CN1170000A - Heri factor coupled material, its preparation, composition containing it and application - Google Patents
Heri factor coupled material, its preparation, composition containing it and application Download PDFInfo
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Abstract
A coupled substance of HRG and EGF, its preparing process, and the cosmetics or medicinal composition containing it for resisting skin ageing are disclosed. The coupled substance HRG/EGF is generated by using disuccinimide bicarboxylate as coupling agent, and can stimulate and rejuvenate the activity of aged epithelial cells. Its storage stability is increased.
Description
The present invention relates to the auspicious factor (heregulin of lotus, HRG) conjugate, particularly with the conjugate of Urogastron (EGF), the preparation method who relates to this conjugate, the makeup or the pharmaceutical composition that contain their resisting age of skin, and their application in makeup and medicine manufacturing.
Aging is a kind of cell processes that is controlled in advance basically, but also have multiple factor such as ultraviolet ray strong quicken HE aging.Aging all the showing on cell and the molecular level that genetics predetermined extent and endogenous external source " wearing and tearing " are caused.Prevent to wear out, particularly skin aging is people's targets of effort for it always.The progress of modern molecule and cytobiology provides necessary means and possibility for this reason.
In vitro study has proved that the program of donor tissue wears out and can be reflected by the behavior of skin culturing cell.The ability that skin fibroblast is replied the external source mitogen reduces (Carlin CR etc. gradually, Nature, 306:617-620,1983), keratinocyte is to the reduction (DanesBS of the reactive of mitogen and formation clone ability, J.Clin.Invest.50:2000-2003,1971) all with aging relevant.Similarly, the forfeiture and aging relevant (GichrestBA etc., J.Invest Dermatol, 83:370-376,1984) of the melanophore multiplication capacity cultivated have been confirmed.Therefore, it seems that understanding will finally cause intervening weathering process with the molecular mechanism that the relevant multiplication capacity that wears out reduces related process, cause the rejuvenation of retarding ageing or aged cells.
From human genetics and oncology studies, unexpectedly obtain recently the understanding of weathering process, wherein studied the regulation mechanism of cell proliferation.The outer source signal of somatomedin form is bred as trigger cells such as Urogastron, the auspicious factor of lotus, transforming growth factors.Because aging relevant with the cell proliferation reduction, it seems wears out will influence some genetic expression.Find that each cell produces in the cultivation of the elderly's keratinocyte epidermic cell deutero-thymocyte activation factor (ETAF) and other factors significantly are lower than newborn infant or young man's keratinocyte.Take these research into consideration, prompting is aging to be a kind of genetic program process, it with the cell proliferation gene close or negative adjusting the (this causes the cellular sensitivity forfeiture to the growth signal) relevant, reach with the generation reduction of somatomedin and signal pathway in protein phosphorylation less relevant.
Part combines with the extracellular part of tyrosine kinase receptor and brings out receptor dimerizationization, and the signal of generation is striden the film transmission, Tyrosylprotein kinase functional zone in the activating cells.This causes the autophosphorylation on the tyrosine residues, and the function of these residues is the anchor position points as signal transducers.Each receptor tyrosine kinase seemingly has distinctive signal-transduction molecule unique combination, is responsible for the special and various effect of each factor in target cell together.Thereby the recent development of molecule and cytobiology opened up the frontier of ageing research, and the basis of present known weathering process is the sequencing generegulation.
The auspicious factor of lotus (HRG) is a member in the human growth factor family, it is acting on its acceptor (ErbB2, ErbB3 and ErbB4) on human cell such as epithelial cell or the fibroblast surface widely mutually, the irritation cell metabolism is also induced cell proliferation (the Carraway III that comprises epithelial cell, KL etc., J.Biol, Chem.269:14303-14306,1994; Carraway III, KL etc., J.Biol.Chem.270:7111-7116,1995).But there is not the epithelial active report of the auspicious factor pair the elderly of lotus, the report that does not yet have the auspicious factor of lotus in makeup, to use.
An object of the present invention is to provide a kind of bioactive compounds that is suitable for makeup or medicinal application that can stimulate epithelial cell growth and make old and feeble epithelial cell rejuvenation.
The present inventor proves through extensively and profoundly research:
1) this mitogen of recombinant human HRG improves cytokine such as Urogastron and old and feeble epithelial the combination greatly;
2) HRG improves the internalization rate of cytokine in old and feeble epithelial cell;
3) cell growth hormone gene such as the S6 kinases and the map kinase of HRG activation senile cell, these genes propagation and growth active and pair cell in young cell play a crucial role;
4) HRG improves aging HE growth and rate of propagation greatly.
Thereby the present inventor finds the strong activity of recombinant human HRG to the senile cell rejuvenation first.Therefore HRG can delay HE weathering process, and can make aging people's cell such as epithelial cell rejuvenation.
In the process of attempting HRG is used for makeup, the present inventor finds that HRG can not satisfy the requirement of cosmetics additive stability in storage, at room temperature the HRG of aqueous solution state is active descends rapidly, residual activity is the about 40% of original value in the time of 6 months, has reduced to below 10% during by 10th month.
The present inventor is crosslinked by coupling agent with HRG and EGF, the HRG/EGF conjugate that is surprised to find that generation not only keeps even has improved the stimulation epithelial cell growth of HRG and made the activity of aging epithelial cell rejuvenation, and significantly improved stability in storage, thereby satisfied requirement to the cosmetics additive stability in storage.
According to above discovery, the invention provides a kind of following formula HRG/EGF conjugate:
HRG-R-EGF
Wherein R is the difunctionality crosslinked group.
" the auspicious factor of lotus; HRG " used herein refers to people's auspicious factor of lotus of recombinating, be the glycoprotein that a kind of molecular weight is about 44kd, by Harvard Medical School (Harvard MedicalSchool) and (the Titan International Inc.MA of Titan international corporation, USA) generation of the genetic engineering coli strain of development and purifying obtain, and sell (Lot.No.94-1005) by above-mentioned company.
EGF, promptly Urogastron is a somatomedin known in this area, and the wide range of commercial supply is arranged.
The difunctionality crosslinked group of R representative is preferably C
4-C
10The fat diacyl, as 1,4-succinyl, 1; the 5-glutaryl-, 1, the 6-adipyl; 1,7-heptanedioyl, 1; the 8-octanedioyl, 1, the 9-azelaoyl; 1, the 10-sebacoyl wherein can have one or more substituting groups to replace on the polymethylene chain in the R group; as low alkyl group such as methyl, ethyl etc., halogen such as fluorine, chlorine, bromine etc., amino, nitro, phenyl or the like.Most preferably R is 1, the 8-octanedioyl.
Another object of the present invention provides wherein, and R is C
4-C
10The preparation method of the conjugate of the present invention of fat diacyl; it comprises that cooling adds down the dimethyl sulphoxide solution of following formula fat diacid two succinimide esters in the PBS solution of about 1: 1 mixture of HRG and EGF; react and add the glycine termination reaction after 10-50 minute; from reaction mixture, reclaim the gained conjugate then
Wherein R is C
4-C
10The fat diacyl.
The available any ordinary method in reaction back reclaims conjugate from mixture, preferably use HPLC gel filtration chromatography purifying, and cross-linking products is separated with single HRG or EGF.
HRG/EGF conjugate of the present invention can also be by crosslinked the making of other conventional polypeptide coupling methods.For example the bifunctional cross-linking compounds by respectively with HRG and EGF form acid amides, ester, monothioester, C-S ,-chemical bonds such as S-S-realize the coupling of HRG and EGF.
Another object of the present invention provides HRG/EGF conjugate of the present invention and irritates human epithelial cell's growth and make the make-up composition of old and feeble epithelial cell rejuvenation or the application in the pharmaceutical composition in preparation.
A further object of the present invention provides the make-up composition or the pharmaceutical composition of a kind of human epithelial cell's of irritating growth and old and feeble epithelial cell rejuvenation, wherein contains HRG/EGF conjugate of the present invention and conventional carrier and additive as activeconstituents.Carrier in the said composition and additive are makeup or acceptable conventional carrier of medicine and additives known in the art.For example carrier can be water in the liquid cosmetic, and other additives can be water-soluble silicon oil, hyaluronic acid, butyleneglycol, glycerine, polymethacrylate, Tegosept M, thickening material, BHT or the like.The paste makeup can be moisture, EDTA disodium salt, polymethacrylate, two butanols, hyaluronate sodium, Tegosept M, dihydroxymethyl dimethyl hydantoin, trolamine, BHT, polyglyceryl methacrylic acid ester or the like.
Composition of the present invention can for example be sneaked into conjugate of the present invention by the preparation of the ordinary method in this area after mixing unclassified stores, perhaps conjugate of the present invention and unclassified stores are mixed together.
Fig. 1, people recombinate aminoacid sequence and the DNA sequences encoding thereof of HRG.
Fig. 2, donor age (A and B) and population doubling level (C and D) are to the reactive influence of EGF.
Fig. 3, HRG, EGF, HRG/EGF are to the influence of youth and the elderly's cell proliferation.
The HRG of Fig. 4, different concns is to the influence of youth and the elderly's cell internalization.
The influence of Fig. 5, HRG on cell proliferation.
The different activated channels of Fig. 6, EGF and HRG.
The HRG of Fig. 7, different concns is to the activation of MAPK.
Fig. 8, HRG stimulate S6 the kinases immune labeled and electron microscopic analysis of back to abduction delivering.
The stable storing linearity curve of Fig. 9, HRG/EGF conjugate and HRG.
Further describe the present invention below in conjunction with embodiment and experiment.
With suberic acid two succinimido ester (DSS) couplings
(Pierce Corp.Rockford III) is dissolved in the dimethyl sulfoxide (DMSO), and preparation concentration is that the solution of 100 μ g/ml is stored on ice with DSS.The reorganization HRG and the EGF of equivalent are mixed in PBS solution, reach final concentration 50mg/ml.At this protein solution of cooled on ice, slowly stirring down then, adding DSS solution reaches final concentration 500 μ g/ml.Mixture is placed on ice, kept 30 minutes.Add 5mM glycine termination reaction then.Then mixture is passed through a HPLC gel-filtration column purification, remove uncrosslinked HRG or EGF and obtain the HRG/EGF conjugate.
Substantially operate by the method for embodiment 1, but wherein replace DSS with Succinic Acid two succinimido esters.Obtain wherein that R is 1, the HRG/EGF conjugate of the present invention of 4-succinyl.
Substantially operate by the method for embodiment 1, but wherein replace DSS with sebacic acid two succinimido esters.Obtain wherein that R is 1, the HRG/EGF conjugate of the present invention of 10-sebacoyl.
Beauty liquid
Earlier make conventional beauty liquid with the following ingredients of following content (by weight percentage):
Water-soluble silicon oil 5%
Butyleneglycol 3%
Tegosept M 0.2%
Sudan Gum-arabic 1.5%
BHT 0.05％
Pure water adds to 100%
In this beauty liquid, add the HRG/EGF conjugate for preparing among the embodiment 1 by every ml 20ng, make the purpose composition.
Ointment
Make conventional ointment by the following ingredients of following weight percent:
EDTA disodium salt 0.05%
Butyleneglycol 5%
Tegosept M 0.15%
0.2% uride in the dihydroxymethyl dimethyl is own
Trolamine 0.75%
BHT 0.05％
Polyglyceryl methacrylic acid ester 10%
Pure water to 100%
Add the HRG/EGF conjugate of preparation among the embodiment 1 then in this ointment, mixture evenly reaches 50ng/g concentration.
Originally experimental results show that aging epithelial propagation and the reaction reduction that mitogen is stimulated.
In experiment of the present invention, use the human keratinized cell and the fibroblast of the skin living tissue (1-3mm) of picking up from three age bracket volunteer upper arm.Three age brackets are respectively: newborn group; Youth's group in 22-27 year; The group in old age in 60-82 year.The living tissue sample carries out tissue culture and has among the DMEM of 10% bovine serum (CS) or in benefit in benefit that 1ng/ml Regular Insulin, 1.4 * 10 is arranged
-6Keep 2-4 generation in the DMEM minimum medium (BM) of M hydrocortisone and 10 μ g/ml Transferrins,iron complexess.In population doublings research, by following calculating population doubling level (PDL): PDL=log (N/No) * (log2)
-1, wherein the N=final cell is counted, N
0=initiator cell counting.The cell continuous passage of elementary back, PDL2,9 and 14 o'clock freezing, melt then and with 10
4Cell/cm
2Plant in three plates.The human keratinized cell culture is grown in to mend to be had in the substratum of hormone: M199 (GibCo, USA), 10 μ g/ml Regular Insulin (INS, MO, USA), 10 μ g/ml Transferrins,iron complexes (TRS, MA), 0.1pg/ml hydrocortisone, known rough ox hypothalamus extract and 2% dialysis foetal calf serum (the Biological Industries that contains keratinocyte growth factor of 150 μ g/ml, BeitHaemek, Isael).
At first, we determine that several leading generation of human skin fibroblast (elementary back PDL 2-5) from the different ages donor is to the responsibility of EGF or 10% bovine serum (CS).Preliminary experiment with different EGF concentration in the conditioned medium proves: identical EGF concentration produces optimum growh reaction (Fig. 2 A) in all age groups.With the replying of this best EGF concentration and 10%CS in observed the propagation marked difference relevant (Fig. 2 B) with the age.In 6 days processs of the test, from (P＜0.0001) of neonatal epithelial cell proliferation faster than young adult, the latter is again faster than (Fig. 2 C) in old age.In the test of end user's keratinocyte, gone out to observe similar age relevant difference.
We have also tested new life, youth and the old donor epithelial cell of external survival for about 20-40 elementary back population doublings (PDL increases progressively).Find that EGF is in conjunction with sharply decline, particularly newborn infant's cell between PDL3-10.When PDL10, young adult below cell levels when EGF reduces to PDL3 with combining of newborn infant's cell.EGF also descends with PDL with combining of young and old adult's cell but is so not rapid.Therefore, the acceptor number increases along with PDL and is tending towards close in three age groups.Observe mitogen reactivity parallel reduction (Fig. 1 D) along with the PDL increase to serum and EGF.Therefore, the variation and the respective change that increases owing to donor age in the early stage PDL cell, basically identical on quality and quantity of the growth velocity that stimulates of EGF binding ability that increases owing to PDL and EGF.Therefore, the elderly's cell shows the restrictive cell multiplication capacity, the reaction of growth-stimulating token stimulus is reduced, and the behavior of aged cells has all been represented in the ability reduction of combination and internalization somatomedin etc. substantially.
Experimental result as shown in Figure 2, solid signal be the newborn infant (●) of PDL3-5, young adult (24-32 year) ▲) and the elderly 68-82 year, the ■) culture of donor (N=16, every group).A): EGF 5ng/ml supports all age group optimum growhs, greater than this concentration then between three groups the cell yield produce difference (p＜0.001).B): (-) among the DMEM of 10%CS arranged or having in the basic medium of 5ng/ml EGF (---) to cultivate simultaneously at interval with 2 days in benefit.The speed of growth different with the final cell yield (p＜0.001) between three groups.C). from the PDL3 of same newborn infant, youth and old donor (solid,-) and PDL10 (hollow ...) 125I-EGF of cell and different concns cultivates.When EGF＞10ng/ml, total EGF combination rate is respectively organized different (p＜0.0001).D) with PDL3 (●), PDL10
And PDL15
Newborn infant's cell in three plates, remain on (-) among the increase serum DMEM or add in the minimum medium of 25ng/ml EGF (...) counted 6 days.After 2 days between three PDL group cell yield create a difference (p＜0.0001).Data point is mean value SD.
Originally experimental results show that HRG and HRG/EGF conjugate stimulate old and feeble epithelial metabolism and propagation.
In order to determine under the stimulation of somatomedin such as EGF and HRG, can aging epithelial cell rejuvenation, carry out this experiment.In this experiment, from the epithelial cell (skin fibroblast) (1 * 10 of young and old donor
5) benefit have 10%FBS and final concentration be among the DMEM of 1ng/ml HRG, EGF or HRG/EGF conjugate 37 ℃ cultivated 62 hours.Cultivate the back and measure cell quantity (* 10
5).The result as shown in Figure 3, EGF only stimulates the cell proliferation of young adult's cell effectively, and HRG stimulates the growth of the elderly and two kinds of cells of young adult.The HRG/EGF conjugate is not only keeping the activity of HRG aspect stimulation youth and the elderly's cell proliferation, and has significance to improve.These results also point out: though HRG and EGF have the ability of inducing cell propagation, HRG has utilized different stimulation approach, and preferentially stimulates and induce aging epithelial propagation.
Receptor-mediated internalization is a kind of cell function of key, selects the indication of the internalization of EGF as somatomedin processing back cell function here.Therefore we have measured the influence of different concns HRG to young and the internalization of the elderly's epithelial cell in research subsequently.The internalization measuring method is as follows: be bordering on the tranquillization fibroblast that converges in one 24 orifice plates with 25ng/ml
1254 ℃ of insulations of I-EGF 1 hour.With PBS hole flushing three times, cultivate the specified time in 37 ℃ with DME and 25ng/ml EGF.Wash the EGF that removed surface bonding in 5 minutes with 0.1M acetate.Use 2N NaOH lysing cell subsequently, the parallel off thing is counted on Beckman γ calculating instrument.The result as shown in Figure 4, after HRG handled, the internalization of EGF molecule improved greatly.Be low to moderate under the 1ng/ml HRG, on young and old adult's cell, observing the significant stimulation of pair cell internalization process.These results suggest HRG stimulates old and feeble epithelial metabolism, and makes old and feeble epithelial cell rejuvenation by receptors bind special with it and the unique signal pathway of activation.
We have measured the optimum concn that HRG stimulates young and old adult's epithelial cell proliferation subsequently, utilize HRG or HRG/EGF conjugate to delay the aging process and the important information of aged cells rejuvenation to provide.Epithelial cell (1 * 10 from young and old donor
5) have among the DMEM of HRG of 10%FBS and various concentration 10ng, 1ng, 80pg, 400pg, 100pg or 10pg/ml 37 ℃ to cultivate 62 hours in benefit.Measure the cell count (* 10 in the cell culture of HRG cultivation back then
5).Each data is three mean values in the hole.Show as the result among Fig. 5: HRG stimulates old and young adult's cell, and when HRG concentration was low to moderate 1ng/ml in substratum, the cell propagation effect of HRG all reached maximal stimulation on young and old adult's cell.
Stimulate epithelial cell by activating MAPK
In order further to study the mechanism that HRG stimulates the elderly's cell proliferation, we measure whether activated the signal pathway kinases, and these kinases on cell proliferation, growth and adjusting all are critical.Receptor tyrosine kinase (PTK) activates the signal transduction cascade reaction that causes as cell response keys such as propagation and differentiation through part.Wherein mitogen activity protein kinase (MAPK)/extracellular signal-regulated kinase approach is one of most important approach, and is activated by various extracellulars signal such as EGF and (see Egan S and WinbergR, Nature, 365:781-783,1993; Sliwkowski MX etc., J.Biol.Chem., 269:14661-14665,1994).
In order to determine to induce the active optimum concn of MAPK, analyzed EGF and HRG activation to young and the elderly's cell MAPK.Will be from the epithelial cell (skin fibroblast) (1 * 10 of young and old donor
6) have the HRG of 10%FBS and various concentration or EGF to cultivate hour in 37 ℃ 48 in benefit.With the lysis after cultivating, press Lane HA then, Nature, 363,170-172,1993 method is measured the MAPK activity.Fig. 5 has represented the relative reactivity (mean value of twice experiment) of mapk kinase, and under 1 μ g/ml HRG or EGF, HRG and EGF all induce the MAPK activity in young and old adult skin cell.But EGF preferentially induces the MAPK activity of young donorcells obviously; And HRG induces the MAPK activity of young and the elderly's skin cells on an equal basis.
In order to determine differential expression and HGR the influence that map kinase expressed of map kinase in young and the elderly's cell, immune labeled and superthin layer electron microscopy have been used.Old and young adult's cell with or do not have 37 ℃ of processing of 100pg concentration HGR after 4 hours, collection, embedding and tomography.Each layer and the reaction of goat-anti AMP kinase antibody are then with the anti-sheep IgG of gold grain link coupled rabbit (Oncogenes, MA, USA) reaction.Under electron microscope, observe gold mark layer then.Find a large amount of gold mark particles in the youth is grown up cell, the indication map kinase is expressed active; And a small amount of gold grain only appears in the elderly's cell, indicate MAPK genetic expression silence.And in the elderly's cell of handling with HRG, MAPK mark gold grain increases greatly, illustrates that the MAPK gene is activated by HRG in the elderly's cell.
In order to determine that HRG stimulates the active optimum concn of MAPK in youth or the elderly's cell, with youth and old donor epithelial cell (keratinocyte) (1 * 10
6) in benefit has the DMEM of 10%FBS, cultivate, stimulate the MAPK activity of measuring cell culture after 48 hours with 37 ℃ of the HRG of various concentration.The MAPK relative reactivity is shown among Fig. 7 with the average of twice experiment.HRG stimulates the map kinase activity of old and young cell effectively under 0.1-1 μ g/ml concentration, reach maximum value during 1 μ g/ml HRG.
Originally experimental results show that HRG activates the S6 kinase pathways, EGF then can not.
Studies show that recently to have p70/p85 S6 kinase pathways, it regulates cell proliferation and differentiation, and (Egan and Winberg, 1993 are activated in newborn infant and young adult's cell; Blume-Jensen etc. 1993).Compare with young adult's cell, the S6 kinase activity reduces greatly in the elderly's cell.The reduction of S6 kinase activity reduces directly related with the propagation of the elderly's cell.
A. from the epithelial cell (1 * 10 of young and old donor
6) benefit have 10%FBS and final concentration be among the DMEM of the HRG of 1ng/ml or EGF 37 ℃ cultivated 48 hours.With lysis, press currently known methods (Lane etc. 1993) mensuration S6 kinase activity then after cultivating with HRG or EGF.Mean value with twice experiment represents that the kinase whose relative reactivity of S6 is shown among Fig. 6.
The result shows, when the HRG with different concns handles the elderly's cell, observes that the S6 kinase activity improves greatly in the elderly's cell.On the contrary, handle the back with EGF and in young and the elderly's cell, do not induce the S6 kinase activity.The cell that HRG handles also showed cell is bred significantly increase, and not any significance raising (Fig. 6) of showed cell propagation of the cell that EGF handles.These results suggest HRG priority activation S6 kinase pathways, thereby the propagation and the cellular metabolism of stimulation aged cells (the elderly's cell), EGF can both stimulate the youth to become human cell multiplication with HRG.
B. also carried out immune labeled and electron microscopic analysis, whether produced by the HRG stimulation really to determine the kinase whose expression of S6.Epithelial cell (1 * 10 from young and old donor
6) benefit have 10%FBS and final concentration be among the DMEM of HRG of 1ng/ml 37 ℃ cultivated 48 hours.After the HRG cultivation, with cell harvesting, embedding and ultra-thin tomography.Each layer hatched with the anti-S6 kinase antibody of rabbit (from MIT, MA, the Dr.Lane of USA), cultivates with the anti-rabbit igg antibody of golden mark again.Under electron microscope, observe gold mark layer.As shown in Figure 8, HRG handles back young (A) and the elderly (B) epithelial cell shows strong mark S6 kinase signal, and the elderly's epithelial cell that does not stimulate through HRG shows S6 kinase expression very limited (C).G represents Golgi complex among the figure.This immune labeled electron microscopy result proves: HRG induced strong S6 kinase activity, this causes irritating young and the elderly's cellular metabolism, and makes the rejuvenation of aged the elderly cell.
Take all factors into consideration above result of experiment,,, and make the epithelial cell rejuvenation of wearing out so HRG can improve young epithelial metabolism always because HRG stimulates and opens in the epithelial cell signal pathway such as map kinase and the kinase whose gene of S6.HRG/EGF conjugate of the present invention keeps and has improved these activity of HRG.
Replace the HRG/EGF conjugate at room temperature to store 2 years with the beauty liquid (adding the HRG/EGF conjugate) of preparation in the experimental example 4 with HRG by the beauty liquid that embodiment 4 makes, take a sample at interval with certain hour therebetween, and by the method working sample of experiment in 2 to the epithelial hormesis of aging.Separately the percentage ratio of biological activity value is represented the residual activity of each time point when storing beginning.The result as shown in Figure 9, the stability in storage that the HRG/EGF conjugate is compared in make-up composition with HRG improves greatly, also keeps activity more than 40% at 2 years terminal hours, meets the requirement to the cosmetics additive stability in storage.
Without departing from the premise in the spirit of the present invention, one skilled in the art will realize that and to change the present invention program, as use maintenance active other coupling methods of HRG and coupling object so that HRG is stable, or otherwise come to stablize HRG, as long as final purpose is to utilize HRG to aging epithelial rejuvenation activity, these schemes should fall within the scope of the invention.
Claims (6)
1. people's following formula conjugate of HRG and EGF of recombinating:
HRG-R-EGF
Wherein R is the difunctionality crosslinked group.
2. the conjugate of claim 1, wherein R is C
4-C
10The fat diacyl.
3. the conjugate of claim 2, wherein R is 1, the 8-octanedioyl.
4. the preparation method of the conjugate of claim 2 comprises that cooling adds C down in the PBS solution of about 1: 1 mixture of HRG and EGF
4-C
10The dimethyl sulphoxide solution of fat diacid two succinimide esters reacts adding glycine termination reaction after 10-50 minute, reclaims the gained conjugate then from reaction mixture.
5. the conjugate of claim 1 stimulates human epithelial cell's growth, the make-up composition of old and feeble epithelial cell rejuvenation or the application in the pharmaceutical composition in preparation.
6. one kind stimulates the human epithelial cell to grow and the make-up composition or the pharmaceutical composition of old and feeble epithelial cell rejuvenation, wherein contains as each conjugate and conventional carrier and additive among the claim 1-3 of activeconstituents.
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| CN96108633A CN1063185C (en) | 1996-07-04 | 1996-07-04 | Heri factor coupled material, its preparation, composition containing it and application |
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