CN105616445A - NK cell secreted protein eye drops for treating viral keratitis and preparation method and application thereof - Google Patents
NK cell secreted protein eye drops for treating viral keratitis and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biomedicine, in particular to an NK cell secreted protein eye drop for treating viral keratitis and a preparation method and application thereof.
Description
Technical field
The present invention relates to field of biomedicine technology, be specifically related to a kind of protein eye drops of NK emiocytosis treating viral keratitis and its preparation method and application.
Background technology
Viral keratitis is the inflammation caused by viral pathogenesis original sense dye cornea, and when passive protective physical fitness goes down, such as flu or high heat etc., it is easy to bring out this disease recurrent exerbation, outbreak vision just declines a bit every time, and serious finally causing is blind. In clinical treatment, conventional antiviral collyrium, coordinates antibiotics collyrium, in case secondary bacterial or fungal infection, when having reaction of iris, then adds mydriatic, according to patient's body constitution situation, decline person or easy catching a cold person, is often aided with and improves the medicine exempted from service. It is usually used in the collyrium that the collyrium of viral keratitis Prevention mainly comprises antibiotics at present, including: oxygen Buddhist sand star, acyclovir, ribavirin etc., prior art exists and adopts the Chinese herbal medicine such as Flos Lonicerae, Flos Chrysanthemi Indici to make smoked ophthalmic preparation to treat the scheme of viral keratitis, but, the medicinal liquid obtained by decoction for Chinese medicine added, a lot of effective ingredient can be allowed to inactivate, it is generally composition indefinite, preparation method is excessively simple, high temperature easily makes effective ingredient inactivate, and therapeutic effect is inconspicuous.
Viral keratitis: viral keratitis is the inflammation caused by viral pathogenesis original sense dye cornea. Cornea shallow-layer has abundant nervi trigeminus tip, therefore this disease often has obvious irritation, has photophobia, sheds tears, aches. Viral keratitis can be caused by multiple virus, and the extent of damage of vision not etc., is not looked lesion locations, inflammation weight, course of disease length, recurrent number and different with or without mixed infection by its clinical manifestation weight. Viral keratitis common clinically has herpes simplex keratitis, vaccinial keratitis, herpes zoster keratitis etc.
The protein factor of NK cell and secretion: NK cell and natural killer cell (naturalkillercell), it it is the important immunocyte of body, not only relevant with antitumor, viral infection resisting and immunomodulating, and participate in the generation of allergy and autoimmune disease in some cases. The killing activity of NK cell limits without MHC, is independent of antibody, because of referred to herein as Nk Cell Activity. NK cell cytosol enriches, and the killing activity containing bigger azurophilic granule, the content of granule and NK cell is proportionate. NK cytosis lethal effect after target cell occurs early, 1 hour in vitro, internal 4 hours i.e. visible lethal effect. The target cell of NK cell mainly has some tumor cell (including part cell line), virus infected cell, some autologous tissue's cell (such as hemocyte), parasite etc., therefore NK cell is body antitumor, the important immune factor of anti-infective, also assists in the IIth type allergy and graft versus host disease. In vitro, the target cell of NK solubilized herpesvirus, vaccinia virus, Measles virus, mumps virus, cytomegalovirus and influenza infection.
Summary of the invention
The present invention is directed to problems of the prior art, thering is provided the protein eye drops of a kind of NK emiocytosis treating viral keratitis, the therapeutic modality being main by antiinflammatory, antiviral in treating for viral keratitis is prepared, definite ingredients, with strong points, effect is obvious.
The preparation method that it is a further object of the present invention to provide the protein eye drops of a kind of NK emiocytosis treating viral keratitis.
Another object of the present invention is to provide the protein eye drops of a kind of NK emiocytosis for treating the purposes of viral keratitis.
The present invention is achieved through the following technical solutions this purpose:
A kind of protein eye drops of the NK emiocytosis treating viral keratitis, including following components:
The preparation method of the protein eye drops of a kind of NK emiocytosis treating viral keratitis, comprises the following steps:
The amplification cultivation of NK cell:
1) upper plasma is collected after gathering the centrifugal 10min of patient self peripheral blood 20��40mL, 200��600g standby;
2) being diluted by lower floor's hemocyte normal saline of 2 times of volumes, then dilute blood is slowly added to lymphocyte separation medium upper strata, make layering clear, the volume ratio of dilute blood and lymphocyte separation medium is 2:1;
3) the centrifugal 30min of 400��700g, tunica albuginea confluent monolayer cells in the middle of extracting, adds normal saline and cleans twice, and 200-500g is centrifuged 5min, counting;
4) X-VIVO15 serum-free medium is added so that cell density is 1��10 �� 105Cell/mL, adds the CD3 monoclonal antibody of 5% autologous plasma, the IL-2 of 100��1000U/mL, the IL-15 of 100��1000U/mL, the IL-21 of 100��1000U/mL, 50��500U/mL;
5) in incubation, fluid infusion in every 3 days once, and adds the IL-15 of the IL-2 of 100��1000U/mL, 100��1000U/mL so that cell density maintains 1��10 �� 105Cell/mL;
6) cultivating 12��14 days, collect NK cell suspension, 200��500g is centrifuged 10min, collects supernatant;
The purification of NK cell culture fluid and lyophilizing:
7) supernatant collected being adopted slipstream device, circulating filtration concentrates at least 3h, removes large particulate matter through 0.45 ��m of membrane filtration, and filtrate removes below 3kDa finely ground particle substance then through 0.22 ��m of membrane filtration;
8) by step 7) the lyophilised machine of concentrated solution collected makes NK cell and cultivates lyophilized powder;
Preparation containing NK cell protein factor eye drop:
9) take sterile water for injection heated and boiled, after adding boric acid, Borax dissolving, be cooled to 40 DEG C;
10) add chloromycetin, ethyl hydroxybenzoate stirring and dissolving, add NK cell and cultivate lyophilized powder, finally add sterile water for injection constant volume, prepare the protein eye drops of the NK emiocytosis for the treatment of viral keratitis.
The protein eye drops of a kind of NK emiocytosis is for treating the purposes of viral keratitis.
Relative to prior art, the invention have the benefit that the protein eye drops of the NK emiocytosis of the treatment viral keratitis of the present invention, antiinflammatory is passed through in treating for viral keratitis, the therapeutic modality that antiviral is main is prepared, definite ingredients, with strong points, effect is obvious, first NK cell is carried out amplification cultivation by preparation method, then the albumen of NK emiocytosis is regathered, albumen is purified with lyophilizing after the protein eye drops of prepared NK composite with auxiliary agent emiocytosis, preparation method is simple, cost is low, the protein eye drops of the NK emiocytosis of the present invention has significant effect in the application for the treatment of viral keratitis, and not easily recur.
Detailed description of the invention
Describe the present invention below in conjunction with specific embodiment.
Embodiment 1.
The preparation method that the present embodiment provides the protein eye drops of a kind of NK emiocytosis treating viral keratitis, comprises the following steps:
The amplification cultivation of NK cell:
1) upper plasma is collected after gathering the centrifugal 10min of patient self peripheral blood 20mL, 200g standby;
2) being diluted by lower floor's hemocyte normal saline of 2 times of volumes, then dilute blood is slowly added to lymphocyte separation medium upper strata, make layering clear, the volume ratio of dilute blood and lymphocyte separation medium is 2:1;
3) the centrifugal 30min of 400g, tunica albuginea confluent monolayer cells in the middle of extracting, adds normal saline and cleans twice, and 200g is centrifuged 5min, counting;
4) X-VIVO15 serum-free medium is added so that cell density is 1 �� 105Cell/mL, adds the CD3 monoclonal antibody of IL-21,50U/mL of IL-15,100U/mL of IL-2,100U/mL of 5% autologous plasma, 100U/mL;
5) in incubation, fluid infusion in every 3 days once, and adds the IL-15 of IL-2,500U/mL of 500U/mL so that cell density maintains 1��10 �� 105Cell/mL;
6) cultivating 12��14 days, collect NK cell suspension, 200g is centrifuged 10min, collects supernatant;
The purification of NK cell culture fluid and lyophilizing:
7) supernatant collected being adopted slipstream device, circulating filtration concentrates at least 3h, removes large particulate matter through 0.45 ��m of membrane filtration, and filtrate removes below 3kDa finely ground particle substance then through 0.22 ��m of membrane filtration;
8) by step 7) the lyophilised machine of concentrated solution collected makes NK cell and cultivates lyophilized powder;
Preparation containing NK cell protein factor eye drop:
9) take sterile water for injection heated and boiled, after adding boric acid, Borax dissolving according to quantity, be cooled to 40 DEG C;
10) add chloromycetin, ethyl hydroxybenzoate stirring and dissolving according to quantity, add NK cell and cultivate.
Embodiment 2.
The preparation method that the present embodiment provides the protein eye drops of a kind of NK emiocytosis treating viral keratitis, comprises the following steps:
The amplification cultivation of NK cell:
1) upper plasma is collected after gathering the centrifugal 10min of patient self peripheral blood 30mL, 400g standby;
2) being diluted by lower floor's hemocyte normal saline of 2 times of volumes, then dilute blood is slowly added to lymphocyte separation medium upper strata, make layering clear, the volume ratio of dilute blood and lymphocyte separation medium is 2:1;
3) the centrifugal 30min of 500g, tunica albuginea confluent monolayer cells in the middle of extracting, adds normal saline and cleans twice, and 300g is centrifuged 5min, counting;
4) X-VIVO15 serum-free medium is added so that cell density is 5 �� 105Cell/mL, adds the CD3 monoclonal antibody of IL-21,3500U/mL of IL-15,500U/mL of IL-2,500U/mL of 5% autologous plasma, 500U/mL;
5) in incubation, fluid infusion in every 3 days once, and adds the IL-15 of IL-2,500U/mL of 500U/mL so that cell density maintains 1��10 �� 105Cell/mL;
6) cultivating 12��14 days, collect NK cell suspension, 300g is centrifuged 10min, collects supernatant;
The purification of NK cell culture fluid and lyophilizing:
7) supernatant collected being adopted slipstream device, circulating filtration concentrates at least 3h, removes large particulate matter through 0.45 ��m of membrane filtration, and filtrate removes below 3kDa finely ground particle substance then through 0.22 ��m of membrane filtration;
8) by step 7) the lyophilised machine of concentrated solution collected makes NK cell and cultivates lyophilized powder;
Preparation containing NK cell protein factor eye drop:
9) take sterile water for injection heated and boiled, after adding boric acid, Borax dissolving, be cooled to 40 DEG C;
10) add chloromycetin, ethyl hydroxybenzoate stirring and dissolving, add NK cell and cultivate.
Embodiment 3.
The preparation method that the present embodiment provides the protein eye drops of a kind of NK emiocytosis treating viral keratitis, comprises the following steps:
The amplification cultivation of NK cell:
1) upper plasma is collected after gathering the centrifugal 10min of patient self peripheral blood 40mL, 600g standby;
2) being diluted by lower floor's hemocyte normal saline of 2 times of volumes, then dilute blood is slowly added to lymphocyte separation medium upper strata, make layering clear, the volume ratio of dilute blood and lymphocyte separation medium is 2:1;
3) the centrifugal 30min of 700g, tunica albuginea confluent monolayer cells in the middle of extracting, adds normal saline and cleans twice, and 500g is centrifuged 5min, counting;
4) X-VIVO15 serum-free medium is added so that cell density is 10 �� 105Cell/mL, adds the CD3 monoclonal antibody of IL-21,500U/mL of IL-15,1000U/mL of IL-2,1000U/mL of 5% autologous plasma, 1000U/mL;
5) in incubation, fluid infusion in every 3 days once, and adds the IL-15 of IL-2,500U/mL of 500U/mL so that cell density maintains 1��10 �� 105Cell/mL;
6) cultivating 12��14 days, collect NK cell suspension, 500g is centrifuged 10min, collects supernatant;
The purification of NK cell culture fluid and lyophilizing:
7) supernatant collected being adopted slipstream device, circulating filtration concentrates at least 3h, removes large particulate matter through 0.45 ��m of membrane filtration, and filtrate removes below 3kDa finely ground particle substance then through 0.22 ��m of membrane filtration;
8) by step 7) the lyophilised machine of concentrated solution collected makes NK cell and cultivates lyophilized powder;
Preparation containing NK cell protein factor eye drop:
9) take sterile water for injection heated and boiled, after adding boric acid, Borax dissolving, be cooled to 40 DEG C;
10) add chloromycetin, ethyl hydroxybenzoate stirring and dissolving, add NK cell and cultivate.
Embodiment 4, compliance test result are tested
Choosing 80,146 oculopathy toxicity keratitis patients are individual, wherein divide according to gradient of infection: point-like light-duty infiltration viral keratitis 54 example 98, shallow-layer infiltration viral keratitis 14 example 28, deep layer infiltration viral keratitis 12 example 20; Being divided into matched group and treatment group to carry out medication contrast test, wherein matched group carries out hospital's general Symptomatic medicine treatment; Treatment group is treated with the protein eye drops medication of the present invention, and compliance test result comparative test result is as shown in table 1.
Matched group: point-like infiltration viral keratitis 26 example 49;
Shallow-layer infiltration viral keratitis 7 example 14;
Deep layer infiltration viral keratitis 6 example 10;
Treatment group: point-like infiltration viral keratitis 28 example 49;
Shallow-layer infiltration viral keratitis 7 example 14;
Deep layer infiltration viral keratitis 6 example 10;
Table 1 compliance test result comparative test result
Result illustrates:
Curing: irritation disappears, keratitis infiltration all absorbs or is formed nebula, kp "-", and fluorescent staining is negative;
Taking a turn for the better: symptom alleviates, have and remain kp on a small quantity, ulcer surface reduces, shoals.
From comparing result shown in table 1, the treatment group of the protein eye drops of the application present invention is better than matched group at the effectiveness of viral keratitis in various degree, show that the protein eye drops of NK emiocytosis of the present invention is in the therapeutic process of viral keratitis, effect with strong points, significant, and not easily recur.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention. It should be pointed out that, for the person of ordinary skill of the art, without departing from the inventive concept of the premise, it is also possible to making some deformation and improvement, these broadly fall into protection scope of the present invention. Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (3)
1. treat a protein eye drops for the NK emiocytosis of viral keratitis, including following components:
2. the preparation method treating the protein eye drops of the NK emiocytosis of viral keratitis, comprises the following steps:
The amplification cultivation of NK cell:
1) upper plasma is collected after gathering the centrifugal 10min of patient self peripheral blood 20��40mL, 200��600g standby;
2) being diluted by lower floor's hemocyte normal saline of 2 times of volumes, then dilute blood is slowly added to lymphocyte separation medium upper strata, make layering clear, the volume ratio of dilute blood and lymphocyte separation medium is 2:1;
3) the centrifugal 30min of 400��700g, tunica albuginea confluent monolayer cells in the middle of extracting, adds normal saline and cleans twice, and 200-500g is centrifuged 5min, counting;
4) X-VIVO15 serum-free medium is added so that cell density is 1��10 �� 105Cell/mL, adds the CD3 monoclonal antibody of 5% autologous plasma, the IL-2 of 100��1000U/mL, the IL-15 of 100��1000U/mL, the IL-21 of 100��1000U/mL, 50��500U/mL;
5) in incubation, fluid infusion in every 3 days once, and adds the IL-15 of the IL-2 of 100��1000U/mL, 100��1000U/mL so that cell density maintains 1��10 �� 105Cell/mL;
6) cultivating 12��14 days, collect NK cell suspension, 200��500g is centrifuged 10min, collects supernatant;
The purification of NK cell culture fluid and lyophilizing:
7) supernatant collected being adopted slipstream device, circulating filtration concentrates at least 3h, removes large particulate matter through 0.45 ��m of membrane filtration, and filtrate removes below 3kDa finely ground particle substance then through 0.22 ��m of membrane filtration;
8) by step 7) the lyophilised machine of concentrated solution collected makes NK cell and cultivates lyophilized powder;
Preparation containing NK cell protein factor eye drop:
9) take sterile water for injection heated and boiled, after adding boric acid, Borax dissolving, be cooled to 40 DEG C;
10) add chloromycetin, ethyl hydroxybenzoate stirring and dissolving, add NK cell and cultivate lyophilized powder, finally add sterile water for injection constant volume, prepare the protein eye drops of the NK emiocytosis for the treatment of viral keratitis.
3. the protein eye drops of a NK emiocytosis is for treating the purposes of viral keratitis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475196A (en) * | 2017-10-09 | 2017-12-15 | 天津长和生物技术有限公司 | The amplification cultivation method of NK culture matrix and NK |
| CN112587632A (en) * | 2020-12-28 | 2021-04-02 | 海南启研干细胞抗衰老医院有限公司 | Biological preparation for preventing and treating HPV (human papillomavirus) infection and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104593324A (en) * | 2014-11-28 | 2015-05-06 | 广州赛莱拉干细胞科技股份有限公司 | Natural killer cell culture medium and natural killer cell amplification culture method |
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- 2016-01-15 CN CN201610029136.4A patent/CN105616445A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593324A (en) * | 2014-11-28 | 2015-05-06 | 广州赛莱拉干细胞科技股份有限公司 | Natural killer cell culture medium and natural killer cell amplification culture method |
Non-Patent Citations (2)
| Title |
|---|
| 宋文刚主编: "《医学免疫学》", 31 August 2013 * |
| 赵家良主编: "《眼科诊疗常规》", 31 October 2012 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475196A (en) * | 2017-10-09 | 2017-12-15 | 天津长和生物技术有限公司 | The amplification cultivation method of NK culture matrix and NK |
| CN112587632A (en) * | 2020-12-28 | 2021-04-02 | 海南启研干细胞抗衰老医院有限公司 | Biological preparation for preventing and treating HPV (human papillomavirus) infection and preparation method thereof |
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Application publication date: 20160601 |