CN104622904A - Skin stem cell active component and application of active component - Google Patents
Skin stem cell active component and application of active component Download PDFInfo
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- CN104622904A CN104622904A CN201510060414.8A CN201510060414A CN104622904A CN 104622904 A CN104622904 A CN 104622904A CN 201510060414 A CN201510060414 A CN 201510060414A CN 104622904 A CN104622904 A CN 104622904A
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Abstract
The invention discloses a skin stem cell active component. The active component is secreted by skin stem cells, exists in supernatant generated by culturing skin stem cells and is expressed in a form of DIk1+/Scal-. The skin stem cell active component is applied to treatment on alopecia and can be used for activating the stem cells of the body, accelerating growth and proliferation of the hairs; the hair growing effects of the active component in the normal model and the pathological model are superior to those of commercially-available minoxidil or finasteride; and the skin stem cell active component is relatively high in safety.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of skin progenitor cell active component, and the acquisition methods of this skin progenitor cell active component.The skin progenitor cell active component that the present invention obtains can be applicable to hair growth.
Background technology
Alopecia refers to that hair pathologic comes off and to cause hair sparse or form alopecia speckle, and serious caused hair all comes off, and pathogenic factor is unclear, is the commonly encountered diseases of department of dermatologry, frequently-occurring disease.In west, within 30 years old, male's sickness rate is about 30%, is 40% when 40 years old, be 50%, and the white man at least 80% of 70 years old suffers from alopecia when 50 years old.In China, alopecia prevalence about 30.29%, wherein 25% has androgenetic alopecia in 25 years old to 55 years old male.Along with the increase of social pressure, alopecia is to the development that becomes younger, and patient groups rises year by year.Although the life of alopecia not entail dangers to people, obviously impact is attractive in appearance, brings very large stress and psychological burden, especially for the child of trophophase to patient.
Over nearly 30 years, Bureau of Drugs Supervision of the U.S. only have approved the medicine of minoxidil and finasteride two kinds of hair growths, and is first-line drug after approval always, has good hair-growing effects to Most patients.But two kinds of medicines, in long-term application, expose some shortcomings part gradually, as, onset invalid to small part patient slowly, easily recurrence etc. after drug withdrawal.What more allow patient perplex is then two kinds of side effects of pharmaceutical drugs.Minoxidil has phenomena of cutaneous irritation occurred frequently, serious meeting causes palpitating speed, the side effect such as blood pressure rising, also have report with use the unspecific allergic that is still not clear of cause effect relation react as welt, allergic rhinitis, facial swelling, allergy breathe hard, have a headache, the untoward reaction such as neuritis.Finasteride treatment androgenetic alopecia, be only applicable to male patient, the common sexual dysfunction of long-term taking (sexual impotence, hyposexuality, defective ejaculation), breast discomfort (mammary gland increase, mammary gland pain) and erythra, still have other to comprise the untoward reaction such as anaphylaxis and testicular pain such as Pruritus sense, rubella and face lip swelling.Some patients is in the unsatisfied situation of medication effect, have employed the hair transplantation operation of somewhat expensive, but for the patient of exiguous human hair own, hair transplantation operation is difficult to carry out, the complication such as easy appearance infection, epidermoid cyst, and outward appearance is often unnatural.
Stem cell (Stem Cell) is the cell colony that a class has self renewal, hyperproliferation and multi-lineage potential, is to form the various tissue of human body, organ " forefathers' cell ", " omnipotent cell ".Stem cell can produce and secrete a large amount of bioactie agents, these stem cell biological active factorses can Effective Regulation body cell intracellular signaling, activating human body stem cell, and then physiological reparation or alternative body injury, pathological changes and senile cell.Such as, stem cell can produce stem cell factor (SCF), nerve growth factor (NGF), stromal cell source property somatomedin (SDF), vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin like growth factor (IGF), epidermal growth factor (EGF), interleukin-6 and IL-7 (IL-6 and IL-7), megakaryocyte colony stimulating factor (M-CSF), tumor necrosis factor (TNF), the factors such as interferon (IFN), these cytokines have promotion cell proliferation, differentiation, the functions such as anti-apoptotic, stem cell can also produce natural immunity albumen, such as IgG, IgA, IgM, IgD, IgE etc., can resist or repair the damage self caused body cell with extraneous factor.
The bioactie agent activating machine somatic stem cell utilizing stem cell secretion to produce, and then repair or substitute the cell of body injury, pathological changes and aging by autologous stem cells physiological, thus the problem of hair growth, have broad application prospects in diseases prevention and treatment and health and beauty field.
Summary of the invention
The object of this invention is to provide a kind of skin progenitor cell active component, and the acquisition methods of this skin progenitor cell active component.
A kind of skin progenitor cell active component, described active component is secreted by skin progenitor cell, is present in and cultivates in supernatant that skin progenitor cell produces, with Dlk1
+/ Sca1
?form is expressed, obtained by following method: be 1:3 according to the volume ratio of skin histology and enzyme liquid, the skin histology shredded is added in the collagenase III enzyme liquid be dissolved in containing the concentration 1.0mg/ml in dual anti-DMEM and place 2h, with 200 order filter screen filtration cells, collect filtrate, by the activity contained with enzyme in the DMEM of 10wt%FBS of equivalent; Residue piece of tissue 37 DEG C of digestion, with 200 order filter screen filtration cells, collects filtrate with 0.25wt% pancreatin/EDTA; Merge twice filtrate, apply Dlk antibody and the Sca1 antibody of marked by magnetic bead respectively, filter out Dlk1 with magnetic bead screening method
+/ Sca1
?skin progenitor cell.
Wherein, the skin histology shredded described in, after cleaning with PBS, is cut into the skin histology of 1mm × 1mm × 1mm size.
With Dlk1 after low cytometric analysis evaluation and screening
+/ Sca1
?the purity of skin progenitor cell, result shows, the Dlk1 of extraction
+/ Sca1
?skin progenitor cell purity can reach 92.0%.
The present invention also provides the cultural method of the skin progenitor cell active component of above-mentioned acquisition simultaneously, comprising:
1), the skin progenitor cell sub-elected is inoculated in culture bottle, adds 5ml skin progenitor cell serum-free special culture media, at humidity>=95%, temperature 37 DEG C, CO
2cultivate under volumetric concentration 5% environment, changed liquid every 2 days;
2), after cell 80% merges, with 0.25wt% pancreatin and 0.02wt%EDTA had digestive transfer culture, by going down to posterity, the cell produced moves in new culture bottle;
3), repeat above cultivation, amplification procedure, best cell quantity in incubation, can be adjusted as required.
Wherein, described skin progenitor cell serum-free special culture media is the DMEM containing 50ug/ml cattle pituitary extract and 5ng/ml hEGF, pH=7.0 ~ 7.4.
Collect the skin progenitor cell supernatant after above-mentioned cultivation, the centrifugal 5min of 1000r/min, discard cell precipitation, supernatant moves in 50ml centrifuge tube, and the centrifugal 15min of 9000r/min, discards liquid, with precipitate at the bottom of the resuspended centrifuge tube of 1ml PBS liquid, namely the skin progenitor cell active component in skin progenitor cell supernatant, moves into the PBS containing skin progenitor cell active component collected in 1.5ml EP pipe, is placed in-20 DEG C of preservations.
The skin progenitor cell active component of the above-mentioned acquisition of the present invention can, as a kind of effective ingredient of medicine, be applied in the medicine of hair growth, achieves significant effect through test.
The present invention is by In vitro culture, amplification, maintain high proliferation ability and the stable character of skin progenitor cell, and skin progenitor cell supernatant is collected in the period that skin progenitor cell has high proliferation ability and stable character, low-speed centrifugal discards cell, by the modern biotechnology such as cryogenic separation, purification means, obtain highly purified skin progenitor cell active component.
Bioactie agent of the present invention or material can be developed to the product with activity of cell biology function, and acting on after human body can activation equipment somatic stem cell, thus promotes the growth propagation of hair.The hair-growing effects of the present invention under normal model and pathological model is all better than street drug minoxidil or finasteride, illustrates that hair-growing effects of the present invention is remarkable.Safety testing shows, the present invention does not show skin irritation and anaphylaxis, has higher safety.The present invention adopts regeneration medicine technology to complete beautifying skin reparation, has that operational approach is simple, safety, and patient is easy to accept, and save time, curative effect is fast, the advantage such as evident in efficacy, thus realizes promoting hair propagation, eliminates the object of hypotrichosis crowd puzzlement.
Accompanying drawing explanation
Fig. 1 is Dlk1 of the present invention
+/ Sca1
?the flow cytometry results figure of skin progenitor cell.
Fig. 2 is Dlk1 of the present invention
+/ Sca1
?the upgrowth situation figure of skin progenitor cell.
Detailed description of the invention
Below by way of specific embodiment, the present invention is further illustrated description, but following embodiment does not form any limitation of the invention.Under the prerequisite not deviating from the technology of the present invention solution, any change that those of ordinary skill in the art made for the present invention easily realize or change, all will fall within right of the present invention.
Embodiment 1: the separation of skin progenitor cell.
1. skin histology is cleaned up with PBS, put into 5ml Ep pipe, be cut into 1mm × 1mm × 1mm size with eye scissors, add in 3 ~ 4ml collagenase III enzyme liquid, containing 1.0mg/ml collagenase III in this enzyme liquid, and dissolve in containing dual anti-DMEM.The mixed liquor of above-mentioned enzyme liquid and cell is placed 2h.
2. with 200 order filter screen filtration cells in round bottom polystyrene tube, collect filtrate, with equivalent containing in the DMEM of 10%FBS and the activity of enzyme.Residue piece of tissue 0.25% pancreatin/0.02%EDTA 37 DEG C of digestion 10min.With 200 order filter screen filtration cells in round bottom polystyrene tube, merge twice filtrate, cell counting.
3., from above-mentioned cell, first apply the Dlk antibody (SIGMA company) of marked by magnetic bead, filter out Dlk1 with magnetic bead screening method
+cell.Concrete steps are counted by the cell counting chamber collected, and control cell number 10
7~ 10
8between, then operate according to sorting test kit description.
4. from the Dlk1 sub-elected
+in cell, the Sca1 antibody (SIGMA company) of application marked by magnetic bead, filters out Dlk1 with magnetic bead screening method
+/ Sca1
?cell.Concrete steps are counted by the cell counting chamber collected, and control cell number 10
7~ 10
8between, then operate according to sorting test kit description.
5. with Dlk1 after low cytometric analysis evaluation and screening
+/ Sca1
?the purity of skin progenitor cell, result shows, the Dlk1 of extraction
+/ Sca1
?skin progenitor cell purity can reach 92.0%(Fig. 1).
Embodiment 2: the cultivation of skin progenitor cell.
1. be inoculated on 25mm culture bottle by the cell after sorting, add skin stem cell serum-free special culture media, culture medium consists of DMEM, wherein containing 50ug/ml cattle pituitary extract and 5ng/ml hEGF, and pH=7.0 ~ 7.4.Changed liquid every 2 days, culture environment remains on humidity>=95%, temperature 37 DEG C, CO
2volumetric concentration 5%.
2. can go down to posterity, by 0.25% pancreatin and 0.02%EDTA had digestive transfer culture after cell 80% merges.
3. repeat this cultivation, amplification procedure for many times, obtain a large amount of epidermal stem cells (cell growth condition is shown in Fig. 2).
Embodiment 3: the acquisition of skin progenitor cell active substance.
1. collect skin progenitor cell supernatant, the centrifugal 5min of 1000r/min, discards cell precipitation.
2. the supernatant collected is moved in 50ml centrifuge tube, use low-temperature and high-speed Centrifugal Machine Control temperature, the skin progenitor cell active substance in collected by centrifugation skin progenitor cell supernatant, centrifugal speed 9000r/min, centrifugation time 15min.
3. discard the liquid in centrifugal rear 50ml centrifuge tube, with the precipitate bottom the resuspended centrifuge tube of 1ml PBS liquid, the PBS containing skin progenitor cell active substance is moved in 1.5ml EP pipe, and-20 DEG C of preservations.
Application examples 1: the efficacy experiment of the skin progenitor cell active substance that the above embodiment of the present invention obtains.
1.1 skin progenitor cell active substances of the present invention are to the effect of depilation C57BL/6 mice.
Male C57BL/6 mice, in 6 week age, hair is in resting stage (skin pinkiness).Adapt to raising one week, losing hair or feathers in back Colophonium paraffin (1:1), is divided into 6 groups at random, often organizes 10.Blank group, does not award any tested material; Adjuvant group, gives 70% ethanol, and each consumption 0.lml/ only; Dosage group, skin progenitor cell active substance high dose group in skin progenitor cell active substance low dose group, skin progenitor cell active substance, difference each dosage 0.05mg/, 0.lmg/ and 0.2mg/; Minoxidil tincture group, gives commercially available minoxidil tincture, and each dosage 0.lml/ only.
Each group of mice is administered once every day sooner or later, and get appropriate uniform application in epilating area, continuous use to skin enters resting stage (observation terminal).Record every mice by entering the fringe time of trophophase resting stage and observing terminal time.In observation terminal time, mice break neck put to death, get the new piliation of back same area area 2.5cm × 2.5cm, weigh.Statistical analysis each tested material convert mice time, observation terminal time and gross weight difference, the results are shown in Table 1.
Fringe time is the time of changing trophophase resting stage into, and the time is shorter, shows that inducing action is stronger, promotes that hair is stronger to the transformation ability of trophophase.As shown in Table 1, skin progenitor cell active substance of the present invention compares with minoxidil tincture group, and fringe time significantly shortens (P<0.05).
Observe terminal time and the time of anagen phase closely related, the time is longer, and the hair growth time is longer, and growth promoting effects effect is stronger.Table 1 shows, all medication groups compare with adjuvant group and blank group, observes terminal time and is all significantly longer than adjuvant group and blank group (P<0.05); There are increase trend skin progenitor cell active substance low dose group of the present invention, middle dosage group and high dose group time, but there was no significant difference (P<0.05); Low dose group and middle dosage group and minoxidil tincture group effect basically identical (P>0.05), the high dose group time is longer than minoxidil tincture group (P<0.05).
Gross weight index weighs hair growth quality condition, and table 1 shows, test empty group and adjuvant group are without remarkable difference, and all medication groups compare with adjuvant group, observes gross weight and is all significantly higher than adjuvant group/blank group (P<0.05); Skin progenitor cell active substance high dose group of the present invention higher than minoxidil tincture group hair weight, low dose group and middle dosage group and minoxidil tincture group effect indistinction (P>0.05); Skin progenitor cell active substance of the present invention each dosage group gross weight is increase trend (P<0.05).
1.2 skin progenitor cell active substances of the present invention are to the effect of androgens psilosis model mice.
6 week age male C57BL/6 mice, 18g to 22g, is divided into 5 groups at random, often organize 10.Sodium chloride solution group, awards 0.9% sodium chloride solution, each to consumption 0.lml/; Dosage group and skin progenitor cell active substance high dose group in skin progenitor cell active substance low dose group, skin progenitor cell active substance, each dosage be respectively 0.05mg/ only, 0.lmg/ only and 0.2mg/ only; Finasteride group, gives finasteride tablet aqueous suspension.
The each group of emerald green ketone injection of mice each intramuscular injection 0.lml propanoic acid, every day 1 time, makes androgenetic alopecia model.Finasteride tablet group, gives finasteride suspension according to 0.17mg/kg finasteride dosage gavage, every day 1 time; All the other groups get each sample uniform application in mouse back, and every day is administered once sooner or later, successive administration 12 weeks.In observation terminal time, mice break neck put to death, clip agents area area is the skin and hair of 2.5cm × 2.5cm, hair weight of weighing.Mice skin of unhairing shears shreds, add PBS tissue refiner high-speed homogenization, homogenate, in the centrifugal 20min of 3000rpm, collects supernatant, measure emerald green ketone (DHT) content of dihydro by test kit description, each group mice is in observing terminal time gross weight measurement result in table 2.
As shown in Table 2, after mice gives skin progenitor cell active substance of the present invention, in skin, DHT content significantly declines, and hair weight is high, has good anticreep effect (P<0.05).Skin progenitor cell active substance high dose group of the present invention reduces skin DHT and increase gross weight effect is all better than positive drug finasteride group (P<0.05); Skin progenitor cell active substance group of the present invention increases with dosage, and gross weight is in significantly strengthening (P<0.05), and in skin, DHT content increases with dosage and reduces; The low middle dosage group of skin progenitor cell active substance of the present invention and the effect of positive drug finasteride group are close to (P>0.05).
1.3 skin progenitor cell active substances of the present invention are to the effect of ring phosphorus phthalein amine alopecia model mice.
Male C57BL/6 mice, in age week in 6 week age to 8, hair is in resting stage (skin pinkiness).Adapt to raising one week, with Colophonium paraffin (1:1) depilation, be divided into 5 groups at random, often organize 10.Adjuvant group, gives 70% ethanol, and each consumption 0.lml/ only; Dosage group, skin progenitor cell active substance high dose group in skin progenitor cell active substance low dose group, skin progenitor cell active substance, each dosage be respectively 0.05mg/ only, 0.lmg/ only and 0.2mg/ only; Encircle plain group, give 0.5% and only encircle plain 0.15ml/.
After mice epilation, epilation district hair follicle was changed to trophophase by resting stage, when epilating area hair is close to growth
first two days of phase, get appropriate each tested material uniform application in epilating area, every day is administered once sooner or later, and continuous use to skin enters resting stage (observation terminal).Each group of mice is in growth
disposablely during the phase press 150mg/kg dosage lumbar injection ring phosphorus phthalein amine.The neck that breaks in time observing terminal puts to death mice, and getting back same area area is that the new piliation of 2.5cm × 2.5cm is weighed.The results are shown in Table 3.
Mouse hair follicles gives ring phosphorus phthalein amine when being in trophophase can stop hair growth, and gross weight significantly declines.As can be seen from Table 3, mice gives skin progenitor cell active substance of the present invention and encircles element, all can improve new piliation amount, have good anticreep effect (P<0.05); Skin progenitor cell active substance of the present invention increases gross weight with dosage and increases, and high dose group effect is better than encircling plain group (P<0.05).
1.4 skin progenitor cell active substances of the present invention are to the effect of seborrheic alopecia patient.
Dermatology Outpatient Department patient, 16 ~ 60 years old age.The alopecia that radiotherapy, chemotherapy cause or have except medicament contact allergies person.Select seborrheic alopecia patient 78 example, be divided into treatment group and matched group, ensure all there is harmony between the sex of two groups of seborrheic alopecia patients, age, the course of disease.Treatment group external skin progenitor cell active substance of the present invention, dips in medicine with banister brush or absorbent cotton and wipes affected part, is coated with all over affected part for degree with medicinal liquid, 3 times/day.Matched group patient external 3% minoxidil shampoo.Medication period two groups of patients wash hair 1 time/3 days with warm water, and wind, cold stimulation are avoided in affected part, and period is without other hair growth promoting therapies.Within 60 days, be a course for the treatment of, carry out 3 courses for the treatment of continuously.
Curative effect judging standard: effective: alopecia obviously reduces or recovers normal, and dandruff, pruritus disappear, affected part has the long or former fine hair on birds or animals galley proof of obvious kainogenesis to send out thicker, black, hard, long, and outward appearance is obviously improved; Effective: alopecia, dandruff reduce, and pruritus alleviates, and affected part has kainogenesis long, and outward appearance makes moderate progress; Invalid: alopecia, dandruff slightly minimizing or unchanged, pruritus is slightly light or the same, and long without kainogenesis, appearance investigation is not obvious.
The curative effect of seborrheic alopecia patient is higher, and onset natural law more early, illustrates that treatment is more effective.As can be seen from Table 4, the seborrheic alopecia patient curative effect of skin progenitor cell active substance group of the present invention is applied apparently higher than matched group 3% minoxidil (P<0.05).Meanwhile, the onset natural law of the table 5 also seborrheic alopecia patient of application skin progenitor cell active substance of the present invention group is obviously early than the onset natural law (P<0.05) of matched group 3% minoxidil.
1.5 skin progenitor cell active substances of the present invention are to the effect of patients with alopecia areata.
Dermatology Outpatient Department patient, 16 ~ 60 years old age.The alopecia that radiotherapy, chemotherapy cause or have except medicament contact allergies person.Select patients with alopecia areata 72 example, be divided into treatment group and matched group, ensure all there is harmony between two groups of patients with alopecia areata sexes, age, the course of disease.Treatment group external skin progenitor cell active substance of the present invention, dips in medicine with banister brush or absorbent cotton and wipes affected part, is coated with all over affected part for degree with medicinal liquid, 3 times/day.Matched group patient external 3% minoxidil shampoo.Medication period two groups of patients wash hair 1 time/3 days with warm water, and wind, cold stimulation are avoided in affected part, and period is without other hair growth promoting therapies.Within 60 days, be a course for the treatment of, carry out 3 courses for the treatment of continuously.
Curative effect judging standard: cure: alopecia district has terminal hair to grow, outward appearance recovers normal, and around alopecia district, hair does not loosen, and new sending out no longer comes off; Effective: alopecia district generally has fine hair on birds or animals hair to grow, and the terminal hair area of coverage exceedes alopecia district 1/2, and around alopecia district, hair does not loosen, new sending out does not come off; Effective: alopecia district generally has fine hair on birds or animals hair to grow, there is no terminal hair or terminal hair does not reach alopecia district 1/2, alopecia obviously reduces; Invalid: alopecia district does not have or only has a small amount of fine hair on birds or animals hair to grow, and hair continues to come off.
The curative effect of patients with alopecia areata is higher, and onset natural law more early, illustrates that treatment is more effective.As can be seen from Table 6, the patients with alopecia areata curative effect of skin progenitor cell active substance group of the present invention is applied apparently higher than matched group 3% minoxidil (P<0.05).Meanwhile, the onset natural law of the table 7 also patients with alopecia areata of application skin progenitor cell active substance of the present invention group is obviously early than the onset natural law (P<0.05) of matched group 3% minoxidil.
Should 3 kinds of models be adopted to study, with the drug action of thoroughly evaluating skin progenitor cell active substance of the present invention by use-case.1) adopt normal C57BL/6 mice Colophonium paraffin to lose hair or feathers, induce its hair to enter new growth cycle period, investigate the effect that skin progenitor cell active substance of the present invention grows it.Result of the test shows, skin progenitor cell active substance of the present invention can shorten by the transformation of resting stage to trophophase, extends the trophophase time, thus effectively increases new piliation weight.2) for investigating the effect under pathological model, establish androgen depilation model and ring phosphorus phthalein amine depilation model, skin progenitor cell active substance of the present invention shows the suppression depilation under two kinds of pathological models and promotes hair growth effect; Skin progenitor cell active substance of the present invention is totally in dose dependent, and with the increase of dosage, drug action strengthens, and effect is better than existing hair growth medicine.3) situation being applied to seborrheic alopecia and patients with alopecia areata illustrates, it is still remarkable that skin progenitor cell active substance of the present invention is applied to human body curative effect.
Application examples 2: the safety evaluatio of skin progenitor cell active substance of the present invention.
Study the safety of skin progenitor cell active substance of the present invention with reference to national drug method for evaluating safety, adopt rabbit to investigate zest to skin, utilize Cavia porcellus to investigate its anaphylaxis to skin.
2.1 skin progenitor cell active substances of the present invention are to the stimulation of skin.
2.1.1 single-dose irritation test.
Male New Zealand rabbit 10, body weight 2.0kg to 3.0kg, 24h before experiment, chooses lateral symmetry position in the middle part of spinal column, and by the careful unhairing of hair scissors, every side 10cm × 10cm, depilation position should without erythema, edema and breakage.
Adopt consubstantiality left and right sides self-contrast method, every rabbit left dorsal unhairing district is coated with skin progenitor cell active substance 0.5mg of the present invention, and unhairing district, right side is coated with 0.9% sodium chloride solution 0.5ml in contrast, and cover with antiseptic gauze after administration, adhesive tape is fixed; Remove residual smear with warm water cleaning after 4h, after removal medicine, 1h, 24h, 48h and 72h observe local response respectively.Result shows, each treated animal recipient site is all without the irritation such as erythema, edema, and in experiment, each animal general state, behavior, sign normally, illustrate that skin progenitor cell active substance of the present invention is to no skin irritation.
2.1.2 multiple dosing irritation test.
Male New Zealand rabbit 10, body weight 2.0kg to 3.0kg, 24h before experiment, chooses lateral symmetry position in the middle part of spinal column, and by the careful unhairing of hair scissors, every side 10cm × 10cm, depilation position should without erythema, edema and breakage.
Adopt consubstantiality left and right sides self-contrast method, every rabbit left dorsal unhairing district is coated with skin progenitor cell active substance 0.5mg of the present invention, and unhairing district, right side is coated with 0.9% sodium chloride solution 0.5ml in contrast, and after administration, antiseptic gauze covers, and adhesive tape is fixed; Every day 2 times, each 1 time sooner or later, each administration time is identical, removes residual smear, smear 4 weeks continuously after 4h with warm water cleaning.1h after medicine is removed in 7th day, the 14th day and the 21st day at every turn, before each administration and last medication remove 1h, 24h, 48h and 72h after medicine, observe local response respectively.Result shows, each treated animal recipient site is all without the irritation such as erythema, edema, and in experiment, each animal general state, behavior, sign normally, illustrate that skin progenitor cell active substance of the present invention is to skin multiple dosing nonirritant.
2.2 skin progenitor cell active substances of the present invention are to the anaphylaxis of skin.
Cavia porcellus, body weight 250g to 300g, is divided into 0.9% sodium chloride solution group, skin progenitor cell active substance group and DNFB positive controls at random, often organizes 6; 24h before experiment, chooses lateral symmetry position in the middle part of spinal column, and by the careful unhairing of hair scissors, every side 3cm × 3cm, depilation position should without erythema, edema and breakage.
Sensitization contact: respectively at the 0th day, the 7th day and 14 days, lose hair or feathers district's uniform application 0.9% sodium chloride solution, skin progenitor cell active substance 0.5mg/ of the present invention only and 1% 2 on the left of each group of Cavia porcellus, 4-dinitrochlorobenzene 0.5ml/ only, cover with antiseptic gauze after administration, adhesive tape is fixed, and contacts 6h at every turn.
Excite contact: after last administration 14 days, lose hair or feathers district's uniform application 0.9% sodium chloride solution, skin progenitor cell active substance 0.5mg/ of the present invention only and 0.1% 2 on the right side of guinea pig back, 4-dinitrochlorobenzene 0.5ml/ only, excite, tested material is removed after 6h, at once observe, and again observe skin allergy situation in 24h, 48h, 72h.
Result shows, 0.9% sodium chloride solution group, skin progenitor cell active substance group of the present invention to guinea pig skin there are no the skin allergic reaction such as edema, erythema, also have no the whole body allergy phenomenons such as Experimental Asthma In Guinea-pigs, astasia or shock to occur, namely without sensitization.There is moderate erythema or edema phenomenon, sensitization rate 100% in positive control DNFB group guinea pig skin.Prove that skin progenitor cell active substance of the present invention does not show skin irritation and anaphylaxis, there is higher safety.
Claims (6)
1. a skin progenitor cell active component, described active component is secreted by skin progenitor cell, is present in and cultivates in the supernatant that produces of skin progenitor cell, with Dlk1
+/ Sca1
?form is expressed, obtained by following method: be 1:3 according to the volume ratio of skin histology and enzyme liquid, the skin histology shredded is added in the collagenase III enzyme liquid be dissolved in containing the concentration 1.0mg/ml in dual anti-DMEM and place 2h, with 200 order filter screen filtration cells, collect filtrate, by the activity contained with enzyme in the DMEM of 10wt%FBS of equivalent; Residue piece of tissue 37 DEG C of digestion, with 200 order filter screen filtration cells, collects filtrate with 0.25wt% pancreatin/EDTA; Merge twice filtrate, apply Dlk antibody and the Sca1 antibody of marked by magnetic bead respectively, filter out Dlk1 with magnetic bead screening method
+/ Sca1
?skin progenitor cell.
2. skin progenitor cell active component according to claim 1, is characterized in that the wherein said skin histology shredded is with after PBS cleaning, is cut into the skin histology of 1mm × 1mm × 1mm size.
3. the cultural method of the skin progenitor cell active component of claim 1 acquisition, comprising:
1), the skin progenitor cell sub-elected is inoculated in culture bottle, adds 5ml skin progenitor cell serum-free special culture media, at humidity>=95%, temperature 37 DEG C, CO
2cultivate under volumetric concentration 5% environment, changed liquid every 2 days;
2), after cell 80% merges, with 0.25wt% pancreatin and 0.02wt%EDTA had digestive transfer culture, by going down to posterity, the cell produced moves in new culture bottle;
3), repeat above cultivation, amplification procedure, in incubation, adjust best cell quantity as required;
Wherein, described skin progenitor cell serum-free special culture media is the DMEM containing 50ug/ml cattle pituitary extract and 5ng/ml hEGF, pH=7.0 ~ 7.4.
4. the skin progenitor cell active component that obtains of claim 1 is as the application of the medicine of hair growth.
5. the skin progenitor cell active component that obtains of claim 1 is as the application of medicine for the treatment of seborrheic alopecia.
6. the skin progenitor cell active component that obtains of claim 1 is as the application of medicine for the treatment of alopecia areata.
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| CN201510060414.8A Pending CN104622904A (en) | 2015-02-05 | 2015-02-05 | Skin stem cell active component and application of active component |
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| CN (1) | CN104622904A (en) |
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| CN106491393A (en) * | 2016-12-13 | 2017-03-15 | 云南农业大学 | Dog stem cell factor shampoo and preparation method thereof |
| CN106591222A (en) * | 2016-12-26 | 2017-04-26 | 山西医科大学 | Method for extracting active components of skin stem cells |
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| CN103142447A (en) * | 2013-03-18 | 2013-06-12 | 山西医科大学 | Epidermal stem cell active component and cosmetic preparation containing same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106491393A (en) * | 2016-12-13 | 2017-03-15 | 云南农业大学 | Dog stem cell factor shampoo and preparation method thereof |
| CN106491393B (en) * | 2016-12-13 | 2019-02-26 | 云南农业大学 | Dog stem cell factor shampoo and preparation method thereof |
| CN106591222A (en) * | 2016-12-26 | 2017-04-26 | 山西医科大学 | Method for extracting active components of skin stem cells |
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