Sox-3在食管鳞癌淋巴转移中的作用及机制研究

发布时间：2018-08-13 09:16

【摘要】：背景与目的在全世界最常见的恶性肿瘤中,食管癌的发病率位居第8,死亡率位居第6位。在过去的40年中,随着诊断、手术和放化疗水平的不断提高,食管癌的5年生存率缓慢升高,但是依然低于19%。食管癌有两种主要的病理类型:食管腺癌和食管鳞癌。中国是世界上食管癌的高发地区之一,其中食管鳞癌占95%,是国内发病率第3、死亡率第4位的恶性肿瘤。和腺癌相比较,食管鳞癌的预后更差,其根治性手术后的10年生存率长期徘徊在20%左右。目前已经证实,淋巴结转移是食管鳞癌最重要的预后因素之一,食管癌淋巴结转移的状态决定肿瘤的分期、治疗方式的选择和预后。但是目前食管鳞癌淋巴结转移的机制尚不清楚,临床上也缺乏有效的预测和治疗食管鳞癌淋巴结转移的方法。Sox-3是一种转录因子,归属于Sox蛋白家族B1亚组。在脊椎动物胚胎发育早期调控神经细胞的发育和分化,并在发育过程中表达逐步降。近年来的研究发现,Sox-3是一种原癌基因,可能和肿瘤的发生发展相关。我们在前期的研究中,发现食管鳞癌组织Sox-3的表达与区域淋巴结转移和预后密切相关,表现为Sox-3高表达患者的淋巴结转移率明显升高,并且预后不良。提示Sox-3的表达可能促进了食管鳞癌的淋巴转移,并影响患者的预后。已有的证据表明,食管鳞癌患者肿瘤相关淋巴管的密度与淋巴结转移和预后相关。而VEGF-C/D-VEGFR3是调控淋巴管生成、淋巴管扩张和通透性主要的信号通路。VEGF-C或VEGF-D的高表达与肿瘤侵犯的深度、肿瘤分期、肿瘤淋巴管的密度、淋巴管受侵和淋巴结转移相关。综上,我们推测Sox-3的表达可能促进食管鳞癌淋巴结的转移,并且和淋巴管生成以及VEGF-C/D-VEGFR3信号系统相关。为了阐明Sox-3在食管鳞癌淋巴转移中的作用及可能的机制,我们利用本实验室已经构建的不同Sox-3表达水平的人食管鳞癌细胞模型,通过体外和体内实验,选择Western blot、RT-PCR、ELISA、免疫组化研究等方法研究,探讨Sox-3在食管鳞癌的发生、发展和淋巴结转移中的作用及其可能的机制,为探索食管鳞癌淋巴结转移的诊断和靶向治疗提供新的方向。方法1.利用本实验室构建的不同Sox-3表达水平的食管鳞癌细胞模型,通过Western blot和q RT-PCR验证其Sox-3的差异表达后,通过细胞培养、MTT增殖实验、流式细胞术、划痕实验、Transwell侵袭实验和条件培养基淋巴管内皮细胞成管实验考察在体外Sox-3的表达,对于人食管鳞癌TE-10细胞增殖、凋亡、侵袭、迁移,特别是诱导淋巴管内皮细胞成管能力的影响。研究Sox-3对于食管鳞癌发生发展和淋巴转移可能的调控作用。2.通过裸鼠右侧腋下注射不同Sox-3表达水平的TE-10细胞,构建裸鼠移植瘤模型。对移植瘤的发生、发展过程、成瘤的大小和同侧腋窝淋巴结的大小进行比较,组织切片HE染色,检查比较淋巴结转移情况;通过Western blot和免疫组化,检测不同Sox-3表达水平的TE-10细胞移植瘤样本中VEGF-C/D的表达,来进一步研究Sox-3对于食管鳞癌淋巴转移的影响。3.在体外细胞培养,通过Western blot和ELISA实验检测抑制Sox-3的表达,对于TE-10细胞VEGF-C/D表达和分泌的影响。在淋巴管内皮细胞成管实验中,加入VEGFR-3的抗体SAR131675阻断淋巴管内皮细胞VEGFR-3的功能,来研究VEGF-C/D通路在Sox-3促进食管鳞癌淋巴结转移过程中可能的作用机制。结果1.通过Western blot实验和q RT-PCR实验证实,TE-10细胞经Sox-3 si RNA慢病毒感染后构建的不同Sox-3表达的人食管鳞癌细胞模型是成功的,对照组TE-10细胞和空载组TE-10细胞Sox-3的表达类似,均显著高于干扰组TE-10细胞。MTT、流式细胞术、划痕实验、transwell实验和条件培养基淋巴管内皮细胞成管实验证实:Sox-3可显著促进人食管鳞癌细胞的增殖、侵袭、迁移和淋巴管内皮细胞成管的能力,而对细胞凋亡有抑制作用。2.成功构建裸鼠移植瘤模型,测量结果显示:和Sox-3低表达的干扰组相比,Sox-3高表达的空载组小鼠移植瘤的生长速度和成瘤大小类似,但是移植瘤同侧腋窝淋巴结的体积显著增大。病理学检查显示Sox-3高表达组小鼠淋巴结的转移率显著增高。Western blot和免疫组化检测显示,空载组移植瘤组织VEGF-C和VEGF-D的表达均显著强于干扰组。表明表明Sox-3对食管鳞癌TE-10细胞体内成瘤和生长的影响不大,但是可能促进食管鳞癌的淋巴结转移,并且和VEGF-C和VEGF-D的表达相关。3.Western blot和ELISA实验结果显示,抑制TE-10细胞Sox-3的表达,可使其VEGF-C和VEGF-D的表达和分泌显著下降;在淋巴管内皮细胞成管实验中,使用SAR131675阻断淋巴管内皮细胞VEGFR-3的功能后,各组TE-10细胞成管的数目均显著下降,并且组间无差异。表明Sox-3在食管鳞癌TE-10细胞可显著上调VEGF-C和VEGF-D的表达和分泌,Sox-3促进淋巴管的生成,是通过VEGF-C/VEGF-D/VEGFR-3通路实现的。结论1.在体外Sox-3可显著促进人食管鳞癌细胞的增殖、侵袭和迁移,抑制细胞凋亡,但是在体内Sox-3对于食管鳞癌细胞成瘤的速度和大小影响不大。2.在体外Sox-3可显著增强人食管鳞癌细胞诱导淋巴管内皮细胞成管的能力,在体内Sox-3可促进食管鳞癌细胞成瘤后的淋巴结转移。3.Sox-3在食管鳞癌细胞的表达,上调肿瘤VEGF-C和VEGF-D的表达和分泌,作用于淋巴管内皮细胞,通过VEGF-C/VEGF-D/VEGFR-3通路,促进淋巴管的生成,是Sox-3促进食管鳞癌的淋巴结转移可能的机制。
[Abstract]:BACKGROUND & OBJECTIVE Among the most common malignant tumors in the world, esophageal cancer ranks eighth in incidence and sixth in mortality. Over the past 40 years, the 5-year survival rate of esophageal cancer has slowly increased, but is still lower than 19% with the improvement of diagnosis, surgery, radiotherapy and chemotherapy. There are two major pathological types of esophageal cancer: adenocarcinoma and carcinoma of the esophagus. Esophageal squamous cell carcinoma is one of the most common esophageal cancers in the world. Esophageal squamous cell carcinoma accounts for 95% of all esophageal cancers in China. It is the third most common malignancy and the fourth leading cause of death in China. Compared with adenocarcinoma, esophageal squamous cell carcinoma has a worse prognosis, and its 10-year survival rate after radical surgery has long hovered around 20%. One of the most important prognostic factors of esophageal squamous cell carcinoma is the state of lymph node metastasis, which determines the stage, treatment and prognosis of esophageal squamous cell carcinoma. Subgroup B1 of the protein family regulates the development and differentiation of neural cells in early embryonic development of vertebrates and gradually decreases their expression during development. Recent studies have shown that Sox-3 is a proto-oncogene and may be associated with the development of tumors. In our previous studies, we found that the expression of Sox-3 in esophageal squamous cell carcinoma tissue and regional lymph nodes. The expression of Sox-3 may promote lymph node metastasis of esophageal squamous cell carcinoma and affect the prognosis of the patients. There is evidence that the density of tumor-associated lymphatic vessels is associated with lymph node metastasis and prognosis in patients with esophageal squamous cell carcinoma. The high expression of VEGF-C or VEGF-D is associated with the depth of tumor invasion, tumor stage, density of tumor lymphatic vessels, lymphatic invasion and lymph node metastasis. In conclusion, we speculate that the expression of Sox-3 may promote lymph node metastasis in esophageal squamous cell carcinoma. In order to elucidate the role of Sox-3 in lymphatic metastasis of esophageal squamous cell carcinoma and its possible mechanism, we used human esophageal squamous cell carcinoma cell models with different levels of Sox-3 expression to select Western blot, RT-PCR, ELISA in vitro and in vivo. To explore the role of Sox-3 in the occurrence, development and lymph node metastasis of esophageal squamous cell carcinoma (ESCC) and its possible mechanism by immunohistochemical methods, and to provide a new direction for the diagnosis and targeted therapy of ESCC with lymph node metastasis. Methods 1. Using the different expression levels of Sox-3 in our laboratory, we constructed a cell model of ESCC. Western blot and Q RT-PCR confirmed the differential expression of Sox-3 in human esophageal squamous cell carcinoma TE-10 cells. The expression of Sox-3 in vitro was examined by cell culture, MTT proliferation, flow cytometry, scratch test, Transwell invasion test and conditioned medium lymphatic endothelial cell tube formation test, especially induction of proliferation, apoptosis, invasion, migration of human esophageal squamous cell carcinoma TE-10 cells. To study the effect of Sox-3 on the development and lymphatic metastasis of esophageal squamous cell carcinoma (ESCC). The effect of Sox-3 on lymph node metastasis of esophageal squamous cell carcinoma was further studied by Western blot and ELISA. The effect of inhibition of Sox-3 expression on the expression and secretion of VEGF-C/D in TE-10 cells was investigated. In the lymphatic endothelial cell tube formation experiment, anti-VEGFR-3 antibody SAR131675 was added to block the function of lymphatic endothelial cells in order to study the possible mechanism of VEGF-C/D pathway in promoting lymph node metastasis of esophageal squamous cell carcinoma. 1. Western blot assay and Q RT-PCR assay confirmed that the human esophageal squamous cell carcinoma cell models with different expression of Sox-3 were successfully constructed by TE-10 cells infected with Sox-3 Si RNA lentivirus. The expression of Sox-3 in TE-10 cells of control group and TE-10 cells of no-load group were similar, which were significantly higher than that of TE-10 cells of interference group. MTT, flow cytometry, scratch test, tra. Nswell test and conditioned medium lymphatic endothelial cell tubing test confirmed that Sox-3 can significantly promote the proliferation, invasion, migration and lymphatic endothelial cell tubing ability of human esophageal squamous cell carcinoma cells, but inhibit cell apoptosis. 2. Successful construction of nude mice transplantation tumor model, the results showed that: compared with the low expression of Sox-3 interfering group, The growth rate and tumor size of the transplanted tumor were similar in the no-load group with high expression of Sox-3, but the volume of axillary lymph nodes on the ipsilateral side of the transplanted tumor was significantly increased. The results of Western blot and ELISA showed that inhibiting the expression of Sox-3 in TE-10 cells could promote the lymph node metastasis of esophageal squamous cell carcinoma. The expression and secretion of VEGF-C and VEGF-D were significantly decreased in the lymphatic endothelial cells. In the lymphatic vessel endothelial cells tube-forming experiment, the number of tubes of TE-10 cells in each group was significantly decreased after blocking the function of VEGF R-3 by SAR131675, and there was no difference between groups. Lymphangiogenesis is achieved through the VEGF-C/VEGF-D/VEGFR-3 pathway. Conclusion 1. Sox-3 can significantly promote the proliferation, invasion and migration of human esophageal squamous cell carcinoma cells in vitro, and inhibit cell apoptosis, but in vivo Sox-3 has little effect on the tumor formation rate and size of esophageal squamous cell carcinoma cells. 2. Sox-3 can significantly enhance the fineness of human esophageal squamous cell carcinoma cells in vitro. Sox-3 can promote lymphatic metastasis of esophageal squamous cell carcinoma cells after tumor formation. 3. Sox-3 can up-regulate the expression and secretion of VEGF-C and VEGF-D in esophageal squamous cell carcinoma cells. It acts on lymphatic endothelial cells and promotes lymphatic angiogenesis through the VEGF-C/VEGF-D/VEGFR-3 pathway. Possible mechanisms of lymph node metastasis in esophageal squamous cell carcinoma.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1

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