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Cancer cell xenograft zebrafish model and construction method and application thereof

A technology of xenotransplantation and construction method, which is applied in the field of cancer cell xenotransplantation zebrafish model, can solve problems such as judging the proliferation of cancer cells, and achieve the effects of reducing damage, ensuring normal survival, and improving survival

Pending Publication Date: 2021-12-10
苏州木芮生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Only when the reporter gene is stably expressed in cancer cells can it increase with the proliferation of cancer cells, which requires enough time to obtain human cancer cells that can stably transduce viruses carrying fluorescent reporter genes; the second is Directly use fluorescent dyes to label cancer cells, but the disadvantage is that the fluorescent dyes will not increase with the proliferation of cells, and the proliferation of cancer cells cannot be judged entirely based on the fluorescence intensity

Method used

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  • Cancer cell xenograft zebrafish model and construction method and application thereof
  • Cancer cell xenograft zebrafish model and construction method and application thereof
  • Cancer cell xenograft zebrafish model and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Obtaining fluorescently labeled cancer cells

[0029] 1) Cell recovery: Take out a tube of frozen liver cancer 468 cells from the -80°C refrigerator, quickly place them in a 37°C water bath, place the thawed liver cancer 468 cells in a centrifuge tube, add 1ml of cell culture medium, and centrifuge , aspirate the supernatant and continue to add cell culture medium, fully resuspend, transfer to a culture dish, make up 7ml of cell culture medium, and culture in a cell culture incubator;

[0030] 2) Cell subculture: When the cells have covered about 70-80% of the bottom of the culture dish, first suck off the cell culture medium, wash the cells 2-3 times with 2ml 1×PBS, and cover the bottom with 0.25% trypsin Put it in the cell culture incubator for 3-4 minutes, take out the cell culture medium, then transfer it to a centrifuge tube, centrifuge, suck out the supernatant and continue to add the cell culture medium, fully resuspend, and culture in the cell culture...

Embodiment 2

[0032] Example 2: Construction of cancer cell xenograft zebrafish model

[0033] 1) Cell counting: Take a small amount of cells to count before injection, resuspend the labeled liver cancer 468 cells in 2 mL 1×PBS, take out 10 μL of the suspension and add 90 μL of 1×PBS to dilute 10 times, mix well, and inhale 10 μL of the diluted suspension Gently blow the solution into the cell counting plate and count under the microscope: (33+40+36+25) / 4×10×10 4 =3.35×10 6 / mL, the average cell density of the cell suspension is 3.35×10 6 / mL.

[0034]2) Injection: Collect fertilized eggs of the AB strain, wait for the embryos to grow to 48hpf (hours after fertilization), anesthetize with tricaine, and place them on a gel plate made of agarose. Manually peel off the film. Adjust the cell culture medium for the fluorescently labeled cells in Example 1 to 2~3×10 4 / μL density. Prepare a microinjection capillary needle for microinjection into the pericardial space of 48hpf zebrafish embr...

Embodiment 3

[0036] Drug treatment after screening: Observe the injected embryos carrying green fluorescence under a fluorescent microscope, select embryos with normal embryonic development and good fluorescence status, and randomly divide them into three groups. Wherein two groups adopt the drug treatment of 0.675nM and 1.350nM concentration respectively, remaining a group is as blank control group (control), and drug treatment 24h and 48h are sampled respectively. The medicine is represented by X, and Apatinib is selected to carry out drug treatment to the zebrafish in the embodiment of this case.

[0037] 1. Directly observe the inhibitory effect of drugs on tumors through fluorescence microscopy, such as figure 2 and image 3 Schematic diagrams of the fluorescence expression of liver cancer 468 cells in zebrafish after 24 hours and 48 hours of drug treatment, respectively. The fluorescence expression of liver cancer 468 cells with fluorescent labels in the pericardial space of zebraf...

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Abstract

The invention relates to a cancer cell xenograft zebrafish model, a construction method thereof and application of the cancer cell xenograft zebrafish model in quantitative evaluation of cancer cell proliferation in a zebrafish model body and screening of effective anti-tumor drugs according to evaluation results. The construction method comprises the following steps: carrying out culture passage on cancer cells in a cell culture medium, carrying out fluorescence labeling by using a CFDA-SE fluorescent dye, injecting the cells into a zebrafish embryo pericardial space through micro-injection, and screening out zebrafish which develops normally and carries the fluorescence label so as to obtain the cancer cell xenograft zebrafish model. The injection site is in the zebrafish embryo pericardial space, so that the damage of allogenic cancer cell transplantation to the embryo can be reduced, and the survival of the transplanted cancer cells is improved, so that the success of tumor allograft is ensured; according to the scheme, the growth and proliferation conditions of the transplanted cancer cells in vivo can be quantitatively evaluated; and the inhibition or elimination effect of a target drug on the cancer cells is analyzed according to the relative expression quantity of humanized genes, and further an effective anti-tumor drug is screened out.

Description

technical field [0001] The invention belongs to the field of biotechnology model construction, in particular to a cancer cell xenograft zebrafish model, its construction method and application. Background technique [0002] Cancer has caused a serious burden to China and the world. In order to provide clinical symptom detection and treatment decision-making for cancer patients, patient-derived tumor xenograft models are an important in vivo method for biological research of tumors, search for diagnostic markers, and drug screening. Model. [0003] At present, mice / rats are the most commonly used patient-derived tumor xenograft model animals, but because the mouse / rat patient-derived tumor xenograft model is a time-consuming and costly model, from tumor inoculation to drug treatment As well as drug efficacy analysis, the time is usually 2-4 months, which makes it unsuitable for clinical real-time guidance of individualized medication. More and more studies have proved that ...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12Q1/6886
CPCA01K67/0271C12Q1/6886A01K2207/12A01K2227/40A01K2267/03C12Q2600/158
Inventor 王明勇郭辉
Owner 苏州木芮生物科技有限公司
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