WO2021114256A1 - Drug for treating pulmonary fibrosis - Google Patents

Abstract

Disclosed is a drug for treating pulmonary fibrosis in a subject. The drug contains a vector and a polynucleotide encoding a PHB2 protein, wherein the vector contains or carries the polynucleotide encoding a PHB2 protein. Not only can PHB2 have an anti-fibrotic effect with regard to pulmonary fibrosis, same can also inhibit the apoptosis of primary fibroblasts and epithelial cells, inhibit the migration of primary fibroblasts, combat the death of alveolar epithelial cells, and improve the survival rate of cells. These results indicate can treat pulmonary fibrotic diseases by means of targeted PHB2.

Description

用于治疗肺纤维化的药物Drugs used to treat pulmonary fibrosis 技术领域Technical field

本发明涉及抑制素2(prohibitin 2,PHB2)的医药用途，具体涉及PHB2在肺纤维化的治疗中的应用。The present invention relates to the medical use of prohibitin 2 (PHB2), and specifically relates to the application of PHB2 in the treatment of pulmonary fibrosis.

背景技术Background technique

肺纤维化(pulmonary fibrosis,PF)是一种慢性、渐进性的肺间质性疾病，由持续的肺泡上皮细胞损伤和病理性损伤修复所致，可通过影响肺换气导致严重的肺功能障碍，从而引起致命性的后果。肺纤维化是多种弥漫性间质性肺疾病的最终结局，以不可逆的肺泡结构破坏和细胞外基质过度沉积为主要特征。临床上，肺纤维化多表现为进行性呼吸困难伴有刺激性干咳、肺功能的进行性降低及低氧血症等症状。病情呈进行性发展，患者最终因呼吸衰竭而死亡。肺纤维化患者的中位生存时间一般为3年。Pulmonary fibrosis (PF) is a chronic and progressive pulmonary interstitial disease. It is caused by continuous alveolar epithelial cell damage and pathological damage and repair. It can cause severe lung dysfunction by affecting lung ventilation. , Causing fatal consequences. Pulmonary fibrosis is the final outcome of a variety of diffuse interstitial lung diseases, characterized by irreversible alveolar structure destruction and excessive extracellular matrix deposition. Clinically, pulmonary fibrosis is mostly manifested as progressive dyspnea with irritating dry cough, progressive decrease in lung function, and hypoxemia. The condition progressed progressively, and the patient eventually died of respiratory failure. The median survival time of patients with pulmonary fibrosis is generally 3 years.

特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)是一种病情更严重的致命性的肺纤维化，由于缺乏灵敏的早期诊断和有效的治疗手段，其致死率在世界范围内排名前五。Idiopathic pulmonary fibrosis (IPF) is a more serious and fatal pulmonary fibrosis. Due to the lack of sensitive early diagnosis and effective treatment, its fatality rate ranks among the top five in the world.

目前，IPF的病因尚不明确。在IPF患者或者肺纤维化动物模型的肺组织中出现了显著的线粒体功能障碍和线粒体自噬不足。IPF的发生与年龄密切相关，通常60岁以后患病的几率较大。线粒体功能在正常衰老的生物体发生紊乱的进程较为缓慢，而在IPF患者的肺组织中变化却比较显著。IPF中代谢功能紊乱将会影响肺组织内细胞的功能和分化从而启动纤维化反应，然后通过激活TGFβ加剧纤维化进程。肺纤维化中出现线粒体自噬不足也是导致纤维化进程加速的重要原因之一，研究发现：博莱霉素诱导的肺纤维化模型小鼠中敲除自噬关键蛋白ATG4B后，其纤维化程度加剧，阐明了自噬受损在肺纤维化发展中的作用。At present, the cause of IPF is still unclear. Significant mitochondrial dysfunction and insufficient mitochondrial autophagy appeared in the lung tissues of IPF patients or animal models of pulmonary fibrosis. The occurrence of IPF is closely related to age, and the chance of getting the disease is usually greater after 60 years of age. Mitochondrial function disorder progresses slowly in normal aging organisms, but changes significantly in the lung tissue of IPF patients. The metabolic dysfunction in IPF will affect the function and differentiation of cells in the lung tissue to initiate the fibrotic response, and then aggravate the fibrotic process by activating TGFβ. Insufficient mitochondrial autophagy in pulmonary fibrosis is also one of the important reasons for the acceleration of the fibrosis process. The study found that the degree of fibrosis after knocking out the key autophagy protein ATG4B in bleomycin-induced pulmonary fibrosis model mice Intensified, elucidating the role of impaired autophagy in the development of pulmonary fibrosis.

自噬(autophagy)是一种进化保守、遗传程序化的细胞内降解途径，是真核细胞内一种重要的细胞稳态机制，在调节细胞生长、代谢和存活，在维持细胞内环境平衡中起着重要作用。在IPF的患者肺组织中由于线粒体受损而导致其功能紊乱，出现自噬受阻，从而加重了纤维化的进程，研究发现PHB2参与线粒体自噬是通过与自噬相关蛋白LC3发生相互作用来实现的。LC3，也称作LC3-II，它可以附着到自噬体的膜上，是自噬体的结构蛋白。在降解过程中，位于自噬溶酶 体外膜的LC3-II被半胱氨酸蛋白酶ATG4B移除后回收，位于内膜的LC3-II则与包裹的内容物一起，被溶酶体降解。Autophagy (autophagy) is an evolutionary conserved and genetically programmed intracellular degradation pathway. It is an important cell homeostasis mechanism in eukaryotic cells. It regulates cell growth, metabolism and survival, and maintains the balance of the intracellular environment. Plays an important role. In the lung tissue of IPF patients, mitochondria are damaged due to their dysfunction, and autophagy is blocked, which aggravates the process of fibrosis. Studies have found that PHB2 participates in mitochondrial autophagy by interacting with the autophagy-related protein LC3. of. LC3, also known as LC3-II, can be attached to the membrane of autophagosomes and is a structural protein of autophagosomes. In the degradation process, the LC3-II located in the outer membrane of autophagylysin is removed by the cysteine protease ATG4B and then recovered, while the LC3-II located in the inner membrane is degraded by the lysosome along with the contents of the package.

PHB首先作为细胞增殖的抑制剂而被发现，它的结构相当保守且普遍存在。其广泛分布于哺乳动物、原虫、植物、细菌以及真菌等生物体中。人类基因组编码两种PHB蛋白质，即prohibitin 1(PHB1)和prohibitin 2(PHB2)，PHB1基因位于染色体17q21上，PHB2基因位于染色体12p13上，二者由高度同源的亚基组成，是真核生物细胞的增殖和发育所必须的。PHB主要分布在线粒体、细胞核、以及细胞膜等，PHB的功能依据其在细胞内的定位不同而不同，在细胞膜和细胞核中主要参与细胞质膜中的信号转导和细胞核中转录调节。PHB与线粒体膜电位的去极化、氧化磷酸化***(OXPHOS)的组装和活性、线粒体的生物合成、线粒体网络、线粒体DNA的稳定性、线粒体的形态、凋亡和自噬均有密切关系，过表达PHB将会提高呼吸频率、电子传递链、以及ATP的生成，而敲除PHB2基因后，将会造成线粒体嵴形态异常、ATP合成、活性氧和mtDNA的表达下调，从而造成线粒体功能障碍。PHB was first discovered as an inhibitor of cell proliferation, and its structure is quite conservative and ubiquitous. It is widely distributed in organisms such as mammals, protozoa, plants, bacteria and fungi. The human genome encodes two PHB proteins, namely prohibitin 1 (PHB1) and prohibitin 2 (PHB2). The PHB1 gene is located on chromosome 17q21 and the PHB2 gene is located on chromosome 12p13. The two are composed of highly homologous subunits. They are eukaryotic organisms. Necessary for cell proliferation and development. PHB is mainly distributed in mitochondria, cell nuclei, and cell membranes. PHB's functions vary according to its intracellular location. In cell membranes and nuclei, it mainly participates in signal transduction in the plasma membrane and transcriptional regulation in the nucleus. PHB is closely related to the depolarization of mitochondrial membrane potential, the assembly and activity of oxidative phosphorylation system (OXPHOS), mitochondrial biosynthesis, mitochondrial network, mitochondrial DNA stability, mitochondrial morphology, apoptosis and autophagy. Overexpression of PHB will increase respiratory rate, electron transport chain, and ATP production, while knocking out PHB2 gene will cause abnormal mitochondrial cristae morphology, ATP synthesis, down-regulation of reactive oxygen species and mtDNA expression, resulting in mitochondrial dysfunction.

近年来，针对IPF的临床试验药物包括糖皮质激素、秋水仙碱、环孢素A、乙酰半胱胺酸、波生坦、依那西普、抗凝剂、尼达尼布、吡非尼酮及伊马替尼等等，但IPF患者对激素和各种药物的治疗反应普遍较差，目前尚无能确切改善患者最终预后的药物，美国胸科医师学会(ATS)的IPF指南中指出，IPF病人到病情晚期唯一值得推荐且有效的治疗便为肺移植，但由于手术治疗费用昂贵、技术程度复杂、供者肺组织来源紧缺、器官移植伦理学及医疗法律法规不健全等因素的存在，限制了肺移植作为IPF患者治疗方法在我国的开展。In recent years, clinical trial drugs for IPF include glucocorticoids, colchicine, cyclosporine A, acetylcysteine, bosentan, etanercept, anticoagulants, nintedanib, pirfenib Ketones and imatinib, etc., but IPF patients generally respond poorly to hormones and various drugs. There is currently no drug that can definitely improve the patient’s final prognosis. The American College of Chest Physicians (ATS) IPF guidelines point out, The only recommended and effective treatment for IPF patients in the late stage of the disease is lung transplantation. However, due to the high cost of surgical treatment, complicated technology, shortage of donor lung tissue sources, incomplete organ transplant ethics and medical laws and regulations, etc., the existence of factors, This limits the development of lung transplantation as a treatment method for IPF patients in China.

目前针对该病的上市药物只有两个，分别是吡非尼酮和尼达尼布，但其确切作用机制尚未完全明确，且都不能有效提高患者生存期，治疗费用高，且临床需求远未得到满足。因此，有必要提供更多能够有效治疗肺纤维化的方法和手段。At present, there are only two drugs on the market for this disease, namely pirfenidone and nintedanib, but the exact mechanism of action is not yet fully clarified, and none of them can effectively improve the survival of patients, the treatment costs are high, and the clinical needs are far from Be satisfied. Therefore, it is necessary to provide more effective methods and means to treat pulmonary fibrosis.

发明内容Summary of the invention

本发明的一方面提供一种在对象中治疗肺纤维化的药物，其中该药物包含载体及编码PHB2蛋白的多核苷酸，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述病毒载体是慢 病毒。One aspect of the present invention provides a drug for treating pulmonary fibrosis in a subject, wherein the drug comprises a vector and a polynucleotide encoding a PHB2 protein, wherein the vector contains or carries the polynucleotide encoding the PHB2 protein. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus.

本发明的另一方面，提供了一种治疗对象的肺纤维化的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的药物，其中该药物包含载体及编码PHB2蛋白的多核苷酸，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述病毒载体是慢病毒。In another aspect of the present invention, there is provided a method for treating pulmonary fibrosis in a subject, the method comprising administering to the subject in need a therapeutically effective amount of the drug of the present invention, wherein the drug comprises a carrier and a polynuclear protein encoding PHB2 protein. Wherein the vector contains or carries the polynucleotide encoding the PHB2 protein. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus.

本发明的另一方面，提供了本发明的药物在制备用于治疗对象的肺纤维化的药物中的应用，其中该药物包含载体及编码PHB2蛋白的多核苷酸，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。在一些实施方式中，该载体是病毒载体或非病毒载体。在一些实施方式中，该病毒载体是腺相关病毒。在一些实施方式中，该病毒载体是慢病毒。Another aspect of the present invention provides the application of the medicament of the present invention in the preparation of a medicament for the treatment of pulmonary fibrosis in a subject, wherein the medicament comprises a carrier and a polynucleotide encoding the PHB2 protein, wherein the carrier comprises or carries The polynucleotide encoding the PHB2 protein. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus.

本发明的一方面提供一种在对象中治疗肺纤维化的药物组合物，其中该药物组合物包含含有载体及编码PHB2蛋白的多核苷酸的药物和药学上可接受的赋形剂，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述病毒载体是慢病毒。One aspect of the present invention provides a pharmaceutical composition for treating pulmonary fibrosis in a subject, wherein the pharmaceutical composition comprises a drug containing a carrier and a polynucleotide encoding a PHB2 protein and a pharmaceutically acceptable excipient, wherein The vector contains or carries the polynucleotide encoding the PHB2 protein. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus.

本发明的另一方面，提供了一种治疗对象的肺纤维化的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的药物组合物，其中该药物组合物包含含有载体及编码PHB2蛋白的多核苷酸的药物和药学上可接受的赋形剂，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述病毒载体是慢病毒。In another aspect of the present invention, there is provided a method for treating pulmonary fibrosis in a subject, the method comprising administering to the subject in need a therapeutically effective amount of the pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises a carrier and A medicament and a pharmaceutically acceptable excipient for a polynucleotide encoding a PHB2 protein, wherein the carrier contains or carries the polynucleotide encoding a PHB2 protein. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus.

本发明的另一方面，提供了本发明的药物组合物在制备用于治疗对象的肺纤维化的药物中的应用，其中该药物组合物包含含有载体及编码PHB2蛋白的多核苷酸的药物和药学上可接受的赋形剂，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述病毒载体是慢病毒。In another aspect of the present invention, there is provided the application of the pharmaceutical composition of the present invention in the preparation of a medicament for treating pulmonary fibrosis in a subject, wherein the pharmaceutical composition comprises a medicament containing a carrier and a polynucleotide encoding a PHB2 protein and A pharmaceutically acceptable excipient, wherein the carrier contains or carries the polynucleotide encoding the PHB2 protein. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus.

在一些实施方式中，本发明的药物或药物组合物被施用于对象后引起PHB2 蛋白在所述对象的细胞中表达增加。在一些实施方式中，本发明的药物或药物组合物被施用于对象后引起PHB2在所述对象的细胞中表达升高。在一些实施方式中，本发明的药物或药物组合物被施用于对象后抑制纤维化因子的表达。在一些实施方式中，本发明的药物或药物组合物被施用于对象后显著增加所述对象细胞的存活率。In some embodiments, the medicament or pharmaceutical composition of the present invention, after being administered to a subject, causes an increase in the expression of PHB2 protein in the cells of the subject. In some embodiments, the medicament or pharmaceutical composition of the present invention, after being administered to a subject, causes the expression of PHB2 to increase in the cells of the subject. In some embodiments, the medicament or pharmaceutical composition of the present invention inhibits the expression of fibrotic factors after being administered to a subject. In some embodiments, the medicament or pharmaceutical composition of the present invention significantly increases the survival rate of the cells of the subject after being administered to the subject.

附图说明Description of the drawings

图1：在纤维化状态下，PHB2表达下调。A.肺纤维化病人和健康供体肺组织中，Fibronectin、Collagen Ⅰ和α-SMA的表达情况。B.肺纤维化病人和健康供体肺组织中，PHB1和PHB2的表达情况。C.博莱霉素(BLM)诱导的C57小鼠的肺纤维化模型中，PHB1和PHB2的表达情况。D.BLM诱导的SD大鼠的肺纤维化模型中，PHB1和PHB2的表达情况。E.TGFβ刺激人胚肺成纤维细胞后，PHB2和以及各纤维因子的表达情况。Figure 1: In the fibrotic state, PHB2 expression is down-regulated. A. The expression of Fibronectin, Collagen I and α-SMA in lung tissues of patients with pulmonary fibrosis and healthy donors. B. The expression of PHB1 and PHB2 in lung tissues of patients with pulmonary fibrosis and healthy donors. C. The expression of PHB1 and PHB2 in the pulmonary fibrosis model of C57 mice induced by bleomycin (BLM). D. The expression of PHB1 and PHB2 in the pulmonary fibrosis model of SD rats induced by BLM. E. After TGFβ stimulates human embryonic lung fibroblasts, the expression of PHB2 and various fibrous factors.

图2：PHB2在小鼠体内的抗纤维化作用。A.肺组织中羟脯氨酸的含量。B.肺组织中PHB2、Fibronectin和Collagen的表达情况。C.各组肺组织的CT扫描图。D.各组肺组织形态变化的HE染色示意图。E.各组肺组织形态变化的天狼星红染色示意图。Figure 2: Anti-fibrosis effect of PHB2 in mice. A. The content of hydroxyproline in lung tissue. B. The expression of PHB2, Fibronectin and Collagen in lung tissues. C. CT scans of lung tissues in each group. D. Schematic diagram of HE staining of lung tissue morphological changes in each group. E. Schematic diagram of Sirius red staining of lung tissue morphological changes in each group.

图3：PHB2对TGFβ诱导的成纤维细胞中纤维化因子及非经典通路的影响。A.PHB2抑制TGFβ诱导48h的HFL1细胞中Fibronectin、Collagen Ⅰ、以及α-SMA的表达。B.PHB2抑制TGFβ诱导1h的HFL1细胞中pakt、p38、以及perk的表达。C.PHB2抑制HFLC细胞中Fibronectin、Collagen Ⅰ、以及α-SMA的表达。Figure 3: The effect of PHB2 on fibrosis factors and non-classical pathways in fibroblasts induced by TGFβ. A. PHB2 inhibits the expression of Fibronectin, Collagen I, and α-SMA in HFL1 cells induced by TGFβ for 48 hours. B. PHB2 inhibits the expression of pakt, p38, and perk in HFL1 cells induced by TGFβ for 1 h. C. PHB2 inhibits the expression of Fibronectin, Collagen I, and α-SMA in HFLC cells.

图4：PHB2对细胞功能的影响。A.BLM对细胞存活率的抑制效率。B.PHB2对抗BLM诱导的MLE12肺泡上皮细胞的死亡，显著上调细胞存活率。C.流式细胞术检测PHB2抑制BLM诱导的细胞凋亡。D-E.流式细胞术检测PHB2抑制TGFβ刺激引起的细胞凋亡。F.PHB2抑制细胞的迁移。Figure 4: The effect of PHB2 on cell function. A. The inhibitory efficiency of BLM on cell viability. B.PHB2 counteracts the death of MLE12 alveolar epithelial cells induced by BLM and significantly increases cell survival rate. C. Flow cytometry detection PHB2 inhibits BLM-induced apoptosis. D-E. Flow cytometry detected PHB2 to inhibit apoptosis caused by TGFβ stimulation. F. PHB2 inhibits cell migration.

图5：PHB2对线粒体功能的影响。A.在体内，PHB2对自噬相关蛋白LC3表达的影响。B.在体外，PHB2对自噬相关蛋白LC3表达的影响。C.PHB2对BLM诱导的MLE12肺泡上皮细胞线粒体膜电势的影响。Figure 5: The effect of PHB2 on mitochondrial function. A. In vivo, the effect of PHB2 on the expression of autophagy-related protein LC3. B. In vitro, the effect of PHB2 on the expression of autophagy-related protein LC3. C. The effect of PHB2 on the mitochondrial membrane potential of MLE12 alveolar epithelial cells induced by BLM.

具体实施方式Detailed ways

定义definition

在本发明中，术语“治疗”是指疗法上的以及预防性的措施，其阻止或减缓对象发生不期望的生理学改变或病症，例如肺部纤维化的发生或癌症进展。有利或期望的临床效果包括，但不限于，症状的缓解、疾病程度的降低、疾病状态的稳定化(即不恶化)、疾病进展的延迟或减缓、疾病状态的减轻或缓和以及疾病的部分或全部治愈，而不论上述效果是否可检测到。“治疗”也可指与不治疗相比生存期延长。需要治疗的对象包括已患有该疾病或病症的对象，以及有可能患有该疾病或病症的对象，或要预防该疾病或病症的对象。In the present invention, the term "treatment" refers to therapeutic and preventive measures that prevent or slow down the occurrence of undesirable physiological changes or conditions in a subject, such as the occurrence of pulmonary fibrosis or cancer progression. Favorable or desired clinical effects include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (that is, no deterioration), delay or slowdown of disease progression, alleviation or alleviation of disease state, and partial or partial disease All are cured, regardless of whether the above effects are detectable. "Treatment" can also refer to prolonged survival compared to no treatment. The objects in need of treatment include those who have already suffered from the disease or condition, as well as those who are likely to suffer from the disease or condition, or those who want to prevent the disease or condition.

“对象”或“患者”、“个体”是指任何期望进行诊断、预后或治疗的对象，特别是哺乳动物对象。哺乳动物包括人、家畜、农畜、动物园动物、竞技动物或宠物，例如狗、猫、猪、兔、大鼠、小鼠、马、牛、奶牛等。本文所称的对象优选是人。"Subject" or "patient" or "individual" refers to any subject for which diagnosis, prognosis, or treatment is desired, especially a mammalian subject. Mammals include humans, domestic animals, farm animals, zoo animals, sports animals, or pets, such as dogs, cats, pigs, rabbits, rats, mice, horses, cows, cows, and the like. The object referred to herein is preferably a human.

本文所用的术语“有治疗需要的患者”或“有治疗需要的对象”包括因施用本发明用于例如检测、诊断和/或治疗用途的多肽或其组合物而受益的对象，如哺乳动物对象。As used herein, the term "patient in need of treatment" or "subject in need of treatment" includes subjects who benefit from the administration of the polypeptides or compositions thereof of the present invention for, for example, detection, diagnosis, and/or therapeutic purposes, such as mammalian subjects .

如本文中所使用的，术语“治疗有效量”或“有效量”是指当将本发明的药物或药物组合物单独给予或与另外的治疗剂联合给予细胞、组织或受治疗者时，其有效防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指所述化合物足以导致症状减缓的量，所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态，或提高对所述病征的治疗率、治愈率、防止率或减缓率。当施用给个体单独给予的活性成分时，治疗有效量是指该单独的成分。当施用组合时，治疗有效量是指产生治疗效果的活性成分的联合的量，而不论其是联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10％；通常至少20％；优选至少约30％；更优选至少40％和最优选至少50％。As used herein, the term "therapeutically effective amount" or "effective amount" means that when the drug or pharmaceutical composition of the present invention is administered alone or in combination with another therapeutic agent to a cell, tissue or subject, it Effectively prevent or slow down the amount of the disease or condition to be treated. A therapeutically effective dose further refers to the amount of the compound sufficient to cause alleviation of symptoms, such as treating, curing, preventing or alleviating related medical conditions, or improving the treatment rate, cure rate, prevention rate, or alleviation rate of the symptoms . When administered to an individual administered active ingredient alone, the therapeutically effective amount refers to the individual ingredient. When a combination is administered, the therapeutically effective amount refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether it is administered in combination, continuous or simultaneous. A therapeutically effective amount will reduce symptoms usually by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.

在本发明中，“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，对于生物学***或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的 可接受误差范围内。In the present invention, "about" means that the index value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value partly depends on how it is measured or determined (ie, the limit of the measurement system). For example, in every practice in the art, "about" can mean within one or more than one standard deviation. Alternatively, "about" or "substantially comprising" can mean up to 20% of the range. In addition, for biological systems or processes, the term can mean at most an order of magnitude or at most 5 times the value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "substantially includes" should be assumed to be within the acceptable error range of the specific value.

如本文所用，术语“PHB2”是指抑制素2(prohibitin 2)。PHB2基因位于染色体12p13上。PHB2的氨基酸序列可以从NCBI获得，登录号为NP_001254629.1或NP_001138303.1。编码PHB2蛋白产物的核苷酸序列如SEQ ID NO:4或SEQ ID NO:5所示。PHB2的信使RNA(mRNA)的NCBI序列登陆号为NM_001267700.1(SEQ ID NO:2)或NM_001144831.2(SEQ ID NO:3)。PHB2的DNA序列的NCBI序列登陆号为NC_000012.12(SEQ ID NO:1)，基因ID为11331。PHB2是一种普遍表达的多效蛋白质，在调节线粒体的正常生长发育、核转录调控、细胞增殖和凋亡、细胞周期调节和衰老中是至关重要的。此外，它主要定位于线粒体中，但也存在于细胞质、细胞核和质膜中。有研究表明，还与线粒体内膜中的PHB1形成大的多聚体环状复合物，该复合物是维持线粒体的功能所必需的，其在不同的细胞过程(如细胞周期进程和衰老)、炎症以及许多疾病(如肥胖、糖尿病和癌症等)中都发挥着重要作用。As used herein, the term "PHB2" refers to prohibitin 2 (prohibitin 2). The PHB2 gene is located on chromosome 12p13. The amino acid sequence of PHB2 can be obtained from NCBI, and the accession number is NP_001254629.1 or NP_001138303.1. The nucleotide sequence encoding the PHB2 protein product is shown in SEQ ID NO: 4 or SEQ ID NO: 5. The NCBI sequence accession number of the messenger RNA (mRNA) of PHB2 is NM_001267700.1 (SEQ ID NO: 2) or NM_001144831.2 (SEQ ID NO: 3). The NCBI sequence accession number of the PHB2 DNA sequence is NC_000012.12 (SEQ ID NO:1), and the gene ID is 11331. PHB2 is a ubiquitously expressed pleiotropic protein, which is essential in regulating the normal growth and development of mitochondria, nuclear transcription regulation, cell proliferation and apoptosis, cell cycle regulation and aging. In addition, it is mainly located in the mitochondria, but also exists in the cytoplasm, nucleus and plasma membrane. Studies have shown that it also forms a large multimer ring complex with PHB1 in the inner mitochondrial membrane. This complex is necessary to maintain the function of mitochondria. It is involved in different cellular processes (such as cell cycle progression and aging), Inflammation and many diseases (such as obesity, diabetes and cancer, etc.) play an important role.

本文可互换使用的术语“多核苷酸”和“核酸”是指任何长度的核苷酸(核糖核苷酸或脱氧核糖核苷酸)的聚合形式。这些术语包括单链、双链或三链DNA，基因组DNA、cDNA、基因组RNA、mRNA、DNA-RNA杂交体、或聚合物；该聚合物包含嘌呤和嘧啶碱基，或其他天然的，化学、生物化学修饰的，非天然或衍生的核苷酸碱基。多核苷酸的骨架可包含糖和磷酸基团(通常可在RNA或DNA中发现)，或经修饰或取代的糖或磷酸基团。或者，多核苷酸的主链可以包含合成亚单位的聚合物(例如氨基磷酸酯(phosphoramidate))，并且因此可以是寡聚脱氧核苷氨基磷酸酯(P-NH2)或混合的氨基磷酸酯-磷酸二酯寡聚体。因此，在本发明的一个方面，本发明的药物包含载体及编码PHB2蛋白的多核苷酸，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码PHB2蛋白的DNA或RNA序列。在一些实施方式中，编码PHB2蛋白的DNA序列为SEQ ID NO:4或SEQ ID NO:5所示的核苷酸序列。在一些实施方式中，编码PHB2蛋白的DNA序列为SEQ ID NO:1所示的核苷酸序列。在一些实施方式中，编码PHB2蛋白的RNA序列为SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列。The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to a polymerized form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. These terms include single-stranded, double-stranded or triple-stranded DNA, genomic DNA, cDNA, genomic RNA, mRNA, DNA-RNA hybrids, or polymers; the polymer contains purine and pyrimidine bases, or other natural, chemical, Biochemically modified, non-natural or derived nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (usually found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide may contain polymers of synthetic subunits (e.g. phosphoramidates), and thus may be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidates- Phosphodiester oligomers. Therefore, in one aspect of the present invention, the medicament of the present invention comprises a vector and a polynucleotide encoding a PHB2 protein, wherein the vector contains or carries the polynucleotide encoding the PHB2 protein. In some embodiments, the polynucleotide is a DNA or RNA sequence encoding PHB2 protein. In some embodiments, the DNA sequence encoding the PHB2 protein is the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the DNA sequence encoding the PHB2 protein is the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the RNA sequence encoding the PHB2 protein is the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

病毒载体Viral vector

病毒载体可将遗传物质带入细胞，原理是利用病毒具有传送其基因组进入其他细胞，进行感染的分子机制。病毒载体也可以称为载体、载体病毒粒子或载体粒子。病毒载体的实例包括但不限于：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒或慢病毒。Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism of viruses to transmit their genomes into other cells for infection. Viral vectors can also be referred to as vectors, vector virus particles or vector particles. Examples of viral vectors include, but are not limited to: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, or lentivirus.

逆转录病毒载体可以使衍生自或能够衍生自任何适合的逆转录病毒。大量的不同的逆转录病毒已经被鉴定。例子包括但不限于：鼠白血病病毒(MLV)、人T-细胞白血病病毒(HTLV)、小鼠乳腺肿瘤病毒(MMTV)、劳氏肉瘤病毒(RSV)、Fujinami肉瘤病毒(FuSV)、莫洛尼鼠白血病病毒(Mo MLV)、FBR鼠骨肉瘤病毒(FBR MSV)、莫洛尼鼠肉瘤病毒(Mo-MSV)、Abelson鼠白血病病毒(A-MLV)、禽骨髓细胞瘤病毒-29(MC29)和禽红细胞增多症病毒(AEV)。The retroviral vector can be derived or capable of being derived from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include but are not limited to: murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse breast tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney Murine Leukemia Virus (Mo MLV), FBR Murine Osteosarcoma Virus (FBR MSV), Moloney Murine Sarcoma Virus (Mo-MSV), Abelson Murine Leukemia Virus (A-MLV), Avian Myeloma Virus-29 (MC29) And avian polycythemia virus (AEV).

腺病毒是双链的线性DNA病毒，其不通过RNA中间体复制。腺病毒是双链的DNA无包膜病毒，其能够在体内、离体和体外转导大范围的人和非人来源的细胞类型。这些细胞包括呼吸道气道上皮细胞、肝细胞、肌肉细胞、心肌细胞、滑膜细胞、原代乳腺上皮细胞和有丝***后终末分化的细胞(例如神经元)。腺病毒已被用作用于基因治疗和异源基因表达的载体。大(36kb)基因组可以容纳高达8kb的外源***DNA，并且能够在互补细胞系中有效地复制以产生每毫升高达1012个转导单位的非常高的效价。腺病毒因此是研究原代非复制细胞中基因表达的最佳***之一。来自腺病毒基因组的病毒基因或外源基因的表达不需要复制细胞。腺病毒载体通过受体介导的内吞作用进入细胞。一旦进入细胞内，腺病毒载体很少整合到宿主染色体中。相反，它们作为附加体(独立于宿主基因组)存在，作为宿主细胞核中的线性基因组。Adenoviruses are double-stranded linear DNA viruses that do not replicate through RNA intermediates. Adenovirus is a double-stranded DNA non-enveloped virus that can transduce a wide range of human and non-human cell types in vivo, in vitro and in vitro. These cells include airway epithelial cells, hepatocytes, muscle cells, cardiomyocytes, synovial cells, primary breast epithelial cells, and terminally differentiated cells (such as neurons) after mitosis. Adenovirus has been used as a vector for gene therapy and heterologous gene expression. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and can replicate efficiently in complementary cell lines to produce very high titers of up to 1012 transduction units per milliliter. Adenovirus is therefore one of the best systems for studying gene expression in primary non-replicating cells. The expression of viral genes or foreign genes from the adenoviral genome does not require replicating cells. Adenovirus vectors enter cells through receptor-mediated endocytosis. Once inside the cell, the adenovirus vector rarely integrates into the host chromosome. Instead, they exist as episomes (independent of the host genome) as a linear genome in the host cell nucleus.

腺相关病毒(adeno-associated virus，AAV)，也称腺伴随病毒，属于微小病毒科依赖病毒属，是目前发现的一类结构最简单的单链DNA缺陷型病毒。重组的AAV载体已成功地被用于标记基因和涉及人类疾病的基因的体外、离体和体内的转导。已经开发了某些AAV载体，其可以有效地结合大的有效载荷(高达8-9kb)。Adeno-associated virus (adeno-associated virus, AAV), also known as adeno-associated virus, belongs to the genus of dependent viruses in the Parvoviridae family, and is the simplest type of single-stranded DNA-deficient virus found so far. Recombinant AAV vectors have been successfully used for the transduction of marker genes and genes involved in human diseases in vitro, in vitro and in vivo. Certain AAV vectors have been developed that can effectively bind large payloads (up to 8-9 kb).

单纯性疱疹病毒(HSV)是包膜的双链DNA病毒，其天然地感染神经元。它可以容纳外源DNA的大区段，并且已经被采用为用于对神经元的基因递送的载体。在治疗过程中HSV的使用需要使毒株减毒，从而它们不能建立裂解周期。特别地，如果HSV载体用于人类的基因治疗，则优选地将多核苷酸***必需基因 中。这是因为如果病毒载体遭遇野生型病毒，则可以通过重组将异源基因转移至野生型病毒。然而，如果以防止其复制的方式构建重组病毒，则这可以通过将寡核苷酸***对复制必需的病毒基因来实现。Herpes simplex virus (HSV) is an enveloped double-stranded DNA virus that naturally infects neurons. It can accommodate large segments of foreign DNA and has been adopted as a carrier for gene delivery to neurons. The use of HSV during treatment requires attenuating the strains so that they cannot establish a lytic cycle. In particular, if the HSV vector is used for gene therapy in humans, it is preferable to insert the polynucleotide into the essential gene. This is because if the viral vector encounters a wild-type virus, the heterologous gene can be transferred to the wild-type virus by recombination. However, if the recombinant virus is constructed in a way that prevents its replication, this can be achieved by inserting oligonucleotides into viral genes necessary for replication.

本发明的病毒载体可以是牛痘病毒载体，例如MVA或NYVAC。牛痘载体的替代物包括例如称为ALVAC的鸡痘或金丝雀痘的禽痘(avipox)载体，以及由其衍生的毒株，该毒株可以在人类细胞中感染和表达重组蛋白但不能复制。应当理解的是，在重组基因的***之后病毒基因组的部分可以保持完整。这意味着病毒载体可以保留感染细胞并随后表达额外的基因的能力的概念，该额外的基因支持其复制并可能促进被感染细胞的裂解和死亡。The viral vector of the present invention may be a vaccinia virus vector, such as MVA or NYVAC. Alternatives to vaccinia vectors include, for example, fowlpox or canarypox (avipox) vectors called ALVAC, and strains derived therefrom, which can infect and express recombinant proteins in human cells but cannot replicate . It should be understood that part of the viral genome can remain intact after the insertion of the recombinant gene. This means that viral vectors can retain the concept of the ability to infect cells and subsequently express additional genes that support their replication and may promote the lysis and death of infected cells.

慢病毒是更大群体的逆转录病毒的一部分。可以分为灵长类动物和非灵长类动物群体。灵长类慢病毒的例子包括但不限于：人免疫缺陷病毒(HIV)、人自身免疫缺陷综合症(AIDS)的病原体以及猿猴免疫缺陷病毒(SIV)。非灵长类动物慢病毒群体包括原型“慢病毒”visna/maedi病毒(VMV)，以及相关的山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)和牛免疫缺陷病毒(BIV)。Lentiviruses are part of a larger group of retroviruses. Can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to: human immunodeficiency virus (HIV), the pathogen of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The group of non-primate lentiviruses includes the prototype "lentivirus" visna/maedi virus (VMV), as well as related goat arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and feline immunodeficiency virus ( FIV) and Bovine Immunodeficiency Virus (BIV).

非病毒载体Non-viral vector

非病毒载体是利用非病毒的载体材料的物化性质来介导基因的转移。任何合适的非病毒载体可被用于将PHB2基因引入对象的细胞中。非病毒载体的例子包括但不限于，质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。Non-viral vectors use the physical and chemical properties of non-viral carrier materials to mediate gene transfer. Any suitable non-viral vector can be used to introduce the PHB2 gene into the cell of the subject. Examples of non-viral vectors include, but are not limited to, plasmids, liposomes, reverse transcription elements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes.

质粒是小型环状DNA分子，在基因工程中作为最常用，最简单的载体，必须包括三部分：遗传标记基因，复制区，目的基因。质粒在所有的细菌类群中都可发现，它们是独立于细菌染色体外自我复制的DNA分子。质粒载体的例子包括但不限于：大肠杆菌质粒载体、枯草杆菌质粒载体、酵母质粒载体、农杆菌质粒载体和蓝细菌质粒载体。Plasmid is a small circular DNA molecule. As the most commonly used and simplest vector in genetic engineering, it must include three parts: genetic marker gene, replication region, and target gene. Plasmids can be found in all bacterial groups. They are DNA molecules that replicate themselves independently of the bacteria's chromosomes. Examples of plasmid vectors include, but are not limited to: Escherichia coli plasmid vectors, Bacillus subtilis plasmid vectors, yeast plasmid vectors, Agrobacterium plasmid vectors, and cyanobacteria plasmid vectors.

脂质体由卵磷脂和神经酰胺等制得的脂质体(空心)，具有的双分子层结构，是一种人工膜。脂质体的组成通常是磷脂(特别是高相转变温度的磷脂)的组合，通常与类固醇(尤其是胆固醇)组合。也可以使用其他磷脂或其他脂质。脂质体的物理特性取决于pH、离子强度和二价阳离子的存在。通过在转导期间通过使用 二油酰磷脂酰乙醇胺可以增加脂质体的转导效率。高效脂质体可商购获得。脂质体的例子包括但不限于：中性脂质体、负电荷脂质体和正电荷脂质体。Liposome Liposome (hollow) made from lecithin and ceramide, etc., has a bilayer structure and is an artificial membrane. The composition of liposomes is usually a combination of phospholipids (especially high phase transition temperature phospholipids), usually in combination with steroids (especially cholesterol). Other phospholipids or other lipids can also be used. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations. The transduction efficiency of liposomes can be increased by using dioleoylphosphatidylethanolamine during transduction. High-efficiency liposomes are commercially available. Examples of liposomes include, but are not limited to: neutral liposomes, negatively charged liposomes, and positively charged liposomes.

附加型载体是一种允许和帮助水溶性物质渗入并穿过细胞膜的脂质层而转移到细胞中去的物质。它是膜上的一种蛋白质，调节着离子主动吸收的一种成分。附加型载体可以包括一种或多种多能性基因，其可操作地连接至少一个用于表达因子的调控序列。本发明的附加型载体还可包括允许该载体在细胞中复制的组分。例如，Epstein Barr oriP/核抗原-1(EBNA-1)组合可以支持哺乳动物细胞(特别是灵长类动物细胞)中载体的自我复制。来自于EBV基因组的EBNA1反式元件和OriP顺式元件使得简单质粒能够在增殖的人类细胞中作为附加体进行复制和维持。Additive carrier is a substance that allows and helps water-soluble substances to penetrate and pass through the lipid layer of the cell membrane to be transferred to the cell. It is a kind of protein on the membrane, a component that regulates the active absorption of ions. The episomal vector may include one or more pluripotency genes, which are operably linked to at least one regulatory sequence for expression of the factor. The episomal vector of the present invention may also include components that allow the vector to replicate in cells. For example, the Epstein Barr oriP/nuclear antigen-1 (EBNA-1) combination can support the self-replication of the vector in mammalian cells (especially primate cells). The EBNA1 trans-elements and OriP cis-elements derived from the EBV genome enable simple plasmids to replicate and maintain as episomes in proliferating human cells.

用于基因转移的阳离子多聚物包括但不限于：多聚赖氨酸、聚乙烯亚胺和树突状聚合物。Cationic polymers used for gene transfer include, but are not limited to: polylysine, polyethyleneimine, and dendritic polymers.

壳聚糖作为一种天然阳离子聚合物，通过与DNA以静电方式作用使壳聚糖-DNA体系不被降解，完全进入细胞。作为基因载体，壳聚糖具有细胞毒性低、生物相容性好、基因免疫性低和转染效率较高等特点。壳聚糖-DNA复合物按制备方法主要分为壳聚糖及其衍生物的DNA复合物、壳聚糖-DNA纳米微球和壳聚糖自聚体-DNA。As a natural cationic polymer, chitosan interacts with DNA in an electrostatic manner to prevent the chitosan-DNA system from being degraded and completely enter the cell. As a gene carrier, chitosan has the characteristics of low cytotoxicity, good biocompatibility, low gene immunity and high transfection efficiency. Chitosan-DNA complexes are mainly divided into chitosan and its derivatives DNA complexes, chitosan-DNA nanospheres and chitosan self-polymer-DNA according to the preparation method.

无机纳米粒子主要通过穿过细胞膜将药物或生物分子转运到生物体中而起到治疗疾病的作用。应用于基因转运的无机纳米粒子的例子包括但不限于硅、铁氧化物、碳纳米管、磷酸钙、金属纳米粒子、量子点等。Inorganic nanoparticles play a role in treating diseases by transporting drugs or biomolecules into organisms through cell membranes. Examples of inorganic nanoparticles used in gene delivery include, but are not limited to, silicon, iron oxide, carbon nanotubes, calcium phosphate, metal nanoparticles, quantum dots, and the like.

药物组合物Pharmaceutical composition

本发明的一个方面提供包含含有载体及编码PHB2蛋白的多核苷酸的药物以及药学上可接受的赋形剂的药物组合物，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。该药物组合物可用于治疗对象的肺纤维化。One aspect of the present invention provides a pharmaceutical composition comprising a medicament containing a carrier and a polynucleotide encoding a PHB2 protein and a pharmaceutically acceptable excipient, wherein the carrier contains or carries the polynucleotide encoding the PHB2 protein. The pharmaceutical composition can be used to treat pulmonary fibrosis in a subject.

药学上可接受的赋形剂是本领域熟知的。如本文所用，“药学上可接受的赋形剂”包括当与组合物的活性成分组合时允许该成分保持生物活性并且不会引起与对象的免疫***的破坏性反应的材料。这些可以包括稳定剂、防腐剂、盐或糖配合物或晶体等。Pharmaceutically acceptable excipients are well known in the art. As used herein, "pharmaceutically acceptable excipients" include materials that when combined with the active ingredients of the composition allow the ingredients to maintain biological activity and do not cause a destructive reaction with the subject's immune system. These may include stabilizers, preservatives, salts or sugar complexes or crystals and the like.

本文使用的术语“载体”包括任何或所有的溶剂、分散介质、载体、包衣、稀释剂、抗菌剂、抗真菌剂、等渗剂、吸附延缓剂、缓冲液、载体溶液、悬浮液、 乳液、胶体等。实施例包括但不限于标准药物赋形剂，例如磷酸盐缓冲盐水溶液、水、乳液(例如油/水乳液)、以及各种类型的润湿剂。非水溶剂的实施例包括但不限于丙二醇、聚乙二醇、植物油(例如橄榄油)、以及可注射的有机酯(例如油酸乙酯)。水性载体包括水、醇/水溶液、乳液或悬浮液，包括盐水和缓冲介质。肠胃外载体包括氯化钠溶液、林格氏葡萄糖、右旋糖和氯化钠、乳酸林格氏液或固定油。静脉内载体包括液体和营养补充剂、电解质补充剂(例如基于林格氏葡萄糖的)等。在其他实施方式中，组合物将被结合进固体基质中，包括缓释颗粒、玻璃珠、绷带、眼睛上的***物、以及局部形式。这些用于药物活性成分的介质和试剂的使用在本领域是已知的。除非已知任何常规介质或试剂不能与该活性成分配伍，否则本发明预期可在组合物中使用任何上述载体。The term "carrier" as used herein includes any or all solvents, dispersion media, carriers, coatings, diluents, antibacterial agents, antifungal agents, isotonic agents, adsorption delaying agents, buffers, carrier solutions, suspensions, emulsions , Colloid, etc. Examples include, but are not limited to, standard pharmaceutical excipients, such as phosphate buffered saline, water, emulsions (e.g., oil/water emulsions), and various types of wetting agents. Examples of non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (for example, based on Ringer's dextrose), and the like. In other embodiments, the composition will be incorporated into a solid matrix, including sustained release particles, glass beads, bandages, inserts on the eye, and topical forms. The use of these media and agents for pharmaceutical active ingredients is known in the art. Unless any conventional medium or agent is known to be incompatible with the active ingredient, the present invention contemplates that any of the above-mentioned carriers can be used in the composition.

“药学上可接受的”是指当施用至人体时不会产生过敏反应或类似的不期望的反应的分子和成分。本领域已知如何制备包含作为活性组分的蛋白的水性组合物。通常，这些组合物被制备成注射剂，例如液态溶液或悬浮液；也可以制备成适于在注射之前配制成溶液或悬浮液的固体形式。"Pharmaceutically acceptable" refers to molecules and ingredients that do not produce allergic reactions or similar undesirable reactions when administered to the human body. It is known in the art how to prepare an aqueous composition containing protein as an active ingredient. Generally, these compositions are prepared as injections, such as liquid solutions or suspensions; they can also be prepared in solid forms suitable for formulating solutions or suspensions prior to injection.

方法和治疗Methods and treatment

本发明的一个方面提供一种治疗对象的肺纤维化的方法，该方法包括向有此需要的对象施用治疗有效量的本发明的药物，其中该药物包含载体及编码PHB2蛋白的多核苷酸，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。进一步地，本发明提供了本发明的药物在治疗或缓解肺纤维化或与肺纤维化有关的症状的方法中的应用，其中该药物包含载体及编码PHB2蛋白的多核苷酸，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。One aspect of the present invention provides a method for treating pulmonary fibrosis in a subject, the method comprising administering a therapeutically effective amount of the medicament of the present invention to a subject in need thereof, wherein the medicament comprises a carrier and a polynucleotide encoding a PHB2 protein, Wherein the vector contains or carries the polynucleotide encoding the PHB2 protein. Further, the present invention provides the application of the medicament of the present invention in a method for treating or alleviating pulmonary fibrosis or symptoms related to pulmonary fibrosis, wherein the medicament comprises a carrier and a polynucleotide encoding a PHB2 protein, wherein the carrier Comprising or carrying the polynucleotide encoding the PHB2 protein.

本发明的另一个方面提供一种治疗对象的肺纤维化的方法，该方法包括向有需要的对象施用治疗有效量的本发明的药物组合物，其中该药物组合物包含含有载体及编码PHB2蛋白的多核苷酸的药物和药学上可接受的赋形剂，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。进一步地，本发明提供了本发明的药物组合物在治疗或缓解肺纤维化或与肺纤维化有关的症状的方法中的应用，其中该药物组合物包含含有载体及编码PHB2蛋白的多核苷酸的药物和药学上可接受的赋形剂，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。Another aspect of the present invention provides a method for treating pulmonary fibrosis in a subject, the method comprising administering to the subject in need a therapeutically effective amount of the pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises a carrier and a protein encoding PHB2 And a pharmaceutically acceptable excipient for the polynucleotide, wherein the carrier contains or carries the polynucleotide encoding the PHB2 protein. Further, the present invention provides the use of the pharmaceutical composition of the present invention in a method for treating or alleviating pulmonary fibrosis or symptoms related to pulmonary fibrosis, wherein the pharmaceutical composition comprises a polynucleotide containing a carrier and a PHB2 protein And a pharmaceutically acceptable excipient, wherein the carrier contains or carries the polynucleotide encoding the PHB2 protein.

合适的给药途径包括胃肠外给药(例如肌内、静脉内或皮下给药)及口服给 药。可按多种常规方式给予本发明方法的药物或药物组合物，这些方法例如有经气管插管给予、经口摄取、吸入、局部施用或经皮肤、皮下、腹膜内、胃肠外、动脉内或静脉内注射。此外，人们可以在靶向药物递送***中给予本发明的药物或药物组合物。在一个实施方式中，经气管插管给予本发明的药物或药物组合物。Suitable routes of administration include parenteral administration (e.g. intramuscular, intravenous or subcutaneous administration) and oral administration. The drugs or pharmaceutical compositions of the method of the present invention can be administered in a variety of conventional ways, such as transtracheal intubation, oral ingestion, inhalation, topical application, or transdermal, subcutaneous, intraperitoneal, parenteral, and intraarterial administration. Or intravenous injection. In addition, one can administer the drug or pharmaceutical composition of the present invention in a targeted drug delivery system. In one embodiment, the medicament or pharmaceutical composition of the present invention is administered via tracheal intubation.

由临床医生例如用本领域已知或怀疑影响治疗或预期影响治疗的参数或因子来测定合适的剂量。通常，开始剂量比最佳剂量稍低，此后少量增加直到达到相对于任何不良副作用所要的或最佳的作用效果。重要的诊断测量包括测量例如炎性症状或所产生的炎性细胞因子的水平。The appropriate dosage is determined by the clinician, for example, using parameters or factors known or suspected to affect the treatment or expected to affect the treatment in the art. Generally, the starting dose is slightly lower than the optimal dose, and thereafter a small increase until the desired or optimal effect is achieved relative to any adverse side effects. Important diagnostic measures include measuring, for example, inflammatory symptoms or the level of inflammatory cytokines produced.

可通过连续给药或通过以一定间隔(例如一天、一周或每周1-7次)给药来施用本发明的药物或药物组合物。可通过气管插管、静脉内、皮下、腹膜内、经皮肤、局部、经口、经鼻、经直肠、肌内、大脑内或脊柱内来提供剂量。优选剂量方案是包括避免显著的不合乎需要的副作用的最大剂量或给药频率的方案。The medicament or pharmaceutical composition of the present invention can be administered by continuous administration or by administration at certain intervals (for example, one day, one week, or 1-7 times a week). The dose can be provided by tracheal intubation, intravenous, subcutaneous, intraperitoneal, transdermal, topical, oral, transnasal, transrectal, intramuscular, intracerebral, or intraspine. A preferred dosage regimen is a regimen that includes the maximum dosage or dosing frequency that avoids significant undesirable side effects.

在一些实施方式中，本发明的治疗肺纤维化的方法阻止了肺纤维化的进展或由肺纤维化导致的疾病的发作。因此，在一些实施方式中，本发明提供一种阻止肺纤维化的进展和/或由肺纤维化导致的疾病的发作的方法，该方法包括向有此需要的对象施用有效量的本发明的药物或药物组合物。在一些实施方式中，这些治疗肺纤维化的方法阻止了与肺纤维化有关的症状的发作、进展和/或复发。因此，在一些实施方式中，本发明提供了治疗与肺纤维化有关的症状的方法，该方法包括向有此需要的对象施用有效量的本发明的药物或药物组合物。In some embodiments, the method for treating pulmonary fibrosis of the present invention prevents the progression of pulmonary fibrosis or the onset of diseases caused by pulmonary fibrosis. Therefore, in some embodiments, the present invention provides a method for preventing the progression of pulmonary fibrosis and/or the onset of diseases caused by pulmonary fibrosis, the method comprising administering to a subject in need thereof an effective amount of the present invention Medicine or pharmaceutical composition. In some embodiments, these methods of treating pulmonary fibrosis prevent the onset, progression, and/or recurrence of symptoms associated with pulmonary fibrosis. Therefore, in some embodiments, the present invention provides a method of treating symptoms associated with pulmonary fibrosis, the method comprising administering an effective amount of the medicament or pharmaceutical composition of the present invention to a subject in need thereof.

实施例1 PHB2在肺纤维化模型中的表达Example 1 Expression of PHB2 in a pulmonary fibrosis model

Western blotting测蛋白Western blotting protein

用RIPA裂解液从细胞/肺组织中提取蛋白，BCA法检测蛋白含量，取待测的样品蛋白20-50含量进行SDS-PAGE胶电泳，100V恒压转移2h后5％脱脂奶粉室温封闭1h，将膜与一抗(α-SMA、actin、FLAG、Fibronectin、Collagen Ⅰ、PHB1、PHB2、perk、erk、pp38、p38、p-akt和akt)室温孵育1h或4℃过夜；TBST缓冲液洗膜5次，每次5分钟，加入二抗室温孵育1h，再次洗膜5次，加入发光液，凝胶成像***拍照分析。Extract protein from cell/lung tissue with RIPA lysate, detect protein content by BCA method, take 20-50 protein content of sample to be tested for SDS-PAGE gel electrophoresis, transfer at 100V constant pressure for 2h, then 5% skimmed milk powder is sealed at room temperature for 1h. Incubate the membrane with primary antibodies (α-SMA, actin, FLAG, Fibronectin, Collagen I, PHB1, PHB2, perk, erk, pp38, p38, p-akt and akt) at room temperature for 1 hour or overnight at 4°C; wash the membrane with TBST buffer 5 times, 5 minutes each time, add the secondary antibody and incubate at room temperature for 1 hour, wash the membrane again 5 times, add luminescent solution, and take photos with the gel imaging system for analysis.

qPCR测基因表达qPCR measurement of gene expression

TRIzol法提取RNA，测浓度后每组吸出2μg进行反转录，根据试剂盒要求 加入20μl的反应体系进行qPCR。RNA was extracted by TRIzol method, and after measuring the concentration, 2μg of each group was aspirated for reverse transcription, and 20μl of reaction system was added for qPCR according to the requirements of the kit.

纤维化因子和PHB在纤维化病人和健康供体肺组织中的表达The expression of fibrotic factors and PHB in lung tissues of fibrotic patients and healthy donors

利用western blotting和qPCR法检测肺纤维化病人(8例)和健康供体(6例)肺组织中Fibronectin、Collagen Ⅰ、α-SMA、以及PHB(PHB1和PHB2)的表达情况。Western blotting and qPCR were used to detect the expression of Fibronectin, Collagen I, α-SMA, and PHB (PHB1 and PHB2) in lung tissues of pulmonary fibrosis patients (8 cases) and healthy donors (6 cases).

大鼠和小鼠的肺纤维化模型Pulmonary fibrosis models in rats and mice

为了观察PHB在动物模型中的表达变化，我们利用BLM诱导了SD大鼠和C57小鼠的肺纤维化模型。In order to observe the changes in PHB expression in animal models, we used BLM to induce lung fibrosis models in SD rats and C57 mice.

提取肺组织中的成纤维细胞Extract fibroblasts from lung tissue

收集肺组织，将组织块剪碎，胶原酶消化过夜。第二天将细胞悬液分别经70μm和40μm细胞滤网，制备单细胞悬液，离心后，将细胞悬浮于含10％FBS的DMEM培养基中，传至第5代左右，开始进行下一步实验。Collect the lung tissues, cut the tissue pieces into small pieces, and digest them with collagenase overnight. On the second day, the cell suspension was passed through a 70μm and 40μm cell strainer to prepare a single cell suspension. After centrifugation, the cells were suspended in DMEM medium containing 10% FBS and passed to the fifth generation or so, and then proceed to the next step. experiment.

纤维化因子和PHB在成纤维细胞中的表达The expression of fibrosis factor and PHB in fibroblasts

用TGFβ刺激获得的人胚肺成纤维细胞，分别于24h、48h和72h利用western blotting和qPCR法检测Fibronectin、Collagen Ⅰ、α-SMA、以及PHB的表达情况。The human embryonic lung fibroblasts obtained by stimulation with TGFβ were used to detect the expression of Fibronectin, Collagen I, α-SMA, and PHB at 24h, 48h and 72h by western blotting and qPCR methods.

结果：与健康供体相比，肺纤维化病人的肺组织中，Fibronectin、Collagen Ⅰ和α-SMAd的表达显著上调(图1A)，而PHB(PHB1和PHB2)的表达显著下调(图1B)。为了确定PHB在体内的表达情况，利用BLM诱导了SD大鼠和C57小鼠的肺纤维化模型。如图1C所示，从小鼠造模后的第一周开始，PHB1和PHB2开始下调，抑制一直持续到第四周。如图1D所示，大鼠造模后的第21天，PHB2的表达显著下调，且与BLM的浓度具有剂量依赖性。为了明确PHB2在纤维化状态下的变化趋势，体外利用TGFβ刺激成纤维细胞。如图1E所示，TGFβ刺激48h后，Fibronectin、Collagen Ⅰ、以及α-SMA的表达均有显著上调，且这种作用一直持续到72h，而PHB2经TGFβ刺激后48h显著下调。这些数据表明，PHB2在纤维化状态下表达下调。Results: Compared with healthy donors, the expression of Fibronectin, Collagen I and α-SMAd was significantly up-regulated in lung tissues of patients with pulmonary fibrosis (Figure 1A), while the expression of PHB (PHB1 and PHB2) was significantly down-regulated (Figure 1B) . In order to determine the expression of PHB in vivo, BLM was used to induce pulmonary fibrosis models in SD rats and C57 mice. As shown in Figure 1C, from the first week after the mice were modeled, PHB1 and PHB2 began to be down-regulated, and the inhibition continued until the fourth week. As shown in Figure 1D, on the 21st day after the rats were modeled, the expression of PHB2 was significantly down-regulated, and it was dose-dependent with the concentration of BLM. In order to clarify the change trend of PHB2 in the fibrotic state, TGFβ was used to stimulate fibroblasts in vitro. As shown in Figure 1E, after TGFβ stimulation for 48h, the expressions of Fibronectin, Collagen I, and α-SMA were significantly up-regulated, and this effect continued until 72h, while PHB2 was significantly down-regulated 48h after TGFβ stimulation. These data indicate that PHB2 is down-regulated in the fibrotic state.

实施例2 PHB2在小鼠体内的抗纤维化作用Example 2 Anti-fibrosis effect of PHB2 in mice

建模Modeling

构建PHB2腺相关病毒AAV-PHB2，每只C57小鼠经气管插管给予2*10 ¹¹个腺相关病毒AAV-PHB2，对照组给予同等剂量的GFP对照病毒。感染后2周，每 只小鼠经气管插管给予BLM造模。第6周收取肺组织进行western blotting、qPCR、HE染色、天狼星红染色。 The PHB2 adeno-associated virus AAV-PHB2 was constructed. Each C57 mouse was given 2*10 ¹¹ adeno-associated virus AAV-PHB2 through tracheal intubation, and the control group was given the same dose of GFP control virus. Two weeks after infection, each mouse was intubated with BLM for modeling. In the 6th week, the lung tissues were collected for western blotting, qPCR, HE staining, and Sirius red staining.

羟脯氨酸含量测定Hydroxyproline content determination

羟脯氨酸在胶原蛋白中占13.4％，在弹性蛋白中占极少量，其它蛋白中均不存在，因此羟脯氨酸的含量能反映肺组织的胶原代谢情况。取于-70℃冰箱冻存的肺组织样本，按南京建成生物工程研究所羟脯氨酸测试盒说明书操作。羟脯氨酸含量结果以μg/g表示。Hydroxyproline occupies 13.4% of collagen, a very small amount in elastin, and does not exist in other proteins. Therefore, the content of hydroxyproline can reflect the collagen metabolism of lung tissue. Take the lung tissue samples frozen in the refrigerator at -70℃, and operate according to the instructions of the hydroxyproline test kit of Nanjing Jiancheng Institute of Bioengineering. The results of hydroxyproline content are expressed in μg/g.

HE染色HE staining

10％中性甲醛固定液固定24h后的肺组织，做抽气处理，去除肺内的空气之后，从肺尖至肺底每隔3mm将肺脏横切成6-12块薄片，10％中性甲醛继续固定72h，乙醇梯度脱水，石蜡定向包埋，连续切片，厚度6μm，选择每块标本的连续切片中第10片，用二甲苯脱去切片中的石蜡，经由高浓度到低浓度酒精后入蒸馏水；放入苏木***溶液中染色数分钟，酸水及氨水中分色，流水冲洗1h后入蒸馏水片刻，入70％和90％酒精中脱水各10分钟，入酒精伊红染色液染色2-3分钟；染色后的切片经纯酒精脱水，再经二甲苯使切片透明，滴上树胶，盖上盖玻片封固；待树胶略干后，进行定性定量观察：每张切片观察5个视野，即切片上下左右四个固定视野和中部一个视野。The lung tissue after 24h fixed with 10% neutral formaldehyde fixative solution is used to extract air. After removing the air in the lungs, the lungs are cut into 6-12 slices every 3mm from the tip of the lung to the bottom of the lung, 10% neutral Formaldehyde continues to be fixed for 72 hours, ethanol gradient dehydration, paraffin embedding, serial sectioning, thickness 6μm, select the 10th piece of the serial section of each specimen, use xylene to remove the paraffin in the section, after passing high concentration to low concentration alcohol Put in distilled water; dye in hematoxylin aqueous solution for a few minutes, separate colors in acid water and ammonia water, rinse with running water for 1h, put in distilled water for a while, put in 70% and 90% alcohol for dehydration for 10 minutes each, put in alcohol and eosin dyeing solution for dyeing 2-3 minutes; the stained sections are dehydrated with pure alcohol, and then xylene is used to make the sections transparent, drip with gum, and cover with a cover glass for sealing; after the gum is slightly dry, perform qualitative and quantitative observations: Observe 5 for each section One field of view, that is, four fixed fields of view up, down, left, and right, and one field of view in the middle of the slice.

结果：与对照组(GFP)相比，过表达PHB2组的羟脯氨酸含量、Fibronectin和Collagen表达量显著降低(图2A-2B)，肺组织中的纤维化程度明显减弱(图2C-2E)。这些数据表明，PHB2在肺纤维化中能够起到抗纤维化的作用。Results: Compared with the control group (GFP), the hydroxyproline content, Fibronectin and Collagen expression in the PHB2 overexpression group were significantly reduced (Figure 2A-2B), and the degree of fibrosis in the lung tissue was significantly weakened (Figure 2C-2E) ). These data indicate that PHB2 can play an anti-fibrosis effect in pulmonary fibrosis.

实施例3 PHB2对TGFβ诱导的成纤维细胞中纤维化因子及非经典通路的影响构建稳转细胞Example 3 The influence of PHB2 on fibrosis factors and non-classical pathways in fibroblasts induced by TGFβ. Construction of stable transfected cells

构建过表达PHB2的慢病毒LV-PHB2，感染原代成纤维细胞HFL1，对照组感染GFP对照病毒。在感染后的第4天，利用嘌呤霉素筛选出稳转细胞。The lentivirus LV-PHB2 overexpressing PHB2 was constructed to infect primary fibroblasts HFL1, and the control group was infected with GFP control virus. On the 4th day after infection, stable transfected cells were selected by puromycin.

检测PHB2对纤维化因子表达的影响Detect the effect of PHB2 on the expression of fibrotic factors

分别将稳定表达PHB2的细胞和对照组细胞接种于6孔板，密度约为90％时，饥饿24h，TGFβ刺激48h。收集细胞，利用western blotting法检测Fibronectin、Collagen Ⅰ和α-SMA的表达情况。Cells stably expressing PHB2 and control cells were respectively seeded on 6-well plates at a density of about 90%, starved for 24 hours, and stimulated with TGFβ for 48 hours. Collect the cells and detect the expression of Fibronectin, Collagen I and α-SMA by western blotting method.

分别提取健康供体肺组织和纤维化肺组织的原代成纤维细胞，然后过表达 PHB2。收集细胞，利用western blotting和qPCR法检测Fibronectin、Collagen Ⅰ和α-SMA的表达情况。The primary fibroblasts from healthy donor lung tissue and fibrotic lung tissue were extracted, and then PHB2 was overexpressed. The cells were collected, and the expressions of Fibronectin, Collagen I and α-SMA were detected by western blotting and qPCR methods.

检测PHB2对TGFβ通路的影响Detect the influence of PHB2 on the TGFβ pathway

分别将稳定表达PHB2的细胞和对照组细胞接种于6孔板，密度约为90％时，饥饿24h，TGFβ刺激1h。收集细胞，利用western blotting法检测AKT、p38、ERK的磷酸化。Cells stably expressing PHB2 and control cells were respectively inoculated into 6-well plates at a density of about 90%, starved for 24 hours, and stimulated by TGFβ for 1 hour. Collect the cells and detect the phosphorylation of AKT, p38 and ERK by western blotting.

结果：经慢病毒介导的PHB2在HFL1细胞中过表达以后，利用TGFβ诱导48h，发现纤维化因子Fibronectin、Collagen Ⅰ、和α-SMA的表达量与未过表达组相比显著下调(图3A)。经慢病毒介导的PHB2在HFL1细胞中过表达以后，利用TGFβ诱导1h，发现pakt、p38、perk的表达量与未过表达组相比显著下调(图3B)，说明PHB2能够抑制TGFβ激活的非经典通路。在健康供体肺组织和纤维化肺组织的原代成纤维细胞中过表达PHB2，发现Fibronectin、Collagen Ⅰ、和α-SMA的表达量与未过表达组相比显著下调(图3C)，说明过表达PHB2能够显著抑制纤维化因子的表达。Results: After the lentivirus-mediated PHB2 was overexpressed in HFL1 cells, TGFβ was used to induce 48h, and it was found that the expression levels of fibrotic factors Fibronectin, Collagen I, and α-SMA were significantly down-regulated compared with the non-overexpression group (Figure 3A). ). After the lentivirus-mediated PHB2 was overexpressed in HFL1 cells, TGFβ was used to induce 1h, and it was found that the expression of pakt, p38, and perk was significantly down-regulated compared with the non-overexpression group (Figure 3B), indicating that PHB2 can inhibit the activation of TGFβ Non-classical access. PHB2 was overexpressed in the primary fibroblasts of healthy donor lung tissue and fibrotic lung tissue, and it was found that the expression of Fibronectin, Collagen I, and α-SMA was significantly down-regulated compared with the non-overexpression group (Figure 3C), indicating Overexpression of PHB2 can significantly inhibit the expression of fibrotic factors.

实施例4 PHB2对细胞功能的影响Example 4 The influence of PHB2 on cell function

CCK8法测细胞存活率CCK8 method to measure cell viability

构建PHB2慢病毒，感染MLE12肺泡上皮细胞，对照组感染GFP对照病毒。于感染后的第4天，嘌呤霉素筛选出稳转细胞。The PHB2 lentivirus was constructed to infect MLE12 alveolar epithelial cells, and the control group was infected with GFP control virus. On the 4th day after infection, puromycin selected stable transfected cells.

分别将稳定表达PHB2的MLE12肺泡上皮细胞和对照组细胞接种于96孔板，密度约为80％时，100μg/ml的BLM作用24h，每孔加入100μl不含血清的DMEM培养基及10μl的CCK8，2h后450nm测吸光度，计算细胞存活率。MLE12 alveolar epithelial cells stably expressing PHB2 and control cells were respectively inoculated into 96-well plates, at a density of about 80%, 100μg/ml BLM was used for 24h, and 100μl serum-free DMEM medium and 10μl CCK8 were added to each well. Measure the absorbance at 450nm after 2h, and calculate the cell viability.

流式细胞术检测细胞凋亡Flow cytometry to detect apoptosis

分别将稳定表达PHB2的MLE12肺泡上皮细胞和对照组细胞接种于12孔板，密度约为80％时，10μg/ml的BLM作用48h，收取细胞加入Annexin-FITC/PI双染试剂进行流式细胞术测细胞凋亡。MLE12 alveolar epithelial cells stably expressing PHB2 and control cells were respectively inoculated into a 12-well plate at a density of about 80%, 10μg/ml BLM was used for 48h, and the collected cells were added with Annexin-FITC/PI double staining reagent for flow cytometry Surgical measurement of cell apoptosis.

分别将稳定表达PHB2的HFL1原代成纤维细胞和对照组细胞接种于6孔板，密度约为90％时，饥饿24h，TGFβ刺激48h后收集细胞。PBS洗两遍，加入Annexin-FITC/PI双染试剂，15分钟后上样，流式细胞仪测细胞凋亡率。HFL1 primary fibroblasts stably expressing PHB2 and control cells were respectively seeded in 6-well plates at a density of about 90%, starved for 24h, and TGFβ stimulated for 48h and harvested the cells. Wash twice with PBS, add Annexin-FITC/PI double staining reagent, load the sample 15 minutes later, and measure the apoptosis rate by flow cytometry.

划痕实验Scratch test

6孔板用直尺画横线，分别将稳定表达PHB2的HFL1原代成纤维细胞和对照组细胞接种于6孔板，密度约为100％时，饥饿24h，用200μl枪头在6孔板里垂直于板外预先画好的线做划痕，TGFβ刺激48h；分别在刺激的第0h、24h、48h进行拍照，计算划痕距离。In the 6-well plate, draw horizontal lines with a ruler, and inoculate HFL1 primary fibroblasts stably expressing PHB2 and the control cells in the 6-well plate. When the density is about 100%, starve for 24 hours, and use 200μl pipette tip on the 6-well plate. The inside is perpendicular to the pre-drawn line outside the board to make a scratch, and TGFβ is stimulated for 48h; photographs are taken at the 0h, 24h, and 48h of the stimulation to calculate the scratch distance.

迁移实验Migration experiment

分别将稳定表达PHB2和对照组细胞计数后，将100μl(无血清培养基)含有约1*10 ⁵个细胞铺在transwell上室中，下室加入无血清培养基600μl，上下室均加入10ng/ml的TGF，作用48h后弃掉孔内培养基，用面签擦去小室上层细胞，PBS洗两遍，4％多聚甲醛固定，结晶紫染色，镜下拍照，计算迁移细胞数。 After counting the cells stably expressing PHB2 and the control group, 100μl (serum-free medium) containing about 1*10 ⁵ cells were plated in the upper chamber of the transwell, 600μl serum-free medium was added to the lower chamber, and 10ng/into both the upper and lower chambers. After 48 hours of treatment with ml of TGF, discard the medium in the wells, wipe the upper cells of the chamber with a face swab, wash twice with PBS, fix with 4% paraformaldehyde, stain with crystal violet, take pictures under the microscope, and count the number of migrated cells.

结果：如图4所示，过表达PHB2后能够抑制原代成纤维细胞和上皮细胞的凋亡，抑制原代成纤维细胞的迁移，CCK8实验显示过表达PHB2以后能够对抗博莱霉素诱导的MLE12肺泡上皮细胞的死亡，显著上调细胞存活率，Results: As shown in Figure 4, overexpression of PHB2 can inhibit the apoptosis of primary fibroblasts and epithelial cells, and inhibit the migration of primary fibroblasts. CCK8 experiments show that overexpression of PHB2 can resist bleomycin-induced The death of MLE12 alveolar epithelial cells significantly increases the cell survival rate,

实施例5 PHB2影响线粒体的功能Example 5 PHB2 affects the function of mitochondria

PHB2对线粒体自噬的影响的测定Determination of the effect of PHB2 on mitochondrial autophagy

用腺相关病毒介导的方式在C57小鼠中过表达PHB2，对照组感染GFP对照病毒。然后用BLM对各组小鼠进行作用。利用慢病毒介导的方式在HFL1细胞中过表达PHB2，TGFβ刺激48h。收取细胞/组织样品进行RNA提取，qPCR法检测自噬相关基因LC3的表达The adeno-associated virus-mediated method was used to overexpress PHB2 in C57 mice, and the control group was infected with GFP control virus. Then use BLM to act on each group of mice. Lentivirus-mediated overexpression of PHB2 in HFL1 cells was used to stimulate TGFβ for 48h. Collect cell/tissue samples for RNA extraction, qPCR method to detect the expression of autophagy-related gene LC3

PHB2对线粒体膜电势影响的测定Determination of the influence of PHB2 on mitochondrial membrane potential

对构建过表达PHB2后经TGFβ/博莱霉素诱导的细胞进行线粒体膜电势变化的测定。分别将各组细胞接种于24孔板中，细胞密度为80％进行TGFβ/博莱霉素干预，48h后弃去培养基，PBS洗两遍，将mitotracker按照1：10000的比例进行稀释，每孔加入200μl孵育20min，PBS洗两遍，4％多聚甲醛固定10min，PBS洗两遍，DAPI复染10min，PBS洗两遍，显微镜下拍照。The changes of mitochondrial membrane potential were measured on the cells induced by TGFβ/bleomycin after constructing overexpression of PHB2. The cells of each group were inoculated into 24-well plates, and the cell density was 80% for TGFβ/bleomycin intervention. After 48 hours, the medium was discarded, washed twice with PBS, and the mitotracker was diluted at a ratio of 1:10000. Add 200 μl to the wells and incubate for 20 minutes, wash twice with PBS, fix with 4% paraformaldehyde for 10 minutes, wash twice with PBS, counter-stain with DAPI for 10 minutes, wash twice with PBS, and take pictures under the microscope.

结果：如图5所示，无论是体内还是体外，PHB2均能够上调自噬相关蛋白LC3的表达(图5A和图5B)。并且，过表达PHB2能够对抗博莱霉素诱导的MLE12肺泡上皮细胞线粒体膜电势的降低(图5C)。Results: As shown in Figure 5, PHB2 can up-regulate the expression of autophagy-related protein LC3 whether in vivo or in vitro (Figure 5A and Figure 5B). Moreover, overexpression of PHB2 can counteract the reduction of mitochondrial membrane potential of MLE12 alveolar epithelial cells induced by bleomycin (Figure 5C).

Claims (12)

1. 一种用于在对象中治疗肺纤维化的药物，所述药物包含载体及编码PHB2蛋白的多核苷酸，其中所述载体包含或携带所述编码PHB2蛋白的多核苷酸。A medicament for treating pulmonary fibrosis in a subject, the medicament comprising a vector and a polynucleotide encoding a PHB2 protein, wherein the vector contains or carries the polynucleotide encoding the PHB2 protein.
2. 根据权利要求1所述的药物，其中所述多核苷酸是编码PHB2蛋白的DNA或RNA序列。The medicament according to claim 1, wherein the polynucleotide is a DNA or RNA sequence encoding PHB2 protein.
3. 根据权利要求1所述的药物，其中所述载体是病毒载体或非病毒载体。The medicine according to claim 1, wherein the vector is a viral vector or a non-viral vector.
4. 根据权利要求3所述的药物，其中所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。The drug according to claim 3, wherein the non-viral vector is selected from the group consisting of plasmids, liposomes, reverse transcription elements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles And exosomes.
5. 根据权利要求3所述的药物，其中所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒或慢病毒。The medicament according to claim 3, wherein the viral vector is selected from: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus or lentivirus.
6. 根据权利要求5所述的药物，其中所述病毒载体是腺相关病毒。The drug according to claim 5, wherein the viral vector is an adeno-associated virus.
7. 根据权利要求5所述的药物，其中所述病毒载体是慢病毒。The drug according to claim 5, wherein the viral vector is a lentivirus.
8. 根据权利要求1所述的药物，其中给药后所述PHB2蛋白在所述对象的细胞中被稳定表达。The medicament according to claim 1, wherein the PHB2 protein is stably expressed in the cells of the subject after administration.
9. 根据权利要求1所述的药物，其中所述对象是哺乳动物。The medicament according to claim 1, wherein the subject is a mammal.
10. 根据权利要求9所述的药物，其中所述哺乳动物是人。The medicament according to claim 9, wherein the mammal is a human.
11. 根据权利要求1所述的药物，所述肺纤维化是特发性肺纤维化。The medicament according to claim 1, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.
12. 一种药物组合物，其包含权利要求1-11中任一项所述的药物和药学上可接受的赋形剂。A pharmaceutical composition comprising the drug according to any one of claims 1-11 and a pharmaceutically acceptable excipient.

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