TW200804592A - Compositions and methods for dual therapies of hair graying and balding in follicular - Google Patents

Abstract

The present invention provides comprehensive compositions for treating problems associated with hair graying and balding via the incorporation of: (i) the cell growth factor of HSCF to induce the migration of melanocyte stem cells and keratinocyte stem cells and then to increase the growth of melanocytes and keratinocytes in hair follicles, (ii) a formula of amino acids and vitamins to provide the nutritional factors for hair growth and pigmentation, and (iii) minoxidil to enhance the function of HSCF on hair re-growth. The compositions comprising at least one of (i), (ii) or (iii) are administered on skin and/or scalp through liposome in the follicular delivery systems, including penetration enhancers and suitable carrier bases. The compositions packaged in liposome in the follicular delivery systems in this invention has been proven to reach the dermis from the skin surface within 15-30 min.

Description

200804592 IX. INSTRUCTIONS: [Technical Field] The present invention provides a main compound composition for treating gray hair and alopecia with double effects, and the main active ingredients include (1) hair stem cell factor ( HSCF) induces melanocyte stem cells (MSC) and keratinocyte stem-alls (KSC) migration, thereby increasing melanocytes and epidermis in hair fo〇mdes Growth of cells (keratinocyte); (8) special solutions containing a variety of amino acids and multivitamins to provide the nutrients needed for hair growth and pigmentation; (out) minoxidil to increase HSCF in hair regeneration Any one of the above (i), (9) or (iii) is embedded in a liposome or a basal colloid, and can be delivered into the hair follicle via a hair follicle delivery system such as scalp application or ultrasonic introduction. Therapeutic effect, the present invention has been noted: The liposome can reach the dermis layer within 15-30 minutes via skin application, and HSCF is The liposome can retain activity for at least U years. [Prior Art] In the hair care market, there are no double-effect products such as gray hair and baldness. Although there are many products for treating baldness, the products for treating gray hair are almost zero. When the hair is dry and lacks the pigment component, gray hair is formed, and there are many reasons for the formation, including: lack of tyrosinase activity in melanocytes in the hairy sputum, melanocytes and keratin, ', month The interaction between the father has decreased, or the melanin stem cells in the pot area migrated to the mastoid area. The study has indicated that stem cell factor (SCF) and insulin growth factor (insulin) Growth fact〇r, iGF4) can stimulate the melanin dry fine 200804592 cells and keratinocytes to migrate to the mastoid region for proliferation (1'2), respectively, forming melanocytes (3,6,9) and keratinocytes (12 '13'14,15). The melanin of hair is secreted by melanocytes (12, (6), the dried keratin protein is secreted by keratinocytes. When the mouse SCF gene is mutated, CSZ7ST), it will show Albino, infertility, anemia (1,5), thus understanding the importance of SCF to hair pigmentation (7,13). In view of this, the present invention uses genetic engineering methods to select human SCF gene and IGF. -I base For the first time, the two were chimeric together to form the HSCF-II gene, which was expressed in vitro and purified to simultaneously stimulate the migration and proliferation of melanin stem cells and keratinocytes in the hair follicle. However, the SCF gene and protein were purified. There are US patent cases passed (3) Publication No. : 20050080250, Application Serial No. 10/620?6425 filed: July 16? 2003· Methods of stimulating growth 〇f str〇mal ah Ming is advanced SCF and IGF] The HSCF4I gene was synthesized and deepened, and then its effect on hair treatment was confirmed by animal experiments. At present, there are many products for the treatment of money, including the topical _ and the mouth and clothing are more effective, and the health products are Contains the sensitive gamma inGxidil) compared to ^ just, but the effect (10) ^. Stem has been shown in the pattern of albino and bald animals (10): HSCF improves filling and significantly enhances -. Interesting alone makes Guan Xiaozhuang (four) side, in addition, can also improve the gray hair. The invention has three types of age: (i) HSCF; (8) - Juyi, contains ammonia" Wei Weisheng «formula; and (d) minGXidil, Sanguanlang _, for the first time Prove that the dual efficacy of = t, can be used to treat the main problems of human hair follicle aging. It can be seen that there are still many shortcomings in the above-mentioned methods of treating gray hair money. It is not a designer of 200804592, and it needs to be improved. In view of the above-mentioned advantages of the above-mentioned conventional methods, the inventors of the present invention have made innovations and innovations, and after years of painstaking efforts and research, they have finally successfully developed and completed the treatment of gray hair and baldness through the hair follicle conduction system. Things and methods. [References] 1. Broudy, V. C. (1997) Stem cell factor and hematopoiesis· Blood 90: 1345-1364 2. Botchkareva, NV? M. Khlgatian, BJ Longley, VA Botchkarev, BA Gilchrest, 2001. SCF/ F-BIT signaling is required for cyclic regeneration of the hair pigmentation unit. FASEB J. 15(3): 645-658. 3. Botchkareva, NV, VA Botchkarev, BA^Gilchrest. 2003. Fate of melanocytes during development of the hair Follicle pigmentary unit. J. Investig. Dermatol. Symp. Proc, 8(1):76-79. 4. Frankel, SK? BM Moats-Staats, CD Cool, MW Wynes, AD Stiles, DW Riches. 2005. Human insulin -like growth factor-IA expression on transgenic mice promotes adenomatous hyperplasia but not pulmonary fibrosis. Am. J. Physiol. Lung Cll Mol. Physiol. 288(5): L805-812. 5. Guyonneau, L.5 F. Murisier, A. Rossier, A. Moulin, F. Beermann. 2004. Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice. Mol. Cell Biol. 24:3396-3403. 6. Hemesath, TJ? ER Price, C. Take Moto, T. Badalian, DE Fisher. 1998. MAP kinase links the transcription factor microphthalmia to c-kit signaling in melanocytes. Nature 391:298-301. 7· Ito, M·, Y· Kawa, Η· Ono, M. Okixra, T. Baba, Y. Kubao, S. I. Nishikawa, M. Mizoguchi, 200804592 1999. Removal of stem cell factor or addition of monoclonal anti-c-kit antibody induces apoptosis in murine melanocyte precursors. J. Invest. Dermatol 112(5): 796-801. 8. Jiang, X·, 0· Gurel, EA Mendiaz, GW Stearns, C. L. Clogston, H. S. Lu, TD Osslund, RS Syed, KE Langley, WA Hendrickson 2000. Structure of the active core of human stem cell factor and analysis of binding to its receptor kit. EMBO J. 19:3192-3203. 9. Nishimura, EK, SA Jordan, H. Oshima, H. Yoshida, M. Noriyama, IJ Jackson, Y. Barrandon, Y. Miyachi, SI Nishikawa. 2002. Dominant role of the niche in melanocyte stem-cell fate determination. Nature 416: 854-860. 10. Mager, M·, R· Pause, N Farjo, S. Muller-Rover, EMJ Peters, K. Foitzik, D. J. Tobin. 2004. Limitations of human occipital scalp hair follicle organ culture for studying the effects of minoxidil as a hair growth enhancer. Exp. Dermatol. 13:635-642. 11. Mol, C. D·, K· B. Lim, V. Sridhar, H. Zou,, E. Υ· T. Chien,, B. C. Sang, J. Nowakpwski, DB Kassel, 3 CN Cronin, D.E. McRee. 2003. Structure of a c-kit product complex reveals the basis for kinase transactivation. J. Biol. Chem. 278:31461-31464. 12. Panteleyev, AA, CAB Jahoda, AM Christiano. 2001. Hair follicle predetermination. J Cell Sci. 114:3419-3431. , 13. Peters. EMJ5 DJ Tobin, N. Botchkareva, M. Maurer, R. Paus. 2002. Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit Expression. Histochem. Cytochem. 50:751-766. 14. Peters, EM J., M. Maurer, V. A. Botchkarev, K. deM. Jensen, P. Welker, GA Scott, R. Paus. 2003. Kit Is expressed by epithelial cells in vivo. J. Inves t. Dermatol. 121:976-984. 15. Philpott, MP5 DA Sander, T. Kealey. 1994. Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an 200804592 important J. Invest. Dermatol. 102(6): 857-861. 16. Sulaimon, SS, B. E. Kitchell. 2003. The biology of melanocytes. Vet. Dermatol. 14(2) :57-65. 17. Zhang, Z.? R. Zhang, A. Joachimiak, J. Schlessinger, XP Kong. 2000. Crystal structure of human stem cell factor: Implication for stem cell factor receptor dimerization and activation. Proc. Natl Acad. Sci. USA 97:7732-7737. 18. Park, WS, CH Lee, BG Lce? IS Chang. 2003. The extract of ThujQe occideutalis semen inhibited 5 a-reductase and androchronogenetic alopecia of B6CBAFl/j hybrid mouse. J. Dermatol. Sci. 31:91-98. SUMMARY OF THE INVENTION The present invention is the first dual treatment of hair gray hair and baldness. The main components are divided into three types: (i) HSCF; (ii) - special solution, Contain Amino acids and vitamins and other recipes; and that is) mmoxuM. In vitro culture mode of mouse hair follicles, as well as smear in male alopecia mouse mode and albino mouse model proved that the three groups were used in the best time, which can solve the problem of filling and white hair reduction at the same time. It can treat the main problems of human hair follicle aging. The invention recombines three human growth factor proteins f by gene-virtual technology, including: milk, IGFI HSCF-II' colonization from several gene sequences, protein-poor performance and pure-like diagrams. These purified protein activities were tested in different cell lines, respectively (Figures 5 and 9). HSCF_n is the first invented by the invention. 'SCF and coffee two _ shouting, can (10) E. coli load - Nida Dali expresses egg self-quality, and separates TF4 and 3Τ3 cell milk and protein activity, confirming that HSCF-II is present By means of the door, the work, and the white (Figure 13). Furthermore, HSCF4I has better advantages in industrial production, because it can save Chengtai, inserts, and cost, and the time is good. HSCF_n on hair follicles The main function is 200804592. At the same time, the melanin stem cells and keratinocytes in the hair follicles are induced to migrate to the mastoid, which promotes the proliferation of melanocytes and horn cells, and forms pigmented hair shaft. It can be confirmed by animal model. White-haired mice regenerate pigmented hair (Fig. 14). i is the first of its kind in the present invention, which is obtained by hybridizing two different strains of small gas (C3HXBalb/C), whose color is private, which is simulated. The genotype of human aging. Because the melanocytes in the aging of the hair follicles, the important key enzymes of the ammonia-cycling, the secret money, the silkworm, the aging of the stomach, or the treatment of whitening drugs, the shaped Lysinase gene +/ Tyr) The white hairs are produced by the fine enzymes of the valley, which is similar to the whitening drugs used by humans due to aging. The whitening drugs used in the month are 1-2〇0/. For example, (4)(10)(10) and _c 〇lic acid sigma scale (with whitening gel _), applied to the back skin of albino mice hair removal, even for 47 days will make the regenerated hair appear white, because whitening gum will inhibit the tyrosinase Activity. This is already clear. And Bei·HSCF-II does change the color of the hair significantly, returning the white color to black (Fig. 14) 'and testing the content of melanin in the hair, also significantly improved (Figure 15) The active ingredient min〇xidil contained in the Luojian quotient has been used for the treatment of androchronogenetic alopecia (AGA) for many years. It is now theoretically inferred that minoxidil can open the potassium channel on the cell membrane to promote nutrients or messages. The input of the factor. Although it is effective for male baldness, it can only prevent the alopecia from continuing, has limited effect on increasing the amount of hair and density, and has no effect on the treatment of white hair. The present invention utilizes HSCF and an amino acid-containing Vitamin The special solution of the formula can enhance the effect of using min〇xidil alone, which is confirmed by the hair follicle culture in vitro and in the experiment of the hairy mouse (Fig. 13 and Fig. 16d). Because the function of HSCf on the hair follicle 10 200804592 is to stimulate dryness _ migration And proliferation, not only effective, but also effective in the treatment of white hair. In order to prove that the above three components have a positive impact on the male charge, refer to the report of Park et al. 8) to establish a male _ nuclear mode to C57BL/6: The female mouse (female) χ CBA/; the male mouse _4 hybrid mouse (named B6CBAFl / j hybrid mouse singular injection male (10) mg / mice / day) up to 3, the local injection site will be _ hair. Applying the ingredients in three compositions to the bald hair does significantly grow new hair (Figure 16). The HSCF growth factor of the present invention belongs to a protein and is highly susceptible to loss of activity at normal temperature. If it is to be stored in a room temperature as a skin care product, it must be embedded in a special material. The present invention uses a simple and inexpensive material to make a liposome. HSCF can be wrapped into micro-nano size to help absorb and penetrate the skin. It can also help to penetrate (4) such as ultrasonic waveguides or micro-current devices, and accelerate the penetration of HSCF micro-lipid into the dermis. Hair follicle parts. These micro-lipid materials (including lipid lipids, lipoproteins, fatty acids and fatty alcohol fatty acids and fatty alc〇hd, surfactants detergents, Sf-type alcohols, glycerin giyc〇is, mineral oil mineral 〇il, etc.) and help infiltration The apparatus and the like all contribute to the absorption and utility of the three component formulations of the present invention applied to the skin, and are referred to as a hair follicle delivery system. The hair follicle delivery system of the present invention has been confirmed that the microlipid particle size is 50 nm - 2 μηι (Fig. 17a), and it can penetrate into the dermis layer after 15-30 min application on the skin (Fig. 17b) 'and HSCF is slightly lipid. The body can retain activity for at least 3 years. [Examples] · Materials and methods of Example 1 (1) Expression and purification of human stem cell growth factor (SCF) Α · gene construction 200804592 Human stem cell growth factor (SCF) is derived from human chromosome 12q22-12q24 The S1 locus encodes that soluble SCF and transmembrane forms of SCF are derived from different RNA splicing, both forms of SCF are biologically active, and SCF248 contains exon 6 (exon 6) There are a total of 165 amino acids, which can encode a proteolytic cleavage site, which is after Alanine, thus giving soluble SCF. The SCF220 lacking exon 6 encodes a transmembrane form of SCF. In SCF220, the amino acid 149-177 is replaced by glycine (Glycine) (as shown in Figure 1 (8)). The soluble SCF is obtained from the total RNA of the human placenta by reverse transcription polymerase chain reaction (RT-PCR). The primer used is: SCF primer - F (BamHI) 55cgggatccatgaagaagacacaaacttggattc 35 R (Xhol) ·· 5 'ccgctcgagaaccacacaatcactagtttcag 3, PCR reaction conditions and products shown in Figure 丨(b), the cdnA (495 bp) sequence of soluble sCF is shown in Figure 2 and SEQ ID NO: 1, which encodes the amine of 165 The base acid sequence is shown in Figure 3 and SEq ID NO: 2. 〇·protein expression (pr〇tein expression) The results of protein expression of soluble SCF are shown in SDS_PAGE and western blot of Figure 4. C. Protein purification (pr〇tein purification) The plastid pET24a-SCF carrying the soluble SCF cDNA sequence was transfected into competent E. coli cells for expression (see 2/-C〇donplus_£:·(7)/ζ·), The cells were placed in 5 ml of LB medium containing % 12 200804592 kanamydn, shake cultured overnight at 37 ° C, and the 5 mi medium was added to 500 ml of LB medium containing 35 pg/ml kanamycin, and continued at 37°C. The armpits were shaken for about 3 hours until the ODwo absorbance reached 0.5-1; then the protein expression was induced with isopropylthiogalactoside (IPTG), placed at 3T>CTM, and then collected by centrifugation. The cells were resuspended in 50 ml of phosphate buffered saline (PBS, pH 7.5) containing 1 〇〇pg/mi lysinase, and the suspension was placed at 4 C. After 30 minutes, it was shocked by ultrasound on the ice (shock 5 sec, 1 second interval, 15 times in total), and then centrifuged at 10,000 g for 10 minutes. The pellet was resuspended in 50 ml of IB Wash Buffer (20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100) and centrifuged again at 10,000 g for 10 minutes. The above procedure was repeated three times. The supernatant was removed, and the cells were resuspended in 25 ml of 8 Μ urea buffer containing 20 mM Tris-HCl, 0.5 M NaCl, pH 7.8, and the suspension was placed at 4 ° C overnight to dissolve the solution. The cells were centrifuged for 30 minutes at 15, 〇〇〇g, the supernatant was removed, and the protein was purified by a His-Bind resin column, and the purified protein was buffered with 9 times volume of rapid renaturation. Diluted with a solution (rapid refolding buffer, 2·5 M urea, 20 mM Tris_HCl, 0·01 mM EDTA, 2 mM GSH, 0.2 mM GSSG, 0.02% sodium azide, 0.2 M arginine, pH 8.5), placed at room temperature 48 After an hour, the mixture was concentrated 10 times by ultrafiltration, and 1000 ml of refolding buffer (20 mM Tris-) containing a descending gradient of urea (2-0 Μ) at 4 °C. Dialysis with HCl, 0·01 mM EDTA). D. Activity analysis The purified protein was dissolved in PBS and added to each well of microtiter plates containing ΙΟΟμΙΤΚΙ cells (1 x 13 200804592 l〇4 cells/ml), each well was ΐμΐ The protein was purified, and the cells were washed 3 times with PBS beforehand and contained 10% fetal calf serum (FCS) and 〇_〇8 ng/ml interleukin-3 (IL-3). RPMI 1640 medium was resuspended; after culturing the cells for 48 hours, each well was added to 10 μΐ of Alamarblue for further 4 hours, after which the fluorescence was measured at an excitation wavelength of 530-560 nm and an emission wavelength of 590 nm. The results of SCF activity test in the D-FI cell line are shown in Fig. 5. (2) Expression and purification of human Insulin-like Growth Facto I (IGF-I) A·gene construction The human IGF-I cDNA sequence of 210 bp in length was obtained by RT-PCR of total RNA from human placenta. The primer used was : IGF-I primer : F (BamHI) : 5' cgggatccggaccggagacgctctgcg 3, R (Xhol) : 5' ccgctcgagagctgacttggcaggcttga 3' PCR reaction conditions and products as shown in Figure 1 (c), human IGF-I cDNA (210bp) sequence As shown in Figure 6 and SEQ ID NO: 3, the amino acid sequence of the encoded 70 is shown in Figure 7 and SEQ ID NO: 4. Β·protein expression (pr〇tein eXpressi〇n) The results of protein expression of human IGF-I are shown in SDS-PAGE and western blot of Figure 8. C·Protein purification (pr〇tein pUrmcat|〇n) The plastid pET24a-IGF-I carrying the human IGF_I CDNA sequence was transfected into E. coli competent cells for expression (and L27-codonplus 〇/〇, the cell Placed in 5 ml of LB medium containing 35 pg/ml 14 200804592 kanamydn. Incubate overnight at 37 ° C, then add 5 ml of the culture solution to 500 ml of LB medium containing 35 pg / ml kanamycin, and continue at 37 The culture was shaken at °C for about 3 hours until the ODwo absorbance reached 0.5-1; then the protein expression was induced with 〇5 mM IPTG, and after 6 hours at 37 ° C, the cells were collected by centrifugation and the cells were resuspended at 5 〇. Ml In PBS (pH 7.5) containing 100 pg/ml lysozyme, place the suspension at 4 ° C for 30 minutes, then vortex with ultrasound on ice (shock for I·5 seconds, interval 1 second, 15 times in total) ), centrifuge again for 10 minutes at 10,000 g. Precipitate with 50 ml IB wash buffer (20 mM Tris_HCl, pH 7.5, 10 〆)

mMEDTA, l0 / 〇 TritonX-100) resuspended, and centrifuged again for 5 minutes, then repeat the above steps three times, remove the supernatant, and contain 20 mM Tris-HCl, 0.5 M NaCl, pH 25 ml. The bacteria were resuspended in 7.8 of 8 M urea buffer, and the suspension was placed in a seperator to dissolve the cells; after centrifugation at 15,000 g for 30 minutes, the supernatant was removed and passed through a His_Bind resin tube. The column was used to purify the protein. The purified protein was eluted with 9 times the volume of rapid refolding buffer (2.5 M urea, 20 mM Tris-HCl, 0.01 mM EDTA3 2 mM GSH, 0.2 mM GSSG, 0.02% sodium). Azide, 0·2 M arginine, pH 8.5) diluted, placed under the '48 hours after phoenix' to concentrate the mixture in a film ultra-transition, and at 1000 ° containing a drop gradient of urea at 4 ° C (descending gradient, 2- 0 Μ) was dialyzed against a refolding buffer (20 mM Tris-HCl, 0.01 mM EDTA). D·activity analysis The purified protein was dissolved in PBS and added to a microtiterpia (four) containing 1 μl of 3T3 cells (1 χ l〇4 cells/ml) per well. Purified protein, the cells were washed 3 times with PBS and resuspended in DMEM medium containing 1% FCS; 15 200804592 After incubating the cells for 48 hours, each well was added with 10 μΐ Alamarblue for 4 hours, then Fluorescence was measured at 530-560 nm excitation wavelength and 590 nm emission wavelength; human IGF-I activity test results in 3T3 cell line are shown in Figure 9. (3) Human hair stem cell factor _II (Human hair stem cell factor-II, HSCF-II) expression and purification Α·Gene constructs a 759 bp human hair stem cell factor _n (HSCF-II) cDNA sequence that binds to a 510 bp human SCF cDNA, 39 bp Length of binding peptide (linking peptide), and 210 bp length of human IGF-I cDNA, and insert these DNA into pET24a vector to express protein; PCR reaction conditions and products shown in Figure 1 (4), human hscf-h The cDNA (759 bp) sequence is shown in Figure 1 and S. As shown in EQ ID NO: 5, the amino acid sequence of the encoded 253 is shown in Figure 11 and SEQ ID Να 6. Β·protein expression (pr〇tein expression) The result of protein expression of human HSCF-II is as follows: Figure 12 shows SDS-PAGE and western blot. C. Protein purification (pr〇tein pUrificatjj〇n) The plastid pET24a-HSCF-II carrying the human HSCF-II cDNA sequence was transfected into expression. The E. coli competent cells were used (see 2hcodon plus pent. co/〇, the cells were placed in 5 ml of LB medium containing 35 Kg/ml kanamycin, shaken overnight at 37 ° C, and then added to 5 ml of the culture solution. 500 ml of LB medium containing 35 Pg/ml kanamycin, and continued at 37. (: shaking for about 3 hours until the QD5% absorbance reached 〇·5-ι; then induced by 〇5mM IpTG 16 200804592 protein expression, set After 6 hours at 37 ° C, the cells were collected by centrifugation, and the cells were resuspended in 50 ml of PBS (pH 7.5) containing 100 pg/ml lysozyme, and the suspension was placed at 4 ° C for 30 minutes, then on ice. Surge with ultrasound (oscillation for 1.5 seconds, 1 second interval, 15 times in total), then at 10,000 g Heart for 10 minutes. The sediment was resuspended in 5 〇 ml EB wash buffer (20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100), and centrifuged again for 1 〇g, 〇〇〇g, and the above steps were repeated. Three times, the supernatant was removed, and the cells were resuspended in 25 ml of 8 M urea buffer (ureabuffer) containing 20 mM Tris-HCl, 0.5 M NaCl, pH 7.8, and the suspension was placed at 4 ° C overnight to dissolve the solution. The cells were removed by centrifugation at 15, 〇〇〇g for 3 minutes, and the supernatant was removed and purified by His-Bind resin column to purify the protein with 9 times volume of rapid refolding buffer (rapid) Refolding buffer, 2.5 M urea, 20 mM Tris-HCl, 0.01 EDTA, 2 mM GSH, 0.2 mM GSSG, 0.02% sodium azide, 0·2 M arginine, pjj 8.5) diluted, placed at room temperature 48 (four), _ bribe The mixture was concentrated iQ times and dialyzed against 4 C in 1000 ml refolding buffer (2 fold mM Tris_Ha, 〇〇1 should be EDTA) containing a urea gradient (descending inhibition, 2_μμ) . (4) In vitro (9)W (4) culture of hair follicles F〇1Ude 〇 _ Preparation (10) F1 mice that were crossed with BALB/C were used for hair follicle test at 8th week; hair follicles could be induced by removing scorpion Long-term hair growth (an-rush (four), 3 days after pulling out the mouse, 'sacrifice the money and take its hair follicles, confirm that each hair follicle is in the early stage of anagen, carefully remove the subcutaneous fat and the connection around the view (eapsule) _ after the hair follicle The upper hair (4) quasi-hair follicle edge line is aligned for the subsequent measurement of hair growth (as shown in Figure 13). The above hair _ each material contains · μ1 _ % _ _ _ 嶋 , , , 核 2008 2008 2008 2008 100 cells/ml penicillin and 0.1 mg/ml chlortetracycline (Sigma) were incubated for 12 days at 37 ° C, 5% CO 2 plus 95% air in a saturated humidity environment; negative control hair follicles Cultured in PBS (pH 7.4) containing 8 g/L NaQ, 0.2 g/L KC Bu1·44 g/L Na2HP04 and 0.24 g/L KHJO4; positive control hair follicles were cultured in propylamine containing 5-100 mg/L Alanine, arginine, asparagine, aspartic acid, cysteine e), cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine (lysine), methionine, phenylalanine, proline, serine, threonine, trypt〇phan, tyramine Acid (tyrosine), valine, and 〇i_i〇mg/L ascorbic acid (vitamin c, asc〇rbic acid), biotin (vitamin h, biotin), pantothenic acid (vitamin B5, pantothenate), chlorination Choline chloride, ergot calciferol (vitamin D2, erg〇calcifer〇1), folic acid (f〇lic add), inositol (inositol), sulphate (hydrogen carbamazepine) (vitamin K3, menadione sodium Bisulfate), nicotinamide (vitamin B3, niacinamide), pyridoxal hydrochloride (vitamin B6, pyridoxai HC1), riboflavin (vitamin B2, riboflavin), tocopherol (vitamin E, t〇c〇pher〇1) , thiamine (vitamin Bl, tlnamine), vitamin A (vitamin A), vitamin B12 (vitamin B12) 〇5 test component for individual treatment 1〇_1〇〇 〇 μΜπ^ηοχϋ 1〇-1〇〇〇 ng/mi SCF, 11〇〇〇 ng/ml IGF-I, 1-1000 ng/ml HSCF, and HSCF combined with 诎. Each set of experiments was repeated 10 times, the fresh medium of the same formula was changed every other day, and the length of hair growth was measured from the edge line of the hair follicle on the 12th day; the whole experiment was repeated 3 times. Hair follicles were obtained under inverted optical microscope (Lieca DM, IL) and captured by CCD 18 200804592 like 'connected to a computer with Northern Eclipse image system software, hair length using SigmaStat pr〇gram (2〇〇 2) Duncan, s new multiple range test by one way analysis of variance (ANOVA) o (5) Living gray-haired animal model gray-haired animal model is used The hair was stimulated to become gray, and the effect of the formulation of the present invention on hair pigmentation in vivo was tested; the animal model was C1H and BALB/C hybrid mouse. The F1 progeny 'δ玄 F1 progeny after hair removal' to contain u〇 After 7 days of continuous application of % hydroquinone and 1-20% glycolic acid whitening gel, the regenerated hair of the treated area appeared white, and the melanin cells in the hair follicle aging were prone to lack of tyrosinase activity, and tyrosinase mainly formed melanin. An important key enzyme, and the whitening gum inhibits the activity of tyrosinase. The gray-haired model animal established by the inventor of the present invention, the parental BALB/C mouse has the same type of shouting rying gene / Ty〇 and white minus the other - parental C3H mouse has the same nutrient reduction enzyme gene. However, it appears brown, so its F1 narrative generation is difficult __fine (T〆 / plus _), the fi progeny in the treatment of whitening _, is extremely lack of ammonia - and white hair, the situation is similar to human White hair produced by aging; therefore, if hscf can make gray hair animal model hair color Ai HSCF should also have the same effect on human gray hair. In this test, a total of 3 groups including: untreated wild species (four) coffee, including unused whitening gum and untreated liposome, were used as a negative control group; whitening gum was treated as well. The surface of the pBs was used as a positive control group; and the gray-white model animals (2〇〇pg/ml) which were still excised by Qiu were treated as the hscf treatment group after the treatment of whitening gum. Each group had $19 200804592 mice' total experiment repeated 3 times. Before topical application of the drug on the third day, the back hair of the test animal (about 2x2 cm2 size) was depilated with tweezers, and the whitening gel was treated on the second day of the second day. The treatment week was 'once the same day' in the morning with 5〇μ1 For example, (10) coffee treatment of hair removal site, once a day for a total of 2 weeks, more than a week of whitening gum treatment. In order to analyze the melanin content in the hair fiber, the back hair of each test animal was braked, 2 mg, cut into small pieces, dissolved in 1 ml of 1 M Na〇H, and heated in a pit for 4 hours to separate melanomas (mdanosome). The melanin is released, and the procedure requires a ship. The dissolved liquid is measured by a spectrophotometer at a wavelength of 475 nm to measure the melanin content, and a standard curve is prepared by measuring the commercially available melanin. (6) Living male alopecia (AGA) animal model C57BL/6J spontaneous mutation gas has a superficial appearance, with the relevant gene (as shown in Figure %), with mutant mice as the female parent and CBA/J mice as the male parent. The progeny obtained by cross-breeding are called B6CBAF1/j hybrid mice; it has been confirmed that the cross-breed female rats can cause alopecia by injection of testosterone (1 mg/mice/day) for 3 weeks, because the cross-breeding females are more than the crossed rats. The mouse is more sensitive to fluorenone, and thus the crossed mouse rat is used as the male alopecia (AGA) model animal of the present invention (18). (7) Permeation rate of liposome in the hair follicle delivery system In order to confirm whether the hair follicle delivery system penetrates and distributes to the dermis layer, after partially applying the liposome containing HSCF, the skin of the back of the test mouse is taken; after the application, 丨5, Minutes, skin samples were embedded in an Optimal Cutting Temperature (OCT, Tissue-Tek 5 4583) compound, cold-sold and cut into 8 μηι thick, then 'this sample was fixed with 4% diformic acid (paraformaldehyde) at 0.5°/ . The skim milk powder was pre-hybridized overnight for immunohistochemistry 20 200804592 staining (Immimohistochemistry) (Fig. 17). To calibrate the liposome containing HSCF-II, a rabbit antibody against SCF (stem cell factor) was used as the primary antibody. (primary sister 0) rabbit anti-SCF, 1:500) (PeproTech), secondary antibody is fluorescent (FITC) goat anti-rabbit IgG (l:1000) (JacksonImmunoReasearch) 〇(8) Stability of growth factors in liposome In order to demonstrate the maintenance of growth factor HSCF activity in liposome, the present inventors reported that reporter protein-enhanced green fluorescent protein (enhanced green fluorescent protein) , EGFP) - mixed with HSCF in liposome and stored in 37 incubator for 3 years. The green fluorescence of EGFP was observed by fluorescent microscope every month to prove the activity of HSCF. The results showed that EGFP protein activity was 37t can last for at least 1-3 years. Example 2 Effect of HSCF, special solution formulation, and Mindray (10) 匕(10) 丨仙) on hair growth outside the house. Hair follicles were cultured in vitro to test the effects of different components on hair regrowth, and the results are shown in Fig. 13. The treatment with the most significant effect is the treatment of HSCF, the special solution formulation of the present invention and mh〇xidil; the hair follicles cultured in PBS solution have no hair growth at all; and hscf is more effective in hair regeneration than SCF and IGF-I is better, demonstrating the effect of HSCF, and the effect of 'mixing HSCF and minoxidil is also significantly greater than the effect of using HSCF alone and ρ<0·05. The components in the formula include ΚΜ000 μΜ minoxidU, 10-1000 ng/mi SCF, Η000 ng/ml IGF-I, 1-1000 ng/ml HSCF; and the medium formula contains amino acids and vitamins, including: 5- 100 mg/L alanine, arginine, asparagine 21 200804592 (asparagine), aspartic acid, cysteine, cystine Cystine), glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine Methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine 'proline ( Valine), and 0.1-10 mg/L ascorbic acid, biotin (vitamin H, biotin), pantothenate (vitamin B5, pantothenate), choline chloride, ergot masculinization Alcohol (vitamin D2, ergocalciferol), folic acid, inositol, sodium bisulfite, methyl bromide (vitamin K3, menadione sodium) Bisulfate), niacinamide, vitamin B3, pyridoxal HC1, riboflavin, tocopherol, tocopherol Vitamin Bi, thianiine), vitamin A (vitamin A), vitamin B12 (vitamin B12). Example 3 Effect of topical application of HSCF on hair melanin formation in a gray-haired animal model Based on the effect of HSCF on melanin stem cells, the inventors tested the effect of HSCF on the treatment of gray hair in a gray-haired animal mode via topical application; HSCF The protein was embedded in liposome's and applied locally to the back of the test animal via the hair follicle delivery system; the results are shown in Figure 14. The hair color of the treatment group (Fig. 14C) was much darker than that of the positive control group (Fig. 14B), while the treatment group (Fig. 4C) The coat color is similar to that of the wild species (Fig. 14A); afterwards, the test hair follicle sheath (hairsha_ melanin containing ϊ' results also showed that the melanin content of the test group was higher than that of the positive control group (p<〇〇5) The same as the melanin content of the wild species (Fig. 15), demonstrating the effect of HSCF on the recovery of melanin production from gray hair 0 22 200804592 Example 4 (10) Alopecia (AGA) animal model test Local application of HSCF and theory (10) The effect on hair regeneration is based on The effect of HSCF on epidermal stem cells, the inventors tested the effect of HSCF on the treatment of hair loss by topical administration in a male (5) animal model; HSCF protein was in lip〇 The s_embedded 'sub-surface was applied to the back of the test animals via the hair follicle delivery system; the results showed that the test group treated with HSCF and/or minoxidil had hairless hair follicles regenerating new hair faster than the control group (Fig. 16), although The use of HSCF alone is sufficient to stimulate hair regeneration, but the effect of HSCF combined with minoxidil is better. Example 5 The liposome in the hair follicle delivery system (iip〇s〇me) The inventor of the present invention uses gray hair and male alopecia mode. Testing the therapeutic effect on alopecia and gray hair in a hair follicle delivery system via topical application of a formula containing HSCF, having an amino acid and a vitamin, minoxidil; wherein the hair follicle delivery system comprises a penetration enhancer and appropriate "bases" and/or devices; "permeation enhancers" are components that promote the movement of substances into and/or through the dermis. Permeation enhancers include, but are not limited to, lipids, lipoproteins ), fatty acids and fatty alcohols, detergents, alcohols (alc〇h〇ls), glycerol (glycols) ), mineral oil, liposome, and devices or devices that help to penetrate, and "appropriate basal gel" means suitable for topical application to mammalian skin without causing abnormal toxicity, irritation, or allergies. The carrier of the reaction, etc.; the penetration enhancer and the appropriate basal colloid all contribute to the absorption and utility of the three component formulations of the present invention applied to the skin. 23 200804592 The hair follicle delivery system of the present invention has confirmed that the microlipid particle size is % trace 2 _ (the image is permeable to the human dermis layer after application on the skin (10) min (Fig. and mcF can retain activity in the liposome at least up to U years. The invention provides the singularity of the singularity of the singularity of the singularity of the singularity of the singularity of the invention, and the method of comparing the above-mentioned citing and other techniques, which have the following advantages: L The invention utilizes biotechnology The combination of two growth factors SCF and khm into HSCF-Π, together with cell expression and purification' not only saves cost and time, but also 璐邙π acts on hair follicle keratinocytes and melanocytes simultaneously, which can treat gray hair and alopecia 2. The microlipid used in the present invention is simple in process and low in cost, and can embed proteins such as growth factors to preserve activity for a long time, and can be applied not only to medicines and cosmetics, but also to foods. The detailed description above is a detailed description of the possible embodiments of the present invention, but it is not intended to be used in the scope of the patents, and is not departing from the spirit of the present invention. Implementation or change shall be included in the patent scope of this case. In summary, the case is not only innovative in terms of method, but also can improve the above-mentioned multiple functions compared with the manufactured articles, and should fully comply with the law of _ sex and progress# Sending a daily transfer, 提出 file an application according to law, and ask your office to approve the invention patent application, in order to invent the invention, to the sense of virtue. [Simplified schematic] Figure 1 (4) is a two-lion human stem cell growth factor (SCF); (9) Human stem cell growth factor (SCF said PCR conditions and products used in the present invention; (c) insulin growth factor (IGF·PCR conditions and products used in the present invention; and (4) hair stem cell growth used in the present invention 24 200804592 -II (HSCF-II) PCR conditions and products; Figure 2 is the cDNA sequence of soluble human stem cell growth factor (SCF) (495 bp); Figure 3 is the amino acid sequence of soluble human stem cell growth factor (SCF) (165 Aa); Figure 4 shows the results of soluble human stem cell growth factor (SCF) protein expression by SDS-PAGE (a) and Western knockdown analysis; Figure 5 shows human stem cell growth factor (SCF) in TF- I fine Figure 6 shows the cDNA sequence of insulin growth factor (IGF-I) (210 bp); Figure 7 shows the amino acid sequence of insulin growth factor (IGF-I) (70a.a.); Figure 8 shows the results of insulin growth factor (IGF-I) protein expression by SDS-PAGE; Figure 9 shows the protein activity of insulin growth factor (Κ}ΙΜ) in 313 cell line; Figure 1 shows hair stem cell growth factor -iI (HSCF4i) cDNA sequence (759 bp); Figure 11 is the amino acid sequence of hair stem cell growth factor_II (HSCF-II) (253 aa); Figure 12 shows hair stem cell growth factor by SDS-PAGE ( The results of HSCF4I) protein expression; Figure 13 is the in vitro test of the coffee SCF, HSCF and / or - ο on the hair growth: (8) the entire hair follicles were removed from the mouse culture, the hair regrowth measured from The top of the hair follicle (where the hoe is pointed) is purely placed; (9) the length of the hair of the stalk is different at different places, and the display is not shown. There was a significant difference between the Hai treatment group and the positive control group (4) 〇 5), t indicates that the treatment group and the mi idil treatment group have significant differences (4) 〇 5); 〆, θ is 乂 文The animal model tests the effect of HSCF on the coat color, and the HSCF-treated tear (C) is similar to the color of the yang, and the group (the color is deep, but the wild is similar); 25 200804592 Figure i5 is tested in the gray animal mode The effect of HSCF on melanin production, * indicates that the treatment group or wild species is significantly different from the positive control group (4) still), Figure 16 (4) is a C57BL/6J mutant mouse with a teacher gene before lactation; (9) The same mutant mouse after weaning; (4) male off-GA model before treatment, N01: hair-filled mice as a control group; Νο·2: treated with HSCF and min. • hair-filling; N〇3: a squirrel that treats HSCF, (4) a male-to-GA) animal pattern after 4 days of treatment, (5) a known number is the same as (4); and Figure 17 (8) shows an HP 〇S_ package The particle size of the buried HSCF; (9) It can be seen by glory staining. 'It is good to embed HSCF with liposome. It can penetrate into the dermis after 15 〇 _ on the skin. [Main component symbol description] 26

Claims (1)

200804592 X. Patent application scope: 1. (hair stem cdi fact〇r iij HSCF.n) t|^ Column ' has a nuclear microsequence as in the sequence listing SEQ ID NO: 5_. 2. Shishen μ specializes in the hair stem cell growth factor·] (brain Ying) recombinant gene sequence described in item 1, wherein the nucleotide sequence is ligated with a nuclear complex sequence as listed in the sequence table seqidn〇:i 'and has the sequence of nucleotides listed in SEQ ID NO: 3 of the Sequence Listing. 3. Hair dry, cell growth g_n (HSCF_n) recombinant protein, as shown in Sequence Listing I. NO: The amino acid sequence listed in 6. 4. The hair stem cell growth factor π as described in claim 3 of the patent scope (brain (four) recombinant protein greek towel δ hai recombinant protein system binds to humans having an amino group sequence as listed in the sequence table SEqIDn 〇: 2 Stem cell growth factor (10) melon (four) color (10) (four) wide and insulin growth factor (inshuiin gr〇wth factor, IGF-Ι) with the amino acid sequence listed in the sequence list Q Ν〇 · 4. 5. As stated in the patent scope The hair stem growth factor_n (hscf-h) recombinant protein 枭' according to item 3, wherein the recombinant protein is embedded in a expression vector by a composition of the nucleotide sequences listed in SEQ ID NO: 1 and SEQ ID NO: 3 of the Sequence Listing a formulation for treating baldness and gray hair, comprising a hair stem cell factor (HSCF) having a monthly female sequence as set forth in SEQ ID NO: 6 of the Sequence Listing, A solution comprising a complex amino acid and a multivitamin, and a minoxidil. 7. A method for treating baldness and gray hair according to claim 6 wherein the complex amino acid comprises all 2 Essential amino group The method of treating baldness and gray hair according to claim 6, wherein the multivitamin comprises ascorbic acid, vitamins, vitamins, and pantothenic acid. (vitamin B5, pantothenate), choline chloride, ergot alcohol (vitamin D2, ergocaldferol), folic acid, in〇sitd, sodium bisulfite S Kun (vitamin K3, menadione sodium bisulfate), acetaminophen (vitamin B3 niacinamide), σ ratio alpha polyacid (vitamin Eg, pyrid〇xai HC1), riboflavin (vitamin B2 dboflavin), tocopherol (vitamin E, toc〇pher〇1), thiamine (vitamin β1, this coffee (6)), vitamin A (vitamin A), and vitamin B12 (vitamin B12). 9. A method for treating baldness and gray hair, including ·· a) using at least one penetration enhancer (penetrati〇n(9)^(10) coffee) and at least one carrier bases to package a formulation for treating baldness and gray hair as described in claim 6 of the patent application to obtain a Packaging drugs; (b) Topically applying the packaged drug obtained by (a) to the skin or scalp. The method of treating baldness and gray hair according to claim 9, wherein the base of the carrier is selected. From the following groups: phospholipid (ph〇sph〇Upid), lecithin (lehicm), cholesterol (cholesterol), ph〇Sphatidylethanolamine (PE), polyethylene glycol (PolyEthylene Glycol, PEG) , Tween 2〇, Tween 80, and Triton X-100 〇11· A formula for treating gray hair comprising a hair stem cell factor (HSCF) having an amino acid sequence as set forth in SEQ ID NO: 6 of the Sequence Listing And a solution containing a complex amino acid and a multivitamin. The method for treating gray hair according to claim 11, wherein the complex amino acid comprises all 20 essential amino acids. 13. The formula for treating gray hair according to claim 11, wherein the multivitamin comprises ascorbic acid, biotin (vitamin H, biotin), pantothenate (vitamin B5, pantothenate), Choline chloride, ergocalciferol, folic acid, inositol, menadione sodium bisulfate, tobacco Alkaline amide (vitamin B3, niacinamide), furfural hydrochloride (vitamin B6, pyridoxal HC1), riboflavin (vitamin B2, rib〇flavin), tocopherol (vitamin E, tocopherol), thiamine (vitamin Bl, Thiamine), vitamin A (vitamin A), and vitamin B12 (vitamin B12). A method of treating gray hair comprising: (a) packaging at least one penetration enhancer and at least one base colloid to carry a formulation for treating gray hair as described in claim 11 to obtain a packaged drug; And (b) topically applying the packaged drug obtained by (8) to the skin or scalp. The method for treating gray hair according to claim 14, wherein the gum base system is selected from the group consisting of phospholipid, lecithin, cholesterol, and lipolysis. Ethylamine (ph〇Sphatidylethanolamine, PE), PolyEthylene Glycol (PEG), Tween 20, Tween 80, and Triton X-100. 16. A method for measuring the therapeutic effect of a component on baldness in vivo, comprising using a male alopecia model in which a C57BL/6J female mouse is crossed with a CBA/J male mouse (C57BL/6J). Female X CBA/J male) hybrid motherhood. 29 200804592 17. A method for measuring the therapeutic effect of a component on gray hair in vivo, comprising a whitening mouse model, which is a hybrid mouse of C3H mice and BALB/C mice. 30 200804592 Sequence Listing <110>林昀正<120> Composition and method for dual treatment of gray hair and baldness through hair follicle conduction system <140> TW095141484 <141> 2006-11-09 <150> US 60/735925 <151> 2005-11-10 <160> 6 <210> 1 <211> 495 <212> DNA <213> Human Soluble SCF (stem cell factor) <220><221> CDS <222> 1..495 <400> 1 atg aag aag aca caa act tgg att etc act tgc att tat ett cag 45 Met Lys Lys Thr Gin Thr Trp lie Leu Thr Cys lie Tyr Leu Gin 1 5 10 15 ctg etc eta tet aat cct etc gtc aaa act gaa ggg ate tgc agg 90 Leu Leu Leu Ser Asn Pro Leu Val Lys Thr Glu Gly lie Cys Arg 20 25 30 aat cgt gtg act aat aat gta aaa gac gtc act aaa ttg gtg gca 135 Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 35 40 45 aat ett cca aaa gac tac atg ata acc etc aaa tat gtc ccc ggg 180 Asn Leu Pro Lys Asp Tyr Met lie Thr Leu Lys Tyr Val Pro Gly 50 55 60 atg gat gtt ttg cca agt cat tgt tgg ata age gag atg gta g Ta 225 Met Asp Val Leu Pro Ser His Cys Trp lie Ser Glu Met Val Val 65 70 75 caa ttg tea gac age ttg act gat ett ctg gac aag ttt tea aat 21Q Gin Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn 80 85 90 att tet gaa ggc ttg agt aat tat tcc ate ata gac aaa ett gtg 315 lie Ser Glu Gly Leu Ser Asn Tyr Ser lie lie Asp Lys Leu Val 95 100 105 aat ata gtg gat gac ett gtg gag tgc gtg aaa gaa Aac tea tet 360 200804592 Asn lie Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser 110 115 aag gat eta aaa aaa tea ttc aag age cca gaa ccc agg etc ttt 405 Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe 125. 130 135 act cct gaa gaa ttc ttt aga att ttt aat aga tcc att gat gcc 450 Thr Pro Glu Glu Phe Phe Arg lie Phe Asn Arg Ser lie Asp Ala 140 145 150 ttc aag gac ttt gta gtg gca tet Gaa act agt gat tgt gtg gtt 495 Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 155 160 165 <210〉 2 <211> 165 <212> PRT <213> Human Soluble SCF * &Lt400> 2 Met Lys Lys Thr Gin Thr Trp lie Leu Thr Cys lie Tyr Leu Gin 15 10 15 Leu Leu Leu Ser Asn Pro Leu Val Lys Thr Glu Gly lie Cys Arg 20 25 30 Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 35 40 45 Asn Leu Pro Lys Asp Tyr Met lie Thr Leu Lys Tyr Val Pro Gly 50 55 60 Met Asp Val Leu Pro Ser His Cys Trp lie Ser Glu Met Val Val 65 70 75 Gin Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn 80 85 90 lie Ser Glu Gly Leu Ser Asn Tyr Ser lie lie Asp Lys Leu Val 95 100 105 Asn lie Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser 110 115 120 Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe 125 130 135 Thr Pro Glu Glu Phe Phe Arg lie Phe Asn Arg Ser lie Asp Ala 140 145 150 Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 155 160 165 <210> 3 <211> 210 <212> DNA <213> Human Worker GF-I <220> 200804592 <221> CDS <222> 1..210 <400> 3 Gga ccg gag acg etc tgc ggg get gag ctg gtg gat get ett Cag 45 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gin 15 10 15 ttc gtg tgt gga gac agg ggc ttt tat ttc aac aag ccc aca ggg 90 Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly 20 25 30 tat ggc tee age agt egg agg geg cct cag aca ggc ate gtg gat 135 Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val Asp 35 40 45 gag tgc tgc ttc egg age tgt gat eta agg agg ctg Gag atg tat 180 Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr 50 55 60 tgc gca ccc etc aag cct gee aag tea get 210 Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala * 65 70 <210> 4 <211> 70 <212> PRT <213> Human IGF-I <400> 4 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gin 15 10 15 Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly 20 25 30 Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val Asp 35 40 45 Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr 50 55 60 Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala 65 70 <2 10<211> 759 <212> DNA <213> Human HSCF-II <220><221> CDS <222> 1..759 200804592 <400> 5 atg aag aag aca caa act Tgg att etc act tgc att tat ett cag 45 Met Lys Lys Thr Gin Thr Trp lie Leu Thr Cys lie Tyr Leu Gin 15 10 15 ctg etc eta tet aat cct etc gtc aaa act gaa ggg ate tgc agg 90 Leu Leu Leu Ser Asn Pro Leu Val Lys Thr Glu Gly lie Cys Arg 20 25 30 aat cgt gtg act aat aat gta aaa gac gtc act aaa ttg gtg gca 135 Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 35 40 45 aat ett cca aaa Gac tac atg ata acc etc aaa tat gtc ccc ggg 180 Asn Leu Pro Lys Asp Tyr Met lie Thr Leu Lys Tyr Val Pro Gly 50 55 60 atg gat gtt ttg cca agt cat tgt tgg ata age gag atg gta gta 225 Met Asp Val Leu Pro Ser His Cys Trp lie Ser Glu Met Val Val 65 70 75 caa ttg tea gac age ttg act gat ett ctg gac aag ttt tea aat 270 Gin Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn 80 85 90 att tet Gaa ggc ttg agt aat tat tcc ate at a gac aaa ett gtg 315 lie Ser Glu Gly Leu Ser Asn Tyr Ser lie lie Asp Lys Leu Val 95 100 105 aat ata gtg gat gac ett gtg gag tgc gtg aaa gaa aac tea tet 360 Asn lie Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser 110 115 120 aag gat eta aaa aaa tea ttc aag age cca gaa ccc agg etc ttt 405 Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe 125 130 135 act cct gaa gaa ttc ttt aga att Ttt aat aga tcc att aat gcc 450 Thr Pro Glu Glu Phe Phe Arg lie Phe Asn Arg Ser lie Asn Ala 140 145 150 ttc aag gac ttt gta gtg gca tet gaa act agt gat tgt gtg gtt 495 Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 155 160 165 tet tea aca tta agt gga gga gga gga aag ett gga gga gga ggc 540 Ser Ser Thr Leu Ser Gly Gly Gly Gly Lys Leu Gly Gly Gly Gly' 170 175 180 tcc ggc ggc gga ccg Gag aeg etc tgc ggg get gag ctg gtg gat 585 Ser Gly Gly Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp 185 190 195 get ett cag ttc gtg tgt gga gac agg ggc ttt tat ttc aac aag 630 Al a Leu Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys 200 205 210 ccc aca ggg tat ggc tcc age agt egg agg geg cct cag aca ggc 675 Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly 215 220 225 ate gtg gat gag tgc tgc ttc egg age tgt gat eta agg agg ctg 720 200804592 work le Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu 230 235 240 gag atg tat tgc gca ccc etc aag cct gcc aag tea Get 759 Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala 245. 250 <210> 6 <211> 253 <212> PRT <213> Human HSCF-II <400> 6 Met Lys Lys Thr Gin Thr Trp lie Leu Thr Cys lie Tyr Leu Gin 15 10 15 Leu Leu Leu Ser Asn Pro Leu Val Lys Thr Glu Gly lie Cys Arg 20 25 30 Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala 35 40 45 Asn Leu Pro Lys Asp Tyr Met lie Thr Leu Lys Tyr Val Pro Gly 50 55 60 Met Asp Val Leu Pro Ser His Cys Trp lie Ser Glu Met Val Val 65 70 75 Gin Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn 80 85 90 lie Ser Glu Gly Leu Ser Asn Tyr Ser lie L. Asp Lys Leu Val 95 100 105 Asn lie Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser 110 115 120 Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe 125 130 135 Thr Pro Glu Glu Phe Phe Arg lie Phe Asn Arg Ser Le Asn Ala 140 145 150 Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 155 160 165 Ser Ser Thr Leu Ser Gly Gly Gly Gly Lys Leu Gly Gly Gly 170 175 180 Ser Gly Gly Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp 185 190 195 Ala Leu Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys 200 205 210 Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly 215 220 225 lie Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu 230 235 240 Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala 245 250 5