WO2021104086A1 - 一种克服肿瘤耐药的方法 - Google Patents

一种克服肿瘤耐药的方法 Download PDF

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WO2021104086A1
WO2021104086A1 PCT/CN2020/129165 CN2020129165W WO2021104086A1 WO 2021104086 A1 WO2021104086 A1 WO 2021104086A1 CN 2020129165 W CN2020129165 W CN 2020129165W WO 2021104086 A1 WO2021104086 A1 WO 2021104086A1
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tumor
cyanobacteria
cancer
drug
laser
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French (fr)
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蔡林涛
何华美
刘兰兰
梁锐晶
周海梅
潘宏
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

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  • the invention belongs to the field of biomedicine, and specifically relates to a method for overcoming the drug resistance of tumors.
  • Chemotherapy is one of the main methods for the treatment of malignant tumors.
  • the biggest advantage of chemotherapy compared with surgery and radiotherapy is that chemotherapy is a systemic treatment method.
  • chemotherapy drugs can reach the whole body through the human circulatory system, so they have a killing effect on the primary cancer lesions and metastatic lesions and even free circulating tumor cells.
  • drugs Through natural extraction, artificial synthesis and a large number of screenings, many drugs have been approved by the U.S. Food and Drug Administration to enter clinical applications.
  • common chemotherapy drugs are doxorubicin, 5-fluorouracil, camptothecin, paclitaxel, and etoposide. , Cis-molybdenum and so on.
  • chemotherapeutic drugs kill tumor cells, they will inevitably kill normal cells, affect the function of normal tissues and organs, produce greater toxic side effects, and cause physical and psychological trauma to the patient during the treatment.
  • most traditional chemotherapy drugs have poor water solubility and small molecular weight. These drugs have poor pharmacokinetics in the body and short blood circulation time, which limits the chemotherapy time window. The complex environment in the body will also change the structure and properties of the drugs. It is difficult to give full play to the anti-cancer effect. What's more serious is that during chemotherapy, tumors can adjust themselves to defend against or adapt to the killing effect of chemotherapy drugs on tumors, resulting in resistance to a single drug or multiple drugs.
  • Tumor drug resistance often leads to chemotherapy failure.
  • Tumor drug resistance can be inherent (primary drug resistance), or it can be caused by tumor cells repeatedly exposed to chemotherapy drugs during chemotherapy (acquired drug resistance). Once tumor drug resistance is formed, the drug cannot exert its anti-cancer effect. Even if most of the tumor cells are killed, the remaining small part of drug-resistant cells will continue to grow, causing cancer recurrence and failure of subsequent chemotherapy. Drug tolerance is a big problem in cancer chemotherapy. Therefore, in order to completely eliminate cancer cells, we must first study the biological principles of tumor cell resistance and reveal the mystery of tumor cells' immortality. The formation mechanism of multidrug resistance is very complicated, and its occurrence is the result of multiple effects of tumor cells from the cell membrane, cytoplasm and nucleus.
  • Tumor drug resistance is an important defense mechanism for tumors against drugs.
  • Tumor drug resistance mechanisms are relatively complex and can be roughly summarized into the following categories: decreased intake of chemotherapeutic drugs; increased efflux of chemotherapeutic drugs; decreased drug activation.
  • Enhanced cell detoxification Enhanced DNA damage repair function
  • inhibition of cell apoptosis enhanced activation of anti-apoptotic system
  • drug vesicle isolation etc.
  • the effect of drugs in killing tumors depends on their accumulated concentration in the tumor.
  • the decrease in the intake of chemotherapeutic drugs by tumor cells weakens the drug's killing effects on tumors. Many drugs enter the tumor by free diffusion or active transportation.
  • the strategies for reversing tumor drug resistance mainly include the following aspects.
  • the first is the structural transformation and modification of existing anti-tumor drugs, and the development of new anti-tumor drugs that are not prone to drug resistance.
  • the limitation of this strategy is that its implementation is limited by the structure of existing drugs, which makes it difficult to implement.
  • the second is biotechnology-related methods to reverse tumor resistance, such as siRNA therapy, immunotherapy and so on.
  • These biotechnologies also have many shortcomings, such as the short half-life of biopharmaceuticals, instability in the body, difficulty in enriching in tumor tissues, etc., and the development cycle of biotechnology is long and the production cost is high.
  • the more commonly used methods are the application of chemical methods to reverse tumor resistance, such as the development of ABC transporter inhibitors.
  • reversal inhibitors that block the function of P glycoprotein have been developed more, such as verapamil, quinidine, and cyclosporine. Supplement A, etc., this type of reversal inhibitor can block the pumping mechanism of glycoprotein by binding to P glycoprotein, increase the accumulation of intracellular drugs, and reverse the efflux of drugs.
  • these reversal inhibitors are difficult to achieve the effective reversal concentration of in vitro experiments in in vivo experiments, and the drug utilization rate is low; more importantly, due to poor targeting, reversal inhibitors will also interact with P glycoprotein in healthy tissues. It can cause the drug to produce toxic and side effects on healthy tissues, so it cannot be put into clinical use.
  • the invention discloses a cyanobacteria that exists in nature itself, which can improve tumor hypoxia through photosynthesis under laser irradiation, and can effectively overcome the problem of drug resistance such as doxorubicin. Since cyanobacteria photosynthesize and produce oxygen under light, under aerobic conditions, tumor cells can suppress multidrug resistance genes and inhibit the expression of P-glycoprotein to inhibit adriamycin by downregulating hypoxia and hypoxia factors. The efflux function can overcome the resistance of cancer cells to adriamycin and improve the anti-tumor efficacy.
  • An object of the present invention is to provide a composition for improving and/or overcoming tumor drug resistance, said composition comprising live cyanobacteria, said composition being capable of generating oxygen when irradiated with light, and said light irradiation is Laser irradiation with a wavelength of 400-700 nm; preferably, the light irradiation is laser irradiation with a wavelength of 660 nm.
  • Another object of the present invention is to provide an application of cyanobacteria in preparing a composition for improving and/or overcoming tumor drug resistance.
  • Another object of the present invention is to provide an application of cyanobacteria in the preparation of a reagent for improving the efficacy of an anti-tumor drug that causes tumor drug resistance due to hypoxia.
  • Another object of the present invention is to provide an application of cyanobacteria in the preparation of reagents or drugs for combined treatment of tumor resistance.
  • the combined treatment includes any of photothermal therapy, photodynamic therapy, chemotherapy, and radiotherapy.
  • One or more combination therapy One or more combination therapy.
  • the cyanobacteria can produce oxygen under laser irradiation, which can improve and/or overcome tumor drug resistance caused by hypoxia, the laser wavelength is 400-700 nm; preferably, the laser wavelength is 660 nm.
  • the drug in the tumor resistance is a chemotherapeutic drug
  • the chemotherapeutic drug is selected from doxorubicin or its derivatives, 5-fluorouracil, camptothecin, paclitaxel, vinblastine, etoposide, Any one or more of cisplatin, platinum drugs, methotrexate (MTX), CTX, ACNU, BCNU, CCNU, irinotecan (CPT11), and more preferably, the chemotherapeutic drug is adriamycin or its derivative.
  • the tumor is selected from basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, Pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma.
  • Another object of the present invention is to provide a method for improving and/or overcoming the drug resistance of tumor cells in vitro, which includes the following steps:
  • the culture medium is BG11;
  • step (2) Take the cyanobacteria obtained in step (1), co-culture the cyanobacteria and the hypoxic tumor cell line. After irradiating with a wavelength of 400-700 nm laser, continue the culture to detect the infiltration status of anti-tumor drugs by increasing oxygen .
  • the laser is irradiated with a laser with a wavelength of 660 nm for 5 minutes; the density of the cyanobacteria is 2 ⁇ 10 7 cfu/mL, the tumor cells are breast cancer cells, and the Anti-tumor drugs are selected from doxorubicin or its derivatives, 5-fluorouracil, camptothecin, paclitaxel, vinblastine, etoposide, cisplatin, platinum drugs, methotrexate (MTX), CTX, ACNU, BCNU Any one or more of, CCNU, and irinotecan (CPT11); more preferably, the anti-tumor drug is adriamycin or its derivatives.
  • Another object of the present invention is to provide an application of cyanobacteria in studying the mechanism of tumor resistance.
  • the invention develops a new function of cyanobacteria that naturally exists in nature, and aims to solve the technical problem of drug resistance of cancer cells to drugs caused by hypoxia. details as follows:
  • Cyanobacteria themselves can produce oxygen under 660 nm laser irradiation, which is an ideal "living drug", which can be used to increase oxygen in the tumor's hypoxic microenvironment, overcome tumor resistance to adriamycin to enhance The killing effect of adriamycin on tumors;
  • Cyanobacteria are widely distributed in freshwater and marine environments. It is very simple and easy to obtain materials. Moreover, cyanobacteria have very simple nutritional requirements. They only need light, water, carbon dioxide and inorganic salts. The growth rate is fast, the cultivation is convenient, the cost is low, and it is relatively easy. The advantages of genetic manipulation methods make it suitable for a wide range of clinical applications.
  • cyanobacteria (4) The use of cyanobacteria is safe and controllable. Without light, cyanobacteria will not grow. It is easy to control and can ensure safe use in the body.
  • Figure 1 is a laser confocal image of cyanobacteria in vitro to increase oxygenation and increase doxorubicin into cells.
  • the cyanobacteria and laser irradiation group can significantly increase doxorubicin into cells, compared with the normal breast cancer cell doxorubicin.
  • the initiation is very similar.
  • Figure 2 is a laser confocal image of cyanobacteria increasing oxygenation in the body to increase the depth of adriamycin into the tumor.
  • the fluorescence intensity and area of the cyanobacteria/adriamycin plus laser group are significantly higher than those of the adriamycin plus laser group alone. large area.
  • Preparation for experiment prepare a 500 ml Erlenmeyer flask, pour cyanobacteria and sterilized BG11 medium into the aseptic operation table, seal the bottle mouth with a ventilable sealing film, and place it in the sun to cultivate and expand.
  • MCF-7 Place hypoxic breast cancer cells (MCF-7) and normal breast cancer cells (MCF-7) in a confocal dish and put them in an incubator (hypoxic cells are placed in a hypoxic incubator, normal Put the cells in a normal incubator) and culture for 24 hours.
  • an incubator hyperoxic cells are placed in a hypoxic incubator, normal Put the cells in a normal incubator
  • DOX and incubate for 1 hour. Stain the nucleus and shoot confocal.
  • MCF-7 breast cancer cells with a density of 1 ⁇ 10 7 cells/mL were resuspended in 100 ⁇ l of serum-free DMEM medium and planted under the skin of Balb/c nude mice.
  • the tumor volume was about 200mm 3
  • the tail vein was injected with 2 ⁇ 10 8 cfu/mL cyanobacteria 100 ⁇ l (the other group did not need to be injected with cyanobacteria).
  • the mice in the cyanobacteria group were given 660nm laser irradiation for 30 minutes. After 24 hours, the tumor was taken out, sectioned, stained with cell nucleus, and confocal shot.

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Abstract

提供了一种用于改善和/或克服肿瘤耐药性的组合物，该组合物包括活蓝细菌，该组合物在光照射时能够产氧气。还提供了一种蓝细菌在在制备用于提高因缺氧导致肿瘤耐药性的抗肿瘤药物的药效的试剂中的应用，以及在制备用于肿瘤耐药性联合治疗的试剂或药物中的应用。

Description

一种克服肿瘤耐药的方法 技术领域
本发明属于生物医药领域，具体涉及一种克服肿瘤耐药的方法。
 背景技术
化疗是治疗恶性肿瘤的主要方法之一，化学疗法与手术疗法、放射疗法相比最大的优点在于化疗是一种全身性治疗方法。通过口服、注射等方式，化疗药物可以通过人体循环系统到达全身，因此对癌症原发病灶和转移病灶甚至游离的循环肿瘤细胞都有杀伤作用。通过自然提取、人工合成和大量的筛选，许多药物己被美国食品与药品监督管理局批准进入临床应用，目前常见的化疗药物有阿霉素、５－氟尿嘧啶、喜树碱、紫杉醇、依托泊苷、顺钼等。然而化疗药物在杀伤肿瘤细胞的同时，也会不可避免杀伤正常细胞，影响正常组织器官的功能，产生较大的毒副作用，并使患者在治疗过程中产生生理和心理上的创伤。此外，多数传统化疗药物水溶性差，分子量小，这些药物在体内的药代动力学状况差，血液循环时间短，限制了化疗时间窗，体内复杂的环境也会改变药物的结构和性质，使其难以充分发挥抗癌效果。更严重的是，在化疗过程中，肿瘤可通过自身调整以防御或适应化疗药物对肿瘤的杀伤作用，产生对单一药物或多种药物的耐药性。
肿瘤的耐药性常常导致化疗失败，肿瘤耐药性可以是固有的（原发性耐药），也可以是化疗过程中肿瘤细胞屡次接触化疗药物的过程中产生的（获得性耐药）。肿瘤耐药一旦形成，药物就不能发挥抗癌作用。即使大部分的肿瘤细胞被杀死了，剩余的小部分耐药细胞依然会继续生长，造成癌症的复发，并使得后续的化疗失败。药物耐受性是癌症化疗的大难题。因此，要想彻底消灭癌细胞，首先要研究肿瘤细胞产生耐药性的生物学原理，揭示瘤细胞的不死之谜。多药耐药性的形成机制非常复杂，它的发生是肿瘤细胞从细胞膜、细胞质和细胞核内产生的多种影响综合作用的结果。到目前为止，仍然没有能够应用于临床的克服肿瘤耐药性的药物和方法。肿瘤的耐药性是肿瘤对药物产生的重要防御机制，肿瘤耐药机制的产生比较复杂，大致可归纳为以下几类：化疗药物摄取量减少；化疗药物外排量增加；药物活化作用减弱，细胞解毒作用增强；DNA损伤修复功能增强；细胞凋亡的抑制，抗凋亡系统活化增强；药物的囊泡隔离等。药物杀伤肿瘤的作用依赖于其在肿瘤内的积累浓度，肿瘤细胞对化疗药物摄取量的减少使药物杀伤肿瘤作用减弱。许多药物以自由扩散或主动运输的方式进入肿瘤，研究者从对甲氨蝶呤的递送实验中发现，载体蛋白的结构改变使药物与载体蛋白的结合能力下降导致细胞对化疗药物摄取减少从而致使肿瘤耐药性产生。而且研究发现，机体的解毒机制和清除机制会随着药物的进入被激活，导致药物代谢加快，使细胞摄取药物量减少，导致耐药。许多以DNA为作用靶点的抗肿瘤药物如阿霉素、甲氨蝶呤、环磷酰胺等，作用机制是破坏DNA的结构和功能，抑制DNA复制和转录，使肿瘤细胞不能正常增殖导致死亡。但是随着化疗药物的广泛使用，DNA修复能力不断增强，致使肿瘤耐药性产生。
目前，逆转肿瘤耐药性的策略主要有以下几个方面，首先是对已有的抗肿瘤药物进行结构的改造及修饰，开发不容易产生耐药性的新的抗肿瘤药物。但该策略的局限在于实施受已有药物结构的限制，难度较大。其次是与生物技术相关的逆转肿瘤耐药性的方法，如siRNA治疗、免疫治疗等。这些生物技术也存在很多不足，如生物药物的半衰期较短、在体内不稳定、难以富集到肿瘤组织等，而且生物技术开发周期长、生产成本高。比较常用的手段是应用化学手段逆转肿瘤耐药性，如ABC转运蛋白抑制剂的开发，其中阻断P糖蛋白功能的逆转抑制剂开发较多，如维拉帕米、奎尼丁、环孢素A等，该类逆转抑制剂可以通过与P糖蛋白结合，阻断糖蛋白的泵药机制，增加细胞内药物的积聚，逆转药物的外排。但是，在进一步研究中发现这些逆转抑制剂在体内实验中难以达到体外实验的有效逆转浓度，药物利用率低；更重要的是由于靶向性差，逆转抑制剂也会与健康组织的P糖蛋白作用，导致药物对健康组织产生毒副作用，因此还不能投入临床使用。如何提高化疗药物的生物利用度，增加其靶向肿瘤的机会，降低在非靶向部位的分布是提高抗肿瘤药物疗效的关键点。近年来，发展纳米技术，尤其是集中于开发纳米药物递送系统以作为逆转肿瘤耐药性的治疗策略，正引起广泛关注。我们通过一种在激光下光合作用产氧的蓝细菌改善肿瘤缺氧微环境克服阿霉素化疗耐药来提高肿瘤治疗效率，为癌症治疗提供新的选择。
 技术问题
本发明公开了一种自然界本身存在的蓝细菌，在激光照射下通过光合作用产氧改善肿瘤缺氧，可以有效克服阿霉素等化学药耐药问题。由于蓝细菌在光下进行光合作用产氧，在有氧条件下，肿瘤细胞下调低氧缺氧因子可以抑制多药耐药基因，抑制P-糖蛋白的表达来抑制癌细胞对阿霉素的外排作用，从而克服癌细胞对阿霉素的耐药性，提高抗肿瘤疗效。
 技术解决方案
本发明的一个目的在于提供一种用于改善和/或克服肿瘤耐药性的组合物，所述组合物包括活蓝细菌，所述组合物在光照射时能够产生氧气，所述光照射为400-700 nm波长的激光照射；优选地，光照射为660 nm波长的激光照射。
本发明的另一目的在于提供一种蓝细菌在制备改善和/或克服肿瘤耐药性的组合物中的应用。
本发明的另一目的在于提供一种蓝细菌在制备用于提高因缺氧导致肿瘤耐药性的抗肿瘤药物的药效的试剂中的应用。
本发明的另一目的在于提供一种蓝细菌在制备用于肿瘤耐药性联合治疗的试剂或药物中的应用，所述联合治疗包括光热治疗、光动力治疗、化疗、放疗中的任一种或多种的联合治疗。
优选地，所述蓝细菌在激光照射下能够产生氧气，可以改善和/或克服因缺氧导致的肿瘤耐药性，所述激光波长为400-700 nm；优选地，所述激光波长为660 nm。
优选地，所述肿瘤耐药性中的药物为化疗药物，较佳地所述化疗药物选自阿霉素或其衍生物、５－氟尿嘧啶、喜树碱、紫杉醇、长春碱、依托泊苷、顺钼、铂类药物、甲氨蝶呤（MTX）、CTX、ACNU、BCNU、CCNU、伊立替康（CPT11）中任一种或多种，更佳地所述化疗药物为阿霉素或其衍生物。
优选地，所述肿瘤选自基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤。
本发明的另一目的在于提供一种体外改善和/或克服肿瘤细胞耐药性的方法，包括如下步骤：
（1）体外培养和扩增蓝细菌，所述培养的培养基为BG11；
（2）取步骤（1）获得的蓝细菌，将蓝细菌和缺氧的肿瘤细胞系共培养，经波长400-700 nm激光照射后，继续培养，检测增氧改善抗肿瘤药物的入胞状况。
优选地，所述步骤（2）中所述激光为波长为660 nm的激光照射5分钟；所述蓝细菌的密度为2×10 7 cfu/mL，所述肿瘤细胞为乳腺癌细胞，所述抗肿瘤药物选自阿霉素或其衍生物、５－氟尿嘧啶、喜树碱、紫杉醇、长春碱、依托泊苷、顺钼、铂类药物、甲氨蝶呤（MTX）、CTX、ACNU、BCNU、CCNU、伊立替康（CPT11）中任一种或多种；更佳地，所述抗肿瘤药物为阿霉素或其衍生物。
本发明的另一目的在于提供一种蓝细菌在研究肿瘤耐药性机理中的应用。
 有益效果
本发明开发了一种自然界天然存在的蓝细菌的新功能，旨在解决因缺氧导致的癌细胞对药物的耐药性的技术问题。具体如下：
（1）首次报道了利用蓝细菌的增氧作用来克服肿瘤细胞对化疗药物，尤其是阿霉素的耐药；
（2）蓝细菌本身在660 nm激光照射下能够产生氧气，是一种理想的“活体药物”，可以用于肿瘤缺氧微环境的增氧，克服肿瘤对阿霉素产生的耐药来增强阿霉素对肿瘤的杀伤作用；
（3）蓝细菌广泛分布淡水和海洋环境，取材非常简单容易，而且蓝细菌具有营养需求非常简单，仅需光、水、二氧化碳和无机盐，增长速度快、培养方便、成本低廉及相对容易的遗传操作手段等优点，使其适宜在临床中广泛应用。
（4）蓝细菌使用安全可控，不给予光照蓝细菌就不会生长，容易控制，能够保证体内使用安全。
 附图说明
图1 是蓝细菌在体外实现增氧增加阿霉素入胞的激光共聚焦图，其中蓝细菌及激光照射组能够显著增加阿霉素的入胞，与正常状态下的乳腺癌细胞阿霉素入胞非常相近。
图2 是蓝细菌在体内增氧增加阿霉素进入肿瘤深度的激光共聚焦图，其中蓝细菌/阿霉素加激光组的荧光强度和面积明显比单纯阿霉素加激光组的荧光强度和面积大。
 本发明的实施方式
以下通过具体实施例对本发明作进一步详细说明，以使本领域技术人员能够更好地理解本发明并予以实施，但实施例并不作为本发明的限定。
以下实施例中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例
准备实验：准备一个500毫升的三角瓶，在无菌操作台中倒入蓝细菌及灭菌的BG11培养基，用可换气的封口膜封住瓶口，放在太阳光下培养扩增。
实验一：体外增氧增加阿霉素入胞实验
在共聚焦皿中铺入缺氧的乳腺癌细胞（MCF-7）及正常状态的乳腺癌细胞（MCF-7）并放入培养箱（缺氧的细胞放在缺氧培养箱，正常状态的细胞放入正常的培养箱）中培养24h，缺氧状态的细胞加入2×10 7cfu/mL蓝细菌或者没有加蓝细菌，660nm激光照射或者没有激光照射并培养24h，加入DOX共孵育1h，染细胞核，拍共聚焦。
共聚焦的结果如图1所示，蓝细菌及激光照射组，改善乳腺癌细胞的缺氧状态，增加阿霉素的入胞，其结果与正常状态下的乳腺癌细胞阿霉素入胞非常相近。
实验二：体内增氧增加阿霉素进入肿瘤深度实验
在体内，取密度为1×10 7个/mL的MCF-7乳腺癌细胞用100µl无血清的DMEM培养基重悬，种植在Balb/c nude小鼠皮下中，待肿瘤体积大概200mm 3时，尾静脉注射2×10 8cfu/mL蓝细菌100µl（另外一组不用注射蓝细菌），24h后注射蓝细菌组的小鼠给予660nm激光照射肿瘤30分钟，两组小鼠都尾静脉注射阿霉素，24h后，取出肿瘤，切片，染细胞核，拍共聚焦。
共聚焦的结果如图2所示，蓝细菌/阿霉素加激光组的结果相比单纯阿霉素组的肿瘤，其阿霉素进入肿瘤深度更多，体内实验结果再次证明蓝细菌在660nm激光照射下能够显著改善阿霉素的肿瘤耐药性。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之。

Claims (10)

  1. 一种用于改善和/或克服肿瘤耐药性的组合物，所述组合物包括活蓝细菌，所述组合物在光照射时能够产氧气，所述光照射为400-700 nm波长的激光照射；优选地，光照射为660 nm波长的激光照射。
  2. 一种蓝细菌在制备改善和/或克服肿瘤耐药性的组合物中的应用。
  3. 一种蓝细菌在制备用于提高因缺氧导致肿瘤耐药性的抗肿瘤药物的药效的试剂中的应用。
  4. 一种蓝细菌在制备用于肿瘤耐药性联合治疗的试剂或药物中的应用，所述联合治疗包括光热治疗、光动力治疗、化疗、放疗中的任一种或多种的联合治疗。
  5. 根据权利要求1所述的组合物，或权利要求2-4任一项所述的应用，其中所述蓝细菌在激光照射下能够产生氧气，可以改善和/或克服因缺氧导致的肿瘤耐药性，所述激光波长为400-700 nm；优选地，所述激光波长为660 nm。
  6. 根据权利要求1所述的组合物，或权利要求2-4任一项所述的应用，所述肿瘤耐药性中的药物为化疗药物，较佳地所述化疗药物选自阿霉素或其衍生物、５－氟尿嘧啶、喜树碱、紫杉醇、长春碱、依托泊苷、顺钼、铂类药物、甲氨蝶呤（MTX）、CTX、ACNU、BCNU、CCNU、伊立替康（CPT11）中任一种或多种，更佳地，所述化疗药物为阿霉素或其衍生物。
  7. 根据权利要求1所述的组合物，或权利要求2-4任一项所述的应用，其中所述肿瘤选自基底细胞癌、鳞状细胞癌、食管癌、恶性胶质瘤、膀胱癌、宫颈癌、乳腺癌、肺癌、肝癌、胃癌、结肠癌、直肠癌、鼻咽癌、胰腺癌、甲状腺癌、前列腺癌、白血病、淋巴瘤、肾脏肿瘤、肉瘤、母细胞瘤。
  8. 一种体外改善和/或克服肿瘤细胞耐药性的方法，包括如下步骤：
    （1）体外培养和扩增蓝细菌，所述培养的培养基为BG11；
    （2）取步骤（1）获得的蓝细菌，将蓝细菌和缺氧的肿瘤细胞系共培养，经波长400-700 nm激光照射后，继续培养，检测增氧改善抗肿瘤药物的入胞状况。
  9. 根据权利要求8所述的方法，所述步骤（2）中所述激光为波长为660 nm的激光照射5分钟；所述蓝细菌的密度为2×10 7 cfu/mL，所述肿瘤细胞为乳腺癌细胞，所述抗肿瘤药物选自阿霉素或其衍生物、５－氟尿嘧啶、喜树碱、紫杉醇、长春碱、依托泊苷、顺钼、铂类药物、甲氨蝶呤（MTX）、CTX、ACNU、BCNU、CCNU、伊立替康（CPT11）中任一种或多种；更佳地，所述抗肿瘤药物为阿霉素或其衍生物。
  10. 一种蓝细菌在研究肿瘤耐药性机理中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101433535A (zh) * 2007-11-16 2009-05-20 华东理工大学 一种用于肿瘤化学治疗中的新型增敏和增效物质
CN110755457A (zh) * 2019-11-28 2020-02-07 深圳先进技术研究院 一种克服肿瘤耐药的方法
CN110893196A (zh) * 2019-11-28 2020-03-20 深圳先进技术研究院 一种改善肿瘤缺氧的新方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101433535A (zh) * 2007-11-16 2009-05-20 华东理工大学 一种用于肿瘤化学治疗中的新型增敏和增效物质
CN110755457A (zh) * 2019-11-28 2020-02-07 深圳先进技术研究院 一种克服肿瘤耐药的方法
CN110893196A (zh) * 2019-11-28 2020-03-20 深圳先进技术研究院 一种改善肿瘤缺氧的新方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHENGDONG QIN, ZHENGGANG REN, ZHAOYOU TANG: "The role of hypoxic microenvironment in the progress of tumor Review", TUMOR, vol. 36, 1 January 2016 (2016-01-01), pages 96 - 102, XP055814486, DOI: 10.3781/j.issn.1000-7431.2016.55.563 *
HUO MINFENG, WANG LIYING, ZHANG LINLIN, WEI CHENYANG, CHEN YU, SHI JIANLIN: "Photosynthetic Tumor Oxygenation by Photosensitizer‐Containing Cyanobacteria for Enhanced Photodynamic Therapy", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, VERLAG CHEMIE, DE, vol. 59, no. 5, 27 January 2020 (2020-01-27), DE, pages 1906 - 1913, XP055813201, ISSN: 1433-7851, DOI: 10.1002/anie.201912824 *

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