A method for constructing a human immune cell transplanted zebrafish model and its application
By injecting human immune cells into zebrafish juveniles, the problem of large demand for immune cells and low detection throughput in the mouse model was solved, and efficient and low-cost tumor research and evaluation was achieved.
Patent Information
- Application Number
- CN202310240690.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-14
- Publication Date
- 2025-07-04
- Estimated Expiration
- 2043-03-14
AI Technical Summary
The existing mouse models have high demand for immune cells, long reconstruction cycles and low detection throughput when building the human immune system, resulting in high research costs and low efficiency.
Zebrafish was used as the host, and human immune cells were injected into zebrafish juveniles 45 to 50 hours after fertilization to construct a zebrafish transplantation model. It was used to study immune cells and tumors with high homology, small size, fast reproduction and low price with zebrafish.
It has achieved low use of immune cells, high detection throughput, short experimental cycle, no immunosuppression treatment required, which is easy to observe and take photos, reduces research costs, and is suitable for the evaluation of anti-tumor effects of human immune cells and immune detection point inhibitors.
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Figure CN116439167B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of immunotherapy, and particularly relates to a method for constructing a human immune cell transplanted zebrafish model and its application. Background Art
[0002] Since 2010, the incidence and mortality of cancer have been continuously increasing, and it has become one of the main causes of death and major public health problems in the world. Traditional cancer treatment methods, such as surgery, radiotherapy, and chemotherapy, are difficult to completely eradicate cancer cells. The emerging immunotherapy is revolutionizing the clinical management of multiple tumors. The main mechanism of immunotherapy is to change the tumor microenvironment or immune cells so that the immune system can achieve the purpose of killing tumors. Immunotherapy targeting auxiliary immune cells and adoptive immunotherapy with gene-edited immune cells are considered promising strategies for treating various cancers.
[0003] Mouse models are commonly used models for studying and evaluating the efficacy of tumor immunotherapy. Among them, reconstructing the human immune system in mice is the key to establishing animal models. However, there is no unified method and standard for reconstructing the human immune system in the currently widely used mouse models. In addition, using mice as hosts for immune reconstruction has many disadvantages. For example, the demand for immune cells during the reconstruction process is huge, the immune reconstruction period is long, and the detection throughput is low. Therefore, it greatly increases the time and economic costs of researchers and poses certain obstacles to the preclinical research and efficacy evaluation of tumor immunotherapy.
[0004] Therefore, providing a human immune cell transplanted animal model with low immune cell usage, high detection throughput, and short experimental period is of great significance for the research on the anti-tumor effects of immune cells and immune checkpoint inhibitors. Summary of the Invention
[0005] In order to solve the technical problems of the existing mouse models with huge demand for immune cells, long immune reconstruction period, and low detection throughput, the present invention provides a method for constructing a human immune cell transplanted zebrafish model and its application. This method for constructing a human immune cell transplanted zebrafish model has the advantages of low immune cell usage and dosage, high detection throughput, short experimental period, no need for immunosuppression induction, easy observation and photography, and can be used for evaluating the anti-tumor effects of human immune cells and immune checkpoint inhibitors.
[0006] The specific technical solution of the present invention is as follows:
[0007] In the first aspect, the present invention provides a method for constructing a human immune cell transplanted zebrafish model, including the following steps:
[0008] (1) Take zebrafish larvae 45 - 50 h after fertilization;
[0009] (2) Inject human immune cells into the sinus venosus of zebrafish larvae to obtain a zebrafish model transplanted with human immune cells.
[0010] Zebrafish has a homology of up to 85% with human genes, and many signaling pathways related to tumors are similar to those in humans. Moreover, like humans, it can also develop cancer spontaneously. In addition, compared with mouse models, using zebrafish for tumor research has the following advantages: small size, requiring less breeding space and being easy to breed; convenient for breeding offspring with a large number, which can provide sufficient sample quantity for experiments and is inexpensive; requiring fewer tumor cells to be transplanted; less drug dosage and convenient drug administration methods; no need for immunosuppressive treatment, as its adaptive immune function is not fully formed in the first 30 days of embryonic development; being transparent throughout the juvenile period, which is convenient for microscopic observation and photographing, etc.
[0011] The research team of the present invention studied the developmental characteristics of zebrafish at different stages and, after combining a large number of experiments, found that after injecting human immune cells (such as human T cells, human natural killer cells, human macrophages) around 2 days post-fertilization (i.e., 2 dpf), the human immune cells migrate in the zebrafish blood vessels, and their number first increases and then decreases over time. The number of human immune cells in zebrafish remains at a relatively high level around 3 - 5 days post-fertilization (i.e., 3 - 5 dpf). Moreover, if tumor cells are injected into zebrafish around 3 days post-fertilization, the subsequent 2 days of continuous culture are sufficient for human immune cells to exhibit anti-tumor effects; if tumor cells and immune checkpoint inhibitors are injected into zebrafish around 3 days post-fertilization, the subsequent 2 days of continuous culture are also sufficient for immune checkpoint inhibitors to exhibit anti-tumor effects. Therefore, the present invention constructs a zebrafish model transplanted with human immune cells by injecting human immune cells around 2 dpf, which can be used to evaluate the anti-tumor effects of human immune cells and immune checkpoint inhibitors.
[0012] Preferably, in step (2), the human immune cells are one or more of human T cells, human natural killer cells, and human macrophages.
[0013] Preferably, in step (2), the injection amount of the human immune cells is 80 - 250 per zebrafish.
[0014] In a second aspect, the present invention provides the application of the method in evaluating the anti-tumor effects of immune cells.
[0015] Preferably, the application includes the following steps:
[0016] S1: Set a control group and an experimental group; the zebrafish in the control group are not injected with human immune cells; the zebrafish in the experimental group are constructed into a zebrafish model transplanted with human immune cells according to the method.
[0017] S2: Continue to culture the zebrafish in the control group and the experimental group until 70 - 75 h after fertilization, and transplant human tumor cells into the zebrafish.
[0018] S3: Continue to culture the zebrafish until 115 - 150 h after fertilization, and quantify the tumor cells in the zebrafish. Compare the number of tumor cells in the zebrafish in the experimental group and the control group, and evaluate whether the human immune cells have a killing effect on the tumor cells based on this.
[0019] When the number of tumor cells in the zebrafish in the experimental group is significantly less than that in the control group, it indicates that the human immune cells have a killing effect on the tumor cells. When there is no significant difference in the number of tumor cells in the zebrafish in the experimental group and the control group, it indicates that the human immune cells do not have a killing effect on the tumor cells.
[0020] Furthermore, the human immune cells are human T cells and / or human macrophages; in step S3, continue to culture the zebrafish until 115 - 125 h after fertilization, and quantify the tumor cells in the zebrafish.
[0021] The present invention finds that when human T cells or human macrophages are injected at about 2 dpf, then during 3 - 5 dpf, the human T cells or human macrophages in the zebrafish remain at a relatively high level and are relatively stable. Therefore, the tumor cells in the zebrafish can be quantified at 115 - 125 h after fertilization. For human natural killer cells, during 3 - 6 dpf, the human natural killer cells in the zebrafish remain at a relatively high level and are relatively stable. Therefore, the tumor cells in the zebrafish can be quantified at 115 - 150 h after fertilization.
[0022] Furthermore, in step S2, the transplantation amount of the human tumor cells is 250 - 350 per zebrafish.
[0023] In the third aspect, the present invention provides the application of the method in evaluating the anti - tumor effect of immune checkpoint inhibitors.
[0024] Preferably, the application includes the following steps:
[0025] S1: Set up a human immune cell transplantation group and a human immune cell + immune checkpoint inhibitor group. The zebrafish in both groups are constructed into a human immune cell - transplanted zebrafish model according to the method.
[0026] S2: Continue to culture the zebrafish in the human immune cell transplantation group and the human immune cell + immune checkpoint inhibitor group until 70 - 75 h after fertilization, transplant human tumor cells into the zebrafish, and inject the immune checkpoint inhibitor into the zebrafish in the human immune cell + immune checkpoint inhibitor group.
[0027] S3: Cultivate the zebrafish until 115 - 150 h after fertilization, and quantify the tumor cells in the zebrafish; compare the number of tumor cells in the zebrafish of the human immune cell transplantation group and the human immune cell + immune checkpoint inhibitor group, and evaluate whether the immune checkpoint inhibitor can promote the killing effect of the human immune cells on the tumor cells based on this.
[0028] When the number of tumor cells in the zebrafish of the human immune cell + immune checkpoint inhibitor group is significantly less than that of the human immune cell transplantation group, it indicates that the immune checkpoint inhibitor can promote the killing effect of the human immune cells on the tumor cells. When the number of tumor cells in the zebrafish of the human immune cell + immune checkpoint inhibitor group has no significant difference from that of the human immune cell transplantation group, it indicates that the immune checkpoint inhibitor cannot promote the killing effect of the human immune cells on the tumor cells.
[0029] Further, in step S1, a control group is set up, and the zebrafish in this group are not injected with human immune cells; in steps S2 and S3, the treatment method of the control group is the same as that of the human immune cell transplantation group.
[0030] Further, in step S1, an immune checkpoint inhibitor group is set up, and the zebrafish in this group are not injected with human immune cells; in steps S2 and S3, the treatment method of the immune checkpoint inhibitor group is the same as that of the human immune cell + immune checkpoint inhibitor group.
[0031] Further, in step S2, the transplantation amount of the human tumor cells is 250 - 350 per zebrafish.
[0032] Compared with the prior art, the present invention has the following advantages:
[0033] The present invention constructs a human immune cell - transplanted zebrafish model by injecting human immune cells into the zebrafish in a specific period. This method has the advantages of less usage amount and dosage of immune cells, high detection throughput, short experimental period, no need for immunosuppression induction, and being convenient for observation and photographing. It can greatly reduce the time and economic costs and play a good role in the anti - tumor effect evaluation of human immune cells and immune checkpoint inhibitors. Description of the Drawings
[0034] Figure 1 It shows the change of the number of human immune cells in the zebrafish. Among them, Figure 1 A is the fluorescence microscope photo of the zebrafish transplanted with human T cells at 3 spf; Figure 1 B shows the change of the number of human T cells in the zebrafish from 3 to 7 dpf; Figure 1C is the fluorescence microscope photo of zebrafish transplanted with human natural killer cells at 3 spf; Figure 1 D shows the change in the number of human natural killer cells in zebrafish from 3 to 7 dpf; Figure 1 E is the fluorescence microscope photo of zebrafish transplanted with human macrophages at 3 spf; Figure 1 F shows the change in the number of human macrophages in zebrafish from 3 to 7 dpf.
[0035] Figure 2 are the experimental results of the anti-tumor effect of human T cells.
[0036] Figure 3 are the experimental results of the anti-tumor effect of human NK cells.
[0037] Figure 4 are the experimental results of the anti-tumor effect of human macrophages.
[0038] Figure 5 are the experimental results of the anti-tumor effect of PD-1 antibody.
[0039] Figure 6 are the experimental results of the anti-tumor effect of CD47 antibody. Specific embodiments
[0040] The present invention will be further described below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims and any equivalents thereof are the scope of protection of the present invention.
[0041] General embodiment
[0042] A method for constructing a zebrafish model transplanted with human immune cells includes the following steps:
[0043] (1) Take zebrafish larvae 45 - 50 h after fertilization;
[0044] (2) Inject human immune cells into the sinus venosus of zebrafish larvae to obtain a zebrafish model transplanted with human immune cells.
[0045] As a specific embodiment, in step (2), the human immune cells are one or more of human T cells, human natural killer cells, and human macrophages.
[0046] As a specific embodiment, in step (2), the injection amount of the human immune cells is 80 - 250 per zebrafish.
[0047] A method for evaluating the anti-tumor effect of immune cells, comprising the following steps:
[0048] S1: Set up a control group and an experimental group; the zebrafish in the control group are not injected with human immune cells; the zebrafish in the experimental group are used to construct a human immune cell-transplanted zebrafish model according to the above method;
[0049] S2: Continue to culture the zebrafish in the control group and the experimental group until 70-75 h after fertilization, and transplant human tumor cells into the zebrafish;
[0050] S3: Continue to culture the zebrafish until 115-150 h after fertilization, and quantify the tumor cells in the zebrafish; compare the number of tumor cells in the zebrafish in the experimental group and the control group, and evaluate whether the human immune cells have a killing effect on the tumor cells accordingly.
[0051] As a specific implementation manner, the human immune cells are human T cells and / or human macrophages; in step S3, the zebrafish are continued to be cultured until 115-125 h after fertilization, and the tumor cells in the zebrafish are quantified.
[0052] As a specific implementation manner, in step S2, the transplantation amount of the human tumor cells is 250-350 per zebrafish.
[0053] A method for evaluating the anti-tumor effect of immune checkpoint inhibitors, comprising the following steps:
[0054] S1: Set up a human immune cell transplantation group and a human immune cell + immune checkpoint inhibitor group, and the zebrafish in both groups are used to construct a human immune cell-transplanted zebrafish model according to the above method;
[0055] S2: Continue to culture the zebrafish in the human immune cell transplantation group and the human immune cell + immune checkpoint inhibitor group until 70-75 h after fertilization, transplant human tumor cells into the zebrafish, and inject the immune checkpoint inhibitor into the zebrafish in the human immune cell + immune checkpoint inhibitor group;
[0056] S3: Continue to culture the zebrafish until 115-150 h after fertilization, and quantify the tumor cells in the zebrafish; compare the number of tumor cells in the zebrafish in the human immune cell transplantation group and the human immune cell + immune checkpoint inhibitor group, and evaluate whether the immune checkpoint inhibitor can promote the killing effect of the human immune cells on the tumor cells accordingly.
[0057] As a specific implementation manner, in step S1, a control group and an immune checkpoint inhibitor group are set up, and the zebrafish in these two groups are not injected with human immune cells; in steps S2 and S3, the treatment method of the control group is the same as that of the human immune cell transplantation group, and the treatment method of the immune checkpoint inhibitor group is the same as that of the human immune cell + immune checkpoint inhibitor group.
[0058] As a specific implementation manner, in step S2, the transplantation amount of the human tumor cells is 250 - 350 per zebrafish.
[0059] Example 1: Construction of a zebrafish model transplanted with human immune cells
[0060] According to the following steps, construct a zebrafish model transplanted with human immune cells, and study the quantity change situations of three kinds of human immune cells (human T cells, human natural killer cells (NK cells), human macrophages) in zebrafish:
[0061] (1) Anesthetize the 2 dpf Tg(fli1:EGFP) strain zebrafish larvae with tricaine and arrange them on the microinjection stage;
[0062] (2) Conduct experiments using three kinds of human immune cells (human T cells, human natural killer cells, human macrophages) respectively. After labeling the human immune cells with CM - Dil, inject about 100 cells into the zebrafish sinus venosus through a microinjector;
[0063] (3) Place the injected zebrafish in the culture water to wake up, and then place them in an incubator at 35°C for cultivation.
[0064] (4) Take pictures of the larvae with a fluorescence microscope 1 to 7 days after injection, and quantify the human immune cells in the larvae.
[0065] As Figure 1 shown in A and B, human T cells migrate in the larvae blood vessels, and the cell quantity first increases and then decreases with time. At 3 - 5 dpf, the quantity of immune cells is relatively stable.
[0066] As Figure 1 shown in C and D, human natural killer cells migrate in the larvae blood vessels, and the cell quantity first increases and then decreases with time. At 3 - 6 dpf, the quantity of immune cells is relatively stable.
[0067] As Figure 1 shown in E and F, human macrophages migrate in the larvae blood vessels, and the cell quantity first increases and then decreases with time. At 3 - 5 dpf, the quantity of immune cells is relatively stable.
[0068] Example 2: Evaluation of the anti - tumor effect of human T cells
[0069] Using the human immune cell transplanted zebrafish model in Example 1, the anti-tumor effect of human T cells was evaluated through the following steps:
[0070] (1) Set up a control group and a T cell transplantation group, with 20 zebrafish larvae in each group;
[0071] Control group: Human T cells were not transplanted at 2 dpf, and approximately 300 human tumor cells (tumor cells obtained by digesting the surgical specimens of gastric cancer patients, the same below) were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0072] T cell transplantation group: Approximately 200 human T cells were transplanted at 2 dpf (according to the method in Example 1), and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0073] (2) The transplanted zebrafish were placed in an incubator at 35 °C until 5 dpf, and the tumor cells were quantified.
[0074] As Figure 2 shown, the number of tumor cells in the T cell transplantation group was significantly less than that in the control group, indicating that human T cells have a killing effect on tumor cells.
[0075] Example 3: Evaluation of the anti-tumor effect of human natural killer cells
[0076] Using the human immune cell transplanted zebrafish model in Example 1, the anti-tumor effect of human natural killer cells was evaluated through the following steps:
[0077] (1) Set up a control group and an NK cell transplantation group, with 20 zebrafish larvae in each group;
[0078] Control group: Human NK cells were not transplanted at 2 dpf, and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0079] NK cell transplantation group: Approximately 200 human NK cells were transplanted at 2 dpf (according to the method in Example 1), and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0080] (2) The transplanted zebrafish were placed in an incubator at 35 °C until 5 dpf, and the tumor cells were quantified.
[0081] As Figure 3 shown, the number of tumor cells in the NK cell transplantation group was significantly less than that in the control group, indicating that human NK cells have a killing effect on tumor cells.
[0082] Example 4: Evaluation of the anti-tumor effect of human macrophages
[0083] Using the human immune cell transplanted zebrafish model in Example 1, the anti-tumor effect of human macrophages was evaluated through the following steps:
[0084] (1) A control group and a macrophage transplantation group were set up, with 20 zebrafish larvae in each group;
[0085] Control group: Human macrophages were not transplanted at 2 dpf, and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0086] Macrophage transplantation group: Approximately 200 human macrophages were transplanted at 2 dpf (according to the method in Example 1), and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0087] (2) The transplanted zebrafish were placed in an incubator at 35 °C until 5 dpf, and the tumor cells were quantified.
[0088] As Figure 4 shown, the number of tumor cells in the macrophage transplantation group was significantly less than that in the control group, indicating that human macrophages have a killing effect on tumor cells.
[0089] Example 5: Evaluation of the anti-tumor effect of PD-1 antibody
[0090] Using the human immune cell transplanted zebrafish model in Example 1, the anti-tumor effect of PD-1 antibody was evaluated through the following steps:
[0091] (1) A control group, a PD-1 antibody group, a T cell transplantation group, and a T cell transplantation + PD-1 antibody group were set up, with 20 zebrafish larvae in each group;
[0092] Control group: Human T cells were not transplanted at 2 dpf, and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0093] PD-1 antibody group: Human T cells were not transplanted at 2 dpf, and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf. Meanwhile, 10 ng of PD-1 antibody was injected intravenously;
[0094] T cell transplantation group: Approximately 200 human T cells were transplanted at 2 dpf (according to the method in Example 1), and approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection at 3 dpf;
[0095] T cell transplantation + PD-1 antibody group: At 2 dpf, approximately 200 human T cells were transplanted (according to the method in Example 1). At 3 dpf, approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection. Meanwhile, 10 ng of PD-1 antibody was injected intravenously.
[0096] (2) The transplanted zebrafish were placed at 35°C and cultured until 5 dpf, and the tumor cells were quantified.
[0097] As Figure 5 shown, the number of tumor cells in the PD-1 antibody group was comparable to that in the control group. The number of tumor cells in the T cell transplantation group was significantly less than that in the control group. The number of tumors in the T cell transplantation + PD-1 antibody group was less than that in other groups, indicating that the PD-1 antibody can promote the killing effect of human T cells on tumor cells.
[0098] Example 6: Evaluation of the anti-tumor effect of CD47 antibody
[0099] Using the human immune cell transplanted zebrafish model in Example 1, the anti-tumor effect of CD47 antibody was evaluated through the following steps:
[0100] (1) A control group, a CD47 antibody group, a macrophage transplantation group, and a macrophage transplantation + CD47 antibody group were set up, with 20 zebrafish larvae in each group.
[0101] Control group: No human macrophages were transplanted at 2 dpf. At 3 dpf, approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection.
[0102] CD47 antibody group: No human macrophages were transplanted at 2 dpf. At 3 dpf, approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection. Meanwhile, 10 ng of CD47 antibody was injected intravenously.
[0103] Macrophage transplantation group: Approximately 200 human macrophages were transplanted at 2 dpf (according to the method in Example 1). At 3 dpf, approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection.
[0104] Macrophage transplantation + CD47 antibody group: Approximately 200 human macrophages were transplanted at 2 dpf (according to the method in Example 1). At 3 dpf, approximately 300 human tumor cells were transplanted into the yolk sac of zebrafish larvae by microinjection. Meanwhile, 10 ng of CD47 antibody was injected intravenously.
[0105] (2) The transplanted zebrafish were placed at 35°C and cultured until 5 dpf, and the tumor cells were quantified.
[0106] As Figure 5As shown, the number of tumor cells in the CD47 antibody group was comparable to that in the control group. The number of tumor cells in the macrophage transplantation group was significantly less than that in the control group. The number of tumors in the macrophage transplantation + CD47 antibody group was less than that in other groups, indicating that the CD47 antibody can promote the killing effect of macrophages on tumor cells.
[0107] Unless otherwise specified, the reagents, biomaterials, and equipment used in the present invention are all common reagents, biomaterials, and equipment in the art that can be obtained commercially; unless otherwise specified, the methods used in the present invention are all conventional methods in the art.
[0108] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims
1. A method for evaluating the anti-tumor effect of immune cells, characterized in that, It includes the following steps: S1: Set up a control group and an experimental group; the zebrafish in the control group are not injected with human immune cells; the zebrafish in the experimental group are used to construct a human immune cell transplanted zebrafish model by injecting human immune cells into zebrafish larvae at 45 - 50 h after fertilization. S2: Continue to culture the zebrafish in the control group and the experimental group until 70 - 75 h after fertilization, and transplant human tumor cells into the zebrafish. S3: Continue to culture the zebrafish until 115 - 150 h after fertilization, and quantify the tumor cells in the zebrafish; compare the number of tumor cells in the zebrafish in the experimental group and the control group, and evaluate whether the human immune cells have a killing effect on the tumor cells accordingly.
2. The method according to claim 1, wherein In step S1, the human immune cells are one or more of human T cells, human natural killer cells, and human macrophages.
3. The method according to claim 2, wherein The human immune cells are human T cells and / or human macrophages; in step S3, continue to culture the zebrafish until 115 - 125 h after fertilization, and quantify the tumor cells in the zebrafish.
4. The method according to claim 1 or 2, characterized in that, In step S1, the injection amount of the human immune cells is 80 - 250 per zebrafish.
5. A method for evaluating the anti-tumor effect of immune checkpoint inhibitors, characterized in that, It includes the following steps: S1: Set up a human immune cell transplantation group and a human immune cell + immune checkpoint inhibitor group. The zebrafish in both groups are used to construct a human immune cell transplanted zebrafish model by injecting human immune cells into zebrafish larvae at 45 - 50 h after fertilization. S2: Continue to culture the zebrafish in the human immune cell transplantation group and the human immune cell + immune checkpoint inhibitor group until 70 - 75 h after fertilization, transplant human tumor cells into the zebrafish, and inject the immune checkpoint inhibitor into the zebrafish in the human immune cell + immune checkpoint inhibitor group. S3: Continue to culture the zebrafish until 115 - 150 h after fertilization, and quantify the tumor cells in the zebrafish; compare the number of tumor cells in the zebrafish in the human immune cell transplantation group and the human immune cell + immune checkpoint inhibitor group, and evaluate whether the immune checkpoint inhibitor can promote the killing effect of the human immune cells on the tumor cells accordingly.
6. The method according to claim 5, wherein In step S1, the human immune cells are one or more of human T cells, human natural killer cells, and human macrophages.
7. The method according to claim 6, wherein The human immune cells are human T cells and / or human macrophages; in step S3, continue to culture the zebrafish until 115 - 125 h after fertilization, and quantify the tumor cells in the zebrafish.
8. The method according to claim 5 or 6, characterized in that, In step S1, the injection amount of the human immune cells is 80 - 250 per zebrafish.
9. The method according to claim 5, characterized in that, In step S1, set up a control group, and the zebrafish in this group are not injected with human immune cells; in steps S2 and S3, the treatment method of the control group is the same as that of the human immune cell transplantation group.
10. The method according to claim 5, characterized in that, In step S1, set up an immune checkpoint inhibitor group, and the zebrafish in this group are not injected with human immune cells; in steps S2 and S3, the treatment method of the immune checkpoint inhibitor group is the same as that of the human immune cell + immune checkpoint inhibitor group.
Citation Information
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