NK cell secreted protein eye drops used for treating viral keratitis as well as preparation method and application thereof

A viral keratitis and NK cell technology, applied in the field of biomedicine, can solve the problems of unclear ingredients, inactivation of active ingredients, and insignificant therapeutic effect, and achieve the effect of obvious effect, not easy to recur, and obvious effect.

Inactive Publication Date: 2016-06-01
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, eye drops commonly used in the treatment and prevention of viral keratitis are mainly eye drops containing antibiotics, including: offofloxacin, acyclovir, ribavirin, etc. Chinese herbal medicines are used to make smoked eye preparations to treat viral keratitis. However, the addition is the liquid obtained by decocting traditional Chinese medicines, which will inactivate many active ingredients. Generally, the ingredients are not clear, and the preparation method is too simple. High temperature is easy to inactivate the active ingredients, and the therapeutic effect is not obvious

Method used

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  • NK cell secreted protein eye drops used for treating viral keratitis as well as preparation method and application thereof
  • NK cell secreted protein eye drops used for treating viral keratitis as well as preparation method and application thereof
  • NK cell secreted protein eye drops used for treating viral keratitis as well as preparation method and application thereof

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Experimental program
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Embodiment 1

[0029] The present embodiment provides a method for preparing protein eye drops secreted by NK cells for the treatment of viral keratitis, comprising the following steps:

[0030] Expansion and culture of NK cells:

[0031] 1) Collect 20mL of the patient's own peripheral blood, centrifuge at 200g for 10min, and collect the upper plasma for later use;

[0032] 2) Dilute the blood cells in the lower layer with 2 times the volume of normal saline, then slowly add the diluted blood to the upper layer of the lymphocyte separation medium to make the layers clear, and the volume ratio of the diluted blood to the lymphocyte separation medium is 2:1;

[0033] 3) Centrifuge at 400g for 30min, extract middle buffy coat cells, add normal saline to wash twice, centrifuge at 200g for 5min, and count;

[0034] 4) Add X-VIVO15 serum-free medium to make the cell density 1×10 5 cell / mL, add 5% autologous plasma, 100U / mL IL-2, 100U / mL IL-15, 100U / mL IL-21, 50U / mL CD3 monoclonal antibody;

[0...

Embodiment 2

[0044] The present embodiment provides a method for preparing protein eye drops secreted by NK cells for the treatment of viral keratitis, comprising the following steps:

[0045] Expansion and culture of NK cells:

[0046] 1) Collect 30 mL of the patient's own peripheral blood, centrifuge at 400 g for 10 min, and collect the upper layer of plasma for later use;

[0047] 2) Dilute the blood cells in the lower layer with 2 times the volume of normal saline, then slowly add the diluted blood to the upper layer of the lymphocyte separation medium to make the layers clear, and the volume ratio of the diluted blood to the lymphocyte separation medium is 2:1;

[0048] 3) Centrifuge at 500g for 30min, extract middle buffy coat cells, add normal saline to wash twice, centrifuge at 300g for 5min, and count;

[0049] 4) Add X-VIVO15 serum-free medium to make the cell density 5×10 5 cell / mL, add 5% autologous plasma, 500U / mL IL-2, 500U / mL IL-15, 500U / mL IL-21, 3500U / mL CD3 monoclonal a...

Embodiment 3

[0059] The present embodiment provides a method for preparing protein eye drops secreted by NK cells for the treatment of viral keratitis, comprising the following steps:

[0060] Expansion and culture of NK cells:

[0061] 1) Collect 40 mL of the patient's own peripheral blood, centrifuge at 600 g for 10 min, and collect the upper layer of plasma for later use;

[0062] 2) Dilute the blood cells in the lower layer with 2 times the volume of normal saline, then slowly add the diluted blood to the upper layer of the lymphocyte separation medium to make the layers clear, and the volume ratio of the diluted blood to the lymphocyte separation medium is 2:1;

[0063] 3) Centrifuge at 700g for 30min, extract middle buffy coat cells, wash twice with normal saline, centrifuge at 500g for 5min, and count;

[0064] 4) Add X-VIVO15 serum-free medium to make the cell density 10×10 5 cell / mL, add 5% autologous plasma, 1000U / mL IL-2, 1000U / mL IL-15, 1000U / mL IL-21, 500U / mL CD3 monoclonal ...

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Abstract

The invention relates to the technical field of biomedicines and in particular relates to NK cell secreted protein eye drops used for treating viral keratitis as well as a preparation method and application thereof. The NK cell secreted protein eye drops used for treating viral keratitis are prepared according to a main anti-inflammation and antiviral treatment method in viral keratitis treatment, ingredients are definite, the pertinence is strong, and the effect is obvious. The preparation method of the NK cell secreted protein eye drops comprises the following steps: firstly carrying out amplification culture on NK cells, then collecting protein secreted by the NK cells, purifying and freeze-drying the protein, and then compounding with an auxiliary preparation, so that the NK cell secreted protein eye drops are obtained. The preparation method is simple, the cost is low, the NK cell secreted protein eye drops have an obvious effect in application of treatment on viral keratitis, and the relapse is hardly caused.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a protein eye drop secreted by NK cells for treating viral keratitis and a preparation method and application thereof. Background technique [0002] Viral keratitis is an inflammation caused by the infection of the cornea by a viral pathogen. When the body’s resistance is weakened, such as a cold or high fever, it is easy to induce repeated attacks of the disease, and the vision will decrease a little every time it occurs. In severe cases, it will eventually lead to blindness. In clinical treatment, antiviral eye drops are often used together with antibiotic eye drops to prevent secondary bacterial or fungal infections. When there is iris reaction, dilated pupils are added. Drugs that boost immunity. At present, eye drops commonly used in the treatment and prevention of viral keratitis are mainly eye drops containing antibiotics, including: offofloxacin, acyclovir, ribaviri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/14A61K38/00A61K9/08A61K47/14A61P27/02A61P31/12A61K31/165A61K33/22
CPCA61K9/0048A61K9/08A61K31/165A61K33/22A61K35/17A61K38/00A61K47/14A61K2300/00
Inventor 王一飞陈海佳葛啸虎应杰
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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