CN102657845A - Applications of odorrana grahami polypeptide AH90 and CW49 for promoting skin regeneration - Google Patents
Applications of odorrana grahami polypeptide AH90 and CW49 for promoting skin regeneration Download PDFInfo
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Abstract
The invention belongs to the technical field of polypeptide drugs in biochemistry and particularly relates to applications of odorrana grahami polypeptide AH90 and CW49 for promoting skin regeneration. The polypeptide AH90 which has the molecular weight of 2657.18 Da, the isoelectric point of 10.26 and the complete sequence primary structure of ATAWDFGPHGLLPIRPIRIRPLCG is used as a drug to be applied to treatments of body-surface traumas, burns and skin ulcer, reduction of scar formation and acceleration of scar repair. The polypeptide CW49 which has the molecular weight of 1210.43 Da, the isoelectric point of 8.29 and the complete sequence primary structure of APFRMGICTTN is used as a drug to be applied to treatments of body-surface traumas, burns and skin ulcer, reduction of scar formation and acceleration of scar repair. The odorrana grahami polypeptide AH90 and CW49 for promoting skin regeneration can promote skin regeneration and reduce epidermis proliferation thickness during the skin regeneration and can be used for preparing drugs for treating body-surface traumas, burns and skin ulcer, reducing scar formation and accelerating scar repair.
Description
Technical field:
The invention belongs to field of polypeptide medicine technology in biochemistry, be specifically related to the short skin repair polypeptide A H90 of Rana grahami and promoting skin repair, reducing the application in cicatrix and the relevant skin diseases of treatment with CW49.
Background technology:
Skin has the effect of mechanical stop pathogen as the natural protection barrier of body.In case the natural cover for defense effect of skin is damaged, body will be opened the injury repairing that the physiology switch carries out skin immediately automatically.Healing behind the human body skin damnification exists above-mentioned tissue to replenish and two processes of cicatrix healing simultaneously.Epithelial cell is by the growth of creeping of normal skin edge centration, or outwards expands growth by the epithelium of skin appendages sweat gland, hair follicle, accomplishes the wound surface epithelium at last and covers.Dermal tissue is to the reaction of damage; Be to form granulation tissue; Granulation tissue is actually a kind of inflammation tissue, and from cytology's angle, granulation tissue comprises: newborn capillary endothelial cell; By next fibroblast and the synthetic thereof of the primary mesoblastema differentiation in part, like collagen fiber, mucopolysaccharide etc.Cicatrix is to be grown by granulation tissue to form, grow into stop voluntarily behind the certain phase and gradually absorption disappear.Cicatrix absorbs gradually thereafter and calms down still in hypertrophy in general wound healing later six months to one year.But this process can continue several years, the more than ten years still can not accomplish.
Rana grahami is the Amphibian of Ranidae Rana, is the peculiar species of China.Be distributed in ground such as Sichuan, Guizhou, Yunnan, often move in the luxuriant and dark moist place of medium and small mountain stream implants.The height above sea level scope of its existence is 720 to 3200 meters.Rana grahami adapts to it from the natural environment of existence and body injury such as avoid injury, and in the evolution of long period of time process, the skin of Rana grahami develops into the organ with multiple function, as: secrete a large amount of antibacterial peptides and resist microbiological attack; The secretory nerve toxin resists all kinds of natural enemies; The secretion somatomedin promotes wound healing etc.
Short skin repair polypeptide A H90 of the Rana grahami of the present invention research and CW49 be to the skin repair function, the public reported that does not have this two peptide species can repair skin so far as yet.
Summary of the invention:
The object of the present invention is to provide short skin repair polypeptide A H90 of Rana grahami and CW49 to produce, accelerate the application in the cicatrix repair medicine at preparation treatment body surface wound, burn, skin ulcer, minimizing cicatrix.
Used short skin repair polypeptide A H90 of Rana grahami of the present invention and CW49 adopt the method for conventional chemosynthesis to obtain; Wherein:
1. the molecular weight of the short skin repair polypeptide A H90 of Rana grahami is 2657.18 Da, and isoelectric point, IP is 10.26, and its complete sequence primary structure is ATAWDFGPHGLLPIRPIRIRPLCG.
2, the molecular weight of the short skin repair peptide C W49 of Rana grahami is 1210.43 Da, and isoelectric point, IP is 8.29, and the complete sequence primary structure is APFRMGICTTN.
Short skin repair polypeptide A H90 of Rana grahami of the present invention and CW49 produce, accelerate the application in the cicatrix repair medicine at preparation treatment body surface wound, burn, skin ulcer, minimizing cicatrix.
Content of the present invention is tested on the animal model of mice, and its testing program is conventional method, and is specific as follows:
I. short skin repair polypeptide A H90 of Rana grahami and CW49 are to the healing effect of wound
After mice was anaesthetized with 2% pentobarbital sodium, both sides, back skin was used the shaver defeathering, behind the alcohol disinfecting; Open the wound of two diameter 7mm at its back, sample dissolution is (the concentration 0.05mg/ml of AH, CW concentration is 1mg/ml) in PBS; Be administered twice every day, each 30ul.Sample is added in the right side wound surface, and the PBS that the left side wound surface adds respective volume makes negative control.Take wound surface with camera every three days and change, i.e. DAY0, DAY2, DAY4, DAY6, DAY8 is till the complete decrustation healing of dosing wound surface.Make scale with gage according to wound when taking pictures.With mapping computed in software wound surface size variation, adopt the marking system scoring of histological score at last.
II. short skin repair polypeptide A H90 of Rana grahami and CW49 are to the proliferative effect of corium, epidermis.
A: paraffin section
1. draw materials
Mice is put to death back clip wound surface skin histology
2. fixing
The hepatic tissue that cuts is washed with the normal saline tissue, dropped into immediately in the neutral formalin fixative
3. washing
Material after fixing, flowing water flushing, several hours or spend the night.
4. dehydration
Material is successively through the dehydration of 70%, 80%, 90% alcoholic solution at different levels, and each 30min puts into each 20min again 95%, 100% each for 2 times.
5. transparent
Absolute alcohol, xylene equivalent mixed liquor 15min, xylene I 15min, II 15min (till transparent).
6. pass through wax
Put into the mixed liquor 15min of xylene and paraffin half and half, put into the paraffin I again, the paraffin II was passed through each 50-60 of wax minute.
7. embedding
Hot a little on alcohol burner with tweezers gripping paraffin mould (metal quality), be placed on the flat desktop, from incubator, take out the wax cup that holds paraffin refined wax, pour a little paraffin into.Again that tweezers are warm a little on alcohol burner, the gripping material is put into wax-pattern, marshalling down with tangent plane.Put embedded box again, pour dewaxing gently into.
8. section
1. the paraffin mass that will fix and fix is contained on the folder thing platform of microtome.
2. microtome knife is fixed on the cutter holder, the edge of a knife upwards.
3. shake the promotion spiral, the paraffin mass and the edge of a knife are pressed close to, but can not surpass the edge of a knife.
4. adjust angle and position between the paraffin mass and the edge of a knife, blade becomes about 15 degree with paraffin section approximately.
5. adjust caliper profiler to required slice thickness, be generally the 4-10 micron.
6. the master can begin section after all were adjusted.This moment, the right hand shook runner, let wax stone be cut into the wax band, and left hand is held brush pen the wax band is mentioned, and rocking-turn speed can not be too anxious, usually with 40-50r/min.
7. the wax that is cut into takes the 20-30cm long time to, and the right hand is provoked the wax band with another brush pen gently, in order to avoid curl, and be distracted into band, and lie on the wax tape drum, lean on the one side of knife face more smooth, down, one of wrinkle face up.
8. it is a bit of to cut wax disk(-sc) with single-edge blade, is placed on to carry on the glass to add one in water, and whether place magnifier or microscopically to observe section good.
9. cut into slices behind the end-of-job, should microtome knife be taken off with chloroform and wipe the paraffin of being stained with on the cutter, preserve the microtome wiped clean.
9. open up sheet, paster
The bath of fetching boiling water makes water temperature maintain 40-45 ℃, and other prepares 30% alcoholic solution.
When 1. cutting into slices, one bowl of 30% alcoholic solution is put on the other desktop of microtome.
2. the wax band of severing with blade in advance with the pincet gripping is placed on the water surface of alcoholic solution, makes section developing.
3. the pincet section that will connect together lightly separately, it is complete to cut into slices with a microscope slide, unfolded section is dragged for to warm water, makes it abundant expansion.
4. get clean microscope slide in addition, pick up unfolded section, be located at section 1/3 place, the other end (edging, a coarse end) flour milling marked or labelled is put on the section frame.
10. dewaxing rehydration
The water-bath temperature is transferred to 60 ℃, when treating that water temperature is controlled at 60 ℃, will cut into slices and put into an exsiccant color jar together with the section frame, put into water-bath, cover lid (salable), 30min melt to wax.Afterwards, paraffin section is put into 100%, 95%, 90%, 80%, 70% each 3-5min of alcoholic solutions at different levels then through xylene I, each 5min of II dewaxing, puts into distilled water 3min again.
B:HE dyeing
1. the about 10-30min of hematoxylin dyeing is put in section.Dyeing time should suitably shorten or prolong according to the maturity and the room temperature height of stain.Put into calorstat when room temperature is low and dye winter.
2. washing
With tap water flowing water flushing 15min.Make the section color become blue (or putting into alkaline water), but will notice that flowing water can not be excessive, in case section comes off.
3. differentiation
1% hydrochloride ethanol liquid is put in section faded, saw to tens of seconds that section reddened lighter color in 2 seconds.
4. rinsing
Tap water flowing water is put in section again makes it recover blue.
5. dehydration I
Each 3-5min in 50% ethanol → 70% ethanol → 80% ethanol is gone in section.
6, redye with 0.5% Yihong ethanol liquid counterstaining 1-3min.
7. the dehydration II is put into the unnecessary redness of 95% ethanol flush away with section, puts into dehydrated alcohol 3-5min then.Blot unnecessary ethanol with absorbent paper at last.
8. transparent
Xylene I, each 3-5min of II are put in section.
9. envelope is hidden: neutral gum is sealed up for safekeeping
C: epidermal thickness calculates.
Section after the dyeing is taken pictures, use software I mageJ computer chart skin thickness at last, adopt the marking system scoring of histological score.
Short skin repair polypeptide A H90 of Rana grahami of the present invention and CW49 can promote the reparation of skin, can reduce the hypertrophy thickness of skin repair process mesocuticle simultaneously, can be used in the medicine of preparation treatment body surface wound, burn, skin ulcer.
Description of drawings:
Fig. 1-A, Fig. 1-B, Fig. 1-C, Fig. 1-D, Fig. 1-E show contrast and the therapeutical effect of the short skin repair polypeptide A H90 of 0 day, 2 days, 4 days, 6 days, 8 days Rana grahamis to the mouse skin damage model respectively.
Fig. 2-A, Fig. 2-B, Fig. 2-C, Fig. 2-D, Fig. 2-E show contrast and the therapeutical effect of the short skin repair peptide C W49 of 0 day, 2 days, 4 days, 6 days, 8 days Rana grahamis to the mouse skin damage model respectively.
Fig. 3-A, Fig. 3-B, Fig. 3-C, Fig. 3-D, Fig. 3-E, Fig. 3-F are respectively contrast and the therapeutical effect slice map of the short skin repair polypeptide A H90 of Rana grahami to mouse skin.
Fig. 4-A, Fig. 4-B, Fig. 4-C, Fig. 4-D, Fig. 4-E, Fig. 4-F are respectively contrast and the therapeutical effect effect slice map of the short skin repair peptide C W49 of Rana grahami to mouse skin.
Fig. 5-A, Fig. 5-B are respectively the short skin repair polypeptide A H90 of Rana grahami and the CW49 scoring to skin healing.
Fig. 6-A, Fig. 6-B are respectively the short skin repair polypeptide A H90 of Rana grahami and CW49 to the epidermis proliferation function.
Fig. 7-A, Fig. 7-B are respectively the short skin repair polypeptide A H90 of Rana grahami and CW49 marks to epidermis propagation.
The specific embodiment:
Used short skin repair polypeptide A H90 of Rana grahami of the present invention and CW49 adopt the method for conventional chemosynthesis to obtain; Wherein:
1. the molecular weight of the short skin repair polypeptide A H90 of Rana grahami is 2657.18 Da, and isoelectric point, IP is 10.26, and its complete sequence primary structure is ATAWDFGPHGLLPIRPIRIRPLCG.
2, the molecular weight of the short skin repair peptide C W49 of Rana grahami is 1210.43 Da, and isoelectric point, IP is 8.29, and the complete sequence primary structure is APFRMGICTTN.
Short skin repair polypeptide A H90 of Rana grahami of the present invention and the CW49 application in preparation treatment body surface wound, burn, skin ulcer medicine.
Content of the present invention is tested on the animal model of mice, and its testing program is a conventional method, and concrete steps are with the description of summary of the invention part.
Embodiment 1:
The short skin repair polypeptide A H90 of Rana grahami is to the repair of mouse skin damage model.
Getting the short skin repair polypeptide A H90 of 0.05mg/ml Rana grahami makes an experiment; The result is: repair function (Fig. 1-A, Fig. 1-B, Fig. 1-C, Fig. 1-D, Fig. 1-E) that can promote skin rapidly; Second day repairing effect is apparent in view; Incrustation in the 4th day recovered on the 6th day basically fully, and with the skin repair of control mice evident difference was arranged.
Embodiment 2:
The short skin repair peptide C W49 of Rana grahami is to the repair of mouse skin damage model.
Getting the short skin repair peptide C W49 of 1mg/ml Rana grahami makes an experiment; The result is: repair function (Fig. 1-A, Fig. 1-B, Fig. 1-C, Fig. 1-D, Fig. 1-E) that can promote skin rapidly; Second day repairing effect is apparent in view; Incrustation in the 4th day recovered on the 8th day basically fully, and with the skin repair of control mice evident difference was arranged.
Embodiment 3:
The short skin repair polypeptide A H90 of Rana grahami is to mouse skin repair slice map.
Mouse skin in the process of recovering, with every day recovery effects make section, observe the epiderm skin growing state.Fig. 3-A, Fig. 3-B, Fig. 3-C, Fig. 3-D, Fig. 3-E, Fig. 3-F show that the 3rd day granulation tissue after the adding AH90 treatment forms, and epidermal tissue recovered basically fully in the 5th day, and epidermal tissue's thickness of the 9th day treatment group is compared obvious minimizing with matched group.
Embodiment 4:
The short skin repair peptide C W49 of Rana grahami is to mouse skin repair slice map.
The section result shows (Fig. 4-A, Fig. 4-B, Fig. 4-C, Fig. 4-D, Fig. 4-E, Fig. 4-F); The short skin repair peptide C W49 treatment of Rana grahami back formed thicker hypertrophy epidermal tissue on the 3rd day; Outgrowth epidermal tissue thickness reduced in the 5th day, and epidermal thickness recovered normal and had body of gland to produce in the 9th day.
Embodiment 5:
Short skin repair polypeptide A H90 of Rana grahami and CW49 are to the scoring of granulation tissue healing.
Smear short skin repair polypeptide A H90 of Rana grahami and CW49 after the wound of mouse skin, the healing state of mice wound is taken pictures to every day, and the marking system of employing histological score heals to granulation tissue and marks.The result show (Fig. 5-A, Fig. 5-B), use the short skin repair polypeptide A H90 of Rana grahami and CW49 after, degree that granulation tissue heals and contrast have obvious significant difference (* P < 0.01).
Embodiment 6:
Short skin repair polypeptide A H90 of Rana grahami and CW49 are to the epidermis proliferation function.
Shown in Fig. 6-A, Fig. 6-B; The thickness of the process mesocuticle of mouse skin injury recovery obviously increases; Smear behind the short skin repair polypeptide A H90 of Rana grahami and the CW49 thickness increase of epidermis and be significantly less than matched group and with matched group obvious significant difference (* P < 0.01) arranged, AH90 and CW49 have the effect of prevention of scar generation.
Embodiment 7:
Short skin repair polypeptide A H90 of Rana grahami and CW49 are to corium and the scoring of epidermis propagation.
Section is made at the position that mouse skin recovers, take pictures then and observe the propagation thickness of epidermis, adopt the marking system of histological score that skin repair is marked.The result shows (Fig. 7-A, Fig. 7-B), use short skin repair polypeptide A H90 of the smelly frog of graduated dial and CW49 epidermis and corium scoring and matched group that the difference of significance is arranged.
SEQUENCE LISTING
< 110>Kunming Institute of Zoology, Chinese Academy of Sciences
< 120>application of short skin repair polypeptide A H90 of Rana grahami and CW49
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 216
<212> DNA
<213> Rana grahami
<400> 2
AH90
atgttcacct tgaagaaatt cctgttgctc cttttctttc ttgggatcgt ctcctcatct 60
ccctgtctta gaaagagaga tgccgatgaa gaaggaaatg aagaaaatgg aggggaagct 120
aaaatggaag acataaaaag agcaacagcc tgggacttcg gaccacacgg gctgctacca 180
atacgcccta ttcggataag accactttgt ggataa 216
CW49
atgttcacct tgaagaaacc cctgttactc cttttctttc ttggaaccat ctccttatct 60
ctctgtgagg aagagaggga tgcagatgaa gaaagaagag atgatgaagt ggaagaaaca 120
aaaagagcgc cgtttcgtat gggtatatgt accacaaact ga 162
<210> 2
<211> 24
<212> PRT
<213> Rana grahami
<400> 2
Ala Thr Ala Trp Asp Phe Gly Pro His Gly Leu Leu Pro Ile Arg Pro
1 5 10 15
Ile Arg Ile Arg Pro Leu Cys Gly
20
CW49
Ala Pro Phe Arg Met Gly Ile Cys Thr Thr Asn
1 5 10
Claims (2)
1. molecular weight is 2657.18 Da; Isoelectric point, IP is 10.26, and the short skin repair polypeptide A H90 of the Rana grahami that its complete sequence primary structure is ATAWDFGPHGLLPIRPIRIRPLCG produces, accelerates the application in the cicatrix repair medicine at preparation treatment body surface wound, burn, skin ulcer, minimizing cicatrix.
2. molecular weight is 1210.43 Da; Isoelectric point, IP is 8.29, and the complete sequence primary structure is that the short skin repair peptide C W49 of the Rana grahami of APFRMGICTTN produces, accelerates the application in the cicatrix repair medicine at preparation treatment body surface wound, burn, skin ulcer, minimizing cicatrix.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101742724A CN102657845B (en) | 2012-05-31 | 2012-05-31 | Applications of odorrana grahami polypeptide AH90 and CW49 for promoting skin regeneration |
| PCT/CN2012/080221 WO2013177866A1 (en) | 2012-05-31 | 2012-08-16 | Use of rana grahami skin repair-promoting polypeptides ah90 and cw49 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101742724A CN102657845B (en) | 2012-05-31 | 2012-05-31 | Applications of odorrana grahami polypeptide AH90 and CW49 for promoting skin regeneration |
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| Publication Number | Publication Date |
|---|---|
| CN102657845A true CN102657845A (en) | 2012-09-12 |
| CN102657845B CN102657845B (en) | 2013-09-11 |
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| CN2012101742724A Active CN102657845B (en) | 2012-05-31 | 2012-05-31 | Applications of odorrana grahami polypeptide AH90 and CW49 for promoting skin regeneration |
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| CN (1) | CN102657845B (en) |
| WO (1) | WO2013177866A1 (en) |
Cited By (8)
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| CN109320591A (en) * | 2018-10-26 | 2019-02-12 | 昆明医科大学 | A kind of polypeptide OA-GL12 and its application |
| CN110563811A (en) * | 2019-09-25 | 2019-12-13 | 昆明医科大学 | skin protection peptide HW-P1 and preparation method and application thereof |
| CN110577588A (en) * | 2019-09-29 | 2019-12-17 | 苏州大学 | Rana amurensis immunoregulation peptide and application thereof |
| CN110935003A (en) * | 2019-12-30 | 2020-03-31 | 河北北方学院附属第一医院 | Biological polypeptide composition for promoting skin injury repair |
| CN113698459A (en) * | 2021-08-04 | 2021-11-26 | 广州格拉姆生物科技有限公司 | Antibacterial peptide mutant and application thereof |
| CN113827706A (en) * | 2021-09-27 | 2021-12-24 | 南通大学 | Application of GIP and peptide derived from GIP in preparation of skin wound treatment drug |
| WO2022042590A1 (en) * | 2020-08-26 | 2022-03-03 | 四川好医生攀西药业有限责任公司 | Polypeptides for repairing skin wounds or mucosal injuries, and applications thereof |
| WO2023015533A1 (en) * | 2021-08-12 | 2023-02-16 | 深圳市维琪医药研发有限公司 | Synthetic peptide, and cosmetic composition or pharmaceutical composition and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109320591A (en) * | 2018-10-26 | 2019-02-12 | 昆明医科大学 | A kind of polypeptide OA-GL12 and its application |
| CN109320591B (en) * | 2018-10-26 | 2021-06-01 | 昆明医科大学 | Polypeptide OA-GL12 and application thereof |
| CN110563811B (en) * | 2019-09-25 | 2022-05-10 | 昆明医科大学 | Skin protection peptide HW-P1 and preparation method and application thereof |
| CN110563811A (en) * | 2019-09-25 | 2019-12-13 | 昆明医科大学 | skin protection peptide HW-P1 and preparation method and application thereof |
| CN110577588A (en) * | 2019-09-29 | 2019-12-17 | 苏州大学 | Rana amurensis immunoregulation peptide and application thereof |
| US11884706B2 (en) | 2019-09-29 | 2024-01-30 | Soochow University | Immunomodulating peptide derived from concave-eared torrent frog and use thereof |
| WO2021057279A1 (en) * | 2019-09-29 | 2021-04-01 | 苏州大学 | Odorrana tormota immunomodulatory peptide and use thereof |
| CN110935003A (en) * | 2019-12-30 | 2020-03-31 | 河北北方学院附属第一医院 | Biological polypeptide composition for promoting skin injury repair |
| CN110935003B (en) * | 2019-12-30 | 2021-01-08 | 北医博大(北京)皮肤病医学研究院有限公司 | Biological polypeptide composition for promoting skin injury repair |
| WO2022042590A1 (en) * | 2020-08-26 | 2022-03-03 | 四川好医生攀西药业有限责任公司 | Polypeptides for repairing skin wounds or mucosal injuries, and applications thereof |
| CN113698459A (en) * | 2021-08-04 | 2021-11-26 | 广州格拉姆生物科技有限公司 | Antibacterial peptide mutant and application thereof |
| WO2023015533A1 (en) * | 2021-08-12 | 2023-02-16 | 深圳市维琪医药研发有限公司 | Synthetic peptide, and cosmetic composition or pharmaceutical composition and application thereof |
| CN116829169A (en) * | 2021-08-12 | 2023-09-29 | 深圳市维琪科技股份有限公司 | Synthetic peptides, and cosmetic or pharmaceutical compositions and uses thereof |
| CN113827706A (en) * | 2021-09-27 | 2021-12-24 | 南通大学 | Application of GIP and peptide derived from GIP in preparation of skin wound treatment drug |
| CN113827706B (en) * | 2021-09-27 | 2023-12-26 | 南通大学 | Application of GIP and its derivative peptide in preparing skin wound treating medicine |
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| WO2013177866A1 (en) | 2013-12-05 |
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