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Method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissue

A technology for lymphoma cells and lymphocytes, which is applied in the field of extracting high-quality B cells, can solve the problems of inability to treat, limit the DNA spectrum and RNA spectrum of lymphoma cells, limit the origin of lymphoma cell clones, etc., and achieve high production efficiency and large Potential and application value, the effect of simple and easy preparation method

Pending Publication Date: 2021-05-28
天津市环湖医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This also limits the use of single-cell sequencing technology to obtain the DNA and RNA profiles of lymphoma cells, and at the same time limits the understanding of the clonal origin and clonal evolution of lymphoma cells, resulting in the inability to treat the etiology of lymphoma cells clinically.

Method used

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  • Method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissue
  • Method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissue
  • Method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Obtaining Peripheral Blood Cells of Lymphoma Patients

[0024] Take 5ml of fresh peripheral blood from lymphoma patients, add hydroxyethyl starch (peripheral blood: hydroxyethyl starch = 5:1), and let stand at room temperature for 1 hour. Take the supernatant (white blood cell layer) and remove the lower layer of red blood cells.

[0025] Add the same volume of lymphocyte separation medium as the supernatant to the centrifuge tube, and carefully transfer the supernatant to the separation medium (operate gently to avoid breaking the interface liquid); centrifuge at 1800rpm, 30 degrees Celsius, for 15 minutes; after centrifugation, the solution is separated , the supernatant is serum, which is transferred to a new centrifuge tube (for spare); the middle is the buffy coat layer (lymphocyte layer), which is transferred to a new centrifuge tube after aspiration. Wash three times with PBS at room temperature and resuspend with 1ml PBS. (Table 1 shows the status of...

Embodiment 2

[0032] Example 2 Measuring the concentration of B cells and other blood cells in the peripheral blood of patients with lymphoma

[0033] Take the above 1x10 5 White blood cells, add 50ul PBS and mix well, add 1ul Antibody-human CD45(Percp-cy5.5), Antibody-CD3(APC-CY7), Antibody-CD19(APC), Antibody-CD33(PE) and mix well, incubate at room temperature for 30 Minutes later, wash once with PBS, resuspend in 300ul PBS, and detect the ratio of various cells by flow cytometry. For experimental results, see figure 1 , 2 (CD45 is a marker on the surface of white blood cells, CD3 is a marker on the surface of T cells, CD33 is a marker on the surface of myeloid cells, and CD19 is a marker on the surface of B cells). It can be seen that through this method, complete B cells (CD19+), T cells (CD3+), and myeloid cells (CD33+) can be obtained from peripheral blood.

Embodiment 3

[0034] Example 3 Obtaining Blood Cells in the Bone Marrow of a Lymphoma Patient

[0035] Take 3ml of fresh bone marrow from lymphoma patients, add hydroxyethyl starch (peripheral blood: hydroxyethyl starch = 5:1), and let stand at room temperature for 1 hour. Take the supernatant (white blood cell layer) and remove the lower layer of red blood cells.

[0036] Add the same volume of lymphocyte separation medium as the supernatant to the centrifuge tube, and carefully transfer the supernatant to the separation medium (operate gently to avoid breaking the interface liquid); centrifuge at 1800rpm, 30 degrees Celsius, for 15 minutes; after centrifugation, the solution is separated , the middle is the buffy coat layer (ie, the lymphocyte layer), which is transferred to a new centrifuge tube after suction. Wash three times with PBS at room temperature and resuspend with 1ml PBS. (Table 3 is the situation of various cells in the fresh bone marrow of patient No. 1, and Table 4 is the...

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Abstract

The invention discloses a method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissue, which comprises the steps of respectively taking the peripheral blood and the bone marrow of a lymphoma patient, adding hydroxyethyl starch, standing and taking supernatant; taking a lymphocyte separation medium with the same volume as the supernatant, adding the supernatant into the lymphocyte separation medium, and centrifuging; taking the centrifuged white film layer, washing and resuspending; splitting tumor tissue of the lymphoma patient, cutting the tissue and incubating; transferring the supernatant into PBS containing 10% of serum, and repeatedly washing the tissue; and enabling the obtained supernatant to pass through a cell sieve, centrifuging, collecting cell precipitate, washing and resuspending. By changing the experimental method for separating and purifying the bone marrow and the peripheral blood, the B cells from the bone marrow and the peripheral blood are extracted more efficiently, and impurity cells are reduced; and by changing a lysis method of lymphoma tissues, more lymphoma cells are mildly and effectively extracted, and a necessary cell basis is provided for subsequent cell culture and single cell sequencing.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissues. Background technique [0002] Malignant lymphoma is an immune cell tumor that occurs in lymph nodes or some extranodal lymphoid tissues, and is derived from the malignant transformation of lymphocytes or histiocytes. In recent years, the incidence of malignant lymphoma has been increasing year by year. According to the statistics of the number of patients, it accounts for the 4th to 6th of all malignant tumors, and it is more common in adolescents and middle-aged and elderly people. Diffuse large B-cell lymphoma (DLBCL) is a large B-cell malignancy with a diffuse growth pattern. DLBCL is the most common non-Hodgkin's lymphoma (NHL), accounting for 20-30% of the total incidence of NHL. In the past 20 years, a number of phase III clinical studies have shown that rituximab co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/09A01N1/02
CPCC12N5/0635C12N5/0694A01N1/0221C12N2509/00C12N2509/10
Inventor 王政张学斌冯珂珂姜炜
Owner 天津市环湖医院
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