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Drug-resistant multiple myeloma animal model and construction method thereof

A multiple myeloma and animal model technology, applied in the field of animal models, can solve the problems of inability, great difference, and inability to observe drug effects, etc.

Inactive Publication Date: 2015-08-12
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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Problems solved by technology

[0004] Research on multiple myeloma is still limited to in vitro cell line experiments and in vivo subcutaneous tumor model inhibition experiments, among which, in vitro cell line experiments can be used for screening of anti-multiple myeloma drugs or research on multiple myeloma-related molecular mechanisms , but the conditions of cell lines cultured in vitro and the actual situation in vivo are very different, and the drugs screened in vitro may not be effective in the treatment of multiple myeloma in vivo
[0005] In addition, animal experiments after transplanting multiple myeloma cells into mice subcutaneously can effectively determine the effect of drugs on the size of subcutaneous tumors and the lifespan of mice, as well as toxicity experiments, but this model ignores the impact of the mouse bone marrow microenvironment on malignant plasma cells. The effect of the drug on other complications such as bone destruction cannot be observed, and this model cannot be used to study the pathogenesis of multiple myeloma in vivo

Method used

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  • Drug-resistant multiple myeloma animal model and construction method thereof
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  • Drug-resistant multiple myeloma animal model and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0032] Drug resistance analysis of embodiment 1:8226-Dox40 drug-resistant cell line

[0033] The drug resistance of 8226-Dox40 drug-resistant cell line was verified by MTT method, and the IC50 value analysis showed that 8226-Dox40 drug-resistant cell line had obvious drug resistance to doxorubicin.

[0034] 1). The experimental method for detecting the drug resistance of 8226-Dox40 drug-resistant cell line by MTT method

[0035] (1). Take out the frozen 8226-Dox40 drug-resistant cell line and 8226 cell line from liquid nitrogen. Thaw at 37°C immediately after taking out, centrifuge at 1000 rpm for 5 minutes, place in RPMI 1640 medium for culture and add 10% fetal bovine serum (the amount of fetal bovine serum added is 10% of the volume of RPMI 1640 medium). The culture condition is 5% CO 2 , cultured in a 37°C cell culture incubator;

[0036] (2). Collect the cells and add them to a 96-well plate at 8000 cells / 200 μL per well;

[0037] (3). Doxorubicin concentrated stock s...

Embodiment 2

[0045] A method for constructing a drug-resistant multiple myeloma animal model, comprising the steps of:

[0046] (1). Take out the frozen RPMI 8226-Dox40 drug-resistant cell line from liquid nitrogen. Immediately after taking it out, it was melted at 37°C, centrifuged at 1000 rpm for 5 minutes, and then cultured in RPMI 1640 medium with 10% fetal bovine serum added. The culture condition is 5% CO 2 , cultured in a 37°C cell culture incubator;

[0047] (2) When the cell density grows to 3×10 5 -1×10 6 When the cell density of / mL was reached, the cells were collected, placed in a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and washed with PBS solution at 4×10 6 Resuspend at a density of cells per mL.

[0048] (3) The cell solution resuspended in the previous step was injected into the tail vein of NOD-SCID immunodeficiency mice according to the amount of 250 μL solution per mouse with a 1 mL syringe, and the animal model was obtained.

Embodiment 3

[0049] Embodiment 3: the flow cytometry detection of multiple myeloma cells in the drug-resistant multiple myeloma mouse model that embodiment 2 obtains

[0050] The expression of myeloma cell surface specific marker CD38 and CD138 is analyzed by flow cytometry, and the detection result shows that there is obvious expression of myeloma cell surface specific antigen in the mouse myeloma animal model (in vivo model), illustrating that the model mouse of the present invention There are specific myeloma cells in the bone marrow.

[0051] 1). An experimental method for analyzing specific markers on the surface of myeloma cells by flow cytometry

[0052] (1). Bone marrow cells were washed out with a 1 mL syringe from the myeloma model mice obtained in Example 2.

[0053] (2). Put 2×10 6 Each bone marrow cell was placed in 50 μL of PBS (referred to as PBE) supplemented with 2% fetal bovine serum and 2 mM EDTA, and mixed well. In addition, a group of 8226-Dox40 cultured in vitro wa...

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Abstract

The invention relates to a drug-resistant multiple myeloma animal model and a construction method thereof. The animal model is constructed by the a method comprising the steps of: 1) taking multiple myeloma drug-resistant cell line from patients with multiple myeloma for standby; 2) culturing the multiple myeloma drug-resistant cell line in an RPMI1640 medium, adding 10% fetal bovine serum and culturing in a cell incubator under the conditions of 5% CO2, 37 DEG C; and 3) dissolving the multiple myeloma drug-resistant cells in a PBS solution and injecting the solution into a tail vein of an NOD-SCID immunodeficient mice by using a syringe, so as to obtain the animal model. The invention successfully establishes an in vivo mice model of drug-resistant multiple myeloma, helps in the future research in the mechanism of myeloma, and has important clinical application prospect.

Description

technical field [0001] The present invention relates to an animal model, in particular to an animal model for the growth of multiple myeloma. The animal model can be used for in vivo drug activity and toxicity experiments against multiple myeloma, and can be used for studying the pathogenesis of multiple myeloma. Background technique [0002] Multiple myeloma, also known as plasma cell myeloma, is a malignant B-cell lymphoma. Plasma cells in patients with multiple myeloma undergo malignant transformation and excessive secretion of monoclonal immunoglobulin. Complications of multiple myeloma include bone destruction, hypercalcemia and other diseases, among which bone destruction is the most common complication, and patients often have Osteolysis, bone pain and fractures. [0003] Existing methods for treating multiple myeloma mainly use steroids, chemotherapy, protease inhibitors or radiotherapy for remission. Multiple myeloma is one of the most common hematological malign...

Claims

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Application Information

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IPC IPC(8): C12N5/09A01K67/027
Inventor 高瀛岱解向群杨铭李聪纪庆
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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