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Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof

A technology of NKT cells and aconite polysaccharides, which is applied in the field of preparation of aconite polysaccharides to induce the proliferation of natural killer T cells, and can solve problems affecting the treatment of patients

Inactive Publication Date: 2014-06-04
李金珍
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the source of obtaining NKT cells is mainly to isolate mononuclear cells from peripheral blood or bone marrow of patients, and induce them alone or in combination with various cytokines, such as recombinant human interleukin 2, recombinant human interleukin 15, recombinant human interleukin 4, Granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant human interleukin 12, recombinant human interleukin 18, or combined induction with α-galactose sphingosine (α-Galcer) to obtain NK1.1 + and TCRβ + NKT cells, the order of cells is 5×10 8 It only meets 1-2 times of clinical treatment, which greatly affects the treatment of patients, and blood needs to be collected again during multi-course treatment, which puts great mental pressure on patients.

Method used

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  • Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof
  • Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof
  • Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof

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preparation example Construction

[0022] The invention relates to a preparation method for the proliferation of natural killer T cells induced by traditional Chinese medicine aconite polysaccharide. Cultured to obtain a large number of natural killer T cells expressing NK1.1 + and CD8 + , and secrete interferon gamma, accounting for more than 12.61% of lymphocytes on the 14th day, the order of magnitude can reach 5×10 9 Above, there are more than 30 interferon gamma enzyme-linked immunospots; it has effective anti-tumor and anti-viral Th1 cell immune response functions.

[0023] About 50 mL of human peripheral blood collected was separated and purified in vitro, red blood cells, platelets and granulocytes were removed, and mononuclear cells were obtained. The mononuclear cells were resuspended in RPMI1640 medium, stained with trypan blue and counted, and RPMI1640 complete medium (containing 1 μg / mL-1000ug / mL aconite polysaccharide, 1IU-200IU / mL IL-2, 10% (v / v) fetal calf serum) adjusted to a cell concentrat...

Embodiment 1

[0036] NKT Cells Obtained from Human Peripheral Blood-derived Mononuclear Cells

[0037] Breast cancer patient, female, 24 years old, blood routine test result was 3.7×10 9 white blood cells per liter, 50ml peripheral blood was collected from the patient (signed informed consent with the patient); the collected peripheral blood was centrifuged at 1500rpm for 10 minutes, the upper layer plasma was collected, inactivated in a water bath at 56°C for 30 minutes, and then centrifuged at 3000rpm for 10 minutes Obtain autologous plasma for use. After the blood cell solution is resuspended with 1 times the volume of 1×PBS (pH=7.4), add 1 / 4 of 0.6% hydroxyethyl starch in normal saline, mix well and let it stand for 30 minutes to absorb the white cell layer as much as possible; The layer was centrifuged by Ficoll-Hypaque density gradient, and the mononuclear cells were resuspended in RPMI1640 medium, and the trypan blue staining count was 1.62×10 8 .

[0038] Peripheral blood mononuc...

Embodiment 2

[0044] Detection of IFN-γ secreted by NKT cells by enzyme-linked immunospot assay (Elispot)

[0045] Coat Elispot 96-well plate with IFN-gamma antibody:

[0046] Add 25 ul of 1 mg / mL anti-human IFN-γ antibody R4-6A2 (Pharmingen Company-18181D) to 5 ml PBS to make the concentration reach 5 ug / ml, add 50 ul to each well, and cover overnight at 4°C. Discard the coated antibody, wash once with complete medium (RPMI-10) containing 10% fetal bovine serum, add 200ul complete medium to each well, cover and block for 2 hours at room temperature, and discard the medium.

[0047] NKT cell activation:

[0048] (1) Take the NKT cells cultured to the 14th day, count the placenta blue, and dilute 3 dilutions with R-5 phenol red-free medium (1×10 6 , 3.3×10 5 and 1×10 5). They were added to the IFN-γ antibody-coated Elispot 96-well plate, so that the volume of each well was 100ul.

[0049] (2) In the experimental group, 100 ul of R1640-10 containing MHC-1-restricted 9-peptide (final con...

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Abstract

The invention provides a culture method for monkshood polysaccharide-induced nature killer T cell (Nature Killer T cell, NKT) proliferation. The method comprises the characteristics as follows: human peripheral blood, marrow or a mononuclear cell with an umbilical cord blood source are continuously cultivated by traditional Chinese medicine monkshood polysaccharide and recombinant human interleukin 2, a lot of nature kill T cells are obtained, NK<1.1+> and CD<8+> are expressed, interferon gamma is secreted, the ratio in lymphocyte at the 14th day is more than 16.15%, the order of magnitude can be up to over 5*10<9>, and the nature kill T cell have effective anti-tumor and anti-virus functions. A clinical application of the NKT cell obtained by the method is also provided by the invention.

Description

technical field [0001] The invention relates to a preparation method and application of aconite polysaccharides for inducing proliferation of natural killer T cells. The mononuclear cells involved in the present invention are isolated and purified in vitro by using human peripheral blood, bone marrow or umbilical cord blood, and continuously cultured under the conditions of traditional Chinese medicine aconite polysaccharide and human recombinant interleukin 2 to obtain a large number of NKT cells that express NK1.1 + and CD8 + , and secretes IFN-γ, has anti-tumor and anti-viral functions, and can be used for various types of cancer, including solid tumors and blood tumors, as well as multiple courses of immunotherapy for various viral infections. Background technique [0002] Anti-tumor and anti-virus immune cells have been widely used clinically, especially cytokine-induced killer cells, which do not have the toxic side effects caused by current conventional treatments, a...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/14A61P35/00A61P31/12A61K35/15
Inventor 金光瑞王琳
Owner 李金珍
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