WO2017071379A1 - Tumor vaccine for use in treating gastric cancer and preparation method for vaccine - Google Patents

Tumor vaccine for use in treating gastric cancer and preparation method for vaccine Download PDF

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WO2017071379A1
WO2017071379A1 PCT/CN2016/096035 CN2016096035W WO2017071379A1 WO 2017071379 A1 WO2017071379 A1 WO 2017071379A1 CN 2016096035 W CN2016096035 W CN 2016096035W WO 2017071379 A1 WO2017071379 A1 WO 2017071379A1
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cells
gastric cancer
exosomes
tumor
saha
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PCT/CN2016/096035
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French (fr)
Chinese (zh)
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李淑兰
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李淑兰
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants

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  • the invention belongs to the field of tumor vaccines, and particularly relates to a tumor vaccine for treating gastric cancer, and a preparation method of the gastric cancer vaccine.
  • Gastric cancer is the second most common cancer in the world. In the past 50 years, although the incidence and mortality of gastric cancer have declined in the global scale, especially in some developed countries, there are still about 1 million new cases every year. Due to the lack of early diagnosis, most patients with symptomatic gastric cancer are advanced at the time of diagnosis. At present, the traditional treatment methods are surgery, chemotherapy and radiotherapy. These treatments are invasive or toxic and side effects, and they can not effectively solve the problems of tumor metastasis and recurrence. Multidrug resistance often occurs after chemotherapy. In short, under traditional treatment, the prognosis of gastric cancer is still poor. The production of malignant tumors is not only due to the malignant transformation of cells, but also because tumor cells can escape the surveillance of the host immune system.
  • Tumor immunotherapy aims to control tumors by regulating the patient's immune system and exerting its own immune surveillance and defense functions.
  • special emphasis has been placed on the development and application of tumor vaccines, which have broad application prospects due to their induction of active specific immune responses.
  • the body's immune system specifically kills tumor cells by identifying antigenic targets of tumor cells.
  • Exosomes are a class of bilayer membranous vesicles that originate in the endocytic system and are excreted outside the cell, between 40 and 100 nm in diameter. Exosomes can be secreted by a variety of cells including dendritic cells, tumor cells, etc., containing a large number of proteins and lipid components closely related to its source and function, as an important carrier of information transmission between cells, participating in a variety of pathophysiological processes. . Tumor cell-derived exosomes contain important immune molecules such as tumor common antigen and thermophilin, which can exhibit anti-tumor effects through various pathways, and as a new type of tumor vaccine, it has obvious advantages over DC vaccine.
  • tumor-derived exosomes can inhibit or even destroy immune cells that play a role in tumors, such as down-regulating the expression of some NK receptors, affecting the activation of some innate immune cells in tumor immunity.
  • Others can significantly inhibit IL-2 and inhibit the proliferation of human lymphocytes, thus playing a negative role in the immunotherapy of tumors.
  • These tumor-derived exosomes may be the key factors for tumor tissue to escape the immune system clearance, which brings many difficulties and challenges to tumor immunotherapy. Therefore, how to improve the immunostimulatory ability of tumor cells derived from exosomes, and reduce its immunosuppressive ability has great practical significance in tumor immunotherapy.
  • a gastric cancer vaccine with improved therapeutic effect comprising an exosomes body secreted by gastric cancer cells, wherein the gastric cancer cells are treated by a combination of Salpichrolide U and SAHA, and the molar ratio of Salpichrolide U to SAHA is 0.8 to 1.2:1. .
  • gastric cancer vaccine with improved therapeutic effect further includes an adjuvant.
  • the adjuvant is an aluminum adjuvant.
  • the preparation method of the gastric cancer vaccine comprises the following steps: gastric cancer cells are cultured in a CO 2 incubator containing DMEM high-sugar medium containing fetal bovine serum, and the cells are monolayer adherent growth, and passaged once every 3 to 4 days. After the cells were grown to log phase, the cells were inoculated. After 24 hours of inoculation, the cells were treated with Salpichrolide U and SAHA. After 24 hours of culture, the culture supernatant was collected and stored at low temperature. The collected culture supernatant of gastric cancer cells was removed by centrifugation.
  • the cells are taken, and the supernatant is removed; the supernatant is removed by centrifugation, the supernatant is collected, concentrated by ultrafiltration, and the concentrate is centrifuged to transfer the separated and purified concentrate to a centrifuge tube, and ultracentrifuged at a low temperature to obtain an exosomes. Small body.
  • the preparation method comprises the steps of: gastric cancer cells are cultured in a DMEM high glucose medium containing 100 ml/L fetal bovine serum in a 50 ml/L CO 2 incubator at 37 ° C, and the cells are monolayer adherent growth. Passage once every 3 to 4 days, inoculate 3 ⁇ 10 6 /100 ml when cells are grown to log phase; use 1 ⁇ 10 -6 mol/L Salpichrolide U and 1 ⁇ 10 -6 mol/L after 24 hours of inoculation The cells were treated with SAHA. After 24 hours of culture, the culture supernatant was collected and stored at 4 ° C.
  • the collected gastric cancer cell culture supernatant was centrifuged for 10 min to remove the cells, and the supernatant was taken.
  • the cell debris was removed by centrifugation at 1500 g for 30 min.
  • the supernatant was concentrated by ultrafiltration through a 100 kU MWCO Centriplus centrifugal ultrafiltration tube, and concentrated at 1500 g for 30 min to obtain a concentrate.
  • the separated and purified concentrate was transferred to a centrifuge tube, and centrifuged at 100 kg for 60 min at a horizontal angle of 4 ° C.
  • the precipitate contains the exosomes body.
  • the gastric cancer vaccine is used in the preparation of a medicament for preventing gastric cancer.
  • nano-sized small vesicle exosomes secreted by untreated gastric cancer cells and soluble immune molecules thereof have significant inhibitory effects on lymphocyte proliferation function.
  • the present invention creatively uses Salpichrolide U and SAHA to treat gastric cancer cells, and the results show that the exosomes secreted by the treated gastric cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition.
  • the invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.
  • MFC cell line was cultured in a culture medium containing 10% fetal bovine serum, penicillin 100 IU/mL, streptomycin 100 ⁇ g/mL (DMEM high sugar), and cultured in a 37 ° C 5% CO 2 incubator. When in the logarithmic phase, inoculate at 3 ⁇ 10 6 /100 ml.
  • Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
  • Experimental grouping The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experiment Group 3 (Salpichrolide U and SAHA combined dosing).
  • control group no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1 ⁇ 10 -6 mol/L 24 h after inoculation, after 24 h of dosing
  • the culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1 ⁇ 10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation.
  • 1 ⁇ 10 -6 mol/L of Salpichrolide U and 1 ⁇ 10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
  • Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
  • Experimental group The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Salpichrolide U and SAHA combined dosing).
  • control group no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1 ⁇ 10 -6 mol/L 24 h after inoculation, after 24 h of dosing
  • the culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1 ⁇ 10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation.
  • 0.8 ⁇ 10 -6 mol/L of Salpichrolide U and 1 ⁇ 10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
  • Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
  • Experimental group The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Salpichrolide U and SAHA combined dosing).
  • control group no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1 ⁇ 10 -6 mol/L 24 h after inoculation, after 24 h of dosing
  • the culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1 ⁇ 10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation.
  • 1.2 ⁇ 10 -6 mol/L of Salpichrolide U and 1 ⁇ 10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
  • HMAC-CP7OG cryogenic ultracentrifuge 100 KU MW COMilliporeAmicon high recovery high flow tangential flow ultrafiltration centrifuge tube (Millipore).
  • Example 7 Detection of proliferation of PBMC cells by H3-TdR incorporation assay
  • Experimental group blank control group (plant lectin and PBMC cells only), exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experiment Group 3 (Salpichrolide U and SAHA combined dosing), exosomes extracted supernatant control group (no drug group), exosomes extraction supernatant group 1 (dosing Salpichrolide U), exosomes extraction supernatant group 2 ( Dosing SAHA), exosomes extraction supernatant group 3 (Salpichrolide U and SAHA combined dosing), each group of three holes.
  • PBMC cells were added to 5 ml of fresh RPMI 1640 medium containing 10% fetal bovine serum. After cell counting, the dilution was 5000/50 ⁇ l. According to the previous grouping, 50 ⁇ l/well was added to the 96-well plate. At the same time, a mixture of penicillin 100 IU/mL and streptomycin 100 ⁇ g/mL was added in a ratio of 1:1000, 50 ⁇ l of the sample was further added, and finally 100 ⁇ l of IPHA was added.
  • the drug-treated supernatant extracted by exosomes and the PBMC cells co-cultured had little effect on cell proliferation, and there was no statistically significant difference compared with the blank control group (P>0.05), without drug treatment.
  • the supernatant had a significant effect on lymphocyte proliferation, and there was a statistically significant difference compared with the blank control group or the supernatant group (P ⁇ 0.05).
  • the medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.
  • mice healthy Kunming mice, MFC gastric cancer mice, mouse gastric cancer cells (MFC), DMEM high sugar medium (purchased from GIBCO), fetal bovine serum.
  • Example 2 Mouse gastric cancer cell (MFC) culture was carried out according to the above Example 1; the cultured cells were divided into 4 groups, the first group was not administered, the second group was administered Salpichrolide U, the third group The drug SAHA was added, and the fourth group of Salpichrolide U and SAHA was combined.
  • the specific drug treatment was referred to Example 2; the collected cell culture supernatants were prepared according to Example 5 to prepare exosomes tumor vaccine.
  • the tumor inhibition rate (%) (control group tumor size - treatment group tumor size) / Control group tumor size ⁇ 100%.
  • the growth of gastric cancer xenografts was inhibited after treatment with exosomes tumor vaccine.
  • the tumor masses of exosomes group 1, exosomes group 2 and exosomes group 3 were (1.76 ⁇ 0.24) g, (1.67 ⁇ 0.26) g, (0.84 ⁇ 0.17), respectively. g, compared with the blank control group (3.37 ⁇ 0.30) g and the exosomes control group (2.74 ⁇ 0.17) g, the difference was significant (P ⁇ 0.05).
  • the combination of Salpichrolide U and SAHA was the strongest inhibitory effect, and the tumor quality was (0.84 ⁇ 0.17) g, the tumor inhibition rate reached 75.07%.
  • the medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.
  • Adjuvants generally refer to a class of substances that specifically bind to an antigen by physical or chemical means to enhance their specific immunity.
  • the most widely used adjuvant in the world is the aluminum adjuvant.
  • An adsorptive vaccine is formed in a mixture of alumina and aluminum phosphate or in alumina.
  • the aluminum phosphate adjuvant and the aluminum hydroxide adjuvant are positively charged at physiological pH, and the negatively charged protein is adsorbed in the lattice structure of the aluminum salt, and can be used for the treatment of the gastric cancer vaccine in the preparation of the gastric cancer vaccine of the present invention. effect.
  • Liposomes can also be used as adjuvants for the gastric cancer vaccine of the present invention to improve the therapeutic effect of gastric cancer vaccines.
  • the present invention creatively uses Salpichrolide U and SAHA to treat gastric cancer cells, and the results show that the exosomes secreted by the treated gastric cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition.
  • the invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.

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Abstract

A tumor vaccine for use in treating gastric cancer and a preparation method for the vaccine. The gastric cancer vaccine comprises exosomes corpuscles secreted by gastric cancer cells, where the gastric cancer cells underwent a combined dosing treatment with Salpichrolide U and SAHA, and the molar concentration ratio of Salpichrolide U to SAHA is 0.8-1.2:1.

Description

一种用于治疗胃癌的肿瘤疫苗及其制备方法Tumor vaccine for treating gastric cancer and preparation method thereof 技术领域Technical field
本发明属于肿瘤疫苗领域，具体涉及一种用于治疗胃癌的肿瘤疫苗，以及该胃癌疫苗的制备方法。The invention belongs to the field of tumor vaccines, and particularly relates to a tumor vaccine for treating gastric cancer, and a preparation method of the gastric cancer vaccine.
背景技术Background technique
胃癌是世界上第二常见的癌症。近50年来，虽然在全球范围内，特别是部分发达国家中，胃癌的发病率和病死率呈下降趋势，但每年仍有约100万新发病例。由于缺乏早期诊断的手段，大部分有症状的胃癌患者在确诊时已属晚期。目前传统的治疗方法为手术、化疗、放疗，这些治疗为侵入性或毒副反应明显，且均不能有效解决肿瘤转移、复发等问题。化疗后常发生多药耐药现象。总之，在传统治疗下，胃癌的预后仍然较差。恶性肿瘤的产生不仅是因为细胞的恶性转化，也是因为肿瘤细胞能逃避宿主免疫系统的监视。肿瘤的免疫治疗旨在通过调节患者的免疫系统，发挥自身的免疫监视和防御功能来控制肿瘤。近年来尤其强调肿瘤疫苗的发展和应用，因其诱导产生主动的特异性免疫反应而有广阔的应用前景。机体的免疫系统通过辨认肿瘤细胞的抗原靶标特异性地杀伤肿瘤细胞。Gastric cancer is the second most common cancer in the world. In the past 50 years, although the incidence and mortality of gastric cancer have declined in the global scale, especially in some developed countries, there are still about 1 million new cases every year. Due to the lack of early diagnosis, most patients with symptomatic gastric cancer are advanced at the time of diagnosis. At present, the traditional treatment methods are surgery, chemotherapy and radiotherapy. These treatments are invasive or toxic and side effects, and they can not effectively solve the problems of tumor metastasis and recurrence. Multidrug resistance often occurs after chemotherapy. In short, under traditional treatment, the prognosis of gastric cancer is still poor. The production of malignant tumors is not only due to the malignant transformation of cells, but also because tumor cells can escape the surveillance of the host immune system. Tumor immunotherapy aims to control tumors by regulating the patient's immune system and exerting its own immune surveillance and defense functions. In recent years, special emphasis has been placed on the development and application of tumor vaccines, which have broad application prospects due to their induction of active specific immune responses. The body's immune system specifically kills tumor cells by identifying antigenic targets of tumor cells.
虽然国内外对肿瘤疫苗的相关研究很多，但到目前为止取得突破性进展的却不多见，存在的主要问题有：将DNA导入到特定细胞进行表达这一技术尚不成熟，并且使用外源DNA的安全性问题尚未解决；由于肿瘤细胞呈现高度异质性，属于同一类型肿瘤的多个瘤细胞上可以表达不同的抗原，由一种肿瘤抗原所激活的T细胞只能杀伤一部分的肿瘤细胞，不表达该抗原的瘤细胞则不能被杀伤；肿瘤细胞疫苗虽然可以包含几乎全部的肿瘤抗原，但目前的研究表明其激活T细胞的作用有限，将其作为疫苗的效果并不理想。因此，如何提供一种肿瘤疫苗，不存在使用安全性问题并且能对几乎全部的肿瘤抗原有效，成为有待解决的问题。Although there are many researches on tumor vaccines at home and abroad, but there have been few breakthroughs so far. The main problems are: the introduction of DNA into specific cells for expression is not yet mature, and the use of foreign sources The safety of DNA has not been solved; because tumor cells are highly heterogeneous, different antigens can be expressed on multiple tumor cells belonging to the same type of tumor, and T cells activated by one tumor antigen can only kill a part of tumor cells. Tumor cells that do not express the antigen cannot be killed; although tumor cell vaccines can contain almost all tumor antigens, current studies have shown that their role in activating T cells is limited, and the effect as a vaccine is not satisfactory. Therefore, how to provide a tumor vaccine, which does not have a safety problem of use and can be effective against almost all tumor antigens, has become a problem to be solved.
Exosomes是一类起源于内吞体系统并被排出于细胞外、直径在40-100nm之间的双层膜性囊泡。Exosomes可以由包括树突状细胞、肿瘤细胞等在内的多种细胞分泌，含有大量与其来源和功能密切相关的蛋白质和脂质成分，作为细胞间传递信息的重要载体，参与多种病理生理过程。肿瘤细胞来源的exosomes含有肿瘤共同抗原、热体克蛋白等重要的免疫分子，可通过多种途径表现出抗肿瘤作用，且其作为一种新型的肿瘤疫苗，较DC疫苗有明显的优势。Exosomes are a class of bilayer membranous vesicles that originate in the endocytic system and are excreted outside the cell, between 40 and 100 nm in diameter. Exosomes can be secreted by a variety of cells including dendritic cells, tumor cells, etc., containing a large number of proteins and lipid components closely related to its source and function, as an important carrier of information transmission between cells, participating in a variety of pathophysiological processes. . Tumor cell-derived exosomes contain important immune molecules such as tumor common antigen and thermophilin, which can exhibit anti-tumor effects through various pathways, and as a new type of tumor vaccine, it has obvious advantages over DC vaccine.
然而，近年来却有一些相关实验显示，一些肿瘤来源的exosomes可以抑制甚至是破坏在肿瘤中发挥作用的免疫细胞，比如下调一些NK受体的表达，影响到肿瘤免疫中一些固有免疫细胞的激活，还有的可以显著抑制IL-2从而抑制人类淋巴细胞的增殖，因而在肿瘤的免疫治疗中起到一些负面作用。这些由肿瘤来源的exosomes可能就是肿瘤组织逃逸机体免疫系统清除的关键因素，给肿瘤的免疫治疗带来很多困难和挑战。因此，如何提高肿瘤细胞来源exosomes的免疫刺激能力，而减少它的免疫抑制能力在肿瘤的免疫治疗中有重大的实际意义。 However, in recent years, some related experiments have shown that some tumor-derived exosomes can inhibit or even destroy immune cells that play a role in tumors, such as down-regulating the expression of some NK receptors, affecting the activation of some innate immune cells in tumor immunity. Others can significantly inhibit IL-2 and inhibit the proliferation of human lymphocytes, thus playing a negative role in the immunotherapy of tumors. These tumor-derived exosomes may be the key factors for tumor tissue to escape the immune system clearance, which brings many difficulties and challenges to tumor immunotherapy. Therefore, how to improve the immunostimulatory ability of tumor cells derived from exosomes, and reduce its immunosuppressive ability has great practical significance in tumor immunotherapy.
发明内容Summary of the invention
本发明的目的在于提供一种对淋巴细胞增殖功能没有抑制作用的胃癌疫苗。It is an object of the present invention to provide a gastric cancer vaccine which does not inhibit lymphocyte proliferation function.
本发明的上述目的是通过下面的技术方案得以实现的：The above object of the present invention is achieved by the following technical solutions:
一种治疗效果改进的胃癌疫苗，包括胃癌细胞分泌的exosomes小体，所述的胃癌细胞是经过Salpichrolide U和SAHA联合加药处理的，Salpichrolide U和SAHA的摩尔浓度之比为0.8～1.2∶1。A gastric cancer vaccine with improved therapeutic effect, comprising an exosomes body secreted by gastric cancer cells, wherein the gastric cancer cells are treated by a combination of Salpichrolide U and SAHA, and the molar ratio of Salpichrolide U to SAHA is 0.8 to 1.2:1. .
进一步地，Salpichrolide U和SAHA的摩尔浓度之比为1∶1。Further, the molar ratio of Salpichrolide U to SAHA is 1:1.
进一步地，所述的治疗效果改进的胃癌疫苗还包括佐剂。Further, the gastric cancer vaccine with improved therapeutic effect further includes an adjuvant.
进一步地，所述佐剂为为铝佐剂。Further, the adjuvant is an aluminum adjuvant.
所述的胃癌疫苗的制备方法，包括如下步骤：胃癌细胞用含胎牛血清的DMEM高糖培养基在CO2孵箱中培养，细胞呈单层贴壁生长，每3～4天传代1次，待细胞生长至对数期时接种；接种24h后，用Salpichrolide U和SAHA加药处理细胞，继续培养24h后收集培养上清液，低温保存；将收集到的胃癌细胞培养上清液离心去除细胞，取上清液；再离心去除细胞碎片，收集上清液，浓缩超滤，离心得到浓缩液，将分离纯化的浓缩液移至离心管中，低温条件下超速离心，所得沉淀即含有exosomes小体。The preparation method of the gastric cancer vaccine comprises the following steps: gastric cancer cells are cultured in a CO 2 incubator containing DMEM high-sugar medium containing fetal bovine serum, and the cells are monolayer adherent growth, and passaged once every 3 to 4 days. After the cells were grown to log phase, the cells were inoculated. After 24 hours of inoculation, the cells were treated with Salpichrolide U and SAHA. After 24 hours of culture, the culture supernatant was collected and stored at low temperature. The collected culture supernatant of gastric cancer cells was removed by centrifugation. The cells are taken, and the supernatant is removed; the supernatant is removed by centrifugation, the supernatant is collected, concentrated by ultrafiltration, and the concentrate is centrifuged to transfer the separated and purified concentrate to a centrifuge tube, and ultracentrifuged at a low temperature to obtain an exosomes. Small body.
进一步地，所述的制备方法包括如下步骤：胃癌细胞用含100ml/L胎牛血清的DMEM高糖培养基，在37℃50ml/L CO2孵箱中培养，细胞呈单层贴壁生长，每3～4天传代1次，待细胞生长至对数期时按3×106/100ml接种；接种24h后用1×10-6mol/L的Salpichrolide U和1×10-6mol/L的SAHA处理细胞，继续培养24h后收集培养上清液，4℃保存；将收集到的胃癌细胞培养上清液300g离心10min去除细胞，取上清液；再以1500g离心30min去除细胞碎片，收集上清液，通过100kU MWCO Centriplus离心超滤管浓缩超滤，以1500g离心30min得到浓缩液，将分离纯化的浓缩液移至离心管中，4℃条件下用水平转角以100kg超速离心60min，所得沉淀即含有exosomes小体。Further, the preparation method comprises the steps of: gastric cancer cells are cultured in a DMEM high glucose medium containing 100 ml/L fetal bovine serum in a 50 ml/L CO 2 incubator at 37 ° C, and the cells are monolayer adherent growth. Passage once every 3 to 4 days, inoculate 3×10 6 /100 ml when cells are grown to log phase; use 1 × 10 -6 mol/L Salpichrolide U and 1×10 -6 mol/L after 24 hours of inoculation The cells were treated with SAHA. After 24 hours of culture, the culture supernatant was collected and stored at 4 ° C. The collected gastric cancer cell culture supernatant was centrifuged for 10 min to remove the cells, and the supernatant was taken. The cell debris was removed by centrifugation at 1500 g for 30 min. The supernatant was concentrated by ultrafiltration through a 100 kU MWCO Centriplus centrifugal ultrafiltration tube, and concentrated at 1500 g for 30 min to obtain a concentrate. The separated and purified concentrate was transferred to a centrifuge tube, and centrifuged at 100 kg for 60 min at a horizontal angle of 4 ° C. The precipitate contains the exosomes body.
所述的胃癌疫苗在制备抗胃癌的药物中应用。The gastric cancer vaccine is used in the preparation of a medicament for preventing gastric cancer.
本发明的优点：已知未经处理的胃癌细胞分泌的纳米级小囊泡exosomes及其可溶性免疫分子对淋巴细胞增殖功能具有显著的抑制作用。本发明创造性地使用Salpichrolide U和SAHA加药处理胃癌细胞，结果表明，处理后的胃癌细胞分泌的exosomes及其可溶性免疫分子则能显著改善前述的抑制作用。本发明显著提高了exosomes肿瘤疫苗治疗效果，具有重要的临床应用价值。Advantages of the invention: It is known that nano-sized small vesicle exosomes secreted by untreated gastric cancer cells and soluble immune molecules thereof have significant inhibitory effects on lymphocyte proliferation function. The present invention creatively uses Salpichrolide U and SAHA to treat gastric cancer cells, and the results show that the exosomes secreted by the treated gastric cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition. The invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.
具体实施方式detailed description
下面结合实施例进一步说明本发明的实质性内容，但并不以此限定本发明保护范围。尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。实施例中未注明具体条件的实验方法，通常按照常规条件，例如教科书和实验指南中所述的条件，或按照制造厂商所建议的条件。Salpichrolide U的化学结构和制备方法参见文献：Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa：S.origanifolia and S.tristis var.lehmannii，J.Nat.Prod.，2013，76，2219-2225。 The substantial content of the present invention is further illustrated by the following examples, but is not intended to limit the scope of the present invention. While the present invention has been described in detail with reference to the preferred embodiments of the present invention, it is understood that the invention may be modified or equivalently modified without departing from the spirit and scope of the invention. Experimental methods in which no specific conditions are indicated in the examples are generally carried out according to conventional conditions, such as those described in the textbooks and experimental guides, or in accordance with the conditions recommended by the manufacturer. The chemical structure and preparation method of Salpichrolide U can be found in the literature: Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225.
实施例1：细胞培养Example 1: Cell Culture
实验材料：MFC细胞株、10％胎牛血清、青链霉素混合液(北京泰格美公司)。Experimental materials: MFC cell strain, 10% fetal bovine serum, and streptomycin mixture (Beijing Tiger Beauty Co., Ltd.).
实验方法：将MFC细胞株培养于含10％胎牛血清、青霉素100IU/mL、链霉素100×μg/mL的培养液中(DMEM高糖)，于37℃5％CO2孵箱中培养，待生长至对数期时，按3×106/100ml接种。Experimental method: MFC cell line was cultured in a culture medium containing 10% fetal bovine serum, penicillin 100 IU/mL, streptomycin 100×μg/mL (DMEM high sugar), and cultured in a 37 ° C 5% CO 2 incubator. When in the logarithmic phase, inoculate at 3 × 10 6 /100 ml.
实施例2：药物处理Example 2: Drug treatment
实验材料：Salpichrolide U自制，制备方法同文献(Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa：S.origanifolia and S.tristis var.lehmannii，J.Nat.Prod.，2013，76，2219-2225)，经鉴定为Salpichrolide U；SAHA购自sigma公司。Experimental material: Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
]实验分组：细胞培养液体积每组严格一致，空白对照组、exosomes对照组(不加药组)、exosomes实验组1(加药Salpichrolide U)、exosomes实验组2(加药SAHA)、exosomes实验组3(Salpichrolide U和SAHA联合加药)。Experimental grouping: The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experiment Group 3 (Salpichrolide U and SAHA combined dosing).
实验方法：对照组，接种后不加药物，24h后收集上清液150ml作为对照；加药组1，接种24h后用浓度1×10-6mol/L的Salpichrolide U处理，在加药24h后收集培养上清液150ml；加药组2，接种24h后用浓度1×10-6mol/L的SAHA处理，在加药24h后收集培养上清液150ml；加药组3，接种24h后用1×10-6mol/L的Salpichrolide U和1×10-6mol/L的SAHA处理，在加药24h后收集培养上清液150ml，并4℃保存。Experimental method: control group, no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1×10 -6 mol/L 24 h after inoculation, after 24 h of dosing The culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1×10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation. 1×10 -6 mol/L of Salpichrolide U and 1×10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
实施例3：药物处理Example 3: Drug treatment
实验材料：Salpichrolide U自制，制备方法同文献(Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa：S.origanifolia and S.tristis var.lehmannii，J.Nat.Prod.，2013，76，2219-2225)，经鉴定为Salpichrolide U；SAHA购自sigma公司。Experimental material: Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
实验分组：细胞培养液体积每组严格一致，空白对照组、exosomes对照组(不加药组)、exosomes实验组1(加药Salpichrolide U)、exosomes实验组2(加药SAHA)、exosomes实验组3(Salpichrolide U和SAHA联合加药)。Experimental group: The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Salpichrolide U and SAHA combined dosing).
实验方法：对照组，接种后不加药物，24h后收集上清液150ml作为对照；加药组1，接种24h后用浓度1×10-6mol/L的Salpichrolide U处理，在加药24h后收集培养上清液150ml；加药组2，接种24h后用浓度1×10-6mol/L的SAHA处理，在加药24h后收集培养上清液150ml；加药组3，接种24h后用0.8×10-6mol/L的Salpichrolide U和1×10-6mol/L的SAHA处理，在加药24h后收集培养上清液150ml，并4℃保存。Experimental method: control group, no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1×10 -6 mol/L 24 h after inoculation, after 24 h of dosing The culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1×10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation. 0.8×10 -6 mol/L of Salpichrolide U and 1×10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
实施例4：药物处理Example 4: Drug treatment
实验材料：Salpichrolide U自制，制备方法同文献(Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa：S.origanifolia and S.tristis var.lehmannii，J.Nat.Prod.，2013，76，2219-2225)，经鉴定为Salpichrolide U；SAHA购自sigma公司。Experimental material: Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
实验分组：细胞培养液体积每组严格一致，空白对照组、exosomes对照组(不加药组)、exosomes实验组1(加药Salpichrolide U)、exosomes实验组2(加药SAHA)、exosomes实验组3(Salpichrolide U和SAHA联合加药)。 Experimental group: The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Salpichrolide U and SAHA combined dosing).
实验方法：对照组，接种后不加药物，24h后收集上清液150ml作为对照；加药组1，接种24h后用浓度1×10-6mol/L的Salpichrolide U处理，在加药24h后收集培养上清液150ml；加药组2，接种24h后用浓度1×10-6mol/L的SAHA处理，在加药24h后收集培养上清液150ml；加药组3，接种24h后用1.2×10-6mol/L的Salpichrolide U和1×10-6mol/L的SAHA处理，在加药24h后收集培养上清液150ml，并4℃保存。Experimental method: control group, no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1×10 -6 mol/L 24 h after inoculation, after 24 h of dosing The culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1×10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation. 1.2×10 -6 mol/L of Salpichrolide U and 1×10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
实施例5：exosomes的分离与纯化Example 5: Isolation and purification of exosomes
实验仪器：HMAC-CP7OG低温超高速离心机，100KU MW COMilliporeAmicon高回收率高流速切向流超滤离心管(Millipore公司)。Experimental equipment: HMAC-CP7OG cryogenic ultracentrifuge, 100 KU MW COMilliporeAmicon high recovery high flow tangential flow ultrafiltration centrifuge tube (Millipore).
实验方法：将实施例2～4收集到的实验组和对照组细胞培养上清液以300g离心10min去除细胞，取上清液；以1500g离心30min去除细胞碎片，收集上清液，通过100kUMWCO Centriplus离心超滤管浓缩超滤，以1500g离心30min得到6ml浓缩液，将分离纯化的浓缩液移至1.5ml的离心管中，4℃下用水平转角以100kg超速离心60min所得沉淀即含有exosomes小体。Experimental methods: The experimental and control cell culture supernatants collected in Examples 2 to 4 were centrifuged at 300 g for 10 min to remove the cells, and the supernatant was taken; the cell debris was removed by centrifugation at 1500 g for 30 min, and the supernatant was collected and passed through 100 kUMWCO Centriplus. Centrifugal ultrafiltration tube concentrated ultrafiltration, centrifuged at 1500g for 30min to obtain 6ml concentrate, the separated and purified concentrate was transferred to a 1.5ml centrifuge tube, and the precipitate obtained by ultracentrifugation at 100kg for 60min at a horizontal angle of 4°C contained exosomes .
实施例6：exosomes的电镜鉴定Example 6: Electron microscopic identification of exosomes
滴20-30μlexosomes悬液于载样铜网上，室温静置lmin用滤纸从侧而吸干液体，滴加20ml/L磷钨酸溶液(pH6.8)约30μl于铜网上，室温负染lmin滤纸吸干负染液，室温下晾干约10min透射电镜下观察照相。结果显示，电镜下MFC细胞分泌的exosomes小体直径为30-80nm的膜性微囊结构，呈圆形或椭圆形，腔内为低电子密度成分。Drop 20-30μlexosomes suspension on the loaded copper mesh, let stand for 1min at room temperature, use the filter paper to absorb the liquid from the side, add 20ml/L phosphotungstic acid solution (pH6.8) about 30μl on the copper net, and negatively dye the lmin filter paper at room temperature. The negative dye solution was blotted, and air-dried at room temperature for about 10 minutes to observe the photograph under transmission electron microscope. The results showed that the exosomes secreted by MFC cells under electron microscope showed a membrane-like microcapsule structure with a diameter of 30-80 nm, which was round or elliptical, and the cavity was low in electron density.
实施例7：H3-TdR掺入法检测PBMC细胞增殖状况Example 7: Detection of proliferation of PBMC cells by H3-TdR incorporation assay
实验材料：H3-TdR(上海原子核研究所)、青链霉素混合液(北京泰格美)、胎牛血清Experimental materials: H3-TdR (Shanghai Institute of Nuclear Research), Streptomycin Mixture (Beijing Taigemei), fetal bovine serum
实验仪器：β-液体闪烁计数器测量(FJ-2107G型)Experimental instrument: β-liquid scintillation counter measurement (FJ-2107G type)
实验分组：空白对照组(只加植物凝集素和PBMC细胞)、exosomes对照组(不加药组)、exosomes实验组1(加药Salpichrolide U)、exosomes实验组2(加药SAHA)、exosomes实验组3(Salpichrolide U和SAHA联合加药)、exosomes提取后上清对照组(不加药组)，exosomes提取后上清实验组1(加药Salpichrolide U)、exosomes提取后上清实验组2(加药SAHA)、exosomes提取后上清实验组3(Salpichrolide U和SAHA联合加药)，每组三复孔。Experimental group: blank control group (plant lectin and PBMC cells only), exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experiment Group 3 (Salpichrolide U and SAHA combined dosing), exosomes extracted supernatant control group (no drug group), exosomes extraction supernatant group 1 (dosing Salpichrolide U), exosomes extraction supernatant group 2 ( Dosing SAHA), exosomes extraction supernatant group 3 (Salpichrolide U and SAHA combined dosing), each group of three holes.
实验方法：分离好的PBMC细胞加入5ml含10％胎牛血清的新鲜RPMI 1640培养基，细胞计数后，稀释为5000个/50μl，按照先前做好的分组，以50μl/孔加入96孔板中，同时以1∶1000的比例加入青霉素100IU/mL、链霉素100×μg/mL的混合液，再加入50μl样本，最后加入100μlIPHA。放入37℃50％CO2孵箱过夜，第二天可在10×的倒置显微镜下观察到PBMC细胞呈团状增殖，将H3-TdR加入培养板中，每孔3.7×104个Bq继续培养6h后，去培养基，用1×PBS洗3次，1mol/L的NaOH破细胞膜，加入闪烁液及反淬灭剂，用β-液体闪烁计数器测量，记录cpm值(分钟计数)。结果如表1所示。Experimental method: Separated PBMC cells were added to 5 ml of fresh RPMI 1640 medium containing 10% fetal bovine serum. After cell counting, the dilution was 5000/50 μl. According to the previous grouping, 50 μl/well was added to the 96-well plate. At the same time, a mixture of penicillin 100 IU/mL and streptomycin 100×μg/mL was added in a ratio of 1:1000, 50 μl of the sample was further added, and finally 100 μl of IPHA was added. Placed in a 50 ° CO 2 incubator at 37 ° C overnight, the next day, PBMC cells were observed to grow in a pellet under a 10 × inverted microscope, and H3-TdR was added to the culture plate at 3.7 × 10 4 Bq per well. After 6 hours of culture, the medium was removed, washed 3 times with 1×PBS, cell membrane was broken with 1 mol/L NaOH, scintillation liquid and anti-quenching agent were added, and the cpm value (minute count) was recorded with a β-liquid scintillation counter. The results are shown in Table 1.
表1经联合药物处理前后的exosomes及提取后的上清对PBMC细胞增殖的影响Table 1 Effect of exosomes before and after combined drug treatment and supernatant after extraction on proliferation of PBMC cells
组别Group 组数Number of groups cpm值(x±S)Cpm value (x±S)
空白对照Blank control 33 24044±2202.4524044±2202.45
exosomes对照组Exosomes control group 33 5018±24.665018±24.66
exosomes实验组1Exosomes experimental group 1 33 13503±769.1613503±769.16
exosomes实验组2Exosomes experimental group 2 33 13705±770.4413705±770.44
exosomes实验组3Exosomes experimental group 3 33 23988±2037.3123988±2037.31
exosomes提取后上清对照组Supervised control group after exosomes extraction 33 4972±413.454972±413.45
exosomes提取后上清实验组1Supervised experimental group 1 after exosomes extraction 33 14931±5514.2914931±5514.29
exosomes提取后上清实验组2Exosomes extracted after supernatant experiment group 2 33 14722±5711.5214722±5711.52
exosomes提取后上清实验组3Supervised experimental group 3 after exosomes extraction 33 23842±5201.1723842±5201.17
结果显示：与空白对照组相比，未经过药物处理的exosomes与PBMC细胞共培养后，对淋巴细胞增殖有显著影响，明显降低(P＜0.05)；经过药物处理，可明显改善exosomes对淋巴细胞增殖功能的抑制，其中经药物组合物处理后获得的exosomes(exosomes实验组3)对淋巴细胞增殖功能的抑制最小，明显小于单独药物处理后获得exosomes(exosomes实验组1和exosomes实验组2)对淋巴细胞增殖的抑制，PBMC增殖值与exosomes对照组比较统计学存在显著差异(P＜0.05)，与空白对照组比较无显著差异(P＞0.05)。在实验组，经exosomes提取后的药物处理的上清液与PBMC细胞共培养后，对细胞增殖几乎无影响，与空白对照组比较无显著统计学差异(P＞0.05)，未经药物处理的上清液则对淋巴细胞增殖功能有显著性影响，与空白对照组或上清实验组相比统计学上均有显著差异(P＜0.05)。实施例3、4与实施例2中的加药组3能制备得到治疗活性类似的疫苗。The results showed that compared with the blank control group, the untreated drug treated exosomes and PBMC cells had a significant effect on lymphocyte proliferation, which was significantly decreased (P<0.05). After drug treatment, the exosomes could be significantly improved. Inhibition of proliferative function, in which exosomes (exosomes experimental group 3) obtained after treatment with the pharmaceutical composition had the least inhibition of lymphocyte proliferation function, which was significantly less than that obtained by exosomes (exosomes experimental group 1 and exosomes experimental group 2). The inhibition of lymphocyte proliferation, PBMC proliferation value and the exosomes control group were statistically significant (P<0.05), and there was no significant difference compared with the blank control group (P>0.05). In the experimental group, the drug-treated supernatant extracted by exosomes and the PBMC cells co-cultured had little effect on cell proliferation, and there was no statistically significant difference compared with the blank control group (P>0.05), without drug treatment. The supernatant had a significant effect on lymphocyte proliferation, and there was a statistically significant difference compared with the blank control group or the supernatant group (P<0.05). The medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.
实施例8：体内抑瘤实验Example 8: In vivo anti-tumor experiment
实验材料：健康昆明系小鼠，MFC胃癌小鼠，小鼠胃癌细胞(MFC)，DMEM高糖培养基(购自GIBCO)，胎牛血清。Experimental materials: healthy Kunming mice, MFC gastric cancer mice, mouse gastric cancer cells (MFC), DMEM high sugar medium (purchased from GIBCO), fetal bovine serum.
实验方法：experimental method:
(1)建立荷瘤鼠模型：无菌操作取MFC胃癌小鼠腹水，台盼蓝染色，光镜下瘤细胞计数，活瘤细胞9000，调整细胞浓度为1×107个/ml，于每只健康昆明系小鼠右腋皮下接种0.2ml，制成实体型荷瘤鼠模型。(1) Establish a tumor-bearing mouse model: aseptically take MFC gastric cancer mice ascites, trypan blue staining, tumor cell count under light microscope, live tumor cells 9000, adjust the cell concentration to 1 × 10 7 /ml, in each Only healthy Kunming mice were inoculated with 0.2 ml of the right iliac crest to make a solid tumor-bearing mouse model.
(2)制备肿瘤疫苗：根据所述实施例1进行小鼠胃癌细胞(MFC)培养；将培养的细胞分为4组，第一组不加药，第二组加药Salpichrolide U，第三组加药SAHA，第四组Salpichrolide U和SAHA联合加药，具体加药处理参照实施例2；将收集到的各组细胞培养上清液参照实施例5制备exosomes肿瘤疫苗。(2) Preparation of tumor vaccine: Mouse gastric cancer cell (MFC) culture was carried out according to the above Example 1; the cultured cells were divided into 4 groups, the first group was not administered, the second group was administered Salpichrolide U, the third group The drug SAHA was added, and the fourth group of Salpichrolide U and SAHA was combined. The specific drug treatment was referred to Example 2; the collected cell culture supernatants were prepared according to Example 5 to prepare exosomes tumor vaccine.
(3)分组和治疗：将健康昆明系小鼠30只随机分为5组，每组各6只。空白对照组：肿瘤细胞接种当天，于小鼠腿根部皮下注射生理盐水；exosomes对照组：肿瘤细胞接种当天，于小鼠腿根部皮下注射第一组制备的肿瘤疫苗(10μg/只)；exosomes实验组1：肿 瘤细胞接种当天，于小鼠腿根部皮下注射第二组制备的肿瘤疫苗(10μg/只)；exosomes实验组2：肿瘤细胞接种当天，于小鼠腿根部皮下注射第三组制备的肿瘤疫苗(10μg/只)；exosomes实验组3：肿瘤细胞接种当天，于小鼠腿根部皮下注射第四组制备的肿瘤疫苗(10μg/只)。每间隔1天重复1次，共3次。所有小鼠接种肿瘤后第15天处死，取瘤体，称重、测肿瘤体积，计算瘤重抑制率、肿瘤体积抑制率。(3) Grouping and treatment: 30 healthy Kunming mice were randomly divided into 5 groups, 6 in each group. Blank control group: On the day of tumor cell inoculation, physiological saline was injected subcutaneously into the root of the mouse; exosomes control group: the day of tumor cell inoculation, the first group of prepared tumor vaccine (10 μg/mouse) was injected subcutaneously into the leg root of the mouse; exosomes experiment Group 1: swollen On the day of tumor cell inoculation, a second group of prepared tumor vaccines (10 μg/mouse) was injected subcutaneously into the roots of the mice; exosomes group 2: on the day of tumor cell inoculation, a third group of tumor vaccines were injected subcutaneously into the roots of the mice ( 10 μg/only); exosomes experimental group 3: On the day of tumor cell inoculation, a fourth group of prepared tumor vaccine (10 μg/head) was subcutaneously injected into the root of the mouse leg. Repeat 1 time every 1 day for 3 times. All mice were sacrificed on the 15th day after tumor inoculation. The tumors were taken, weighed, tumor volume was measured, and tumor weight inhibition rate and tumor volume inhibition rate were calculated.
(4)称取肿瘤重量并计算肿瘤抑制率(4) Weigh the tumor weight and calculate the tumor inhibition rate
小鼠处死后，分别取出肿瘤瘤体，剥离干净后，以滤纸拭净血污，电子天平称取瘤重，计算抑瘤率：抑瘤率(％)＝(对照组肿瘤大小-治疗组肿瘤大小)/对照组肿瘤大小×100％。After the mice were sacrificed, the tumor tumors were taken out separately, and the blood was stained with filter paper. The tumor weight was weighed and the tumor inhibition rate was calculated. The tumor inhibition rate (%) = (control group tumor size - treatment group tumor size) / Control group tumor size × 100%.
表2肿瘤疫苗对胃癌移植瘤的抑制作用Table 2 Inhibition of tumor vaccine on gastric cancer xenografts
实验分组Experimental grouping 肿瘤平均Tumor average
空白对照组Blank control group 3.37±0.303.37±0.30
exosomes对照组Exosomes control group 2.74±0.172.74±0.17
exosomes实验组1Exosomes experimental group 1 1.76±0.241.76±0.24
exosomes实验组2Exosomes experimental group 2 1.67±0.261.67±0.26
exosomes实验组3Exosomes experimental group 3 0.84±0.170.84±0.17
采用exosomes肿瘤疫苗治疗后胃癌移植瘤生长受到抑制，exosomes实验组1、exosomes实验组2、exosomes实验组3瘤质量分别为(1.76±0.24)g、(1.67±0.26)g、(0.84±0.17)g，与空白对照组(3.37±0.30)g、exosomes对照组(2.74±0.17)g比差异显著(P＜0.05)，Salpichrolide U和SAHA联合加药制备的肿瘤疫苗抑制作用最强，瘤质量为(0.84±0.17)g，抑瘤率达75.07％。实施例3、4与实施例2中的加药组3能制备得到治疗活性类似的疫苗。The growth of gastric cancer xenografts was inhibited after treatment with exosomes tumor vaccine. The tumor masses of exosomes group 1, exosomes group 2 and exosomes group 3 were (1.76±0.24) g, (1.67±0.26) g, (0.84±0.17), respectively. g, compared with the blank control group (3.37±0.30) g and the exosomes control group (2.74±0.17) g, the difference was significant (P<0.05). The combination of Salpichrolide U and SAHA was the strongest inhibitory effect, and the tumor quality was (0.84±0.17) g, the tumor inhibition rate reached 75.07%. The medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.
实施例9：胃癌疫苗佐剂选择Example 9: Selection of gastric cancer vaccine adjuvant
佐剂泛指一类能特异性地通过物理或化学的方式与抗原结合而增强其特异性免疫的物质。现在世界上最广泛使用的佐剂为铝佐剂。铝佐剂疫苗主要有2种制备方法：一种是将铝佐剂添加到抗原溶液中形成铝酸盐蛋白沉淀；另一种是将抗原溶液添加到预先制备的氢氧化铝、磷酸铝、氢氧化铝和磷酸铝混合物或氧化铝中形成吸附性疫苗。磷酸铝佐剂和氢氧化铝佐剂在生理pH值下为正电荷，带负电的蛋白质被吸附在铝盐的网架结构中，用于本发明的胃癌疫苗的制备中可以提高胃癌疫苗的治疗效果。Adjuvants generally refer to a class of substances that specifically bind to an antigen by physical or chemical means to enhance their specific immunity. The most widely used adjuvant in the world is the aluminum adjuvant. There are two main preparation methods for the aluminum adjuvant vaccine: one is to add an aluminum adjuvant to the antigen solution to form an aluminate protein precipitate; the other is to add the antigen solution to the previously prepared aluminum hydroxide, aluminum phosphate, hydrogen. An adsorptive vaccine is formed in a mixture of alumina and aluminum phosphate or in alumina. The aluminum phosphate adjuvant and the aluminum hydroxide adjuvant are positively charged at physiological pH, and the negatively charged protein is adsorbed in the lattice structure of the aluminum salt, and can be used for the treatment of the gastric cancer vaccine in the preparation of the gastric cancer vaccine of the present invention. effect.
还可以选用脂质体作为本发明胃癌疫苗的佐剂，提高胃癌疫苗的治疗效果。Liposomes can also be used as adjuvants for the gastric cancer vaccine of the present invention to improve the therapeutic effect of gastric cancer vaccines.
本发明创造性地使用Salpichrolide U和SAHA加药处理胃癌细胞，结果表明，处理后的胃癌细胞分泌的exosomes及其可溶性免疫分子则能显著改善前述的抑制作用。本发明显著提高了exosomes肿瘤疫苗治疗效果，具有重要的临床应用价值。The present invention creatively uses Salpichrolide U and SAHA to treat gastric cancer cells, and the results show that the exosomes secreted by the treated gastric cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition. The invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.
上述实施例的作用在于说明本发明的实质性内容，但并不以此限定本发明的保护 范围。本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和保护范围。 The above embodiments are intended to illustrate the substantial content of the present invention, but do not limit the protection of the present invention thereto. range. A person skilled in the art should understand that the technical solutions of the present invention may be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

  1. 一种治疗效果改进的胃癌疫苗，其特征在于：包括胃癌细胞分泌的exosomes小体，所述的胃癌细胞是经过Salpichrolide U和SAHA联合加药处理的，Salpichrolide U和SAHA的摩尔浓度之比为0.8～1.2∶1。A gastric cancer vaccine with improved therapeutic effect is characterized in that it comprises an exosomes body secreted by gastric cancer cells, wherein the gastric cancer cells are treated by a combination of Salpichrolide U and SAHA, and the molar concentration ratio of Salpichrolide U and SAHA is 0.8. ~1.2:1.
  2. 根据权利要求1所述的治疗效果改进的胃癌疫苗，其特征在于：Salpichrolide U和SAHA的摩尔浓度之比为1∶1。The gastric cancer vaccine with improved therapeutic effect according to claim 1, wherein the molar ratio of Salpichrolide U to SAHA is 1:1.
  3. 根据权利要求1或2所述的治疗效果改进的胃癌疫苗，其特征在于：还包括佐剂。The gastric cancer vaccine with improved therapeutic effect according to claim 1 or 2, which further comprises an adjuvant.
  4. 根据权利要求3所述的治疗效果改进的胃癌疫苗，其特征在于：所述佐剂为为铝佐剂。The gastric cancer vaccine with improved therapeutic effect according to claim 3, wherein the adjuvant is an aluminum adjuvant.
  5. 权利要求1或2所述的胃癌疫苗的制备方法，其特征在于包括如下步骤：胃癌细胞用含胎牛血清的DMEM高糖培养基在CO2孵箱中培养，细胞呈单层贴壁生长，每3～4天传代1次，待细胞生长至对数期时接种；接种24h后，用Salpichrolide U和SAHA加药处理细胞，继续培养24h后收集培养上清液，低温保存；将收集到的胃癌细胞培养上清液离心去除细胞，取上清液；再离心去除细胞碎片，收集上清液，浓缩超滤，离心得到浓缩液，将分离纯化的浓缩液移至离心管中，低温条件下超速离心，所得沉淀即含有exosomes小体。The method for preparing a gastric cancer vaccine according to claim 1 or 2, comprising the steps of: gastric cancer cells are cultured in a CO 2 incubator using DMEM high-sugar medium containing fetal bovine serum, and the cells are grown in a single layer. The cells were passaged every 3 to 4 days, and the cells were inoculated until the logarithmic phase. After 24 hours of inoculation, the cells were treated with Salpichololide U and SAHA. After 24 hours of culture, the culture supernatant was collected and stored at low temperature; The cells of the gastric cancer cell culture supernatant are centrifuged to remove the cells, and the supernatant is taken; the cell debris is removed by centrifugation, the supernatant is collected, the ultrafiltration is concentrated, the concentrate is centrifuged, and the separated and purified concentrate is transferred to a centrifuge tube under low temperature conditions. After ultracentrifugation, the resulting precipitate contains exosomes bodies.
  6. 根据权利要求5所述的制备方法，其特征在于包括如下步骤：胃癌细胞用含100ml/L胎牛血清的DMEM高糖培养基，在37℃50ml/L CO2孵箱中培养，细胞呈单层贴壁生长，每3～4天传代1次，待细胞生长至对数期时按3×106/100ml接种；接种24h后用1×10-6mol/L的Salpichrolide U和1×10-6mol/L的SAHA处理细胞，继续培养24h后收集培养上清液，4℃保存；将收集到的胃癌细胞培养上清液300g离心10min去除细胞，取上清液；再以1500g离心30min去除细胞碎片，收集上清液，通过100kU MWCO Centriplus离心超滤管浓缩超滤，以1500g离心30min得到浓缩液，将分离纯化的浓缩液移至离心管中，4℃条件下用水平转角以100kg超速离心60min，所得沉淀即含有exosomes小体。The preparation method according to claim 5, comprising the steps of: gastric cancer cells are cultured in a DMEM high glucose medium containing 100 ml/L fetal bovine serum in a 50 ml/L CO 2 incubator at 37 ° C, and the cells are single. The layer was adherently grown and passaged once every 3 to 4 days. When the cells were grown to log phase, they were inoculated at 3×10 6 /100 ml; after inoculation for 24 hours, 1 × 10 -6 mol/L of Salpichrolide U and 1×10 were used. The cells were treated with -6 mol/L SAHA, and the culture supernatant was collected for 24 hours. The culture supernatant was collected and stored at 4 ° C. The collected gastric cancer cell culture supernatant was centrifuged for 10 min to remove the cells, and the supernatant was taken. The cells were centrifuged at 1500 g for 30 min. The cell debris was removed, and the supernatant was collected, concentrated by ultrafiltration through a 100 kU MWCO Centriplus centrifugal ultrafiltration tube, centrifuged at 1500 g for 30 min to obtain a concentrate, and the separated and purified concentrate was transferred to a centrifuge tube at a horizontal angle of 100 kg at 4 ° C. After ultracentrifugation for 60 min, the resulting precipitate contained exosomes bodies.
  7. 权利要求1或2所述的胃癌疫苗在制备抗胃癌的药物中应用。 The gastric cancer vaccine according to claim 1 or 2 is used for the preparation of a medicament for preventing gastric cancer.
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