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Preparation method and application of nervous system neoplasm cell autophagosome to preparing vaccine

A nervous system and tumor cell technology, which is applied to the preparation method of nervous system tumor cell autophagosomes and in the field of vaccine preparation and dendritic cell tumor vaccines, can solve the problems of different cross-presentation capabilities, and achieve the promotion of proliferation, Promote the maturity of DC and improve the effect of obtaining efficiency

Pending Publication Date: 2019-03-01
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulties faced by DC autophagosome tumor vaccines, in addition to the above-mentioned difficulties in extraction, also lie in: 1. The short-lived proteins (half-life of about 10 minutes) produced by the tumor cells themselves are mainly presented on the surface of tumor cells, and the autophagy pathway The obtained antigen is the longevity protein of the tumor, and the two do not necessarily match. This is one of the problems of the "longevity protein"-based tumor vaccine; 2. DCs have different cross-presentation capabilities in different states, and the cross-presentation efficiency The level of DC will directly affect the efficacy of DC tumor vaccines. How to make DCs cross-present tumor antigens with maximum efficacy is the second problem of DC-based tumor vaccines.

Method used

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  • Preparation method and application of nervous system neoplasm cell autophagosome to preparing vaccine
  • Preparation method and application of nervous system neoplasm cell autophagosome to preparing vaccine
  • Preparation method and application of nervous system neoplasm cell autophagosome to preparing vaccine

Examples

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Embodiment 1

[0050] Example 1 Preparation of autophagosomes from tumor cells derived from the nervous system, using C6 / GL261 glioma cells as tumor cells derived from the nervous system,

[0051] Include the following steps:

[0052] (1) Cultivation of C6 / GL261 glioma cell line:

[0053] Resuscitate the frozen C6 / GL261 glioma cells, thaw quickly in a 37°C water bath, shake it from time to time, and try to reach room temperature within 1 minute, and transfer it to 5ml of DMEM complete medium containing 10% fetal bovine serum under aseptic conditions. Centrifuge at a speed of 1000r / min for 5 minutes, discard the supernatant, add an appropriate amount of the above-mentioned complete medium, adjust the inoculation density to about 1×10^6 / ml, inoculate in a T72 culture bottle, and place in a 37°C, 5% carbon dioxide atmosphere. Place the culture in the incubator, change the medium once after the cells adhere to the wall the next day, and continue the culture, and judge whether to pass passage ac...

Embodiment 2

[0109] In the application of preparing tumor vaccines, the embodiment of the present invention obtains mouse DCs and DCs loaded with autophagosomal antigens:

[0110] 1) Acquisition of mouse DC

[0111] 1. Execute the C57 mice by neck breaking, soak them in 75% alcohol for about 10 minutes, and fix the mice on a sterile operating table;

[0112] 2. Take out the mouse tibia and femur aseptically and soak them in PBS;

[0113] 3. Use a 1ml syringe to extract PBS to rinse the bone marrow cavity until the bone becomes white. After mixing, filter with a 200-mesh filter and add to a 50ml centrifuge tube;

[0114] 4. 1000rpm, 5 minutes, centrifuge at room temperature, remove the supernatant;

[0115] 5. Add 2ml of erythrocyte lysate and let stand at room temperature for 3 minutes;

[0116] 6. Add 10ml PBS and mix well to stop the lysis. 1000rpm, 5 minutes, centrifuge at room temperature, discard the supernatant;

[0117] 7. Add 3 ml of 1640 medium (DC induction medium) containin...

Embodiment 3

[0132] Example 3 Autophagosome Vaccine-Induced Specific Immune Response Experiment

[0133] 1) Preparation of mouse peripheral blood-derived lymphocytes

[0134] 1. After fixing the mouse, collect about 1ml of peripheral blood from the mouse through orbital blood collection, and put it into a 5ml centrifuge tube pre-coated with 100ul EDTA anticoagulant;

[0135] 2. Take a 15ml centrifuge tube, add 3ml of mouse lymphocyte separation medium at room temperature, add the obtained anticoagulant blood sample on the liquid surface of the separation medium, centrifuge at 400g, 20min, at room temperature;

[0136] 3. Use a 1ml pipette tip to carefully suck out the supernatant 5mm above the upper layer of the separation solution, and discard it; then use a 1ml pipette tip to carefully absorb the milky white lymphocyte layer into a new centrifuge tube;

[0137] 4. Add an appropriate amount of cell washing solution into the centrifuge tube, mix well, 300g, 10min, and centrifuge at room t...

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Abstract

The invention belongs to the technical field of biologics and relates to a preparation method and application of nervous system neoplasm cell autophagosome to preparing a vaccine. Sirolimus and ammonium chloride are adopted to induce and culture nervous tumor cells, autophagosome generation is promoted, and autophagosome is extracted through repeated blowing and beating and high-speed centrifugation at different steps. A great deal of autophagosome can be rapidly prepared by using the method provided by the invention, after the autophagosome is carried by DCs (Dendritic Cells), DC maturation can be effectively promoted, T lymphocyte can be activated, proliferation of the T lymphocyte can be promoted, a remarkable killing function on tumor cells can be achieved, and the killing effect is remarkably better than tumor freeze thawing splitting antigen prepared in the prior art. The autophagosome prepared by using the method can be further adopted to prepare a nervous system tumor vaccine,and the vaccine is a dendritic cell tumor vaccine for nervous system derived tumor cell autophagosome.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to nervous system tumor cell autophagosomes and uses thereof, in particular to a method for preparing nervous system tumor cell autophagosomes and its use in preparing vaccines, and the vaccine is directed against nervous system-derived tumors Autophagosome-based Dendritic Cell Tumor Vaccines. Background technique [0002] The prior art discloses that glioma is a tumor originating from glial cells, and is the most common primary intracranial tumor. Among them, glioblastoma (WHO grade IV) has the highest incidence rate, accounting for 46.1% , the incidence rate is about 3.20 / 100,000, and the case fatality rate of young and middle-aged people ranks second. Clinical practice shows that therapeutic interventions, including surgery, adjuvant concurrent chemoradiotherapy, can only delay the progress of the disease, and the above-mentioned diseases all inevitably recur. According to re...

Claims

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Application Information

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IPC IPC(8): C12N5/09A61K39/00A61P35/00
CPCA61K39/0011C12N5/0693A61K2039/5154C12N2500/05
Inventor 姚瑜周良辅孙泽林余双全唐超
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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