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Preparation method and application of autologous natural killer cell proliferation

A technology of natural killer cells and cells, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of long culture time, low killing function of NK cells, telomerase activity Lowering and other issues

Active Publication Date: 2015-09-09
ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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Problems solved by technology

The number of NK cells and the killing function are directly related to the curative effect. The problems in the treatment are the limited number of NK cells and the long culture time
Therefore, the traditionally used low-dose interleukin-2 activation growth factor is limited, the activity of telomerase is reduced, and the long culture time causes the low killing function of NK cells

Method used

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  • Preparation method and application of autologous natural killer cell proliferation
  • Preparation method and application of autologous natural killer cell proliferation
  • Preparation method and application of autologous natural killer cell proliferation

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[0036] The invention relates to a preparation method for proliferation of autologous natural killer cells, which is characterized in that K562 cells transfected and stably expressed by PDK1 and CD122 and interleukin 2 are used to amplify and activate the natural killer cells, so that the natural killer cells can proliferate in large quantities.

[0037] The K562 cell line of the present invention is transfected with an expression vector that stably expresses 3-phosphoinositide-dependent protein kinase 1 and CD122. The vector contains a viral promoter and a selection marker gene, and PDK1 and CD122 polypeptides on the K562 cell membrane can be obtained , K562 cells still stably express 3-phosphoinositide-dependent protein kinase 1 and CD122 after culture and proliferation. The cells expressing 3-phosphoinositide-dependent protein kinase 1 and CD122 on the cultured K562 cell membrane are collected by strong acid, strong alkali and / or irradiation, and high-speed centrifugation for...

Embodiment 1

[0061] Preparation of transfected K562 cell line stably expressing PDK1 and CD122

[0062] The K562 cell line is used to transcribe and stably express the expression vector of PDK1 and CD122. The vector contains a viral promoter and a selection marker gene; when the exogenous expression vector enters the host cell, activate the promoter, and cultivate the K562 cell for a period of time, the cell membrane can be obtained 3-Phosphoinositide-dependent protein kinase 1 and CD122 polypeptide on the cultured K562 cell membrane with cells expressing PDK1 and CD122, collected by strong acid, strong alkali and / or irradiation, and by high-speed centrifugation to collect K562 cells Purified collection.

Embodiment 2

[0064] Preparation of autologous natural killer cells

[0065] Collect 30ml of patients with advanced breast cancer and normal healthy people (with informed consent), add an equal amount of 1×PBS to dilute, gently superimpose on the lymphocyte separation medium along the wall of the centrifuge tube to form a clear interface, and centrifuge at 2000rpm for 20min at room temperature , Aspirate the PBMCs in the interface layer and wash twice with 1×PBS (pH 7.4). After counting orchids, use CellGro SCGM to adjust the concentration of mononuclear cells to 1×10 7 / ml.

[0066] K562 cells stably expressing 3-phosphoinositide-dependent protein kinase 1 and CD122 were first co-cultured with natural killer cells for 5 days, and the dosage ratio of K562 cells to lymphocytes was the number of K562 cells: the number of mononuclear cells = 1:5 .

[0067] After 5 days of co-cultivation, the natural killer cells were aspirated into a new culture bottle with a 5 mL pipette, and 20 mL of fres...

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Abstract

The invention provides a preparation method of autologous natural killer cell proliferation and culture. The preparation method of the autologous natural killer cell proliferation and culture is characterized in that mononuclear cells of a cancer patient are amplified and activated to obtain natural killer cells after the mononuclear cells are transfected by 3- phosphoinositide-dependent protein kinase-1 and CD122 and under the effect of K562 cells which are expressed stably serve as trophoblast cells and interleukin 2, proliferation times of the natural killer cells are above 2500, phenotype of CD16+ / CD56+ is above 90%, and in-vivo and in-vitro tests show that the natural killer cells have an anti-tumor and have quite high killer toxicity. The invention also provides a kit which is used for autologous natural killer cell proliferation and culture and is obtained by using the preparation method. The kit has an efficient anti-tumor function.

Description

technical field [0001] The present invention relates to a preparation method for the proliferation of autologous natural killer cells, which includes the following features: the mononuclear cells isolated from the peripheral blood of cancer patients are transfected with 3-phosphoinositide-dependent protein kinase 1 and CD122 and Stable expressed K562 cells are amplified and activated under the action of interleukin 2 to obtain natural killer cells; the present invention also provides a kit containing autologous natural killer cells obtained by the above method, which can be used for various types of cancers including solid tumors and Multiple courses of immunotherapy including hematological malignancies. Background technique [0002] The abnormal number and function of natural killer cells (Natural Killer cells, NK) are closely related to the occurrence of tumors and persistent viral infections. At present, after NK cells are cultured and expanded in vitro with interleukin-...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85A61K35/17A61P35/00A61P35/02
Inventor 陈建勋
Owner ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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