研究背景： 近年来，我国结直肠癌发病率呈上升趋势，严重影响国民健康。慢性应激与肿瘤的发生、发展密切相关，直接影响肿瘤患者的疗效和预后。β肾上腺素能系统作为心理应激的信号传递途径参与肿瘤的进展。其中，去甲肾上腺素(NE)作为重要的交感神经递质，逐渐引起了学术界的重视。 NE在外周与靶细胞膜上β肾上腺素能受体(β-AR)结合后，通过Gsα蛋白活化腺苷酸环化酶，使第二信使cAMP含量升高而活化蛋白激酶A，进而引发多条信号转导通路促进肿瘤进展。在肿瘤治疗中，针对NE的干预未见报道。目前，常用的抗抑郁药包括选择性5-HT再摄取抑制剂和5-HT和NE再摄取抑制剂(如文拉法辛，VEN)等。其中，VEN的抑制靶点为5-HT再摄取转运体和NE再摄取转运体(NET)，提示其在肿瘤的治疗中可能发挥作用。尽管国外临床回顾性研究报道抗抑郁药除改变肿瘤患者精神症状外，亦会影响患者的预后，但目前机制不清。本研究拟针对VEN影响NE促进结肠癌进展的作用和具体分子机制展开初步研究，为抗抑郁药用于结直肠癌临床辅助治疗提供理论基础。 研究目的： 1.分析NE对小鼠结肠癌细胞在体皮下移植瘤和体外对小鼠结肠癌细胞增殖及促血管生成的影响。 2.探究VEN对NE促进小鼠结肠癌细胞在体皮下移植瘤和体外促进小鼠结肠癌细胞增殖及促血管生成的影响及作用机制。 3.研究人结肠癌细胞中，NE处理和VEN处理对血管生成的影响及作用机制。 研究方法： 1.利用小鼠CT26皮下移植瘤实验模型，观察NE和VEN对小鼠CT26皮下移植瘤的影响。应用免疫组织化学和Western Blot实验检测CD34、VEGF在各组瘤组织中的表达情况。 2.以小鼠结肠癌细胞CT26为对象，应用MTT、流式细胞技术、Western Blot实验、ELISA实验和人脐静脉内皮细胞(HUVEC)体外成管实验观察NE和／或VEN对CT26细胞增殖和促HUVEC成管的影响及其作用机制。 3.运用RNA干扰技术，设计并合成特异靶向NET的siRNA。应用上述技术观察siNET对NE促进CT26细胞旁分泌作用和对HUVEC成管的影响及其作用机制。 4.以人结肠癌细胞系HCT116和RKO为对象，应用上述技术观察VEN、sihNET对NE促进人结肠癌细胞旁分泌作用和对HUVEC成管的影响及其作用机制。 结果： 1.NE促进小鼠CT26皮下移植瘤的形成，且瘤组织内微血管生成增多；VEN减缓NE在体对小鼠CT26皮下移植瘤的促进作用，且瘤组织内微血管生成减少。 2.NE促进CT26细胞增殖，VEN抑制NE促进CT26细胞增殖的作用。 3.NE使CT26细胞合成并分泌VEGF增多，旁分泌作用使HUVEC体外成管增多； VEN抑制NE促进VEGF的合成和分泌以及HUVEC体外成管的作用。 4.转染siNET抑制NE促进CT26细胞中VEGF的合成和分泌以及HUVEC体外成管的作用。 5.CT26中，NE引起Akt活化，VEN和转染siNET均使NE活化的Akt水平下降。 6.HCT116和RKO中，NE处理引起Akt活化、VEGF含量升高和HUVEC体外成管；VEN和转染sihNET均抑制NE引起的上述变化。 结论： 1.VEN通过靶向NET，抑制NE引起的Akt活化、VEGF合成分泌增多和微血管生成增多，从而减缓NE对小鼠结肠癌细胞皮下移植瘤的促进作用。 2.在人结肠癌细胞中，NE引起Akt活化、VEGF含量升高和血管内皮细胞成管； VEN处理靶向NET而抑制NE引起的Akt活化、VEGF含量升高和血管内皮细胞成管。 关键词：去甲肾上腺素；文拉法辛；结肠癌；肿瘤进展；慢性应激 论文类型：应用基础

 Backgrounds: In recent years, the incidence of colorectal cancer is increasing in China, which seriously affects national health. Chronic stress is closely related to the occurrence and development of tumor, and affects the prognosis of cancer patients. The beta adrenergic system is involved in the progression of tumors as a signaling pathway for psychological stress. As a critical sympathetic neurotransmitter, norepinephrine (NE) has gradually attracted the attention of the academic community. After binding to the beta-adrenergic receptor(β-AR), NE activates adenylate cyclase through Gsα and increases cAMP to activate protein kinase A, thus triggering a number of signalling pathways to promote tumor progression. In the treatment of cancer, the intervention for NE has not been reported. Currently, Antidepressants commonly used include selective 5-HT reuptake inhibitors and 5-HT and NE reuptake inhibitors (such as venlafaxine, VEN). The target of venlafaxine is 5-HT reuptake transporter and NE reuptake transporter (NET), suggesting that it may play a role in the treatment of tumors. Although clinical investigations have reported that antidepressants may affect the prognosis of cancer patients, the mechanism is unclear. The aim of this study is to explore the effect and specific mechanism of venlafaxine on the NE-promoted colon cancer progression, and to provide a theoretical basis for the clinical adjuvant treatment of antidepressants in colorectal cancer. Objectives: 1.To explore the effect of NE on the proliferation and angiogenesis of mouse colon cancer cells in vivo and in vitro. 2.To explore the effect and mechanisms of VEN on NE-promoted cell proliferation and angiogenesis of mouse colon cancer cell lines in vivo and in vitro. 3.To explore the effect and mechanisms of NE treatment and VEN treatment on angiogenesis in human colon cancer cells. Methods: 1.The effect of NE and VEN on tumorigenesis in BALB/c mice was observed by using CT26 subcutaneous xenograft model. Immunohistochemistry and Western Blot were used to detect the expression of CD34 and VEGF in tumor tissues. 2.Using MTT, flow cytometry, Western Blot, ELISA and in vitro human umbilical vein endothelial cells (HUVEC) tube-formation assay, the effect of NE and / or VEN on CT26 cell proliferation and HUVEC tube-formation and its preliminary mechanism were observed. 3.Using RNA interference, siNET was synthesized; and the effect and mechanism of siNET on NE-mediated in vitro HUVEC tube-formation was observed. 4.Using human colon cancer cells HCT116 and RKO, we observed the effects and mechanisms of VEN and sihNET on NE-mediated in vitro HUVEC tube-formation. Results: 1.NE promoted CT26 xenograft formation in BALB/c mice and increased angiogenesis in tumor tissues. VEN retarded the promotion of NE on CT26 xenograft formation in vivo and angiogenesis of tumor tissues. 2.NE promoted the proliferation of CT26 cells. VEN inhibited the NE-promoted cell proliferation of CT26. 3.NE elevated VEGF synthesis and secretion, and increased in vitro HUVEC tube-formation paracrinely.VEN retarded the increase of VEGF level, and in vitro HUVEC tube-formation by NE. 4.Transfection of siNET inhibited the NE-promoted increase of VEGF level and in vitro HUVEC tube-formation in CT26 cells. 5.NE activated Akt; VEN and siNET transfection inhibited NE-activated Akt in CT26 cells. 6.In HCT116 and RKO cells, NE treatment made Akt activated, VEGF level elevated, and in vitro HUVEC tube-formation increased; VEN and sihNET transfection inhibited these changes induced by NE. Conclusion: 1.By targeting NET, VEN inhibits the NE-induced Akt activation, elevation of VEGF synthesis and secretion, and increase of microvasculature, thus retarding the effect of NE on the development of colon cancer in mice. 2.In human colon cancer cell lines, NE causes Akt activation, elevated VEGF level, and increased HUVEC tube-formation; VEN targets NET and inhibits NE-induced Akt activation, elevated VEGF level, and increased HUVEC tube-formation. KEY WORDS: Norepinephrine; Venlafaxine; Colon cancer; Tumor progression; Chronic stress TYPE OF DISSERTATION: Application Fundamentals