CN101642563A - Pilatory containing Wnt protein - Google Patents
Pilatory containing Wnt protein Download PDFInfo
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- CN101642563A CN101642563A CN200910306586A CN200910306586A CN101642563A CN 101642563 A CN101642563 A CN 101642563A CN 200910306586 A CN200910306586 A CN 200910306586A CN 200910306586 A CN200910306586 A CN 200910306586A CN 101642563 A CN101642563 A CN 101642563A
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Abstract
The invention provides a pilatory containing Wnt protein. The pilatory contains Wnt protein which enhances the growth of hair follicle by a Wnt/Beta-catenin signal channel. The pilatory leads the hairfollicle to have a growing period phenotype, and the hair follicle is transformed to the growing period from a stationary period. The pilatory has the advantages of obvious effect, fast effect, clearmechanism and the like and has important social and economical significance to develop drugs for enhancing the growth of hair.
Description
Technical field
The present invention relates to a kind of hair growth promoter, relate in particular to a kind of hair growth promoter, relate in particular to the hair growth promoter of the proteic promotion hair follicle growth of a kind of Wnt3a of containing by the proteic promotion hair follicle growth of Wnt that Wnt/ β-the catenin signal path plays a role.
Background technology
Hair and send out peculiarly by mammal is the filament of growing in the keratinization on whole skin surface, is the deutero-appendages of epidermis.The hair of animal skin skin is extremely many, plays an important role to keeping body temperature and the infringement of antagonism mechanicalness.Human hair is quite degenerated, but hair follicle has abundant teleneuron, is sensitive touch sensor.Simultaneously, human hair affects people's appearance, has great social significance.
Stretch out extracutaneous hair and claim hair shaft, be embedded in the title Rhizoma Imperatae in the skin, Rhizoma Imperatae wraps in the sheath shape hair follicle of being made up of epithelium and connective tissue.The lower end of Rhizoma Imperatae and hair follicle combines, and forms the ball top that expands.The ground indent of ball top is held hair papilla in it, also be dermal papilla.
Hair follicle is to have a periodically special inferior organ of growth characteristics, is made up of the three-decker (hair shaft, inner root sheath, external root sheath) that is the concentric circular arrangement, comprises ten various kinds of cell monoid (see figure 1)s.Hair follicle is arranged in corium and subcutaneous tissue, and short hair follicle is arranged in corium.On longitudinal section, the hair follicle of trophophase can be divided into three sections: 1. infundibulum, and its upper end be the opening of hair follicle, the sebaceous gland opening is arrived in the lower end; 2. isthmus, this section is shorter, and the upper end is to the sebaceous gland opening, and lower end to arrectores pilorum adheres to the place; 3. hypomere is adhered to by arrectores pilorum and to locate the hair follicle bottom, comprises inner root sheath, external root sheath and ball top.Whole hair follicle is held by connective tissue sheath.Connective tissue sheath also claims fibrous sheath, wherein is rich in III Collagen Type VI and elastic fiber, and abundant sensory nerve ending and blood vessel are arranged.
Hair follicle stem cells is positioned at the knuckle district of hair follicle epimere and arrectores pilorum junction, shows as germinal cell on morphology and biochemistry, and the cyto-dynamics feature is that labelling keeps cell, and some clear and definite molecular marked compounds are arranged.Hair follicle stem cells has the three-dimensional differentiation potential: the descending cell that is divided into the various monoids of hair follicle, upwards migration and differentiation is sebaceous gland cell and the epidermis cell that is divided into skin under specific circumstances.At present, the knuckle district is that the theory of hair follicle stem cells habitat is generally admitted.
Mammiferous hair follicle is periodically growth, and (see figure 2) moved in circles between the mammiferous whole vital stage.The hair follicle cycle is divided into three phases: the hair follicle of trophophase and the knit growth that jumps; The hair of catagen stops growing, and hair follicle and ball top are degenerated; The hair follicle of resting stage and ball top atrophy, hair easily comes off.
In the growth end of term, a series of degenerative take place ball top changes.When catagen began, the hair matrix cell stopped propagation, and simultaneously, the external root sheath epithelium is shunk back into the last leather hose of keratinization, wraps the spherical lower end of hair shaft, formed club hair.
In resting stage, the epithelial cell post atrophy of club hair below also is moved upward, and free hair papilla is followed in epithelium post lower end, and has connective tissue to trail the epithelium post back of shrinking back.At last, move on to arrectores pilorum on the club hair and adhere to the hair follicle place.In resting stage, some factor such as hormone, medicine etc. activate hair follicle stem cells, and then hair follicle enters next trophophase.
When trophophase began, hair papilla was rebuild around its undifferentiated cell with a part.Hair bud generates new hair matrix, and increase and elongated, and the connective tissue sheath that the edge is left over to moving down, is differentiated to form inner root sheath and hair shaft with hair papilla simultaneously.Newborn hair shaft is growth upwards, and original club hair is released.
The hair of human body comes off not obvious, also irregular, because adjacent hair is in the different times of growth cycle, is unfavorable for research.And the hair follicle undulate of mice growth, promptly the hair follicle of same cross section is in same one-period, and the cycle of entire back differed the model of providing convenience for the research of hair follicle end to end only about 2 days.
The Wnt gene is a kind of proto-oncogene that clones in the inductive mouse breast cancer of muroid breast cancer virus (MMTV), can be found and report in 1982 by people such as Nusse, is called the int gene at that time.The aptery gene wingless (wg) of the fruit bat of discovering int gene and Sharma report afterwards is a homologous genes, thereby both are collectively referred to as Wnt.Over past ten years to fruit bat, amphibian, mammals to human Wnt family gene, coded product and Biological Effects thereof show, the Wnt signal pathway has participated in a series of control cell growth promoter from embryo to the adult and the regulation and control of differentiation, has particularly more and more given prominence to its potential value in stem cell and oncobiology research.
The Wnt protein family is the glycoprotein of the secreted of a series of high conservatives.In the animal body early development, a series of critical events such as the formation of Wnt signal deciding dorsoventral axis, germinal layer foundation, body segment differentiation, tissue or organ formation, and directly controlling propagation, differentiation, polarization, apoptosis and the isocellular destiny of anti-apoptosis.Simultaneously, the Wnt signal pathway also takes place closely related with tumor.It all is by its classical signals approach that many Wnt protein moleculars play a role, and the classical Wnt signal pathway is also referred to as Wnt/ β-catenin signal pathway.The molecular mechanism of Wnt/ β-catenin signal transduction has high conservative in different plant species.
Wnt/ β-catenin signal path has all played important effect to the g and D of hair follicle.Continued to express the inhibitor DKK1 of Wnt path during fetal development, hair follicle can not generate fully, and the birth back is crossed and expressed the growth that DKK1 suppresses hair follicle.On the contrary, the β-catenin of continuous expression stable form, the quantity that the mice hair follicle generates increases.
Wnt3a is one of wnt family member, and Wnt3a participates in the early stage formation of organs such as lung, tooth, hair follicle, regulates the self renewal of hematopoietic stem cell.In vitro culture finds that wnt3a can promote the propagation of epidermal stem cells and mescenchymal stem cell, suppresses its differentiation.Dermal papilla cell in the hair follicle can be induced the differentiation of epidermal stem cells, and In vitro culture discovers that Wnt3a can keep this inducing action of dermal papilla cell.
Along with social competition's aggravation, various pressure increase, and high fat diet increases, and the modern life causes sleep to reduce, and all can make the sickness rate of alopecia be rising and the more and more trend of rejuvenation gradually.In recent years, in China's policlinic (particularly big city hospital) the outpatient service amount of hair loss patient in continuous rising.
Early stage hair growth promoter, from prescription, folk prescription from folk medicine is main, reach the promotion hair regrowth with microcirculation improvement, developed into afterwards based on clinical research methods, physiological and pathological reason according to alopecia is developed product targetedly, as use Pimobendane promotion microcirculation, regulate hormone and antiinflammatory bactericidal agent etc.
At present, various hair growth promoters are various in style on the China market, based on herbal species, mostly are medicine for external use greatly.Western medicine and oral medicine are less.The curative effect of most of product is as one wishes not to the utmost on the market, and the patient uses the back doing well,improving not obvious.Relevant investigation also shows, has hair loss patient about 98% to think that traditional treatment alopecia medicine can not bring satisfactory effect on the market, and Most patients can be selected to abandon when invalid in hair growth promoter on probation half a year.Nonetheless, because people's living standard improves constantly, more and more pay attention to the problem of physical beauty, the people who therefore buys hair growth promoter is on the increase.Therefore, how can treat alopecia effectively, be the problem that extensive patients more is concerned about.
Summary of the invention
The purpose of this invention is to provide the proteic hair growth promoter of a kind of Wnt of containing.Advantages such as it makes hair follicle present the trophophase phenotype, and hair follicle transformed to trophophase from resting stage, has rapid-actionly, and effect is obvious, and mechanism is clear have important society and economic implications to developing short hair growth medicine.
Technical scheme of the present invention is:
Described hair growth promoter comprises the Wnt albumen that promotes hair follicle growth by Wnt/ β-catenin signal path.
Described Wnt albumen by Wnt/ β-catenin signal path promotion hair follicle growth is Wnt3a albumen.
Promote the application of Wnt albumen in preparation treatment alopecia medicine of hair follicle growth by Wnt/ β-catenin signal path.
The periodicity growth of hair follicle is subjected to the regulation and control of complex mechanism.The Wnt signal path all plays a significant role in hair follicle development and regeneration, and the wnt albumen that can screen obviously short hair effect is the basis, and exploitation promotes the hair growth promoter of hair follicle growth.As the accessory organ of skin, hair has important physical and sociology function.Hair lacks to the patient and brings very big psychological burden, and the short hair growth medicine of exploitation has important society and economic implications.
Wnt3a albumen makes hair follicle enter trophophase rapidly from resting stage by directly activating wnt classical signals path, thereby promotes hair follicle growth.Kai Fa hair growth promoter based on this, advantage such as it is obvious to have effect, rapid-action, and mechanism is clear is for clinical hair loss patient brings glad tidings.
Preferred embodiment cited below particularly, and conjunction with figs. are described in detail below.
Description of drawings
Fig. 1 is trophophase hair follicle structure figure;
Fig. 2 is the hair follicle periodogram;
Fig. 3 infects the situation of 293 cells for AdWnt3a;
Fig. 4 is that 14 days integral body is taken a picture behind the injected in mice AdWnt3a;
Fig. 5 A and 5B are for injecting AdGFP group and injection AdWnt3a group respectively through the painted hair follicle growth diagram of HE;
Fig. 6 A and Fig. 6 B are that AdWnt3a handled back hair follicle organ culture 1 day and 4 days contrast diagrams; Adding AdWnt3a group hair follicle growth obviously accelerates.
Fig. 7 is that the integral body of 14d behind the injected in mice AdWnt10b is taken a picture;
Fig. 8 A and Fig. 8 B are respectively injection AdWnt10b and inject the diagram of AdGFP group through the painted hair follicle growth of HE.
The specific embodiment
The reagent that present embodiment adopted is the commercially available prod that is used for cell experiment.
The autogamy of WilliamE serum-free medium, prescription is seen embodiment 2.
The C57BL/6 mice is available from Third Military Medical University's Experimental Animal Center.
The structure of embodiment 1AdWnt3a and amplification
The AdEasy system in what logical river of employing etc., the titre that obtains AdWnt3a is 10
9Pfu/ml.With reference to gene order NM_011718.1.(concrete grammar is seen document: 1.He TC, Zhou S, da Costa LT, et al.ASimplified system for generating recombinant adenoviruses.Proc Natl Acad Sci US A, 1998,95:2509-14 2.Luo J, Deng ZL, Luo X, et al.A protocol for rapidgeneration of recombinant adenoviruses using the AdEasy system.Nat Protoc, 2007,2:1236-47.)
Embodiment 2Wnt3a promotes the hair growth test
(1) zoopery
1) female C57BL/6 mice in 8 ages in week is got in injection, and this moment, the mice dorsal body setae was in resting stage, was grouped as follows: 36 of recombinant adenovirus AdWnt3a injection groups, 36 of AdGFP matched groups (empty carrier), 36 of PBS matched groups.
Lumbar injection 100 μ l 1% pentobarbital.After about 5 minutes, mice is in narcotism, with shaver carefully shave except that mouse back by hair, note not shaving and hinder skin and stir hair follicle.
After shaving hair, in the middle of dorsal line, get some intradermal injection 25 μ l AdWnt3a, virus titer 10
8Pfu/ml notes not being expelled to subcutaneous.After the injection, the injection site produces the skin mound of an about 5mm of diameter, with 3% picric acid along edge labelling injection site, skin mound.
2) observe the injection back and observe and photograph every day, notice that the injection site skin color changes and the hair follicle growth situation.
3) draw materials injection back 3 days, 5 days, 7 days, 9 days, 14 days, dead 6 mices in 28 natural gift other places are got injection site skin, after 4% paraformaldehyde fixedly spends the night, and the routine paraffin wax embedding, HE dyeing observation is carried out in 5 μ m thickness sections.
The HE staining procedure:
1. slice gradient hydration: 3 minutes → distillation of dimethylbenzene I15 minute → dimethylbenzene II 15 minutes → 100% ethanol I3 minute → 95% ethanol II3 minute → 85% ethanol 3 minutes → 75% ethanol 3 minutes → 50% ethanol washing 3 minutes.
2. haematoxylin dyeing is 2 minutes, and the tap water flushing is turned blue to cutting into slices.
3. color separation: 1% acidic alcohol color separation 3-5 second (carry insert several down), and section reddens, and color is more shallow to get final product.
4. return indigo plant: tap water immersion 15 minutes or warm water (about 50 ℃) 5 minutes.(microscopically is seen the nucleus blueness, and Cytoplasm and connective tissue are colourless is advisable)
5. redye: Yihong liquid 2 minutes.
6. gradient dehydration: ethanol 3 minutes → 100% ethanol I3 minute → 100% ethanol II of 50% ethanol, 3 minutes → 75% ethanol, 3 minutes → 85% ethanol 3 minutes → 95% blotted unnecessary alcohol with absorbent paper in 3 minutes, dimethylbenzene I3 minute → dimethylbenzene II 3 minutes.
7. mounting: slide is taken out from dimethylbenzene, put dimethylbenzene around the tissue on the skin, drip resinene rapidly in tissue with absorbent paper, get the coverslip of cleaning, on alcohol burner, dry by the fire and dry, cover gently on natural gum, should avoid producing bubble with the one side of not baking.
8. observe: microscopically is observed, and takes a picture.
(2) organ culture
1) get one of newborn 7 days healthy C57BL/6 mice, this moment, hair follicle was in trophophase, was easy to separate.The cervical vertebra dislocation is put to death, cotton ball soaked in alcohol sterilization antenna position.The vertical skin of eye scissors is cut mice both sides antenna skin, puts into the culture dish that D-Hank ' s liquid is housed.Clip skin with tweezers and do cleaning slightly, inhale and remove unnecessary D-Hank ' s liquid;
2) (* 3.2) branch away hair follicle with No. 27 syringe needles under the anatomic microscope together with connective tissue sheath, and D-Hank ' s liquid is cleaned, and move in 24 orifice plates, and 5 hair follicles in every hole add 1ml WilliamE serum-free medium;
3) add AdWnt3a, AdGFP and equivalent PBS respectively, make viral final concentration reach 10
7The pfu/ml titre is put into incubator and is cultivated.
4) changed liquid in per 2 days, porosely all add identical culture medium.
5) observation is examined under a microscope every day, measures hair follicle length and photograph with micrometer.
WilliamE serum-free culture based formulas:
WilliamE：100ml
Hydrocortisone: 10ng/ml
Insulin: 10ug/ml
Glutamine: 2mmol/L
Penicillin/streptomycin: 100U/ml
Embodiment 3 interpretations of result
1.AdWnt3a acquisition
Through repeatedly amplification, the titre that obtains AdWnt3a is 10
9Pfu/ml.Fig. 3 infects 2 days situation of 293 cells for AdWnt3a.As seen, infected back 2 days, 293 cell infection rates reach 100%.
2.Adwnt3a promotion hair growth
(1) zoopery.9d behind injection AdWnt3a, injection site skin begins blackening (18 merely hit 15 appearance); 11 days, there is hair shaft to grow epidermis (12 merely hit 10 appearance); 14 days, the hair shaft total length went out (12 merely hit 11 appearance).And during whole, injection AdGFP and injection PBS group, the injection site still is in resting stage, with the no significant change of injection periphery.See Fig. 4, wherein, 2 on the left side is respectively PBS matched group and empty carrier matched group, and 3 on the right is an AdWnt3a injection group.As seen, injection AdWnt3a position grows new quilt hair (trophophase), and other positions still are in resting stage.
The HE observation of dyeing, AdWnt3a injected back 3 days, and the injection site hair follicle begins to occur the early stage structure of trophophase (6 merely hit 4 appearance); Injected back 5 days, injection rear hair follicle is trophophase phenotype (6 merely hit 6 occurs); Inject back 7 days, 9 days, 14 days, 28 days hair follicles and all be the trophophase phenotype.During whole, AdWnt3a injection group (Fig. 5 B) hair follicle is in the typical growth phase, and AdGFP group (Fig. 5 A) hair follicle still is in resting stage.
(2) organ culture
Hair follicle is cultivated after separation, continued growth (comprising inner root sheath, external root sheath and hair shaft).After adding AdWnt3a, hair follicle growth is obviously accelerated.See Fig. 6 A and Fig. 6 B, experimental group is for adding the AdWnt3a group, and matched group is for adding the AdGFP group.After cultivating hair follicle adding AdWnt3a, hair follicle growth is obviously accelerated.Confirm all that in body zoopery and external organ culture experiment after the AdWnt3a effect, hair follicle presents the trophophase phenotype, hair follicle was transformed to trophophase from resting stage.
Obtain AdWnt10b with above-mentioned same procedure, and adopt above-mentioned same method to handle.The result shows that AdWnt10b has the effect that promotes hair follicle growth equally.Fig. 7 handles back 14d for AdWnt10b, 2 mices in the left side are AdWnt10b processed group (2 points are chosen along dorsal line in the injection site), 1 on the right is a matched group, as seen, AdWnt10b processed group position is in the typical growth phase, and the position of being untreated still is in resting stage, and promptly AdWnt10b has promoted the growth of hair follicle.Fig. 8 A is a HE coloration result behind the AdWnt10b processing back 14d, and Fig. 8 B is HE coloration result behind AdGFP (contrast) the processing back 14d, and is consistent with the result that takes a picture substantially.
Present embodiment is the intradermal injection administration to the processing method of mice, can reach same effect by route of administration such as smearing, oral.
Conclusion: Wnt10b and Wnt3a have the effect that promotes hair follicle growth equally, and they all are to bring into play the effect that promotes hair follicle growth by activating Wnt/ β-catenin signal path.Therefore, other Wnt albumen that play a role by this path have the effect that promotes hair follicle growth equally.
Present embodiment is the intradermal injection administration to the processing method of mice, and hair growth promoter of the present invention adopts other administering modes all can reach same effect of the present invention as smear, oral etc.
Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when can doing a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.
Claims (3)
1. a hair growth promoter is characterized in that described hair growth promoter comprises the Wnt albumen that promotes hair follicle growth by Wnt/ β-catenin signal path.
2. hair growth promoter according to claim 1 is characterized in that described Wnt albumen by Wnt/ β-catenin signal path promotion hair follicle growth is Wnt3a albumen.
3. promote the application of Wnt albumen in preparation treatment alopecia medicine of hair follicle growth by Wnt/ β-catenin signal path.
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| CN200910306586A CN101642563A (en) | 2009-09-04 | 2009-09-04 | Pilatory containing Wnt protein |
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| CN200910306586A CN101642563A (en) | 2009-09-04 | 2009-09-04 | Pilatory containing Wnt protein |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103781799A (en) * | 2011-08-04 | 2014-05-07 | 凯尔格恩有限公司 | WNT family-derived peptide and uses thereof |
| CN107530253A (en) * | 2015-03-31 | 2018-01-02 | 株式会社爱茉莉太平洋 | It is used to promote hair tonic and/or educates the composition of hair containing psoralidine |
| CN111568910A (en) * | 2020-07-06 | 2020-08-25 | 上海沸米生物科技有限公司 | Application of crocin II in preparation of medicine for preventing and treating alopecia by acting on hair follicle tissues |
| CN114306183A (en) * | 2021-12-29 | 2022-04-12 | 香港科技大学 | Novel use of plant-derived alkaline phosphatase expression promoter |
| CN116942847A (en) * | 2023-08-30 | 2023-10-27 | 深圳市华健生物技术有限公司 | Drug-loaded plant exosome targeting hair follicle, and preparation method and application thereof |
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2009
- 2009-09-04 CN CN200910306586A patent/CN101642563A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103781799A (en) * | 2011-08-04 | 2014-05-07 | 凯尔格恩有限公司 | WNT family-derived peptide and uses thereof |
| JP2016029061A (en) * | 2011-08-04 | 2016-03-03 | ケアジェン カンパニー, リミテッドCaregen Co., Ltd. | Wnt family-derived peptides and use thereof |
| CN103781799B (en) * | 2011-08-04 | 2016-12-14 | 凯尔格恩有限公司 | WNT series derivatives peptide and application thereof |
| JP2017008030A (en) * | 2011-08-04 | 2017-01-12 | ケアジェン カンパニー, リミテッドCaregen Co., Ltd. | Wnt family-derived peptide and uses thereof |
| CN107530253A (en) * | 2015-03-31 | 2018-01-02 | 株式会社爱茉莉太平洋 | It is used to promote hair tonic and/or educates the composition of hair containing psoralidine |
| CN107530253B (en) * | 2015-03-31 | 2020-10-23 | 株式会社爱茉莉太平洋 | Composition containing psoralen for promoting hair growth and/or hair growth |
| CN111568910A (en) * | 2020-07-06 | 2020-08-25 | 上海沸米生物科技有限公司 | Application of crocin II in preparation of medicine for preventing and treating alopecia by acting on hair follicle tissues |
| CN111568910B (en) * | 2020-07-06 | 2021-06-01 | 上海沸米生物科技有限公司 | Application of crocin II in preparation of medicine for preventing and treating alopecia by acting on hair follicle tissues |
| CN114306183A (en) * | 2021-12-29 | 2022-04-12 | 香港科技大学 | Novel use of plant-derived alkaline phosphatase expression promoter |
| WO2023125519A1 (en) * | 2021-12-29 | 2023-07-06 | 香港科技大学 | New application of plant-derived alkaline phosphatase expression promoter |
| CN116942847A (en) * | 2023-08-30 | 2023-10-27 | 深圳市华健生物技术有限公司 | Drug-loaded plant exosome targeting hair follicle, and preparation method and application thereof |
| CN116942847B (en) * | 2023-08-30 | 2024-04-09 | 深圳市华健生物技术有限公司 | Drug-loaded plant exosome targeting hair follicle, and preparation method and application thereof |
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Open date: 20100210 |