WO2010003307A1

WO2010003307A1 - Nitric oxide and its information transfer system are used in detecting tumors

Info

Publication number: WO2010003307A1
Application number: PCT/CN2009/000690
Authority: WO
Inventors: 费瑞德·穆拉德; 卞卡; 李桃·洁西卡
Original assignee: 卞化石
Priority date: 2008-07-10
Filing date: 2009-06-22
Publication date: 2010-01-14

Abstract

Nitric oxide and its information transfer system are used in detecting tumors. By the division cycle and differentiation character of the tumor, this method can diagnose the special expression and distribution of nitric oxide and its information transfer system in the tumor qualitatively and quantitatively.On the basis of this method,the directed medicine of tumor based on the nitric oxide biology regulation can be established.

Description

一氧化氮及其信息传递***在制备检测恶性肿瘤的试剂中的应用 Application of Nitric Oxide and Its Information Transmission System in Preparation of Reagents for Detection of Malignant Tumors

技术领域 Technical field

本发明涉及生物技术，具体涉及一氧化氮及其信息传递***在制备检测恶性肿瘤的试剂中的应用。 The present invention relates to biotechnology, and in particular to the use of nitric oxide and its information delivery system for the preparation of a reagent for detecting a malignant tumor.

背景技术 Background technique

恶性肿瘤是危害人类的主要杀手，为防治恶性肿瘤，人们进行了大量的研究。由于一氧化氮 (Nitric Oxide; NO)广泛参与生命活动密切相关的信息传导活动，因此， NO 与肿瘤生长的关联历来倍受国际注目。然而，在一氧化氮诺贝尔奖成果取得以来的近 20 余年， NO 与恶性肿瘤诊断和治疗的关系方面，缺乏根本性的突破。其主要原因在于- Malignant tumors are the main killers of humans. A lot of research has been done to prevent and treat malignant tumors. Since Nitric Oxide (NO) is widely involved in information transmission activities closely related to life activities, the association between NO and tumor growth has always attracted international attention. However, in the nearly 20 years since the Nobel Prize in Nitrogen was achieved, there has been no fundamental breakthrough in the relationship between NO and the diagnosis and treatment of malignant tumors. The main reason is -

1)一氧化氮（N0) 是近年来引起人们注意的无机气体自由基， NO因含有 1个不配对电子，故具有自由基的特点。 NO因具有独特的理化性质，正常情况下具有生物作用的多样性，几乎对全身各***都有影响。 N0脂溶性极强，易扩散到邻近组织细胞中与靶物质反应，产生一系列生物学效应。 NO广泛参与生理信息传递（例如，扩张血管及作为神经递质）。与此同时，在病理情况下 NO 的过量产生则对机体造成广泛损害。 1) Nitric oxide (N0) is an inorganic gas radical that has attracted attention in recent years. NO contains a pair of unpaired electrons, so it has the characteristics of free radicals. Because of its unique physical and chemical properties, NO has a biological diversity under normal conditions and affects almost all systems. N0 is highly soluble in fat and easily diffuses into adjacent tissue cells to react with target substances, producing a range of biological effects. NO is widely involved in the transmission of physiological information (for example, expanding blood vessels and acting as neurotransmitters). At the same time, excessive production of NO in pathological conditions causes extensive damage to the body.

2) NO 生物信息作用途径的多样性， NO 的作用途径可分为两大类： 2) The diversity of NO biological information pathways, the pathways of NO can be divided into two categories:

a) cGMP 依赖性途经（NO/sGC或 pGC/cGMP)和 a) cGMP dependent pathway (NO/sGC or pGC/cGMP) and

b) cGMP 非依赖性途经（NO/氧化硝基化）。 b) cGMP independent pathway (NO/oxidative nitration).

3) 实体恶性肿瘤构造的复杂性，实体恶性肿瘤组织包括两大部分- a)肿瘤细胞 (parenchyma)， b) 外周组织 (stroma) 。这两大部分的一氧化氮生物学有着本质的不同。 3) Complexity of solid malignant tumors, solid malignant tumor tissue consists of two major parts - a) tumor cells (parenchyma), b) peripheral tissue (stroma). These two parts of nitric oxide biology are fundamentally different.

4)肿瘤细胞株的信息***变异：肿瘤实验常用的细胞株经过无数代的繁殖，其特性已起了根本性的变化。特别是由一些小分子参与的信息传递***发生了本质性的改变。 4) Information system variation of tumor cell lines: The cell lines commonly used in tumor experiments have undergone numerous generations of reproduction, and their characteristics have undergone fundamental changes. In particular, the information transfer system involving small molecules has undergone a fundamental change.

5) NO 相关信息分子的分布及表达的复杂性: NO 合成酶至少有 3 种亚型。 cGMP 可以被至少 4 种酶所合成，而 cGMP 的分解体系又十分复杂，外加恶性肿瘤的非生理性特质更增加了分析的困难性。因此，至今为止， NO在恶性肿瘤中的存在和作用情况一直不明。 5) Distribution and expression complexity of NO related information molecules: NO synthase has at least 3 subtypes. cGMP It can be synthesized by at least 4 enzymes, and the decomposition system of cGMP is very complicated, and the non-physiological characteristics of malignant tumors increase the difficulty of analysis. Therefore, the existence and action of NO in malignant tumors have not been known until now.

近年来，炎症与癌症发生发展的相关性已被相当程度的阐明. 趋炎性状态或趋炎性恶性肿瘤环境是恶性肿瘤赖以生长和转移的必要条件（图 1)。趋炎性状态或趋炎性恶性肿瘤环境的一个重要标志就是过量产生活性氧（reactive oxygen species, ROS) /活性氮（reactive nitrogen species, RNS) . 过量 ROS / R S 可导致与肿瘤生物学密切相关的肿瘤细胞生长周期，肿瘤抑制因子，肿瘤相关基因等的调节紊乱。由此可见，一氧化氮对炎症乃至免疫发生发展的各个阶段均起了举足轻重的作用。 In recent years, the correlation between inflammation and the development of cancer has been clarified to a considerable extent. The inflammatory state or the inflammatory malignant environment is a necessary condition for the growth and metastasis of malignant tumors (Fig. 1). An important marker of the inflammatory or inflammatory malignant environment is the excessive production of reactive oxygen species (ROS) / reactive nitrogen species (RNS). Excessive ROS / RS can lead to tumor biology Regulation of tumor cell growth cycle, tumor suppressor, tumor-associated genes, etc. It can be seen that nitric oxide plays an important role in all stages of inflammation and even immune development.

虽然一氧化氮合成酶各型在肿瘤中的表达有过一些报道，然而，国际上从没有对诱导性一氧化氮合成酶（iNOS)表达与肿瘤恶性程度的相关性进行过***性研究。此外，也未见一氧化氮及其信息传递***与恶性肿瘤的相关性和***性研究的报道。 Although there have been some reports on the expression of nitric oxide synthase in tumors, there has been no systematic study on the correlation between the expression of inducible nitric oxide synthase (iNOS) and the degree of malignancy in the world. In addition, there have been no reports of correlations and systematic studies of nitric oxide and its information transmission systems with malignant tumors.

发明内容 Summary of the invention

本发明所要解决的技术问题在于研究设计一氧化氮及其信息传递***在检测恶性肿瘤中的应用。 The technical problem to be solved by the present invention is to study and design the application of nitric oxide and its information transmission system in detecting malignant tumors.

本发明提供了一种一氧化氮及其信息传递***在制备检测恶性肿瘤中的应用，具体提供了一种一氧化氮及其信息传递***在制备检测恶性肿瘤的发现，定性，转移，和治疗效果评价的试剂中的应用。 The invention provides a nitric oxide and an information transmission system thereof for preparing and detecting a malignant tumor, and specifically provides a nitric oxide and an information transmission system thereof for preparing, detecting, metastalyizing, and treating a malignant tumor. Application in reagents for effect evaluation.

本发明人以一氧化氮细胞信息***中 7个关键生物靶点系列，既：诱导型一氧化氮合成酶以及内皮型，神经型一氧化氮合成酶亚型；细胞蛋白质氧化和硝基化；环磷酸鸟苷（cGMP ) ；可溶性鸟甘酸环化酶（sGC ) ；钠尿肽家族受体（Receptors for natriuretic peptides, NPs) 及其他细胞膜性鸟甘酸环化酶（pGC) ；磷酸二酯酶各亚型；丝氨酸 /苏氨酸 -特异性蛋白激酶 [系列；包括蛋白激酶 G (PKG) ]的基因表达及蛋白质功能在恶性肿瘤组织中的特异性变化作为恶性肿瘤的发现，定性，转移，和治疗效果评价的诊断靶点。形成基于一氧化氮细胞信息***的检测肿瘤药物和试剂。 The present inventors have a series of seven key biological targets in the nitric oxide cell information system, namely: inducible nitric oxide synthase and endothelial type, neuronal nitric oxide synthase subtype; cell protein oxidation and nitration; Cyclic guanosine monophosphate (cGMP); soluble ornithine cyclase (sGC); Receptors for natriuretic peptides (NPs) and other cell membrane ornithine cyclase (pGC); phosphodiesterase Subtype; serine/threonine-specific protein kinases [series; including protein kinase G (PKG)] gene expression and protein function in malignant tumor tissue specific changes as malignant tumors, qualitative, metastatic, and Diagnostic target for evaluation of therapeutic effects. The detection of tumor drugs and reagents based on a nitric oxide cell information system is formed.

本发明提供了一种一氧化氮及其信息传递***在制备检测恶性肿瘤的试剂中的应用。 The invention provides a nitric oxide and an information transmission system thereof for preparing a reagent for detecting a malignant tumor Application.

本发明试剂包括以下系列： The reagents of the invention include the following series:

1)诱导型一氧化氮合酶（iNOS 或 N0S-2)的基因及蛋白表达和活性的试剂，内皮型一氧化氮合成酶 (eNOS或 N0S-3)的基因及蛋白表达和活性的试剂,神经型一氧化氮合成酶亚型 (nNOS或 N0S- 1)的基因及蛋白表达和活性的试剂。 1) an agent for inducible nitric oxide synthase (iNOS or NOS-2) gene and protein expression and activity, an agent for endothelial nitric oxide synthase (eNOS or NOS-3) gene and protein expression and activity, An agent for the expression and activity of a gene and protein of a neuronal nitric oxide synthase subtype (nNOS or NOS-1).

2) 可溶性鸟苷酸环化酶 sGC 的 od、 α2 ; β1、 β2亚基的基因及蛋白表达和活性的试剂； 2) soluble guanylate cyclase sGC od, α2; β1, β2 subunit gene and protein expression and activity reagents;

3) 环磷酸鸟苷（cGMP) 在各种生物标本和肿瘤标本中的浓度或含量测定：3) Determination of the concentration or content of cyclic guanosine monophosphate (cGMP) in various biological specimens and tumor specimens:

4) 与肽配体及其结构相似合成物起反应的膜结合鸟苷酸环化酶 A、 B或 C型； GCA、 GCB或 GCC的基因及蛋白表达和活性的试剂； 4) a membrane that binds to a peptide ligand and a structurally similar composition thereof, and binds to a guanylate cyclase A, B or C; a gene and protein expression and activity reagent of GCA, GCB or GCC;

5) 磷酸二酯酶 PDE系列的基因及蛋白表达和活性的试剂： 5) Phosphodiesterase PDE series of genes and protein expression and activity reagents:

6) 丝氨酸 /苏氨酸-特异性蛋白激酶系列；包括蛋白激酶 GPKG的基因及蛋白表达和活性的试剂； 6) a serine/threonine-specific protein kinase series; an agent comprising the gene and protein expression and activity of the protein kinase GPKG;

7)检测恶性肿瘤中蛋白质氧化和硝基化水平的试剂。 7) An agent for detecting the level of protein oxidation and nitration in a malignant tumor.

本发明涉及的丝氨酸 /苏氨酸 -特异性蛋白激酶系列的详细说明如下： A detailed description of the serine/threonine-specific protein kinase series involved in the present invention is as follows:

磷酸化酶激酶（phosphorylase kinase, PHK)是一个丝氨酸 /苏氨酸特异蛋白激酶，能将磷酸化 H b转化为磷酸化酶 a。其中被磷酸化酶激酶活化的一个酶是糖原磷酸化酶。磷酸化酶激酶 α的基因是 PH A1 , ΡΗΚΑ2, 磷酸化酶激酶 β的基因是 ΡΗΚΒ, 磷酸化酶激嗨 γ的基因是 PHKG1， PKHG2。磷酸化酶激酶受变构调节和可逆的磷酸化调节。 Phosphorylase kinase (PHK) is a serine/threonine-specific protein kinase that converts phosphorylated Hb to phosphorylase a. One of the enzymes activated by phosphorylase kinase is a glycogen phosphorylase. The gene for phosphorylase kinase α is PH A1 , ΡΗΚΑ2, the gene for phosphorylase kinase β is ΡΗΚΒ, and the gene for phosphorylase γ is PHKG1, PKHG2. Phosphorylase kinases are regulated by allosteric regulation and reversible phosphorylation.

蛋白激酶 A (PKA) 是一个全酶，由两个调节亚基和两个催化亚基组成。 cAMP水平较低时，全酶保持完整性，不被接触反应活化。当 cAMP浓度升高时（如 G蛋白偶联受体偶联 Gs后活化腺苷酸环化酶，抑制降解 cAMP的磷酸二酯酶）， cAMP结合到调节亚基的两个位点上，'从而引起构象变化，释放催化亚基。 Protein kinase A (PKA) is a holoenzyme consisting of two regulatory subunits and two catalytic subunits. When the level of cAMP is low, the whole enzyme remains intact and is not activated by the contact reaction. When the cAMP concentration is increased (eg, G-protein coupled receptors bind Gs to activate adenylate cyclase, inhibit cAMP-degrading phosphodiesterase), cAMP binds to two sites of regulatory subunits, ' This causes a conformational change that releases the catalytic subunit.

蛋白激酶 B (PKB)也称为 AKT激酶。人类 Akt家族有三个基因： Aktl， Akt2和 Akt3。 Protein kinase B (PKB) is also known as AKT kinase. The human Akt family has three genes: Aktl, Akt2 and Akt3.

Aktl通过抑制凋亡进程而参与细胞生存传导路径。 Aktl 同样能诱导蛋白合成通路，因此是细胞通路的关键信号蛋白，能导致骨骼肌过度增殖，引起组织生长。因其能阻断凋亡，因此能促进细胞存活， Aktl也是导致多种类型癌症的主要因素。 Akt (现在又名 Aktl ) 最初被认为是逆转录酶病毒转换中的致癌基因。 Aktl participates in the cell survival pathway by inhibiting the process of apoptosis. Aktl also induces protein synthesis pathways and is therefore a key signaling protein in the cellular pathway that causes excessive proliferation of skeletal muscle and tissue growth. Because it blocks apoptosis, it promotes cell survival. Aktl is also a major cause of many types of cancer. Akt (now The nickname Aktl) was originally thought to be an oncogene in retroviral transformation.

Akt2 是胰岛素信号通路中的重要的信号分子，在诱导葡萄糖转运时必需的调节酶。 Akt2 is an important signaling molecule in the insulin signaling pathway, a regulatory enzyme necessary for inducing glucose transport.

通过对 Aktl或 /和 Akt2基因敲除小鼠的研究证明， Aktl和 Akt2具有不同的作用。在 Aktl无效而 Akt2正常的小鼠体内，葡萄糖动态平衡不受干扰，但动物体形较小，这与 Aktl主要对生长起作用的功能一致。与之相反， Aktl正常而 Akt2被敲除的小鼠仅有轻微的生长缺陷，而表现为糖尿病显型（胰岛素抵抗），这又与 Akt2在信号通路中对胰岛素受体具有特异性的观点一致。 Akt3的作用尚不明确，但其在大脑中具有明显表达。有报道显示， Akt3缺乏的小鼠的大脑较小。 Studies on Aktl or / and Akt2 knockout mice have demonstrated that Aktl and Akt2 have different roles. In mice with null Aktl and normal Akt2, glucose homeostasis is undisturbed, but animals are smaller in size, which is consistent with Aktl's primary role in growth. In contrast, mice with normal Aktl and knocked out Akt2 had only slight growth defects, but showed a diabetic phenotype (insulin resistance), which is consistent with the view that Akt2 is specific for the insulin receptor in the signaling pathway. . The role of Akt3 is not known, but it is clearly expressed in the brain. It has been reported that mice lacking Akt3 have smaller brains.

蛋白激酶 C (PKC) 实际上是一个蛋白激酶家族，由大约 10个同工酶组成。基于其第二信使需要可分为三个亚家族：经典型、新型和非经典型。 Protein kinase C (PKC) is actually a family of protein kinases composed of approximately 10 isozymes. Based on its second messenger needs can be divided into three subfamilies: classic, new and non-classical.

经典型 PKC -包括 α， βΙ， βΠ和 γ亚基。其活化需要 Ca2+，甘油二酯（DAG) 以及磷脂如卵磷脂。 Classical PKC - includes alpha, beta, beta and gamma subunits. Its activation requires Ca2+, diglycerides (DAG) and phospholipids such as lecithin.

PKC-α (PR CA) PKC-α (PR CA)

PKC-βΙ (PRKCB1) PKC-βΙ (PRKCB1)

PKC-βΙΙ PKC-βΙΙ

P C-γ (PR CG) P C-γ (PR CG)

新型（n) PKCs包括 δ， ε， η和 Θ亚基，活化时需要 DAG, 但不需要 Ca2+。因此，经典型和新型的 PKCs与磷脂酶 C一样，通过同样的信号转导途径活化。 Novel (n) PKCs include δ, ε, η and Θ subunits, which require DAG for activation but do not require Ca2+. Therefore, both classical and novel PKCs are activated by the same signal transduction pathway as phospholipase C.

PKC-δ (PRKCD) PKC-δ (PRKCD)

PKC-ε (PRKCE) PKC-ε (PRKCE)

PKC-η (PRKCH) PKC-η (PRKCH)

P C-Θ (PRKCQ) P C-Θ (PRKCQ)

非经典型 PKC -活化时不需要 Ca2+和 DAG。 Non-classical PKC - Ca2+ and DAG are not required for activation.

PKC- 1 (PR CI) PKC- 1 (PR CI)

PKC— ζ (PR Z) PK-N1 (PKN1) PKC— ζ (PR Z) PK-N1 (PKN1)

PK-N2 (PKN2) PK-N2 (PKN2)

Ca2+/钙调素依赖蛋白激酶或 CaM激酶，主要由 Ca2+/钙调素复合物调节。 CaM激酶有两类： Ca2+/calmodulin-dependent protein kinase or CaM kinase is mainly regulated by the Ca2+/calmodulin complex. There are two types of CaM kinases:

特异性 CaM激酶：例如肌球蛋白轻链激酶（MLCK) ，其能磷酸化肌球蛋白，弓 I起肌肉收缩。 Specificity CaM kinase: For example, myosin light chain kinase (MLCK), which phosphorylates myosin, causes muscle contraction.

多功能 CaM激酶：同样也称为 CaM激酶 II，在许多进程中起重要作用，如神经递质释放，转录因子调节和糖代谢。大脑中约 1%到 2%的蛋白是 CaM激酶 II。 The multifunctional CaM kinase: also known as CaM kinase II, plays an important role in many processes, such as neurotransmitter release, transcription factor regulation, and glucose metabolism. About 1% to 2% of the protein in the brain is CaM kinase II.

CaMK I - CAMK1, CAM 1D, CAMK1G CaMK I - CAMK1, CAM 1D, CAMK1G

CaMK II - CAMK2A, CAM 2B CAMK2D, CA K2G CaMK II - CAMK2A, CAM 2B CAMK2D, CA K2G

CaMK IV - CAMK4 CaMK IV - CAMK4

丝裂原活化蛋白激酶（MAPKs ) 在细胞外刺激 (丝裂原）下起作用，调节多种细胞活动，如基因表达、有丝***、分化和细胞生存 /凋亡。 Mitogen-activated protein kinases (MAPKs) act under extracellular stimuli (mitogens) and regulate a variety of cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis.

MAPK与多数非核的致癌基因活动相关，其对生长因子如 BDNF或神经生长因子作用下细胞反应密切相关。 MAPK is associated with most non-nuclear oncogene activities and is closely related to cellular responses to growth factors such as BDNF or nerve growth factor.

细胞外刺激通过信号级联（MAPK级联）导致 MAP激酶活化， MAPK级联由 MAP激酶、 MAP激酶激酶 (MKK或 MAP2K)和 MAP激酶激酶激酶（MKKK或 MAP3K) 组成。 Extracellular stimulation leads to activation of MAP kinase by a signaling cascade (MAPK cascade) consisting of MAP kinase, MAP kinase kinase (MKK or MAP2K) and MAP kinase kinase kinase (MKKK or MAP3K).

细胞外刺激弓 f起 MAP2K丝氨酸和苏氨酸残端磷酸化，导致 MAP3K活化;而后 MAP2K 通过磷酸化其丝氨酸和酪氨酸残端活化 MAP激酶。从酵母到哺乳动物， MAP激酶信号级联发展完善。 Extracellular stimulation of the MAP2K serine and threonine residues phosphorylates, resulting in activation of MAP3K; MAP2K then activates MAP kinase by phosphorylating its serine and tyrosine residues. From yeast to mammals, the MAP kinase signal cascade is well developed.

目前，哺乳动物身上共表现出六种不同的 MAPKs: Currently, there are six different MAPKs in mammals:

细胞外信号调节激酶（ERK1，ERK2) ：在生长因子和佛波酯（一种肿瘤生长促进剂）的刺激下， ERKs (又名 MAP激酶）信号通路被首先活化，调节细胞增殖和分化。 Extracellular signal-regulated kinase (ERK1, ERK2): ERKs (aka MAP kinase) signaling pathway is first activated, modulating cell proliferation and differentiation, stimulated by growth factors and phorbol esters (a tumor growth promoter).

c-Jun N-末端激酶（JNKs) (MAPK8, APK9, MAPK10)也是应激激活蛋白激酶（SAPKs)。 p38 亚基（MAPK11, MAPK12 (= ERK6) , MAPK13, MAPK14) ： JNK和 p38信号通路在应激如细胞因子，紫外线照射，热休克，渗透休克刺激下反应，与细胞分化和凋亡相关。 c-Jun N-terminal kinases (JNKs) (MAPK8, APK9, MAPK10) are also stress-activated protein kinases (SAPKs). P38 subunit (MAPK11, MAPK12 (= ERK6), MAPK13, MAPK14): JNK and p38 signaling pathways under stress such as cytokines, UV irradiation, heat shock, osmotic shock, cell differentiation and apoptosis Related.

ERK5 ERK5 ( APK7) 是最近发现的，可被生长因子和应激刺激活化，参与细胞增殖。 ERK5 ERK5 (APK7) was recently discovered and is activated by growth factors and stress stimuli and is involved in cell proliferation.

ERK3/4 ERK3 (MAPK6)和 ERK4 ( APK4) 在结构上与非典型的 APKs相关，在活化环路上有 SEG基序，主要的区别仅表现在 C端延伸处。 ERK3和 EKR4主要是胞浆蛋白，其结合、转位和活化 MK5 (PPAR, MAP2K5) 。与 ERK4的相对稳定不同， ERK3是非稳定性的。 ERK3/4 ERK3 (MAPK6) and ERK4 (APK4) are structurally related to atypical APKs, with SEG motifs on the active loop, the main difference being only in the C-terminal extension. ERK3 and EKR4 are mainly cytosolic proteins that bind, translocate and activate MK5 (PPAR, MAP2K5). Unlike the relative stability of ERK4, ERK3 is unstable.

ERK7/8 (MAPK15) 是 MAPKs家族的最新成员，作用与非典型的 MAPKs类似， ERK7/8 有一个类似与 ERK3/4的 C末端。 ERK7/8 (MAPK15) is the newest member of the MAPKs family and functions similarly to atypical MAPKs. ERK7/8 has a C-terminus similar to ERK3/4.

Mos/Raf 激酶是组成 MAPKK激酶家族的一部分，被生长因子活化。该酶的功能是刺激细胞生长。目前对 Raf 的抑制已成为新型抗肿瘤转移药物的靶点，药物能通过抑制 MAPK级联以减少细胞增殖。 Mos/Raf kinase is part of the MAPKK kinase family and is activated by growth factors. The function of this enzyme is to stimulate cell growth. The current inhibition of Raf has become a target for novel anti-tumor metastatic drugs that can reduce cell proliferation by inhibiting the MAPK cascade.

Pelle是一个丝氨酸 /苏氨酸激酶，能自身磷酸化， Tube和 Toll也是如此。 Pelle is a serine/threonine kinase that phosphorylates itself, as does Tube and Toll.

其他成员包括： Rhol, Racl, VE-cadherin, Tiaml, Epacl, Ra l, pll5-RhoGEF, phospho-Vav2, and pan~Vav2 , di-phospho— MLC， phospho— PKA substrate, phospho-PAKl, and PAK1 本发明所述检测恶性肿瘤的试剂中的恶性肿瘤包括： Other members include: Rhol, Racl, VE-cadherin, Tiaml, Epacl, Ra l, pll5-RhoGEF, phospho-Vav2, and pan~Vav2, di-phospho-MLC, phospho-PKA substrate, phospho-PAKl, and PAK1 The malignant tumor in the reagent for detecting a malignant tumor of the invention includes:

脑恶性肿瘤：星形细胞瘤，少突胶质细胞瘤，室管膜瘤，脑（脊）膜瘤，以及胚 (组织）瘤。 Brain malignant tumors: astrocytoma, oligodendroglioma, ependymoma, brain (spine), and embryo (tissue) tumors.

肺癌：是引起人类因肿瘤死亡的首要因素。 Lung cancer: It is the primary cause of human death from cancer.

甲状腺癌：甲状腺癌有四型，其中一型是滤泡状癌。 Hurthle细胞甲状腺癌与滤泡状甲状腺癌不同，其在甲状腺癌中的发生率约为 4%。 Thyroid cancer: There are four types of thyroid cancer, one of which is follicular carcinoma. Hurthle cell thyroid cancer, unlike follicular thyroid cancer, has an incidence of about 4% in thyroid cancer.

膀胱癌是泌尿道最常见的恶性肿瘤。 Bladder cancer is the most common malignant tumor of the urinary tract.

乳腺癌 Breast cancer

*** 卵巢癌 Cervical cancer Ovarian cancer

子宫癌 Uterine cancer

霍奇金（氏）病是源于***的恶性肿瘤。它发生在任何年龄段，但以儿童和青少年为多见。 Hodgkin's disease is a malignant tumor derived from lymph nodes. It occurs at any age, but is more common in children and adolescents.

非霍奇金（氏）淋巴瘤 non-Hodgkin's lymphoma

骨癌：绝大多数骨肿瘤是继发性肿瘤，由身体其他部位的原发性恶性肿瘤细胞扩散至骨而发展而来。 Bone cancer: Most bone tumors are secondary tumors that develop from the spread of primary malignant tumor cells in other parts of the body to the bone.

骨髓癌：绝大多数骨髓癌是继发性肿瘤，因其由发生于身体其他部位扩散、转移而来。 Bone marrow cancer: The vast majority of myeloma is a secondary tumor that is caused by the spread and metastasis that occurs in other parts of the body.

黑色素瘤 Melanoma

胆囊癌：美国癌症学会认为，胆囊癌是发生在胃肠道的第四大常见恶性肿瘤，约占美国所有癌症发生率的 2〜3%。 Gallbladder cancer: According to the American Cancer Society, gallbladder cancer is the fourth most common malignant tumor in the gastrointestinal tract, accounting for about 2 to 3% of all cancers in the United States.

结肠和直肠癌是男性第三大常见恶性肿瘤，也是女性第二大常见恶性肿瘤。食管癌多发生在 50到 70岁之间。 Colon and rectal cancer is the third most common malignant tumor in men and the second most common malignant tumor in women. Esophageal cancer occurs mostly between the ages of 50 and 70.

胃肠道肿瘤：胃肠道，或称为消化***，消化我们吃入的食物，吸收营养物质，并把残渣作为粪便形式排出体外。 Gastrointestinal tumors: The gastrointestinal tract, or the digestive system, digests the food we eat, absorbs nutrients, and excretes the residue as a form of feces.

肾癌: "肾"意味着与肾脏相关的部分； "癌"是指发生在腺体、皮肤以及粘膜上皮细胞的恶性肿瘤的一种。 Kidney cancer: "Kidney" means a part related to the kidney; "Cancer" refers to a type of malignant tumor that occurs in glandular, cutaneous, and mucosal epithelial cells.

白血病是造血组织的恶性肿瘤，造血组织包括骨髓、 ***、脾脏。虽然白血病是引起 3〜14岁儿童死亡的主要病因，白血病在成人中也很常见。 Leukemia is a malignant tumor of hematopoietic tissue. Hematopoietic tissues include bone marrow, lymph nodes, and spleen. Although leukemia is the leading cause of death in children aged 3 to 14 years, leukemia is also common in adults.

肝癌多发生在 45岁之后，男性高发于女性。 Liver cancer occurs mostly after 45 years of age, and males are more likely to develop in women.

淋巴瘤是淋巴***的恶性肿瘤。淋巴***包括***，淋巴腺以及脾脏，淋巴腺遍布全身，通过毛细***彼此连接。 Lymphoma is a malignant tumor of the lymphatic system. The lymphatic system includes lymph nodes, lymph glands, and spleen, which are distributed throughout the body and are connected to each other by capillary lymphatic vessels.

口腔、咽与喉癌包括白斑或红斑形成，口腔和喉咙黏膜溃烂。 Oral, pharyngeal and laryngeal cancers include leukoplakia or erythema, mucosal ulceration of the mouth and throat.

胰腺癌是位于胃后面的腺体。胰腺分泌的胰液内包括可以消化食物的酶，也包括胰岛细胞分泌胰岛素，胰岛素可以帮助调节血糖水平。直肠癌：美国癌症学会认为，美国是世界上直肠癌发病率最高的国家之一，仅次于***癌、肺癌和皮肤癌。 Pancreatic cancer is a gland located behind the stomach. The pancreatic juice secreted by the pancreas includes enzymes that can digest food, as well as insulin secretion from islet cells. Insulin can help regulate blood sugar levels. Rectal Cancer: The American Cancer Society believes that the United States is one of the countries with the highest incidence of rectal cancer in the world, second only to prostate cancer, lung cancer and skin cancer.

***癌 Prostate cancer

睾丸癌 Testicular cancer

肉瘤是发生在肌肉、骨、软骨及纤维组织中的恶性肿瘤。肉瘤的命名是源于其最初生长的组织类型。 Sarcomas are malignant tumors that occur in muscle, bone, cartilage, and fibrous tissue. The sarcoma is named after the type of tissue it was originally grown on.

皮肤癌 skin cancer

胃癌在世界范围内普遍发生，而近四十年来，美国的胃癌发病率显著降低。咽癌：最常见的咽喉肿瘤是喉癌。 Gastric cancer is common worldwide, and in the past 40 years, the incidence of gastric cancer in the United States has decreased significantly. Pharyngeal cancer: The most common throat tumor is laryngeal cancer.

输尿管肿瘤：输尿管是连接肾脏和膀胱的管道，通过肌肉收縮将尿液输送至膀胱。眼癌相对罕见。其影响眼睛的许多区域：眼窝及其保护组织，眼球，眼膜和眼睑。本发明所述检测恶性肿瘤试剂包括口服、胃肠外给药、经皮给药、外用、直肠给药、皮下给药、肌肉给药、静脉给药、 ***给药、鼻内给药、支气管给药、直接浸润、腹膜给药或局部灌流的药物。 Ureteral Tumor: The ureter is a conduit that connects the kidneys to the bladder and delivers urine to the bladder through muscle contraction. Eye cancer is relatively rare. It affects many areas of the eye: the eye socket and its protective tissue, eyeballs, eye mask and eyelids. The detection of malignant tumor agents of the present invention includes oral, parenteral, transdermal, topical, rectal, subcutaneous, intramuscular, intravenous, vaginal, intranasal, bronchial Drugs for administration, direct infiltration, peritoneal administration or local perfusion.

本发明涉及的鸟苷酸环化酶 / 利钠肽受体的配体或活化剂及其主要分布的细胞和组织总结于表 1 : 表 1. 鸟苷酸环化酸 /利钠肽受体的配体或活化剂及其主要分布的细胞和组织 The ligands or activators of the guanylate cyclase/nagin peptide receptors and their major distributions of cells and tissues of the present invention are summarized in Table 1: Table 1. Guanylate cyclized acid/sodium peptide receptors Ligand or activator and its predominantly distributed cells and tissues

GC/NP (鸟苷酸环

GC/NP (guanylate ring

配体分布组织 Ligand distribution organization

化酶 /利钠肽）受体 Chemase/Natriuretic Peptide Receptor

(鸟苷 /尿鸟苷） (C型利钠肽受体） (guanosine / guanosine) (C-type natriuretic peptide receptor)

Orphan (根据美国 the Orphan Drug Orphan (according to the United States the Orphan Drug

GC-D GC-D

Act of January 1983 ("ODA"发展的嗅神经上皮 Act of January 1983 ("ODA" development of the olfactory nerve epithelium

(D型利钠肽受体） (D-type natriuretic peptide receptor)

药物） Drug)

GC-E GC-E

Act of January 1983 ("ODA"发展的视网膜，松果体 Act of January 1983 ("ODA" development of the retina, pineal gland

(E型利钠肽受体） (E-type natriuretic peptide receptor)

药物） Drug)

GC-F GC-F

Act of January 1983 ("ODA"发展的视网膜 Act of January 1983 ("ODA" development of the retina

(F型利钠肽受体） (F-type natriuretic peptide receptor)

药物） Drug)

GC-G GC-G

Act of January 1983 ("ODA"发展的骨骼肌，肺，肠，肾及其它组织Act of January 1983 ("ODA" development of skeletal muscle, lung, intestine, kidney and other tissues

(G型利钠肽受体） (G-type natriuretic peptide receptor)

药物） Drug)

ROS-GC 素（无中 ROS-GC (no middle)

钙调素结合蛋白杆体外节 Calmodulin-binding protein

文） Text)

Act of January 1983 ("ODA"发展的 GC-Y-X1(无中文） C. elegans的感觉神经 Act of January 1983 ("ODA" development of GC-Y-X1 (no Chinese) C. elegans sensory nerve

药物） Drug)

NO (—氧化氮), CO (—氧化碳）可溶性环化酶平滑肌，血小板，肾，肺及其它组织 NO (-nitrogen oxide), CO (carbon monoxide) soluble cyclase smooth muscle, platelets, kidney, lung and other tissues

本发明方法中所述的 cGMP (环磷酸鸟苷）类似物包括 PKG (蛋白激酶 G) 的直接活化剂如 8-溴 -cGMP及相关 cGMP类似物。 The cGMP (cyclic guanosine monophosphate) analogs described in the methods of the invention include direct activators of PKG (protein kinase G) such as 8-bromo-cGMP and related cGMP analogs.

本发明在国际上巳阐明 N0信息***与恶性肿瘤的实质性关系的基础上，发展出与 N0诺贝尔奖理论相关的恶性肿瘤发现、定性、转移和治疗效果评价的核心技术。该核心技术结合恶性肿瘤细胞***周期和分化特点，将对恶性肿瘤中 N0相关分子的分布及表达进行定性和定量诊断。为实施一氧化氮生物学调节为基础的恶性肿瘤靶向性药物综合治疗奠定基础。揭示了一氧化氮及其信息传递***具有使用的靶向性，广泛性、特异性、敏感性、有效性及无毒无副作用性，有广泛临床应用价值。附图说明 The present invention internationally clarifies the substantial relationship between the N0 information system and malignant tumors, and develops the core technology for the discovery, characterization, metastasis and therapeutic effect evaluation of malignant tumors related to the N0 Nobel Prize theory. This core technology combines the cell division cycle and differentiation characteristics of malignant tumors to qualitatively and quantitatively diagnose the distribution and expression of N0-related molecules in malignant tumors. To lay the foundation for the implementation of comprehensive treatment of malignant tumor-targeted drugs based on the biological regulation of nitric oxide. It reveals that nitric oxide and its information transmission system have the specificity, specificity, sensitivity, effectiveness, non-toxic and no side-effect, and have wide clinical application value. DRAWINGS

图 1 炎症、 iNOS (诱导型一氧化氮合酶)、蛋白质酪氨酸硝基化与恶性肿瘤关系示意图。 Figure 1. Schematic diagram of the relationship between inflammation, iNOS (inducible nitric oxide synthase), protein tyrosine nitration and malignancy.

图 2恶性肿瘤中 sGC (可溶性鸟苷酸环化酶）刺激的非反应性及 cGMP水平降下。图 3和图 4 恶性肿瘤中 sGC (可溶性鸟苷酸环化酶） mRNA表达和 sGC (可溶性鸟苷酸环化醜）蛋白表达的特异性（M 恶性）。 Figure 2. Non-reactive and cytosolic levels of sGC (soluble guanylate cyclase) stimulation in malignant tumors. Figure 3 and Figure 4 Specificity of sGC (soluble guanylate cyclase) mRNA expression and sGC (soluble guanylate cyclin) protein expression in malignant tumors (M malignant).

图 5 实验所用良、恶性肿瘤样本的人端粒酶逆转录酶表达。 Figure 5 Human telomerase reverse transcriptase expression in a good, malignant tumor sample used in the experiment.

图 6 良性和恶性肿瘤样本中 NPR-A (GC-A, A型利钠肽受体), NPR-B (GC-B, B型利钠肽受体） and GC-C (C型利钠肽受体) mRNA表达（BM=良性脑膜瘤; 性脑膜瘤； Figure 6 NPR-A (GC-A, type A natriuretic peptide receptor), NPR-B (GC-B, B-type natriuretic peptide receptor) and GC-C (type C natriuretic peptide) in benign and malignant tumor samples Peptide receptor) mRNA expression (BM = benign meningioma; meningioma;

G-神经胶质瘤)。 G-glioma).

图 7钠尿肽家族和受体示意图。 Figure 7. Schematic diagram of the natriuretic peptide family and receptors.

图 8和图 9 ANP (A型利钠肽)、 BNP (B型利钠肽）加上磷酸二脂酶抑制剂 IBMX对神经胶质瘤细胞不同浓度处理，在第 4天和第 6天分别检测其增生情况。 CON为空白对照组。 Figure 8 and Figure 9 ANP (type A natriuretic peptide), BNP (type B natriuretic peptide) plus phosphodiesterase inhibitor IBMX treatment of different concentrations of glioma cells, on days 4 and 6, respectively Detect its proliferation. CON is a blank control group.

具体实施方式实例 1 恶性肿瘤中变异的诱导性一氧化氮合成酶表达 DETAILED DESCRIPTION OF THE INVENTION Example 1 Inducible expression of nitric oxide synthase in malignant tumors

本发明人使用经过美国肿瘤病理学执照医师检验认可的人类良性和恶性肿瘤手术采取样本，仅使用肿瘤原代培养细胞或肿瘤组织进行一氧化氮生物学分析. 揭示了恶性肿瘤中变异的诱导性一氧化氮合成酶表达，为将 iNOS 检测用于恶性肿瘤诊断，肿瘤扩散追踪，及治疗效果评价的方法和试剂奠定了基础. 实验结果显示良性肿瘤没有或仅有非常少量的诱导性一氧化氮合成酶（iNOS)的表达。但随着肿瘤恶性程度的提高，诱导性一氧化氮合成酶 (iNOS)的表达也呈相关性提高（图 1 ) 。 The present inventors performed a sample of human benign and malignant tumors approved by a tumor pathologist in the United States, and used only primary tumor cells or tumor tissues for biological analysis of nitric oxide. Revealed the inducibility of variation in malignant tumors. Nitric oxide synthase expression lays the foundation for the use of iNOS detection for malignant tumor diagnosis, tumor spread tracking, and evaluation of therapeutic effects. Experimental results show that there is no or only a very small amount of inducible nitric oxide in benign tumors. Expression of synthetase (iNOS). However, as the degree of malignancy of the tumor increased, the expression of inducible nitric oxide synthase (iNOS) was also correlated (Fig. 1).

蛋白质酪氨酸硝基化趋炎性恶性肿瘤环境中 iNOS 大量表达导致了过量产生活性氧 (reactive oxygen species, R0S) /活性氮 (reactive nitrogen species, RNS) .其中引起细胞损害主要由 0N00—介导。 NO 与 0₂—结合形成 0N00-, 而 0N0CT具有极强氧化性，可与蛋白质、脂质、碳水化合物和核酸反应，在休克、炎症、缺血再灌注损伤、呼吸***疾病及神经退行性病变中起重要作用。 0Ν0( 也可与体内活性分子发生反应，如内源性抗氧化剂 (抗坏血酸，维生素 Ε)、巯基团和芳香族化合物。 0N0CT导致蛋白质的氧化，使含硫基团氧化、羟基化和硝化，也可使蛋白质酪氨酸残基硝化The expression of iNOS in the environment of protein tyrosine nitration-induced inflammatory malignant tumors leads to excessive production of reactive oxygen species (ROS) / reactive nitrogen species (RNS), which causes cell damage mainly by 0N00- guide. NO combines with 0 ₂ to form 0N00-, while 0N0CT is highly oxidizing and can react with proteins, lipids, carbohydrates and nucleic acids in shock, inflammation, ischemia and reperfusion It plays an important role in injury, respiratory diseases and neurodegenerative diseases. 0Ν0 (also reacts with active molecules in the body, such as endogenous antioxidants (ascorbic acid, vitamin oxime), sulfonium groups and aromatic compounds. 0N0CT leads to oxidation of proteins, oxidation, hydroxylation and nitration of sulfur-containing groups, also Nitrification of protein tyrosine residues

(图 1)，这可改变细胞内信号通路（例如，对酪氨酸磷酸化的影响）。实例 2 恶性肿瘤中环磷酸鸟苷（cGMP) 和可溶性鸟甘酸环化酶（sGC) 的变异试验环磷酸鸟苷（cGMP) 可以被可溶性鸟甘酸环化酶（sGC) 或细胞膜性鸟甘酸环化酶（pGC) 所合成. 可溶性鸟甘酸环化酶由 α和 β两个亚基协同产生酶的活力. 环磷酸鸟苷（cGMP) —旦被合成，可通过 cGMP-依赖性蛋白激酶（PKGs)，环苷介导的离子通道 (CNGs), cGMP 调节的磷酸二脂酶（PDEs) 等途径发挥作用。 (Figure 1), this changes the intracellular signaling pathway (for example, the effect on tyrosine phosphorylation). Example 2 Mutation of cyclic guanosine monophosphate (cGMP) and soluble ornithine cyclase (sGC) in malignant tumors Cyclic guanosine monophosphate (cGMP) can be soluble in ornithine cyclase (sGC) or membranous ornithine cyclase (pGC) synthesized. Soluble ornithine cyclase synergistically produces enzyme activity from two subunits, alpha and beta. Cyclic guanosine monophosphate (cGMP), once synthesized, can pass cGMP-dependent protein kinases (PKGs), Cyclic mediated ion channels (CNGs), cGMP-regulated phosphodiesterase (PDEs), and other pathways play a role.

上述实验结果显示，随着肿瘤恶性程度的提高，诱导性一氧化氮合成酶 (iNOS) 的表达也呈相关性提高。在恶性肿瘤中，高表达的 iNOS 所合成的大量一氧化氮生产 (N0)是如何对其下游信息***起作用的？为了解明机理,在实验中分别使用了 NONOate (—氧化氮供给药）和 Bay412272 (可溶性鸟甘酸环化酶特异性刺激剂)，如图 2所示，与良性肿瘤不同，恶性肿瘤中的可溶性鸟甘酸环化酶对一氧化氮及可溶性鸟甘酸环化酶特异性刺激剂的刺激没有反应. 环磷酸鸟苷（cGMP) 的水平很低。 . 为了进一步解密恶性肿瘤中环磷酸鸟苷（cGMP) 水平低下的原因，使用了 The above experimental results show that the expression of inducible nitric oxide synthase (iNOS) is also correlated with the increase of tumor malignancy. In malignant tumors, how does the large amount of nitric oxide production (N0) synthesized by highly expressed iNOS contribute to its downstream information system? To understand the mechanism, NONOate (-nitrogen oxide supply) and Bay412272 (soluble ornithine cyclase specific stimulator) were used in the experiment, as shown in Figure 2. Unlike benign tumors, soluble birds in malignant tumors. Glycolate cyclase does not respond to stimulation by nitric oxide and soluble ornithine cyclase-specific stimulators. The level of cyclic guanosine monophosphate (cGMP) is very low. To further decipher the cause of low levels of cyclic guanosine monophosphate (cGMP) in malignant tumors,

SDSPAGE蛋白质电泳及特异性抗体，对良性和恶性肿瘤中可溶性鸟甘酸环化酶 α和 β两个亚基的蛋白表达进行了检测，结果证明恶性肿瘤中可溶性鸟甘酸环化酶 α和 β 两个亚基的蛋白表达均消失或极弱（图 3、 4)。 SDSPAGE protein electrophoresis and specific antibodies were used to detect the protein expression of soluble ornithine cyclase alpha and beta subunits in benign and malignant tumors. The results showed that soluble ornithine cyclase α and β were found in malignant tumors. The protein expression of the subunits disappeared or was extremely weak (Fig. 3, 4).

为了更严密的进行分子生物学的分析，对良性和恶性肿瘤中可溶性鸟甘酸环化酶 α和 β两个亚基的基因（mRNA)表达进行了实时 RT-PCR检测 (7700 Prizm Sequence Detector System), 如图 3 所表明，恶性肿瘤中可溶性鸟甘酸环化酶 α和 β两个亚基的基因表达均消失或极弱。结果揭示了恶性肿瘤中环磷酸鸟苷（cGMP) 和可溶性鸟甘酸环化酶（sGC) 的变异（图 2, 3, 4) ,使用肿瘤原代培养细胞或肿瘤组织进行一氧化氮生物学分析. 揭示了恶性肿瘤中环磷酸鸟苷（cGMP) 和可溶性鸟甘酸环化酶 (sGC) 的变异（图 2, 3, 4) , 为将 cGMP和 sGC 的检测用于恶性肿瘤诊断，肿瘤扩散追踪的试剂奠定了基础。 For more stringent molecular biology analysis, real-time RT-PCR detection of the gene (mRNA) expression of soluble ornithine cyclase alpha and beta subunits in benign and malignant tumors (7700 Prizm Sequence Detector System) As shown in Figure 3, the gene expression of soluble ornithine cyclase alpha and beta subunits in malignant tumors disappeared or was extremely weak. The results revealed mutations in cyclic guanosine monophosphate (cGMP) and soluble ornithine cyclase (sGC) in malignant tumors (Fig. 2, 3, 4). The primary cultured cells or tumor tissues were used for biological analysis of nitric oxide. Reveals the variation of cyclic guanosine monophosphate (cGMP) and soluble ornithine cyclase (sGC) in malignant tumors (Fig. 2, 3, 4). For the detection of cGMP and sGC for malignant tumor diagnosis, tumor spread The traced reagents laid the foundation.

人端粒酶逆转录酶本发明人还将所使用的良性和恶性肿瘤样本进行了人端粒酶逆转录酶 (hTERT)的 RT-PCR (7700 Prizm Sequence Detector System)检测。如（图 5 ) 所表明，恶性肿瘤中人端粒酶逆转录酶的基因表达非常高，而良性肿瘤样本则均极弱。进一步证明本发明人使用的，经过美国肿瘤病理学执照医师检验认可的人类良性和恶性肿瘤样本的可靠性。 Human telomerase reverse transcriptase The present inventors also used benign and malignant tumor samples for human telomerase reverse transcriptase (hTERT) RT-PCR (7700 Prizm Sequence Detector System). As shown in (Fig. 5), human telomerase reverse transcriptase gene expression is very high in malignant tumors, while benign tumor samples are extremely weak. The reliability of human benign and malignant tumor samples approved by the inventors of the United States for examination by a tumor pathologist is further demonstrated.

实例 3 钠尿肽家族受体及其细胞膜性鸟甘酸环化酶 Example 3 Natriuretic peptide family receptor and its membrane membranous ornithine cyclase

钠尿肽家族 (natriuretic peptides, NPs)主要包括心房钠尿肽（ atrial natriuretic peptide, ANP)^ 脑钠尿肽( brain natriuretic peptide, BNP)、 C-型钠尿肽 (c-type natriuretic peptide,CNP)及人工合成的血管钠肽（ vasonatrin peptide, VNP)等，在维持机体水盐平衡、血压稳定、心血管及肾脏等器官功能中具有重要意义的一组多肽. 钠尿肽家族作用于其受体产生作用，其中两种受体是细胞膜性鸟甘酸环化酶（pGC) g|3: NPR-A/GC-A and NPR-B/GC-B. (图 6) Natriuretic peptides (NPs) mainly include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), c-type natriuretic peptide, CNP And synthetic natriuretic peptide (VAP), a group of peptides that are important in maintaining the body's water and salt balance, blood pressure stability, cardiovascular and kidney functions. The natriuretic peptide family acts on it. The two receptors are cell membrane ornithine cyclase (pGC) g|3: NPR-A/GC-A and NPR-B/GC-B. (Fig. 6)

为了对环磷酸鸟苷（cGMP) 的产生背景更严密的进行分子生物学的分析，本发明人对良性和恶性肿瘤中钠尿肽家族的受体基因（mRNA)的表达进行了全面分析。如图 5 所表明，实时 RT-PCR检测（7700 Prizm Sequence Detector System) 结果没有发现钠尿肽家族受体的表达与肿瘤的恶性程度呈相关性，这组实验数据为使用钠尿肽家族受体配体来上调环磷酸鸟苷（cGMP) 提供了依据，钠尿肽家族受体及其细胞膜性鸟甘酸环化酶与恶性肿瘤关系的研究表明，由于恶性肿瘤可溶性鸟甘酸环化酶的表达变异，从而，肿瘤是否表达钠尿肽家族受体或表达哪一类型钠尿肽家族受体，不仅直接关系到肿瘤组织自身环磷酸鸟苷（cGMP) 的产生水平，而且为将钠尿肽家族受体配体用于检测恶性肿瘤药制备提供基础。 In order to more closely analyze the molecular biology of cyclic guanosine monophosphate (cGMP), the present inventors have comprehensively analyzed the expression of the receptor gene (mRNA) of the natriuretic peptide family in benign and malignant tumors. As shown in Figure 5, real-time RT-PCR (7700 Prizm Sequence Detector System) results showed no correlation between the expression of the natriuretic peptide family receptor and the malignancy of the tumor. The experimental data used the natriuretic peptide family receptor. Ligand-mediated upregulation of cyclic guanosine monophosphate (cGMP) provides a basis for the relationship between the natriuretic peptide family receptor and its membrane membranous ornithine cyclase and malignant tumors, suggesting that the expression variation of soluble ornithine cyclase in malignant tumors Thus, whether the tumor expresses a natriuretic peptide family receptor or which type of natriuretic peptide family receptor is expressed is not only directly related to the level of production of cyclic guanosine monophosphate (cGMP) in the tumor tissue, but also to the natriuretic peptide family. The ligands provide a basis for the detection of malignant drug preparation.

实例 4 通过磷酸二酯酶信息***调节细胞内环磷酸鸟苷（cGMP)水平与恶性肿瘤治疗试验 Example 4 Regulation of intracellular cyclic guanosine monophosphate (cGMP) levels and malignant tumor treatment trials by phosphodiesterase information system

磷酸二酯酶的活性直接关系到细胞内第二信使, cAMP和 cGMP 的浓度. 现己解明的磷酸二酯酶亚型有 11 种。根据各个磷酸二酯酶亚型分布的细胞和组织的不同, 以及各病理状况下表达的改变，磷酸二酯酶抑制剂的使用为多种疾病的治疗带来了新的希望。总结近年来的临床应用情况，并对其作用机制进行分析，为进一步的开发应用提供依据。 The activity of phosphodiesterase is directly related to the intracellular second messenger, cAMP and cGMP concentrations. There are 11 phosphodiesterase subtypes. Depending on the cell and tissue distribution of each phosphodiesterase subtype, As well as changes in expression under various pathological conditions, the use of phosphodiesterase inhibitors has brought new hope for the treatment of various diseases. Summarize the clinical application situation in recent years, and analyze its mechanism of action to provide a basis for further development and application.

虽然心房钠尿肽 (ANP)或脑钠尿肽 (BNP)作用于其受体即： NPR-A/GC-A and NPR-B/GC-B可使细胞内环磷酸鸟苷（cGMP)水平提高（图 7) ,但当本发明人单独使用心房钠尿肽 (ANP)或脑钠尿肽 (BNP)时，对恶性肿瘤生长的抑制作用不明显。然而, 我们用心房钠尿肽 (ANP)和脑钠尿肽 (BNP)再加上磷酸二脂酶抑制剂 IBMX对恶性肿瘤细胞进行处理，如（图 8， 9) 结果显示，心房钠尿肽 (ANP)和脑钠尿肽 (BNP) 均浓度依赖性的抑制了恶性肿瘤细胞的生长（5X10— ⁵M和 5X10_⁶M, 统计学 P<0. 01) 。 Although atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) acts on its receptors: NPR-A/GC-A and NPR-B/GC-B can cause intracellular cyclic guanosine monophosphate (cGMP) levels Increased (Fig. 7), but when the inventors used atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) alone, the inhibitory effect on the growth of malignant tumors was not significant. However, we treated malignant tumor cells with atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) plus phosphodiesterase inhibitor IBMX, as shown (Figure 8, 9). Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were concentration-dependent inhibition of the growth of malignant tumor cells (5X10- ⁵ M and 5X10_ ⁶ M, statistically P <0. 01).

结果表明通过确定磷酸二酯酶亚型在恶性肿瘤的表达变异，为靶向性提高肿瘤组织自身环磷酸鸟苷（cGMP) 的产生水平提供依据，并为将磷酸二酯酶亚型的检测作为靶向性抑制剂用于检测恶性肿瘤的药物提供基础。 The results indicate that by determining the expression variation of phosphodiesterase subtypes in malignant tumors, it provides a basis for targeting the increase of the level of cGMP production in tumor tissues, and the detection of phosphodiesterase subtypes is Targeted inhibitors provide a basis for drugs for detecting malignant tumors.

Claims

权利要求 Rights request

1、一种一氧化氮及其信息传递***在制备检测恶性肿瘤的试剂中的应用。 1. Use of a nitric oxide and an information delivery system thereof for preparing a reagent for detecting a malignant tumor.

2、根据权利要求 1所述一氧化氮及其信息传递***在制备检测恶性肿瘤的试剂中的应用，其特征在于该试剂包括以下系列- 2. Use of nitric oxide and an information delivery system thereof according to claim 1 for the preparation of a reagent for detecting a malignant tumor, characterized in that the reagent comprises the following series -

1) 诱导型一氧化氮合酶（iNOS 或 N0S- 2)的基因及蛋白表达和活性的试剂，内皮型一氧化氮合成酶 (eNOS或 N0S-3)的基因及蛋白表达和活性的试剂，神经型一氧化氮合成酶亚型 (nNOS或 N0S-1)的基因及蛋白表达和活性的试剂； 1) an agent for inducible nitric oxide synthase (iNOS or NOS-2) gene and protein expression and activity, an agent for endothelial nitric oxide synthase (eNOS or NOS-3) gene and protein expression and activity, An agent for the expression and activity of a gene and protein of a neuronal nitric oxide synthase subtype (nNOS or NOS-1);

2) 可溶性鸟苷酸环化酶 sGC 的 α1、 α2 ; β1、 β2亚基的基因及蛋白表达和活性的试剂； 2) soluble guanylate cyclase sGC α1, α2; β1, β2 subunit gene and protein expression and activity reagents;

3) 环磷酸鸟苷（cGMP)在各种生物标本和肿瘤标本中的浓度或含量测定的试剂; 3) reagents for determining the concentration or content of cyclic guanosine monophosphate (cGMP) in various biological specimens and tumor specimens;

5) 磷酸二酯酶 PDE系列的基因及蛋白表达和活性的试剂； 5) Phosphodiesterase PDE series of genes and protein expression and activity reagents;

3、根据权利要求 1所述一氧化氮及其信息传递***在制备检测恶性肿瘤的试剂中的应用，其特征在于所述恶性肿瘤包括脑恶性肿瘤、肺癌、甲状腺癌、膀胱癌、乳腺癌、 ***、卵巢癌、子宫癌、霍奇金（氏）病、非霍奇金（氏）淋巴瘤、骨癌、骨髓癌、黑色素瘤、胆囊癌、结肠和直肠癌、食管癌、胃肠道肿瘤、肾癌、白血病、肝癌、淋巴瘤、口腔、咽与喉癌、胰腺癌、直肠癌、 ***癌、睾丸癌、肉瘤、皮肤癌、胃癌、输尿管肿瘤或眼癌。 3. The use of nitric oxide according to claim 1 and an information delivery system thereof for the preparation of a medicament for detecting a malignant tumor, characterized in that the malignant tumor comprises a brain malignant tumor, a lung cancer, a thyroid cancer, a bladder cancer, a breast cancer, Cervical cancer, ovarian cancer, uterine cancer, Hodgkin's disease, non-Hodgkin's lymphoma, bone cancer, bone marrow cancer, melanoma, gallbladder cancer, colon and rectal cancer, esophageal cancer, gastrointestinal tract Tumor, kidney cancer, leukemia, liver cancer, lymphoma, oral, pharyngeal and laryngeal cancer, pancreatic cancer, rectal cancer, prostate cancer, testicular cancer, sarcoma, skin cancer, stomach cancer, ureteral tumor or eye cancer.

4、根据权利要求 1-3任一项所述一氧化氮及其信息传递***在制备检测恶性肿瘤的试剂中的应用，其特征在于所述检测恶性肿瘤试剂包括口服、胃肠外给药、经皮给药、外用、直肠给药、皮下给药、肌肉给药、静脉给药、 ***给药、鼻内给药、支气管给药、直接浸润、腹膜给药或局部灌流的药物。 The use of the nitric oxide according to any one of claims 1 to 3 and the information transmission system thereof for preparing a reagent for detecting a malignant tumor, characterized in that the reagent for detecting malignant tumor comprises oral administration, parenteral administration, Transdermal, topical, rectal, subcutaneous, intramuscular, intravenous, vaginal, intranasal, bronchial, direct infiltration, peritoneal or topical perfusion.