CN102657845B - 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 for the treatment of 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, finishes 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, from cytology's angle, granulation tissue comprises: newborn capillary endothelial cell, by original next fibroblast and the synthetic thereof of mesoblastema differentiation in part, as 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 finish.
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 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.
The short skin repair polypeptide A H90 of the Rana grahami of the present invention's research and the skin repair function of CW49 do not have this two peptide species can repair the open report of skin so far as yet.
Summary of the invention:
The object of the present invention is to provide the 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.
The 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.
The 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 at the animal model of mice, and its testing program is conventional method, and is specific as follows:
I. the healing effect of the short skin repair polypeptide A H90 of Rana grahami and the wound of CW49
After mice was anaesthetized with 2% pentobarbital sodium, both sides, back skin shaver defeathering was 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. the proliferative effect of the short skin repair polypeptide A H90 of Rana grahami and the corium of CW49, 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, a few 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, dimethylbenzene equivalent mixed liquor 15min are till dimethylbenzene I 15min, II 15min(are extremely transparent).
6. saturating wax
Put into the mixed liquor 15min of dimethylbenzene and paraffin half and half, put into paraffin I, the saturating wax of paraffin II again each 50-60 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, 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 paraffin mass and the edge of a knife, blade becomes about 15 degree approximately with paraffin section.
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, allowed 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 gently with another brush pen, in order to avoid curl, and be distracted into band, and lie on the wax tape drum, more smooth by the one side of knife face, 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, microtome knife should be taken off with chloroform and wipe the paraffin of being stained with on the cutter, the microtome wiped clean is preserved.
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, the section that has launched is dragged for to warm water, makes it abundant expansion.
4. get clean microscope slide in addition, pick up the section of expansion, be located at section 1/3 place, labelling or labelled on the other end (edging, a coarse end) flour milling is put on the section frame.
10. dewaxing rehydration
The water-bath temperature is transferred to 60 ℃, treat water temperature control in the time of 60 ℃, section is put into the color jar of a drying 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 dimethylbenzene I, II each 5min that dewaxes, and 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 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 in 2 seconds, color is more shallow.
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
Dimethylbenzene 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.
The 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 0 day, 2 days, 4 days, 6 days, the 8 days short mouse skin damage models of skin repair polypeptide A H90 of Rana grahami respectively.
Fig. 2-A, Fig. 2-B, Fig. 2-C, Fig. 2-D, Fig. 2-E show contrast and the therapeutical effect of 0 day, 2 days, 4 days, 6 days, the 8 days short mouse skin damage models of skin repair peptide C W49 of Rana grahami 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 mouse skin of skin repair polypeptide A H90 of Rana grahami.
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 mouse skin of skin repair peptide C W49 of Rana grahami.
Fig. 5-A, Fig. 5-B are respectively the scoring of the short skin repair polypeptide A H90 of Rana grahami and the skin healing of CW49.
Fig. 6-A, Fig. 6-B are respectively the short skin repair polypeptide A H90 of Rana grahami and the epidermis proliferation function of CW49.
Fig. 7-A, Fig. 7-B are respectively the short skin repair polypeptide A H90 of Rana grahami and the scoring of the epidermis propagation of CW49.
The specific embodiment:
The 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.
The 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 at the animal model of mice, and its testing program is conventional method, and concrete steps are with the description of summary of the invention part.
Embodiment 1:
The repair of the short mouse skin damage model of skin repair polypeptide A H90 of Rana grahami.
Getting the short skin repair polypeptide A H90 of 0.05mg/ml Rana grahami tests, the result is: repair function (Fig. 1-A, Fig. 1-B, Fig. 1-C, Fig. 1-D, the Fig. 1-E) that can promote skin rapidly, second day repairing effect is apparent in view, incrustation in the 4th day, basically recovered fully in the 6th day, and and the skin repair of control mice evident difference is arranged.
Embodiment 2:
The repair of the short mouse skin damage model of skin repair peptide C W49 of Rana grahami.
Getting the short skin repair peptide C W49 of 1mg/ml Rana grahami tests, the result is: repair function (Fig. 1-A, Fig. 1-B, Fig. 1-C, Fig. 1-D, the Fig. 1-E) that can promote skin rapidly, second day repairing effect is apparent in view, incrustation in the 4th day, basically recovered fully in the 8th day, and and the skin repair of control mice evident difference is arranged.
Embodiment 3:
Rana grahami is urged the mouse skin repair of skin repair polypeptide A H90 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:
Rana grahami is urged the mouse skin repair of skin repair peptide C W49 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, epidermal tissue's thickness of the 5th day hypertrophy reduces, and epidermal thickness recovered normal and had body of gland to produce in the 9th day.
Embodiment 5:
The scoring of the short skin repair polypeptide A H90 of Rana grahami and the granulation tissue healing of CW49.
Smear the 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 shows that (Fig. 5-A, Fig. 5-B), behind the short skin repair polypeptide A H90 of use Rana grahami and the CW49, degree and the contrast of granulation tissue healing have obvious significant difference (* P＜0.01).
Embodiment 6:
The short skin repair polypeptide A H90 of Rana grahami and the epidermis proliferation function of CW49.
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 be significantly less than matched group and and matched group obvious significant difference (* P＜0.01) is arranged, AH90 and CW49 have the effect that prevention of scar produces.
Embodiment 7:
The short skin repair polypeptide A H90 of Rana grahami and the corium of CW49 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 that (Fig. 7-A, Fig. 7-B), the short skin repair polypeptide A H90 of the smelly frog of use graduated dial and CW49 epidermis and corium scoring and matched group have the difference of significance.
SEQUENCE LISTING
＜110〉Kunming Institute of Zoology, Chinese Academy of Sciences
＜120〉application of the 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, minimizing cicatrix.
2. molecular weight is 1210.43 Da, and 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, 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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| CN102657845A CN102657845A (en) | 2012-09-12 |
| CN102657845B true CN102657845B (en) | 2013-09-11 |
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| WO (1) | WO2013177866A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110577588B (en) * | 2019-09-29 | 2020-12-29 | 苏州大学 | Rana amurensis immunoregulation peptide and application thereof |
| 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 |
| CN114106100B (en) * | 2020-08-26 | 2024-05-17 | 四川好医生攀西药业有限责任公司 | Polypeptide for repairing skin wound or mucous membrane injury and application thereof |
| CN113698459B (en) * | 2021-08-04 | 2023-06-27 | 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 |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896097A (en) * | 2006-03-22 | 2007-01-17 | 中国科学院昆明动物研究所 | Peptide for promoting insulin release of frog and its use in production of drugs |
| CN101037473A (en) * | 2007-02-12 | 2007-09-19 | 中国科学院昆明动物研究所 | R. grahami rnmunoregulating polypeptide, gene and variant and its application in medicine production |
| CN100522993C (en) * | 2006-05-30 | 2009-08-05 | 中国科学院昆明动物研究所 | Odorranagrahami antimicrobialpeptides and application thereof |
-
2012
- 2012-05-31 CN CN2012101742724A patent/CN102657845B/en active Active
- 2012-08-16 WO PCT/CN2012/080221 patent/WO2013177866A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896097A (en) * | 2006-03-22 | 2007-01-17 | 中国科学院昆明动物研究所 | Peptide for promoting insulin release of frog and its use in production of drugs |
| CN100522993C (en) * | 2006-05-30 | 2009-08-05 | 中国科学院昆明动物研究所 | Odorranagrahami antimicrobialpeptides and application thereof |
| CN101037473A (en) * | 2007-02-12 | 2007-09-19 | 中国科学院昆明动物研究所 | R. grahami rnmunoregulating polypeptide, gene and variant and its application in medicine production |
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| WO2013177866A1 (en) | 2013-12-05 |
| CN102657845A (en) | 2012-09-12 |
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