TWI345571B - Acidic fibroblast growth factor 1 (fgf-1) used for skin caring - Google Patents

Description

1345571 IX. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a composition comprising human acid fibroblast growth factor (FGF-1) for use in the manufacture of a skin care product. [Prior Art] It is well known that the age of each person is related to the replication and shedding of epidermal cells, such as cell turnover, and the reduction of epidermal cells causing aging. This process usually produces a dull and aging skin appearance. In addition, the number of microvessels in the skin decreases with age, and the skeleton of the sputum under the epidermis also undergoes structural disintegration, followed by aging and light damage, so elastic tissue degeneration, which causes skin wrinkles. And the result of relaxation. "Aging" at the cellular level refers to the improper repair of DNA, resulting in a defect in cell replication or non-existence. In addition, it refers to the loss of control factors located in the nucleus and cytoplasm of mitosis, resulting in the exchange of substances including abnormal nuclear and cytoplasm, permanent closure of the micro-vascular bed, detachment of the apical cells, and ultimately the loss of cells and organelle membranes. As time progresses, the effects of time effects include wrinkles and hardening of the skin. The skin is composed of impurities including elastin and collagen. Collagen is the main protein component of white fibers in connective tissue, such as the connective tissue of cartilage and bone. White elastin is the main blood vessel in connective tissue. Protein, white elastin, makes these tissues elastic with 1345571 and maintains the original configuration of these tissues. Both the protoprotein and the elastin have fibers, which are known to connect with each other; it is believed that the aging of mammals and humans includes the complication of compounding, making money and skin. The shackles of sexual reduction. The theory of free radicals includes the negative development of oxidation products, which causes aging of skin wrinkles. The fibroblast growth factor family is her large family of growth factors, the fibroblast growth factor family records the enemy length and the regenerative Wei side, which contains several members with different degrees of recording on the egg (four) level, with a few exceptions. These growth factor members appear to have a promoting pulse for a wide range of cells, for example, _ sub-promoting various cell proliferation of the progenitor and neuroectodermal origin, or/and promoting vascular proliferation. The performance pattern of the growth factor family members is not changed, and the __ limitation is expressed in some of the developmental wealth. It seems that the heparin with good content on the extracellular matrix, heparin sulfate protein (IV), (10) Cong combined strongly concentrated in the extracellular matrix. US Pat. No. 4,695,59, describes a method for delaying aging. The body-species chemical synthesis is recorded by the secret diphenyl sulphate. The preferred branch is oral, and I is convenient for oral administration. Riding Kelin into a fine gamma, giving a chemical composition within the touch. ^45571 A variety of protein factors are known to be necessary for cell growth and differentiation, including epidermal cells. Many proteins similar to this function have been extracted from tissues and identified for functions such as epidermal growth factor (EGF), lysin growth factor (IGF), platelet-derived growth factor (PDGF), and fibroblast growth. Factor (FGF), and fibroblast growth factor. Plaque epidermal growth factor treatment of corneal wounds is described in U.S. Patent No. 4,959,353 (US Pat. No. 4, 959, 353), and U.S. Patent No. 5,13,298 (US Pat. , 298) describes that the epidermal growth factor composition 'has a stabilizing effect against metal cation decomposition and can be used to treat wounds. US Patent No. 5,104,977, U.S. Patent No. 5,104,977, discloses the use of transgenic cell growth gj y? (TGF-beta) or transformed cell growth factor alpha (TGF_alpha) to treat injured tissue. _, in the examples of these patents, the protein growth factor is not used to reduce the aging of undisturbed skin and uninjured skin cells: before the method is considered to be like the growth factor read large eggs, can not penetrate without Injured or finished skin reaches the appropriate basal cell layer to increase cell replication, thus reducing cell aging. So finding a simple ingredient to achieve skin care is still needed today. SUMMARY OF THE INVENTION Unless otherwise defined in the present invention, all the technical and scientific terms used in the present invention are the same as those defined by those skilled in the art to which the patent belongs, although , , and methods and materials are the same as those mentioned in the present invention for practicing and testing the present invention, but the best method and material for the 1345571 will be described in detail later in the article "For convenience, the following names The 斶 definition will provide a description of the invention to make the invention easier to understand. Definitions of proper nouns Growth factors: including natural and recombinant protein growth factors, and biologically active protein fragments and analogs; these protein growth factors can reduce the aging of epidermal cells and skin. Individual protein growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), transformed cell growth factor (TGF_alpha), or protein fragments known to reduce epidermal cells and skin aging. analog. Collagen: is the main protein component of bones, cartilage and other connective tissues. Collagen is a microfibrous protein, a subunit of collagen and procollagen, including three polypeptides and its amino acid sequence is (glycine-XY)n. X refers to proline and Y refers to hydroxyproline. Skin: A film covering the human body, the skin layer including the epidermis layer and the dermis layer. Skin care products: Health and youth products used to keep human skin, or used to repair wounds and maintain skin integrity, including cosmetics and medicines; skin care products can be defined as daily use. 1345571 Effective in reducing aging (4): Fibroblast growth factor or a composition containing a cosmetically or pharmaceutically acceptable carrier, which can be applied to the skin to achieve the desired effect, for example, delaying the growth of the second type of skin, at this time The dose of the factor or composition. Antioxidant: in the sequence of the wild-type human acid fibroblast growth factor (IV) ν〇. The sulfhydryl group on cysteine (C-SiO is oxidized to produce a negatively charged Guji (4) (6), if substituted with serine Reduction, silk (4) record base (s_(6) and can no longer be oxidized. The complex structure of human skin is regulated by cytokines to maintain its softness, smoothness and elasticity. However, as the age increases, the cytokines will be less balanced and will lose money. Loss of elasticity and wrinkles. Skin care usually relies on signal factors to transmit messages. These include fluorescing fibroblasts (FGF_2 Qr bFGF)^f_growth liver (10)0ΓFGF-7) The main functions of the parent cell growth factor include: the regenerative capacity of aging tissue, the synthesis of collagen eggs from (four) force eggs, the growth of live retinoblasts, the promotion of epidermal cell growth and movement, the synthesis of hyaluronic acid and the promotion of wound healing. The main functions include: promoting cell-cell binding, promoting cell proliferation and differentiation, and activating epidermal cell growth and wound healing. The reason for the loss is collagen loss. Collagen is a structurally heavy egg of 1345571 in the animal body. The egg is compiled from the egg. The human protein, (10) of the county protein will be affected by the Base damage _, free private will only interrupt the original protein of the knee, it will also damage the fibroblasts that produce collagen; in addition, with the increase of age, the rate of collagen synthesis is far from the county. In order to reduce skin aging, it is a very important and common strategy to provide collagen; however, the molecular weight of collagen is too pure, and it does not pass through the skin of human skin, so the direct application of collagen on the skin cannot be added. Collagen, unless the county protein has been specially treated. The safety of collagen obtained from other animals is a must-have _ because there are many people and animals sharing _ disease 'repair: Niu Hai Zai __ Qing) or It is Japanese encephalitis of pigs; in addition, the antibiotics and residuals in economic animals are another worrying consideration. The collagen-rich amine sequence is divided into 21 types, and different types of prion protein are formed in different tissues. Collagen is produced by fibroblasts, so the development of A's strategy for the supply of collagen is to provide growth factor-stimulated collagen-producing fibroblast growth, which in turn produces protein to complement the prokaryotic protein. the goal of. The present invention provides a composition of the strip which is suitable for skin care, and which comprises an effective amount of human acid retinoic acid contact (FGF-1). 1345571 Acidic fibroblast growth factor, is an acidic fiber · Cytochrome has the activity of promoting fibroblast growth and mitosis; acid fibroblast growth factor will also stimulate other proliferation of __, and ship cell Egg self and elastin. A preferred embodiment is a composition comprising human-acid fibroblast growth factor, wherein human acid fibroblast growth comprises wild-type human fibroblast growth factor, recombinant human acid fiber _: _ growth g, mutant human fibrosis _ mother slit length factor; wherein, the wild-type human acid fibroblast-born amino acid sequence listed in seq id I provides human acid fibroblast growth factor to human county - High safety considerations, providing human acid fibroblast growth factor ship-female president, and further producing bioactive collagen cells to produce collagen; a preferred example is human acid fibroblast growth factor stimulation (1) Quantitative fibroblast growth target signal transmission pathway initiation, step-by-step promotion of fibroblast proliferation (2) keratinocyte growth factor signal transmission pathway initiation, and promote keratinocyte proliferation. In a preferred embodiment, the compositions of the present invention comprise collagen having the ability to regenerate skin, stimulate collagen synthesis, eliminate wrinkles, reduce aging, and maintain skin elasticity. BEST MODE FOR CARRYING OUT THE INVENTION The composition of the present invention further comprises a pharmaceutically or cosmetically acceptable carrier which allows the composition of the present invention to be in the form of an aqueous solution, an emulsion, a mask, a gel, an emulsion, a balm, an ointment, a liquid, a powder. Shape or other type that can be used for the subject or for internal use. The human acid fibroblast growth factor referred to in the present invention is analyzed by a polypropylene guanamine gel electrophoresis in the state of reduction of 1345571, and the result is a protein having a molecular weight of about 33 kDa and an acid value of about 7. Human acid fibroblast growth factor not only stimulates the growth of fibroblasts, but also stimulates the growth of epidermal cells. The proliferation of fibroblasts and epidermal cells is caused by the basic fibroblast growth factor signal transmission pathway and the signal transduction pathway of keratinocyte growth factor, respectively. The proliferation of fibroblasts is positively related to the production of collagen in the human body. Stimulating the proliferating fibroblasts by human acid fibroblast growth factor can effectively provide collagen, maintain the elasticity of human skin and reduce skin aging. Further promote the proliferation of epidermal cells, and enhance the regeneration ability of skin cells. The present invention further provides a shortened human acid fibroblast growth factor, that is, the second to fifteen amino acids in the sequence are absent, and this form of human acid fibroblast growth factor can be found in humans, that is, The amino acid sequence of human acid fibroblast growth factor, which is shortened by human acid fibroblast growth factor via protein degradation, is listed in SEQ ID N〇. The present invention also provides a mutant human acidic fibroblast growth factor which is highly resistant to oxidation and comprises amino acid sequences as set forth in SEQ ID NO. 3 and SEQ ID NO. The mutant preparation is based on the growth of wild-type human acid fibroblasts by substitution of cysteine in the amino acid sequence to serine. The amino acid sequence represented by SEQ ID NO. 3 is a cysteine at position 131 of the amino acid sequence of SEQ ID NO. 1 substituted with a serine, designed as C131S; SEQ ID 4 is represented by The three cysteines at positions 30, 97 and 131 of the amino acid sequence of SEQ ID NO. 1 were substituted with serine, and the design was called CUTS. Amino acid-substituted mutant human acid fiber 12 mother cell growth g) can reduce the attack of free radicals, add antioxidant capacity, and the half-life is longer than the wild type, that is, the speed of the surface decomposition is slow. . Compositions comprising mutant human acid fibroblast growth factor are applied to skin care to maintain longer and longer lasting fibroblast and epidermal cell proliferation. Accordingly, the present invention provides a polypeptide of human acid fibroblast growth factor, comprising the amino acid sequence represented by the Qing ID N0'4 (CHS) and a derivative thereof, which is an amino acid sequence of _ID Ν〇ι The three cysteine-substituted amino acids at the 30th, 97th, and 131st positions are highly resistant to oxidation. Human 6sl fibroblastic meristems, including wild-type, recombinant, and transgenic and derivative-based reds, are prepared by the following methods, which comprise (4) making recombinant recombination with the human heterofibroblast growth factor gene (6) The preparation of the host cell which can reproduce the recombination fineness and (6) the use of the heparin affinity column to purify the human fibrillar Liner melon from the scale of the host. The original human acid fibroblast growth factor gene is derived from humans. This gene can be a complete gene or a partial gene, as long as it can express the function of acidic fibroblast growth factor. The human acid fibroblasts are grown in a liver-based human-recombinant carrier, and the vector can be selected from bacteria, viruses, or rhymes. The recombinant vector carrying the human acid fibroblast growth factor gene is then transformed or transfected into a suitable host cell, such as a bacterium, yeast, insect cell or mammalian cell. The transformed or transfected host cells are cultured in an appropriate environment and exhibit a long history of human acid fiber. Separation and reduction of human face _ mother fine finances, using 1345571 is like the well-known seam and recorded column, such as · liver and sex column. The composition comprising the human acid fibroblast growth factor provided by the present invention can be applied to a skin care product. This skin care product can be applied to cosmetics or pharmaceuticals, including daily skin care. The composition may further comprise a component which is selected from the list of acceptable minerals. This product contains aqueous solutions, semi-solids and solids; such as lotions, lotions, masks, condensed 'emulsions, balms, ointments, liquids, powders or other types that can be used by secret agents. . Because of the younger and more pleasing appearance, it is the result expected by the method provided by the present invention. The main carrier is the recorded product or the money accepting body. Here, it refers to the non-toxic carrier of the conventional cosmetic or pharmaceutical. It can be more convenient when used on cosmetics or medicines, and can maintain the qualitative and bioavailability of acidic fibroblast growth factor when it is directly on the skin surface. For example, the acidic fibroblast growth factor is dissolved in an aqueous solution, dispersed or emulsified in a matrix, prepared into an aqueous solution according to the convenience of use, or mixed with a semi- (gelatin) or (iv) carrier to form a gelatinous or powdery form. , cream, lotion, lotion or the like. Suitable cosmetically or pharmaceutically acceptable carriers are the same as those known to those of ordinary skill in the art, including cosmetically or pharmaceutically acceptable liquids, creams, oils, lotions, lotions, gels. Or solid forms include cosmetic night creams, foundation creams, sunscreen creams, sunscreen lotions, hand lotions, foundations and foundations, masks and the like. This composition may contain other therapeutically active ingredients such as epithelial cell growth factor (EGF), platelet-derived growth factor (PDGF), or transformed cell growth factor 1.345571 (TGF-alpha). On the other hand, 'this composition may contain other ingredients conventionally added to cosmetics, such as flavors, estrogens, vitamins A, C, E and alpha hydrocarbyl keto acids such as pyroic acid, lactic acid or glycolic acid, lanolin , aloe vera, methylation or propane-based preservatives, pigments and the like. Suitable cosmetic or pharmaceutically acceptable carriers include water, petrolatum, petrolatum oil, mineral oil, vegetable oil, animal oil, organic and inorganic waxes, such as microcrystalline waxes, paraffin waxes, ceresin waxes; natural polymers like xanthanes, several Butyl, cellulose, collagen, starch and gum arabic, synthetic polymers like alcohol, polyols and the like discussed below. Since the non-toxic host carrier traits are mostly composed of a carrier which is easy to mix with water, such as a host cosmetic or pharmaceutically acceptable carrier which is easily mixed with water, it is not only included that one or more other suitable ingredients may be mixed, and Contains surfaces that can be transferred to a reduced rate, including hydrates, water-dispersible or water-soluble ingredients like 'lipids, tiny sponges, microparticles, micro-capsules, water-based ointments, water-soluble oils or An oil-soluble emulsion, gel or the like. If it is pre- _... or a plurality of added ingredients, it is usually found in the composition of the main age makeup. The types including the carrier include moisturizing lotion, moisturizing cream, perfume, buffer, pigment, preservative, vitamin ACE, Emulsifiers, dispersants, wetting agents, perfumes, emollients, apologies, coatings, antibacterial agents, anti-Japanese ingredients, enzymes and the like. Those skilled in the art can select the appropriate specific additions, including the type and quantity. [Embodiment] 15 1345571 The following examples are provided to explain the present invention and are not intended to limit the present invention. Example 1 (Α) Construction of wild-type acidic weimen-derived cell growth factor expression Characterization of human and bovine acidic fibroblast growth factor Gene coding, using oligonucleotide primers with restriction enzyme cleavage sites, to amplify existing complementary DNA clones via polymerase chain reaction; these restriction enzyme sites facilitate the insertion of genes into prokaryotic expression vectors In pET20b(+) (Novagen), the acid fibroblast growth factor for the selection, performance and purification of snails has been previously described (Partie ei aA Growth factor (1997) 14: 39-57). Human Acidic Fibroblast Growth Factor Complementary Deoxyribonucleic Acid (Chiu ei a/. Oncogene (1990) 5: 755-762) utilizes the forward primer BEF1 (5-TGA AGCCAT ATG GCTGAAGGG GA-3) and the reverse primer HBGF605 (5-AGA TCT CTT TAA TCA GAA GAG ACT-3) for proliferation;

The bovine acidic fibroblast growth factor complements deoxyribonucleic acid (also known as Nucleic Acid Res. (1988) 16: 10913), using the forward primer BEF1 (5-TGA AGC CAT ATG GCT GAA GGG GA-3) and Reverse primer BER3 (5-CAA CAG ATC TCT TTA ATC AGA GGA GAC-3) Zoom in. The conditions of the polymerase chain reaction were 10 mM Tris-HCh 50 raM potassium chloride, 1.5 mM magnesium sulphate, 〇· 〇i% Tween 20, 0.01% Triton X-100, 〇. 25 mM deoxynucleoside triphosphate 'primer 10 pm 〇 1 and 2 units of polymerase; reaction starts at denaturation temperature 95 ° C for 2 minutes' followed by 30 cycles of reaction, denaturation at 95 ° C for 1 minute, The primer is bonded at 42 °C 16 . The next i minutes (human _ fibroblast growth factor "5g. 〇, is the extension of the product at 1 C knives a c and the last 72 〇 c7 minutes extended. From the complement of bovine sputum fibroblast growth factor The deoxygenated nuclear nucleic acid is amplified to obtain the product of the shed test, and the gelatin gel is purified, and the (four) surface and the sputum cutting are cut, and the expression of the restriction enzyme 1 and the finely cut expression carrier b (1) is used first, thereby generating performance. The growth of bovine fibroblasts is pre-fine, called PETbQVFGF丨. In addition, the complementary deoxyribonucleic acid of human fibroblast growth factor is used as a template to amplify the products of 485 test-bases, which are then purified from acacia gum. First, access to the _escript vector after restriction enzymes such as I cleavage, and the recombinant vector received is then cleaved with restriction enzymes and lysin j, and the expression vector pET2 先 is used to access the restriction enzyme ship and the fine sputum J. In b(1), a vector expressing human fibroblast growth factor, called pEThiimFGF-1, is produced. (B) Constructing a mutant fibroblast growth factor 丨 expression vector in the 139th amine of the acid fibroblast growth factor amino acid sequence Base acid position, The tyrosine dog becomes stupid aminopropionic acid (human Y139F); in the homologous amino acid sequence of bovine and snail, it is mutated from phenylaminopropionic acid to tyrosine (burdock 39Υ and 蝾螈F139Y). The mutated step of the previously described human fibrinogen growth factor expression vector for human bovine and scorpion using the site-directed mutagenesis technology provided by ci〇ntech (Clontech) (Myers jm〇1 Chem

(1995) 270: 8257-8266), the positive selection primer used in these vectors was to change the position of the restriction enzyme enzymatic cleavage on the pET20b(1) vector to Spel using 5, GCC 17 1345571 ACT AGT AAC AGG ΑΤΤ-3 '; To mutate the reverse mutation primers of human, bovine and scorpion acid fibroblast growth factor, respectively, using 5'-CTTTCT GGC CM AGT GAG TCC-3, 5-CTT TCT GGC CGT AGT GAGTCC-3', 5' - CTT TTT GGC CAT AGT GGG TCC-3'. The selection primer was also used to change the cysteine at position i3 of human acid fibroblast growth factor to serine (human C131S), and to change serine at position 131 of bovine acid fibroblast growth factor to In the mutation reaction of cysteine (bovine S131C), the mutation was used for human C131S and bovine S131C', and its sequence was 5, -GGA CCG CGT TTG GAG CTC CCA TTC-3 , and 5' -GGA CCG AGT TTA CAC CTT CCG TTC-3, the vector after mutation is determined by restriction enzyme cleavage analysis, or directly determines the correctness of the vector's DNA sequence. The amino acid sequence of wild-type human acid fibroblast growth factor is listed in SEQ ID NO. 1, however, the human acid fibroblast growth factor of SEQ ID NO. 2 is also found in humans, that is, the shortened wild type. Human acid fibroblast growth factor, a product of human acid fibroblast growth factor decomposed by proteases, whose second to fifteen amino acids are deleted; SEq ID NO. 3 refers to SEQ ID The cysteine at position 131 in NO. 1 is substituted with serine and is designed to be called C131S; SEQ ID NO. 4 shows the position at positions 3, 97 and 131 of the amino acid sequence of SEQ ID NO. Cysteine is substituted for serine and is designed to be called CIIIS. 18 1345571 Example 2: Purification of the expression vector of acidic fibroblast growth factor protein acidic fibroblast growth factor, which was used to transform into the expression host of bacteria BL21(DE3)pLysS, and then according to the previous quotation (Partie ei < 3乂Growth factor (1997) 14: 39-57) Inducing protein expression and purifying it, in addition to 15 / zL extraction samples from different tubes separated by heparin affinity column at different time points, Perform 15% polyacrylamide oxime electrophoresis and silver staining to determine the protein content and purity. The first 29 amino acid sequences of the wild-type acid fiber of the snail, using the protein sequencing machine Pr〇tein

Sequencer 475A (PE Applied Biosystems, Foster City, CA) determined that the N-terminal amino acid sequence, as expected, was initiated by methionine and was cleaved during protein formation; Desorption-mass spectrometry (MALDI) to determine the molecular weight of the purified recombinant protein, LASERMAT 2000 mass spectrometer mass spectrometer (Finnigan MAT, San

Jose, CA) was also used for the analysis of the above molecular weight. The results of pre-estimated molecular weight, observed molecular weight and percentage error are shown below: human wild-type acid fibroblast growth factor (17 330. 0; 17 331 · 3; +0· 008%), human mutant acid fiber Maternal growth factor was 31 from 17 314 · 0; 17 311 · 2; -0· 016%) and _ mutant acid fibroblast growth factor f (1) 414.8; 17 41G.3; -G.G26%). Two protein sequence analyses and molecular weight _ mass spectrometers are used to determine and confirm the phase of each recombinant protein. Tianlang cattle's acid fibroblasts (10) DSyst·, Minne_is, MN), in the form of fine amino acids, ie amines with _155 positions in bovine acidic fibroblast growth factor Base acid (Norino acids 16-155). The results showed that the purified acid fibroblast growth factor 1345571 was 98% pure. Example 3: Cell mitosis assay

Swiss/3T3 cells were cultured in Dulbecco's-modified Eagle's (DMEM) medium supplemented with 1% bovine serum and antibiotics lincillin and streptomycin (penicillin/streptomycin), and 20,000 cells were separately dispensed. In a 24-well culture dish, when the cells reach 8 minutes, wash once with double the phosphate buffer (PBS) and add the low serum content of the culture medium (dmem, 0.5% calf serum, penicillin/ After 24 hours of culture with streptomycin, the cells were stimulated with recombinant human or bovine fibroblast growth factor for 2 hours in the presence of heparin (10 g/mL) and then added to the [3H] isotope-labeled thymus of 1 ACi. Pyrimidine (NEN), cultured for another six hours; bovine serum is 10% concentration as a positive control group, labeled cells are washed twice with one milliliter of phosphate buffered saline (PBS), and then 5% Rinse with cold tri-acetic acid for two times, and finally wash it twice with one milliliter of phosphate buffered saline (PBS), then dissolve the cells in 8. 8 ml of 〇·25 NaOH, and take 〇.1 ml. Beckman Scintillation counter The amount of radiation contained in its cells. Human fibroblast growth factor has the ability to cause Swiss/3T3 mouse fibroblasts to have a sickle split and is comparable to the recombinant bovine acidic fibroblast growth factor used in these studies. The most appropriate person, the thief, the virgin, the fascinating factor, can cause the Swlss/3T3 old __ cells to have a silk score (10) concentration per ml. 20 1345571 Example 4: Acidic lin Cell Growth Factor Media Fibroblast Growth Factor II and Keratinocyte Growth Factor Signal Delivery Paths Expressed with Spirulina and Human Keratinocyte Growth Factor Receptor (KGFR) The proliferation of cells is tailored to the occupational lining cell growth, but the sulphur-like heteroblast cell growth factor FGF-2 does not respond to the keratinocyte growth factor receptor. The keratinocyte growth factor KGF (also known as FGF-7) can inhibit the proliferation of 14 bone-path muscle cells that exhibit snails and humans. (4) 心肌 心肌 肌 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ α α α α α α α α α α α α α α α α α α α α α α α α The vector is transfected into the sputum myogenic cells. The vector containing the snail keratinocyte growth factor receptor in the ffib PBJ5 expression vector is pNkgfr, or the vector carrying the human keratinocyte growth factor receptor is pHKGFR. Transfected cells are cultured in culture medium with additional growth factors (heteroblastic growth factor, immunofibroblast growth factor or keratinocyte growth factor) or without additional growth factors. (4) Phototin test and tens of galactose activity test. Luciferin reports activity-resulting alpha-cardiac actin/luciferin activity, in addition to measuring the activity of the cell-derived galactosidase enzyme, which can be measured from the cell-free extract. Each group of transfected cells were cultured in the absence of additional growth factors. When 1.0' transfected cells were lined with (4) cytoplasmic receptor carriers, or 21 1345571 human keratinocytes were grown. The factor-gitter carrier pHKGFR, but obtained in the culture condition without growth factors, is compared with the postal itself, which is 72% and 62%, this, '. The MM14 cells of the non-proliferated towel will express the horn cell growth factor, the keratinocyte growth factor expressed by the sputum cells; i can activate the transfected „cell growth factor junction, and will partially inhibit the reporter _activation. County _ In Figure 3, it is the result of the experiment from the three catches. The results show that the mature mother _ will be activated when growing (four) fine charm fairy receptor and the inferior fibrillation fine factor long factor; but secret _ mother cell Fairy does not activate the keratinocyte growth factor receptor, and keratinocyte growth factor does not activate the fibroblast growth factor receptor. Therefore, 'acid fibroblast growth factor has clear activated fibroblast growth factor and keratinocyte growth. The activity effect of the factor-messogenic pathway. Example 5: Field (4) and dope-dimensional growth factor-mg of acidic fibroblast growth factor, and acid value 7.4 of Tris_HCl (maximum, / / chen Degree 20 rituals' and the final concentration of 1 〇 mM # gasification copper mixed in the total volume of 2 hurricane, in the room 'drink for 30 minutes' reaction in the addition of double SDS no reduction sample Liquid (including i〇mM EDTA) is terminated, and directly added to 15% of the polypropylene-amine gel, without boiling, colloidal electricity, the secret point of the ink dot method, miscellaneous The sub-protein can be seen on the nitric acid fiber paper. In some experimental towels, the gas-treated egg self-mass, the protein reducing agent disulfide domain is added to the final concentration (10) _, and 15% of the poly (four) is added Amine gel 22 1345571 before boiling 're-execution of Western blotting method. Fruits are shown in the 4' wild-type human and bovine acid fibroblasts. Liver and human mutant (10)s, bovine mutant (10) c-fibroblast growth factor, The ability to form a dimer can be tested by treating the egg with chlorinated steel. Because previous studies have shown that copper chloride induces human fibrosis - the mother's age (four) is divided into two (fine coffee) view

Chem (1992) 267:11307-11315) 'As expected, wild-type human acid fibroblast growth factor is in the absence of gasification, on-the-job miscellaneous _ molecular weight position, but in the presence of copper chloride Will be transferred to the molecular weight of the dimer (Figure 4, line i and line 2); interesting is the human mutant type cms, under the treatment of copper chloride, will form few dimers (such as the third Line and line 4) 'The 131st Wei-level of the silk field is the amino acid position of the human vitamin cell under the gasification copper treatment; in other words, 'these results also prove __Yueyiyi, town-level 3Q and cysteine 97, did not make a meaningful contribution in the formation of di-hybrids; and this observation county - regardless of wild-type and mutant smc bovine acid fibroblast growth factor , with an additional cysteine at the 61st position, forms a degree of dimerism similar to that of the human-type human acidic fibroblast growth factor (line 5 Na 8). Next, the fine egg is converted from the original sulphur thiosulfate to reduce the acidified fibroblast-treated long-factor protein, and the process of the dimer is a reversible reaction (lines 9 to 12). [Simple diagram] 23 1345571 Figure 1 shows the results of polyacrylamide gel electrophoresis (SDS_PAGE) analysis and immunostaining of recombinant acid fibroblast growth factor. (A) Recombinant acidic fibroblast growth factor FGF-1, which increased from 100 ng to 500 ng (100, 250, and 500 ng), was mixed with the same volume of bovine serum albumin and then executed. 15% polypropylene guanamine gel electrophoresis, followed by silver staining. (B) The figure is to transfer the 15% polyacrylamide film of (A) onto the master fiber paper, and use the anti-bovine peptide chain antibody (1:1000) for the immune reaction, the target protein Secondary antibody detection with wasabi catalase-bonded. The triangle indicates the slow-moving sputum and human recombinant FGF-1, and the arrow indicates the bovine recombinant FGF-丨, and the molecular weight of the protein is indicated on the left. Figure 2 shows that bovine and human recombinant acidic fibroblast growth factor cation (1) stimulates fibroblasts to undergo mitotic responses. When the Swiss/TO and Namu fine charms reach the eight-point full growth in the 24 holes, the weaving serum domain is 24, when Wei is not given serum; the cow's (solid circle) and the human (heart) The secret Η is directly added to the cells after the serum is hung according to the concentration of the silk, and then the cells are further cultured for 20 hours, then added! The radioactivity of the cells after 6 hours of ACi[3H] isotope calibration of the thymic nucleus was the average cpm value (soil SE) of the two replicate samples. Figure 3 shows that the acidic fibroblast growth factor mediator inhibits the MMU skeletal muscle muscle cell differentiation of the snail and human keratinocyte growth factor receptor KGFR; however, the fibroblast growth factor FGF-2 does not respond to KGFR. The cell culture of the machine was carried out without additional growth due to the publication of the heart column, plus FGM (open bar chart), additional wealth 2 (dot histogram) or the addition of 24 1.345571 group. Transfection of fine FGF-7 (straight-striped histogram) 'Every cell when 1. Bu Figure 4 shows fine shouting human and bovine, wild-type and money-type hybrid fibroblast growth factor FGF-Bu human wild Type (lines 2 and 9), human mutant C131S (lines 3, 4 and 1), bovine wild type (lines 5, 6 and 11) and bovine mutant S131c (lines 7, 8 and 12) In the absence of copper chloride (lines 1, 3, 5 and 7) or in the presence of copper chloride (lines 2, 4, 6 and 8 to 12). The reaction is carried out in the absence of dithiothreitol (DTT) (rows 1 to 8) or with dithiothreitol (lines 9 to 12), and the reaction product is brewed using unreduced 15% polypropylene. Amine gel electrophoresis analysis and transfer onto nitrocellulose paper 're-immunization with anti-FGF-1 antibody' target protein and then detected by ECL chemiluminescence reagent' protein molecular weight indicated as kDa. 25 1345571 Sequence Listing <110> Qiu Yingming <120> Acidic Vegivorous Growth Factor (FGF-1) for Skin Care <130> No <160> 4 <170>Patentln Version 3.1 <210&gt ; 1 <211> 155 <212> PRT <213> Human <220>

<221> PEPTIDE <222> (1)..(155) <223> FGF-1 wild type protein <400>

Met Ala Glu Gly Glu lie Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 15 10 15

Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30

Asn Gly Gly His Phe Leu Arg lie Leu Pro Asp Gly Thr Val Asp Gly 35 40 45

Thr Arg Asp Arg Ser Asp Gin His lie Gin Leu Gin Leu Ser Ala Glu 50 55 60 80

Ser Val Gly Glu Val Tyr lie Lys Ser Thr Glu Thr Gly Gin Tyr Leu 65 70 75

Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn Glu 85 90 95

Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 lie Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125

Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys Ala 130 135 140 lie Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 1 1345571 145 150 155 <210> 2 <211> 141 <212> PRT <213>Human<220><221> VARIANT <222> (1)..(141) <223> hFGF-Ι truncated type <400>2

Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr 1 5 10 15

Cys Ser Asn Gly Gly His Phe Leu Arg lie Leu Pro Asp Gly Thr Val 20 25 30

Asp Gly Thr Arg Asp Arg Ser Asp Gin His lie Gin Leu Gin Leu Ser 35 40 45

Ala Glu Ser Val Gly Glu Val Tyr lie Lys Ser Thr Glu Thr Gly Gin 50 55 60 80

Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro 65 70 75

Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn 85 90 95

Thr Tyr lie Ser Lys Lys His Ala Glu Lys Asn Tip Phe Val Gly Leu 100 105 110

Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gin 115 120 125

Lys Ala lie Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 <210> 3 <211> 155 <212> PRT <213>human <220><221> MUTAGEN <222> 1)..(155) <223> hFGF-1 C131S <400> 3 1345571

Met Ala Glu Gly Glu lie Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15

Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30

Asn Gly Gly His Phe Leu Arg lie Leu Pro Asp Gly Thr Yal Asp Gly 35 40 45

Thr Arg Asp Arg Ser Asp Gin His lie Gin Leu Gin Leu Ser Ala Glu 50 55 60

Ser Val Gly Glu Val Tyr lie Lys Ser Thr Glu Thr Gly Gin Tyr Leu 65 70 75 80

Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn Glu 85 90 95

Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 lie Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125

Asn Gly Ser Ser Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys Ala 130 135 140 lie Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 <210> 4 <211> 155 <212> PRT <213>Human<220><221> MUTAGEN <222> (1)..(155) <223> hFGF-lCmS <400>4

Met Ala Glu Gly Glu lie Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 15 10 15

Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Ser Ser 20 25 30

Asn Gly Gly His Phe Leu Arg lie Leu Pro Asp Gly Thr Val Asp Gly 35 40 45

Thr Arg Asp Arg Ser Asp Gin His lie Gin Leu Gin Leu Ser Ala Glu 50 55 60

Ser Val Gly Glu Val Tyr lie Lys Ser Thr Glu Thr Gly Gin Tyr Leu 65 70 75 80

Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn Glu 3 95 1345571 85 90

Glu Ser Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110

He Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125

Asn Gly Ser Ser Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys Ala 130 135 140

He Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 4

Claims (1)

1345571 w years, months / ρ days repair (more) original ten, application w patent range 1. - a cosmetic composition for skin care, including an effective dose of human acid fibroblast growth factor " human acid fibroblast growth The factor is selected from the group consisting of recombinant human acid fibroblast growth factor SEQ ID NO. 2 and mutant human acid fibroblast growth factor 1]} NO. 3 and SEQ ID NO. 2. The cosmetic composition according to the patent scope 帛i, wherein the human acid fibroblast growth factor is a protein f having a molecular weight of about 17.3 kDa, which is analyzed by a polyacrylamide gel electrophoresis and analyzed under reduced conditions. . 3. The cosmetic composition according to item 1 of the patent application, which further comprises a conjugated protein. 4. According to the scope of the patent application! A cosmetic composition having skin regenerating ability, stimulating collagen synthesis, eliminating wrinkles, reducing aging, and maintaining skin elasticity. 5. The cosmetic composition according to claim 1 of the patent application, which further comprises a cosmetic or a pharmaceutically acceptable carrier. 6. The cosmetic composition according to claim 1 of the patent application, which is in the form of an aqueous solution, an emulsion, a mask, a gel, an emulsion, a fragrance, an ointment, a liquid, a powder or the like, which can be used for the main body or the internal use. The type of acceptance. 7. The cosmetic composition according to claim 1, wherein the human acid fibroblast growth factor is produced by the following method: (a) producing a recombinant vector carrying a human acid fibroblast growth factor gene (b) Preparing a host cell expressing the recombinant vector and (c) purifying the human acidic fibroblast growth factor from the cultured host cell. 8. The cosmetic composition according to item 7 of the patent application, wherein the recombinant vector is selected from the group consisting of bacteria, 1345571 virus or phage vector. 9. According to the patent application No. 7 Lion Cosmetic Composition, (4) the main cell line is selected from the group consisting of bacteria, yeast, insect cells or mammalian cells. 10. A polypeptide of human acid fibroblast growth factor having the sequence of SEQ ID NO. 4; wherein SEQ ID NO. 4 and a derivative thereof are amino acid sequence of SEQ Π) NO. The substitution of three cysteines at 97 and 131 positions into serine, designed as nns, is highly resistant to oxidation. U. According to the patent of the scope of the patent application, the polypeptide of the invention has a higher anti-oxidation property than the wild-type human acid fibroblast growth factor SEQ Π) NO. i, and thus has a longer half-life.