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Tumor specificity gamma delta T cell preparation method

A tumor-specific, tumor cell-based technology, applied in the field of preparation of tumor-specific γδT cells, can solve problems such as complex steps, uncontrollable conditions, and high cost

Active Publication Date: 2018-06-01
湖南东冈生物技术合伙企业(有限合伙)
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Problems solved by technology

[0007] The main purpose of the present invention is to provide a method for preparing tumor-specific γδT cells, aiming at solving the problem that conventional γδT cell culture methods have low toxicity to prostate cancer cells, the method steps are complicated, the cost is high, there are many procedures, and the conditions are uncontrollable. Moreover, the requirements for the equipment environment are relatively high, and it is not easy to produce in large quantities, which brings inconvenience to the scientific research and medical work of medical workers.

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preparation example Construction

[0050] The invention provides a method for preparing tumor-specific γδT cells, comprising the following steps:

[0051] S1, extracting autophagosomes derived from tumor cells;

[0052] As mentioned above, in the present invention, the selected tumor cells are prostate cancer cells.

[0053] As mentioned above, it needs to be understood that the autophagosome is formed by a small part of the cytoplasm surrounded by a double membrane, and the digested substances are various components contained in the cytoplasm, such as mitochondria, fragments of the endoplasmic reticulum, etc. It is one of the components of the cell vacuole system by fusing with lysosomes to degrade their contents. Also known as autolysosomes. Under pathological atrophy, autophagosomes increase and lipofuscin granules appear in the cytoplasm of atrophic cells such as cardiomyocytes and hepatocytes.

[0054] S2, collecting peripheral venous blood and obtaining mononuclear cells therefrom;

[0055] As mention...

Embodiment

[0096] The present invention provides a method for preparing tumor-specific γδT cells. Prostate cancer DU145 cells are cultured, cells are induced by factors to produce autophagosomes, and they are extracted and identified, including the following steps:

[0097]1. For the cultivation of prostate cancer cells, cultivate them with MEM medium containing 10% FBS+100U / mL penicillin-streptomycin. The area of ​​-90% shall prevail), add 100nM rapamycin (autophagy inducer), 100nM bortezomib (proteasome inhibitor) and 10nM NH4Cl (lysosomal chemoattractant, prevent lysosome and autophagy After 18-24h treatment, the cells were collected.

[0098] 2. Pour the cell culture supernatant into a 50mL centrifuge tube, centrifuge at 1600r / min for 10min. The adherent cells were digested with trypsin and centrifuged at 1000r / min for 10min. After centrifugation, the pelleted cells were resuspended in saline and centrifuged after repeated pipetting. The supernatant obtained by centrifugation was t...

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Abstract

The invention provides a tumor specificity gamma delta T cell preparation method which comprises the following steps: S1, extracting autophagosome sourced from tumor cells; S2, collecting peripheral venous blood and obtaining mononuclear cells from the peripheral venous blood; S3, adding phosphoric acid antigen zoledronic acid (Zol) factors into the mononuclear cells to stimulate and induce multiplication culture of the gamma delta T cells to obtain gamma delta T cells; S4, performing co-culture on the autophagosome and the gamma delta T cells within preset time and preparing to obtain the tumor specificity gamma delta T cells. The specificity gamma delta T cells have stronger killing capacity on varieties of tumor cells, cytotoxic activity is obviously stronger than that of gamma delta Tcells prepared by a general method and from antigen; furthermore, the preparation method disclosed by the invention has the advantages of low preparation cost, simple work procedures, conditions easyto control, lower requirement in equipment and easiness in scale production.

Description

technical field [0001] The invention relates to the technical field of cellular immunology, in particular to a method for preparing tumor-specific γδT cells. Background technique [0002] Tumor refers to the neogrowth formed by the proliferation of local tissue cells in the body under the action of various tumorigenic factors, because this neogrowth is mostly in the form of space-occupying blocky protrusions, also known as neoplasm (neoplasm) . In recent years, with the change of lifestyle, the deterioration of the natural environment, and the improvement of medical technology, the number of tumors detected is on the rise, and tumors have become one of the important diseases that seriously threaten human health. [0003] Due to the high damage and high mortality rate of tumors, the research and application of anti-tumor treatment methods have always been the focus of attention. Tumor immunotherapy is the fourth largest therapy for tumor treatment in clinical practice, among...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2501/999C12N2502/30
Inventor 邹畅赵盼郭吉楠王建红
Owner 湖南东冈生物技术合伙企业(有限合伙)
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