) group(C).At the 1th,2th,3th week after the treatment with normal saline or UFT,5 in each group were sacrificed.The morphological characteristics of slides of tumor specimens stained with H&E and PAS were examined in microscope, and counted the numbers of vasculogenic mimicry(VM).Immunohistochemical staining was used to analyze the expression of CD31.At 3th week after the treatment,the mouse sera were collected and measured for human VEGF by ELISA.Results1.In immunodeficient mice bearing MKN-45 and SGC 7901 human gastric carcinoma cell lines,Huh-7 human hepatoma cell lines and U87 human glioblastoma cell lines,increases in the levels of CEPs were observed.Nude mice bearing U87 human glioblastoma cell lines expressed the highest numbers of CEPs.In contrast,nude mice bearing Huh-7 human hepatoma cell lines were found to have much lowest numbers of CEPs.Moreover,we also observed a correlation between the number of CEPs and cancer cell lines.Using CEPs as a biomarker,we determined that the OBD of metronomic UFT administered daily was 20mg/kg.2.In immunodeficient mice bearing MKN-45 human gastric carcinoma cell lines, metronomic UFT inhibited the mobilization of CEPs and reduced MVD,but metronomic CTX didn’t,although in group CTX LMD,inhibiton rate of tumor was higher than in group UFT LMD.We observed a correlation between the number of CEPs and MVD.3.MKN-45 tumor cells were ability to form VM in vivo.We found metronomic UFT inhibited the VM formations by MKN-45 gastric cancer cells,but MTD UFT didn’t.Conclusion1.There are different levels of CEPs in the nude mice bearing different cancer cells.2.We determine the OBD of metronomic UFT by detection of CEPs was 20mg/kg.3.Metronomic UFT has antiangiogenic effect in human MKN-45 gastric cancer.4.Metronomic UFT can inhibited VM formations by MKN-45 gastric cancer cells.更多还原