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A PDX model establishment method of granulocytic tumor

A technology of model establishment and establishment method, which is applied in the field of PDX model establishment of granulocytic tumors, which can solve the problems of small number of cells, single tumor type, and increased cost of new drug research and development, so as to reduce the number of cells, shorten the time of onset, and have a good application prospect Effect

Active Publication Date: 2022-03-29
XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • A PDX model establishment method of granulocytic tumor
  • A PDX model establishment method of granulocytic tumor
  • A PDX model establishment method of granulocytic tumor

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 method for establishing a PDX model of granulocytic tumors, and relates to the technical field of animal models. The PDX model established by the present invention for AML with different FAB types has a clear onset, can be stably passed down, and the cytogenetic changes are consistent with those of the patient. It is an ideal platform for studying the pathogenesis of AML and screening targeted drugs, and can satisfy various hematological experiments Base and pharmaceutical companies' needs will generate huge economic and social benefits. The PDX models of M2a, M4 and M5 three kinds of AML are established by using the method of the invention, and the pathogenesis is clear, and can be passed down stably, with good uniformity, can be used in large-scale drug screening experiment, and has good application prospect.

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 Patents(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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