WO2020103531A1 - 熊脱氧胆酸或其药用盐在制备抗肿瘤药物中的应用及抗肿瘤药物 - Google Patents

熊脱氧胆酸或其药用盐在制备抗肿瘤药物中的应用及抗肿瘤药物

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WO2020103531A1
WO2020103531A1 PCT/CN2019/104831 CN2019104831W WO2020103531A1 WO 2020103531 A1 WO2020103531 A1 WO 2020103531A1 CN 2019104831 W CN2019104831 W CN 2019104831W WO 2020103531 A1 WO2020103531 A1 WO 2020103531A1
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tumor
ursodeoxycholic acid
cells
drug
tumor drug
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蔡志坚
王建莉
沈颖颖
路超杰
宋正波
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Zhejiang University
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Zhejiang University
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

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  • the invention relates to the technical field of biomedicine, in particular to the application of ursodeoxycholic acid or its medicinal salts in the preparation of antitumor drugs and antitumor drugs.
  • tumor immunotherapy has achieved rapid development in basic and clinical research, especially immune checkpoint inhibitors such as programmed death receptor 1 (PD-1) / programmed death ligand (PD-L1) antibody has brought long-term survival benefits to patients with various solid tumors such as advanced melanoma and non-small cell lung cancer.
  • immune checkpoint inhibitors such as programmed death receptor 1 (PD-1) / programmed death ligand (PD-L1) antibody has brought long-term survival benefits to patients with various solid tumors such as advanced melanoma and non-small cell lung cancer.
  • PD-1 programmed death receptor 1
  • P-L1 programmed death ligand
  • immune checkpoint inhibitors can significantly improve the survival rate of patients, only 10% to 30% of patients with solid tumors can benefit from monotherapy. The reason is that the immunosuppressive microenvironment inside the tumor is an important factor in the ineffectiveness of anti-tumor immunity, in which the infiltration of inhibitory cells has an indispensable role.
  • Common inhibitory cells include myeloid-derived inhibitory cells (MDSC), tumor-associated macrophages (TAM), and regulatory T cells (Treg cells for short).
  • MDSC myeloid-derived inhibitory cells
  • TAM tumor-associated macrophages
  • Treg cells regulatory T cells
  • Treg cell-mediated immunosuppression has become a major obstacle to effective treatment of tumors. Woo et al.
  • Treg cells were clustered in melanoma, breast cancer, colorectal cancer, lung cancer and pancreatic cancer and other cancer tissues and cancer tissues (Zou W. Regulatory Cells, tumor, immunity, and immunotherapy. Nat Rev Immunol , 2006, 6: 295-307).
  • Treg cells are conducive to the immune escape of tumors.
  • the increase of Treg cells in the tumor microenvironment of breast cancer, ovarian cancer, gastric cancer and liver cancer is associated with poor prognosis.
  • Treg cells not only have the ability to inhibit a wide range of anti-tumor immune responses, but can also promote angiogenesis in the tumor microenvironment. Therefore, selective removal or suppression of Treg cells in the tumor microenvironment has become a new direction for tumor immunotherapy.
  • Ursodeoxychilic acid as a natural bile salt in human bile, is produced by the reduction of secondary cholic acid in the intestinal tract. It has the effect of choleretic and protects liver cells. It is commonly used in clinical to prevent and treat Cholecystitis, cholangitis, bile dyspepsia, and jaundice caused by cholesterol stones and stones. Recent studies have shown that ursodeoxycholic acid can inhibit TNF- ⁇ , IL-1 ⁇ , IL6 and other cytokines involved in the regulation of inflammatory response (Ko, WK., Kim, SJ, Jo, MJ. Et al. Mol Neurobiol 2018.) .
  • the present invention has found through research that ursodeoxycholic acid has an anti-tumor effect and can be used to prepare anti-tumor drugs.
  • the present invention first provides the application of ursodeoxycholic acid or its pharmaceutically acceptable salts in the preparation of antitumor drugs.
  • the ursodeoxycholic acid or its pharmaceutically acceptable salt works by reducing the proportion of regulatory T cells in tumor-infiltrating lymphocytes.
  • the ursodeoxycholic acid or a pharmaceutically acceptable salt thereof is used in combination with an anti-PD-1 drug.
  • the types of tumors targeted by the anti-tumor drugs are: melanoma, rectal cancer or lung cancer.
  • the invention also provides an anti-tumor drug, including ursodeoxycholic acid or a pharmaceutically acceptable salt thereof.
  • the anti-tumor drugs also include anti-PD-1 drugs.
  • the anti-PD-1 drug is an anti-PD-1 monoclonal antibody drug.
  • SHR-1210 produced by Hengrui Medicine is a humanized anti-PD-1 antibody.
  • the types of tumors targeted by the anti-tumor drugs are: melanoma, rectal cancer or lung cancer.
  • ursodeoxycholic acid can inhibit the differentiation of endogenous Treg cells.
  • ursodeoxycholic acid can significantly inhibit the growth of tumors in tumor-bearing mouse models, using flow cytometry to infiltrate the tumors
  • flow cytometry to infiltrate the tumors
  • lymphocytes revealed that in the tumor-bearing mouse model, the proportion of Treg cells in tumor-infiltrating lymphocytes in the group treated with ursodeoxycholic acid was significantly reduced.
  • ursodeoxycholic acid and anti-PD-1 antibody in the treatment of tumor, it was found that ursodeoxycholic acid can greatly improve the therapeutic effect of anti-PD-1 antibody.
  • Ursodeoxycholic acid and its medicinal salts have a good inhibitory effect on a variety of tumor cells and can be used to prepare anti-tumor drugs.
  • Ursodeoxycholic acid has been used clinically as a choleretic drug, does not require clinical safety assessment, has good application prospects, and can be used as an auxiliary drug to improve the therapeutic effect of anti-PD-1 drugs.
  • Figure 1 is a graph of the test results of the effect of ursodeoxycholic acid on the differentiation of endogenous Treg cells, where Figure A is the flow cytometry test result graph, and Figure B is the statistical result graph, where *** represents P ⁇ 0.001, the same below .
  • Figure 2 is a graph showing the results of the anti-tumor effect of ursodeoxycholic acid in a mouse model of melanoma, where A is the effect of ursodeoxycholic acid on the change in tumor volume, and B is the change in the proportion of Treg cells in tumor-infiltrating lymphocytes.
  • Figure C is a graph of statistical results, where ** represents P ⁇ 0.01, the same below.
  • Figure 3 is the detection of the anti-tumor effect of ursodeoxycholic acid in a mouse model of lung cancer, where A is the result of the effect of ursodeoxycholic acid on the change of tumor volume, and B is the change of the proportion of Treg cells in tumor infiltrating lymphocytes by flow cytometry Results graph, Figure C is a graph of statistical results, where * represents P ⁇ 0.05, the same below.
  • Figure 4 is the detection of the anti-tumor effect of ursodeoxycholic acid in a mouse model of colon cancer, where A is the effect of ursodeoxycholic acid on the change of tumor volume, and B is the change of the proportion of Treg cells in tumor infiltrating lymphocytes by flow cytometry Test result graph, Figure C is a statistical result graph.
  • Figure 5 is a graph showing the effect of ursodeoxycholic acid combined with humanized anti-programmed cell death receptor (PD-1) on tumor volume changes in a colon cancer mouse model.
  • PD-1 humanized anti-programmed cell death receptor
  • Figure 6 is a graph showing the effect of ursodeoxycholic acid combined with humanized anti-programmed cell death receptor (PD-1) on tumor volume changes in a lung cancer mouse model.
  • PD-1 humanized anti-programmed cell death receptor
  • FIG. 7 is a graph showing the effect of ursodeoxycholic acid combined with humanized anti-programmed cell death receptor (PD-1) on tumor volume changes in a melanoma mouse model.
  • PD-1 humanized anti-programmed cell death receptor
  • Figure 8 is a graph showing the results of ursodeoxycholic acid reducing the differentiation of human endogenous Treg, where A is the flow cytometry test result, and Figure B is the statistical result chart, where * represents P ⁇ 0.05, *** represents P ⁇ 0.001.
  • mice Female C57BL / 6 (6-8 weeks) mice were purchased from Shanghai Slake Experimental Animal Co., Ltd. Humanized PD-1 transgenic mice were purchased from Nanjing University-Nanjing Institute of Biomedicine, all mice were bred at SPF level facility. Mouse colon cancer cell line MC38, lung cancer cell line LLC-luci, and melanoma cell line B16 were all purchased from the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • T cell sorting sorting mouse T cells by immunomagnetic bead method. The mice were sacrificed by cervical dislocation, the spleen and lymph nodes were taken aseptically, the filter was ground, and the cells were resuspended in sorting buffer after centrifugation at 400g for 5 minutes. After counting the total number of cells with 3% glacial acetic acid, follow the instructions of the sorting kit strictly for subsequent operations.
  • CD4 + T cells (CD4 positive initial T cells) were used with the CD4 negative selection kit (EasySep TM Mouse CD4 + T Cell Isolation Kit, # 19852A) in combination with the Biotin sorting kit (EasySep TM Mouse Biotin Positive Selection Kit, # 18556) .
  • CD4 + T cells were sorted by negative selection, followed by CD62L + T cells by positive selection. After sorting, CD4 + CD62L + purity was greater than 95%, the cells were used for subsequent experiments.
  • Treg cells not only have the ability to inhibit a wide range of anti-tumor immune responses, they can also promote the regeneration of tumor microenvironment blood vessels.
  • female SPF grade C57BL / 6 was used as the experimental object, and tumor-bearing mice were constructed using mouse colon cancer cell line MC38, lung cancer cell line LLC-luci, and melanoma cell line B16 model.
  • the ursodeoxycholic acid sodium salt URSO (SANTA CRUZ BIOTECHNOLOGY, # 2898-95-5) is used instead of the ursodeoxycholic acid.
  • control group and control group were injected with ddH 2 O and URSO (30 mg / kg) every day, and the tumor size was observed and recorded.
  • the results are shown in Figure 2A, Figure 3A, and Figure 4A.
  • the volume of tumor-bearing mice in the treatment group was significantly smaller.
  • the tumor tissue is concentrated and shredded in 1ml 1640 culture medium containing 10% serum;
  • ursodeoxycholic acid can reduce the proportion of Treg cells in tumor-infiltrating lymphocytes.
  • Ursodeoxycholic acid inhibits tumor growth mainly by reducing the proportion of Treg cells in the tumor microenvironment, and SHR-1210 as a humanized anti-PD-1 antibody (Hengrui Pharmaceutical), which can specifically block PD- 1 Binding to PD-L1 terminates the PD-1 immunosuppressive signal caused by the interaction between PD-1 and PD-L1 in T cells.
  • colon cancer cells MC38
  • melanoma cell lines B16
  • lung cancer cell lines LLC
  • SHR-1210 SHR-1210, URSO, or SHR-1210 and URSO in combination, water for injection and human-derived IgG4 (SHRR-1210 isotype control antibody)
  • SHR-1210 was used once every two days at a dosage of 10 mg / kg; URSO once a day at a dosage of 30 mg / kg, and the volume of each tumor was recorded.
  • URSO and SHR-1210 can significantly inhibit tumor growth, but the combined treatment of the two is significantly better than the single treatment group.
  • 1 represents URSO alone treatment group and URSO and SHR-1210 combination treatment group
  • 2 represents SHR-1210 alone treatment group and URSO and SHR-1210 combination treatment group
  • *** represents P ⁇ 0.001
  • ** represents P ⁇ 0.01
  • * represents P ⁇ 0.05.
  • the bottom of the tube is red blood cells
  • the middle layer is the separation liquid
  • the uppermost layer is the plasma / tissue homogenate layer.
  • a thin and dense white membrane that is: a single nucleus Layer of cells (including lymphocytes and monocytes). Carefully pipette the white film layer into another centrifuge tube.
  • Ursodeoxycholic acid can reduce the differentiation of human endogenous Tregs.

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Abstract

一种包括熊脱氧胆酸及其药用盐的抗肿瘤药物及其在制备抗肿瘤药物中的应用。熊脱氧胆酸通过降低肿瘤浸润淋巴细胞中调节性T细胞的比例对多种肿瘤细胞具有抑制作用，其还可以作为辅助药物，提高抗PD-1药物的治疗效果。

Description

熊脱氧胆酸或其药用盐在制备抗肿瘤药物中的应用及抗肿瘤药物 技术领域
本发明涉及生物医药技术领域，特别是涉及熊脱氧胆酸或其药用盐在制备抗肿瘤药物中的应用及抗肿瘤药物。
背景技术
随着肿瘤发生率和死亡率的逐年上升，恶性肿瘤已经成为严重威胁人类健康的重大慢性疾病，是我国乃至全球最严重的公共卫生问题之一，肿瘤控制已成为世界各国政府的卫生战略重点。对于早、中期患者而言，手术是首选的治疗方案。但由于肿瘤起病隐匿，早期诊断困难，多数患者首诊时病变已属局部晚期或已出现远处转移，针对这类患者，放疗、化疗及靶向治疗是目前主要治疗手段，然而它们并未给晚期实体瘤患者带来长期的生存获益。随着肿瘤学、免疫学以及分子生物学等相关学科的不断发展和交叉渗透，肿瘤免疫治疗在基础和临床研究中取得了迅猛发展，尤其是免疫检查点抑制剂，如程序性死亡受体1(PD-1)/程序性死亡配体(PD-L1)抗体，为晚期黑色素瘤和非小细胞肺癌等多种实体瘤患者带来了长期的生存获益。2018年诺贝尔生理或医学奖也颁发给了James P.Allison教授和Tasuku Honjo教授以表彰他们在人类肿瘤免疫治疗方面做出的贡献。
虽然免疫检查点抑制剂可以显著提高患者的生存率，然而仅有10％～30％的实体瘤患者可以从单药治疗中获益。究其原因，肿瘤内部的免疫抑制性微环境是抗肿瘤免疫无效的重要因素，其中抑制性细胞的浸润具有不可或缺的作用。常见的抑制性细胞包括髓系来源的抑制性细胞(MDSC)、肿瘤相关巨噬细胞(TAM)以及调节性T细胞(简称Treg细胞)。其中，Treg细胞介导的免疫抑制成为有效治疗肿瘤的主要障碍。Woo等首次报道在非小细胞肺癌和卵巢癌患者肿瘤组织中有大量Treg细胞浸润(Woo EY，Chu CS，Goletz TJ，et al.Regulatory CD4+CD25+T cell in tumors from patients with early-stage non-small cell lung cancer and late-satge ovarian cancer.Cancer Res,2001,61:4766-72)。随后，一系列报道表明Treg细胞在黑色素瘤、乳腺癌、结直肠癌、肺癌和胰腺癌等多种肿瘤癌旁和癌组织中聚集(Zou W.Regulatory T cells,tumour immunity and immunotherapy.Nat Rev Immunol,2006,6:295-307)。大量实验表明，Treg细胞增多有利于肿瘤的免疫逃逸，就临床病例来讲，乳腺癌、卵巢癌、胃癌和肝癌等肿瘤微环境中Treg细胞的增多和预后不良相关。从机制上来说，Treg细胞不仅 有能力抑制广泛的抗肿瘤免疫反应，同时还可以促进肿瘤微环境血管再生。所以，选择性去除或抑制肿瘤微环境中的Treg细胞成为肿瘤免疫治疗的新方向。
熊脱氧胆酸(ursodeoxychilic acid，UDCA)作为人胆汁中的天然胆汁盐，是由次级胆酸在肠道还原生成，具有利胆和保护肝细胞的作用，临床上常用来用来预防和治疗胆囊炎、胆管炎、胆汁消化不良和由胆固醇结石和结石引起的黄疸。最近有研究表明，熊脱氧胆酸能够抑制TNF-α，IL-1β，IL6等细胞因子参与炎症反应的调控(Ko,WK.,Kim,S.J.,Jo,MJ.et al.Mol Neurobiol 2018.)。
发明内容
本发明经研究发现熊脱氧胆酸具有抗肿瘤作用，能够用于制备抗肿瘤药物。
熊脱氧胆酸的化学式如式Ⅰ所示：
Figure PCTCN2019104831-appb-000001
本发明首先提供了熊脱氧胆酸或其药用盐在制备抗肿瘤药物中的应用。
所述熊脱氧胆酸或其药用盐通过降低肿瘤浸润淋巴细胞中调节性T细胞的比例起作用。
所述熊脱氧胆酸或其药用盐与抗PD-1药物联用。
所述抗肿瘤药物所针对的肿瘤种类为：黑色素瘤、直肠癌或肺癌。
本发明还提供了一种抗肿瘤药物，包括熊脱氧胆酸或其药用盐。
所述的抗肿瘤药物，还包括抗PD-1药物。
优选的，所述抗PD-1药物为抗PD-1单抗药物。比如恒瑞医药生产的SHR-1210，为一种人源化抗PD-1抗体。
所述抗肿瘤药物所针对的肿瘤种类为：黑色素瘤、直肠癌或肺癌。
本发明体外试验发现熊脱氧胆酸能够抑制内源性Treg细胞分化。为了探究该药物在体内的效应，通过三种肿瘤模型(黑色素瘤，直肠癌，肺癌)，发现熊脱氧胆酸能够明显抑制荷瘤小鼠模型中肿瘤的生长，利用流式细胞术对肿瘤浸润淋巴细胞进一步分析发现，在荷瘤小鼠模型中，用熊脱氧胆酸治疗组肿瘤浸 润淋巴细胞中Treg细胞的比例明显降低。通过熊脱氧胆酸与抗PD-1抗体联合治疗肿瘤，发现熊脱氧胆酸能够极大提高抗PD-1抗体的治疗效果。
本发明研究发现熊脱氧胆酸及其药用盐对多种肿瘤细胞具有良好的抑制作用，能够用于制备抗肿瘤药物。熊脱氧胆酸作为一种利胆药物已经应用于临床，不需要进行临床安全评估，具有很好的应用前景，而且能够作为辅助药物，提高抗PD-1药物的治疗效果。
附图说明
图1为熊脱氧胆酸对内源性Treg细胞分化的影响检测结果图，其中图A为流式细胞仪检测结果图，图B为统计结果图，其中***代表P<0.001，下同。
图2为黑色素瘤小鼠模型中熊脱氧胆酸抗肿瘤作用检测结果图，其中A为熊脱氧胆酸对肿瘤体积变化的影响结果图，B为肿瘤浸润淋巴细胞中Treg细胞的比例变化流式细胞仪检测结果图，图C为统计结果图，其中**代表P<0.01，下同。
图3为肺癌小鼠模型中熊脱氧胆酸抗肿瘤作用检测，其中A为熊脱氧胆酸对肿瘤体积变化的影响结果图，B为肿瘤浸润淋巴细胞中Treg细胞的比例变化流式细胞仪检测结果图，图C为统计结果图，其中*代表P<0.05，下同。
图4为结肠癌小鼠模型中熊脱氧胆酸抗肿瘤作用检测，其中A为熊脱氧胆酸对肿瘤体积变化的影响结果图，B为肿瘤浸润淋巴细胞中Treg细胞的比例变化流式细胞仪检测结果图，图C为统计结果图。
图5为熊脱氧胆酸联合人源化抗程序性细胞死亡受体(PD-1)对结肠癌小鼠模型肿瘤体积变化的影响结果图。
图6为熊脱氧胆酸联合人源化抗程序性细胞死亡受体(PD-1)对肺癌小鼠模型肿瘤体积变化的影响结果图。
图7为熊脱氧胆酸联合人源化抗程序性细胞死亡受体(PD-1)对黑色素瘤小鼠模型肿瘤体积变化的影响结果图。
图8为熊脱氧胆酸降低人源内源性Treg的分化检测结果图，其中A为流式细胞仪检测结果图，图B为统计结果图，其中*代表P<0.05，***代表P<0.001。
具体实施方式
雌性C57BL/6(6-8周)小鼠购买于上海斯莱克实验动物有限公司，人源化PD-1转基因小鼠购买于南京大学-南京生物医药研究院，所有小鼠均培育于SPF级设施。小鼠结肠癌细胞系MC38、肺癌细胞系LLC-luci、黑色素瘤细胞系B16 均购买于美国菌种保藏中心(ATCC)。
实施例1
Treg细胞表面能够表达CD4和CD25分子，除此之外，其特征标志为其高表达转录因子Foxp3，利用上述特征，采用流式细胞术在体外探究熊脱氧胆酸对Treg细胞分化的影响，具体步骤如下：
T细胞分选：用免疫磁珠法分选小鼠T细胞。颈椎脱臼法处死小鼠，无菌取脾脏和淋巴结，研磨后过滤网，400g离心5min后用分选缓冲液重悬细胞。3％冰醋酸计数细胞总数后，严格按照分选试剂盒说明书进行后续操作。
分选
Figure PCTCN2019104831-appb-000002
CD4+T细胞(CD4阳性初始T细胞)采用CD4阴选试剂盒(EasySep TMMouse CD4+T Cell Isolation Kit，#19852A)联合Biotin分选试剂盒(EasySep TMMouse Biotin Positive Selection Kit，#18556)使用。先阴选分选出CD4+T细胞，再接着阳选分选CD62L+T细胞，分选后流式检测CD4+CD62L+纯度大于95％时，细胞用于后续实验。
在96孔板中，用高压灭菌的PBS稀释anti-CD3、anti-CD28(BioXcell，#145-2C11)至终浓度2μg/ml，按照200μl/孔进行包板，37℃静置2小时以上。将上述分选出来的
Figure PCTCN2019104831-appb-000003
CD4+T细胞，4×10 5cells/孔，铺到96孔板里。分别加入浓度为50μM的DMSO或熊脱氧胆酸U5127(SIGMA-ALDRICH，#128-13-2)，第4天，通过流式细胞仪检测Treg细胞分化情况。具体步骤如下：
(1)收集细胞至流式管，PBS缓冲液冲洗一遍；
(2)添加0.2μl anti-mouse CD4 FITC(Biolegend，#100406)和anti-mouse CD25 APC(Biolegend，#101909)流式抗体，室温避光染色20分钟；
(3)PBS缓冲液冲洗一遍，用1ml eBioscience TMFoxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent(invitrogen，#00-5521-00)破核固定1小时；
(4)用1×eBioscience TMPermeabilization Buffer(invitrogen，#00-8333-56)中和，离心弃上清；
(5)添加0.5μl anti-mouse Foxp3 PE(eBioscience，#12-5773-82)流式抗体，4℃染色1小时；
(6)用1×Permeabilization Buffer缓冲液中和，洗2遍，弃上清，适量PBS缓冲液重悬细胞上机，通过流式细胞仪检测Treg分化情况。
结果显示，熊脱氧胆酸能够明显降低内源性Treg细胞的比例(图1)。
实施例2
由于Treg细胞不仅有能力抑制广泛的抗肿瘤免疫反应，同时还可以促进肿瘤微环境血管再生。为了探究体内熊脱氧胆酸对Treg细胞的影响，以雌性SPF级C57BL/6为实验对象，利用小鼠结肠癌细胞系MC38、肺癌细胞系LLC-luci、黑色素瘤细胞系B16构建荷瘤小鼠模型。为了更好地促进熊脱氧胆酸在体内吸收，用熊脱氧胆酸钠盐URSO(SANTA CRUZ BIOTECHNOLOGY，#2898-95-5)代替上述熊脱氧胆酸。
对照组(Control)和实验组每天注射ddH 2O和URSO(30mg/kg)，观察肿瘤大小并记录。结果如图2A，图3A，图4A所示，相比于对照组，治疗组荷瘤小鼠的体积明显偏小。
两周后，分离肿瘤浸润淋巴细胞，采用流式细胞术对其进行分析。具体步骤如下：
(1)肿瘤组织于1ml含10％血清的1640培养集中并剪碎；
(2)转移到10ml eppendorf管并补加培养基至5ml；
(3)添加四型胶原酶2mg/ml(Sangon Biotech，#A004186)、脱氧核糖核酸酶Ⅰ20μg/ml(SIGMA-ALDRICH，#10104159001)，37℃消化2小时；(4)消化结束后通过200目尼龙网过滤，得到单细胞悬液，1500rpm，4℃离心沉淀细胞悬液4-5分钟，弃掉上清；
(4)配制密度为1.082g/ml、1.075g/ml、1.07g/ml、1.06g/ml的Percoll(GE，#17-0819-09)分离液，并用高密度Percoll液重悬细胞，按照密度从小到大依次叠加到15ml离心管里，1500rpm，4℃离心20分钟；
(5)观察细胞分层，取中间白膜层细胞并用2倍体积PBS中和洗涤，1500rpm，4℃离心5分钟，弃掉上清；
(6)将细胞转移到流式管中，并按照实施例1中细胞染色步骤进行。
根据流式结果(图2B，图3B，图4B)，熊脱氧胆酸能够降低肿瘤浸润淋巴细胞内Treg细胞的比例。
实施例3
熊脱氧胆酸抑制肿瘤生长主要是降低肿瘤微环境中Treg细胞的比例发挥作用，而SHR-1210作为一种人源化抗PD-1抗体(恒瑞医药)，其可以特异性阻滞PD-1与PD-L1的结合，终止T细胞中PD-1与PD-L1相互作用引起的PD-1免疫抑制信号。
为了验证熊脱氧胆酸和抗PD-1抗体联合治疗是否能够提高抗肿瘤疗效，将 结肠癌细胞(MC38)，黑色素瘤细胞株(B16)，肺癌细胞株(LLC)皮下接种到人源PD-1转基因小鼠上，当肿瘤体积达到100mm 3，随机分成四组，治疗组分别用SHR-1210、URSO或SHR-1210和URSO联合治疗，注射用水和人源IgG4(SHRR-1210同型对照抗体)作为对照组，其中SHR-1210每两天一次，用量为10mg/kg；URSO每天一次，用量为30mg/kg，记录每次肿瘤的体积。结果如图5、图6、图7所示，URSO和SHR-1210能够明显抑制肿瘤生长，但两者联合治疗效果要明显优于单独治疗组。其中1代表URSO单独治疗组和URSO和SHR-1210联合治疗组比较；2代表SHR-1210单独治疗组和URSO和SHR-1210联合治疗组比较；***代表P<0.001，**代表P<0.01，*代表P<0.05。
实施例4
为了探究熊脱氧胆酸对人源性Treg细胞分化是否有影响，采集志愿者的全血(伦理审批：经浙江大学医学院医学伦理委员会审批，编号：申伦第(2019-013)号；健康志愿者共5名，每位采集外周静脉血10ml，血液采集由浙江省医疗健康集团杭州医院具体操作实施。)，分选
Figure PCTCN2019104831-appb-000004
CD4+T细胞，具体步骤如下：
1、外周血单核细胞分离：
(1)取新鲜抗凝全血(EDTA、枸橼酸钠或肝素抗凝剂均可)。用等体积等渗溶液(PBS或生理盐水)稀释全血。
(2)在离心管中加入一定体积的分离液(达优，#7111011)，将稀释后的血样平铺到分离液液面上方，保持两液面界面清晰。分离液、抗凝未经稀释全血、等渗溶液(PBS或生理盐水)体积为1∶1∶1。
(3)室温，水平转子700g-800g离心20min-30min。
(4)离心结束后，管底是红细胞，中间层是分离液，最上层是血浆/组织匀浆层，血浆层与分离液层之间是一层薄较致密的白膜，即：单个核细胞(包括淋巴细胞和单核细胞)层。小心吸取白膜层到另一离心管中。
(5)用等渗溶液(PBS、生理盐水或培养基)稀释到一定体积，颠倒混匀。室温，水平转子250g，离心10min弃上清。重复洗涤1-2次。
(6)用等渗溶液(PBS、生理盐水或培养基将细胞重悬备用。
2、人的
Figure PCTCN2019104831-appb-000005
CD4+T细胞分选：
(1)使用Human
Figure PCTCN2019104831-appb-000006
CD4+T Cell Kit(STEMCELL，#19155)，按照试剂盒标准方法，从人外周血单核细胞中分选
Figure PCTCN2019104831-appb-000007
CD4+T细胞按照实施例1中的方法进行铺板。
(2)加入Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Gibco，#11161D)；
(3)第4天，通过流式细胞仪检测Treg细胞分化情况。
结果如图8所示，熊脱氧胆酸能够降低人源内源性Treg的分化。

Claims (8)

  1. 熊脱氧胆酸或其药用盐在制备抗肿瘤药物中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述熊脱氧胆酸或其药用盐通过降低肿瘤浸润淋巴细胞中调节性T细胞的比例起作用。
  3. 如权利要求1所述的应用，其特征在于，所述熊脱氧胆酸或其药用盐与抗PD-1药物联用。
  4. 如权利要求1所述的应用，其特征在于，所述抗肿瘤药物所针对的肿瘤种类为：黑色素瘤、直肠癌或肺癌。
  5. 一种抗肿瘤药物，其特征在于，包括熊脱氧胆酸或其药用盐。
  6. 如权利要求5所述的抗肿瘤药物，其特征在于，还包括抗PD-1药物。
  7. 如权利要求6所述的抗肿瘤药物，其特征在于，所述抗PD-1药物为抗PD-1单抗药物。
  8. 如权利要求5所述的抗肿瘤药物，其特征在于，所述抗肿瘤药物所针对的肿瘤种类为：黑色素瘤、直肠癌或肺癌。
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