WO2023246003A1 - 一种嵌合抗原受体巨噬细胞及其制备方法和应用 - Google Patents

一种嵌合抗原受体巨噬细胞及其制备方法和应用 Download PDF

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WO2023246003A1
WO2023246003A1 PCT/CN2022/137819 CN2022137819W WO2023246003A1 WO 2023246003 A1 WO2023246003 A1 WO 2023246003A1 CN 2022137819 W CN2022137819 W CN 2022137819W WO 2023246003 A1 WO2023246003 A1 WO 2023246003A1
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antigen receptor
chimeric antigen
cells
bone marrow
culture medium
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万晓春
陈有海
刘茂玄
戴昆
刘骏晨
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深圳先进技术研究院
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Definitions

  • the invention relates to the field of cell culture, and in particular to a chimeric antigen receptor macrophage and its preparation method and application.
  • CAR-M Chimeric antigen receptor macrophages install a CAR on macrophages so that they can specifically kill/phagocytose tumor cells.
  • CAR-M has the following advantages: it can improve the immune microenvironment of solid tumors; it can serve as antigen-presenting cells to present antigens after engulfing tumor cells; and it is easier to infiltrate into tumors. Internally, it cooperates with other immune cells to infiltrate tumors, etc. Therefore, the research on CAR-M is of great significance.
  • the chassis cells used are mainly mouse or human cell lines (such as RAW 264.7; J774A.1; THP-1 cells), while immortalized or tumor cell lines do not well represent the primary The nature of the cells provides limited role in CAR-M research.
  • some researchers have used human primary macrophages as chassis cells to prepare CAR-M cells.
  • the source of human primary macrophages is limited.
  • the source of blood cells from healthy volunteers is inconvenient, and the mononuclear cells in healthy human PBMCs are The proportion of cells (used to differentiate into macrophages) is low, about 10%, and it is almost impossible to expand. Therefore, using human primary macrophages as the source of CAR-M has a relatively limited number of cells, which also greatly affects CAR-M research.
  • the chassis cells used are mainly mouse or human cell lines (such as RAW 264.7; J774A.1; THP-1 cells), while immortalized or tumor cell lines do not well represent the primary The nature of the cells provides limited role in CAR-M research.
  • some researchers have used human primary macrophages as chassis cells to prepare CAR-M cells.
  • the source of human primary macrophages is limited.
  • the source of blood cells from healthy volunteers is inconvenient, and the mononuclear cells in healthy human PBMCs are The proportion of cells (used to differentiate into macrophages) is low, about 10%, and it is almost impossible to expand. Therefore, using human primary macrophages as the source of CAR-M has a relatively limited number of cells, which also greatly affects CAR-M research.
  • the purpose of the present invention is to provide a chimeric antigen receptor macrophage and its preparation method and application.
  • a first aspect of the present invention provides a chimeric antigen receptor macrophage.
  • the chimeric antigen receptor macrophage is a mouse bone marrow-derived macrophage modified by a chimeric antigen receptor and expressing a chimeric antigen receptor. .
  • the modification includes introducing the nucleic acid or vector encoding the polynucleotide sequence of the chimeric antigen receptor into mouse bone marrow-derived macrophages;
  • the nucleic acid is located on different viral vectors;
  • the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector;
  • the vector is a transposon or an mRNA vector.
  • mouse bone marrow-derived macrophages are mouse primary bone marrow-derived macrophages.
  • a second aspect of the present invention provides a method for preparing chimeric antigen receptor macrophages, including the following steps:
  • nucleic acid or vector encoding the polynucleotide sequence of the chimeric antigen receptor is introduced into mouse primary bone marrow-derived macrophages.
  • mouse primary bone marrow-derived macrophages is:
  • Mouse bone marrow cells are cultured in DMEM cell culture medium. After three days of culture, half of the culture medium is replaced, and after seven days of culture, the entire culture medium is replaced. The resulting adherent cells are BMDM cells;
  • the DMEM cell culture medium is a DMEM high-glucose culture medium containing 10 ⁇ 20% FBS, 10 ⁇ 20 ng/mL M-CSF, and 1% penicillin and streptomycin;
  • the DMEM cell culture medium is DMEM high-glucose culture medium containing 20% FBS, 20 ng/mL M-CSF, and 1% penicillin and streptomycin;
  • the mouse bone marrow cells are cultured in non-TC-treated or TC-treated T25 cell culture bottles;
  • the mouse bone marrow cells are cultured in non-TC-treated T25 cell culture bottles;
  • step 2) also includes digesting the adherent cells with ACCUTASE digestion solution.
  • step 1) to extract mouse bone marrow cells is as follows: separate and remove the femur and tibia of the mouse, place them in a cell culture dish containing 75% alcohol, further separate and remove the tissue around the bones, and then move them into Wash in another cell culture dish containing 1 ⁇ PBS, and finally transfer it to another cell culture dish containing DMEM cell culture medium; remove both ends of the femur and tibia, and use DMEM cell culture medium to remove the two ends of the bone from one end of the bone. Rinse the bone marrow cells from the broken end into a sterile centrifuge tube. Centrifuge the collected cell suspension at 1500 r/min and 4°C for 8 minutes. Discard the supernatant.
  • red blood cell lysis solution pipette repeatedly with a pipette, and then let stand on ice. 5 min; add 4-5 times of red blood cell lysis solution in PBS to stop, centrifuge at 1500 r/min for 8 min at 4°C, discard the supernatant; add 5 mL of DMEM cell culture medium to resuspend the cells, and then filter with a 70 ⁇ m cell mesh. Cells; centrifuge at 1500 r/min, 4°C for 8 min, discard the supernatant, and repeat 3 times to obtain;
  • the DMEM cell culture medium is DMEM high glucose culture medium containing 10 ⁇ 20% FBS, 10 ⁇ 20 ng/mL M-CSF and 1% penicillin and streptomycin.
  • the nucleic acid is located on different viral vectors;
  • the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector;
  • the vector is a transposon or an mRNA vector
  • the introduction method includes electroporation, transduction or transfection.
  • nucleic acid encoding the polynucleotide sequence of the chimeric antigen receptor is preferably introduced into mouse primary bone marrow-derived macrophages; the nucleic acid is located on a lentiviral vector;
  • the preparation method of the lentiviral vector is: combining the lentiviral vector encoding the polynucleotide sequence of the chimeric antigen receptor, psPAX2 plasmid, and pMD2.G plasmid according to the mass ratio (10-15): (7.5-10 ): (3-5) Transfect 293T cells to obtain the lentivirus.
  • the lentiviral vector was introduced into mouse primary bone marrow-derived macrophages by transfection;
  • the MOI is 1 to 1000.
  • the third aspect of the present invention provides that the chimeric antigen receptor macrophages or the chimeric antigen receptor macrophages prepared by the preparation method are used to study the properties of chimeric antigen receptor macrophages in mice or For research on chimeric antigen receptor macrophages in the treatment of tumors and their indications;
  • the tumor is a hematological tumor or a solid tumor
  • the tumor is leukemia, multiple myeloma, malignant lymphoma, glioma, liver cancer, lung cancer, gastric cancer, colon cancer, pancreatic cancer or breast cancer;
  • the indication is liver fibrosis, pulmonary fibrosis or Alzheimer's disease.
  • the chimeric antigen receptor macrophages provided by the present invention are prepared from mouse bone marrow-derived macrophages (BMDM) and have many advantages: (1) As primary macrophages, they can more truly reflect the situation of CAR-M; ( 2) The source of cells is not limited, and the number of cells available for research is large; (3) When studying some properties of CAR-M in vivo, mouse CAR-BMDM was used as the research object to conduct corresponding experiments in mice. It can more truly reflect some situations of CAR-M in the body.
  • the chimeric antigen receptor macrophage of the present invention lays a foundation for subsequent research on CAR-M, especially in vivo research on CAR-M, and is also convenient for research on treating tumors and its indications.
  • Figure 1 Positive rate detection of CAR expression in CAR-M cells.
  • the DMEM cell culture medium is DMEM high glucose medium (Gibco) + 20% FBS (fetal bovine serum) + 20 ng/mL M-CSF+1% double antibody (penicillin-streptomycin).
  • Opti-MEM culture medium was purchased from Thermo Fisher Scientific, product number: 31985088.
  • ACCUTASE digestion solution was purchased from Biolegend.
  • the mouse bone marrow cells were taken and added to the prepared DMEM cell culture medium to induce the bone marrow cells to differentiate into macrophages. Count the cells, adjust the cell concentration to 1 ⁇ 3 ⁇ 10 6 cells/ml, and culture them in two prepared DMEM cell culture media (different concentrations of M-CSF factors of 20 ng/mL and 10 ng/mL). In non-TC-treated and TC-treated T25 cell culture bottles (6 mL/bottle), place them in a 37°C, 5% carbon dioxide incubator. After three days of culture, half of the culture medium is replaced, and after seven days of culture, the entire culture medium is replaced. The resulting Adherent cells are BMDM cells.
  • the DMEM cell culture medium was DMEM high-glucose medium (Gibco) + 20% FBS (fetal bovine serum) + 20 ng/mL M-CSF. +1% double antibody (penicillin-streptomycin).
  • the centrifuge must be sprayed with 75% ethanol for disinfection after use); 4 After 72 hours of infection, FcR ⁇ CAR-M and Megf10 CAR-M cells were obtained and analyzed by fluorescence microscope or flow. The positive rate was detected by cytometer.
  • the SPECTROstar Omega microplate reader was used to collect and analyze the fluorescence intensity for cell killing experiments.
  • Raji cells stably expressing luciferase serum-free, M-CSF-free DMEM medium
  • the experiment was divided into 4 groups: Raji blank control group, BMDM, FcR ⁇ CAR-M and Megf10 CAR-M cells.
  • the killing results are shown in Figure 2.
  • the prepared FcR ⁇ CAR-M and Megf10 CAR-M cells were both able to significantly kill Raji target cells, indicating that FcR ⁇ has the targeted killing function.
  • CAR-M and Megf10 CAR-M cells were successfully prepared.

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Abstract

提供了一种嵌合抗原受体巨噬细胞及其制备方法和应用，该嵌合抗原受体巨噬细胞为经嵌合抗原受体修饰且表达嵌合抗原受体的小鼠骨髓来源巨噬细胞。

Description

一种嵌合抗原受体巨噬细胞及其制备方法和应用 技术领域
本发明涉及细胞培养领域，具体涉及一种嵌合抗原受体巨噬细胞及其制备方法和应用。
 背景技术
嵌合抗原受体巨噬细胞（CAR-M）是在巨噬细胞上装上一个CAR，使其可以特异性的杀伤/吞噬肿瘤细胞。CAR-M与CAR-T相比，除了可以直接杀伤肿瘤细胞之外，其优势在于：可以改善实体瘤肿瘤免疫微环境；在吞噬肿瘤细胞后可以作为抗原呈递细胞呈递抗原；更容易浸润到肿瘤内部，协同其他免疫细胞浸润肿瘤等。因此，对于CAR-M的研究具有非常重要的意义。
目前关于CAR-M的制备，所用的底盘细胞主要有用小鼠或人的细胞系（如RAW 264.7；J774A.1；THP-1细胞），而永生化或肿瘤细胞系并不能良好的代表原代细胞的性质，对于CAR-M的研究提供的作用有限。此外，也有研究者采用人原代巨噬细胞作为底盘细胞制备CAR-M细胞，但是人原代巨噬细胞来源受限，首先，健康志愿者的血细胞来源不方便，并且健康人PBMC中单核细胞（用于分化成巨噬细胞）的比例较低，大概10%左右，同时几乎不能扩增。因此，采用人原代巨噬细胞作为CAR-M的来源，细胞数量比较受限，也极大的影响CAR-M的研究。
 技术问题
目前关于CAR-M的制备，所用的底盘细胞主要有用小鼠或人的细胞系（如RAW 264.7；J774A.1；THP-1细胞），而永生化或肿瘤细胞系并不能良好的代表原代细胞的性质，对于CAR-M的研究提供的作用有限。此外，也有研究者采用人原代巨噬细胞作为底盘细胞制备CAR-M细胞，但是人原代巨噬细胞来源受限，首先，健康志愿者的血细胞来源不方便，并且健康人PBMC中单核细胞（用于分化成巨噬细胞）的比例较低，大概10%左右，同时几乎不能扩增。因此，采用人原代巨噬细胞作为CAR-M的来源，细胞数量比较受限，也极大的影响CAR-M的研究。
 技术解决方案
为了解决现有技术中的不足，本发明的目的在于提供一种嵌合抗原受体巨噬细胞及其制备方法和应用。
具体技术方案如下：
本发明第一方面提供一种嵌合抗原受体巨噬细胞，所述嵌合抗原受体巨噬细胞为经嵌合抗原受体修饰且表达嵌合抗原受体的小鼠骨髓来源巨噬细胞。
进一步地，所述修饰包括将编码嵌合抗原受体的多核苷酸序列的核酸或载体导入小鼠骨髓来源巨噬细胞中；
优选地，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
优选地，所述载体为转座子或mRNA载体。
进一步地，所述小鼠骨髓来源巨噬细胞为小鼠原代骨髓来源巨噬细胞。
本发明第二方面提供一种嵌合抗原受体巨噬细胞的制备方法，包括如下步骤：
（1）获取小鼠原代骨髓来源巨噬细胞；
（2）将编码嵌合抗原受体的多核苷酸序列的核酸或载体导入小鼠原代骨髓来源巨噬细胞中，即得。
进一步地，获取小鼠原代骨髓来源巨噬细胞的方法为：
1）提取小鼠骨髓细胞；
2）小鼠骨髓细胞加入DMEM细胞培养基培养，培养三天后半量更换培养基，七天后全换培养基，所得的贴壁细胞即为BMDM细胞；
所述DMEM细胞培养基为含有10~20% FBS、10~20 ng/mL M-CSF以及1%青霉素和链霉素的DMEM高糖培养基；
优选地，所述DMEM细胞培养基为含有20% FBS、20 ng/mL M-CSF以及1%青霉素和链霉素的DMEM高糖培养基；
优选地，所述小鼠骨髓细胞培养在非TC处理或TC处理的T25细胞培养瓶中；
优选地，所述小鼠骨髓细胞培养在非TC处理的T25细胞培养瓶中；
优选地，步骤2）还包括将贴壁细胞用ACCUTASE消化液消化下来。
进一步地，步骤1）提取小鼠骨髓细胞的具体操作为：分离并取下小鼠的股骨和胫骨，放入含有75%酒精的细胞培养皿中，进一步分离去除骨周围组织，然后将其移入含有1×PBS的另一细胞培养皿中清洗，最后再将其转移至另一含有DMEM细胞培养基的细胞培养皿中；去除股骨和胫骨的骨两端，用DMEM细胞培养基从骨的一个断端冲洗骨髓细胞到无菌离心管中，把收集到的细胞悬液1500 r/min，4℃离心 8 min，弃上清液；加入红细胞裂解液，用吸管反复吹打，然后冰上静置 5 min；加入4-5倍红细胞裂解液的PBS终止，1500 r/min，4℃离心 8 min，弃上清液；加入5 mL DMEM 细胞培养基重悬细胞，然后用70 μm细胞筛网过滤细胞；1500 r/min，4℃离心 8 min，弃上清液，重复 3 次，即得；
优选地，所述DMEM细胞培养基为含有10~20% FBS、10~20 ng/mL M-CSF以及1%青霉素和链霉素的DMEM高糖培养基。
进一步地，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
所述载体为转座子或mRNA载体；
所述导入的方式包括电穿孔、转导或转染。
进一步地，优选将编码嵌合抗原受体的多核苷酸序列的核酸导入小鼠原代骨髓来源巨噬细胞中；所述核酸位于慢病毒载体上；
优选地，所述慢病毒载体的制备方法为：将编码嵌合抗原受体的多核苷酸序列的慢病毒载体、 psPAX2质粒、pMD2.G质粒按照质量比（10-15）:（7.5-10）:（3-5）转染293T细胞，获得所述慢病毒。
进一步地，所述慢病毒载体采用转染的方式导入小鼠原代骨髓来源巨噬细胞中；
优选地，所述慢病毒载体转染小鼠原代骨髓来源巨噬细胞时，MOI为1~1000。
本发明第三方面提供所述的嵌合抗原受体巨噬细胞或所述制备方法制备的嵌合抗原受体巨噬细胞用于在小鼠体内研究嵌合抗原受体巨噬细胞的性质或用于嵌合抗原受体巨噬细胞在治疗肿瘤及其适应症中的研究；
优选地，所述肿瘤为血液瘤或实体瘤；
优选地，所述肿瘤为白血病、多发性骨髓瘤、恶性淋巴瘤、脑胶质瘤、肝癌、肺癌、胃癌、结肠癌、胰腺癌或乳腺癌；
优选地，所述适应症为肝纤维化、肺纤维化或老年痴呆。
 有益效果
本发明的有益效果为：
本发明提供的嵌合抗原受体巨噬细胞采用小鼠骨髓来源巨噬细胞（BMDM）制备，具有诸多优势：（1）作为原代巨噬细胞，可以比较真实反映CAR-M的情况；（2）细胞来源不受限，可供研究的细胞数量较多；（3）在研究CAR-M在体内的一些性质时，以小鼠CAR-BMDM作为研究对象在小鼠体内进行相应的实验，可以比较真实的反映CAR-M在体内的一些情况。本发明嵌合抗原受体巨噬细胞为后续CAR-M的研究，尤其是CAR-M的体内研究，奠定了基础，也方便用于治疗肿瘤及其适应症中的研究。
 附图说明
图1：CAR-M细胞CAR表达的阳性率检测。
图2：杀伤48小时后各组的杀伤率统计（效应细胞：靶细胞=3）。
 本发明的实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
本发明所采用培养基及试剂如下：
本发明中所采用的DMEM细胞培养基为，DMEM高糖培养基（Gibco）+10~20% FBS(胎牛血清)+ 10~20 ng/mL M-CSF+1%双抗(青霉素-链霉素)。在具体的实施例中，BMDM细胞的获取步骤中，DMEM细胞培养基为DMEM高糖培养基（Gibco）+20% FBS(胎牛血清)+ 20 ng/mL M-CSF+1%双抗(青霉素-链霉素)。BMDM细胞培养步骤中，DMEM细胞培养基为DMEM高糖培养基（Gibco）+20% FBS(胎牛血清)+ 20 ng/mL M-CSF+1%双抗(青霉素-链霉素)。
红细胞裂解液，购自碧云天公司，产品编号：C3702。
Opti-MEM培养基，购自赛默飞世尔科技，货号：31985088。
ACCUTASE消化液，购自Biolegend公司。
实施例1
本实施例提供采用小鼠骨髓来源巨噬细胞（BMDM）制备CAR-M的方法，具体如下：
（1）BMDM细胞的获取
C57BL/6N小鼠颈椎脱臼法处死，用75％酒精溶液对其充分消毒，固定小鼠；在无菌条件下分离并取下小鼠的股骨和胫骨，小心不要打破骨头，然后放入事先含有75%酒精的75 cm 2的细胞培养皿中；移入生物安全柜中，进一步分离去除骨周围组织，然后将其移入含有1×PBS的另一细胞培养皿中清洗，最后再将其转移至另一 75cm 2细胞培养皿（含有DMEM细胞培养基）中等待下一步处理；用眼科组织剪去除股骨和胫骨的骨两端，然后用含有2 mL DMEM 细胞培养基的注射器从骨的一个断端冲洗骨髓细胞到50 mL无菌离心管中，重复3次；把收集到的细胞悬液用1500 r/min，4℃离心 8 min，弃上清液；加入红细胞裂解液（1 mL/只），用吸管反复吹打，然后冰上静置 5 min；加入4-5倍红细胞裂解液的PBS终止，1500 r/min，4℃离心 8 min，弃上清液；加入5 mL DMEM 细胞培养基重悬细胞，然后用70 μm细胞筛网过滤细胞；1500 r/min，4℃离心 8 min，弃上清液，重复 3 次。获得小鼠骨髓细胞。
（2）BMDM细胞培养
取好的小鼠骨髓细胞加入配制好的DMEM细胞培养基诱导骨髓细胞分化为巨噬细胞。细胞计数，调整细胞浓度至1~3×10 6个／ml，分别用配制好的两种DMEM细胞培养基培养（区别为20 ng/mL和10 ng/mL浓度的M-CSF因子）分别培养在非TC处理和TC处理的T25细胞培养瓶中（6 mL/瓶），放于 37℃、5％二氧化碳培养箱中培养，培养三天后半量更换培养基，七天后全换培养基，所得的贴壁细胞即为BMDM细胞。然后用ACCUTASE消化液消化下来比较不同因子浓度的培养基和不同培养瓶所获得的BMDM细胞数量以及F4/80染色用流式细胞术测纯度。结果显示，20 ng/mL M-CSF因子培养的BMDM细胞收率更高，纯度与10 ng/mL组差别不明显。采用非TC处理的T25细胞培养瓶培养的BMDM细胞收率更高，纯度与TC处理的T25细胞培养瓶组无差别。
最终确定慢病毒感染BMDM细胞培养时，采用非TC处理的T25细胞培养瓶，DMEM细胞培养基为DMEM高糖培养基（Gibco）+20% FBS(胎牛血清)+ 20 ng/mL M-CSF+1%双抗(青霉素-链霉素)。
（3）慢病毒的制备
①准备目的质粒与包装质粒：用质粒大提试剂盒提取所需目的质粒以及二代包装质粒psPAX2和pMD2.G，并用Nano Drop仪器检测相应质粒的浓度。
②准备293T细胞：复苏293T细胞，传2-3代；在包被病毒的前一天，把293T细胞用胰酶消化且计数，接种3.5×10 6左右个细胞于10 cm平皿中，补DMEM完全培养基到10 mL；当细胞融合度达到70-80%左右的时候，更换5 mL新的无抗生素DMEM培养基后即可准备病毒的包被。
③脂质体转染：将20 μL Lipofectamine 3000加入500 μL Opti-MEM培养基于一1.5 mL EP管中，用枪轻轻混匀，不要产生气泡，室温静置5min；接着将10-15 μg目的质粒（pHR CD19-FcRγ CAR或pHR CD19-Megf-10 CAR质粒，均含GFP标签）、7.5-10 μg psPAX2质粒、3-5 μg pMD2.G质粒加入500 μL Opti-MEM培养基于另一1.5 mL EP管中，然后再加入20 μL P3000试剂，用枪轻轻混匀，不要产生气泡；将上述两种混合溶液进行混合，用枪多次轻力吹打均匀，室温静置15 min后将混合后的液体用枪转入之前换液完成备用的293T细胞培养液中，轻轻晃动培养皿即可，不要把293T细胞晃动脱离培养皿底；最后把培养皿放入37℃，5%二氧化碳培养箱中，6-8 h之后，吸出含有脂质体的培养基，换成正常的DMEM完全培养基；
④收毒(生物安全柜内操作)：将培养了48 h的293T细胞的培养液上清全部吸出至15 mL离心管内，然后往培养皿中补加10 mL293T细胞培养液（继续培养细胞，24 h后可以再收毒一次）；接着把吸出的培养液上清置于离心机中3000 rpm离心10 min，用注射器吸出上清，0.45 μm滤器过滤后得到CD19-FcRγ CAR、CD19-Megf-10 CAR慢病毒，备用（可直接使用，也可以放入-80℃冰箱保存）。
（4）慢病毒感染BMDM细胞
①根据前面描述BMDM细胞的获取方法进行小鼠BMDM的分离与培养，待培养至第七日时将生长状态良好的BMDM细胞的培养液吸去，用无抗生素的DMEM清洗一次；②将收取的CD19-FcRγ CAR或CD19-Megf-10 CAR慢病毒分别加入BMDM细胞中，其中，Blank组加入等量的空白培养基。将培养瓶口用封口膜封住，放入37℃，5%二氧化碳培养箱中培养；③48 h之后，将含有病毒液的培养基去除，加入新的BMDM培养基（丢弃的上清和用过的离心管等必须高压灭菌处理，不得乱丢乱放，离心机用后要喷洒75%乙醇消毒）；④感染72 h后，得到FcRγ CAR-M和Megf10 CAR-M细胞，通过荧光显微镜或流式细胞仪检测其阳性率。
（5）慢病毒感染BMDM效率的检测
72 h后，利用ACCUTASE细胞消化液对病毒感染的BMDM细胞进行收集，利用流式细胞术对CAR的表达以及BMDM细胞的病毒感染效率进行检测。结果如附图1所示，CAR成功表达在BMDM的细胞表面。
（6）CAR-M杀伤靶细胞的检测
采用SPECTROstar Omega酶标仪对荧光强度进行采集分析进行细胞杀伤实验。首先把BMDM、FcRγ CAR-M和Megf10 CAR-M细胞以2-5×10 4/孔接种至不透光的96孔细胞培养板中，每组5-10个复孔，静置24-48 h。24 h后按照效应细胞：靶细胞=3：1加入稳定表达luciferase的Raji细胞（无血清，无M-CSF的DMEM培养基），与各组巨噬细胞共培养。实验共分为4组：Raji空白对照组、BMDM、FcRγ CAR-M和Megf10 CAR-M细胞。
37℃，5%二氧化碳培养箱中培养24-48 h后，向不透光96孔板中加入浓度为100-200 μg/mL的D-luciferin，potassium salt底物，37℃避光孵育10 min后，使用SPECTROstar Omega酶标仪对荧光强度进行采集分析，并通过以下公式各巨噬细胞组对Raji(luciferase)靶细胞的溶解进行计算：
%细胞溶解(Lysis%) = 【1 -（共培养细胞的荧光信号-背景荧光信号）/
（单独培养PC-3细胞荧光信号-背景荧光信号）】*100
杀伤结果如附图2所示，与BMDM对照组相比，制备的FcRγ CAR-M和Megf10 CAR-M细胞均能够显著杀伤Raji靶细胞，表明具备靶向杀伤功能的FcRγ CAR-M和Megf10 CAR-M细胞制备成功。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种嵌合抗原受体巨噬细胞，其特征在于，所述嵌合抗原受体巨噬细胞为经嵌合抗原受体修饰且表达嵌合抗原受体的小鼠骨髓来源巨噬细胞。
  2. 根据权利要求1所述的嵌合抗原受体巨噬细胞，其特征在于，所述修饰包括将编码嵌合抗原受体的多核苷酸序列的核酸或载体导入小鼠骨髓来源巨噬细胞中；
    优选地，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
    优选地，所述载体为转座子或mRNA载体。
  3. 根据权利要求1所述的嵌合抗原受体巨噬细胞，其特征在于，所述小鼠骨髓来源巨噬细胞为小鼠原代骨髓来源巨噬细胞。
  4. 一种嵌合抗原受体巨噬细胞的制备方法，其特征在于，包括如下步骤：
    （1）获取小鼠原代骨髓来源巨噬细胞；
    （2）将编码嵌合抗原受体的多核苷酸序列的核酸或载体导入小鼠原代骨髓来源巨噬细胞中，即得。
  5. 根据权利要求4所述的制备方法，其特征在于，获取小鼠原代骨髓来源巨噬细胞的方法为：
    1）提取小鼠骨髓细胞；
    2）小鼠骨髓细胞加入DMEM细胞培养基培养，培养三天后半量更换培养基，七天后全换培养基，所得的贴壁细胞即为BMDM细胞；
    所述DMEM细胞培养基为含有10~20% FBS、10~20 ng/mL M-CSF以及1%青霉素和链霉素的DMEM高糖培养基；
    优选地，所述DMEM细胞培养基为含有20% FBS、20 ng/mL M-CSF以及1%青霉素和链霉素的DMEM高糖培养基；
    优选地，所述小鼠骨髓细胞培养在非TC处理或TC处理的T25细胞培养瓶中；
    优选地，所述小鼠骨髓细胞培养在非TC处理的T25细胞培养瓶中；
    优选地，步骤2）还包括将贴壁细胞用ACCUTASE消化液消化下来。
  6. 根据权利要求5所述的制备方法，其特征在于，步骤1）提取小鼠骨髓细胞的具体操作为：分离并取下小鼠的股骨和胫骨，放入含有75%酒精的细胞培养皿中，进一步分离去除骨周围组织，然后将其移入含有1×PBS的另一细胞培养皿中清洗，最后再将其转移至另一含有DMEM细胞培养基的细胞培养皿中；去除股骨和胫骨的骨两端，用DMEM细胞培养基从骨的一个断端冲洗骨髓细胞到无菌离心管中，把收集到的细胞悬液1500 r/min，4℃离心 8 min，弃上清液；加入红细胞裂解液，用吸管反复吹打，然后冰上静置 5 min；加入4-5倍红细胞裂解液的PBS终止，1500 r/min，4℃离心 8 min，弃上清液；加入5 mL DMEM 细胞培养基重悬细胞，然后用70 μm细胞筛网过滤细胞；1500 r/min，4℃离心 8 min，弃上清液，重复 3 次，即得；
    优选地，所述DMEM细胞培养基为含有10~20% FBS、10~20 ng/mL M-CSF以及1%青霉素和链霉素的DMEM高糖培养基。
  7. 根据权利要求4所述的制备方法，其特征在于，所述核酸位于不同的病毒载体上；所述病毒载体为慢病毒载体、腺病毒载体或逆转录病毒载体；
    所述载体为转座子或mRNA载体；
    所述导入的方式包括电穿孔、转导或转染。
  8. 根据权利要求4所述的制备方法，其特征在于，优选将编码嵌合抗原受体的多核苷酸序列的核酸导入小鼠原代骨髓来源巨噬细胞中；所述核酸位于慢病毒载体上；
    优选地，所述慢病毒载体的制备方法为：将编码嵌合抗原受体的多核苷酸序列的慢病毒载体、 psPAX2质粒、pMD2.G质粒按照质量比（10-15）:（7.5-10）:（3-5）转染293T细胞，获得所述慢病毒。
  9. 根据权利要求8所述的制备方法，其特征在于，所述慢病毒载体采用转染的方式导入小鼠原代骨髓来源巨噬细胞中；
    优选地，所述慢病毒载体转染小鼠原代骨髓来源巨噬细胞时，MOI为1~1000。
  10. 权利要求1-3任一项所述的嵌合抗原受体巨噬细胞或权利要求4-9任一项所述制备方法制备的嵌合抗原受体巨噬细胞用于在小鼠体内研究嵌合抗原受体巨噬细胞的性质或用于嵌合抗原受体巨噬细胞在治疗肿瘤及其适应症中的研究；
    优选地，所述肿瘤为血液瘤或实体瘤；
    优选地，所述肿瘤为白血病、多发性骨髓瘤、恶性淋巴瘤、脑胶质瘤、肝癌、肺癌、胃癌、结肠癌、胰腺癌或乳腺癌；
    优选地，所述适应症为肝纤维化、肺纤维化或老年痴呆。
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