WO2014139456A1 - Application of aluminum hydroxide in preparation of medicament for treatment of liver cancer - Google Patents

Application of aluminum hydroxide in preparation of medicament for treatment of liver cancer Download PDF

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Publication number
WO2014139456A1
WO2014139456A1 PCT/CN2014/073411 CN2014073411W WO2014139456A1 WO 2014139456 A1 WO2014139456 A1 WO 2014139456A1 CN 2014073411 W CN2014073411 W CN 2014073411W WO 2014139456 A1 WO2014139456 A1 WO 2014139456A1
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liver cancer
group
tumor
aluminum hydroxide
mice
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PCT/CN2014/073411
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French (fr)
Chinese (zh)
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王红阳
闻玉梅
王宾
付静
王波
任一彬
朱俊杰
汪萱怡
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复旦大学
中国人民解放军第二军医大学
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Publication of WO2014139456A1 publication Critical patent/WO2014139456A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/08—Oxides; Hydroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of chemical medicines, and relates to a novel use of aluminum hydroxide [ ⁇ 1( ⁇ ) 3 ] compound in pharmacy, in particular to the application of aluminum hydroxide [ ⁇ 1( ⁇ ) 3 ] compound in the preparation of a medicament for treating liver cancer.
  • liver cancer primary liver cancer
  • liver cancer is the sixth most common malignant tumor in the world, with more than 700,000 new cases per year, of which hepatocellular carcinoma accounts for about 80%.
  • the annual incidence of new liver cancer in China accounts for about 55% of the total number of global diseases.
  • Liver cancer has become the second leading cause of death in China. So far, surgical resection is still the best treatment for liver cancer, and it is the first choice for early liver cancer.
  • due to the concealed incidence of primary liver cancer rapid development has resulted in only about 10% of surgical resections at the time of diagnosis.
  • liver cancer Even if surgery is performed, there are still many cases that cannot be completely removed or the tumor is difficult to avoid postoperative recurrence. In addition, because the surgery has a great trauma to the human body, it often reduces the immunity of the patient and causes a series of complications. Therefore, in clinical practice, patients with advanced liver cancer are often unable to undergo surgery due to liver dysfunction. In this part of the patient, it is necessary to treat them by various non-surgical methods, or to perform non-surgical treatment to reduce the size of the mass, and then to perform surgical resection, that is, the second stage of removal, so that patients can obtain better benefits. Therefore, finding effective non-surgical treatment of liver cancer is crucial for improving the survival rate of patients with liver cancer, especially those with advanced liver cancer.
  • Non-specific immunotherapy refers to the use of some immunomodulators to non-specifically enhance the body's immune function and activate the body's anti-tumor immune response to achieve tumor treatment.
  • cytokines L-2, IFN, TNF, etc.
  • microorganisms and their products vitamin K, heat shock protein (HSP) and so on.
  • Active immunotherapy refers to a method for inducing specific immunity by using tumor cells or tumor antigen substances to induce specific immunity, thereby actively killing tumor cells, preventing tumor growth and recurrence and treating tumors; currently used DC vaccines and tumors are commonly used. Cell vaccines and heterologous recombinant alpha-fetoprotein vaccines.
  • Adoptive immunotherapy pointing to liver cancer patients to input immune cells with anti-tumor activity, directly killing tumors or stimulating the body's anti-tumor immune effect, thereby achieving the purpose of treating liver cancer.
  • Aluminum hydroxide [ ⁇ 1( ⁇ )3] is the only human vaccine adjuvant approved by the US FDA and the China Food and Drug Administration. The use of the vaccine not only improves the body's immune response but also has better safety. After more than 80 years of use, aluminum hydroxide adjuvant has been proven and proven in practice and has been approved for commercial use in vaccines such as DTP. Influenza Blood bacillus vaccine, hepatitis B vaccine (HBV), etc.
  • the commercially available aluminum hydroxide adjuvant is essentially an incompletely dehydrated product of ⁇ 1( ⁇ )3, ie, a fibrous crystalline form of particles having a size of 4.5 nm ⁇ 2.2 nm x 10 nm and an isoelectric point of 11.4.
  • the aluminum hydroxide adjuvant is in the form of an aluminum hydroxy group whose hydroxyl group can provide or accept a proton and thus behave as an amphoteric compound.
  • the adjuvant aluminum hydroxide of commercial products is a nearly transparent sol. After adhering to the vaccine antigen, the antigen is confined to a specific lattice structure formed by colloids, and the body can be enhanced by two mechanisms: "repository effect” and "immunostimulation effect". An immune response to an antigen.
  • the reservoir effect refers to the longer the antigen-presenting cell (APC) acts on the antigen during uptake, processing, and treatment, and the longer the antigen and immune cells act, the better the subsequent immune response.
  • APC antigen-presenting cell
  • aluminum hydroxide is highly concentrated on the surface and inside, and is physically presented to the immune cells without changing its chemical structure, so that the immune cells can be fully, high-level, and long-term. Contact with the vaccine antigen, thereby enabling a meaningful immune response to be induced.
  • the immunostimulatory effect mainly refers to the activation of ⁇ cell mother cellization after aluminum hydroxide, thereby promoting antibody production.
  • aluminum hydroxide can stimulate the immune effects of the body in various ways, including 1) promoting the aggregation of inflammatory cells such as neutrophils, inflammatory monocytes and dendritic cells. (DC); 2) acting on human peripheral blood mononuclear cells, inducing monocyte differentiation into mature CD40 + , CD83 + DC; 3) inducing Th2 type immune response in animal models; 4) mediating secretion of inflammatory factors , such as through the Nlrp3 inflammatory body mediated IL- ⁇ secretion; 5) through the cell surface lipids to promote DC uptake of antigen; 6) in vitro induced T cell proliferation, enhance memory CD8 + T cells, induce killer T cells Differentiation.
  • the invention provides the use of aluminum hydroxide [ ⁇ 1( ⁇ ) 3 ] in the preparation of antitumor drugs, in particular to the application of aluminum hydroxide as a separate component in the preparation of a medicament for treating liver cancer.
  • the aluminum hydroxide [ ⁇ 1( ⁇ ) 3 ] of the present invention means an aluminum hydroxide adjuvant widely used in various vaccines.
  • the present invention provides the use of aluminum hydroxide as a separate component in the preparation of a medicament for treating liver cancer.
  • various experimental methods such as tumor inoculation experiment, immunological analysis, proliferation, apoptosis and immunohistochemical detection in the tumor-bearing experimental mice are carried out, and the results show that: the administration of aluminum hydroxide can significantly inhibit the liver cancer cells in vivo. Proliferation, inducing neutrophil infiltration of tumors, tumor cell apoptosis, thereby inhibiting the growth of subcutaneous tumors in vivo and improving the survival rate of tumor-bearing mice, indicating that the use of aluminum hydroxide as the sole component in the present invention can effectively inhibit liver cancer cells. Growth, with anti-liver cancer effect.
  • the present invention utilizes an aluminum hydroxide [ ⁇ 1( ⁇ ) 3 ] compound (obtained by a commercially available channel, which is provided by the Medical Molecular Virology Laboratory of Shanghai Medical College of Fudan University in the embodiment of the present invention) as a separate component in the mouse liver.
  • an aluminum hydroxide [ ⁇ 1( ⁇ ) 3 ] compound obtained by a commercially available channel, which is provided by the Medical Molecular Virology Laboratory of Shanghai Medical College of Fudan University in the embodiment of the present invention
  • Experiments were performed in a cancer-bearing tumor model.
  • a mouse liver cancer tumor-bearing model was established by subcutaneous injection of a mouse-derived liver cancer cell line (Hepal-6 cells, provided by the laboratory of Professor Wang Hongyang of the Second Military Medical University in the embodiment of the present invention), and the growth of the subcutaneous tumor in the mouse was observed.
  • mice the results showed that the subcutaneous tumor volume of the blank group and the saline group was significantly increased and many experimental mice died, while the experimental group administered only aluminum hydroxide [ ⁇ 1( ⁇ ) 3 ] When the mice died, the growth rate of the subcutaneous tumors in the mice was significantly slowed down, and the subcutaneous tumors of several mice were significantly reduced, which was invisible.
  • H&E staining of the mouse organs it was shown that after administration of ⁇ 1( ⁇ ) 3 , as in the blank group and the saline-administered group, no significant damage was observed in the main organs of the mouse.
  • immunohistochemical staining of subcutaneous tumors in mice it was shown that after administration of ⁇ 1( ⁇ ) 3 , the proliferation of tumor cells was significantly slowed down and apoptosis was significantly increased.
  • the present invention has been experimentally shown that the administration of ⁇ 1( ⁇ ) 3 can significantly inhibit the proliferation of liver cancer cells, induce apoptosis, thereby inhibiting the growth of subcutaneous tumors in vivo and improving the survival rate of tumor-bearing mice, indicating that ⁇ 1 of the present invention ( ⁇ ) 3 As a sole and unique component, it can safely and effectively inhibit the growth of liver cancer cells, and has an anti-liver cancer effect.
  • ⁇ 1( ⁇ ) 3 of the present invention can be used as a single active ingredient for preparing a medicament for treating liver cancer, and can also be used as a separate component and other effective liver cancer intervention methods or anti-hepatocarcinoma drugs for comprehensive intervention of liver cancer.
  • ⁇ 1( ⁇ ) 3 is used as a separate active ingredient in combination with surgical or radiological interventions or other anti-hepatocarcinoma drugs for the comprehensive intervention of liver cancer.
  • the invention provides a new idea for the selection of interventional measures for liver cancer and the clinical application of ⁇ 1( ⁇ ) 3 .
  • the present invention will be described in detail below through the specific drawings and embodiments. It is to be understood that the specific embodiments and the drawings are intended for the purpose of illustration Variations are also included within the scope of the invention.
  • Figure 1 is a model of a mouse liver cancer bearing tumor.
  • FIG 2 is a control group, saline group and ⁇ 1 ( ⁇ ) tumor growth graph 3 groups of mice,
  • Fig. 2-1 is a growth curve of tumors of each group of mice
  • Fig. 2-2 is a representative picture of subcutaneous tumors of each group of mice after 6 weeks of treatment.
  • Figure 3 is a survival curve of the blank group, the saline group, and the ⁇ 1 ( ⁇ ) group 3 mice.
  • FIG 4 is a control group, saline group and ⁇ 1 ( ⁇ ) 3 mice organs
  • Fig. 4-1 is a representative picture of the organs of each group after 6 weeks of treatment
  • Fig. 4-2 is a H&E staining diagram of the organs of each group of mice.
  • Figure 5 is a Ki67 immunohistochemical staining of subcutaneous tumors of blank group, saline group and ⁇ 1 ( ⁇ ) group 3 mice.
  • Figure 6 shows the apoptosis of TUNEL cells in subcutaneous tumors of blank group, saline group and ⁇ 1( ⁇ ) group 3 mice.
  • FIG H & E staining is a saline group, the subcutaneous tumor growth curves of FIG Al (OH) 3 7 days beginning 11 days of treatment group, and detection of immune cells in tumor sections and subcutaneous chart of FIG H & E staining,
  • Figure 7-1 is a graph showing the subcutaneous tumor growth of mice in the saline group, ⁇ 1 ( ⁇ ) 3 7 days, and 11 days starting treatment group;
  • Fig. 7-2 is a flow chart showing the flow cytometry and data of neutrophils in the spleen and tumor of the mice in the saline group, ⁇ 1 ( ⁇ ) 3 7 days and 11 days;
  • Figure 7-3 is a H&E staining of subcutaneous tumor sections of mice in the saline group, ⁇ 1 ( ⁇ ) 3 7 days, and 11 days starting treatment group.
  • Example 1 Establishment of a tumor-bearing model of mouse liver cancer
  • Example 2 Tumor growth in mice of blank group, saline group and ⁇ 1 ( ⁇ ) group 3
  • mice in the blank group were not treated, and the mice in the experimental group were given ⁇ 1( ⁇ )3 solution (dissolved in physiological saline), 0.25 mg/mouse, once every 3 days, intraperitoneally.
  • the control group was given an equal amount of normal saline (0.9% NaCl liquid), once every 3 days, intraperitoneally.
  • Example 3 Survival curves of mice in the blank group, saline group, and ⁇ 1 ( ⁇ ) group 3
  • mice The blank group, the saline group, and the ⁇ 1 ( ⁇ ) group 3 mice were treated as described in Example 2, and the survival of the mice was observed daily and the survival curve was drawn. As shown in the results in Fig. 3, as of the end of the experiment, 4 mice died in the empty group, 5 mice died in the saline group, and only one mouse died in the ⁇ 1 ( ⁇ ) group. The results showed that injection of ⁇ 1( ⁇ )3 significantly improved the survival rate of tumor-bearing mice.
  • Example 4 Organs in the blank group, saline group, and ⁇ 1 ( ⁇ ) group 3
  • mice The blank group, the saline group and the ⁇ 1 ( ⁇ ) group 3 mice were treated as described in Example 2, and the mice were sacrificed 6 weeks later, and the organs of each group (heart, liver, spleen, lung and kidney) were collected. , fixed in formalin, embedded in paraffin, sectioned and H&E stained.
  • H&E staining method 1) Organ tissue paraffin section Wax to water; 2) Sumu semen staining for 5 min, water washing; 3) 1% hydrochloric acid ethanol 1-3 s, water washing; 4) 0.5% eosin staining lmin, water washing; 5) dehydration sealing.
  • Ki67 is a proliferating cell-associated nuclear antigen whose function is closely related to mitosis and is indispensable in cell proliferation. Ki67 is an antigen that marks the proliferative state of cells, and its positive staining indicates that cell proliferation is active.
  • Ki67 staining method 1) Deparaffinization of paraffin sections of tumor tissue to water; 2) 3% H202 room temperature lOmin, water washing; 3) antigen retrieval; 4) goat serum blocking for 30 min; 5) addition of Ki67 antibody (1:100, purchased from Cell Signaling Technology), overnight at 4 °C; 6) Adding horseradish peroxidase-labeled secondary antibody (purchased from Shanghai Changdao Biotechnology Co., Ltd.) at 37 ° C for 30 min; 7) DAB color development (purchased from DAKO); 8) Hematoxylin counterstaining, hydrochloric acid alcohol differentiation; 9) Dehydration sealing.
  • mice were treated as described in Example 2. After 6 weeks, the mice were sacrificed, and the subcutaneous tumor tissues were removed, fixed in formalin, embedded in paraffin, and produced. The sections were subjected to TUNEL apoptosis detection (TUNEL FITC staining kit was purchased from Shanghai Rui'an Biotechnology, and the experimental procedure is described in the kit manual).
  • the TUNEL method uses the action of terminal deoxyribonucleotidyl transferase to catalyze the incorporation of fluorescein FITC-labeled deoxyuridine triphosphate at the end of apoptotic cell cleavage DNA, and then directly observe FITC-labeled cells under a fluorescence microscope. Dead cells.
  • mice Twenty-one male C57BL/6J mice, 5-6 weeks old, were randomly divided into 3 groups, 7 rats in each group. Each mouse was inoculated with 1 ⁇ 10 7 Hepal-6 cells by subcutaneous injection.
  • ⁇ 1( ⁇ )3 solution dissolved in physiological saline
  • 0.25 mg/0.25 mL/only once every 3 days, was intraperitoneally injected at 7, 11 days after inoculation.
  • the control group was given an equal amount of normal saline (0.9% NaCl) 7 days after inoculation. Liquid), once every 3 days, intraperitoneal injection.
  • tumor volume tumor long diameter X tumor short diameter 2 / 2
  • spleen was treated with grinding, red cleavage, etc. to obtain single cell suspense, neutrophils, macrophages, NK, CD 4 , CD 8 T , T reg, etc.
  • Cell flow detection The tumor tissue was subjected to shearing, collagenase and DNase digestion treatment to obtain a single cell suspension, and flow cytometry of neutrophils and macrophages was performed. A part of the tumor tissue was taken for pathological sectioning, and HE staining was performed with reference to Example 2.
  • the results of microscopic observation of the tumor were as follows: 1) 100 times of the injection of aluminum adjuvant was found to cause necrosis of cells in the tumor, and the necrosis of the ⁇ 1( ⁇ )3 7 day treatment group was severer than that of the 11-day treatment group. No necrosis was observed in the group; 2) 4001( ⁇ )3 was observed at 400 times. There was a large amount of neutrophil infiltration in the tumor in the 7-day treatment group, but in the saline group and ⁇ 1( ⁇ )3 11-day treatment group.

Abstract

An application of aluminum hydroxide, Al(OH)3, as an individual component in preparation of a medicament for treatment of liver cancer. Inhibition of liver cancer cell growth is allowed, and, use in conjunction with other liver cancer prevention methods or anti-liver cancer medicaments is allowed.

Description

氢氧化铝在制备治疗肝癌药物中的应用  Application of aluminum hydroxide in preparing medicine for treating liver cancer
技术领域 Technical field
本发明涉及化学药物领域， 涉及氢氧化铝 [Α1(ΟΗ)3]化合物在制药中的新的 用途， 具体涉及氢氧化铝 [Α1(ΟΗ)3]化合物在制备治疗肝癌药物中的应用。 The invention relates to the field of chemical medicines, and relates to a novel use of aluminum hydroxide [Α1(ΟΗ) 3 ] compound in pharmacy, in particular to the application of aluminum hydroxide [Α1(ΟΗ) 3 ] compound in the preparation of a medicament for treating liver cancer.
背景材料 Background material
据报道， 原发性肝癌 (简称肝癌)是世界范围内第六位的最常见恶性肿瘤， 每 年新增病例约 70余万， 其中肝细胞癌约占 80%。 调查显示肝癌是中国最常见的 恶性肿瘤， 我国每年新发肝癌病例约占全球发病总数的 55%,肝癌已成为我国第 二大肿瘤致死原因。 至今， 临床治疗中手术切除仍是治疗肝癌的最佳治疗方法， 也是早期肝癌的首选治疗措施。 但由于原发性肝癌发病隐匿， 发展快速， 导致确 诊时能手术切除者仅为 10 %左右， 而且， 即使施行手术， 仍有许多病例无法完 全切除或肿瘤难以避免术后复发。 此外， 由于手术对人体的创伤较大， 常会使患 者的免疫力降低，并且导致一系列并发症， 因此，临床实践中对于晚期肝癌患者， 多因其肝功能不全而无法手术。这一部分病人， 需通过各种非手术切除的方法进 行治疗， 或者先行非手术治疗， 使肿块缩小局限， 再行手术切除治疗， 即二期切 除， 以使患者获得较好的益处。 因此， 寻找有效的肝癌非手术治疗方式对于提高 肝癌患者， 尤其是中晚期肝癌患者的生存率是至关重要的。  According to reports, primary liver cancer (referred to as liver cancer) is the sixth most common malignant tumor in the world, with more than 700,000 new cases per year, of which hepatocellular carcinoma accounts for about 80%. The investigation shows that liver cancer is the most common malignant tumor in China. The annual incidence of new liver cancer in China accounts for about 55% of the total number of global diseases. Liver cancer has become the second leading cause of death in China. So far, surgical resection is still the best treatment for liver cancer, and it is the first choice for early liver cancer. However, due to the concealed incidence of primary liver cancer, rapid development has resulted in only about 10% of surgical resections at the time of diagnosis. Moreover, even if surgery is performed, there are still many cases that cannot be completely removed or the tumor is difficult to avoid postoperative recurrence. In addition, because the surgery has a great trauma to the human body, it often reduces the immunity of the patient and causes a series of complications. Therefore, in clinical practice, patients with advanced liver cancer are often unable to undergo surgery due to liver dysfunction. In this part of the patient, it is necessary to treat them by various non-surgical methods, or to perform non-surgical treatment to reduce the size of the mass, and then to perform surgical resection, that is, the second stage of removal, so that patients can obtain better benefits. Therefore, finding effective non-surgical treatment of liver cancer is crucial for improving the survival rate of patients with liver cancer, especially those with advanced liver cancer.
目前， 肝癌的非手术治疗措施包括针对肿瘤的化学治疗、 放射治疗、 免疫治 疗和基因治疗等。 肿瘤的免疫治疗是近年来兴起的最有前景的新疗法， 该疗法能 增强免疫系统的自我调节能力， 激发特异性免疫反应， 从而达到治疗肿瘤并延缓 和降低肿瘤复发转移的目的。 通常， 肝癌的免疫治疗分为以下几类： （1)非特异 性免疫治疗， 指应用一些免疫调节剂通过非特异性地增强机体的免疫功能， 激活 机体的抗肿瘤免疫应答， 以达到治疗肿瘤的目的； 目前应用于肿瘤治疗的有： 细 胞因子 (lL-2、 IFN、 TNF 等)、 微生物及其产物、 维生素 K、 热休克蛋白 (HSP) 等。 （2)主动免疫治疗， 指利用肿瘤细胞或肿瘤抗原物质诱导机体产生特异性免 疫， 进而主动杀伤肿瘤细胞， 阻止肿瘤的生长扩散和复发以治疗肿瘤的方法； 目 前常用的括扩 DC疫苗、肿瘤细胞疫苗及异种重组甲胎蛋白疫苗等。（3)过继免疫 治疗， 指向肝癌患者输入具有抗瘤活性的免疫细胞， 直接杀伤肿瘤或激发机体抗 瘤免疫效应， 从而达到治疗肝癌的目的。  Currently, non-surgical treatments for liver cancer include chemotherapy for cancer, radiation therapy, immunotherapy, and gene therapy. Tumor immunotherapy is the most promising new treatment in recent years. It can enhance the immune system's self-regulation ability and stimulate specific immune response, so as to achieve the purpose of treating tumors and delaying and reducing tumor recurrence and metastasis. Generally, immunotherapy for liver cancer is divided into the following categories: (1) Non-specific immunotherapy refers to the use of some immunomodulators to non-specifically enhance the body's immune function and activate the body's anti-tumor immune response to achieve tumor treatment. Currently used in cancer treatment are: cytokines (lL-2, IFN, TNF, etc.), microorganisms and their products, vitamin K, heat shock protein (HSP) and so on. (2) Active immunotherapy refers to a method for inducing specific immunity by using tumor cells or tumor antigen substances to induce specific immunity, thereby actively killing tumor cells, preventing tumor growth and recurrence and treating tumors; currently used DC vaccines and tumors are commonly used. Cell vaccines and heterologous recombinant alpha-fetoprotein vaccines. (3) Adoptive immunotherapy, pointing to liver cancer patients to input immune cells with anti-tumor activity, directly killing tumors or stimulating the body's anti-tumor immune effect, thereby achieving the purpose of treating liver cancer.
氢氧化铝 [Α1(ΟΗ)3]作为美国 FDA和中国食品药品监督局批准的唯一人用疫 苗佐剂， 在疫苗中使用不仅可以提高机体的免疫应答而且具有较好的安全性。 氢 氧化铝佐剂经 80余年的使用， 其有效性和安全性已得到了实践的验证和人们的 公认， 并已获得批准应用于商业性的疫苗， 如白百破三联疫苗 (DTP)、 型流感嗜 血杆菌疫苗、乙肝疫苗 (HBV)等。有关商品化的氢氧化铝佐剂，其实质是 Α1(ΟΗ)3 的不完全脱水产物， 即纤维状结晶形态的粒子， 大小 4.5 nm χ 2.2 nm x 10 nm, 等电点 11.4。 这种粒子聚集后以松散的 1 ~ 10 μ ηι大小的形式存在。 在化学上， 氢氧化铝佐剂是以铝羟基形式存在的，其羟基可以提供或接受质子， 从而表现为 两性化合物。 商业产品的佐剂氢氧化铝为近乎透明的溶胶， 黏附疫苗抗原后， 抗 原被限定在胶体形成的特定网格结构中， 可通过 "储存库效应" 和 "免疫刺激 效应"两种机制增强机体对抗原的免疫应答。储存库效应是指抗原提呈细胞 (APC) 在对抗原摄取、 加工、 处理过程中， 抗原与免疫细胞作用时间越长， 就越有利于 后续的免疫应答。 氢氧化铝黏附疫苗抗原后， 在其表面和内部高度浓聚着许多抗 原，物理性的呈递给免疫细胞，而不改变其化学结构， 以便于免疫细胞能充分地、 高水平地、 长时间地与疫苗抗原接触作用， 从而使有意义的免疫应答被诱导。 免 疫刺激效应主要是指氢氧化铝后可激活 Β 细胞母细胞化， 进而促进抗体产生。 近来随着研究的深入， 人们发现氢氧化铝还可通过多种方式刺激机体的免疫效 应， 包括 1)促进炎性细胞的聚集， 如中性粒细胞、 炎性单核细胞和树突状细胞 (DC); 2)作用于人的外周血单核细胞，诱导单核细胞分化为成熟的 CD40+、 CD83+ DC; 3)诱导动物模型的 Th2型免疫应答； 4)介导炎性因子的分泌， 如通过 Nlrp3 炎症小体介导 IL-Ιβ分泌； 5)通过细胞表面脂类的活化促使 DC摄入抗原； 6)体 外诱导 T细胞显著增殖， 增强记忆性 CD8+ T细胞， 诱导杀伤性 T细胞的分化。 Aluminum hydroxide [Α1(ΟΗ)3] is the only human vaccine adjuvant approved by the US FDA and the China Food and Drug Administration. The use of the vaccine not only improves the body's immune response but also has better safety. After more than 80 years of use, aluminum hydroxide adjuvant has been proven and proven in practice and has been approved for commercial use in vaccines such as DTP. Influenza Blood bacillus vaccine, hepatitis B vaccine (HBV), etc. The commercially available aluminum hydroxide adjuvant is essentially an incompletely dehydrated product of Α1(ΟΗ)3, ie, a fibrous crystalline form of particles having a size of 4.5 nm χ 2.2 nm x 10 nm and an isoelectric point of 11.4. This particle aggregates in the form of a loose 1 ~ 10 μ ηι size. Chemically, the aluminum hydroxide adjuvant is in the form of an aluminum hydroxy group whose hydroxyl group can provide or accept a proton and thus behave as an amphoteric compound. The adjuvant aluminum hydroxide of commercial products is a nearly transparent sol. After adhering to the vaccine antigen, the antigen is confined to a specific lattice structure formed by colloids, and the body can be enhanced by two mechanisms: "repository effect" and "immunostimulation effect". An immune response to an antigen. The reservoir effect refers to the longer the antigen-presenting cell (APC) acts on the antigen during uptake, processing, and treatment, and the longer the antigen and immune cells act, the better the subsequent immune response. After adhering to the vaccine antigen, aluminum hydroxide is highly concentrated on the surface and inside, and is physically presented to the immune cells without changing its chemical structure, so that the immune cells can be fully, high-level, and long-term. Contact with the vaccine antigen, thereby enabling a meaningful immune response to be induced. The immunostimulatory effect mainly refers to the activation of Β cell mother cellization after aluminum hydroxide, thereby promoting antibody production. Recently, with the deepening of research, it has been found that aluminum hydroxide can stimulate the immune effects of the body in various ways, including 1) promoting the aggregation of inflammatory cells such as neutrophils, inflammatory monocytes and dendritic cells. (DC); 2) acting on human peripheral blood mononuclear cells, inducing monocyte differentiation into mature CD40 + , CD83 + DC; 3) inducing Th2 type immune response in animal models; 4) mediating secretion of inflammatory factors , such as through the Nlrp3 inflammatory body mediated IL-Ιβ secretion; 5) through the cell surface lipids to promote DC uptake of antigen; 6) in vitro induced T cell proliferation, enhance memory CD8 + T cells, induce killer T cells Differentiation.
目前， 有关氢氧化铝作为疫苗佐剂的作用机制已在体内外进行了大量的实验 研究， 但迄今尚未见氢氧化铝单独用于抗肿瘤及其相关机制研究的报道。  At present, a large number of experimental studies on the mechanism of action of aluminum hydroxide as a vaccine adjuvant have been conducted in vitro and in vivo, but no reports of aluminum hydroxide alone for anti-tumor and related mechanisms have been reported so far.
发明内容 Summary of the invention
本发明的目的是提供氢氧化铝 [Α1(ΟΗ)3]化合物的新的药用用途。 It is an object of the present invention to provide new pharmaceutical uses of aluminum hydroxide [Α1(ΟΗ) 3 ] compounds.
本发明提供了氢氧化铝 [Α1(ΟΗ)3]在制备抗肿瘤药物中的用途， 尤其涉及氢 氧化铝作为单独成分在制备治疗肝癌的药物中的应用。 The invention provides the use of aluminum hydroxide [Α1(ΟΗ) 3 ] in the preparation of antitumor drugs, in particular to the application of aluminum hydroxide as a separate component in the preparation of a medicament for treating liver cancer.
本发明的氢氧化铝 [Α1(ΟΗ)3]是指广泛用于多种疫苗的氢氧化铝佐剂。 The aluminum hydroxide [Α1(ΟΗ) 3 ] of the present invention means an aluminum hydroxide adjuvant widely used in various vaccines.
本发明提供了氢氧化铝作为单独成分在制备治疗肝癌药物中的应用。本发明 中， 进行了荷瘤实验小鼠体内肿瘤接种实验、 免疫学分析、 增殖、 凋亡和免疫组 化检测等多种实验手段， 结果显示： 给予氢氧化铝干预可明显抑制肝癌细胞的体 内增殖， 诱导中性粒细胞浸润肿瘤、 肿瘤细胞凋亡， 进而抑制皮下肿瘤的体内生 长并提高荷瘤小鼠的生存率，表明本发明釆用氢氧化铝作为单独唯一成分能够有 效抑制肝癌细胞的生长， 具有抗肝癌作用。  The present invention provides the use of aluminum hydroxide as a separate component in the preparation of a medicament for treating liver cancer. In the present invention, various experimental methods such as tumor inoculation experiment, immunological analysis, proliferation, apoptosis and immunohistochemical detection in the tumor-bearing experimental mice are carried out, and the results show that: the administration of aluminum hydroxide can significantly inhibit the liver cancer cells in vivo. Proliferation, inducing neutrophil infiltration of tumors, tumor cell apoptosis, thereby inhibiting the growth of subcutaneous tumors in vivo and improving the survival rate of tumor-bearing mice, indicating that the use of aluminum hydroxide as the sole component in the present invention can effectively inhibit liver cancer cells. Growth, with anti-liver cancer effect.
本发明利用氢氧化铝 [Α1(ΟΗ)3]化合物（通过巿购渠道获得， 本发明的实施例 中由复旦大学上海医学院医学分子病毒学实验室提供，）作为单独成分在小鼠肝 癌荷瘤模型中进行了实验。通过皮下注射小鼠来源的肝癌细胞系（Hepal-6细胞， 本发明的实施例中由第二军医大学王红阳教授实验室提供）， 建立小鼠肝癌荷瘤 模型， 观察小鼠皮下肿瘤的生长情况和小鼠的生存情况， 结果显示， 空白组和给 予生理盐水组皮下肿瘤体积明显增大并有多只实验小鼠死亡，而实验组给药氢氧 化铝 [Α1(ΟΗ)3]后仅有一只小鼠死亡， 小鼠皮下肿瘤生长速率明显减慢， 并有数 只小鼠的皮下肿瘤明显缩小， 几不可见。 通过对小鼠器官的 H&E染色， 显示给 药 Α1(ΟΗ)3后， 与空白组和给予生理盐水组相同， 小鼠主要脏器未见明显损伤。 通过对小鼠皮下肿瘤的免疫组化染色， 显示给药 Α1(ΟΗ)3后， 肿瘤细胞的增殖明 显减慢、 细胞凋亡明显增多。 The present invention utilizes an aluminum hydroxide [Α1(ΟΗ) 3 ] compound (obtained by a commercially available channel, which is provided by the Medical Molecular Virology Laboratory of Shanghai Medical College of Fudan University in the embodiment of the present invention) as a separate component in the mouse liver. Experiments were performed in a cancer-bearing tumor model. A mouse liver cancer tumor-bearing model was established by subcutaneous injection of a mouse-derived liver cancer cell line (Hepal-6 cells, provided by the laboratory of Professor Wang Hongyang of the Second Military Medical University in the embodiment of the present invention), and the growth of the subcutaneous tumor in the mouse was observed. And the survival of the mice, the results showed that the subcutaneous tumor volume of the blank group and the saline group was significantly increased and many experimental mice died, while the experimental group administered only aluminum hydroxide [Α1(ΟΗ) 3 ] When the mice died, the growth rate of the subcutaneous tumors in the mice was significantly slowed down, and the subcutaneous tumors of several mice were significantly reduced, which was invisible. By H&E staining of the mouse organs, it was shown that after administration of Α1(ΟΗ) 3 , as in the blank group and the saline-administered group, no significant damage was observed in the main organs of the mouse. By immunohistochemical staining of subcutaneous tumors in mice, it was shown that after administration of Α1(ΟΗ) 3 , the proliferation of tumor cells was significantly slowed down and apoptosis was significantly increased.
本发明经实验表明， 给予 Α1(ΟΗ)3干预后， 可明显抑制肝癌细胞的增殖， 诱 导细胞凋亡，进而抑制皮下肿瘤的体内生长并提高荷瘤小鼠的生存率， 表明本发 明的 Α1(ΟΗ)3作为单独唯一成分能够安全、 有效地抑制肝癌细胞的生长， 具有抗 肝癌作用。 The present invention has been experimentally shown that the administration of Α1(ΟΗ) 3 can significantly inhibit the proliferation of liver cancer cells, induce apoptosis, thereby inhibiting the growth of subcutaneous tumors in vivo and improving the survival rate of tumor-bearing mice, indicating that Α1 of the present invention (ΟΗ) 3 As a sole and unique component, it can safely and effectively inhibit the growth of liver cancer cells, and has an anti-liver cancer effect.
进一步的， 本发明所述的 Α1(ΟΗ)3可作为单独活性成分制备治疗肝癌的药 物，还可作为单独成分与其他有效的肝癌干预方法或抗肝癌药物用于肝癌的综合 干预措施。 如， 将 Α1(ΟΗ)3作为单独活性成分与手术或放射干预方法或其他抗肝 癌药物联合用于肝癌的综合干预。 Further, the Α1(ΟΗ) 3 of the present invention can be used as a single active ingredient for preparing a medicament for treating liver cancer, and can also be used as a separate component and other effective liver cancer intervention methods or anti-hepatocarcinoma drugs for comprehensive intervention of liver cancer. For example, Α1(ΟΗ) 3 is used as a separate active ingredient in combination with surgical or radiological interventions or other anti-hepatocarcinoma drugs for the comprehensive intervention of liver cancer.
本发明为肝癌干预措施的选择和 Α1(ΟΗ)3的临床应用提供了新的思路。 为了便于理解， 以下将通过具体的附图和实施例对本发明进行详细地描述。 需要特别指出的是， 具体实例和附图仅是为了说明， 显然本领域的普通技术人员 可以根据本文说明， 在本发明的范围内对本发明做出各种各样的修正和改变， 这 些修正和改变也纳入本发明的范围内。 The invention provides a new idea for the selection of interventional measures for liver cancer and the clinical application of Α1(ΟΗ) 3 . For ease of understanding, the present invention will be described in detail below through the specific drawings and embodiments. It is to be understood that the specific embodiments and the drawings are intended for the purpose of illustration Variations are also included within the scope of the invention.
附图说明 DRAWINGS
图 1是建立的小鼠肝癌荷瘤模型。 Figure 1 is a model of a mouse liver cancer bearing tumor.
图 2是空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠肿瘤生长关系图， FIG 2 is a control group, saline group and Α1 (ΟΗ) tumor growth graph 3 groups of mice,
其中， 图 2-1是各组小鼠肿瘤的生长曲线； 图 2-2是给予处理 6周后， 各组 小鼠皮下肿瘤的代表性图片。  Fig. 2-1 is a growth curve of tumors of each group of mice; Fig. 2-2 is a representative picture of subcutaneous tumors of each group of mice after 6 weeks of treatment.
图 3是空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠的生存曲线。 Figure 3 is a survival curve of the blank group, the saline group, and the Α1 (ΟΗ) group 3 mice.
图 4是空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠脏器情况图， FIG 4 is a control group, saline group and Α1 (ΟΗ) 3 mice organs FIG case,
其中， 图 4-1是给予处理 6周后， 各组小鼠脏器的代表性图片； 图 4-2是各 组小鼠脏器的 H&E染色图。  Fig. 4-1 is a representative picture of the organs of each group after 6 weeks of treatment; Fig. 4-2 is a H&E staining diagram of the organs of each group of mice.
图 5是空白组、生理盐水组和 Α1(ΟΗ)3组小鼠皮下肿瘤的 Ki67免疫组化染色图。 图 6是空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠皮下肿瘤的 TUNEL细胞凋亡检测 图。 Figure 5 is a Ki67 immunohistochemical staining of subcutaneous tumors of blank group, saline group and Α1 (ΟΗ) group 3 mice. Figure 6 shows the apoptosis of TUNEL cells in subcutaneous tumors of blank group, saline group and Α1(ΟΗ) group 3 mice. Figure.
图 7是生理盐水组、 Al(OH)37天和 11天开始治疗组的小鼠皮下肿瘤生长曲线图， 体内免疫细胞检测及统计图和皮下肿瘤切片的 H&E染色图， 7 is a saline group, the subcutaneous tumor growth curves of FIG Al (OH) 3 7 days beginning 11 days of treatment group, and detection of immune cells in tumor sections and subcutaneous chart of FIG H & E staining,
其中图 7-1是生理盐水组、 Α1(ΟΗ)37天和 11天开始治疗组的小鼠皮下肿瘤 生长曲线图； Figure 7-1 is a graph showing the subcutaneous tumor growth of mice in the saline group, Α1 (ΟΗ) 3 7 days, and 11 days starting treatment group;
图 7-2是生理盐水组、 Α1(ΟΗ)37天和 11天开始治疗组的小鼠脾脏和肿瘤中 中性粒细胞的流式检测图及数据统计图； Fig. 7-2 is a flow chart showing the flow cytometry and data of neutrophils in the spleen and tumor of the mice in the saline group, Α1 (ΟΗ) 3 7 days and 11 days;
图 7-3是生理盐水组、 Α1(ΟΗ)37天和 11天开始治疗组的小鼠皮下肿瘤切片 的 H&E染色图。 Figure 7-3 is a H&E staining of subcutaneous tumor sections of mice in the saline group, Α1 (ΟΗ) 3 7 days, and 11 days starting treatment group.
具体实施方式 detailed description
实施例 1: 小鼠肝癌荷瘤模型的建立  Example 1: Establishment of a tumor-bearing model of mouse liver cancer
选取 4-6周龄、雄性 C57BL/6J小鼠 40只，釆用皮下注射方式每只接种 1 x 107个 Hepal-6细胞， 两周后观察成瘤情况。 共有 29只小鼠成瘤， 随即分为空白 组 （ 9只；)、 对照组给予生理盐水（ 10只）和实验组给予 Α1(ΟΗ)3 ( 10只 )。 实施例 2: 空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠肿瘤生长情况 Forty-five-year-old male C57BL/6J mice were selected and inoculated with 1 x 10 7 Hepal-6 cells by subcutaneous injection. The tumor formation was observed two weeks later. A total of 29 mice were tumorigenic, which were then divided into a blank group (n=9), a control group was given normal saline (10 rats), and an experimental group was given Α1(ΟΗ)3 (10 rats). Example 2: Tumor growth in mice of blank group, saline group and Α1 (ΟΗ) group 3
空白组小鼠不予处理、 实验组小鼠给予 Α1(ΟΗ)3 溶液 （溶于生理盐水）， 0.25mg/只， 每隔 3 天一次， 腹腔注射。 对照组给予等量的生理盐水 (0.9%NaCl 液体)， 每隔 3天一次， 腹腔注射。 给药后每周测量各组小鼠皮下肿瘤大小， 计 算肿瘤体积 （肿瘤体积 =肿瘤长径 X 肿瘤短径 2/ 2 )。 给予处理 6周后， 处死各 组小鼠， 取出皮下肿瘤并绘制肿瘤生长曲线。 The mice in the blank group were not treated, and the mice in the experimental group were given Α1(ΟΗ)3 solution (dissolved in physiological saline), 0.25 mg/mouse, once every 3 days, intraperitoneally. The control group was given an equal amount of normal saline (0.9% NaCl liquid), once every 3 days, intraperitoneally. The subcutaneous tumor size of each group of mice was measured weekly after administration, and the tumor volume (tumor volume = tumor long diameter X tumor short diameter 2 / 2) was calculated. After 6 weeks of treatment, each group of mice was sacrificed, subcutaneous tumors were removed and tumor growth curves were plotted.
如图 2-1和 2-2所示， 空白组与生理盐水组小鼠的皮下肿瘤生长较迅速， 体 积明显增大， 两组间肿瘤体积和生长曲线无明显差异。 给予 Α1(ΟΗ)3 后， 小鼠 皮下肿瘤生长速度明显减慢， 体积明显缩小， 实验证实注射 Α1(ΟΗ)3 可明显抑 制小鼠体内肝癌细胞的生长。  As shown in Figures 2-1 and 2-2, the subcutaneous tumors of the blank group and the saline group grew rapidly and the volume increased significantly. There was no significant difference in tumor volume and growth curve between the two groups. After the administration of Α1(ΟΗ)3, the growth rate of subcutaneous tumors in mice was significantly slowed down and the volume was significantly reduced. It was confirmed that the injection of Α1(ΟΗ)3 significantly inhibited the growth of liver cancer cells in mice.
实施例 3: 空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠的生存曲线 Example 3: Survival curves of mice in the blank group, saline group, and Α1 (ΟΗ) group 3
空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠按实施例 2所述方法给予处理， 每日 观察小鼠的生存情况并绘制生存曲线。 如图 3结果所示， 截止至实验结束时， 空 白组共有 4只小鼠死亡， 生理盐水组有 5只小鼠死亡， 而 Α1(ΟΗ)3组仅有一只 小鼠死亡。 结果表明注射 Α1(ΟΗ)3能明显提高荷瘤小鼠的生存率。  The blank group, the saline group, and the Α1 (ΟΗ) group 3 mice were treated as described in Example 2, and the survival of the mice was observed daily and the survival curve was drawn. As shown in the results in Fig. 3, as of the end of the experiment, 4 mice died in the empty group, 5 mice died in the saline group, and only one mouse died in the Α1 (ΟΗ) group. The results showed that injection of Α1(ΟΗ)3 significantly improved the survival rate of tumor-bearing mice.
实施例 4: 空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠脏器情况 Example 4: Organs in the blank group, saline group, and Α1 (ΟΗ) group 3
空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠按实施例 2所述方法给予处理， 6周 后处死小鼠， 收取各组小鼠脏器（心、 肝、 脾、 肺和肾）， 以福尔马林液固定， 石蜡包埋， 制作切片并进行 H&E染色。 H&E染色方法： 1)脏器组织石蜡切片脱 蜡至水； 2)苏木精液染色 5 min, 水洗； 3)1%盐酸乙醇 1-3 s, 水洗； 4)0.5%伊 红液染色 lmin, 水洗； 5)脱水封片。 The blank group, the saline group and the Α1 (ΟΗ) group 3 mice were treated as described in Example 2, and the mice were sacrificed 6 weeks later, and the organs of each group (heart, liver, spleen, lung and kidney) were collected. , fixed in formalin, embedded in paraffin, sectioned and H&E stained. H&E staining method: 1) Organ tissue paraffin section Wax to water; 2) Sumu semen staining for 5 min, water washing; 3) 1% hydrochloric acid ethanol 1-3 s, water washing; 4) 0.5% eosin staining lmin, water washing; 5) dehydration sealing.
如图 4-1和 4-2所示， 各组小鼠脏器的形态、 体积和组成细胞均未见明显异 常， 表明注射 Α1(ΟΗ)3对小鼠主要脏器没有明显的损伤作用。 As shown in Figures 4-1 and 4-2, there were no obvious abnormalities in the morphology, volume and composition of the organs of each group, indicating that injection of Α1(ΟΗ) 3 had no obvious damage to the main organs of the mice.
实施例 5: 空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠皮下肿瘤的 Ki67免疫组化染 色 Example 5: Ki67 immunohistochemical staining of subcutaneous tumors of blank group, saline group and Α1 (ΟΗ) group 3 mice
空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠按实施例 2所述方法给予处理， 6周 后处死小鼠， 取出皮下肿瘤组织， 以福尔马林液固定， 石蜡包埋， 制作切片并进 行 Ki67免疫组化染色。 Ki67是一种增殖细胞相关的核抗原， 其功能与有丝分裂 密切相关， 在细胞增殖中是不可缺少。 Ki67 作为标记细胞增殖状态的抗原， 其 阳性染色说明细胞增殖活跃。 Ki67 染色方法： 1)肿瘤组织石蜡切片脱蜡至水； 2)3%H202 室温 lOmin,水洗； 3)抗原修复； 4)山羊血清封闭 30min; 5)滴加 Ki67 抗体 ( 1 :100, 购自 Cell Signaling Technology公司 )， 4°C过夜; 6)滴加辣根过氧 化物酶标记的二抗 （购自上海长岛生物公司） 37°C 30min; 7)DAB 显色 （购自 DAKO公司）； 8)苏木素复染， 盐酸酒精分化； 9)脱水封片。  The blank group, the saline group and the Α1 (ΟΗ) group 3 mice were treated as described in Example 2. After 6 weeks, the mice were sacrificed, and the subcutaneous tumor tissues were removed, fixed in formalin, embedded in paraffin, and produced. Sections were taken and subjected to Ki67 immunohistochemical staining. Ki67 is a proliferating cell-associated nuclear antigen whose function is closely related to mitosis and is indispensable in cell proliferation. Ki67 is an antigen that marks the proliferative state of cells, and its positive staining indicates that cell proliferation is active. Ki67 staining method: 1) Deparaffinization of paraffin sections of tumor tissue to water; 2) 3% H202 room temperature lOmin, water washing; 3) antigen retrieval; 4) goat serum blocking for 30 min; 5) addition of Ki67 antibody (1:100, purchased from Cell Signaling Technology), overnight at 4 °C; 6) Adding horseradish peroxidase-labeled secondary antibody (purchased from Shanghai Changdao Biotechnology Co., Ltd.) at 37 ° C for 30 min; 7) DAB color development (purchased from DAKO); 8) Hematoxylin counterstaining, hydrochloric acid alcohol differentiation; 9) Dehydration sealing.
如图 5所示， 与空白组和生理盐水组相比， Α1(ΟΗ)3组小鼠皮下肿瘤中 Ki67 染色阳性细胞明显减少， 证明注射 Α1(ΟΗ)3明显抑制小鼠体内肝癌细胞的增殖。 实施例 6: 空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠皮下肿瘤的 TUNEL细胞凋 亡检测 As shown in Fig. 5, compared with the blank group and the saline group, the Ki67-staining positive cells in the subcutaneous tumors of the Α1(ΟΗ)3 group were significantly reduced, indicating that the injection of Α1(ΟΗ) 3 significantly inhibited the proliferation of hepatoma cells in mice. . Example 6: Detection of apoptosis in TUNEL cells of subcutaneous tumors of blank group, saline group and Α1(ΟΗ)3 group
空白组、 生理盐水组和 Α1(ΟΗ)3组小鼠按实施例 2所述方法给予处理， 6周 后处死小鼠， 取出皮下肿瘤组织， 以福尔马林液固定， 石蜡包埋， 制作切片并进 行 TUNEL细胞凋亡检测 （ TUNEL FITC染色试剂盒购自上海睿安生物， 实验步 骤参见试剂盒说明书）。 TUNEL法是应用末端脱氧核糖核苷酸转移酶的作用在凋 亡细胞断裂 DNA的末端催化掺入荧光素 FITC标记的脱氧三磷酸尿苷， 进而在 荧光显微镜下直接观察 FITC标记细胞， 即为凋亡细胞。  The blank group, the saline group and the Α1 (ΟΗ) group 3 mice were treated as described in Example 2. After 6 weeks, the mice were sacrificed, and the subcutaneous tumor tissues were removed, fixed in formalin, embedded in paraffin, and produced. The sections were subjected to TUNEL apoptosis detection (TUNEL FITC staining kit was purchased from Shanghai Rui'an Biotechnology, and the experimental procedure is described in the kit manual). The TUNEL method uses the action of terminal deoxyribonucleotidyl transferase to catalyze the incorporation of fluorescein FITC-labeled deoxyuridine triphosphate at the end of apoptotic cell cleavage DNA, and then directly observe FITC-labeled cells under a fluorescence microscope. Dead cells.
如图 6 所示， 与空白组和生理盐水组相比， Α1(ΟΗ)3 组小鼠皮下肿瘤中 TUNEL FITC染色阳性细胞明显增多， 证明注射 Α1(ΟΗ)3明显诱导小鼠体内肝癌 细胞的凋亡。 As shown in Fig. 6, compared with the blank group and the saline group, the number of TUNEL FITC-positive cells in the subcutaneous tumor of the Α1(ΟΗ)3 group was significantly increased, indicating that injection of Α1(ΟΗ) 3 significantly induced hepatocarcinoma cells in mice. Apoptosis.
实施例 7： 铝佐剂治疗方案的确立及机制研究 Example 7: Establishment and Mechanism Study of Aluminum Adjuvant Treatment
选取 5-6周龄、 雄性 C57BL/6J小鼠 21只， 随机分为 3组， 每组 7只， 釆 用皮下注射方式每只小鼠分别接种 1 X 107个 Hepal-6细胞， 两组实验组小鼠分 别于接种后 7, 11天开始给予 Α1(ΟΗ)3溶液 （溶于生理盐水）， 0.25mg/0.25mL/ 只，每隔 3天一次，腹腔注射。对照组于接种后 7天给予等量的生理盐水 (0.9%NaCl 液体)， 每隔 3天一次， 腹腔注射。 给药后每周测量各组小鼠皮下肿瘤大小两次， 并计算肿瘤体积（肿瘤体积 =肿瘤长径 X 肿瘤短径 2/ 2 )。最后一次免疫后 3天， 处死所有小鼠， 取其脾脏经研磨， 裂红等处理， 获得单细胞悬疑， 进行中性粒细 胞， 巨噬细胞， N K , C D 4 , C D 8 T , T r e g等细胞流式检测。 肿瘤组织 经剪切， 胶原酶和 DNA酶消化处理后获单细胞悬液， 进行中性粒细胞和巨噬细 胞的流式检测。 并取部分肿瘤组织进行病理切片， 参照实例 2进行 HE染色。 Twenty-one male C57BL/6J mice, 5-6 weeks old, were randomly divided into 3 groups, 7 rats in each group. Each mouse was inoculated with 1×10 7 Hepal-6 cells by subcutaneous injection. In the experimental group, Α1(ΟΗ)3 solution (dissolved in physiological saline), 0.25 mg/0.25 mL/only, once every 3 days, was intraperitoneally injected at 7, 11 days after inoculation. The control group was given an equal amount of normal saline (0.9% NaCl) 7 days after inoculation. Liquid), once every 3 days, intraperitoneal injection. The subcutaneous tumor size of each group of mice was measured twice a week after administration, and the tumor volume (tumor volume = tumor long diameter X tumor short diameter 2 / 2) was calculated. Three days after the last immunization, all the mice were sacrificed, and the spleen was treated with grinding, red cleavage, etc. to obtain single cell suspense, neutrophils, macrophages, NK, CD 4 , CD 8 T , T reg, etc. Cell flow detection. The tumor tissue was subjected to shearing, collagenase and DNase digestion treatment to obtain a single cell suspension, and flow cytometry of neutrophils and macrophages was performed. A part of the tumor tissue was taken for pathological sectioning, and HE staining was performed with reference to Example 2.
如图 7-1所示， 当接种后 7天开始给予 Α1(ΟΗ)3后， 小鼠皮下肿瘤生长速度 明显减慢， 体积明显缩小。 而生理盐水组和 Α1(ΟΗ)311天治疗组小鼠的皮下肿瘤 生长较迅速， 体积明显增大。 实验证实接种后 7天开始给予 Α1(ΟΗ)3 治疗可明 显抑制小鼠体内肝癌细胞的生长。 As shown in Figure 7-1, when Α1(ΟΗ)3 was administered 7 days after inoculation, the growth rate of subcutaneous tumors in mice was significantly slowed down and the volume was significantly reduced. In the saline group and the Α1(ΟΗ) 3 11-day treatment group, the subcutaneous tumors grew rapidly and the volume increased significantly. The experiment confirmed that the administration of Α1(ΟΗ)3 treatment starting 7 days after inoculation significantly inhibited the growth of liver cancer cells in mice.
经过对小鼠脾脏和肿瘤中细胞进行流式分析， 结果显示， 小鼠脾脏中中性粒 细胞（ C D 1 1 b + G r 1 + )随肿瘤的增大而增多， 而肿瘤中的中性粒细胞则 随肿瘤的增大而减少， （如图 7-2 )对切片进行 HE染色可见 Α1(ΟΗ)3 7天治疗组 肿瘤内有大量的中性粒细胞， 其形态为具典型的分叶核的细胞（如图 7-4 )。 在显 微镜下观察肿瘤的切片结果可见： 1 ) 100倍下发现注射铝佐剂会引起肿瘤内细 胞的坏死， Α1(ΟΗ)3 7天治疗组的坏死比 11天治疗组的程度严重， 生理盐水组未 见任何坏死； 2) 400倍下可见 Α1(ΟΗ)3 7天治疗组的肿瘤内有大量的中性粒细胞 的侵润， 而在生理盐水组和 Α1(ΟΗ)3 11天治疗组的小鼠肿瘤内少有中性粒细胞 的侵润， 几乎全是肿瘤细胞； 3 ) Α1(ΟΗ)3 7 天治疗组的肿瘤内除了坏死， 还见 有坏死后的纤维化修复， 而在其他两组则见到大量的肿瘤细胞处于核分裂相， 表 明肿瘤细胞生长旺盛（如图 7-4 )。 鉴于中性细胞具有抗肿瘤的作用， 据文献报道 肿瘤微环境内的 TGF- β可诱导中性粒细胞的功能发生变化， 发挥促肿瘤生长的 功能， 当阻断 TGF- β后， 中性粒细胞则具有抑制肿瘤生长的功能； 从本实验 7 天和 11天治疗组的效果差异可能也是因为肿瘤微环境的变化所致。  Flow cytometry analysis of mouse spleens and tumor cells showed that neutrophils (CD 1 1 b + G r 1 + ) in the spleen of mice increased with tumor enlargement, while neutral in tumors The granulocytes decreased with the increase of tumors (Fig. 7-2). HE staining of the sections showed that Α1(ΟΗ)3 7 days of treatment, there were a large number of neutrophils in the tumor, and the morphology was typical. The cells of the nucleus (Figure 7-4). The results of microscopic observation of the tumor were as follows: 1) 100 times of the injection of aluminum adjuvant was found to cause necrosis of cells in the tumor, and the necrosis of the Α1(ΟΗ)3 7 day treatment group was severer than that of the 11-day treatment group. No necrosis was observed in the group; 2) 4001(ΟΗ)3 was observed at 400 times. There was a large amount of neutrophil infiltration in the tumor in the 7-day treatment group, but in the saline group and Α1(ΟΗ)3 11-day treatment group. There is little neutrophil infiltration in the tumor of the mouse, almost all tumor cells; 3) Α1(ΟΗ)3 7 days in the treatment group, except for necrosis, there is also fibrotic repair after necrosis, but in In the other two groups, a large number of tumor cells were found to be in the mitotic phase, indicating that the tumor cells grew vigorously (Figure 7-4). In view of the anti-tumor effect of neutrophils, it has been reported in the literature that TGF-β in the tumor microenvironment can induce changes in the function of neutrophils and play a role in promoting tumor growth. When blocking TGF-β, neutrophils The cells have the function of inhibiting tumor growth; the difference in the effect of the treatment group from 7 days and 11 days in this experiment may also be due to changes in the tumor microenvironment.

Claims

权利 要 求 Rights request
1.氢氧化铝 [Al(OH)3]化合物作为单独唯一成分在制备治疗肝癌药物中的用途。 1. Use of an aluminum hydroxide [Al(OH) 3 ] compound as a sole sole component in the preparation of a medicament for treating liver cancer.
2.根据权利要求 1所述的用途， 其特征在于， 所述的氢氧化铝抑制肝癌细胞的体 内增殖。  The use according to claim 1, wherein the aluminum hydroxide inhibits in vivo proliferation of liver cancer cells.
3.根据权利要求 1所述的用途， 其特征在于， 所述的氢氧化铝诱导中性粒细胞浸 润肿瘤和诱导肿瘤细胞凋亡。  The use according to claim 1, characterized in that the aluminum hydroxide induces neutrophil infiltration of the tumor and induces apoptosis of the tumor cells.
4.根据权利要求 1所述的用途， 其特征在于， 氢氧化铝作为单独成分联合其他有 效的抗肝癌药物在制备肝癌治疗药物中的应用。  The use according to claim 1, characterized in that aluminum hydroxide is used as a separate component in combination with other effective anti-hepatocarcinoma drugs for the preparation of a medicament for treating liver cancer.
5.根据权利要求 1所述的用途， 其特征在于， 氢氧化铝作为单独成分联合其他有 效的肝癌干预方法在制备肝癌综合治疗措施中的应用。  The use according to claim 1, characterized in that the use of aluminum hydroxide as a separate component in combination with other effective liver cancer intervention methods in the preparation of a comprehensive therapeutic measure for liver cancer.
6.根据权利要求 5所述的用途， 其特征在于， 所述的其他有效的肝癌干预方法选 自手术或放射干预方法。  6. Use according to claim 5, characterized in that said other effective liver cancer intervention method is selected from a surgical or radiological intervention method.
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