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Kit used for identifying hematopoietic stem cell transplantation effect

A technology of hematopoietic stem cells and stem cells is applied in the field of kits for identifying the effect of hematopoietic stem cell transplantation, which can solve the problems of lack of scientific and effective detection methods for the bone marrow hematopoietic microenvironment, and achieve the effect of simple operation.

Inactive Publication Date: 2014-01-29
CHINESE PEOPLES LIBERATION ARMY AIR FORCE GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of scientific and effective detection methods for the reconstruction of the bone marrow hematopoietic microenvironment

Method used

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  • Kit used for identifying hematopoietic stem cell transplantation effect
  • Kit used for identifying hematopoietic stem cell transplantation effect
  • Kit used for identifying hematopoietic stem cell transplantation effect

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0055] The preparation method of low-sugar DMEM medium: take 12.1g low-sugar DMEM powder, 2mmol L-glutamine, 2.4gNaHCO 3 and 25mmol HEPES, dissolved in water and dilute to 1L with water.

[0056] Crystal violet dye: the product of Biyuntian Company, the article number is CO121.

[0057] Ascorbyl phosphate: product of SIGMA Company, the product number is A-8960.

[0058] Dexamethasone: SIGMA Company, the product number is D-2915.

[0059] β-Glycerol phosphate: MERCK company, product number is Art4168.

[0060] Alkaline phosphatase staining kit: SIGMA Company, the article number is 85L3R.

[0061] Insulin: SIGMA Company, the product number is i-6634.

[0062] Oil Red O: Amresco Company, the product number is 0684.

[0063] CFSE, the full name is 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE is a green fluorescent dye that can be marked on cells, and its fluorescence intensity gradually decays with cell division and proliferation. Using this principle, flow cy...

Embodiment 1

[0068] Embodiment 1, culture and identification of bone marrow mesenchymal stem cell colony

[0069] 1. Culture of bone marrow mesenchymal stem cell colonies

[0070] 15 volunteers with informed consent (these 15 volunteers have been followed up for 2 years after hematopoietic stem cell transplantation, and the hematopoietic stem cell transplantation has been successful and in good condition), 8 patients with acute lymphoblastic leukemia , 4 patients with acute myeloid leukemia, and 3 patients with chronic myeloid leukemia. Each volunteer underwent hematopoietic stem cell transplantation, and bone marrow samples were taken before the pretreatment of hematopoietic stem cell transplantation (bone marrow sample-0) , A bone marrow sample was taken 1 month after hematopoietic stem cell transplantation (bone marrow sample-1), a bone marrow sample was taken 3 months after hematopoietic stem cell transplantation (bone marrow sample-3), and a bone marrow sample was taken 6 months after...

Embodiment 2

[0089] Example 2, the ability of bone marrow mesenchymal stem cells to inhibit the proliferation of T lymphocytes

[0090] 1. Human CD3 + T lymphocytes were resuspended in PBS buffer containing 5% (volume ratio) fetal bovine serum to obtain 10 7 cells / ml of cell suspension.

[0091] 2. Take 1 mL of the cell suspension obtained in step 1, add CFSE to make the concentration 10 μM, incubate at room temperature for 25 minutes, then add 10 mL of PBS buffer containing 2% (volume ratio) fetal bovine serum to stop the reaction, centrifuge at 1000 rpm for 10 Minutes, cells were collected and washed with PBS buffer (cells were collected by centrifugation at 1000 rpm for 10 minutes for each wash).

[0092] 3. Lymphoblast transformation test

[0093] The first group: take a 6-well plate, according to 1×10 5 The bone marrow mesenchymal stem cells prepared from bone marrow sample-0 obtained in Example 1 and transferred to the 4th generation were inoculated at a density of 2 cells / cm2, a...

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Abstract

The invention discloses a kit used for identifying hematopoietic stem cell transplantation effect. The kit comprises assemblies A, B, C, D, E, F, G, and H. The assembly A is a substance used for preparing bone marrow mononuclear cells; the assembly B is a substance used for culturing bone marrow mesenchymal stem cells to form colonies; the assembly C is a substance used for counting the bone marrow mesenchymal stem cell colonies; the assembly D is a substance used for identifying whether bone marrow mesenchymal stem cells are capable of being differentiated into osteoblasts; the assembly E is a substance used for identifying whether bone marrow mesenchymal stem cells are capable of being differentiated into adipocytes; the assembly F is a substance used for identifying whether bone marrow mesenchymal stem cells have capacity of inhibiting T lymphocyte proliferation; and the assembly G is a substance used for identifying whether bone marrow mesenchymal stem cells are capable of being feeder cells of hematopoietic stem cells. The kit provided by the invention is convenient and practical, and is simple to operate. The kit can be used for detecting bone marrow hematopoietic microenvironment recovery condition after a hematopoietic stem cell transplantation operation. With the kit, a basis is provided for the researched on mesenchymal stem cells.

Description

technical field [0001] The invention relates to a kit for identifying the effect of hematopoietic stem cell transplantation. Background technique [0002] Hematopoietic stem cell transplantation is one of the most effective means to treat a variety of malignant blood diseases. Before transplanting healthy hematopoietic stem cells, it is usually necessary to remove the malignant hematological cells in the patient with the help of various drugs or radiation (often referred to as pretreatment in medicine). This large dose of drugs or radiation often kills While destroying tumor cells, it also destroyed the hematopoietic microenvironment in the patient's bone marrow. [0003] The hematopoietic microenvironment of the bone marrow is located in the bone marrow cavity, mainly referring to the stromal cells in the bone marrow (including osteoblasts, adipocytes, mesenchymal stem cells and endothelial cells, etc.), which secrete various cytokines (IFN-γ, IL- 1β, IL-17, IL-6, IL-7, V...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 王恒湘丁丽郭子宽朱恒
Owner CHINESE PEOPLES LIBERATION ARMY AIR FORCE GENERAL HOSPITAL
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