Solid tumor patient autologous NK cell separation, excitation, amplification and activity detection method

A technology for NK cells and solid tumors, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., to achieve the effects of multiple amplification, simple operation steps, and low cost of reagents and consumables

Active Publication Date: 2015-09-23
山西大医院
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  • Abstract
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Problems solved by technology

[0011] The present invention aims to overcome the deficiencies of the existing methods for the in vitro separation and culture, activation and expansion, and detection of cytotoxic activity

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  • Solid tumor patient autologous NK cell separation, excitation, amplification and activity detection method
  • Solid tumor patient autologous NK cell separation, excitation, amplification and activity detection method
  • Solid tumor patient autologous NK cell separation, excitation, amplification and activity detection method

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[0070] Example 1

[0071] A method for isolating, activating and expanding autologous NK cells from patients with solid tumors, comprising the following steps:

[0072] (1) Collection and separation of NK cells:

[0073] 1.1. Peripheral blood collection: 40~60ml of peripheral blood from patients with solid tumors, the most suitable for 50ml. The collection device is a disposable 100ml syringe containing 10ml of normal saline and 50unit / ml of heparin sodium anticoagulant, and the amount of heparin sodium is calculated according to the volume of collected blood. 2ml of peripheral blood was drawn to detect lymphocyte subsets by flow cytometry.

[0074] 1.2. NK cell isolation:

[0075] 1.2a. Put the collected peripheral blood into a 50ml centrifuge tube, centrifugation conditions: centrifugal force 500g, centrifugation time 10min. Aspirate the plasma with a pipette and put it into another centrifuge tube, inactivate it in a 56°C water bath for 30 minutes, and then centrifuge...

Example Embodiment

[0105] Example 2

[0106] A method for detecting the cytotoxic effect of autologous NK cells in patients with solid tumors, comprising the following steps:

[0107] 3.1. CFSE-labeled target cells:

[0108] Tumor cell culture: Suspend tumor cell lines (K562 cells) in 1640 medium containing 10% inactivated fetal bovine serum (FBS), and place at 37°C, 5% CO. 2 , subcultured under saturated humidity conditions.

[0109] CFSE-labeled target cells: collect tumor cells in good growth condition, use PBS buffer, centrifuge at a centrifugal force of 300 g, and centrifuge at room temperature for 5 min. Wash twice, count with trypan blue and resuspend cells in PBS buffer. Add the pre-warmed CFSE-PBS solution to the cell pellet (the final concentration of CFSE is 2umol / L), and gently pipette the suspended cells (the cell concentration is 1×10). 6 / mL). Cells were placed at 37°C for 5-10 min. The reaction was terminated by adding 10 mL of 1640 medium containing 10% FBS. Centrifuge th...

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Abstract

The invention provides a solid tumor patient autologous NK cell separation, excitation, amplification and activity detection method. By the adoption of the NK cell separation, culturing and amplification method, highly active lymphocyte populations can be obtained through separation from peripheral blood, through stimulation of a series of in-vitro cell factors, NK cells (CD3-CD56+) are purified, activated and amplified greatly, and the NK cells can be applied to clinic treatment of tumors. Through the NK cell activity detection method, the kill rate of effector cells to target cells can be specifically analyzed in mixed cell sap of immune cells and tumor cells, interference from the effector cells in a detection result is avoided, and early apoptosis target cells and late apoptosis target cells can be analyzed. The method is easy and convenient to implement, the flexibility is high, the kill function of a cell can be detected at the single cell level, and the method has important significance in fundamental research conducted before clinical application of tumor adoptive cellular immunotherapy and quality control conducted in the clinical application process.

Description

technical field [0001] The technology of the present invention mainly relates to a method for in vitro separation and culture, activation and amplification, and detection of cytotoxic activity of NK cells derived from autologous peripheral blood of patients with solid tumors. Background technique [0002] Natural killer cells (NK), also known as natural killer cells, have anti-virus infection and anti-tumor without MHC restriction, and have been used as an important therapeutic method for clinical cell immunotherapy of tumors. As the body's natural immune cells, NK cells mainly kill tumor cells, viruses and clear non-self cells by directly killing tumor cells and secreting cytokines to regulate other immune cells. Rosenberg et al. found in the process of treating tumors with IL-2-activated LAK cells that the cells characterized by CD3-CD16+ cells (natural killer cells, NK) play the main role in anti-tumor, and NK cells are the main effector cells. Since then, researchers ha...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12Q1/02
Inventor 张俊萍郭继强韩亚萍贾原赵晴李芳
Owner 山西大医院
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