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A kind of preparation method and application of NK cell freeze-dried powder

A technology of NK cells and freeze-dried powder, applied in cell dissociation methods, biochemical equipment and methods, freeze-dried transportation, etc., can solve the problems of ineffective chronic pain, affected kidney function, and poor prognosis, and achieve the goal of inhibiting invasion. infection, reduce lesions, and shorten the course of disease

Active Publication Date: 2022-04-08
北京荟科柘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Broad-spectrum antiviral drugs, poor therapeutic effect, long treatment cycle, and impact on renal function
In the acute inflammatory phase, high-dose glucocorticoids can be used to suppress the inflammatory process, but it is basically ineffective for chronic pain, and high-dose glucocorticoids can cause many complications
The existing treatment methods have problems such as poor curative effect, poor prognosis, and easy to cause sequelae such as neuralgia.

Method used

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  • A kind of preparation method and application of NK cell freeze-dried powder
  • A kind of preparation method and application of NK cell freeze-dried powder
  • A kind of preparation method and application of NK cell freeze-dried powder

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 Preparation of NK cell freeze-dried powder

[0028] Step 1: NK cell culture flask pretreatment: Dissolve CD16 monoclonal antibody and RetroNctin in D-PBS, transfer to a T175 culture flask, and store in the dark at 37°C for 3 hours, or overnight at 4°C.

[0029] Step 2: PBMC Isolation

[0030] Sub-step 2.1: Take the lymphocyte separation solution out of the 4°C refrigerator 30 minutes in advance and place it at room temperature, and use it after the temperature rises to room temperature.

[0031] Sub-step 2.2: Pour the blood into a 50ml centrifuge tube, balance, centrifuge at 700g / min for 20 minutes (slowest speed drop), collect the upper layer liquid, inactivate at 56°C for 30 minutes, then place it at 4°C for 15 minutes, and finally centrifuge at 2000g for 20 minutes to absorb the upper layer The liquid is ready for use.

[0032] Sub-step 2.3: Add D-PBS to the lower layer after centrifugation of the above-mentioned blood, mix evenly, and set the volume t...

Embodiment 2

[0048] Example 2: Detection of NK cell exosome content

[0049] According to the method of Example 1, three batches of NK cell exosomes were prepared, and three batches of NK cell exosomes were detected. Specific steps are as follows:

[0050] The NK cell exosomes obtained by the method in Example 1 were further purified. First, the cell debris in the liquid was removed by using a 0.45 μm mPES hollow fiber filter column; the filtrate was further passed through an mPES hollow fiber filter column with a molecular weight cut-off of 300-kDa Concentrate to obtain a crude product; then dilute with 3 times the volume of the crude product of PBS, and concentrate using an mPES hollow fiber filter column with a molecular weight cut-off of 300-kDa to obtain exosomes for detection.

[0051] The obtained exosomes for detection were diluted 100 times with PBS, the particle size distribution of exosomes was detected by Zetasizer Nano ZSE system, and the detection results were analyzed by Di...

Embodiment 3

[0053] Embodiment 3: comparison experiment of virus proliferation inhibition

[0054] Amplification of herpes virus VZV: Spread Vero cells evenly in a 24-well plate, culture to a confluence of 70%-80%, discard the supernatant in the Vero cells, add the virus dilution to the Vero cells, 300 per well microliter, and placed in a 37°C incubator for 2 hours of adsorption. After culturing for 72 hours, if more than 80% of the cell layer has lesions, freeze and thaw repeatedly 3 times, collect the cells and supernatant, centrifuge at low temperature, 4000rpm, 10min, absorb the supernatant, and store at -80°C for later use.

[0055] Prepare agarose: Make 2% agarose in water, autoclave and melt it, then place the agarose in a 42°C water bath to keep it in a molten state. Pre-warm the cell culture medium to 37 °C.

[0056] Infected cell monolayer: 1 x 10 per well 6 Spread the Vero cells evenly in a 24-well plate and culture them to a confluence of 70%-80%. Five groups of drugs (lyoph...

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Abstract

The invention discloses that isolated PBMCs are used to induce culture to obtain NK cells, and after expanded culture, exosomes of NK cells are extracted, mixed with oxytetracycline and extracts of Asparagus japonica, and freeze-dried to obtain freeze-dried powder. Freeze-dried powder can significantly inhibit the infection of VSV, reduce the lesions of herpes zoster, shorten the course of the disease, and improve the prognosis, achieving the unexpected effect of traditional treatment methods.

Description

technical field [0001] The invention belongs to the field of immunotherapy, and specifically relates to the field of medicine in which NK cell exosome freeze-dried powder is used to prepare and treat viral infection. Background technique [0002] Shingles is a viral skin disease caused by varicella-zoster virus (VZV). The affected area often appears first with erythema, followed by miliary to soybean-sized papules, which are distributed in clusters without fusion, and then quickly turn into blisters, with tense and shiny walls, clear blister fluid, and blush around the periphery. The skin in the middle is normal; the lesions are arranged in bands along a peripheral nerve, mostly occur on one side of the body, and generally do not exceed the midline. Herpes zoster will cause obvious pain and affect normal life. If it is not controlled in time, there will be complications such as bacterial infection, postherpetic neuralgia, keratitis, corneal ulcer, conjunctivitis, toxic ence...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078C12N5/0783A61K36/704A61K9/19A61K35/17A61P17/00A61P31/22A61K31/65
CPCA61K35/17A61K31/65A61K36/704A61K9/19A61P31/22A61P17/00C12N5/0646C12N5/0634C12N2509/00C12N2500/90C12N2501/24C12N2501/2302C12N2501/2321C12N2501/2315A61K2300/00
Inventor 朱哲张涛
Owner 北京荟科柘生物科技有限公司
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