【作者】 卢春来；

【导师】 王春生； 葛棣； 纪元； 王群；

【作者基本信息】 复旦大学 ， 心胸外科学， 2009， 博士

【摘要】 第一部分食管鳞形细胞癌组织中CXCR4和相关蛋白MMP-9、VEGF的表达及临床意义研究背景和目的食管癌是一种常见的消化道肿瘤,以外科手术为主、辅以放化疗是该疾病目前的标准治疗模式,但根治性手术带来的巨大创伤以及放化疗导致的严重不良反应限制了传统治疗的发展。研究食管癌侵袭和转移机制、寻找新的分子靶点成为食管癌治疗的新方向。近年研究发现趋化因子受体CXCR4在多种肿瘤中均有表达,并且和肿瘤的侵袭、血管新生、转移及预后相关。基质金属蛋白酶-9(MMP-9)和血管内皮生长因子(VEGF)分别是肿瘤侵袭及血管新生的重要分子。本部分实验主要探讨食管鳞形细胞癌组织中CXCR4、MMP-9和VEGF表达水平及三者相关性,同时分析它们与患者临床病理学特征、预后的关系。方法收集2005年于我院行根治性开胸手术的127例食管鳞形细胞癌患者的病史和随访资料;同时收集他们的蜡块标本,根据免疫组化染色面积和染色强弱判断肿瘤组织中CXCR4、MMP-9和VEGF的表达水平;采用Kaplan-Meier法(单因素分析)及Cox回归(多因素分析)分析相关因素和预后的关系。结果CXCR4、MMP-9和VEGF在食管鳞形细胞癌组织中的表达阳性率分别为88.2%、93.7%及79.5%;CXCR4表达与肿瘤分化、肿瘤大小、肿瘤浸润深度、区域淋巴结转移以及TNM分期相关(P＜0.05);MMP-9表达与患者年龄及肿瘤分化具有相关性(P＜0.05);VEGF表达与肿瘤分化、肿瘤浸润深度及TNM分期相关(P＜0.05):CXCR4与MMP-9(P＜0.01,r=0.365)及CXCR4与VEGF(P＜0.01,r=0.380)表达之间存在正相关,MMP-9与VEGF表达之间不存在相关性(P＞0.05);单因素分析显示CXCR4表达、肿瘤大小、肿瘤浸润深度、区域淋巴结转移、TNM分期与患者预后相关(P＜0.05),多因素分析发现肿瘤大小及区域淋巴结转移是食管癌患者的独立预后不良因素(P＜0.01)。结论CXCR4在食管鳞形细胞癌组织中高表达,并且和MMP-9、VEGF、患者临床病理学特征及预后密切相关,推测CXCR4在食管鳞形细胞发展过程中起着重要作用,并且可能参与了肿瘤细胞对MMP-9、VEGF分泌的调控。第二部分CXCR4表达下调对Eca109细胞株的增殖、凋亡、侵袭及MMP-9、VEGF表达的影响研究背景和目的随着分子生物学的发展,基因靶向治疗成为肿瘤研究的热点。作为基因靶向药物的小分子酪氨酸激酶抑制剂(吉非替尼、厄洛替尼)已经应用于非小细胞肺癌的临床治疗,并且取得了一定的疗效。但至今还没有一种有效的靶向药物应用于食管癌的临床治疗。第一部分的研究发现,CXCR4在食管癌组织中高表达,并且和患者临床病理特征、预后以及MMP-9、VEGF表达密切相关。本部分实验将通过体外细胞实验进一步探讨CXCR4基因下调对食管癌Eca109细胞株增殖、凋亡、侵袭及MMP-9、VEGF表达的影响。方法针对CXCR4基因,构建4条RNA干扰序列,以质粒为载体转染Eca109细胞株,利用荧光实时定量PCR及Western印迹法检测质粒干扰效果,筛选出效果最佳干扰质粒;利用最佳干扰质粒转染Eca109细胞株,下调CXCR4基因表达,之后分别采用MTT法、流式细胞仪、Transwell小室实验、Western印迹法检测细胞增殖、凋亡、侵袭能力以及MMP-9、VEGF的表达水平。结果荧光实时定量PCR及Western印迹法检测发现,2号质粒对食管癌CXCR4基因干扰效果最佳。之后,利用2号干扰质粒转染Eca109细胞株,进行相关实验。结果发现,与阴性对照组(无质粒组)、非特异性干扰组相比较,转染CXCR4干扰质粒的细胞侵袭能力(P＜0.05)以及MMP-9、VEGF表达水平明显下降(P＜0.01):但是,细胞增殖及凋亡没有明显变化(P＞0.05)。结论CXCR4调控MMP-9、VEGF的表达,推测CXCR4在食管癌Eca109细胞侵袭及血管新生过程中起着重要作用;CXCR4将来可能会成为食管癌基因治疗新的靶点。更多还原

【Abstract】 BACKGROUNDEsophageal cancer is a common disease of alimentary tract. The standard treatment is multimodality therapy, which is combined surgery with chemotherapy and radiotherapy. However, the trauma due to operation and the adverse effects of adjuvant treament restrict the development of multimodality therapy. To investigate the mechanism of invasion, metastasis and then find out the molecular target have become the new focus in the treatment of esophageal cancer. In recent years, CXCR4 was found to be expressed in many tumors and significantly correlated with invasion, angiogenesis, metastasis and prognosis. MMP-9 and VEGF were the important fators in tumor invasion and angiogenesis, respectively. In this study, we will investigate the expressions of CXCR4, MMP-9 and VEGF in esophageal squamous cell cancer and analyze their associations with patients’s clinicopathological features and prognosis.METHODSWe collected the paraffin-embedded samples, as well as medical records and follow-up data of 127 patients with esophageal cancer, who were treated by thoracic surgery in Zhongshan Hospital in 2005. The CXCR4, MMP-9 and VEGF expressions in esophageal cancer tissues were evaluated according to the immunohistochemical staining area and intensity. The associations between patients’ prognosis and covariates were analyzed by Kaplan-Meier method (univariate analysis) and Cox regression (multivariate analysis).RESULTSThe overall expression rate of CXCR4, MMP-9 and VEGF was 88.2%, 93.7% and 79.5%, respectively. CXCR4 expression was significantly associated with tumor grade, tumor size, tumor depth, regional lymph node metastasis and TNM stage (P<0.05). MMP-9 expression was significantly associated with age and tumor grade (P<0.05). VEGF expression was significantly associated with tumor grade, tumor depth and TNM stage (P<0.05). CXCR4 expression was positively correlated with MMP-9 expression (P<0.01, r =0.365) and VEGF expression (P<0.01, r=0.380). However, there was no significant association between MMP-9 and VEGF expressions (P>0.05). In univariate analysis, CXCR4 expression, tumor size, tumor depth, regional lymph node metastasis and TNM stage were correlated with patients’ prognosis (P<0.05); in multivariate analysis, tumor size and regional lymph node metastasis were the independent factors of poor prognosis.CONCULSIONSCXCR4 was highly expressed in esophageal cancer and correlated with MMP-9, VEGF, clinicopathological features and prognosis. We speculated CXCR4 play an important role during the progression of this disease and CXCR4 might participate in the regulation of MMP-9 and VEGF secretions. BACKGROUNDTargeted thearpy has become the focus of cancer research with the development of molecular biology. As a typical targeted drug, the small-molecule tyrosine kinase inhibitors (Gefitinib, Erlotinib) have been approved for clinical treatment of no small cell lung cancer and got the promising effect. However, there was no effective targeted drug used in clinical treatment for esophageal caner. Through the previous study of Part 1, we found that CXCR4 was highly expressed in esophageal cancer and significantly associated with patients’ clinicopathological features, prognosis and MMP-9, VEGF expressions. The aim of this study was to investigate the influence of CXCR4 downregulation on proliferation, apopotosis, invasion and MMP-9, VEGF expressions in Eca109 cell line in vitro.METHODSFour RNA interference sequences targeted for CXCR4 were constructed and transfected into Eca109 cell with the vector of plasmid. The best effective RNA interference plasmid was selected by Real time PCR and Western blot. After downregulating the expression of CXCR4 in Eca109 cell by the best effective plasmid transfection, the cell proliferation, apoptosis, invasion and the expressions of MMP-9 and VEGF were detected by MTT assay, flow cytometry, transwell assay and Western blot, respectively.RESULTSNumber 2 plasmid showed the best RNA interference effect according to the detection of Real time PCR and Western blot. And then, Eca109 was transfected with Number 2 plasmid for the experiments. Compared with the negative control (no plasmid transfection) and the nonspecific control, the invasion ability of Eca109 cell transfected with RNA interference plasmid was significantly lower (P<0.05), as well as the expressions of MMP-9 and VEGF (P<0.01). However, no difference was foundin cell proliferation or apoptosis (P>0.05).CONCULTIONSMMP-9、VEGF expressions were regulated by CXCR4, which would act as an important role in Eca109 cell’s invasion and angiogenesis; CXCR4 might be a novel therapeutic target of esophageal cancer in the future.更多还原