Method for efficiently amplifying NK and transfecting NKG2D activated NK cells in vitro

Abstract

The invention belongs to the field of biological medicine and the technical field of cell therapy, and particularly relates to a method for efficiently amplifying NK and transfecting NKG2D activated NK cells in vitro. The method comprises the following specific steps: optimizing an NK cell in-vitro efficient amplification technology and detecting NK immune biological characteristics; the method comprises the following steps: detecting mRNA expression of NKG2D ligands MICA and ULBP 1, 2 and 3 in tumor cells and tumor tissues by using an RT-PCR method; performing construction of an NKG2D cloning vector; performing construction of an NKG2D eukaryotic expression vector; expressing and identifying the eukaryotic expression vector in CHO cells; the eukaryotic expression vector transfects NK cells and influences the biological functions of the NK cells. The invention provides a method for constructing a pEGFP-N1 / NKG2D eukaryotic fluorescent expression vector by using a genetic engineering technology, transfecting NK cells with the constructed pEGFP-N1 / NKG2D plasmids, detecting the proliferation situation of the NK cells before and after NKG2D transfection and the killing effect on tumor cell lines by using an MTT method, detecting the mRNA expression level of related killing molecules IL-2, TNF-a, Perforin and TWEAK by using an RT-PCR method. The method for efficiently amplifying the NK in vitro and transfecting the NKG2D activated NK cells, which is a new thought, is provided for tumor treatment.

Description

technical field [0001] The invention belongs to the field of biomedicine and the field of cell therapy technology, and specifically relates to a method for efficiently amplifying NK in vitro and transfecting NKG2D to activate NK cells. Background technique [0002] On the basis of the technology of successfully and efficiently expanding high-purity NK cells in vitro by adopting a new cytokine combination scheme, continue to optimize the clinical industrialization culture scheme of NK cells and select NKG2D, an activated receptor target molecule, to modify NK cells with NKG2D, It is expected to obtain NK cells targeting tumor cell molecules with better targeting, higher killing activity and persistence, which has important clinical value and scientific significance. [0003] During the occurrence and development of tumors in the body, NK cells can play a "controlling" role, and tumor immunotherapy based on NK cells has a good clinical application prospect. Although the safet...

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Problems solved by technology

[0004] Healthy donors can obtain highly pure NK cells through leukapheresis and immunomagnetic bead sorting, but the process of obtaining NK cells using these two methods will cause cell activation and loss, so it is difficult to obtain a large number of NK cells. NK cells with high purity, in addition, the umbilical cord blood source in the clinical supplement blood source, due to the limitation of the number of umbilical cord blood, apheresis cannot be used to obtain a sufficient amount of NK cells

Traditional NK cell expansion technology has not been widely used due to low expansion efficiency and insufficient purity

In the process of expansion, auxiliary cells are added to improve the expansion efficiency of NK cells, etc., and the cells are used to stimulate the proliferation of NK cells, but the results are not very satisfactory

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