VIP对巨噬细胞SIRPα的影响及其在胃癌免疫逃逸中的作用

发布时间：2018-04-26 17:59

本文选题：VIP + L　；参考：《南昌大学》2016年博士论文

【摘要】：研究背景与目的:胃癌为消化道最常见的恶性肿瘤,在我国癌症发病率及死亡率中排到第2位,较欧美国家具有极高的发病率及死亡率。近年来,胃癌发病率有下降的趋势,但多数患者确诊时已属进展期而失去手术根治机会。研究胃癌的免疫调节和免疫治疗是探索胃癌未来治疗的一个重要的方向。随着对肿瘤的深入研究,发现肿瘤微环境在肿瘤的存活、增殖、侵袭和转移的过程中具有非常重要的作用。肿瘤微环境中浸润的免疫细胞是肿瘤间质的重要组成部分,这些免疫细胞参与了肿瘤免疫调节,而存在于肿瘤微环境中的巨噬细胞往往占据显著优势,这些巨噬细胞也被称为肿瘤相关性巨噬细胞(tumor-associated macrophages,TAMs)。巨噬细胞具有很大的可塑性,在不同微环境中可极化为M1型和M2型。Ml型巨噬细胞的活化通过经典的激活途径,可显著释放细胞炎性因子如TNFα、IL-12等;M2型巨噬细胞活化则通过替代途径激活,信号机制复杂,目前仍不完全清楚。激活的M2型巨噬细胞可分泌具有免疫抑制的细胞炎性因子如IL-10等。肿瘤微环境中的肿瘤细胞可诱导分化多种浸润的免疫细胞,使其成为耐受性免疫细胞,失去对肿瘤的免疫监视功能而促进肿瘤的进展,如形成肿瘤诱导的肿瘤浸润树突细胞(tumor infiltrating DCs,Ti DCs)、肿瘤诱导的调节性T细胞(regulatory T cells,Tregs)、肿瘤诱导的骨髓来源的抑制性细胞(myeloid-derived suppressor cells,MDSCs)和肿瘤诱导的TAMs等。由于在肿瘤间质中TAMs往往占据较大的比例,因此研究TAMs对肿瘤的免疫逃逸具有十分重要的作用。许多研究发现,TAMs具有M2型巨噬细胞的表型特征及细胞功能,可促进多数肿瘤进展和转移。血管活性肠肽(Vasoactive intestinal peptide,VIP)是属于胰泌素/胰高血糖素家族的一种神经肽,在机体广泛表达,具有许多重要的生理功能,包括扩张血管,舒张平滑肌,促进胰液的分泌,抑制胃酸及胃泌素的分泌,调节胃肠道运动等。近年研究发现,VIP可以通过对免疫细胞免疫调节而维持机体的生理功能,如VIP可促进激活的巨噬细胞分泌抑制性炎性因子IL-10,而抑制激活的巨噬细胞的促炎性细胞因子TNF-α、IL-12等的表达,诱导巨噬细胞或T细胞、树突细胞和NK细胞等其他免疫细胞的免疫功能而产生免疫耐受。因此,VIP是一种重要的参与免疫抑制、诱导免疫耐受的胃肠肽激素。在机体的整体调节中,神经-内分泌-免疫调节具有特别重要的作用,肿瘤的发生发展和人体神经压力的关系密不可分,许多神经递质和激素在对肿瘤发生、生长和转移中起到正性和负性的调控作用[1]。研究发现,某些肿瘤细胞可以分泌VIP,如胃腺癌组织表达VIP及某些胃癌细胞系也自分泌VIP,VIP受体在许多肿瘤细胞上有显著表达,VIP在调节多种肿瘤的生长过程中起到重要的作用。因此,在肿瘤免疫调节过程中,VIP可能扮演着重要的角色。VIP为具有免疫抑制功能的胃肠肽激素,能够抑制巨噬细胞的免疫功能,具有类似IL-10和TGF-β等抑制性细胞因子抑制机体免疫的作用。VIP主要通过和G蛋白偶联受体结合而启动c AMP依赖和非c AMP依赖信号通路发挥对激活的巨噬细胞的免疫调节作用。由于调节巨噬细胞免疫功能的信号机制非常复杂,VIP是否还能通过其他信号途径调节巨噬细胞的极化改变目前还没有发现。信号调节蛋白α(Signal regulatory proteinα,SIRPα)是SIRPs一个家族成员。有研究发现,SIRPα能够调节巨噬细胞的免疫功能,使其出现M2样极化改变,其显著特征就是其胞内段ITIMs可以发生酪氨酸磷酸化并募集含有SH2结构域的信号分子SHP-1和SHP-2,启动相关信号机制,负性调节巨噬细胞的免疫功能。胃癌组织和一些特定的胃癌细胞能够表达VIP,如SGC-7901细胞,而胃癌细胞通过自分泌VIP调节巨噬细胞的免疫功能,可能与胃癌细胞的发生发展有关。本研究旨在通过体内和体外细胞实验观察VIP对巨噬细胞极化的影响及VIP通过巨噬细胞对胃癌细胞功能的影响。通过体外细胞实验进一步观察VIP调节巨噬细胞SIRPα表达及相关信号分子的变化,探讨上述的结果是否可能与VIP通过影响巨噬细胞SIRPα的信号机制对巨噬细胞的免疫调节作用有关。为VIP在肿瘤的免疫调节和免疫治疗中的作用提供理论与实验依据。方法:1.收集临床胃癌患者手术切除的肿瘤与正常组织样本,采用免疫组化染色,以CD68为TAMs标记,检测胃癌组织与正常组织中VIP、CD68、IL-10及IL-12的表达差异,分析胃癌组织中CD68、VIP与主要临床病理指标的相关性。进一步分析胃癌组织中VIP分别与CD68、IL-10及IL-12之间的相关性。通过体内实验探讨VIP对巨噬细胞极化的影响。2.使用PMA诱导THP-1单核细胞分化为巨噬细胞,利用流式细胞仪检测诱导后的THP-1细胞CD14和CD68的表达以鉴定巨噬细胞。采用ELISA和实时荧光定量PCR分别检测各组巨噬细胞的相关炎性细胞因子及i NOS mRNA、Arg-1 mRNA的表达。通过体外细胞实验探讨VIP对巨噬细胞极化的影响。3.采用transwell共培养技术将巨噬细胞与SGC-7901细胞共培养,MTT法检测各组巨噬细胞对胃癌细胞增殖活性的影响;采用transwell共培养技术将巨噬细胞与SGC-7901细胞共培养后观察各组巨噬细胞对胃癌细胞侵袭能力的影响。通过体外细胞实验观察VIP通过巨噬细胞对胃癌细胞功能的影响。4.采用实时荧光定量PCR、Western blot及IP的方法检测各组巨噬细胞SIRPα、p-SIRPα、PI3Kp85、SHP-2、Akt及p-Akt的表达。通过体外细胞实验进一步探讨上述的结果是否与VIP通过影响巨噬细胞SIRPα的信号机制对巨噬细胞的免疫调节作用有关。结果:1.肿瘤组织CD68阳性表达率及表达强度显著高于正常组织的巨噬细胞(p0.001);肿瘤组织VIP阳性表达率及表达强度显著高于正常组织的巨噬细胞(p0.05,p0.05);肿瘤组织IL-10阳性表达率稍微高于正常组织(p0.05),表达强度显著高于正常组织(p0.05);肿瘤组织IL-12阳性表达率低于正常组织(p0.05),表达强度显著低于正常组织(p0.01);胃癌组织CD68表达强度与不同性别、年龄和肿瘤部位等均无显著性差异(p0.05),CD68表达强度与分化程度低、淋巴结转移阳性、TNM高分期有一定的相关趋势(p0.05,p0.01,p0.01);胃癌组织VIP表达强度与不同性别、年龄和肿瘤部位等均无显著性差异(p0.05),VIP表达强度与分化程度低、淋巴结转移阳性、TNM高分期有一定的相关趋势(p0.05);胃癌组织中VIP与CD68之间的表达强度呈明显的正相关(p0.001),胃癌组织中VIP与IL-10之间的表达强度呈明显的正相关(p0.001),胃癌组织中VIP与IL-12之间的表达强度呈明显的负相关(p0.001)。2.LPS处理巨噬细胞后TNF-α、IL-12、IL-6和IL-10表达均有显著上升(p0.05);而仅给予VIP对上述的炎性细胞因子表达未见显著影响(p0.05);VIP对LPS处理的巨噬细胞TNF-α、IL-12和IL-6表达有显著抑制作用(p0.05),而对IL-10表达有显著促进作用(p0.05);LPS处理巨噬细胞后i NOS mRNA表达显著上升(p0.05),仅给予VIPi NOS mRNA表达未见显著变化(p0.05);VIP对LPS处理的巨噬细胞i NOS mRNA表达有显著抑制作用(p0.05);Arg-1mRNA表达在各组间比较均无统计意义(p0.05)。3.LPS处理巨噬细胞后抑制胃癌细胞的增殖活性(p0.05),VIP通过LPS处理的巨噬细胞促进胃癌细胞的增殖活性(p0.05);LPS处理巨噬细胞后抑制胃癌细胞的侵袭力(p0.05),VIP通过LPS处理的巨噬细胞促进胃癌细胞的侵袭力(p0.05)。4.LPS处理巨噬细胞后显著下调巨噬细胞SIRPαmRNA及蛋白水平(p0.05),VIP通过LPS处理的巨噬细胞上调巨噬细胞SIRPαmRNA及蛋白水平(p0.05);LPS处理巨噬细胞后上调巨噬细胞SIRPα磷酸化水平,VIP通过LPS处理的巨噬细胞上调巨噬细胞SIRPα磷酸化水平,SIRPα磷酸化后可募集SHP-2及PI3Kp85分子;LPS处理巨噬细胞后显著上调巨噬细胞Akt磷酸化水平,VIP通过LPS处理的巨噬细胞显著下调巨噬细胞Akt磷酸化水平(p0.05),各组Akt蛋白比较无显著性差异(p0.05)。结论:1.通过体内实验证实了VIP促进了巨噬细胞M2样极化改变,VIP与胃癌的免疫逃逸相关。2.通过体外细胞实验进一步验证了VIP促进巨噬细胞M2样极化改变,从而促进胃癌细胞的增殖和侵袭能力。3.上述过程可能与VIP通过上调巨噬细胞SIRPα蛋白及促进SIRPα磷酸化,进而与SHP-2结合,竞争性抑制NF-κB及PI3K-Akt信号的激活,负性调节巨噬细胞免疫功能的机制有关。
[Abstract]:Background and objective: gastric cancer is the most common malignant tumor in the digestive tract. It ranks second in the incidence and mortality of cancer in China. It has a very high incidence and mortality compared with the European and American countries. In recent years, the incidence of gastric cancer has declined, but most of the patients have lost the chance of radical operation at the time of diagnosis. Immunotherapy is an important direction for the exploration of the future treatment of gastric cancer. With the in-depth study of the tumor, it is found that the tumor microenvironment plays an important role in the survival, proliferation, invasion and metastasis of the tumor. The immune cells infiltrated in the tumor microenvironment are an important part of the tumor interstitium. These immune cells are immune to the tumor. Cells are involved in tumor immunomodulation, and macrophages, which exist in tumor microenvironments, often occupy significant advantages. These macrophages are also called tumor-associated macrophages (TAMs). Macrophages have great plasticity. In different microenvironments, the macrophages can be polarized to type M1 and M2 type.Ml macrophages. Activation through classical activation pathways can significantly release inflammatory cytokines such as TNF alpha, IL-12, etc. the activation of M2 type macrophages is activated by alternative pathways, and the signal mechanism is complex and is still not completely clear. Activated M2 macrophages can secrete inflammatory cytokines such as IL-10, such as IL-10. Tumor microenvironment tumors are fine. Cells can induce a variety of infiltrating immune cells, making it a tolerant immune cell, losing the immune surveillance of the tumor and promoting the progress of the tumor, such as the formation of tumor induced tumor infiltrating dendritic cells (tumor infiltrating DCs, Ti DCs), tumor induced T cells (regulatory T cells, Tregs), tumor induced bone Myeloid-derived suppressor cells (MDSCs) and tumor induced TAMs and so on. Because TAMs often occupies a large proportion in the tumor interstitium, the study of TAMs plays an important role in the immune escape of the tumor. Many studies have found that TAMs has the phenotypic characteristics and cell functions of the M2 type macrophages. Vasoactive intestinal peptide (VIP), a neuropeptide belonging to the secretin / glucagon family, is widely expressed in the body and has many important physiological functions, including dilating blood vessels, relaxing smooth muscle, promoting secretion of pancreatic juice, inhibiting the secretion of gastric acid and gastrin, and regulating the secretion of gastric acid and gastrin. Recent studies have found that VIP can maintain the physiological function of the body by immunoregulation of immune cells, such as VIP can promote the secretion of inhibitory inflammatory factor IL-10 by activated macrophages, and inhibit the expression of inflammatory cytokines, TNF- a, IL-12 and so on, and induce macrophage or T cells, and the dendrite is fine. The immune tolerance of other immune cells, such as cell and NK cells, is produced. Therefore, VIP is an important gastrointestinal peptide hormone involved in immunosuppression and induction of immune tolerance. In the overall regulation of the body, neuroendocrine immunoregulation is of special importance. The relationship between the development of tumor and the pressure of human body is closely related. Many neurotransmitters and hormones play a positive and negative regulatory role in the occurrence, growth and metastasis of tumors. [1]. studies have found that some tumor cells can secrete VIP, such as gastric adenocarcinoma tissue expression VIP and some gastric cancer cell lines that also secrete VIP, VIP receptors are expressed in many tumor cells, VIP is regulating a variety of tumors. In the process of growth, VIP may play an important role in the process of immunomodulating,.VIP, a gastrointestinal peptide hormone with immunosuppressive function, which inhibits the immune function of macrophages, and has the effect of inhibiting the immune function, such as IL-10 and TGF- beta, and the effect of.VIP mainly through and G Protein coupled receptors bind to activate C AMP dependent and non C AMP dependent signaling pathways for the immunoregulation of activated macrophages. As the signal mechanism for regulating macrophage immunity is very complex, it is not yet found that VIP can regulate the change of macrophage polarity through other signaling pathways. White alpha (Signal regulatory protein alpha, SIRP a) is a member of a family of SIRPs. Some studies have found that SIRP alpha can regulate the immune function of macrophages and make it M2 like polarization change. Its significant feature is that its intracellular segment ITIMs can produce tyrosine phosphorylation and raise the signal molecules containing SH2 domains, SHP-1 and SHP-2, to initiate related letters. The immune function of macrophages is negatively regulated. Gastric cancer tissue and some specific gastric cancer cells can express VIP, such as SGC-7901 cells, and gastric cancer cells regulate the immune function of macrophages by autocrine VIP, which may be related to the occurrence and development of gastric cancer cells. This study aims to observe the giant VIP by in vivo and in vitro cell experiments. The effect of macrophage polarization and the effect of VIP through macrophage on the function of gastric cancer cells. Through in vitro cell experiment, we further observe the changes in the expression of SIRP alpha in macrophages and the related signal molecules by VIP, and explore whether the above results may be related to the immunoregulation of VIP through the signal mechanism affecting macrophage SIRP alpha. To provide theoretical and experimental basis for the role of VIP in the immunomodulatory and immunotherapy of tumor. Methods: 1. the tumor and normal tissue samples of the patients with gastric cancer were collected and the immunohistochemical staining was used to detect the difference in the expression of VIP, CD68, IL-10 and IL-12 in the gastric cancer tissue and the normal group with the CD68 TAMs markers. The correlation between CD68, VIP and main clinicopathological indexes in gastric cancer tissue. Further analysis of the correlation between VIP and CD68, IL-10 and IL-12 in gastric cancer tissue. The effect of VIP on the polarization of macrophages through the experiment in vivo:.2. using PMA induced THP-1 mononuclear cells to differentiate into macrophages, and the detection of the induced THP- by flow cytometry. 1 cells CD14 and CD68 were expressed to identify macrophages. ELISA and real-time fluorescence quantitative PCR were used to detect the inflammatory cytokines, I NOS mRNA, and the expression of Arg-1 mRNA, respectively. The effect of VIP on the polarization of macrophages in vitro was studied.3. by Transwell co culture technique. The effect of macrophages on the proliferation of gastric cancer cells was detected by MTT method. The influence of macrophages and SGC-7901 cells on the invasion ability of gastric cancer cells was observed by co culture of macrophages and SGC-7901 cells by Transwell co culture technique. The effect of macrophage on the function of gastric cancer cells through macrophage in vitro was observed. .4. used real-time fluorescent quantitative PCR, Western blot and IP to detect the expression of SIRP alpha, p-SIRP a, PI3Kp85, SHP-2, Akt and p-Akt in each group. The results of the in vitro cell test were further explored whether the above results were related to the immunoregulation effect of VIP through the signaling mechanism affecting macrophages. Results: 1. The positive expression rate and expression intensity of CD68 in tumor tissues were significantly higher than those of normal tissue macrophages (p0.001), and the positive expression rate and expression intensity of VIP in tumor tissues were significantly higher than those of normal tissue macrophages (P0.05, P0.05), and the positive rate of IL-10 in tumor tissues was slightly higher than that of normal tissue (P0.05), and the expression intensity was significantly higher than that of normal tissue (P0.05). The positive expression rate of IL-12 in tumor tissue was lower than that of normal tissue (P0.05), and the expression intensity was significantly lower than that of normal tissue (P0.01). The expression intensity of CD68 in gastric cancer tissues was not significantly different from that of different sex, age and tumor site (P0.05), CD68 expression intensity and differentiation degree were low, lymph node metastasis was positive, and TNM high stage had a certain correlation trend (P0.05, P0.01, P0.01); there was no significant difference in the expression intensity of VIP in gastric cancer tissue with different sex, age and tumor site (P0.05), VIP expression intensity and differentiation degree, lymph node metastasis positive, TNM high staging (P0.05); the expression intensity between VIP and CD68 in gastric cancer tissues was positively correlated (p0.001), gastric cancer tissue The expression intensity of VIP and IL-10 was positively correlated (p0.001). The expression intensity of VIP and IL-12 in gastric cancer tissues was negatively correlated (p0.001).2.LPS treated macrophages, TNF- a, IL-12, IL-6 and IL-10 were significantly increased (P0.05). 5); VIP had a significant inhibitory effect on the expression of TNF- alpha, IL-12 and IL-6 in the macrophages treated by LPS (P0.05), but significantly promoted the expression of IL-10 (P0.05). The expression of I NOS mRNA was significantly higher than that of LPS treated macrophages. Inhibition effect (P0.05); Arg-1mRNA expression in each group had no statistically significant (P0.05).3.LPS treatment of macrophages to inhibit the proliferation activity of gastric cancer cells (P0.05), VIP through LPS treated macrophages to promote the proliferation activity of gastric cancer cells (P0.05); LPS treated macrophages to inhibit the invasion of gastric cancer cells (P0.05), VIP through LPS Macrophages promoted the invasion of gastric cancer cells (P0.05).4.LPS to significantly reduce macrophage SIRP alpha mRNA and protein level (P0.05), VIP through LPS treated macrophages up regulation of macrophage SIRP alpha mRNA and protein level (P0.05); LPS treatment of macrophage after macrophage activation of macrophage SIRP alpha phosphorylation level. LPS treated macrophages raised the level of SIRP alpha phosphorylation of macrophages, and SIRP alpha could raise SHP-2 and PI3Kp85 molecules after phosphorylation. LPS treated macrophages significantly up-regulated the level of phosphorylation of Akt in macrophages. VIP through LPS treated macrophages significantly lowered the level of phosphorylation of Akt of macrophage Akt (P0.05). There was no significant difference in Akt protein in each group. Difference (P0.05). Conclusion: 1. in vivo experiments confirmed that VIP promoted the M2 like polarization change of macrophages and the immune escape related.2. of VIP and gastric cancer further verified that VIP promoted M2 like polarization change of macrophages, thus promoting the proliferation and invasion of gastric cancer cells, which may be associated with VIP through up regulation of macrophages. The cell SIRP alpha protein and the promotion of SIRP alpha phosphorylation, then combined with SHP-2, competitively inhibit the activation of NF- kappa B and PI3K-Akt signals, and the mechanism of negative regulation of macrophage immune function.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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