CN113476601B - Medicine or cosmetic prepared by stem cell cytokine and algal polysaccharide - Google Patents

Abstract

The invention relates to a medicine or cosmetic prepared by stem cell cytokine and algal polysaccharide. The invention provides the epidermal stem cell cytokine, the monoclonal antibody for specifically inhibiting tyrosinase and algal polysaccharide, which can inhibit the proliferation of melanoma cells and have a good whitening application effect prospect.

Description

Medicine or cosmetic prepared by stem cell cytokine and algal polysaccharide

Technical Field

The invention relates to the field of biology, in particular to a medicine or cosmetic prepared by combining stem cell cytokines with algal polysaccharides.

Background

The cell factor refers to a kind of high-activity multifunctional small molecule protein produced and secreted by immune cells and related cells, does not include immunoglobulin, complement and general physiological cell products, and has high immunoregulatory activity. There are hundreds of cytokines which are found at present, mainly including interferon, interleukin, tumor necrosis factor, epidermal growth factor, transforming growth factor, nerve growth factor, etc. Cytokines play important roles in immune response, virus resistance, tumor resistance and inflammatory response of the body, play an important role in treating diseases and are essential substances for resisting infection.

The cell factor has very low content in human body, but has very high bioactivity and has a plurality of important biological effects and physiological functions. They are related to the growth, division, differentiation, proliferation and migration of skin cells, can effectively act on the skin cells and play an outstanding role in beautifying and protecting skin. In the prior art, the application of cytokines such as EGF (epidermal growth factor), aFGF (acidic fibroblast growth factor), bFGF (basic fibroblast growth factor), KGF (keratinocyte growth factor), VEGF (vascular endothelial growth factor) and the like in cosmetics has been studied.

EGF can promote growth, division and metabolism of epidermal cells, epithelial cells and fibroblasts of an organism in vivo, promote growth of microvasculature and improve microenvironment of cell growth. Therefore, the skin repair liquid has good repair and nursing effects on damaged skin, sensitive skin, wounded skin and reconstructed skin, particularly repair of local damaged skin after skin changing operation which is popular in beauty parlors at present, and can also prevent postoperative complications. EGF also stimulates the formation of granulation tissue and promotes epithelialization of granulation tissue, and also regulates collagen degradation and renewal, so that collagen fibers are arranged in a linear manner to prevent abnormal proliferation of connective tissue, thereby having the effects of shortening wound healing time and reducing scar formation, and also having good effects of preventing and caring acne. aFGF is a mitogen having a very strong action, has an action of promoting division of a variety of cells derived from mesoderm and neuroectoderm, and can be used for the treatment of wounds, burns, ulcers and the like. The cosmetic effects of bFGF may be manifested in several ways: 1) skin care and repair. The bFGF can improve the microenvironment of cell growth, promote the synthesis of elastic fibers and collagen, enable skin to be elastic and enable the skin to be in a smooth and tender state. 2) Anti-wrinkle and anti-aging effects. Can promote the growth and development of fibroblasts, and continuously replace aged cells with new cells, thereby having the effects of preventing and removing wrinkles.

Cytokines have also been isolated and prepared by previous studies. For example, CN105543313B discloses a method for separating and purifying human mesenchymal stem cell factor, which comprises the following steps: a. cell culture supernatant acquisition: taking human mesenchymal stem cells, placing the human mesenchymal stem cells in a mesenchymal stem cell serum-free culture medium for culture, and collecting supernatant when the cells grow to reach 75-85% of confluence degree; b. and (3) filtering and concentrating: filtering the supernatant in the step a by adopting a 0.22 mu m filter membrane; then passing through 50KD ultrafiltration membrane, collecting dialysate, passing through 1KD ultrafiltration membrane, collecting retentate to obtain cytokine concentrate. CN106701672B discloses a culture method of human adipose-derived mesenchymal stem cell factor, which comprises the following steps: and (3) placing the human adipose-derived mesenchymal stem cells in a high-coenzyme serum-free culture medium, culturing until the cell confluency is 75-85%, and collecting the supernatant to obtain the human adipose-derived mesenchymal stem cells.

However, the methods for separating and preparing the cytokines in the prior art are not enough, and the types of the cosmetics which are prepared better and contain the cytokines are not enough.

Disclosure of Invention

The invention provides technical improvement aiming at the prior art, and provides a whitening cosmetic or a medicament for inhibiting melanoma cells.

In one aspect, the invention provides the use of a cytokine, algal polysaccharides and a monoclonal antibody in the preparation of a pharmaceutical composition for inhibiting melanoma; wherein, the cell factor is prepared by the following method: placing the epidermal stem cells into a cell culture medium for subculture, wherein the cell culture medium is 89% DMEM + 10% FBS + 1% double antibody stem cell culture medium, and the culture conditions are 37 ℃ and 5% CO₂Culturing; taking well-grown stem cells, adjusting cell concentration to 1 × 10⁶The cell is inoculated in a 6-pore plate, and 50 ng-mL-plus is added after the cell is attached to the wall¹Secretagogue peptide SEQ ID NO: 1, culturing for 48h wherein the culturing is in 3% O₂+5％CO₂+92％N₂Collecting cell supernatant, ultrafiltering the supernatant with 1KD ultrafilter membrane to remove small molecular impurities with molecular weight below 1000, and collecting trapped fluid to obtain cell factor;

the monoclonal antibody is a monoclonal antibody for specifically inhibiting tyrosinase: the light chain variable region and the heavy chain variable region of the antibody are respectively shown as follows:

light chain variable region (SEQ ID NO: 2)

DIVITQRPALMAASPGEKVTITCWYAMSGLTMREQWYQQKSGISPKPWIYDDETMHCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCPHISMFDRDFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKLSCKASGYIFSSVCPKWIKQRPGQGLEWIGWSITECSYEKSLRWQAGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGKLHHFSGWGLGTTLAVSS。

Further, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

Further, the pharmaceutical composition also comprises an excipient.

In another aspect of the invention, the use of the cytokine, algal polysaccharides and monoclonal antibody in the preparation of whitening cosmetics is provided; the cosmetic contains a cytokine and a monoclonal antibody, wherein the cytokine is prepared by the following method: placing the epidermal stem cells into a cell culture medium for subculture, wherein the cell culture medium is 89% DMEM + 10% FBS + 1% double antibody stem cell culture medium, and the culture conditions are 37 ℃ and 5% CO₂Culturing; taking well-grown stem cells, adjusting cell concentration to 1X10⁶The cell is inoculated on a 6-well plate, 50 ng/mL is added after the cell is attached to the wall^-1Secretagogue peptide SEQ ID NO: 1, culturing for 48h wherein the culturing is in 3% O₂+5％CO₂+92％N₂Collecting cell supernatant, ultrafiltering the supernatant with 1KD ultrafilter membrane to remove small molecular impurities with molecular weight below 1000, and collecting trapped fluid to obtain cell factor;

the monoclonal antibody is a monoclonal antibody for specifically inhibiting tyrosinase: the light chain variable region and the heavy chain variable region of the antibody are respectively shown as follows:

light chain variable region (SEQ ID NO: 2)

DIVITQRPALMAASPGEKVTITCWYAMSGLTMREQWYQQKSGISPKPWIYDDETMHCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCPHISMFDRDFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKLSCKASGYIFSSVCPKWIKQRPGQGLEWIGWSITECSYEKSLRWQAGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGKLHHFSGWGLGTTLAVSS。

In one aspect, the present disclosure provides a method of inhibiting melanoma cell proliferation, the method comprising administering to a subject in need thereof an effective amount of an exemplary pharmaceutical composition, wherein proliferation of cancer cells is inhibited.

In certain embodiments, the present disclosure provides methods of treating cancer in a subject. The method comprises administering to a subject in need thereof an effective amount of an exemplary antibody described herein.

In another aspect, the present invention also provides a whitening cosmetic comprising a cytokine and the monoclonal antibody.

The skin whitening component used herein may include a whitening component that can improve the area of skin damaged by melanin pigmentation, for example, an extract derived from natural substances such as animals, plants and minerals, amino acids, hormones and vitamins, but the present invention is not limited thereto. For example, the component may be one or more compounds selected from hydroquinone-based compounds; vitamins such as niacinamide and vitamin C and its derivatives; flavonoid compounds such as monatin and hesperidin; resveratrol compounds and derivatives thereof; a curcumin derivative; amino acid-based compounds such as glutathione and methyl undecylenate; arbutin, kojic acid, azelaic acid, -hydroxy acid, -bisabolol, adenosine, ascorbyl glucoside, ascorbyl tetraisopalmitate, ethyl ascorbyl ether, fullerene, acetyl tyrosine, diacetyl benzoyl sorbitol, atractylodes oil, magnesium ascorbyl phosphate, diosgenin, macelignan, acetyl sphingosine, dihydroxymethoxy chalcone, -bisabolol, protocatechualdehyde, oryzanol, licorice extract, a broussonetia extract, a green tea extract, a mulberry bark extract, an ADLAY extract, yeast extract, a white atractylodes rhizome extract, a wheat germ extract, a peony extract, a chinese sage root extract, a ginseng extract, a red ginseng extract, a lizardtail extract, a hairyvein agrimony extract, a canna root extract, a SASA querertaetensis extract, extract of roots of sophora flavescens ait, extract of coix seeds, extract of fruits of cherry, chitosan, polylysine, glucosamine and derivatives thereof, but the present invention is not limited thereto.

The cosmetic composition for skin whitening according to the present invention may include a skin whitening component having a functional group, and thus may be used in the formulation of the cosmetic composition for skin whitening. Examples of skin products may include, in general, skin care products (toners, serums, essences, lotions, creams, etc.) for smoothing and protecting skin, color cosmetics (cosmetic bases, foundations, powders, eye shadows, lip gloss, lipsticks, etc.), cleansing cosmetics (foam cleansers, cleansing oils, cleansing lotions, cleansing creams, cleansing gels, masks, soaps, cleansing towels, etc.), sun protection cosmetics, body cosmetics (body lotions, body washes, body creams, body oils, etc.).

In some embodiments, the pharmaceutical compositions are capsules, tablets, emulsions and aqueous suspensions, aqueous dispersions and solutions, and the like (without limitation), and can be administered orally in any oral form and in any oral form.

In some embodiments, the pharmaceutical composition is a tablet.

In some embodiments, the pharmaceutical composition further comprises an excipient. Excipients, for example microcrystalline wax, lactose, mannitol, ethylcellulose, sorbose, starch, threo-cellulose, calcium phosphate, powdered cellulose, silicified microcrystalline cellulose, iso-martensite or mixtures thereof. In some embodiments, the excipient is microcrystalline cellulose.

Advantageous effects

The invention provides the epidermal stem cell cytokine, the monoclonal antibody for specifically inhibiting tyrosinase and algal polysaccharide, which can inhibit the proliferation of melanoma cells and have a good whitening application effect prospect.

Drawings

FIG. 1 is a graph showing the effect of a secretagogue peptide on cytokine promotion

FIG. 2 is a graph showing the results of growth of fibroblast cells promoted by cytokines

FIG. 3 shows the results of tumor inhibition

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation and culture identification of epidermal stem cells

Removing subcutaneous fat layer of foreskin skin sheet under aseptic condition, repeatedly washing skin sheet with PBS containing penicillin and streptomycin for 3 times, building into small pieces with size of 0.2cm x 0.2cm, digesting with neutral protease at 4 deg.C for 18h, removing supernatant, and separating epidermis and dermis layers. ScissorsBreaking the epidermis, digesting with 0.25% trypsin at 37 deg.C for 15min, adding DMEM containing 10% calf serum to stop digestion, pipetting the suspension and filtering through a 200 mesh screen. The filtrate was centrifuged at 1000r/min for 5min to collect the cells. Discarding the supernatant, adding K-SFM and gently blowing to obtain single cell suspension. Inoculating into culture flask pre-coated with type IV collagen, incubating at 37 deg.C, sucking out cell suspension, adding K-SFM, standing at 37 deg.C and 5% CO₂Placing the mixture in an incubator for continuous culture, changing the culture solution for 1 time every 2 days, digesting the cells in the culture flask by 0.25 percent of trypsin and 0.02 percent of EDTA when the cells are approximately 80 percent fused, and carrying out passage according to the ratio of 1: 1.5.

The cultured cells are taken, washed 3 times by PBS, fixed for 30min at room temperature by 4 percent paraformaldehyde, and chemically stained by the immunocytochemistry of the cells K19 and beta 1 integrin respectively by a two-step immunohistochemical detection method. The results show that β 1 integrin, K19 immunocytochemical staining, both appear in the cytoplasm of the cells and are expressed positively as a brown-yellow stain. Beta 1 and K19 are one of markers of the epidermal stem cells, and the high expression of the two markers indicates that the isolated epidermal stem cells are.

EXAMPLE 2 preparation of epidermal stem cell cytokine

The epidermal stem cells prepared in example 1 were subcultured in a cell culture medium of 89% DMEM + 10% FBS + 1% double antibody at 37 ℃ and 5% CO₂Culturing;

taking well-grown stem cells, adjusting the cell concentration to be 1X10⁶The solution is changed after the cells are attached to the wall, and the experiment groups are respectively added with the solution containing 0, 1, 10, 50 mg.L-¹Complete medium for secretion promoting peptide (SEQ ID NO: 1), complete medium only added to the control group, and cultured for 48h (wherein 50 mg. L-¹Secretory peptide culture was further divided into 0% O₂+5％CO₂+94N₂And 3% of O₂+5％CO₂+92％N₂) Then, collecting cell supernatants of each group, ultrafiltering the two culture supernatants with 1KD ultrafiltration membrane, removing small molecular impurities with molecular weight below 1000, and collecting the trapped fluid for later use.

The cells were washed 3 times with PBS and total RNA was extracted by Trizol. PCR amplification is carried out to identify the relative expression amounts of Hepatocyte Growth Factor (HGF), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1) and Vascular Endothelial Growth Factor (VEGF). The primer sequences are shown in Table 1.

TABLE 1 primer set

Name of primer	Primer sequence (5 '-3')
		HGF upstream primer	AGAGTGGCATCAAATGTCAG
HGF downstream primer	GCTTGTGAAACACCAGGGT
		uPA upstream primer	GGGGAGATGAAGTTTGAGGTG
uPA downstream primer	GGTCTGTATAGTCCGGGATGG
		MMP-1 upstream primer	GATGATGATGAAACCTGGACAA
MMP-1 downstream primer	GCCGGTGTAGGTGTAGATAGGA
		VEGF upstream primer	GCCTTGCCTTGCTGCTCTA
VEGFDownstream primer	CACCAGGGTCTCGATTGGAT

The results are shown in FIG. 1.

As can be seen from the results in FIG. 1, 50 mg. L-¹The secretion promoting peptide can remarkably promote the secretion of several cytokines. Furthermore, the results show that the promoting effects on MMP-1 and VEGF are more significant. In addition, 3% of O was added during the culture₂Can further promote the secretion of cytokines, and as can be seen from the figure, 3% O is added₂Is better than O₂. Therefore, the cytokine is prepared by adding 50 mg. L-¹Secretory peptide at 3% O₂Culturing and preparing under the condition of (1).

EXAMPLE 3 fibroblast proliferation promotion assay

Fibroblast cells (from Saiko Biotech Co., Ltd., product No. HXFB-00001) in logarithmic growth phase are added into each well with 4000/200. mu.l of a serum-free culture medium DMEM + sodium pyruvate + streptomycin mixed solution, the cell concentration is adjusted to 4000/200. mu.l, the cell inoculation amount is 4000/200. mu.l/well, cytokine extracts are added into each well according to different concentrations (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%), three wells are arranged, the culture medium is added, an incubator is used for incubation for 72h observation, CCK-8 solution is added for continuous culture for 2h, an enzyme reader is used for measuring the absorbance value (OD value) of the cells at the wavelength of 450nm, and 0% of the wells are used as control wells, and the cell proliferation activity increase percentage is calculated. Calculating the formula: (OD experimental wells-OD control wells)/OD control wells.

The results are shown in fig. 2, and the fibroblast growth rate is obviously increased after the addition of the cytokine extract, and the proliferation promoting rate is gradually increased along with the increase of the concentration of the cytokine.

Example 4 measurement of inhibitory Effect of cytokines on melanin synthesis

B16 melanocytes at 1 × 10⁵one/mL density was seeded in 3 6-well plates. 3mL per well, 5% CO at 37 ℃₂After incubation in the incubator for 24h,discarding the supernatant, adding the cytokine to be detected, wherein the final mass concentration of the cytokine is 50mg/L, 10mg/L, 1mg/L and 0mg/L respectively after each concentration of 3 holes. After 3 days of cytokine action, the supernatant was discarded, washed with PBS, and 015mL of trypsinized cells were added per well for 3min, and 2mL of maintenance solution per well to stop digestion. After the mixture was blown and mixed, 0.1mL of each concentration was taken out and counted. Centrifuging the rest cell suspension at 1500r/min for 10min, discarding supernatant, adding 200 μ L distilled water and 1:1 volume ratio of ethanol and diethyl ether mixture (removing some opaque substances), shaking, standing at room temperature for 15min, and centrifuging at 3000r/min for 5 min. The supernatant was discarded, and 1mL of a 1mol/L aqueous solution of NaOH containing 10% (by mass) was added to the precipitate. Heating at 80 deg.C for 30min (with stopper), and measuring absorbance at 490nm with ELISA. Melanocyte growth rate ═ (drug well cell density ÷ control well cell density) × 100%, melanin synthesis inhibition rate ═ 1- (drug well absorbance value ÷ drug well cell density) ÷ (control well absorbance value ÷ control well cell density)]×100％。

TABLE 2 Melanin inhibition

The experimental results in Table 2 show that the cytokine has obvious inhibitory effect on melanin synthesis, the inhibitory effect of 50mg/L can reach (53.42 +/-3.41)%, the inhibitory effect is obvious, the concentration is increased, and the inhibitory effect is increased.

Example 4 preparation of monoclonal antibodies that inhibit tyrosinase

Tyrosinase protein (KL238Po01, KALANG) is used as immunogen, and a conventional hybridoma monoclonal antibody preparation method is adopted to prepare and obtain a TYR monoclonal antibody 2D4. which is subjected to sequence identification, wherein the light chain and heavy chain variable region sequence of the monoclonal antibody is shown as follows.

Light chain variable region (SEQ ID NO: 2)

DIVITQRPALMAASPGEKVTITCWYAMSGLTMREQWYQQKSGISPKPWIYDDETMHCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCPHISMFDRDFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKLSCKASGYIFSSVCPKWIKQRPGQGLEWIGWSITECSYEKSLRWQAGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGKLHHFSGWGLGTTLAVSS

The monoclonal antibody has good specificity, only binds to TYR protein, and does not bind to other proteins. And the dissociation constant reaches 11.4nM, and the method has a better binding effect.

Example 5 preparation of melanoma mouse model and corresponding experiment

A375 human melanoma cell line was cultured in DMEM medium containing 10% fetal bovine serum (containing 100U/mL penicillin and 100. mu.g/mL streptomycin) at 37 deg.C with 5% by volume of CO₂Culturing in an incubator. Cells were harvested and counted at approximately 80% -90% confluency, and cell concentration was adjusted to 2X 10 using serum-free DMEM⁶/mL，2×10⁵Mice inoculated with C57BL/6 mice had subcutaneous tumors in the right axilla. On day 3 of tumor implantation, the tumor volume is 100mm³At time, mice were randomly divided into 5 groups: saline group (200 μ L/time, day 0, 3, 6, 9 i.p.), cytokine group (200 μ g/mouse, day 0, 3, 6, 9, 12, 15 i.p.), cabozantinib control group (15mg/kg/D, day 0, 3, 6, 9 i.p.), 2D4 mab group (10mg/kg/D, day 0, 3, 6, 9 i.p.), 2D4 mab group (10mg/kg/D, day 0, 3, 6, 9 i.p.), combined cytokine group (200 μ g/mouse, day 0, 3, 6, 9, 12, 15 i.p.). Tumor volume was measured at day 0 and day 21. Tumor volume (V) ═ a × b2/2(a is major diameter, b is minor diameter). Tumor inhibition ratios were calculated using methods of tumor inhibition ratio calculation routine in the art, and the results are shown in fig. 3.

The results in fig. 3 show that the combination of the cytokine and the 2D4 monoclonal antibody can significantly improve the tumor inhibition rate to 96.1 ± 2.56%. The inhibitor has better inhibiting effect than the control cabozantinib and better effect than the single use of cell factor and monoclonal antibody.

Example 6 Security detection

According to the abnormal toxicity inspection method in the appendix of Chinese pharmacopoeia, 5 mice are taken, and the body mass is about 20 g. The test mouse should be healthy and non-injurious, bright and active in hair color, and the female mouse is non-pregnant. Each mouse was weighed and labeled. Mice were satiated before weighing. And (3) intraperitoneal injection: holding the mouse with one hand, holding the back of the mouse between the thumb and forefinger, fixing the hind limb and tail of the mouse with the ring finger and little finger, the abdomen facing upwards, inserting the syringe needle subcutaneously from the left side of the abdomen of the mouse, and making the needle pass through the midline of the abdomen in parallel subcutaneously, then entering the right abdominal cavity, and slowly injecting 0.1g of the monoclonal antibody and cytokine composition. The immediate response was observed after injection and the status, toxicity performance and number of deaths were recorded in 4h, 24h, 48h animals.

The reaction index of the test animals is shown in Table 3.

TABLE 3 index of abnormal toxic response of test animals

The results are detailed in Table 4.

TABLE 4 examination of abnormal toxicity

As can be seen from Table 4, the limits of abnormal toxicity of the mAb and cytokine meet the specifications. Has better safety.

EXAMPLE 7 experiment on the inhibition of melanocyte proliferation by Combined administration

Culturing mouse B16 melanocyte under aseptic condition, and mixing at a ratio of 1 × 10⁵one/mL of B16 cells were inoculated in a flask of 10% (volume fraction) calf serum in RPMI1640 medium at 5% (volume fraction) CO₂The culture was carried out in an incubator and passaged every 3 days by digestion with 0.25% pancreatin.After the cells had grown to log phase, they were washed with PBS, digested with 0.25% pancreatin and counted on a cell counting plate. And observing the morphological change of the cells by an electron microscope. Conditioning cells 2X 10⁴one/mL. Inoculating into two 96-well plates (100 μ L per well), and placing at 37 deg.C with 5% CO₂After incubation in incubator for 24h, the supernatant was discarded, first group: cytokine final concentration of 0.8% and 2D4 monoclonal antibody of 10 mg.L were added^-1(ii) a Second group: adding 100mg/L algal polysaccharide, final concentration of cytokine of 0.8% and 2D4 monoclonal antibody of 10 mg.L^-1(ii) a 4 wells were set for each concentration, the control group was not dosed with drug, the same amount of maintenance solution was used instead, and the blank wells were not inoculated with cells. Incubated at 37 ℃ in a 5% CO2 incubator for 72 h. The melanocyte proliferation inhibition rate is measured by adopting a tetramethylazo blue colorimetric method (MTT method), namely, 10 mu L of MTT solution of 5g/L is added into each hole after the medicine acts for 72 hours, supernatant is discarded after 4 hours, 100 mu L of DMSO is added into each hole, oscillation is carried out for about 10min, the absorbance value of each hole is detected on an enzyme-labeling instrument, the blank hole is used for zero adjustment, and the wavelength is 490 nm. The melanocyte proliferation inhibition rate was ═ 1 (average absorbance value per concentration ÷ average absorbance value of control group) × 100%.

TABLE 5 melanocyte proliferation inhibition Rate

Group of	Melanocyte proliferation inhibition rate
		First group	85.25±3.42
Second group	94.36±4.03

The experimental results in table 5 show that algal polysaccharide, cytokine and 2D4 monoclonal antibody have good inhibitory effect on melanocytes, and have good synergistic effect. The results further show that the stem cell cytokine, the 2D4 monoclonal antibody and the algal polysaccharide can effectively inhibit the proliferation of melanocytes and have a good whitening effect.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

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Claims (4)

1. The application of the cell factor, the algal polysaccharide and the monoclonal antibody in preparing the pharmaceutical composition for inhibiting the melanoma; wherein, the cell factor is prepared by the following method: placing the epidermal stem cells into a cell culture medium for subculture, wherein the cell culture medium is 89% DMEM + 10% FBS + 1% double antibody stem cell culture medium, and the culture conditions are 37 ℃ and 5% CO₂Culturing; taking well-grown stem cells, adjusting cell concentration to 1X10⁶The cells were seeded in 6-well plates and 50 ng/mL was added after the cells adhered to the plate^{- 1}And (3) carrying out liquid change on a complete culture medium of the secretion promoting peptide, wherein the sequence of the secretion promoting peptide is shown as SEQ ID NO: 1, culturing for 48h, wherein the culture is in 3% O₂+5%CO₂+92%N₂Is carried out under the condition; collecting cell supernatant, ultrafiltering the supernatant with 1KD ultrafilter membrane to remove small molecular impurities with molecular weight below 1KD, and collecting trapped fluid to obtain cytokine;

the monoclonal antibody is a monoclonal antibody for specifically inhibiting tyrosinase, and a light chain variable region and a heavy chain variable region of the monoclonal antibody are respectively as follows:

the sequence of the light chain variable region is shown below:

DIVITQRPALMAASPGEKVTITCWYAMSGLTMREQWYQQKSGISPKPWIYDDETMHCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCPHISMFDRDFGAGTKLELK；

the sequence of the heavy chain variable region is shown below:

EVQLEESATELARPGASVKLSCKASGYIFSSVCPKWIKQRPGQGLEWIGWSITECSYEKSLRWQAGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGKLHHFSGWGLGTTLAVSS。

2. use according to claim 1, characterized in that the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that the pharmaceutical composition further comprises an excipient.

4. The application of the cell factor, the algal polysaccharide and the monoclonal antibody in preparing the whitening cosmetics; the cosmetic contains a cytokine, algal polysaccharides and monoclonal antibodies, wherein the cytokine is prepared by adopting the following method: placing the epidermal stem cells into a cell culture medium for subculture, wherein the cell culture medium is 89% DMEM + 10% FBS + 1% double antibody stem cell culture medium, and the culture conditions are 37 ℃ and 5% CO₂Culturing; taking well-grown stem cells, adjusting cell concentration to 1X10⁶The cells were seeded in 6-well plates and 50 ng/mL was added after the cells adhered to the plate^{- 1}And (3) carrying out liquid change on a complete culture medium of the secretion promoting peptide, wherein the sequence of the secretion promoting peptide is shown as SEQ ID NO: 1, culturing for 48h, wherein the culture is in 3% O₂+5%CO₂+92%N₂Is carried out under the condition; collecting cell supernatant, ultrafiltering the supernatant with 1KD ultrafiltration membrane to remove small molecular impurities with molecular weight below 1KD, and collecting trapped fluid to obtain cytokine;

the monoclonal antibody is a monoclonal antibody for specifically inhibiting tyrosinase, and a light chain variable region and a heavy chain variable region of the monoclonal antibody are respectively as follows:

the sequence of the light chain variable region is shown below:

DIVITQRPALMAASPGEKVTITCWYAMSGLTMREQWYQQKSGISPKPWIYDDETMHCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCPHISMFDRDFGAGTKLELK；

the sequence of the heavy chain variable region is shown below:

EVQLEESATELARPGASVKLSCKASGYIFSSVCPKWIKQRPGQGLEWIGWSITECSYEKSLRWQAGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGKLHHFSGWGLGTTLAVSS。

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