WO2019114791A1 - GENE EXPRESSION AND APPLICATION OF INTRACELLULAR NF-κB ACTIVITY-BASED ACTIVATION EFFECTOR GENE IN NF-κB OVER-ACTIVATED CELLS - Google Patents

Abstract

Provided are an NF-κB specific activation gene expression vector and an application thereof. The gene expression vector comprises the two elements of a promoter sequence that regulates gene expression and a promoter downstream effector gene encoding sequence; the promoter sequence consists of an NF-κB response sequence and a minimal promoter sequence. After the gene expression vector is introduced into an NF-κB over-activated cell, the effector gene on the vector is expressed by the activation of the sequence-specific transcription factor NF-κB, and the effector gene generates effects on cell physiology, such as cell growth inhibition, apoptosis, death, and so on. The gene expression vector may be used for the preparation of gene therapy agents and drugs for treating diseases associated with NF-κB over-activation.

Description

基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达及应用Gene expression and application of NF-κB activation-activated gene in NF-κB over-activated cells based on intracellular NF-κB activity 技术领域Technical field

本发明涉及一种基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达技术及应用，属于医学生物技术领域。The invention relates to a gene expression technology and application based on NF-κB activity-activated effector gene in NF-κB over-activated cells, and belongs to the field of medical biotechnology.

背景技术Background technique

NF-κB是在B淋巴细胞中发现的一种转录因子，因其参与免疫球蛋白κ轻链的转录调控而得名。后来发现NF-κB是一种广泛分布于各种细胞中的真核细胞转录因子，是先天性免疫、炎症、细胞生存、增殖、凋亡的重要调节者。NF-κB家族有RelA(p65)、RelB、c-Rel，p50/p105(NF-κB1)和p52/p100(NF-κB2)五个成员，其N端都含有一个高度保守的Rel同源区(RHD)，负责结合DNA、二聚化、核定位，以及结合NF-κB抑制蛋白(如IκB)。RelA/p50是最常见的NF-κB靶基因调控者，其中只有RelA具有转录激活结构域(AD)。RelA/p50二聚体一般与其抑制蛋白IκBα结合，滞留在细胞质中。当细胞受刺激时，IκB激酶IKK活化，使IκBα经磷酸化和泛素化后降解，RelA/p50得以释放并进入细胞核。在核内RelA/p50结合其DNA靶点(有时称为κB)，调节靶基因的表达。NF-κB是一种可诱导型转录因子，已知肿瘤坏死因子-α(TNFα)、脂多糖(LPS)、佛波酯(PMA)、紫外线、细菌和病毒等许多物理化学因素可诱导激活NF-κB信号通路。因其重要性，NF-κB自发现以来一直被剧烈研究。目前Pubmed数据库中NF-κB相关文献达7万多篇，2015年该数据库收录了6152篇NF-κB相关论文，充分说明其在科学研究中受到的关注程度，也反映其研究具有重要价值。NF-κB is a transcription factor found in B lymphocytes and is named for its involvement in the transcriptional regulation of the immunoglobulin kappa light chain. It was later discovered that NF-κB is a eukaryotic transcription factor widely distributed in various cells and is an important regulator of innate immunity, inflammation, cell survival, proliferation and apoptosis. The NF-κB family has five members, RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1) and p52/p100 (NF-κB2), all of which contain a highly conserved Rel homology region at the N-terminus. (RHD), responsible for binding DNA, dimerization, nuclear localization, and binding to NF-κB inhibitory proteins (such as IκB). RelA/p50 is the most common regulator of NF-κB target genes, of which only RelA has a transcriptional activation domain (AD). The RelA/p50 dimer generally binds to its inhibitory protein IκBα and remains in the cytoplasm. When the cells are stimulated, the IκB kinase IKK is activated, and the IκBα is degraded by phosphorylation and ubiquitination, and RelA/p50 is released and enters the nucleus. In the nucleus, RelA/p50 binds to its DNA target (sometimes referred to as kappa B) and regulates the expression of the target gene. NF-κB is an inducible transcription factor. It is known that many physicochemical factors such as tumor necrosis factor-α (TNFα), lipopolysaccharide (LPS), phorbol ester (PMA), ultraviolet light, bacteria and viruses can induce NF activation. -κB signaling pathway. Because of its importance, NF-κB has been studied violently since its discovery. At present, there are more than 70,000 NF-κB related articles in the Pubmed database. In 2015, the database included 6152 NF-κB related papers, which fully explained the degree of attention it received in scientific research, and also reflected the important value of its research.

大量研究表明，NF-κB与炎症与癌症的发生发展密切相关。几乎在所有炎症、癌症疾病中，NF-κB常过度活化。因此，NF-κB成为炎症及癌症治疗的重要靶点，筛选和发展NF-κB抑制剂也成为新药发展的重要途径，由此产生了形形色色的大量的NF-κB抑制剂。但实践表明，这些NF-κB抑制剂往往由于过度抑制细胞内的NF-κB活性，而损伤了正常细胞内必须的NF-κB基础活性，从而出现难以克服的副作用。因此，直至目前，仍没有一种NF-κB抑制剂成为临床药物。但由于NF-κB过度活化与炎症与癌症发生发展的密切关系，针对NF-κB过度活化这一病理现象仍有发展新的炎症与癌症治疗方法的可行性。Numerous studies have shown that NF-κB is closely related to the development of inflammation and cancer. NF-κB is often over-activated in almost all inflammatory and cancer diseases. Therefore, NF-κB has become an important target for the treatment of inflammation and cancer. Screening and development of NF-κB inhibitors has also become an important pathway for the development of new drugs, resulting in a large number of NF-κB inhibitors. However, it has been shown in practice that these NF-κB inhibitors often impair the NF-κB basal activity necessary for normal cells due to excessive inhibition of NF-κB activity in the cells, thereby causing insurmountable side effects. Therefore, until now, no NF-κB inhibitor has become a clinical drug. However, due to the close relationship between NF-κB overactivation and the development of inflammation and cancer, the pathogenesis of NF-κB overactivation still has the feasibility of developing new methods of inflammation and cancer treatment.

癌症是目前人类尚未完全攻克的重大疾病，严重地困扰着人类的健康与生活。NF-κB与癌症的发生发展密切相关。以我国最广泛的肝炎肝癌为例，说明一下NF-κB在癌症发 生发展中的重要作用。原发性肝癌是全球第二致命的癌症。在肝炎肝癌的大量研究中，发现NF-κB总是被过度激活。炎症反应在肿瘤中所起的重大作用目前已众所皆知，肝细胞癌(HCC)就是一种典型的炎症相关癌症。慢性炎症与HCC的关系极为密切，以致HCC被作为洞察炎症相关癌症发生过程的良好模型。研究发现，在全球所有地区，任何原因所导致的慢性肝病都可能在肝癌的发生和进展过程中起到重要的作用。因此，在HCC的发生过程中，慢性肝炎是最重要的致病因素之一。NF-κB是一种重要的炎症相关转录因子，大量的研究已表明在HCC发生发展中NF-κB呈高表达和过度活化状态。在人肝癌组织及癌旁组织中均能定量检测到NF-κB活性，且癌组织明显高于癌旁组织。Cancer is a major disease that humans have not yet fully overcome, which seriously plagues human health and life. NF-κB is closely related to the development of cancer. Take the most extensive hepatitis liver cancer in China as an example to illustrate the important role of NF-κB in the development of cancer. Primary liver cancer is the second most deadly cancer in the world. In a large number of studies on hepatitis liver cancer, it was found that NF-κB was always overactivated. The important role of inflammatory response in tumors is well known. Hepatocellular carcinoma (HCC) is a typical inflammation-related cancer. Chronic inflammation is closely related to HCC, so that HCC is used as a good model for insight into the process of inflammation-related cancer development. The study found that chronic liver disease caused by any cause in all regions of the world may play an important role in the occurrence and progression of liver cancer. Therefore, chronic hepatitis is one of the most important pathogenic factors in the development of HCC. NF-κB is an important inflammation-related transcription factor, and a large number of studies have shown that NF-κB is highly expressed and over-activated in the development of HCC. NF-κB activity was quantitatively detected in human hepatocarcinoma tissues and adjacent tissues, and the cancer tissues were significantly higher than adjacent tissues.

激活的NF-κB参与癌症的启动、发生及发展过程，在肝细胞炎症与癌变间起桥梁作用。研究已表明，在慢性肝炎致癌的过程中，与慢性肝炎关系最密切的乙肝及丙肝病毒(HBV、BCV)都可以通过机体内介导炎症反应的重要信号通路——NF-κB信号通路而激活NF-κB。不仅病毒，NF-κB在几乎所有的慢性肝病中都存在激活状态，包括病毒性肝炎、酒精性肝病、非酒精性脂肪肝及肝内胆道疾病等。NF-κB的过度活化是炎症介质如TNF-a、IL-1、IL-6等产生的关键环节。肝细胞长期漫性炎症导致肝硬化，最终发展为肝癌。因此，NF-κB的过度活化及与肝癌的发生有着密切的关系。HBV表达的HBx蛋白可通过ras-raf-map激酶通路激活NF-κB，促使病毒复制、肝细胞增生、细胞癌变、癌细胞转移及凋亡抑制。因此，NF-κB的激活不仅造成诱发细胞癌变的炎性环境，而且通过抑制细胞凋亡而促进肝癌细胞增殖。可以说过度激活的NF-κB参与了肝细胞变性、癌变发生的全过程。正因如此，NF-κB被作为肝癌治疗的靶点。很多研究表明，使用NF-κB抑制剂可有效抑制肝癌细胞的增殖、诱导肿瘤细胞凋亡。研究表明，在肝炎肝癌发生发展中发挥重要作用的很多基因都是NF-κB的靶基因。例如，TNFα、IL1、Bcl-2、GADD45β、IAPs都是近实验确证的NF-κB的直接靶基因。NEMO途径显示了生理状态下NF-κB的作用及NEMO敲除后的结局。其他与肝炎肝癌关系密切的细胞因子如IL6、IL8、CCL2(MCP-1)、ICAM、COX2也是NF-κB的直接靶基因；促进肝癌细胞侵袭与转移的uPA也是NF-κB的直接靶基因。Activated NF-κB is involved in the initiation, development and progression of cancer, and acts as a bridge between hepatocyte inflammation and carcinogenesis. Studies have shown that in the process of carcinogenesis of chronic hepatitis, hepatitis B and hepatitis C virus (HBV, BCV), which are most closely related to chronic hepatitis, can be activated by the NF-κB signaling pathway, an important signaling pathway that mediates inflammation in the body. NF-κB. Not only viruses, but NF-κB is activated in almost all chronic liver diseases, including viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease, and intrahepatic biliary tract disease. Over-activation of NF-κB is a key link in the production of inflammatory mediators such as TNF-a, IL-1, and IL-6. Long-term diffuse inflammation of liver cells leads to cirrhosis and eventually to liver cancer. Therefore, the excessive activation of NF-κB is closely related to the occurrence of liver cancer. HBV-expressed HBx protein activates NF-κB via the ras-raf-map kinase pathway, which promotes viral replication, hepatocyte proliferation, cell carcinogenesis, cancer cell metastasis, and apoptosis inhibition. Therefore, activation of NF-κB not only causes an inflammatory environment that induces cancerous cells, but also promotes proliferation of liver cancer cells by inhibiting apoptosis. It can be said that over-activated NF-κB is involved in the whole process of hepatocyte degeneration and carcinogenesis. For this reason, NF-κB is used as a target for the treatment of liver cancer. Many studies have shown that the use of NF-κB inhibitors can effectively inhibit the proliferation of liver cancer cells and induce tumor cell apoptosis. Studies have shown that many genes that play an important role in the development of hepatitis C liver cancer are target genes of NF-κB. For example, TNFα, IL1, Bcl-2, GADD45β, and IAPs are direct target genes for NF-κB confirmed by experiments. The NEMO pathway shows the role of NF-κB in physiological states and the outcome after NEMO knockout. Other cytokines closely related to hepatitis liver cancer such as IL6, IL8, CCL2 (MCP-1), ICAM, and COX2 are also direct target genes of NF-κB; uPA, which promotes invasion and metastasis of hepatoma cells, is also a direct target gene of NF-κB.

NF-κB是一类重要的调控性转录因子，在许多生理和病理过程中起关键作用，如免疫、细胞增殖、凋亡、炎症、尤其是肿瘤发生。一些研究表明许多疾病与NF-κB及其信号通路的异常活化有关。例如，NF-κB在许多人类癌症中异常激活，通过上调抗细胞凋亡基因来促进生存和恶性肿瘤。已经发现，NF-κB的核聚集和高NF-κB靶基因特征导致大多数多发性骨髓瘤细胞系的NF-κB通路富集，并对细胞凋亡敏感。因此，许多制药公司和科 学家一直致力于开发可用于癌症治疗的NF-κB抑制剂。但是，遗憾的是这些化学药物的特异性低，在发挥抑制NF-κB活性的同时，产生严重的副作用。NF-κB is an important class of regulatory transcription factors that play key roles in many physiological and pathological processes such as immunity, cell proliferation, apoptosis, inflammation, and especially tumorigenesis. Several studies have shown that many diseases are associated with abnormal activation of NF-κB and its signaling pathways. For example, NF-κB is abnormally activated in many human cancers, and promotes survival and malignancy by up-regulating anti-apoptotic genes. It has been found that nuclear aggregation of NF-κB and high NF-κB target gene characteristics result in the enrichment of the NF-κB pathway in most multiple myeloma cell lines and are sensitive to apoptosis. Therefore, many pharmaceutical companies and scientists have been working to develop NF-κB inhibitors for cancer therapy. However, it is a pity that these chemicals have low specificity and exert serious side effects while exerting inhibition of NF-κB activity.

目前，针对NF-κB的抑制剂药物研究和开发主要集中在通过抑制细胞内的NF-κB活性进而实现NF-κB过度活化相关疾病的治疗，但是由于细胞内NF-κB激活通路的多样性、复杂性，一种通路的关闭很难彻底抑制NF-κB活性；此外，切断了一种通路，可能造成该通路负责的其他生理过程的干扰，产生副作用。At present, research and development of inhibitors for NF-κB mainly focus on the treatment of diseases related to NF-κB overactivation by inhibiting NF-κB activity in cells, but due to the diversity of NF-κB activation pathways in cells, Complexity, the closure of one pathway is difficult to completely inhibit NF-κB activity; in addition, cutting off a pathway may cause interference with other physiological processes responsible for the pathway, causing side effects.

发明内容Summary of the invention

发明目的：针对现有NF-κB过度活化相关疾病治疗技术存在的问题，本发明提出一种NF-κB激活基因表达技术，该基因表达技术由一种NF-κB特异性激活基因表达载体实现。载体可以感知细胞内的NF-κB活性，可温和而有效地表达载体上携带的效应基因，导致效应基因在NF-κB过度活化细胞中的特异表达。本发明采用与现有技术中NF-κB抑制剂完全不同的策略，通过利用NF-κB过度活化这一看似不利的病理现象，发展了一种不同于现有技术的NF-κB过度活化相关疾病治疗策略。本发明发展了一种针对NF-κB过度活化相关疾病的基因治疗技术。OBJECTS OF THE INVENTION: In view of the problems existing in the prior art NF-κB overactivation-related disease treatment technology, the present invention proposes an NF-κB activating gene expression technique which is realized by an NF-κB-specific activation gene expression vector. The vector can sense the NF-κB activity in the cell, and can express the effector gene carried on the vector gently and effectively, resulting in the specific expression of the effector gene in the NF-κB over-activated cells. The present invention adopts a completely different strategy from the prior art NF-κB inhibitor, and develops a NF-κB overactivation correlation different from the prior art by utilizing the seemingly unfavorable pathological phenomenon of NF-κB overactivation. Disease treatment strategy. The present invention develops a gene therapy technique for diseases associated with NF-κB overactivation.

技术方案：为了实现上述目的，本发明提出一种基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达技术及其在NF-κB过度活化疾病的治疗药物或试剂中的应用，该基因表达技术由一种NF-κB特异性激活基因表达载体实现。其中基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达技术原理如图1所示。Technical Solution: In order to achieve the above object, the present invention provides a gene expression technology based on NF-κB activity-activated effector gene in NF-κB over-activated cells and a therapeutic drug or reagent thereof for NF-κB over-activation disease. In this application, the gene expression technique is achieved by an NF-κB specific activation gene expression vector. The principle of gene expression based on NF-κB activity-activated effector gene in NF-κB over-activated cells is shown in Figure 1.

其中，所述的NF-κB特异性激活基因表达载体，包含两个序列元件，具体为调控基因表达的启动子序列和启动子下游效应基因编码序列。Wherein, the NF-κB specific activation gene expression vector comprises two sequence elements, specifically a promoter sequence for regulating gene expression and a promoter downstream gene encoding sequence.

其中，所述启动子序列由一段NF-κB应答序列和最小启动子序列组成。Wherein the promoter sequence consists of a NF-κB responsive sequence and a minimal promoter sequence.

更进一步地，所述NF-κB应答序列包括各种序列的NF-κB应答序列；所述NF-κB应答序列为一段可与NF-κB蛋白特异性结合的DNA序列，例如5′-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3′(SEQ ID NO.1)；NF-κB应答序列的主要序列特征为含有数量不同的各种NF-κB结合靶点，如GGGACTTTCC(SEQ ID NO.2)。NF-κB应答序列包括包括各种序列的NF-κB应答序列，包括天然及人工筛选的序列。Further, the NF-κB response sequence comprises NF-κB response sequences of various sequences; the NF-κB response sequence is a DNA sequence which specifically binds to the NF-κB protein, such as 5′-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3' (SEQ ID NO. 1); The main sequence of the NF-κB response sequence is characterized by containing a plurality of different NF-κB binding targets, Such as GGGACTTTCC (SEQ ID NO. 2). The NF-κB response sequence includes NF-κB response sequences including various sequences, including natural and artificially screened sequences.

更进一步地，所述最小启动子包括各种来源的最小启动子序列，包括天然及人工筛选的最小启动子序列；优选单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase，HSV-TK)启动子最小启动子；其主要作用是与基础转录因子及RNA聚合酶II结合，形成 通用转录机器，构成基因表达的基本条件。Further, the minimal promoter comprises a minimal promoter sequence from various sources, including a minimal promoter sequence naturally and artificially screened; preferably a herpes simplex virus thymidine kinase (HSV-TK) promoter The minimal promoter; its main function is to bind to the basic transcription factor and RNA polymerase II to form a universal transcriptional machinery, which constitutes the basic condition of gene expression.

其中，所述效应基因包括各种类型、序列和功能的效应基因；包括天然基因及人工突变及合成的基因。优选效应基因为在细胞内表达后其表达产物可引起细胞生理发生改变的基因，如引起细胞生长抑制、凋亡、死亡的基因。Wherein, the effector gene includes various types, sequences and functional effector genes; including natural genes and artificially mutated and synthesized genes. Preferably, the effector gene is a gene whose expression product can cause changes in cell physiology after expression in a cell, such as a gene causing cell growth inhibition, apoptosis, and death.

其中，所述基因表达载体为一线性(linear)或环状(circular)核酸分子。Wherein, the gene expression vector is a linear or circular nucleic acid molecule.

更进一步地，所述核酸分子为脱氧核糖核酸(DNA)或核糖核酸(RNA)分子，包括双链DNA(如腺病毒DNA分子)、单链DNA(腺相关病毒分子)或单链RNA分子(如慢病毒RNA分子)等。Further, the nucleic acid molecule is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule, including double-stranded DNA (such as an adenoviral DNA molecule), single-stranded DNA (adeno-associated virus molecule), or a single-stranded RNA molecule ( Such as lentiviral RNA molecules).

更进一步地，所述线性核酸分子包括普通线性DNA分子(如PCR扩增片段、酶切片段)、病毒DNA分子(如腺病毒DNA分子、腺相关病毒分子)或病毒RNA分子(如慢病毒RNA分子)等；所述环状核酸分子包括质粒DNA等。Further, the linear nucleic acid molecule comprises a common linear DNA molecule (such as a PCR amplified fragment, a fragment), a viral DNA molecule (such as an adenovirus DNA molecule, an adeno-associated virus molecule) or a viral RNA molecule (such as a lentiviral RNA). Molecular) or the like; the circular nucleic acid molecule includes plasmid DNA and the like.

其中，所述NF-κB特异性激活基因表达载体导入NF-κB活性过度活化的细胞内时，细胞内的序列特异性转录因子NF-κB就会激活该载体，使其表达载体上的效应基因；效应基因产物可引发细胞生理的改变，如细胞生长抑制、凋亡或死亡。Wherein, when the NF-κB-specific activation gene expression vector is introduced into a cell in which NF-κB is actively activated, an intracellular sequence-specific transcription factor NF-κB activates the vector to cause an effector gene on the expression vector. The effector gene product can initiate changes in cell physiology, such as cell growth inhibition, apoptosis, or death.

其中，所述导入NF-κB活性过度活化的细胞内的导入方法包括各种类型的核酸细胞导入方法；优选通过病毒载体、纳米载体、脂质体、电转移、基因枪等导入方式。Among them, the intracellular introduction method for introducing an excessive activation of NF-κB activity includes various types of nucleic acid cell introduction methods; preferably, introduction methods such as viral vectors, nanocarriers, liposomes, electrotransfers, gene guns, and the like.

作为优选，细胞导入方法为病毒载体；更进一步地，优选腺相关病毒(AAV)。Preferably, the cell introduction method is a viral vector; further, an adeno-associated virus (AAV) is preferred.

其中，所述效应基因产物包括RNA和蛋白质；所述RNA包括各种功能类型的RNA(如microRNA等)；所述蛋白质包括各种功能类型的蛋白质及多肽等。Wherein, the effector gene product comprises RNA and protein; the RNA comprises various functional types of RNA (such as microRNA, etc.); the protein includes various functional types of proteins and polypeptides and the like.

一种NF-κB特异性激活基因表达载体在NF-κB过度活化疾病治疗药物中的应用；一种NF-κB特异性激活基因表达载体在制备用于NF-κB过度活化密切相关疾病基因治疗的试剂或药物中的应用，尤其是在炎症、肿瘤和自身免疫性疾病治疗药物中的应用。Application of a NF-κB specific activation gene expression vector in the treatment of NF-κB overactivation disease; a NF-κB specific activation gene expression vector for preparing gene therapy for NF-κB overactivation and closely related diseases The use of reagents or drugs, especially in the treatment of inflammatory, neoplastic and autoimmune diseases.

大量针对NF-κB的抑制剂药物研究和开发实践已经表明，通过抑制NF-κB活性实现NF-κB过度活化相关疾病的治疗并不是一个容易行得通的策略。反其道而行之，与其难以通过简单抑制NF-κB活性达到治疗NF-κB过度活化相关疾病(特别是炎症和肿瘤)，不如思考如何利用NF-κB过度活化这一病理现象，开发一种对NF-κB过度活化采用因势利导的疾病治疗新策略。A large number of drug research and development practices for NF-κB inhibitors have shown that treatment of NF-κB overactivation-related diseases by inhibiting NF-κB activity is not an easy strategy. On the contrary, it is difficult to achieve a treatment of NF-κB overactivation-related diseases (especially inflammation and tumors) by simply inhibiting NF-κB activity. It is better to think about how to use NF-κB overactivation to develop this pathological phenomenon. A new strategy for the treatment of NF-κB over-activation is based on the situation.

NF-κB是一种序列特异性DNA结合转录因子。NF-κB发挥其生理和病理作用的分子机制为：NF-κB被激活后可进入细胞核，结合其DNA结合靶点(如经典的结合位点GGGACTTTCC，SEQ ID NO.2)，由此改变其靶基因的表达水平，发挥其生理和病理作用。 大量与炎症相关的基因如细胞因子/趋化因子(MCP-1/JE、CCL17、IL-2)、细胞粘附因子(NCAM、VCAM-1、ELAM-1)、肿瘤坏死因子TNFα等，都是NF-κB的靶基因。利用NF-κB与其DNA结合靶点的特异性结合，可使其发挥效应基因的表达调控作用。例如，构建一种基因表达载体，其启动子为仅由NF-κB DNA结合位点构成的NF-κB应答序列，则该启动子下游的效应基因的表达仅受细胞内NF-κB的调控。从而使这种基因表达载体成为一种NF-κB特异性激活基因表达载体。例如含有这类NF-κB应答序列的报告基因表达载体被发展为检测细胞内NF-κB活性的一种手段。但这类载体能否用于NF-κB过度活化疾病的治疗，还没有被充分研究和开发。NF-κB is a sequence-specific DNA-binding transcription factor. The molecular mechanism by which NF-κB exerts its physiological and pathological effects is that NF-κB can be activated to enter the nucleus and bind its DNA binding target (eg, the classical binding site GGGACTTTCC, SEQ ID NO. 2), thereby changing its The expression level of the target gene exerts its physiological and pathological effects. A large number of genes related to inflammation such as cytokines/chemokines (MCP-1/JE, CCL17, IL-2), cell adhesion factors (NCAM, VCAM-1, ELAM-1), tumor necrosis factor TNFα, etc. It is a target gene of NF-κB. The specific binding of NF-κB to its DNA binding target allows it to exert expression regulation of effector genes. For example, by constructing a gene expression vector in which the promoter is an NF-κB response sequence consisting only of the NF-κB DNA binding site, expression of an effector gene downstream of the promoter is regulated only by intracellular NF-κB. Thus, the gene expression vector becomes an expression vector for NF-κB specific activation gene. For example, a reporter gene expression vector containing such an NF-κB response sequence has been developed as a means of detecting NF-κB activity in cells. However, whether such vectors can be used for the treatment of NF-κB over-activation diseases has not been fully researched and developed.

其中关键的问题是这类NF-κB特异性激活基因表达载体是否只对过度活化的NF-κB做出应答，即表达出载体上的效应基因，而对正常细胞中生理水平(也可以称为基础水平)的NF-κB活性不做出应答，即不表达出载体上的效应基因。如果这类NF-κB特异的基因表达载体是只对过度活化的NF-κB做出应答，而对正常细胞中生理水平的NF-κB活性不做出应答，这类载体就可以作为NF-κB过度活化相关疾病的治疗试剂，用于这类疾病的治疗。炎症(如动脉粥样硬化、慢性肝炎等)和癌症是典型的NF-κB过度活化相关疾病，因此这类NF-κB特异的基因表达载体或将成为治疗这两类目前严重困扰人类健康的重大疾病的新的治疗策略。The key question is whether such NF-κB-specific activating gene expression vectors respond only to over-activated NF-κB, ie, expressing the effector gene on the vector, but also the physiological level in normal cells (also known as The basal level of NF-κB activity does not respond, ie does not express the effector gene on the vector. If such NF-κB-specific gene expression vectors respond only to over-activated NF-κB and do not respond to physiological levels of NF-κB activity in normal cells, such vectors can be used as NF-κB. A therapeutic agent for over-activation of related diseases for the treatment of such diseases. Inflammation (such as atherosclerosis, chronic hepatitis, etc.) and cancer are typical NF-κB overactivation-related diseases, so such NF-κB-specific gene expression vectors may become a major cause of treatment for these two types of human health. New treatment strategies for the disease.

此外，利用NF-κB特异性激活基因表达载体去治疗NF-κB过度活化相关疾病，是一种基因治疗方法，完全不同于目前发展的形形色色的NF-κB抑制剂；NF-κB抑制剂是直接或间接地区抑制细胞内的NF-κB活性，但由于细胞内NF-κB激活通路的多样性、复杂性，一种通路的关闭很难彻底抑制NF-κB活性；此外，切断了一种通路，可能造成该通路负责的其他生理过程的干扰，产生副作用。最严重的问题是，外源导入NF-κB抑制剂很难控制一个恰当的度，使其既灭活了过度的NF-κB活性，又避免伤及生理水平的NF-κB活性。与之相反，NF-κB特异性激活基因表达载体不是去抑制NF-κB活性，而是利用NF-κB活性。显然这是一种因势利导，类似大禹治水的策略。In addition, the use of NF-κB-specific activation gene expression vector to treat NF-κB overactivation-related diseases is a gene therapy method that is completely different from the current development of various NF-κB inhibitors; NF-κB inhibitors are direct Or indirect inhibition of NF-κB activity in cells, but due to the diversity and complexity of the NF-κB activation pathway in cells, it is difficult to completely inhibit NF-κB activity by shutting down one pathway; in addition, one pathway is cut, It may cause interference with other physiological processes responsible for the pathway, causing side effects. The most serious problem is that it is difficult to control an appropriate degree of exogenously introduced NF-κB inhibitors, which both inactivates excessive NF-κB activity and avoids physiological levels of NF-κB activity. In contrast, the NF-κB-specific activation gene expression vector does not inhibit NF-κB activity, but utilizes NF-κB activity. Obviously this is a kind of strategy that is similar to the situation.

本发明构建了一种NF-κB特异性激活基因表达载体，通过大量实验实例，不仅论证了NF-κB RelA(NF-κB基因表达调控作用的主要实现者)在肿瘤细胞内的特异表达，还论证了所构建的NF-κB特异性激活基因表达载体在NF-κB过度活化肿瘤细胞内的表达特性；发现该种NF-κB特异基因表达载体可在在NF-κB过度活化肿瘤细胞内表达效应基因(如绿色荧光蛋白、microRNA、Cas9蛋白等)，而在正常细胞内不能表达效应基因。本发明由此发展了一种基于NF-κB特异性激活基因表达载体进行NF-κB过度活化相关疾病治 疗的新策略。The invention constructs a NF-κB specific activation gene expression vector, and demonstrates not only the specific expression of NF-κB RelA (the main implementer of NF-κB gene expression regulation) in tumor cells, but also a large number of experimental examples. The expression of NF-κB-specific gene expression vector in NF-κB over-activated tumor cells was demonstrated. The expression of NF-κB-specific gene expression vector in NF-κB over-activated tumor cells was found to be expressed. Genes (such as green fluorescent protein, microRNA, Cas9 protein, etc.) cannot express effector genes in normal cells. The present invention thus develops a novel strategy for the treatment of diseases associated with NF-κB overactivation based on NF-κB-specific activation gene expression vectors.

本发明发展了一种NF-κB激活基因表达技术，设计论证了一种NF-κB特异性激活基因表达载体。该载体包含一种由NF-κB应答序列和最小启动子构成的NF-κB应答启动子(DMP)，利用该启动子可以控制下游效应基因在NF-κB过度活化细胞中的特异性表达，效应基因表达产物可以产生对细胞生理的影响，产生诸如细胞生长抑制、凋亡、死亡等效应。本发明设计和论证的基因表达载体可以用于制备NF-κB过度活化相关疾病的基因治疗药物，作为一种新的基因治疗试剂，用于炎症、肿瘤的治疗。本发明设计和论证的NF-κB特异性激活基因表达载体可包装在腺相关病毒(AAV)中，利用AAV病毒作为基因治疗优良载体的特性，用于人体内炎症、肿瘤这类NF-κB过度活化疾病的成像及治疗。目前AAV病毒载体可单针一次性注射进行疾病的基因治疗，因此，腺相关病毒(AAV)搭载了本发明设计和论证的NF-κB特异性激活基因表达载体后，有望成为治疗NF-κB过度活化疾病的简单、无创(一次性静脉注射)、高效的新型生物药物。本发明设计和论证的NF-κB特异性激活基因表达载体因利用NF-κB活性而不同于目前大量的NF-κB活性抑制分子，不会对正常细胞内的生理NF-κB活性产生损伤，因此避免了传统NF-κB抑制剂的副作用。The invention develops a NF-κB activation gene expression technology, and designs and demonstrates an NF-κB specific activation gene expression vector. The vector comprises an NF-κB responsive promoter (DMP) consisting of an NF-κB responsive sequence and a minimal promoter, and the promoter can control the specific expression of a downstream effector gene in NF-κB over-activated cells, and the effect Gene expression products can produce effects on cell physiology, producing effects such as cell growth inhibition, apoptosis, and death. The gene expression vector designed and demonstrated by the present invention can be used for preparing a gene therapy drug for NF-κB overactivation-related diseases, and is a novel gene therapy reagent for the treatment of inflammation and tumor. The NF-κB-specific activating gene expression vector designed and demonstrated by the present invention can be packaged in an adeno-associated virus (AAV), and utilizes AAV virus as a good carrier for gene therapy, and is used for NF-κB overexpression such as inflammation and tumor in human body. Imaging and treatment of activated diseases. At present, the AAV virus vector can be used for single-shot injection for gene therapy of diseases. Therefore, the adeno-associated virus (AAV) is expected to become an NF-κB over-expressed after carrying out the NF-κB-specific activation gene expression vector designed and demonstrated by the present invention. A simple, non-invasive (disposable intravenous), highly effective new biopharmaceutical that activates disease. The NF-κB-specific activating gene expression vector designed and demonstrated by the present invention differs from the current large number of NF-κB activity inhibitory molecules by utilizing NF-κB activity, and does not damage physiological NF-κB activity in normal cells. The side effects of traditional NF-κB inhibitors are avoided.

虽然本发明中NF-κB特异性激活基因表达载体的构建方法为现有的常规手段，但本发明最主要创新之处是NF-κB特异性激活基因表达载体的组成形式，并通过设计实验论证了所构建的载体具有NF-κB过度活化细胞内的特异性表达特性，如本发明实验发现，NF-κB在肿瘤细胞内特异表达，即NF-κB可检测的(本发明中使用了荧光定量PCR技术进行检测)活性仅在肿瘤细胞内出现，而在正常细胞中没有可检测的活性(附图2)。基于这一发现，本发明推测使用NF-κB特异性激活基因表达载体可以实现某种效应基因在NF-κB过度活化细胞(如炎性细胞和肿瘤细胞)内的特异性表达，而避免在正常细胞内的表达。本发明的实验充分证明了这一推测，表明采用NF-κB特异性激活基因表达载体完全可以控制某种效应基因(如本发明中使用的zsGreen、SBP、miRNA、Cas9)仅仅在NF-κB过度活化细胞(如本发明中使用的各种肿瘤细胞)内表达。这种人工控制基因在NF-κB过度活化细胞内的特异表达，具有重要的应用价值，例如肿瘤治疗。Although the construction method of the NF-κB specific activation gene expression vector in the present invention is a conventional conventional method, the most important innovation of the present invention is the composition form of the NF-κB specific activation gene expression vector, and is demonstrated by design experiments. The constructed vector has specific expression characteristics in NF-κB over-activated cells, and as found in the present invention, NF-κB is specifically expressed in tumor cells, that is, NF-κB is detectable (fluorescence quantification is used in the present invention) Detection by PCR technique) activity occurs only in tumor cells, but no detectable activity in normal cells (Fig. 2). Based on this finding, the present invention speculates that the use of NF-κB-specific activation gene expression vector can achieve specific expression of an effector gene in NF-κB over-activated cells (such as inflammatory cells and tumor cells), while avoiding normal Intracellular expression. The experiments of the present invention fully demonstrate this hypothesis, suggesting that the use of NF-κB-specific activation gene expression vector can completely control an effector gene (such as zsGreen, SBP, miRNA, Cas9 used in the present invention) only excessively in NF-κB. The cells are expressed in activating cells (such as various tumor cells used in the present invention). This artificial control gene has specific application value in NF-κB over-activated cells, such as tumor therapy.

附图说明DRAWINGS

图1为本发明基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达技术原理示意图；其中gene expression vector为基因表达载体；NF-κB responsive sequence(NF-κB binding sequences)为NF-κB应答序列(NF-κB结合序列)；minimal promoter为最小启动子；effective gene为效应基因；transfection为转染；over activated  transcription factor NF-κB为过度活化的转录因子NF-κB；NF-κB binding and gene expression activation为NF-κB结合及基因表达激活；effective gene expression为效应基因表达；effective gene products为效应基因产物；cell growth arrest/apoptosis/dead为细胞生长抑制/凋亡/死亡；NF-κB over-activated cell为NF-κB过度活化的细胞。1 is a schematic diagram showing the principle of gene expression technology of NF-κB activation-activated effector gene in NF-κB over-activated cells according to the present invention; wherein gene expression vector is a gene expression vector; NF-κB responsive sequence (NF-κB binding sequences) NF-κB response sequence (NF-κB binding sequence); minimal promoter is the minimal promoter; effective gene is the effector gene; transfection is transfection; over activated transcription factor NF-κB is an over-activated transcription factor NF-κB; NF-κB binding and gene expression activation is NF-κB binding and gene expression activation; effective gene expression is effector gene expression; effective gene products is effector gene product; cell growth arrest/apoptosis/dead is cell growth inhibition/apoptosis/death NF-κB over-activated cell is a cell in which NF-κB is over-activated.

图2为NF-κB在不同细胞中表达的荧光定量PCR检测结果。可见NF-κB在肿瘤细胞中有表达，但在正常细胞中无表达。Figure 2 shows the results of fluorescent quantitative PCR detection of NF-κB expression in different cells. It can be seen that NF-κB is expressed in tumor cells, but not in normal cells.

图3为DMP-zsGreen表达载体转染各种细胞后，细胞中绿色荧光蛋白的流式细胞仪测定结果。可见DMP控制的zsGreen仅在肿瘤细胞中有表达，在正常细胞中无表达。Figure 3 shows the results of flow cytometry of green fluorescent protein in cells after transfection of various cells with DMP-zsGreen expression vector. It can be seen that DMP-controlled zsGreen is expressed only in tumor cells and is not expressed in normal cells.

图4为DMP-zsGreen表达载体转染293T、HeLa、HepG2、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3细胞后，细胞在白场(phase)和绿色荧光(FITC)通道下的显微拍照。可见DMP控制的zsGreen在这些肿瘤细胞中的表达。Figure 4 shows the DMP-zsGreen expression vector transfected into 293T, HeLa, HepG2, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3 cells, cells in white phase and green fluorescence (FITC) Microscopic photographing under the channel. The expression of DMP-controlled zsGreen in these tumor cells can be seen.

图5为DMP-zsGreen表达载体转染Hepa1-6、RAW264.7、MRC-5、HL7702细胞后，细胞在白场(phase)和绿色荧光(FITC)通道下的显微拍照。可见DMP控制的zsGreen仅在肿瘤细胞(Hepa1-6、RAW264.7)中有表达，在正常细胞(MRC-5、HL7702)中无表达。Figure 5 is a photomicrograph of cells in Hepa1-6, RAW264.7, MRC-5, and HL7702 cells transfected with DMP-zsGreen expression vector in white and green fluorescent (FITC) channels. It can be seen that DMP-controlled zsGreen is only expressed in tumor cells (Hepa1-6, RAW264.7) and not in normal cells (MRC-5, HL7702).

图6为DMP-Display-SBP表达载体转染Hepa1-6、HepG2、MRC-5、HL7702、293T细胞后，对细胞进行FITC标记的链霉亲和素蛋白的染色，之后在白场(bright field)和绿色荧光(FITC)通道下对细胞显微拍照；再将白场和绿色荧光通道图像的叠加。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(Hepa1-6、HepG2、293T)中有表达，在正常细胞(MRC-5、HL7702)中无表达。Figure 6 shows that DMP-Display-SBP expression vector was transfected into Hepa1-6, HepG2, MRC-5, HL7702, and 293T cells, and the cells were stained with FITC-labeled streptavidin protein, followed by white field (bright field). Microphotographing of cells under green fluorescent (FITC) channels; superposition of white and green fluorescent channel images. It can be seen that DMP-controlled cell surface display (SBP) is expressed only in tumor cells (Hepa1-6, HepG2, 293T) and not in normal cells (MRC-5, HL7702).

图7为DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像；再对各孔荧光强度进行量化。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达。Figure 7 shows DMP-Display-SBP expression vector transfected with HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10, MRC- 5. After HL7702 cells, the cells were stained with IRDye800CW-labeled streptavidin protein, and then scanned on a near-infrared fluorescence scanner; the fluorescence intensity of each well was quantified. It can be seen that DMP-controlled cell surface display (SBP) is only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7). , expressed in B16F10), no expression in normal cells (MRC-5, HL7702).

图8为DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色；之后在近红外荧光扫描仪上扫描成像并在白场拍照。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、 Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达。Figure 8 shows DMP-Display-SBP expression vector transfected with HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10, MRC- 5. After HL7702 cells, the cells were trypsinized and collected, and then stained with IRDye800CW-labeled streptavidin protein; then scanned on a near-infrared fluorescence scanner and photographed in the white field. It can be seen that DMP-controlled cell surface display (SBP) is only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7). , expressed in B16F10), no expression in normal cells (MRC-5, HL7702).

图9为将DMP-Display-SBP包装成腺相关病毒(AAV)表达载体(AAV-SBP)后，用AAV-SBP转染293T、HepG2、Hepa1-6、MRC-5、HL7702细胞。对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像(A图)；对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像并在白场拍照(B图)。可见包装成AAV病毒载体(AAV-SBP)，DMP控制的细胞表面展示表达的SBP可以高效地展示在肿瘤细胞(HepG2、293T、Hepa1-6)表面，在正常细胞(MRC-5、HL7702)中无表达。Figure 9 shows that DMP-Display-SBP was packaged into an adeno-associated virus (AAV) expression vector (AAV-SBP), and 293T, HepG2, Hepa1-6, MRC-5, and HL7702 cells were transfected with AAV-SBP. The cells were stained with IRDye800CW-labeled streptavidin protein, and then scanned on a near-infrared fluorescence scanner (Panel A); the cells were trypsinized and collected, and then IRDye800CW-labeled streptavidin protein was used. After staining, the image was scanned on a near-infrared fluorescence scanner and photographed on a white field (panel B). It can be seen that it is packaged into AAV virus vector (AAV-SBP), and DMP-controlled cell surface display expressed SBP can be efficiently displayed on the surface of tumor cells (HepG2, 293T, Hepa1-6) in normal cells (MRC-5, HL7702). No expression.

图10为DMP-miR533 _RelA表达载体转染细胞的检测分析。A图为DMP-miR533 _RelA表达载体转染用TNFα处理的HepG2细胞后，用定量定量PCR检测细胞中NF-κB RelA/p65的表达。B图为HepG2及HL7702细胞后，对细胞进行凋亡检测处理，再用流式细胞仪测定细胞凋亡。可见DMP-miR533 _RelA的转染引起肿瘤细胞(HepG2)凋亡，而正常细胞(HL7702)无凋亡。 Figure 10 is a detection analysis of DMP-miR533 _RelA expression vector transfected cells. Panel A shows that the expression of NF-κB RelA/p65 in cells was detected by quantitative quantitative PCR after transfection of HepG2 cells treated with TNFα with DMP-miR533 _RelA expression vector. In the B picture, HepG2 and HL7702 cells were subjected to apoptosis detection, and apoptosis was measured by flow cytometry. It can be seen that transfection of DMP-miR533 _RelA causes apoptosis of tumor cells (HepG2), whereas normal cells (HL7702) have no apoptosis.

图11为U6-TsgRNA-DMP-Cas9表达载体转染细胞后，对细胞进行吖啶橙染色，再在绿色荧光通道下对细胞进行显微拍照。可见U6-TsgRNA-DMP-Cas9转染后，引起肿瘤细胞(HepG2、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6)的死亡，而正常细胞(MRC-5、HL7702)的生长不受影响。Figure 11 shows the cells transfected with U6-TsgRNA-DMP-Cas9 expression vector, the cells were subjected to acridine orange staining, and the cells were micrographed under green fluorescent channels. It can be seen that U6-TsgRNA-DMP-Cas9 transfected, causing the death of tumor cells (HepG2, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6), while normal cells The growth of (MRC-5, HL7702) was not affected.

具体实施方式Detailed ways

以下结合实施例和附图对本发明作进一步说明。The present invention will be further described below in conjunction with the embodiments and the accompanying drawings.

实施例1 NF-κB RelA不同细胞表达Example 1 Different cell expression of NF-κB RelA

实验方法：experimental method:

细胞培养：HEK-293T(人胎肾细胞)、HepG2(人肝癌细胞)、A549(人肺癌细胞)、HT-29(人结肠癌细胞)、HeLa(人***细胞)、SKOV3(人卵巢癌细胞)、PANC-1(胰腺癌细胞)、MDA-MB-453(人乳腺癌)、Hepa1-6(小鼠肝癌细胞)、小鼠巨噬细胞(RAW264.7)、小鼠黑色素瘤细胞(B16F10)、HL7702(人正常肝细胞)及MRC5(人胚胎成纤维细胞)细胞培养。细胞培养使用DEME(Hepa1-6、HEK-293T、HepG2、HeLa、PANC-1、MDA-MB-453、RAW264.7、B16F10、MRC-5)或RPMI 1640培养基(A549、HT-29、SKOV-3、HL7702)、10％胎牛血清(HyClone)、100units/mL青霉素和100μg/mL链霉素培养；培养环境为含有5％(v/v)CO ₂的加湿培养箱中37℃培养。细胞复苏后，按同等密度接种到24孔微孔板(1×10 ⁵/孔)或12孔微孔板(2×10 ⁵/孔)中，培养过夜贴壁后，进行转染。 Cell culture: HEK-293T (human fetal kidney cells), HepG2 (human liver cancer cells), A549 (human lung cancer cells), HT-29 (human colon cancer cells), HeLa (human cervical cancer cells), SKOV3 (human ovarian cancer) Cells), PANC-1 (pancreatic cancer cells), MDA-MB-453 (human breast cancer), Hepa 1-6 (mouse liver cancer cells), mouse macrophages (RAW264.7), mouse melanoma cells ( Cell culture of B16F10), HL7702 (human normal liver cells) and MRC5 (human embryonic fibroblasts). Cell culture using DEME (Hepa 1-6, HEK-293T, HepG2, HeLa, PANC-1, MDA-MB-453, RAW264.7, B16F10, MRC-5) or RPMI 1640 medium (A549, HT-29, SKOV) -3, HL7702), 10% fetal bovine serum (HyClone), 100 units/mL penicillin and 100 μg/mL streptomycin were cultured; the culture environment was cultured at 37 ° C in a humidified incubator containing 5% (v/v) CO ₂ . After the cells were resuscitated, they were seeded at the same density in 24-well microplates (1 × 10 ⁵ /well) or 12-well microplates (2 × 10 ⁵ /well), cultured overnight, and then transfected.

基因表达检测：收集细胞，用Trizol提取总RNA，逆转录合成互补DNA(cDNA)。cDNA制备反应及程序为：10μL反转录反应组分包含2μL 5×PrimeScript RT Master Mix(Takara)、50ng总RNA，用RNase Free ddH ₂O将反应总体积补至10μL；37℃反应15分钟，升温至85℃反应5秒使反转录酶失活，反应液4℃保存。通过qPCR定量分析RelA表达。用于qPCR的上下引物为5′-CCT GGA GCA GGC TAT CAG TC-3′(F)与5′-ATG GGA TGA GAA AGG ACA GG-3′(R)。PCR模板为cDNA。通过qPCR定量分析RelA表达。10μL qPCR反应含有5μL Fast SYBR Green Master Mix(ABI)，0.2μL 10μM F，0.2μL 10μM R和1μL cDNA，用ddH ₂O将反应总体积补至10μL。将配制好的反应体系放于荧光定量PCR仪(StepOne plus，ABI)上进行扩增，设置的扩增程序为：95℃预变性10分钟、45个扩增循环(95℃变性15s，在各退火温度下扩增1分钟)。荧光定量PCR扩增的特异性用溶解曲线来分析确定，用比较CT值法来计算基因表达的相对定量(RQ)，数据最后表示为平均值±标准偏差(SD)，统计显著性用t检验来确定。 Gene expression assay: Cells were harvested, total RNA was extracted with Trizol, and complementary DNA (cDNA) was synthesized by reverse transcription. The cDNA preparation reaction and procedure were as follows: 10 μL of the reverse transcription reaction component contained 2 μL of 5×PrimeScript RT Master Mix (Takara), 50 ng of total RNA, and the total volume of the reaction was supplemented to 10 μL with RNase Free ddH ₂ O; and reacted at 37 ° C for 15 minutes. The reaction was heated to 85 ° C for 5 seconds to inactivate the reverse transcriptase, and the reaction solution was stored at 4 ° C. RelA expression was quantitatively analyzed by qPCR. The upper and lower primers for qPCR were 5'-CCT GGA GCA GGC TAT CAG TC-3' (F) and 5'-ATG GGA TGA GAA AGG ACA GG-3' (R). The PCR template is cDNA. RelA expression was quantitatively analyzed by qPCR. The 10 μL qPCR reaction contained 5 μL of Fast SYBR Green Master Mix (ABI), 0.2 μL of 10 μM F, 0.2 μL of 10 μM R and 1 μL of cDNA, and the total volume of the reaction was supplemented to 10 μL with ddH ₂ O. The prepared reaction system was amplified by a quantitative PCR instrument (StepOne plus, ABI), and the amplification procedure was set: pre-denaturation at 95 ° C for 10 minutes, and 45 amplification cycles (denaturation at 95 ° C for 15 s, in each Amplification at annealing temperature for 1 minute). The specificity of real-time PCR amplification was determined by the dissolution curve. The relative quantification (RQ) of gene expression was calculated by comparative CT value method. The data was finally expressed as mean±standard deviation (SD), and the statistical significance was analyzed by t test. to make sure.

实验结果：Experimental results:

为了考察NF-κB RelA/p65基因在各种肿瘤细胞及正常细胞中的表达，用荧光定量PCR检测了NF-κB RelA/p65在11种肿瘤细胞及正常细胞(HL7702及MRC5)中的表达(图2)。结果表明在所有肿瘤细胞株中NF-κB RelA/p65均有表达，而在正常细胞(HL7702及MRC5)中，检测不得到NF-κB表达。说明用本发明提出的NF-κB激活基因表达载体是一种NF-κB过度活化细胞特异的基因表达载体，如肿瘤细胞中NF-κB激活基因表达载体的表达。To investigate the expression of NF-κB RelA/p65 gene in various tumor cells and normal cells, the expression of NF-κB RelA/p65 in 11 tumor cells and normal cells (HL7702 and MRC5) was detected by real-time PCR. figure 2). The results showed that NF-κB RelA/p65 was expressed in all tumor cell lines, while in normal cells (HL7702 and MRC5), NF-κB expression was not detected. The NF-κB activating gene expression vector proposed by the present invention is a gene expression vector specific for NF-κB over-activation, such as expression of an NF-κB activating gene expression vector in tumor cells.

实施例2 DMP-zsGreen(效应基因胞内表达)Example 2 DMP-zsGreen (effector gene intracellular expression)

实验方法：experimental method:

载体构建：构建一表达载体DMP-zsGreen；该载体含有DMP序列及其下游受其控制的可在细胞内表达的绿色荧光蛋白zsGreen的编码序列。其中DMP含有NF-κB应答序列(5′-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3′，SEQ ID NO.1)以及最小启动子序列(5′-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3′，SEQ ID NO.3)。Vector construction: Construction of an expression vector DMP-zsGreen; this vector contains the DMP sequence and its coding sequence for the green fluorescent protein zsGreen which can be expressed in the cell downstream. Wherein DMP contains the NF-κB response sequence (5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3', SEQ ID NO. 1) and the minimal promoter sequence (5'-TAG) AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3', SEQ ID NO. 3).

细胞培养：HEK-293T(人胎肾细胞)、HepG2(人肝癌细胞)、A549(人肺癌细胞)、HT-29(人结肠癌细胞)、HeLa(人***细胞)、SKOV3(人卵巢癌细胞)、PANC-1(胰腺癌细胞)、MDA-MB-453(人乳腺癌)、Hepa1-6(小鼠肝癌细胞)、小鼠巨噬细胞(RAW264.7)、HL7702(人正常肝细胞)及MRC5(人胚胎成纤维细胞)细胞培养。细胞 培养使用DEME(Hepa1-6、HEK-293T、HepG2、HeLa、PANC-1、MDA-MB-453、RAW264.7、MRC-5)或RPMI 1640培养基(A549、HT-29、SKOV-3、HL7702)、10％胎牛血清(HyClone)、100units/mL青霉素和100μg/mL链霉素培养；培养环境为含有5％(v/v)CO ₂的加湿培养箱中37℃培养。细胞复苏后，按同等密度接种到24孔微孔板(0.5×10 ⁵/孔)中，培养过夜贴壁后，进行转染。 Cell culture: HEK-293T (human fetal kidney cells), HepG2 (human liver cancer cells), A549 (human lung cancer cells), HT-29 (human colon cancer cells), HeLa (human cervical cancer cells), SKOV3 (human ovarian cancer) Cells), PANC-1 (pancreatic cancer cells), MDA-MB-453 (human breast cancer), Hepa 1-6 (mouse liver cancer cells), mouse macrophages (RAW264.7), HL7702 (human normal liver cells) And MRC5 (human embryonic fibroblasts) cell culture. Cell culture using DEME (Hepa 1-6, HEK-293T, HepG2, HeLa, PANC-1, MDA-MB-453, RAW264.7, MRC-5) or RPMI 1640 medium (A549, HT-29, SKOV-3) HL7702), 10% fetal bovine serum (HyClone), 100 units/mL penicillin and 100 μg/mL streptomycin were cultured; the culture environment was cultured at 37 ° C in a humidified incubator containing 5% (v/v) CO ₂ . After the cells were resuscitated, they were seeded at the same density in 24-well microplates (0.5 × 10 ⁵ /well), cultured overnight, and then transfected.

细胞转染：细胞培养液换为无血清培养基培养1h。用DMP-zsGreen转染上述细胞。空脂质体转染的细胞作为转染对照。每孔细胞的总DNA用量及脂质体用量参考脂质体产品说明书进行。DNA-脂质体加入无血清培养基培养4h。换为含血清新鲜培养基，继续培养24h。Cell transfection: The cell culture medium was changed to serum-free medium for 1 h. The above cells were transfected with DMP-zsGreen. Empty lipofected cells were used as transfection controls. The total amount of DNA per cell and the amount of liposome used were determined by reference to the liposome product instructions. DNA-liposomes were added to serum-free medium for 4 h. Switch to serum-containing fresh medium and continue to culture for 24 h.

细胞成像：用倒置荧光显微镜(Olympus IX51-DPI71)观测拍照各种处理的细胞，主要观察细胞是否产生绿色荧光；同时观察细胞生长情况，如生长旺盛、贴壁良好、无污染等。对各种处理的细胞进行多视野明场及绿色荧光观察通道的照相拍摄。Cell imaging: The inverted cells were observed with an inverted fluorescence microscope (Olympus IX51-DPI71) to observe whether the cells produced green fluorescence. At the same time, cell growth was observed, such as vigorous growth, good adherence, and no pollution. Photographic photographing of multi-field brightfield and green fluorescence observation channels was performed on various treated cells.

流式分析：用胰酶消化收集细胞，用流式细胞仪(ACEA NovoCyte公司)检测分析细胞的绿色荧光。Flow cytometry: Cells were harvested by trypsinization and analyzed for green fluorescence by flow cytometry (ACEA NovoCyte).

实验结果：Experimental results:

为了验证NF-κB激活的基因表达载体，构建一个含有DMP序列及其下游绿色荧光蛋白zsGreen的编码序列的表达载体，该载体中zsGreen的编码序列的表达受DMP序列的控制，可将zsGreen蛋白在表达在细胞内。将该载体转染不同的细胞，结果发现在HEK-293T细胞、所有肿瘤细胞(Hepa1-6、HepG2、HeLa、PANC-1、MDA-MB-453、RAW264.7、A549、HT-29、SKOV-3)内均表达zsGreen，而在正常细胞(HL7702及MRC5)内，不表达zsGreen(图3、图4)。进一步的流式细胞分析表明，zsGreen仅在所有肿瘤细胞内表达，而在正常细胞(HL7702及MRC5)内不表达(图5)。本实例转染实验说明，本发明提出的NF-κB激活基因表达载体是一种肿瘤细胞特异的基因表达载体。肿瘤细胞是典型的NF-κB过度活化细胞。NF-κB与肿瘤的发生发展存在密切关系。In order to validate the NF-κB-activated gene expression vector, an expression vector containing the coding sequence of the DMP sequence and its downstream green fluorescent protein zsGreen was constructed. The expression of the coding sequence of zsGreen in the vector was controlled by the DMP sequence, and the zsGreen protein was Expressed in cells. The vector was transfected into different cells and found to be in HEK-293T cells, all tumor cells (Hepa1-6, HepG2, HeLa, PANC-1, MDA-MB-453, RAW264.7, A549, HT-29, SKOV). -3) zsGreen was expressed intra-in vivo, while zsGreen was not expressed in normal cells (HL7702 and MRC5) (Fig. 3, Fig. 4). Further flow cytometric analysis indicated that zsGreen was only expressed in all tumor cells but not in normal cells (HL7702 and MRC5) (Fig. 5). The transfection experiment of the present example demonstrates that the NF-κB activating gene expression vector proposed by the present invention is a tumor cell-specific gene expression vector. Tumor cells are typical NF-κB over-activated cells. NF-κB is closely related to the occurrence and development of tumors.

实施例3 DMP-Display-SBP(效应基因细胞表面展示表达)Example 3 DMP-Display-SBP (effector gene cell surface display expression)

实验方法：experimental method:

载体构建：构建一表达载体DMP-Display-SBP；该载体含有DMP序列及可细胞表达展示链亲和素结合肽(SBP)的编码序列。其中DMP含有NF-κB应答序列(5′-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3′，SEQ ID NO.1)以及最小启动子序列(5′-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3′，SEQ ID  NO.3)。SBP编码序列为：ATG GAC GAG AAG ACC ACC GGG TGG CGG GGC GGC CAC GTT GTG GAG GGT CTC GCT GGC GAG CTG GAG CAG CTC AGG GCC CGC TTG GAG CAC CAT CCC CAG GGG CAA CGC GAG CCT ATC GAT TAA(SEQ ID NO.4)。展示表达的骨架序列克隆自载体pDisplay ^TM(Invitrogen)；pDisplay ^TM将要展示在细胞膜表面的蛋白或多肽融合在大鼠Igκ-链引导序列(Igκ-chain leader sequence)的N端，该引导序列可指引蛋白的分母途径；pDisplay表达蛋白的C末端为血小板衍生生长因子受体(platelet derived growth factor receptor，PDGFR)跨膜区，该跨膜区将蛋白锚定在细胞膜上，从而将展示蛋白展示在细胞外侧。这种膜蛋白可以和细胞培养液中的蛋白发生互作，如本实验中的链霉亲和素与展示在细胞膜表面的链霉亲和素结合肽(SBP间的互作)。 Vector construction: An expression vector DMP-Display-SBP was constructed; the vector contains a DMP sequence and a coding sequence for a cell-expressing display streptavidin-binding peptide (SBP). Wherein DMP contains the NF-κB response sequence (5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3', SEQ ID NO. 1) and the minimal promoter sequence (5'-TAG) AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3', SEQ ID NO. 3). The SBP coding sequence is: ATG GAC GAG AAG ACC ACC GGG TGG CGG GGC GGC CAC GTT GTG GAG GGT CTC GCT GGC GAG CTG GAG CAG CTC AGG GCC CGC TTG GAG CAC CAT CCC CAG GGG CAA CGC GAG CCT ATC GAT TAA (SEQ ID NO .4). It shows the expression of sequences cloned from the vector backbone pDisplay ^TM (Invitrogen); pDisplay ^TM appear to be cell surface protein or polypeptide fused to the leader sequence Igκ- chain rats (Igκ-chain leader sequence) of the N-terminus of the leader sequence may direct The denominator pathway of the protein; the C-terminus of the pDisplay-expressing protein is the platelet derived growth factor receptor (PDGFR) transmembrane region, which anchors the protein to the cell membrane, thereby displaying the display protein in the cell Outside. This membrane protein can interact with proteins in cell culture fluids, such as streptavidin in this experiment and streptavidin-binding peptide (SBP interaction) displayed on the surface of cell membranes.

细胞培养：HEK-293T(人胎肾细胞)、HepG2(人肝癌细胞)、A549(人肺癌细胞)、HT-29(人结肠癌细胞)、HeLa(人***细胞)、SKOV3(人卵巢癌细胞)、PANC-1(胰腺癌细胞)、MDA-MB-453(人乳腺癌)、Hepa1-6(小鼠肝癌细胞)、小鼠巨噬细胞(RAW264.7)、小鼠黑色素瘤细胞(B16F10)、HL7702(人正常肝细胞)及MRC5(人胚胎成纤维细胞)细胞培养。细胞培养使用DEME(Hepa1-6、HEK-293T、HepG2、HeLa、PANC-1、MDA-MB-453、RAW264.7、B16F10、MRC-5)或RPMI 1640培养基(A549、HT-29、SKOV-3、HL7702)、10％胎牛血清(HyClone)、100units/mL青霉素和100μg/mL链霉素培养；培养环境为含有5％(v/v)CO ₂的加湿培养箱中37℃培养。细胞复苏后，按同等密度接种到24孔微孔板(1×10 ⁵/孔)或12孔微孔板(2×10 ⁵/孔)中，培养过夜贴壁后，进行转染。 Cell culture: HEK-293T (human fetal kidney cells), HepG2 (human liver cancer cells), A549 (human lung cancer cells), HT-29 (human colon cancer cells), HeLa (human cervical cancer cells), SKOV3 (human ovarian cancer) Cells), PANC-1 (pancreatic cancer cells), MDA-MB-453 (human breast cancer), Hepa 1-6 (mouse liver cancer cells), mouse macrophages (RAW264.7), mouse melanoma cells ( Cell culture of B16F10), HL7702 (human normal liver cells) and MRC5 (human embryonic fibroblasts). Cell culture using DEME (Hepa 1-6, HEK-293T, HepG2, HeLa, PANC-1, MDA-MB-453, RAW264.7, B16F10, MRC-5) or RPMI 1640 medium (A549, HT-29, SKOV) -3, HL7702), 10% fetal bovine serum (HyClone), 100 units/mL penicillin and 100 μg/mL streptomycin were cultured; the culture environment was cultured at 37 ° C in a humidified incubator containing 5% (v/v) CO ₂ . After the cells were resuscitated, they were seeded at the same density in 24-well microplates (1 × 10 ⁵ /well) or 12-well microplates (2 × 10 ⁵ /well), cultured overnight, and then transfected.

细胞转染：细胞培养液换为无血清培养基培养1h。分别用DMP-Display-SBP转染上述细胞。空脂质体转染的细胞作为转染对照。每孔细胞的总DNA用量及脂质体用量参考脂质体产品(Lipofectamine 2000；ThermoFisher Scientific)说明书进行。DNA-脂质体加入无血清培养基培养4h。换为含血清新鲜培养基，继续培养20h。Cell transfection: The cell culture medium was changed to serum-free medium for 1 h. The above cells were transfected with DMP-Display-SBP, respectively. Empty lipofected cells were used as transfection controls. The total amount of DNA per cell and the amount of liposome used were determined in accordance with the instructions for liposome products (Lipofectamine 2000; ThermoFisher Scientific). DNA-liposomes were added to serum-free medium for 4 h. Change to serum-containing fresh medium and continue to culture for 20 h.

细胞染色：用FITC标记的链亲和素及其IRDye800CW(一种近红外荧光分子；LiCor公司)标记的链亲和素(LiCor)对细胞染色。细胞转染后再新鲜培养基中直接加入用FITC标记的链亲和素或IRDye800CW标记的链亲和素(终浓度均为1μg/mL)。细胞继续培养20h，去除培养基，PBS洗涤细胞2次。细胞用荧光显微镜或近红外荧光扫描仪(Oddysey，LiCor)扫描成像。细胞观察：用倒置荧光显微镜(Olympus IX51-DPI71)观测拍照各种处理的细胞，主要观察细胞表面是否产生绿色荧光；同时观察细胞生长情况，如生长旺盛、贴壁良好、无污染等。对各种处理的细胞进行多视野明场及绿色荧光观察通道的照 相拍摄。Cell staining: Cells were stained with FITC-labeled streptavidin and its IRDye800CW (a near-infrared fluorescent molecule; LiCor)-labeled streptavidin (LiCor). After transfection of the cells, FITC-labeled streptavidin or IRDye800CW-labeled streptavidin (final concentration of 1 μg/mL) was directly added to the fresh medium. The cells were further cultured for 20 h, the medium was removed, and the cells were washed twice with PBS. The cells were scanned with a fluorescence microscope or a near-infrared fluorescence scanner (Oddysey, LiCor). Cell observation: The inverted cells were observed with an inverted fluorescence microscope (Olympus IX51-DPI71) to observe whether the cell surface produced green fluorescence. At the same time, the cell growth was observed, such as vigorous growth, good adherence, and no pollution. Photomicrographs of multi-field brightfield and green fluorescence observation channels were performed on various treated cells.

实验结果：Experimental results:

用DMP-Display-SBP表达载体转染Hepa1-6、HepG2、MRC-5、HL7702、293T细胞；对细胞进行FITC标记的链霉亲和素蛋白的染色，之后在白场(bright field)和绿色荧光(FITC)通道下对细胞显微拍照；再将白场和绿色荧光通道图像的叠加(图6)。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(Hepa1-6、HepG2、293T)中有表达，在正常细胞(MRC-5、HL7702)中无表达(图6)。Hepa1-6, HepG2, MRC-5, HL7702, and 293T cells were transfected with DMP-Display-SBP expression vector; cells were stained with FITC-labeled streptavidin protein, followed by white field (bright field) and green Microscopic photographing of cells under fluorescent (FITC) channels; superposition of white and green fluorescent channel images (Figure 6). It can be seen that DMP-controlled cell surface display SBP is expressed only in tumor cells (Hepa 1-6, HepG2, 293T) and not in normal cells (MRC-5, HL7702) (Fig. 6).

用DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像；再对各孔荧光强度进行量化。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达(图7)。Transfection of HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10, MRC-5 with DMP-Display-SBP expression vector After HL7702 cells, the cells were stained with IRDye800CW-labeled streptavidin protein, and then scanned on a near-infrared fluorescence scanner; the fluorescence intensity of each well was quantified. It can be seen that DMP-controlled cell surface display (SBP) is only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7). In B16F10), it was not expressed in normal cells (MRC-5, HL7702) (Fig. 7).

用DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色；之后在近红外荧光扫描仪上扫描成像并在白场拍照。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达(图8)。Transfection of HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10, MRC-5 with DMP-Display-SBP expression vector After HL7702 cells, the cells were trypsinized and collected, and stained with IRDye800CW-labeled streptavidin protein; then scanned on a near-infrared fluorescence scanner and photographed on a white field. It can be seen that DMP-controlled cell surface display (SBP) is only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7). , expressed in B16F10), was not expressed in normal cells (MRC-5, HL7702) (Fig. 8).

用将DMP-Display-SBP包装成腺相关病毒(AAV)表达载体(AAV-SBP)后，用AAV-SBP转染293T、HepG2、Hepa1-6、MRC-5、HL7702细胞。对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像(图9A)；对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像并在白场拍照(图9B)。可见包装成AAV病毒载体(AAV-SBP)，DMP控制的细胞表面展示表达的SBP可以高效地展示在肿瘤细胞(HepG2、293T、Hepa1-6)表面，在正常细胞(MRC-5、HL7702)中无表达(图9)。After packaging DMP-Display-SBP into an adeno-associated virus (AAV) expression vector (AAV-SBP), 293T, HepG2, Hepa1-6, MRC-5, and HL7702 cells were transfected with AAV-SBP. The cells were stained with IRDye800CW-labeled streptavidin protein, which was then scanned on a near-infrared fluorescence scanner (Fig. 9A); cells were trypsinized and collected, and then IRDye800CW-labeled streptavidin protein was used. After staining, the image was scanned on a near-infrared fluorescence scanner and photographed on a white field (Fig. 9B). It can be seen that it is packaged into AAV virus vector (AAV-SBP), and DMP-controlled cell surface display expressed SBP can be efficiently displayed on the surface of tumor cells (HepG2, 293T, Hepa1-6) in normal cells (MRC-5, HL7702). No expression (Figure 9).

实施例4 DMP-miRNA _RelA转染实验(miRNA为效应基因) Example 4 DMP-miRNA _RelA transfection assay (miRNA is an effector gene)

实验方法：experimental method:

载体构建：构建一质粒表达载体，该载体上克隆DMP片段及其下游的一种靶向NF-κB RelA mRNA的人工设计microRNA编码序列，即miR533，其序列为5′-CAA AGA TGG  GAT GAG AAA GGA-3′(SEQ ID NO.5)；其RelA mRNA靶序列为5′-TCC TTT CTC ATC CCA TCT TTG-3′(SEQ ID NO.6)。其靶序列坐落于RelA基因编码序列内。Vector construction: A plasmid expression vector was constructed, which cloned the DMP fragment and an artificially designed microRNA coding sequence targeting NF-κB RelA mRNA, ie, miR533, whose sequence is 5′-CAA AGA TGG GAT GAG AAA GGA-3' (SEQ ID NO. 5); its RelA mRNA target sequence is 5'-TCC TTT CTC ATC CCA TCT TTG-3' (SEQ ID NO. 6). Its target sequence is located within the coding sequence of the RelA gene.

细胞培养：人肝癌细胞HepG2与人正常肝细胞HL7702用含有10％(v/v)胎牛血清(FBS)(HyClone公司)，100units/mL青霉素和100μg/mL链霉素的DMEM在含有5％(v/v)CO ₂的加湿培养箱中37℃培养。将细胞以5×10 ⁵个细胞/cm ²的密度接种在6孔板，孵育24小时(汇合度约80％)。 Cell culture: human hepatoma cell HepG2 and human normal liver cell HL7702 were contained in DMEM containing 10% (v/v) fetal bovine serum (FBS) (HyClone), 100 units/mL penicillin and 100 μg/mL streptomycin in 5%. (v/v) CO ₂ was incubated at 37 ° C in a humidified incubator. The cells were seeded in a 6-well plate at a density of 5 x 10 ⁵ cells/cm ² and incubated for 24 hours (confluence of about 80%).

细胞转染及诱导：用EndoFree质粒试剂盒(CWBio)分离用于转染的DMP-miR533载体。在转染前，小心地取出培养基，用500μL PBS冲洗细胞。然后，向每个孔中加入500μL OPTI-MEM培养基并孵育2小时。通过使用基于脂质的转染试剂Lipofectamine2000(Invitrogen)将4μg的DMP-miR533载体转染到细胞中。6小时后，除去混合物，加入完全培养基，继续培养细胞48h。培养48h后，细胞用TNF-α(10ng/mL)刺激1小时。Cell transfection and induction: The DMP-miR533 vector for transfection was isolated using the EndoFree Plasmid Kit (CWBio). Prior to transfection, the medium was carefully removed and the cells were washed with 500 μL of PBS. Then, 500 μL of OPTI-MEM medium was added to each well and incubated for 2 hours. 4 μg of DMP-miR533 vector was transfected into cells by using lipid-based transfection reagent Lipofectamine 2000 (Invitrogen). After 6 hours, the mixture was removed, complete medium was added, and the cells were further cultured for 48 h. After 48 hours of culture, the cells were stimulated with TNF-α (10 ng/mL) for 1 hour.

基因表达检测：收集细胞，用Trizol提取总RNA，逆转录合成互补DNA(cDNA)。cDNA制备反应及程序为：10μL反转录反应组分包含2μL 5×PrimeScript RT Master Mix、50ng total RNA，用RNase Free ddH ₂O将反应总体积补至10μL；37℃反应15分钟，升温至85℃反应5秒使反转录酶失活，反应液4℃保存。通过qPCR定量分析RelA表达。用于qPCR的上下引物为5′-CCT GGA GCA GGC TAT CAG TC-3′(F)(SEQ ID NO.7)与5′-ATG GGA TGA GAA AGG ACA GG-3′(R)(SEQ ID NO.8)。PCR模板为cDNA。通过qPCR定量分析RelA表达。10μL qPCR反应含有5μL Fast SYBR Green Master Mix(ABI)，0.2μL 10μM F，0.2μL 10μM R和1μL cDNA，用ddH ₂O将反应总体积补至10μL。将配制好的反应体系放于荧光定量PCR仪(StepOne plus，ABI)上进行扩增，设置的扩增程序为：95℃预变性10分钟、45个扩增循环(95℃变性15s，在各退火温度下扩增1分钟)。荧光定量PCR扩增的特异性用溶解曲线来分析确定，用比较CT值法来计算基因表达的相对定量(RQ)，数据最后表示为平均值±标准偏差(SD)，统计显著性用t检验来确定。 Gene expression assay: Cells were harvested, total RNA was extracted with Trizol, and complementary DNA (cDNA) was synthesized by reverse transcription. The cDNA preparation reaction and procedure were as follows: 10 μL of the reverse transcription reaction component contained 2 μL of 5×PrimeScript RT Master Mix, 50 ng of total RNA, and the total volume of the reaction was supplemented to 10 μL with RNase Free ddH ₂ O; the reaction was carried out at 37 ° C for 15 minutes, and the temperature was raised to 85. The reaction was carried out at ° C for 5 seconds to inactivate the reverse transcriptase, and the reaction solution was stored at 4 ° C. RelA expression was quantitatively analyzed by qPCR. The upper and lower primers for qPCR are 5'-CCT GGA GCA GGC TAT CAG TC-3' (F) (SEQ ID NO. 7) and 5'-ATG GGA TGA GAA AGG ACA GG-3' (R) (SEQ ID NO.8). The PCR template is cDNA. RelA expression was quantitatively analyzed by qPCR. The 10 μL qPCR reaction contained 5 μL of Fast SYBR Green Master Mix (ABI), 0.2 μL of 10 μM F, 0.2 μL of 10 μM R and 1 μL of cDNA, and the total volume of the reaction was supplemented to 10 μL with ddH ₂ O. The prepared reaction system was amplified by a quantitative PCR instrument (StepOne plus, ABI), and the amplification procedure was set: pre-denaturation at 95 ° C for 10 minutes, and 45 amplification cycles (denaturation at 95 ° C for 15 s, in each Amplification at annealing temperature for 1 minute). The specificity of real-time PCR amplification was determined by the dissolution curve. The relative quantification (RQ) of gene expression was calculated by comparative CT value method. The data was finally expressed as mean±standard deviation (SD), and the statistical significance was analyzed by t test. to make sure.

细胞凋亡测定：转染前一天选取状态良好的HepG2及HL7702细胞用0.25％的胰蛋白酶溶液消化、计数并以1×10 ⁵细胞/孔接种于24孔细胞培养板过夜培养，在细胞融合度为80％左右时进行转染实验。取800ng的DMP-miR533质粒分别转染HepG2及HL7702细胞。将培养24h后的HepG2及HL7702细胞用不含EDTA的胰蛋白酶溶液消化离心得到细胞沉淀，用AnnexinV-FITC/PI细胞凋亡检测试剂盒(凯基公司)处理细胞后，用流式细胞仪(ACEANovoCyte公司)中检测细胞凋亡情况。 Apoptosis assay: HepG2 and HL7702 cells in good condition one day prior to transfection were digested with 0.25% trypsin solution, counted and seeded at 1×10 ⁵ cells/well in 24-well cell culture plates overnight for cell fusion. Transfection experiments were performed at around 80%. 800 ng of DMP-miR533 plasmid was transfected into HepG2 and HL7702 cells, respectively. The HepG2 and HL7702 cells cultured for 24 hours were digested with EDTA-free trypsin solution to obtain a cell pellet, and the cells were treated with Annexin V-FITC/PI Apoptosis Detection Kit (KGI), followed by flow cytometry ( Apoptosis was detected in ACEANovoCyte).

实验结果：Experimental results:

为了NF-κB激活RelA miRNA表达载体是否可导致细胞内NF-κB RelA表达下调，用DMP-miR533载体转染人肝癌细胞。用荧光定量PCR检测细胞内NF-κB RelA表达。结果表明靶向NF-κB RelA mRNA的miR533的导入，成功敲低细胞内NF-κB RelA表达水平(图10A)。由于NF-κB经常通过抑制细胞凋亡的靶基因发挥抗凋亡作用。用RelA miRNA敲低NF-κB RelA表达水平，可能引起肿瘤细胞的生长抑制或凋亡。为了验证这一推断，我们检测了DMP-miR533转染对人类肝癌细胞HepG2以及人正常肝细胞HL7702凋亡的影响。In order for NF-κB-activated RelA miRNA expression vector to cause down-regulation of NF-κB RelA expression in cells, human liver cancer cells were transfected with DMP-miR533 vector. The expression of NF-κB RelA in cells was detected by real-time PCR. The results indicated that the introduction of miR533 targeting NF-κB RelA mRNA successfully knocked down the expression level of NF-κB RelA in cells (Fig. 10A). Since NF-κB often exerts an anti-apoptotic effect through a target gene that inhibits apoptosis. Knockdown of NF-κB RelA expression levels with RelA miRNAs may result in growth inhibition or apoptosis of tumor cells. To test this hypothesis, we examined the effect of DMP-miR533 transfection on apoptosis of human hepatoma cell HepG2 and human normal liver cell HL7702.

用AnnexinV-FITC/PI细胞凋亡检测试剂盒操作说明处理细胞后，用流式细胞仪检测了DMP-miR533对细胞凋亡的影响。结果显示，DMP-miR533导致人肝癌细胞发生凋亡(图10B)。与人类肝癌细胞HepG2进行对比，人正常肝细胞HL7702作为NF-κB过度活化的阴性对照，DMP-miR533未能诱导HL7702细胞凋亡(图10B)。这些数据表明，DMP-amiR533具有NF-κB特异性。DMP-amiR533对正常人肝细胞HL7702活性没有影响，这意味着这种新的NF-κB抑制剂可以消除NF-κB过度激活的细胞(如癌症或炎性细胞)中NF-κB的过度活化，但不会对正常细胞产生影响。The effect of DMP-miR533 on apoptosis was examined by flow cytometry after treatment with Annexin V-FITC/PI Apoptosis Detection Kit. The results showed that DMP-miR533 caused apoptosis in human liver cancer cells (Fig. 10B). In contrast to human hepatoma cell line HepG2, human normal liver cell HL7702 served as a negative control for NF-κB overactivation, and DMP-miR533 failed to induce apoptosis in HL7702 cells (Fig. 10B). These data indicate that DMP-amiR533 has NF-κB specificity. DMP-amiR533 has no effect on normal human hepatocyte HL7702 activity, suggesting that this new NF-κB inhibitor can abolish the excessive activation of NF-κB in NF-κB over-activated cells such as cancer or inflammatory cells. But it does not affect normal cells.

实施例5 DMP-Cas9转染实验(Cas9为效应基因)Example 5 DMP-Cas9 transfection experiment (Cas9 is an effector gene)

实验方法：experimental method:

载体构建：构建U6-TsgRNA-DMP-Cas9表达载体，其中有U6启动子控制的靶向人端粒DNA序列的sgRNA表达序列。U6启动子序列为：GAT CCG ACG CCG CCA TCT CTA GGC CCG CGC CGG CCC CCT CGC ACA GAC TTG TGG GAG AAG CTC GGC TAC TCC CCT GCC CCG GTT AAT TTG CAT ATA ATA TTT CCT AGT AAC TAT AGA GGC TTA ATG TGC GAT AAA AGA CAG ATA ATC TGT TCT TTT TAA TAC TAG CTA CAT TTT ACA TGA TAG GCT TGG ATT TCT ATA AGA GAT ACA AAT ACT AAA TTA TTA TTT TAA AAA ACA GCA CAA AAG GAA ACT CAC CCT AAC TGT AAA GTA ATT GTG TGT TTT GAG ACT ATA AAT ATC CCT TGG AGA AAA GCC TTG TTT(SEQ ID NO.9)；TsgRNA靶序列为：5′-TAA CCC TAA CCC TAA CCC TA-3′(SEQ ID NO.10)；sgRNA序列为：5′-TAG GGT TAG GGT TAG GGT TA-3′(SEQ ID NO.11)。DMP序列含有NF-κB应答序列(5′-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3′，SEQ ID NO.1)以及最小启动子序列(5′-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3′，SEQ ID NO.2)；以及Cas9蛋白编码基因。Vector construction: A U6-TsgRNA-DMP-Cas9 expression vector was constructed in which a U6 promoter-controlled sgRNA expression sequence targeting human telomeric DNA sequences was constructed. The U6 promoter sequence is: GAT CCG ACG CCG CCA TCT CTA GGC CCG CGC CGG CCC CCT CGC ACA GAC TTG TGG GAG AAG CTC GGC TAC TCC CCT GCC CCG GTT AAT TTG CAT ATA ATA TTT CCT AGT AAC TAT AGA GGC TTA ATG TGC GAT AAA AGA CAG ATA ATC TGT TCT TTT TAA TAC TAG CTA CAT TTT ACA TGA TAG GCT TGG ATT TCT ATA AGA GAT ACA AAT ACT AAA TTA TTA TTA TAA AAA ACA GCA CAA AAG GAA ACT CAC CCT AAC TGT AAA GTA ATT GTG TGT TTT GAG ACT ATA AAT ATC CCT TGG AGA AAA GCC TTG TTT (SEQ ID NO. 9); TsgRNA target sequence: 5'-TAA CCC TAA CCC TAA CCC TA-3' (SEQ ID NO. 10); sgRNA sequence is: 5 '-TAG GGT TAG GGT TAG GGT TA-3' (SEQ ID NO. 11). The DMP sequence contains the NF-κB response sequence (5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3', SEQ ID NO. 1) and the minimal promoter sequence (5'-TAG) AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3', SEQ ID NO. 2); and Cas9 protein encoding gene.

细胞培养：HepG2(人肝癌细胞)、A549(人肺癌细胞)、HT-29(人结肠癌细胞)、HeLa(人***细胞)、SKOV3(人卵巢癌细胞)、PANC-1(胰腺癌细胞)、MDA-MB-453(人乳腺癌)、Hepa1-6(小鼠肝癌细胞)、HL7702(人正常肝细胞)及MRC5(人胚胎成纤 维细胞)细胞培养。细胞培养使用DEME(Hepa1-6、HEK-293T、HepG2、HeLa，PANC-1、MDA-MB-453)或RPMI 1640培养基(A549、HT-29、SKOV-3、MRC-5、HL7702)、10％胎牛血清(HyClone)、100units/mL青霉素和100μg/mL链霉素培养；培养环境为含有5％(v/v)CO ₂的加湿培养箱中37℃培养。细胞复苏后，按同等密度(0.5×10 ⁵/孔)接种到24孔微孔板中，培养过夜贴壁后，进行转染。 Cell culture: HepG2 (human liver cancer cells), A549 (human lung cancer cells), HT-29 (human colon cancer cells), HeLa (human cervical cancer cells), SKOV3 (human ovarian cancer cells), PANC-1 (pancreatic cancer cells) ), MDA-MB-453 (human breast cancer), Hepa 1-6 (mouse liver cancer cells), HL7702 (human normal liver cells), and MRC5 (human embryonic fibroblasts) cell culture. Cell culture using DEME (Hepa 1-6, HEK-293T, HepG2, HeLa, PANC-1, MDA-MB-453) or RPMI 1640 medium (A549, HT-29, SKOV-3, MRC-5, HL7702), 10% fetal bovine serum (HyClone), 100 units/mL penicillin and 100 μg/mL streptomycin were cultured; the culture environment was cultured at 37 ° C in a humidified incubator containing 5% (v/v) CO ₂ . After the cells were resuscitated, they were inoculated into 24-well microplates at the same density (0.5 × 10 ⁵ /well), cultured overnight, and then transfected.

细胞转染：细胞培养液换为无血清培养基培养1h。分别用U6-TsgRNA-DMP-Cas9转染上述细胞。空脂质体转染的细胞作为转染对照。每孔细胞的总DNA用量及脂质体用量参考脂质体产品(Lipofectamine 2000；ThermoFisher Scientific)说明书进行。DNA-脂质体加入无血清培养基培养4h。换为含血清新鲜培养基，继续培养24h。Cell transfection: The cell culture medium was changed to serum-free medium for 1 h. The above cells were transfected with U6-TsgRNA-DMP-Cas9, respectively. Empty lipofected cells were used as transfection controls. The total amount of DNA per cell and the amount of liposome used were determined in accordance with the instructions for liposome products (Lipofectamine 2000; ThermoFisher Scientific). DNA-liposomes were added to serum-free medium for 4 h. Switch to serum-containing fresh medium and continue to culture for 24 h.

细胞染色：吖啶橙(acridine orange，AO))染色按吖啶橙产品说明(索莱宝；北京)进行(室温染色10分钟)。程序为：用100μg/mL吖啶橙室温染色10分钟。染色后细胞用PBS洗涤。Cell staining: Acridine orange (AO) staining was performed according to the acridine orange product description (Solebao; Beijing) (room temperature staining for 10 minutes). The procedure was: staining with 100 μg/mL acridine orange for 10 minutes at room temperature. After staining, the cells were washed with PBS.

细胞观察：用倒置荧光显微镜(Olympus IX51-DPI71)观测拍照各种处理的细胞；对各种处理的细胞进行多视野绿色荧光通道观察及拍照。Cell observation: Various treated cells were observed by an inverted fluorescence microscope (Olympus IX51-DPI71); multi-field green fluorescent channels were observed and photographed on various treated cells.

实验结果：Experimental results:

通过上述转染及吖啶橙染色操作后，对各种处理的细胞进行多视野绿色荧光通道观察及拍照，获得的代表性实验结果如图11所示。结果表明，转染了U6-TsgRNA-DMP-Cas9表达载体的肿瘤细胞均发生不同程度的死亡，部分癌症种类的肿瘤细胞株发生大量死亡(如HepG2、SKOV3、PANC-1)。而转染U6-TsgRNA-DMP-Cas9表达载体是正常细胞(HL7702、MRC5)为发生死亡。可见，由于上述实施例1、2、3证明的NF-κB在肿瘤细胞中的特异表达，以及NF-κB特异激活基因表达载体(DMP-效应基因)在肿瘤细胞内的表达，U6-TsgRNA-DMP-Cas9表达载体在具有NF-κB过度活化水平的肿瘤细胞中发生表达；After the above transfection and acridine orange staining operation, various treated cells were observed and photographed by multi-field green fluorescent channel, and representative experimental results obtained are shown in FIG. The results showed that the tumor cells transfected with U6-TsgRNA-DMP-Cas9 expression vector all died to varying degrees, and a large number of tumor cell lines of some cancer types died (such as HepG2, SKOV3, PANC-1). The U6-TsgRNA-DMP-Cas9 expression vector was transfected into normal cells (HL7702, MRC5) for death. It can be seen that the specific expression of NF-κB in tumor cells as demonstrated by the above Examples 1, 2, and 3, and the expression of NF-κB specific activation gene expression vector (DMP-effector gene) in tumor cells, U6-TsgRNA- DMP-Cas9 expression vector is expressed in tumor cells with NF-κB overactivation level;

在本实验中，我们在上述DMP载体的基础上，将Cas9基因作为效应基因，构建了U6-TsgRNA-DMP-Cas9载体。当将U6-TsgRNA-DMP-Cas9载体分子转染细胞后，载体表达产生的sgRNA与Cas9结合形成靶向端粒DNA的sgRNA-dCas9复合物。该复合物与细胞端粒DNA解结合可导致端粒DNA的切割，造成端粒DNA损伤，从而引发细胞死亡。In this experiment, we constructed the U6-TsgRNA-DMP-Cas9 vector based on the above DMP vector using the Cas9 gene as an effector gene. When the U6-TsgRNA-DMP-Cas9 vector molecule is transfected into cells, the vector-generated sgRNA binds to Cas9 to form a sgRNA-dCas9 complex that targets telomeric DNA. Decomposition of this complex with cellular telomeric DNA can result in cleavage of telomeric DNA, causing damage to telomeric DNA, thereby triggering cell death.

本实验结果表明，转染U6-TsgRNA-DMP-Cas9载体的细胞中，作为NF-κB阳性细胞的肿瘤细胞株，包括HepG2、PANC-1、A549、HT-29、HeLa、SKOV3、MDA-MB-453、Hepa1-6，均发生不同程度的显著死亡，部分癌症种类的肿瘤细胞株发生大量死亡(如HepG2、SKOV3、PANC-1)。而作为NF-κB阴性细胞的MRC-5及HL7702未发生死亡 (图11)。实验结果符合预期，即NF-κB活性特异的基因转录表达***可用于杀灭肿瘤细胞。The results of this experiment indicate that the tumor cell lines that are NF-κB positive cells, including HepG2, PANC-1, A549, HT-29, HeLa, SKOV3, MDA-MB, are transfected into the U6-TsgRNA-DMP-Cas9 vector. -453, Hepa1-6, all of which showed significant degrees of significant death, and a large number of cancer cell lines of some cancer types (such as HepG2, SKOV3, PANC-1). MRC-5 and HL7702, which are NF-κB-negative cells, did not die (Fig. 11). The experimental results are in line with expectations that a gene transcriptional expression system specific for NF-κB activity can be used to kill tumor cells.

Claims (13)

1. 一种基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达技术及其在NF-κB过度活化疾病的治疗药物或试剂中的应用，该基因表达技术由一种NF-κB特异性激活基因表达载体实现。A gene expression technology based on NF-κB activity-activated effector gene in NF-κB over-activated cells and its application in therapeutic drugs or reagents for NF-κB over-activation disease, the gene expression technique is a kind of NF - kappa B specific activation gene expression vector implementation.
2. 根据权利要求1所述的基因表达技术及其应用，其特征在于，所述NF-κB特异性激活基因表达载体包含两个序列元件，具体为调控基因表达的启动子序列和启动子下游效应基因编码序列。The gene expression technique according to claim 1 and the use thereof, wherein the NF-κB-specific activation gene expression vector comprises two sequence elements, specifically a promoter sequence for regulating gene expression and a promoter downstream effector gene Coding sequence.
3. 根据权利要求2所述的基因表达技术及其应用，其特征在于，所述启动子序列由一段NF-κB应答序列和最小启动子序列组成。The gene expression technique according to claim 2 and the use thereof, wherein the promoter sequence consists of a NF-κB responsive sequence and a minimal promoter sequence.
4. 根据权利要求3所述的基因表达技术及其应用，其特征在于，所述NF-κB应答序列为可与NF-κB蛋白特异性结合的DNA序列，其主要序列特征为含有NF-κB结合靶点；该NF-κB应答序列包括各种序列的NF-κB应答序列。The gene expression technique according to claim 3 and the use thereof, wherein the NF-κB response sequence is a DNA sequence which specifically binds to NF-κB protein, and the main sequence is characterized by containing NF-κB binding target The NF-κB response sequence includes NF-κB responsive sequences of various sequences.
5. 根据权利要求3所述的基因表达技术及其应用，其特征在于，所述最小启动子包括各种来源的最小启动子序列，具体包括天然及人工筛选的最小启动子序列。The gene expression technique according to claim 3 and the use thereof, wherein the minimal promoter comprises a minimal promoter sequence of various origins, specifically comprising a minimal promoter sequence naturally and artificially screened.
6. 根据权利要求2所述的基因表达技术及其应用，其特征在于，所述效应基因包括各种类型、序列和功能的效应基因；包括天然基因序列及人工突变及合成的效应基因。The gene expression technique according to claim 2 and the use thereof, wherein the effector gene comprises various types, sequences and functional effector genes; and includes natural gene sequences and artificially mutated and synthesized effector genes.
7. 根据权利要求1所述的基因表达技术及其应用，其特征在于，所述NF-κB特异性激活基因表达载体为一线性核酸分子或环状核酸分子。The gene expression technique according to claim 1 or the use thereof, wherein the NF-κB specific activation gene expression vector is a linear nucleic acid molecule or a circular nucleic acid molecule.
8. 根据权利要求7所述的基因表达技术及其应用，其特征在于，所述线性核酸分子包括普通线性DNA分子、病毒DNA分子或病毒RNA分子；所述环状核酸分子包括质粒DNA。The gene expression technique according to claim 7 and the use thereof, wherein the linear nucleic acid molecule comprises a general linear DNA molecule, a viral DNA molecule or a viral RNA molecule; and the circular nucleic acid molecule comprises plasmid DNA.
9. 根据权利要求2所述的基因表达技术及其应用，其特征在于，所述NF-κB特异性激活基因表达载体导入NF-κB活性过度活化的细胞内时，细胞内的序列特异性转录因子NF-κB就会激活该载体，使其表达该载体上的效应基因，效应基因产物可引发细胞生理的改变，包括细胞生长抑制、凋亡或死亡。The gene expression technique according to claim 2, wherein the NF-κB specific activation gene expression vector is introduced into a cell in which NF-κB is actively activated, and the sequence-specific transcription factor NF is intracellularly - kappa B activates the vector to express the effector gene on the vector, and the effector gene product can initiate changes in cell physiology, including cell growth inhibition, apoptosis or death.
10. 根据权利要求7所述的基因表达技术及其应用，其特征在于，所述NF-κB特异性激活基因表达载体导入NF-κB活性过度活化的细胞内的导入方法包括各种类型的核酸细胞导入方法，包括病毒载体、纳米载体、脂质体、电转移或基因枪的导入方式。The gene expression technique according to claim 7, and the use thereof, wherein the introduction of the NF-κB-specific activation gene expression vector into a cell in which NF-κB activity is excessively activated includes introduction of various types of nucleic acid cells. Methods include viral vectors, nanocarriers, liposomes, electrotransfers, or introduction methods for gene guns.
11. 根据权利要求9所述的基因表达技术及其应用，其特征在于，所述效应基因产物包括RNA和蛋白质；所述RNA包括各种功能类型的RNA；所述蛋白质包括各种功能类型的蛋白质及多肽等。The gene expression technique according to claim 9 and the use thereof, wherein the effector gene product comprises RNA and a protein; the RNA comprises RNA of various functional types; the protein comprises proteins of various functional types and Peptides and the like.
12. 权利要求1中所述的NF-κB特异性激活基因表达载体在NF-κB过度活化疾病治疗药物中的应用或NF-κB过度活化密切相关疾病基因治疗的试剂或药物中的应用。Use of the NF-κB-specific activation gene expression vector of claim 1 in the treatment of NF-κB over-activation disease or an agent or drug for NF-κB over-activation of a closely related disease gene therapy.
13. 根据权利要求12所述的应用，其特征在于，所述的NF-κB过度活化疾病包括各种与NF-κB过度活化密切相关的疾病，尤其是炎症和肿瘤。The use according to claim 12, wherein said NF-κB over-activation disease comprises various diseases closely related to NF-κB overactivation, particularly inflammation and tumors.

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