CN106265737A - A kind of external used medicine for treating skin appendages and itching skin disease and its preparation method and application - Google Patents

A kind of external used medicine for treating skin appendages and itching skin disease and its preparation method and application Download PDF

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CN106265737A
CN106265737A CN201610994956.7A CN201610994956A CN106265737A CN 106265737 A CN106265737 A CN 106265737A CN 201610994956 A CN201610994956 A CN 201610994956A CN 106265737 A CN106265737 A CN 106265737A
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stock solution
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卢玲琳
李祥宇
王苏鸣
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Shanghai Mingda Biological Technology Co Ltd
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/17Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • A61K47/44Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0014Skin, i.e. galenical aspects of topical compositions

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Abstract

The invention discloses a kind of external used medicine for treating skin appendages and itching skin disease and its preparation method and application, described medicine be mainly composed of NK cell stock solution, described NK cell stock solution is carried out amplification cultivation acquisition by NK cell, NK cell stock solution is mixed from different excipient substances, can be made into liquid preparation for external application or external ointment preparation.Compared with prior art, this external used medicine is to utilize the protein of NK emiocytosis and cytokine to be easily absorbed by the skin, and the feature of target cell can be directly acted on, can be directly used for skin appendages and the treatments of itching skin disease such as acne, seborrheic dermatitis, pruritus and prurigo nodularis, curative effect is quick, the immune system of skin can be improved simultaneously, repair impaired skin.Excreted factor in NK cell stock solution also can kill fungus and antibacterial, reaches the purpose of Comprehensive Treatment acne, dermatitis and pruritus, and therapeutic domain is extensive, can prevent repeatedly increasing the weight of of the state of an illness.

Description

A kind of external used medicine for treating skin appendages and itching skin disease and system thereof Preparation Method and application
Technical field
The present invention relates to field of biological pharmacy, particularly relate to a kind of for treating skin appendages and pruritic skin Sick external used medicine and its preparation method and application.
Background technology
Acne vulgaris is the most common a kind of dermatosis, has 600,000,000 5 thousand ten thousand patients in the whole world.It is due to hair follicle, skin Adipose gland blocks, and causes Propiobacterium amount reproduction present in skin and the inflammation that causes.Be mainly in the more face of sebaceous gland, , often there are blackhead, Whiteheads, red pimple, pustule, cyst etc., and are likely to result in skin in the positions such as cervical region and shirtfront back Skin pigementation or generation cicatrix.Acne impact is attractive in appearance, and the patient that situation is serious often bears huge mental pressure.Really The cause of disease cut is the clearest, may relate to the factors such as androgen, sebum generation and Propiobacterium infection, and heredity is also individual important Aspect.In serious patients with acne blood, androgen precursor substantially increases, and patient with slight symptoms's simply tissue Serum Testosterone Levels raises. Sebaceous gland hyperfunctioning, generates too much sebum, is also a key factor of acne generation.Propiobacterium infects the immunity caused React relevant with the generation of struvite skin lesion.
The treatment of acne typically uses medicine for external use and innerlich anwenden: antibiotic, glucocorticoid, tretinoin etc., but this type of medicine Treatment targeting is single, needs to coordinate other to infect and antimicrobial drug is used in combination, and life-time service exists toxic and side effects.
Seborrheic dermatitis, also known as seborrheic eczema, is to occur at sebaceous gland and enriches the one chronic papulosquamous inflammation at position Disease dermatoses.Primary disease is more common in adult and neonate, is apt to occur in the sebaceous gland such as head, trunk and enriches district.
Pruritus is a kind of subjective symptoms that many dermatosiss are total, and this cause of disease is because of complexity, and skin exists the sensor of gargalesthesia, And it is transmitted to brain by teleneuron.The release of affected tissue amine can promote gargalesthesia.Scratch because tenderness has itching-relieving action, but promote Enter and discharge more histamine, increase the weight of gargalesthesia, form the vicious cycle grabbed of itching, cause even more serious skin injury and inflammation Disease.
Prurigo nodularis is more common in adult female, is apt to occur in four autopodium sides and brothers back.From the beginning of for indefinite light of number Red papules, gradually expands Semen Glycines to Semen Viciae fabae size bronzing or taupe hemispherical tuberosity.Rough surface is excipuliform, paroxysm Property play is itched, and has pigmentation and lichenification because scratching.The chronic course of disease.The cause of disease is not yet clear and definite, patient mostly be allergic constitution with The sting such as mosquito sand fly are relevant, pathobolism, gastrointestinal dysfunction, endocrine dysfunction, namely may be with this Sick relevant.
General treatment skin appendages and itching skin disease many employings external and oral medicine comprehensively use, and medicine for external use is main It is some glucocorticoids, antimicrobial drug, Selsun Blue, vancide ZP lotion, antifungal preparation, sulfur and (or) salicylic acid Lotion etc., oral medicine use glucocorticoid, tripterygium glycosides, antibiotic, these a few class medicines of vitamin B group, with treatment this type of Medicine equally exists that treatment is single, and the course for the treatment of is longer, and permanent use exists toxic and side effects, and therefore one comprehensive medicine of research and development makes Its curative effect is quick, have no side effect in the urgent need to.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that one is used for treating skin appendages and pruritus External used medicine of dermatoses and preparation method thereof, single to solve conventional medication, the course for the treatment of is longer, and permanent use exists poison The technical problems such as side effect.
The present invention is achieved by the following technical solutions:
The invention provides a kind of external used medicine for treating skin appendages and itching skin disease, described medicine for external use Thing includes the NK cell stock solution that mass percent is 20%-100%.
Further, described external used medicine is a kind of liquid preparation, named SNK liquid, the component of described SNK liquid and matter thereof Amount percentage ratio include: NK cell stock solution 70.0-80.0%, glycerol 2.0-5.0%, ethylparaben 0.01-0.03%, Borax 0.03-0.08%, Oleum menthae 0.5-1.5%, solvent is water.
Further, described external used medicine is a kind of ointment preparation, named SNK cream, the component of described SNK cream and matter thereof Amount percentage ratio includes: NK cell stock solution 60.0-70.0%, Carbopol 941 are 0.1-0.5%, glycerol 2.0-5.0%, Aloe powder 0.2-0.5%, ethylparaben 0.1-0.5%, hyaluronic acid 0.1-0.3%, Oleum menthae 0.5-1.5%, surplus is Water.
Present invention also offers the preparation method of the external used medicine of above-mentioned treatment skin appendages and itching skin disease, bag Include following steps:
(1) take NK cell and carry out amplification cultivation, it is thus achieved that NK cell stock solution;
(2) NK cell stock solution is mixed with external used medicine adjuvant, prepare described external used medicine.
Further, if described medicine is liquid preparation, the mass percent of described NK cell stock solution is 70.0- 80.0%, described adjuvant and mass percent thereof include: glycerol 2.0-5.0%, ethylparaben 0.01-0.03%, Borax 0.03-0.08%, Oleum menthae 0.5-1.5%, solvent is water;If described medicine is ointment preparation, described NK cell is former The mass percent of liquid is 60.0-70.0%, and described adjuvant and mass percent thereof include: Carbopol 941 be 0.1-0.5%, Glycerol 2.0-5.0%, Aloe powder 0.2-0.5%, ethylparaben 0.1-0.5%, hyaluronic acid 0.1-0.3%, thin Lotus oil 0.5-1.5%, surplus is water.
Further, the method that described NK cell amplification is cultivated, comprise the following steps:
Step one: take fresh blood, extracts the peripheral blood lymphocytes (hereinafter referred to as PBMC) in blood;
Step 2: the celebrating of interleukin I L-2,80U/ml of adding 200U/ml in RPMI-1640 basal medium is the most mould Element and the hyclone of 10% percentage by weight, it is thus achieved that NK cell culture fluid;
Step 3: take 3.0 × 107-4.0×107Individual PBMC, in a Tissue Culture Flask, adds the NK cell training of 20-30ml The concentration of nutrient solution and 2ml is 1.0 × 107The NK amplifing reagent of individual/ml, it is thus achieved that initial inoculum;
Step 4: NK cell is inoculated in initial inoculum cultivation 1-2 days, adds according to cell growth state during cultivation Add NK cell culture fluid;When cultivate to the 3rd day time, by medium centrifugal, it is thus achieved that cell precipitation in add NK cell culture fluid Continue to cultivate;
Step 5: when NK cell quantity reaches 1.5 × 108Time more than individual/bottle, add 2 NK amplifing reagents and the training of NK cell Nutrient solution is enlarged cultivating, and increases NK cell culture fluid according to cell growth state and carries out splitting cultivation, make in incubation Cell concentration is 1.0 × 106Individual/ml, when cultivating to the 12nd day, controls liquid volume added, and making cell concentration is 2.0 × 106Individual/ml;
Step 6: continue to cultivate after the 14th day, collect cell culture fluid, it is thus achieved that NK cell stock solution.
Present invention also offers above-mentioned external used medicine as the application in skin subsidiarity disease therapeuticing medicine.
Further, NK cell stock solution and different external used medicine adjuvants are mixed, prepare liquid preparation for external application and External ointment preparation, when using above-mentioned external medicine preparation treatment skin appendages and itching skin disease, first uses external use liquid Body preparation, then carry out mixing treatment with external ointment preparation, to keep drug effect can allow skin absorption well, is used in mixed way number After it, symptom can alleviate or disappear.
The principle of the present invention is: NK cell (natural killer cell, natural killer cell) is important the exempting from of body Epidemic disease cell, not only relevant with antitumor, viral infection resisting and immunomodulating, and in some cases participate in allergy and from The generation of body immune disease.NK cell can kill some antibacterial, fungus etc. in vitro, and this discharges some with NK cell and kills Jie Matter is relevant.The medium of NK release mainly has perforin, NK cytotoxic factor and TNF etc..And in NK cell cultivation process, can close Becoming and secrete cytokine profiles, play immunoregulation effect, these cytokines include MIP-1 α, MIP-1 β, RANTES, list Monocyte chemoattractant protein, interleukin, colony stimulating factor, tumor necrosis factor, interferon and perforin etc..This biological preparation Use NK cell stock solution treatment skin appendages and itching skin disease, utilize the egg of NK emiocytosis in NK cell stock solution just White matter and cytokine directly can act on the feature of target cell, meanwhile, containing abundant inflammation in NK cell stock solution by skin permeation Inflammation factor, after this inflammatory factor is absorbed by the skin, induction body produces humoral immunization and cell immune response, improves skin certainly The resistivity of body, and accelerate to repair impaired tissue and cell, reach the effect for the treatment of. simultaneously.
Compared with prior art, the invention provides a kind of external for treating skin appendages and itching skin disease Medicine and its preparation method and application, this external used medicine utilizes the protein of NK emiocytosis and cytokine easily to be inhaled by skin Receive, and the feature of target cell can be directly acted on, can be directly used for acne, seborrheic dermatitis, pruritus and prurigo nodularis Deng skin appendages and the treatment of the skin subsidiarity disease such as pruritic, curative effect is quick, can improve the siberian crabapple of skin simultaneously System, repairs impaired skin.Excreted factor in NK cell stock solution also can kill fungus and antibacterial, reach Comprehensive Treatment acne, Dermatitis and the purpose of pruritus, therapeutic domain is extensive, can prevent repeatedly increasing the weight of of the state of an illness.
Detailed description of the invention
Embodiment 1
1, material:
The conventional method that the present embodiment method therefor is the most all known to those skilled in the art dawn, used The material such as reagent, if no special instructions, be commercially available purchase product.
2, method
2.1 preparation NK cell stock solution
Step one: PBMC extracts:
Fresh blood is centrifuged 10 minutes in 740g, it is thus achieved that the faint yellow blood plasma in upper strata and lower floor's hemocyte, by thin for lower floor's blood Born of the same parents mix according to volume ratio 1:1 with normal saline, take 50ml centrifuge tube, and the blood cell suspension of dilution is layered on 15ml lymphocyte Separating on liquid, 400g is centrifuged 30 minutes.Collecting the membranaceous cellular layer of middle white, add brine, 400g is centrifuged 10 points Clock, abandons supernatant, and repeated washing is once.Finally precipitate with appropriate culture fluid re-suspended cell, i.e. obtain PBMC.
Step 2: the preparation of NK cell culture fluid:
200U/ml is added in Gibco RPMI Medium 1640basic culture fluid (buying in GIBCO company of the U.S.) The gentamycin of interleukin I L-2,80U/ml and the Gibco FBS of 10% percentage by weight (buy in U.S. GIBCO public Department), it is thus achieved that NK cell culture fluid.
Step 3: the preparation of NK cell initial inoculum:
Take 3.0 × 107-4.0×107Individual PBMC, in a T75 culture bottle, adds the NK cell culture fluid and 1 of 20-30ml Propping up NK amplifing reagent, wherein, described NK amplifing reagent (sees Chinese patent for a kind of CD8 α-interleukin-22 1-CD137 complex Document CN102154207A), can directly obtain by the way of market is bought, the product rule of the NK amplifing reagent that the present invention uses Lattice are that 2ml/ props up, and concentration is 1 × 107Individual/ml.
Step 4: NK cell is initial to be cultivated:
NK cell is inoculated in above-mentioned T75 culture bottle cultivation, wherein:
Within 1st, 2 days, note observation of cell state.If cell state well can not process.If culture fluid cell density is obvious Strengthen, NK cell culture fluid 10ml can be added.
3rd day by medium centrifugal, it is thus achieved that cell precipitation be transferred in new T75 culture bottle, and add the NK of 30ml Cell culture fluid continues to cultivate.
4th day observation of cell, if cell state is good, can not process, if cell density is excessive, can take the circumstances into consideration to add NK thin Born of the same parents' culture fluid.
5th day, if the 4th day untreated, and cell state was good, if cell density is big, then added NK cell culture fluid 20ml。
6th day observation of cell, adds culture fluid 30-50ml according to cell density.
Step 5: NK cell expansion is cultivated:
7th day counting, if cell quantity is more than 1.5 × 108Individual/bottle, then add 2 NK amplifing reagents, if cell quantity At 30-60 million, then length added NK amplifing reagent again to the 8th day.Then add NK cell culture fluid, make total cell concentration exist 1.0×106Individual/ml.Incrementally increasing with volume of culture, can proceed to T175 and cultivate the cultivation of square vase extended volume, culture fluid volume is big It is more than 2.0 × 10 in 120ml or cell quantity8Individual/bottle time, then can be transferred to culture bag cultivate.
8-11 days every days observation of cell so that cell concentration is 1.0 × 106Individual/about ml, is added by counting and cultivates Liquid so that cell concentration is 1.0 × 106Individual/ml.
12-14 days every days observed, when cell quantity is more than 3.0 × 109Individual/bag, and when volume is more than 1200ml, will Cell is divided into 2 bags.
Step 6: collection NK cell stock solution:
Can be collected when cell grows to requirement using or frozen.Generally, within 14 or 15 days, collect Cell, because now cell is in exponential phase, in the best state, the slip of cell state meeting the most backward, now cell can To 3 × 109More than individual, NK cell (CD3-CD56+) purity can reach more than 80%.
2.2 preparation SNK liquid:
By weight, NK cell stock solution 80%, glycerol 2.0%, ethylparaben 0.01%, Borax are taken 0.03%, Oleum menthae 0.5%, is substantially soluble in sterilized water, sterilizing subpackage after mixing, it is thus achieved that described SNK liquid.
2.3 preparation SNK cream:
By weight, take NK cell stock solution 70%, Carbopol 941 be 0.1%, glycerol 2.0%, Aloe powder 0.2%, to hydroxyl Yl benzoic acid ethyl ester 0.1%, hyaluronic acid 0.1%, Oleum menthae 0.5%, surplus is sterilized water, sterilizing subpackage after mixing, it is thus achieved that Described SNK cream.
3, clinical verification
Clinical experiment is divided into 3 groups carry out, particularly as follows:
Experimental group 1: select 8 examples to suffer from the patient of acne vulgaris, 6 examples suffer from the patient of seborrheic dermatitis, pruritus 5 example patient With prurigo nodularis patient 4 example, carry out smearing or hydrojet treatment with SNK liquid, use 2-3 time every day, add up clinical treatment outcome.
Experimental group 2: select 8 examples to suffer from the patient of acne vulgaris, 6 examples suffer from the patient of seborrheic dermatitis, pruritus 5 example patient With prurigo nodularis patient 4 example, carry out smearing treatment with SNK cream, use 2-3 time every day, add up clinical treatment outcome.
Experimental group 3: select 8 examples to suffer from the patient of acne vulgaris, 6 examples suffer from the patient of seborrheic dermatitis, pruritus 5 example patient With prurigo nodularis patient 4 example, smear with SNK liquid and cream or hydrojet treatment, use 2-3 time every day, add up clinical treatment outcome.
Result is as shown in table 1 below:
Table 1:SNK liquid and SNK cream clinical trial results statistical table
Result shows, the NK cell stock solution of the present invention, SNK liquid and SNK cream are to acne, seborrheic dermatitis, pruritus and knot Joint property prurigo all has certain therapeutic effect, especially notable to the therapeutic effect of acne vulgaris, can thoroughly cure acne, alleviate fat The symptom of property dermatitis of overflowing, and need not long-term prescription and take innerlich anwenden, greatly reduce toxic and side effects.Wherein: first group and Second component not Wei SNK liquid and SNK cream, SNK liquid is different due to the content of NK cell stock solution with in SNK cream, and curative effect somewhat has difference Not, SNK liquor ratio cream is effective.(first it is coated with SNK liquid, repaste SNK cream) when SNK liquid and SNK cream are used together, both can ensure NK liquid In active factors efficiently and be persistently less readily available for absorption by the skin, curative effect can be improved again, both are used in combination best results.
Embodiment 2
Present embodiments providing a kind of SNK liquid, by weight, described SNK liquid is by NK cell stock solution 70.0%, glycerol 5.0%, ethylparaben 0.03%, Borax 0.08%, Oleum menthae 1.5% and appropriate amounts of sterilized water composition, wherein, described The preparation method of NK cell stock solution is with embodiment 1.
Choosing suffers from patients with acne vulgaris 3 example, pruritus patient 2 example, is directly sprayed on affected part with above-mentioned SNK liquid and controls Treat, use 2-3 time every day, add up clinical treatment outcome.
It was found that above-mentioned SNK liquid is notable to the therapeutic effect of acne vulgaris, 7 days symptoms of severe patient substantially alleviate, even Continuous use can be fully recovered after 2 weeks;Have certain therapeutic effect to pruritus, patients with mild can be fully recovered in 2 weeks, and severe patient is after 2 weeks Symptom is clearly better.
Embodiment 3
Present embodiments providing a kind of SNK cream, by weight, described SNK cream is by NK cell stock solution 60.0%, carbomer 941 be 0.5%, glycerol 5.0%, Aloe powder 0.5%, ethylparaben 0.5%, hyaluronic acid 0.3%, Oleum menthae 1.5% and suitable quantity of water composition.
Choosing suffers from seborrheic dermatitis patient 2 example, utilizes above-mentioned SNK cream to carry out smearing treatment, uses 2-3 time every day, statistics Clinical treatment outcome.
It was found that after using 2 weeks, the symptom of 2 example patients with seborrheic dermatitis all takes an evident turn for the better, and this SNK cream pair is described Seborrheic dermatitis has certain therapeutic effect.
Embodiment 4
Present embodiments providing a kind of SNK liquid, by weight, described SNK liquid is by NK cell stock solution 20%, glycerol 5% He Appropriate amounts of sterilized water forms, and utilizes described SNK liquid to treat acne vulgaris, necessarily controls it was found that acne vulgaris is had by this SNK liquid Therapeutic effect, after patient uses 10 days continuously, symptom all has certain improvement.
Embodiment 5
Present embodiments providing a kind of SNK liquid, by weight, described SNK liquid is by NK cell stock solution 50%, glycerol 5% He Appropriate amounts of sterilized water forms, and utilizes described SNK liquid treatment prurigo nodularis patient after 30 days, and symptom disappears substantially.
Embodiment 6
Present embodiments providing a kind of SNK liquid, described SNK liquid is the NK cell stock solution of 100%, utilizes described SNK liquid to control Treat prurigo nodularis can fully recover after 25 days.
In theory, the content of the NK cell stock solution in medicine is the highest, and active factors content is the highest, and effect is the best, but considers In the case of ease of use and increase percutaneous permeability and protectiveness, need to the addition of on the basis of NK cell stock solution Auxiliary element, uses the NK cell stock solution of 100% to treat, its clinical trial results and the SNK of embodiment 1 in embodiment 6 Liquid and cream are compared, and there is no very big difference on therapeutic effect.
It is more than a kind of detailed embodiment of the present invention and concrete operating process, is to be front with technical solution of the present invention Put and implement, but protection scope of the present invention is not limited to the above embodiments.

Claims (8)

1. the external used medicine being used for treating skin appendages and itching skin disease, it is characterised in that described external used medicine Including the NK cell stock solution that mass percent is 20%-100%.
A kind of external used medicine for treating skin appendages and itching skin disease the most according to claim 1, it is special Levying and be, described medicine is a kind of liquid preparation, and its component and mass percent thereof include: NK cell stock solution 70.0-80.0%, Glycerol 2.0-5.0%, ethylparaben 0.01-0.03%, Borax 0.03-0.08%, Oleum menthae 0.5-1.5%, molten Agent is water.
A kind of external used medicine for treating skin appendages and itching skin disease the most according to claim 1, it is special Levying and be, described medicine is a kind of ointment preparation, and its component and mass percent thereof include: NK cell stock solution 60.0-70.0%, Carbopol 941 is 0.1-0.5%, glycerol 2.0-5.0%, Aloe powder 0.2-0.5%, ethylparaben 0.1-0.5%, Hyaluronic acid 0.1-0.3%, Oleum menthae 0.5-1.5%, surplus is water.
4. the preparation of the external used medicine for treating skin appendages and itching skin disease as described in claim 1-3 Method, it is characterised in that comprise the following steps:
(1) take NK cell and carry out amplification cultivation, it is thus achieved that NK cell stock solution;
(2) NK cell stock solution is mixed with external used medicine adjuvant, prepare described external used medicine.
The preparation of a kind of external used medicine for treating skin appendages and itching skin disease the most according to claim 4 Method, it is characterised in that if described medicine is liquid preparation, the mass percent of described NK cell stock solution is 70.0- 80.0%, described adjuvant and mass percent thereof include: glycerol 2.0-5.0%, ethylparaben 0.01-0.03%, Borax 0.03-0.08%, Oleum menthae 0.5-1.5%, solvent is water;If described medicine is ointment preparation, described NK cell is former The mass percent of liquid is 60.0-70.0%, and described adjuvant and mass percent thereof include: Carbopol 941 be 0.1-0.5%, Glycerol 2.0-5.0%, Aloe powder 0.2-0.5%, ethylparaben 0.1-0.5%, hyaluronic acid 0.1-0.3%, thin Lotus oil 0.5-1.5%, surplus is water.
The preparation of a kind of external used medicine for treating skin appendages and itching skin disease the most according to claim 4 Method, it is characterised in that the method that described NK cell amplification is cultivated, comprises the following steps:
Step one: take fresh blood, extracts peripheral blood lymphocytes PBMC in blood;
Step 2: add in RPMI-1640 basal medium interleukin I L-2,80U/ml of 200U/ml gentamycin and The hyclone of 10% percentage by weight, it is thus achieved that NK cell culture fluid;
Step 3: take 3.0 × 107-4.0×107Individual PBMC, in a Tissue Culture Flask, adds the NK cell culture fluid of 20-30ml It is 1.0 × 10 with the concentration of 2ml7The NK amplifing reagent of individual/ml, it is thus achieved that initial inoculum;
Step 4: NK cell is inoculated in initial inoculum cultivation 1-2 days, adds NK according to cell growth state during cultivation Cell culture fluid;When cultivate to the 3rd day time, by medium centrifugal, it is thus achieved that cell precipitation in add NK cell culture fluid continue Cultivate;
Step 5: when NK cell quantity reaches 1.5 × 108Time more than individual/bottle, add 2 NK amplifing reagents and NK cell culture fluid enters Row amplification culture, increases NK cell culture fluid according to cell growth state in incubation and carries out splitting cultivation, making cell dense Degree is 1.0 × 106Individual/ml, when cultivating to the 12nd day, controls liquid volume added, and making cell concentration is 2.0 × 106Individual/ml;
Step 6: continue to cultivate after the 14th day, collect cell culture fluid, it is thus achieved that NK cell stock solution.
7. the external used medicine as described in claim 1-3 is as the application in skin subsidiarity disease therapeuticing medicine.
A kind of external used medicine the most according to claim 7 as the application in skin subsidiarity disease therapeuticing medicine, its It is characterised by, NK cell stock solution is mixed with external used medicine adjuvant, prepare liquid preparation for external application and external ointment preparation, first With liquid preparation for external application, then using external ointment preparation, immixture is in affected part.
CN201610994956.7A 2016-11-11 2016-11-11 A kind of external used medicine for treating skin appendages and itching skin disease and its preparation method and application Pending CN106265737A (en)

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CN110200996A (en) * 2019-05-16 2019-09-06 安徽瑞达健康产业有限公司 A kind of application of cell composition on skin ulcer
CN111712227A (en) * 2017-12-14 2020-09-25 绿十字生命健康有限公司 Cosmetic and pharmaceutical compositions for relieving atopic dermatitis, alopecia and trauma or reducing skin wrinkles

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