PDX model establishing method for granulocyte tumors

A technology for model establishment and method establishment, applied in the field of PDX model establishment of granulocytic tumors, can solve the problems of increasing the cost of new drug research and development, low number of cells, single tumor type, etc., and achieve good application prospects, reduce the number of cells, and shorten the onset time. Effect

Active Publication Date: 2021-01-22
XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

However, the construction of PDX mouse models of hematological malignancies is limited by the small number of cells and low implantation rate, and there is no systematic application of PDX models.
Although some laboratories at home and abroad have constructed PDX mouse models including AML and acute lymphoblastic leukemia, and very few have been commercialized, most of the tumor types are single and cannot represent the complex genetics and biological phenotype of AML; and The price is expensive, which greatly increases the cost of new drug research and development, and hinders the progress of anti-blood cancer drug research and development in my country

Method used

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  • PDX model establishing method for granulocyte tumors
  • PDX model establishing method for granulocyte tumors
  • PDX model establishing method for granulocyte tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Establishment of AML-M2a mouse PDX model

[0035] 1. AML patient sample processing:

[0036] 1. Collect 20 mL of bone marrow from the patient, centrifuge with Ficoll separation medium, and separate mononuclear cells from the patient. After washing the mononuclear cells twice with normal saline, resuspend in 0.5 mL of normal saline, and count the cells.

[0037] 2. CD34 + Sorting: Add rabbit anti-human CD34 antibody to the cells in step 1, incubate on ice for 20 minutes, wash excess antibody with PBS, resuspend in 0.5mL PBS, and sort out CD34 by flow cytometry + Hematopoietic stem / progenitor cells; the sorted cells were counted, resuspended in 1mL PBS, injected into mice through the tail vein, and the number of inoculated cells was 2×10 6 / only.

[0038] 2. Feeding and inoculation of NSG mice:

[0039] 1. Raising conditions for mice: 8-week-old female NSG mice were raised under SPF grade conditions. The room temperature is 20°C, the relative humidity is 50%, and the...

Embodiment 2

[0047] Establishment of AML-M4 mouse PDX model

[0048] The steps 1 and 2 of constructing the PDX model of the P1 generation M4 mouse are the same as in "Example 1", and the bone marrow cells are derived from the M4 patient. Follow up with the following steps:

[0049] 3. Continuous inoculation and passage of AML cells and genetic testing:

[0050] 1. Preservation and cryopreservation of P1-generation AML cells: the bone marrow and spleen of P1-generation NSG mice were made into single-cell suspension, and long-term cryopreserved in liquid nitrogen with fetal bovine serum + 10% DMSO.

[0051] 2. Continuous inoculation and passage of AML cells: Before passage, the bone marrow and spleen of P1 generation NSG mice were quickly thawed in a water bath at 42°C, washed with PBS, and injected into NSG mice irradiated with X-ray radiation through the tail vein of the rats. , the number of cells is 2×10 6 / only.

[0052] 3. Judgment of the onset of the P2 generation: the observation...

Embodiment 3

[0057] Establishment of AML-M5 mouse PDX model

[0058] Steps 1, 2, and 3 of M5 mouse PDX model construction are the same as in "Example 2", using bone marrow cells derived from M5 patients. The detection indicators for successful modeling of PDX model P1 generation of M5 type AML are as follows: Figure 5 As shown, the spleen was significantly enlarged ( Figure 5 Middle A), observation of hCD45 in bone marrow by flow cytometry + cell ratio ( Figure 5 Middle B), indicating successful modeling and passage.

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Abstract

The invention provides a PDX model establishing method for granulocyte tumors, and relates to the technical field of animal models. According to the PDX model establishing method, PDX models are established for AML of different FAB types, pathogenesis is clear, stable passage is achieved, cytogenetics changes are consistent with a patient, the models are ideal platforms for researching the pathogenesis of the AML and screening targeted drugs, the requirements of various hematology experiment bases and pharmaceutical companies are met, and huge economic benefits and social benefits can be generated. The three PDX models of M2a, M4 and M5 for the AML are established by utilizing the method disclosed by the invention, pathogenesis is clear, stable passage is achieved, homogeneity is good, themodels can be used for large-scale drug screening experiments, and good application prospects are achieved.

Description

technical field [0001] The invention belongs to the technical field of animal models, and in particular relates to a method for establishing a PDX model of granulocytic tumors. Background technique [0002] Acute myeloid leukemia (AML) is a hematological malignancy in which immature granulocytes proliferate extremely due to the blockage of hematopoietic cell differentiation, accounting for about 80% of all acute leukemias. The incidence of AML increases with age. The median age at diagnosis is 67 years old. With the acceleration of my country's population aging process, AML is becoming one of the malignant tumors affecting the health of Chinese adults, especially the elderly. [0003] AML can be divided into several different subtypes, M0-M7, according to cell morphology and histochemical characteristics. Except for M3 type, the clinical treatment of AML has always been based on cytotoxic chemotherapy drugs such as cytarabine, but the long-term effect of chemotherapy is no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N5/0789
CPCA01K67/0271C12N5/0647A01K2207/12A01K2227/105A01K2267/0331
Inventor 卢莹闫金松姚志荣江月杜成坎
Owner XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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