CN108721602A - A kind of joint HA promotes the novel preparation for baldness of stem cell of hair regeneration - Google Patents
A kind of joint HA promotes the novel preparation for baldness of stem cell of hair regeneration Download PDFInfo
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Abstract
The present invention relates to the novel preparations for baldness of stem cell that a kind of joint HA promotes hair regeneration, it is characterised in that：Including KGF, PDGF, VEGF, hyaluronic acid solution, the content of described KGF, PDGF and VEGF are 320 μ g/ml, 120 μ g/ml and 180 μ g/m, a concentration of 0.5wt% of hyaluronic acid solution.The present invention can effectively improve the fuel-displaced situation of alopecia seborrheica mouse hair using the preparation for baldness that HA solution+conditioned medium is formed, the hormonal readiness in alopecia seborrheica and alopecia areata Mice Body can be effectively relieved, reach the level of normal mouse, and it can be obtained by the analysis of the pathological staining of alopecia area skin, alopecia seborrheica and alopecia areata mouse pass through the treatment of novel preparation for baldness, it is compared to the mouse of unused novel preparation for baldness, the grease of its perifollicolar accumulation is reduced, hair follicle quantity increases, hair follicle structure is complete, this provides indispensable nutriment for the continued propagation of hair.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to preparation for baldness, especially a kind of joint HA promote hair regeneration
The novel preparation for baldness of stem cell.
Background technology
The phenomenon that epilation, is named alopecia, and the alopecia of normal person is generally daily in 50-100 roots, alopecia amount and newborn hair
In dynamic equilibrium, hair amount is substantially not in apparent variation.If hair hair amount has apparent reduction, it is contemplated that be pathology
Property alopecia.With the increasingly quickening of people's rhythm of life, among everyone is all in nervous work climate, it is excessive with brain and
The fatigue of body can all cause alopecia.According to incompletely statistics, only just there is the puzzlement that about 1.6 hundred million people endure alopecia to the fullest extent in China.
Alopecia type common at present has：Alopecia seborrheica, physical alopecia, nervous baldness, postpartum alopecia, chemically
Alopecia, alopecia areata etc..Alopecia seborrheica, also known as male pattern alopecia, androgenetic alopecia.This Disease Clinical symptom shows as head more
Hair density gradually decreases, and scalp fat secretion is excessive, and hair and scalp greasy feeling are full, often increase with scabies pain, dandruff etc..Usually
It first falls ill since forehead, the crown and two temples, gradually spreads to entire overhead, alopecia area light is without hair, remaining hair
Withered soft jaundice.
Nervous baldness is the depilation phenomenon usually occurred when stress is excessive, under the action of stress, human body
Arrectores pilorum is shunk, hair is upright, and vegetative nerve or nervous centralis function get muddled, and hair follicle papilla changes with nutrition not
It is good, it is transplanted so as to cause hair growth function, hair enters stand-down and alopecia occurs.
Alopecia areata is a kind of non-scar plaque-like alopecia of autoimmune, shows as the hair of any hair overlay area of body
In spot flaking, most common position is exactly scalp.Patients with alopecia areata, hair follicle, papilla structure are gradually reduced, hair follicle structure
Location is gradually moving up, and in the infiltration of the visible lymphocyte of perifollicolar, therefore it is now recognized that alopecia areata generation
There may be the pathogenesis of autoimmunity.
In recent years, with the gradual intensification that people study umbilical cord mesenchymal stem cells (UC-MSCs), it is thus understood that UC-
MSCs can break up to various kinds of cell, tissue direction.And also demonstrate that UC-MSCs not only can be in the research of our early periods
The hormonal readiness for balancing body, can also effectively improve the immunity of body.This both depend on UC-MSCs secretion cell because
Son-keratinocyte growth factor (KGF), platelet derived growth factor (PDGF), endothelial growth factor (VEGF) etc.
Porcine HGF can participate in hormone and immunoregulation.
Hyaluronic acid (HA) is a kind of acid mucopolysaccharide.The HA of low molecular weight can improve skin-nourishing metabolism, be skin
Meat energy smooth, increase elasticity, prevents aging, while being also good skin penetration enhancer, coordinates with other nutritional ingredients,
The effect for promoting nutrient absorption can be played.
The cell factor that UC-MSCs secretes in the present invention is such as：KGF, PDGF, VEGF etc. can balance the hormonal readiness of body,
And the immunity of body is effectively improved, most importantly the cell factor of its secretion has chemotaxis, can significantly improve machine
Body various functions；The HA of low molecular weight can be effectively promoted absorption of the skin to other nutritional ingredients；Based on their advantage, have
Effect combines the two, for treating clinical common alopecia symptom.
Invention content
The purpose that the present invention carries provides a kind of doing for joint HA promotion hair regenerations in place of overcome the deficiencies in the prior art
The novel preparation for baldness of cell, by clinical volunteers by the use to novel preparation for baldness, the hair at alopecia position obtains the present invention
Regeneration is arrived, and the hair loss quantity of alopecia volunteer is reduced, the fuel-displaced situation of hair is alleviated, and volunteer breaks away from
The puzzlement of dandruff.
The present invention realizes that the technical solution of purpose is as follows：
A kind of joint HA promotes the novel preparation for baldness of stem cell of hair regeneration, including KGF, PDGF, VEGF, hyaluronic acid are molten
Liquid.
Moreover, the content of described KGF, PDGF and VEGF are 320 μ g/ml, 120 μ g/ml and 180 μ g/m.
Moreover, a concentration of 0.5wt% of hyaluronic acid solution.
The method that joint HA promotes the novel preparation for baldness of stem cell of hair regeneration is prepared, steps are as follows：
(1) the separation of umbilical cord mesenchymal stem cells：Sterile umbilical cord tissue is obtained, umbilical cord tissue is removed under aseptic condition
Artery and vein in outer membrane and umbilical cord tissue is isolated umbilical cord China Tong Shi glue and is shredded to 2-3mm3, by the magnificent Tong Shi stickers shredded in
Culture bottle bottom surface, appropriate culture solution is added after 1-4h into culture bottle, and culture solution uses DMEM/HAM ' SF-12+10%FBS；
(2) the digestion, passage of umbilical cord mesenchymal stem cells, when umbilical cord mesenchymal stem cells are grown to 80%, using 0.05%
The EDTA of trypsase+0.02% it is digested, reached with 10000cells/cm2 in new culture bottle, continue to train
It supports；
(3) UC-MSCs was cultivated to P3 generations, is cleaned UC-MSCs with phosphate buffer when cell is grown to 70%, will be cultivated UC-
The culture solution of MSCs is changed to DMEM/HAM ' the SF-12 culture mediums of serum-free, continues to cultivate 72h, after collecting culture UC-MSCs
DMEM/HAM ' SF-12 culture solutions, 1000g centrifuge 10min, remove dead cell, one of the raw material as novel preparation for baldness；
(4) protein quantification detection is carried out to the conditioned medium being collected into, determine KGF, PDGF, VEGF factor in culture medium
Content, a concentration of 156.53 ± 56 μ g/ml of KGF, a concentration of 76.85 ± 18 μ g/ml of PDGF, a concentration of 82.73 ± 27 μ of VEGF
g/ml；
(5) it is concentrated using the ultracentrifugal mode of low temperature, retention 3K molecular weight proteins are selected when ultracentrifugation
Strainer, the content that the content of KGF is 779.43 ± 23 μ g/ml after concentration, the content of PDGF is 289.55 ± 13 μ g/ml, VEGF
For 421.51 ± 11 μ g/ml；
(6) HA solution is prepared：It uses DMEM/HAM ' SF-12 culture solutions to dissolve molecular weight for the HA of 3K, is finally configured to dense
The HA solution that degree is 5%；It is spare as one of novel preparation for baldness raw material；
(7) the HA dissolved dilution conditioned mediums for using a concentration of 0.5%, finally prepare novel preparation for baldness, wherein novel
The content of KGF, PDGF and VEGF in preparation for baldness are 320 μ g/ml, 120 μ g/ml and 180 μ g/ml.
The present invention compared with prior art the advantages of and good effect it is as follows：
1, the present invention can effectively improve alopecia seborrheica mouse hair using the preparation for baldness that HA solution+conditioned medium is formed
Oily situation is sent out, the hormonal readiness in alopecia seborrheica Mice Body can be effectively relieved, reaches the level of normal mouse, and
It can show that alopecia seborrheica mouse passes through the treatment of novel preparation for baldness, phase by the pathological staining analysis of alopecia area skin
It is compared to that the mouse of novel preparation for baldness is not used, the grease of perifollicolar accumulation is reduced, hair follicle quantity increases, hair follicle structure is complete
Whole, this provides indispensable nutriment for the continued propagation of hair.
2, alopecia areata mouse passes through the treatment of novel preparation for baldness in the present invention, and alopecia position grown new hair, and
And the hair of the hair texture and normal mouse newly grown is without any difference；And it is small without the alopecia areata that novel preparation for baldness is treated
Mouse, alopecia position do not regenerate new hair；Alopecia areata Mice Body to alopecia areata mouse and after the treatment of novel preparation for baldness
Internal hormone level is detected, and finds the alopecia areata mouse after the treatment of novel preparation for baldness, internal hormonal readiness with it is normal
Mouse is completely the same；Pathological staining analyzes the skin histology at alopecia areata mouse alopecia position, and discovery is compared to without novel life
The alopecia areata mouse for sending out agent treatment, the hair follicle quantity in alopecia areata mouse its alopecia area skin tissue by the treatment of novel preparation for baldness
Increase, and hair follicle structure is completed, have hair regeneration and is continuously the condition that hair provides nutrition.
3, by clinical volunteers by the use to novel preparation for baldness, the hair at alopecia position has obtained again the present invention
It is raw, and the hair loss quantity of alopecia volunteer is reduced, the fuel-displaced situation of hair is alleviated, and volunteer has broken away from dandruff
Puzzlement.
Description of the drawings
Fig. 1 is that the novel preparation for baldness of the present invention promotes alopecia seborrheica animal model hair regeneration photo.
Fig. 2 is that (different group C57 are small for the novel preparation for baldness recovery alopecia seborrheica animal model hormone in vivo level of the present invention
Mouse hormone in vivo level).
Fig. 3 is that the novel preparation for baldness of the present invention remolds alopecia seborrheica animal hair follicle structure.
Fig. 4 is that the novel preparation for baldness of the present invention promotes alopecia areata animal model hair regeneration photo.
Fig. 5 is that the novel preparation for baldness of the present invention restores horizontal (the different group C3H/HEJ mouse of alopecia areata animal model hormone in vivo
Hormone in vivo level).
Fig. 6 is that the novel preparation for baldness of the present invention remolds alopecia areata animal hair follicle structure.
Fig. 7 is the novel preparation for baldness clinical trial result of the present invention.
Specific implementation mode
The application is described in further detail below by specific embodiment, following embodiment is descriptive, is not limit
Qualitatively, the protection domain of the application cannot be limited successively.
The present invention will collect the cell factor of UC-MSCs secretions, and the UC- of collection is concentrated by the way of low-temperature and high-speed centrifugation
MSCs secretion cell factor, quantitative analysis concentration cell factor in KGF, PDGF, VEGF content；It is 3K with molecular weight,
A concentration of 0.5% HA solution dilutes the cell factor of concentrated UC-MSCs secretions, finally prepares novel preparation for baldness and is used for
Treat alopecia seborrheica and alopecia areata.
Seborrheica and alopecia areata animal is treated using the novel stem cells preparation for baldness that the present invention prepares.Seborrheica and alopecia areata is dynamic
The treatment of stem cell preparation for baldness of the alopecia position of object by the present invention, alopecia position have grown new hair, and newborn
Hair structure and normal hair without any difference.
The stem cell preparation for baldness of the present invention is applied to clinic, treats alopecia seborrheica and alopecia areata, is achieved significant
Effect.Compared to more traditional hair stimulator, it is without any side effects and new to prepare stem cell preparation for baldness using the present invention
Raw hair will not fall off again after stopping using stem cell preparation for baldness.
A kind of joint HA promotes the novel preparation for baldness of stem cell of hair regeneration, preparation method as follows：
(1) the separation of umbilical cord mesenchymal stem cells：Sterile umbilical cord tissue is obtained, umbilical cord tissue is removed under aseptic condition
Artery and vein in outer membrane and umbilical cord tissue is isolated umbilical cord China Tong Shi glue and is shredded to 2-3mm3, by the magnificent Tong Shi stickers shredded in
Culture bottle bottom surface, appropriate culture solution is added after 1-4h into culture bottle, and culture solution uses DMEM/HAM ' SF-12+10%FBS；
(2) the digestion, passage of umbilical cord mesenchymal stem cells, when umbilical cord mesenchymal stem cells are grown to 80%, using 0.05%
The EDTA of trypsase+0.02% it is digested, with 10000cells/cm2It reaches in new culture bottle, continues to train
It supports；
(3) UC-MSCs was cultivated to P3 generations, is cleaned UC-MSCs with phosphate buffer when cell is grown to 70%, will be cultivated UC-
The culture solution of MSCs is changed to DMEM/HAM ' the SF-12 culture mediums of serum-free, continues to cultivate 72h, after collecting culture UC-MSCs
DMEM/HAM ' SF-12 culture solutions, 1000g centrifuge 10min, remove dead cell, one of raw material as novel preparation for baldness (item
Part culture medium)；
(4) BCA protein quantifications are carried out to the conditioned medium being collected into, determine KGF, PDGF, VEGF factor in culture medium
Content, a concentration of 156.53 ± 56 μ g/ml of KGF, a concentration of 76.85 ± 18 μ g/ml of PDGF, a concentration of 82.73 ± 27 μ g/ of VEGF
ml；
(5) use the mode of low temperature ultracentrifugation (low temperature Ultracentrifugation conditions is 4 DEG C, 4000g, 60min) to carry out it dense
The strainer of retention 3K molecular weight proteins is selected in contracting when ultracentrifugation, and the content of KGF is 779.43 ± 23 μ g/ml, PDGF after concentration
Content be 289.55 ± 13 μ g/ml, the content of VEGF is 421.51 ± 11 μ g/ml；
(6) HA solution is prepared：It uses DMEM/HAM ' SF-12 culture solutions to dissolve molecular weight for the HA of 3K, is finally configured to dense
The HA solution that degree is 5%；It is spare as one of novel preparation for baldness raw material；
(7) the HA dissolved dilution conditioned mediums for using a concentration of 0.5%, finally prepare novel preparation for baldness, wherein novel
The content of KGF, PDGF and VEGF in preparation for baldness are 320 μ g/ml, 120 μ g/ml and 180 μ g/ml；
(8) alopecia seborrheica animal model is established using testosterone propionate, the testosterone propionate of the 5mg/ml of 100 μ l is injected into
C57 mouse backs are subcutaneous, once two days, continuous injection 30 days, with the extension C57 mouse backs for injecting the testosterone propionate time
Hair starts to become sparse, and skin of back has a large amount of grease to be precipitated, and stops injection testosterone propionate after 30 days, test mice
Back hair continues to become sparse, and fuel-displaced situation still remains, and it is seborrheica de- to illustrate that we have been successfully established using testosterone propionate
Send out animal model；
(9) alopecia areata animal model is established using imiquimod, 50mg imiquimods are applied to C3H/HEJ mouse back hairs
The root of hair is persistently smeared 15 days once a day, with the extension for smearing the imiquimod time, C3H/HEJ mouse back hairs
It starts shedding off, and the skin of back of C3H./HEJ mouse is gradually fully exposed, forms the circle of an a diameter of 1cm
Alopecia areata stopped smearing imiquimod after 30 days, and still no hair is grown at the position of test mice back smearing imiquimod, is said
It is bright that we have been successfully established alopecia areata animal model using imiquimod；
(10) DMEM/HAM ' SF-12 culture mediums and novel preparation for baldness are applied to alopecia seborrheica animal model back respectively,
Primary, a 100 μ l are smeared daily, persistently smearing 30 days, observe the growing state of alopecia seborrheica back part of animal hair daily；
(11) DMEM/HAM ' SF-12 culture mediums and novel preparation for baldness are applied to alopecia areata animal model back respectively, applied daily
Primary, a 100 μ l are smeared, persistently smearing 30 days, observes the growing state of alopecia areata back part of animal hair daily；
(12) respectively in 0d, 15d and 30d the acquisition animal subject orbital venous plexus peripheral blood and the back of the body for smearing novel preparation for baldness
Portion's skin histology carries out the pathology detection and analysis of animal subject hormone in vivo level and hair follicle structure, further verifies novel
Effect of the preparation for baldness for hair regeneration；
(13) sterile novel preparation for baldness is applied to seborrheica and alopecia areata volunteer's alopecia position, taken at regular intervals alopecia position
Image further verify novel preparation for baldness to compare volunteer in the growing state using hair after novel preparation for baldness and facing
Effect on bed.
Claims (4)
1. a kind of joint HA promotes the novel preparation for baldness of stem cell of hair regeneration, it is characterised in that：Including KGF, PDGF, VEGF,
Hyaluronic acid solution.
2. joint HA according to claim 1 promotes the novel preparation for baldness of stem cell of hair regeneration, it is characterised in that：It is described
The content of KGF, PDGF and VEGF are 320 μ g/ml, 120 μ g/ml and 180 μ g/m.
3. joint HA according to claim 1 promotes the novel preparation for baldness of stem cell of hair regeneration, it is characterised in that：Quality
Score is a concentration of 0.5wt% of the hyaluronic acid solution of 3K.
4. the method that the joint HA described in one of claim 1-3 promotes the novel preparation for baldness of stem cell of hair regeneration is prepared,
It is characterized in that：Steps are as follows：
(1) the separation of umbilical cord mesenchymal stem cells：Sterile umbilical cord tissue is obtained, the outer membrane of umbilical cord tissue is removed under aseptic condition
It with artery and vein in umbilical cord tissue, isolates umbilical cord China Tong Shi glue and shreds to 2-3mm3, by the magnificent Tong Shi stickers shredded in culture
Bottom of bottle face, appropriate culture solution is added after 1-4h into culture bottle, and culture solution uses DMEM/HAM ' SF-12+10%FBS；
(2) the digestion, passage of umbilical cord mesenchymal stem cells, when umbilical cord mesenchymal stem cells are grown to 80%, using 0.05% pancreas
The EDTA of protease+0.02% digests it, is reached with 10000cells/cm2 in new culture bottle, continues to cultivate；
(3) UC-MSCs was cultivated to P3 generations, is cleaned UC-MSCs with phosphate buffer when cell is grown to 70%, will be cultivated UC-MSCs
Culture solution be changed to DMEM/HAM ' the SF-12 culture mediums of serum-free, continue to cultivate 72h, after collecting culture UC-MSCs
DMEM/HAM ' SF-12 culture solutions, 1000g centrifuge 10min, remove dead cell, one of the raw material as novel preparation for baldness；
(4) protein quantification detection is carried out to the conditioned medium that is collected into, determine containing for KGF, PDGF, VEGF factor in culture medium
Amount, a concentration of 156.53 ± 56 μ g/ml of KGF, a concentration of 76.85 ± 18 μ g/ml of PDGF, a concentration of 82.73 ± 27 μ g/ of VEGF
ml；
(5) it is concentrated using the ultracentrifugal mode of low temperature, the filter of retention 3K molecular weight proteins is selected when ultracentrifugation
Net, the content of KGF is 779.43 ± 23 μ g/ml after concentration, the content of PDGF is 289.55 ± 13 μ g/ml, the content of VEGF is
421.51±11μg/ml；
(6) HA solution is prepared：It uses DMEM/HAM ' SF-12 culture solutions to dissolve molecular weight for the HA of 3K, is finally configured to a concentration of
5% HA solution；It is spare as one of novel preparation for baldness raw material；
(7) the HA dissolved dilution conditioned mediums for using a concentration of 0.5%, finally prepare novel preparation for baldness, wherein novel hair tonic
The content of KGF, PDGF and VEGF in agent are 320 μ g/ml, 120 μ g/ml and 180 μ g/ml.
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| CN109464374A (en) * | 2018-11-23 | 2019-03-15 | 北京安溢生物科技有限公司 | Umbilical cord mesenchymal stem cells factor coniplexes are promoting the application in hair restoration |
| CN110227057A (en) * | 2019-06-10 | 2019-09-13 | 北京恒峰铭成生物科技有限公司 | A kind of hair gel cream of hair regeneration and preparation method thereof |
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| CN110585078A (en) * | 2019-08-23 | 2019-12-20 | 镇江大美瑞珍麦迪森生物技术有限公司 | Hair nourishing and developing liquid and preparation method thereof |
| CN110585478A (en) * | 2019-08-23 | 2019-12-20 | 镇江大美瑞珍麦迪森生物技术有限公司 | Hair-planting restoration liquid and preparation method thereof |
| CN110917334A (en) * | 2019-11-27 | 2020-03-27 | 浙江卫未生物医药科技有限公司 | Hair regenerating agent based on hair follicle cell dryness maintenance |
| CN115120550A (en) * | 2021-03-26 | 2022-09-30 | 株式会社现代百朗德 | Hair cosmetic composition comprising a culture of streptococci providing beneficial effects on the hair |
| CN115397458A (en) * | 2020-02-25 | 2022-11-25 | 安普力菲卡公司 | Compositions and methods for stimulating hair growth |
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| CN110585078A (en) * | 2019-08-23 | 2019-12-20 | 镇江大美瑞珍麦迪森生物技术有限公司 | Hair nourishing and developing liquid and preparation method thereof |
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| CN110917334A (en) * | 2019-11-27 | 2020-03-27 | 浙江卫未生物医药科技有限公司 | Hair regenerating agent based on hair follicle cell dryness maintenance |
| CN115397458A (en) * | 2020-02-25 | 2022-11-25 | 安普力菲卡公司 | Compositions and methods for stimulating hair growth |
| CN115120550A (en) * | 2021-03-26 | 2022-09-30 | 株式会社现代百朗德 | Hair cosmetic composition comprising a culture of streptococci providing beneficial effects on the hair |
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