CN115025217A - Use of stem cell lysate in combination with active polysaccharide and tyrosinase inhibitor for preparing medicine or cosmetics - Google Patents

Abstract

The invention relates to the use of stem cell lysates in combination with active polysaccharides and tyrosinase inhibitors for the preparation of a medicament or a cosmetic. The invention aims at the tyrosinase of human beings to prepare a corresponding monoclonal antibody, and the antibody can specifically inhibit the activity of the tyrosinase so as to inhibit the synthesis of melanin and inhibit the activity of melanocytes. The monoclonal antibody, the stem cell factor and the spirulina polysaccharide are prepared into the pharmaceutical composition together, so that the pharmaceutical composition can effectively inhibit the proliferation of melanoma and has a good application prospect.

Description

Use of stem cell lysate in combination with active polysaccharide and tyrosinase inhibitor for preparing medicine or cosmetics

Technical Field

The invention relates to the field of biology, in particular to the use of stem cell lysate combined with active polysaccharides and tyrosinase inhibitors in the preparation of drugs or cosmetics.

Background

Melanoma, usually referred to as malignant melanoma, is a highly malignant tumor of melanocyte origin, abbreviated as malignant melanoma, which frequently occurs in the skin, also visible in mucous membranes and internal organs, accounting for about 3% of all tumors. Malignant melanoma of the skin accounts for the third place (about 6.8% -20%) of malignant tumors of the skin. People who are fair and caucasian have a high incidence rate when they are better than adults, while asians and africans with dark skin have a lower incidence rate and are rarely seen in children. Some patients have familial multiple phenomena. Malignant melanoma may develop from congenital or acquired benign melanocytic nevus, or from dysplastic nevus, or may be a new occurrence. In recent years, the incidence and mortality of malignant melanoma has increased year by year, with a lower lethal age than other solid tumors.

At present, in the world of medical science, the main treatment methods of malignant melanoma include surgical treatment, radiotherapy and chemotherapy treatment, traditional Chinese medicine treatment, biological diagnosis and treatment and the like, and the specific treatment method is determined according to the physical condition of a patient. Surgical excision of lesions is always the main method for treating the disease, and once diagnosis is confirmed, surgical excision should be performed as soon as possible due to the wide extension range of the disease. It is generally accepted that tumors should be extensively resected, and the scope of resection should depend on the type and location of the tumor. The skin grafting is needed after the large-area excision, which brings great pain to the patient, meanwhile, the operation can also accelerate the diffusion and the transfer of the melanoma, and increase the load area of the tumor cells, although the radiotherapy and the chemotherapy can locally or systemically relieve the load of the tumor cells, the tumor cells are easy to recur, the systemic toxic and side effect is large, the nausea, the vomiting and other toxic and side effects are easy to generate, the immune function of the human body is damaged, and the physical and psychological health of the melanoma patient is greatly attacked.

In recent years, with the development of technology, monoclonal antibodies have been widely studied for the treatment of melanoma. anti-PD-1 mab has become the standard first-line treatment of advanced BRAF wild-type melanoma as a monotherapy or in combination with ipilimumab as a regimen that is comparable to targeted treatment of unresectable BRAF mutant melanoma in the context of first-line treatment. Recently, the clinical application of anti-PD-1 formulations has been extended to other diseases such as resected high risk melanoma, non-cutaneous subtypes and brain metastases. How to better resist the drug resistance of the anti-PD-1 monoclonal antibody is still the main focus of clinical research. Recently, nivolumab + high dose interferon- α/pegylated IFN α and ipilimumab were approved in the united states as adjuvant treatment options for patients with high risk melanoma after resection. CHECKMATE-238 test patients with resected stage IIIB, IIIC or IV melanoma were randomized into one year nivolumab,3mg/kg Q2W (n 453), or 4 doses of ipilimumab,10mg/kg Q3W and then into groups of 12 weeks (Q12W) (n 453). The primary endpoint was relapse-free survival (RFS) and the secondary endpoints included OS, safety and quality of life. In the interim analysis with at least 18 months of follow-up, the 12-month RFS rate in the nivolumab group was 70.5% and the ipilimumab group was 60.8% (HR: 0.65, p < 0.001). With respect to disease stage, tumor PD-L1 expression, BRAF mutation, lymph node involvement and primary focal ulcer status, RFS results were consistent in the subgroup analysis. Both groups did not reach the median RFS and no distant metastasis survival (exploratory endpoint). However, the combined use of antibodies and chemical drugs can cause toxic and side effects to the body, so that the body can be harmed unnecessarily while the body is treated.

Therefore, the combination of antibodies with bioactive substances to reduce the harm of chemical drugs is the focus of research. Currently, the infusion of stem cell cytokines, monoclonal antibodies, and active polysaccharides and combinations thereof is an important research direction in the treatment of melanoma.

Spirulina Polysaccharide (PSP) is a water-soluble acidic heteropolysaccharide isolated from Spirulina. PSP has wide biological activities of resisting virus, tumor, radiation, mutation, oxidation, blood sugar and blood fat, regulating immunity and the like, so that PSP becomes one of the hot spots of the research of spirulina at present. Particularly in the aspect of anti-tumor, because the compound has no toxicity to cells, the compound has cytotoxicity different from the traditional anti-cancer drugs, thereby arousing the wide attention of broad scholars, and the research proves that PSP increases the apoptosis rate of BEL7404 liver cancer cells and U251 cells. Suggesting that induction of tumor cell apoptosis is one of the mechanisms of PSP in vivo tumor-inhibiting activity. Experiments show that PSP can reduce the survival rate of MCF27 breast cancer cells, the telomerase activity and the bc122 gene expression, and simultaneously improve the p53 gene expression. The suggestion that the gene can play an anti-tumor role by inhibiting the proliferation of tumor cells, inhibiting the activity of telomerase and the expression of bc122 gene, influencing the signal transduction of gene expression and enhancing the expression of cancer suppressor genes. It is reported that after PSP acts on BELT40412 liver cancer cells for 24 hours, flow cytometry detects that human cysteine proteinase 3(easpase-3) is obviously activated, and the mechanism of PSP for inducing liver cancer cell apoptosis is probably related to the cells through a mitochondrial caspase cascade reaction pathway. Selenium polysaccharide in selenium-rich spirulina can induce tumor cell apoptosis by inhibiting cyclin dependent protein kinases (CDKs) and has antitumor effect. Meanwhile, 9701 cells treated by selenium polysaccharide for 48 hours are detected to have telomerase activity obviously inhibited, and telomeres lost in cell division cannot be supplemented, so that cells are killed, and the growth of tumor cells is inhibited. In addition, the spirulina polysaccharide can reduce lipid peroxidation, reduce the formation of lipofuscin in tissues and keep skin activity by inhibiting free radical oxidation reaction in organisms, thereby achieving the effect of slowing down the aging of the organisms.

Therefore, it is urgent to develop a monoclonal antibody and/or spirulina polysaccharide and/or cytokine for treating melanoma and improving immunity.

Disclosure of Invention

In one aspect of the present invention, a monoclonal antibody capable of inhibiting proliferation and migration of melanocytes is provided.

Specifically, the monoclonal antibody is the variable region sequence of the light chain and the variable region sequence of the heavy chain of the LYSM-3 antibody: the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2:

DLVMTQTAPSVPVTPGESVSISCRSTGDYLDLRQAPCLYWFLQRPGQSPQLLIYFSWNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCEQVAASVPGFGSGTKLEIK

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3:

VKPGGSLKLSCAASMQRNHQETMSWVRQTPDKRLEWVAIIPQIDEYSYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCDICPTDGLGMDWGQGTTVTVS。

the monoclonal antibody can specifically inhibit the activity of tyrosinase by taking the tyrosinase as a target, and further inhibit the synthesis of melanin so as to inhibit the proliferation and the activity of melanocytes.

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDR and framework regions. Amino acid substitutions can be introduced into an antibody of interest and screened for products of desired activity, e.g., retained/improved antigen binding, reduced immunogenicity.

In some embodiments, the heavy chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9 amino acid substitutions (e.g., conservative amino acid substitutions) as compared to the amino acid sequence set forth in one of SEQ ID NOs 2 or 3 in some embodiments, the light chain of the antibody comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9 amino acid substitutions (e.g., conservative amino acid substitutions) as compared to the amino acid sequence set forth in one of SEQ ID NOs 2 or 3.

In some embodiments, an antibody or antigen-binding fragment can include up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) in the framework region of the heavy chain of the antibody, or the light chain of the antibody, or both the heavy and light chains of the antibody, as compared to known framework regions.

Furthermore, the monoclonal antibody is prepared into a pharmaceutical composition for treating melanoma.

In some embodiments, the composition is a pharmaceutical composition and comprises the monoclonal antibody and a pharmaceutically acceptable carrier, diluent or excipient. In preparing pharmaceutical compositions comprising the antibodies described in the teachings herein, various excipients and excipients can be used, as will be apparent to those skilled in the art.

The pharmaceutical compositions generally comprise a pharmaceutically acceptable carrier and a pharmacologically effective amount of an antibody or antibody mixture.

The pharmaceutical composition can be formulated into powder, granule, solution, suspension, aerosol, solid, pill, tablet, capsule, gel, topical cream, suppository, transdermal patch and/or another formulation known in the art.

As used herein, a "pharmaceutically acceptable carrier" includes any standard pharmaceutically acceptable carrier known to one of ordinary skill in the art in formulating pharmaceutical compositions. For example, the antibody may be prepared as a formulation in a pharmaceutically acceptable diluent, including, for example, saline, Phosphate Buffered Saline (PBS), aqueous ethanol, or dextrose, mannitol, dextran, propylene glycol, oils (e.g., vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, solutions of polysorbate 80, or solid formulations in a suitable excipient.

Pharmaceutical compositions will typically also contain one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, and the like), bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents and/or preservatives. Alternatively, the compositions of the present invention may be formulated as a lyophilizate.

Preservatives may be added to retard the growth of microorganisms, and may be added in amounts of 0.2% to 1% (w/v). Suitable preservatives for use with the present disclosure include phenol, benzyl alcohol, m-cresol, methyl paraben, propyl paraben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethylene ammonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes referred to as "stabilizers," may be added to ensure isotonicity of the liquid compositions of the present disclosure, including polyhydric sugar alcohols, such as ternary or higher sugar alcohols, e.g., glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a wide variety of excipients that can range in function from fillers to additives that solubilize the therapeutic agent or help prevent denaturation or adhesion of the container walls. Typical stabilizers may be polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclic alcohols such as inositol; polyethylene glycol; an amino acid polymer; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, -monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizing agent may be present in the range of 0.1 to 10,000 weight/weight active protein.

An effective dose of a monoclonal antibody of the disclosure can be about 0.001 to about 75mg/kg per single (e.g., bolus) administration, multiple administrations, or continuous administration, or up to each single (e.g., bolus) administration, multiple administrations, or continuous administration, depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject. In certain embodiments, each dose may be about 10mg per kilogram of body weight.

Any suitable carrier known to those of ordinary skill in the art may be used in the composition comprising at least one antibody described herein. The antibody composition can be formulated for any suitable mode of administration, including, for example, oral, nasal, mucosal, intravenous, intraperitoneal, intradermal, subcutaneous, and intramuscular administration.

The monoclonal antibody has certain effects of improving the immunity of organisms and enhancing the cancer treatment effect together with stem cell factors and spirulina polysaccharide, so that the two components are used together with the monoclonal antibody, the curative effect is remarkably enhanced, and the application prospect is better.

In another aspect of the invention, the invention provides the use of a tyrosinase monoclonal antibody in combination with stem cell factor and spirulina polysaccharide in the preparation of a medicament for inhibiting melanocyte proliferation; wherein, the stem cell factor is HY StemCell HZ0301C human umbilical cord mesenchymal stem cells, the stem cell factor is obtained by collecting cells after culturing, specifically, removing thallus fragments from a lysate obtained after cracking the cells through ultrasonic disruption, ultrafiltering a supernatant through a 1KD ultrafiltration membrane to obtain a trapped fluid which is a target product containing the stem cell factor, and freeze-drying the trapped fluid to obtain the stem cell factor.

Furthermore, the monoclonal antibody provided by the invention can also inhibit the synthesis of melanin, and can be used for preparing whitening products.

Furthermore, the invention provides an application of the monoclonal antibody for specifically inhibiting melanin synthesis in preparing a whitening product.

Advantageous effects

The invention aims at the tyrosinase of human beings to prepare a corresponding monoclonal antibody, and the antibody can specifically inhibit the activity of the tyrosinase so as to inhibit the synthesis of melanin and inhibit the activity of melanocytes. The monoclonal antibody, the stem cell factor and the spirulina polysaccharide are prepared into the pharmaceutical composition together, so that the pharmaceutical composition can effectively inhibit the proliferation of melanoma and has good application prospect.

Drawings

FIG. 1 is a graph showing the results of the monoclonal antibody inhibiting the proliferation of melanoma cells

FIG. 2 is a graph showing the results of experiments for inhibiting melanin synthesis using monoclonal antibodies

FIG. 3 is a graph showing the results of experiments on the inhibition of melanoma cell migration by monoclonal antibodies

FIG. 4 shows the results of in vivo anti-tumor experiments with monoclonal antibodies and combinations thereof

Detailed Description

The following examples are provided to illustrate certain specific features and/or embodiments. These embodiments should not be construed as limiting the disclosure to the particular features or embodiments described.

EXAMPLE 1 preparation of human tyrosinase monoclonal antibody

(1) Preparation of recombinant human tyrosinase

According to the method for preparing recombinant human tyrosinase disclosed in CN104662149B, the optimized human tyrosinase gene SEQ ID NO: 1 into a pFastbacTM vector, bacmid was prepared and the target protein was expressed in sf9 cells. Western blotting of insect cells expressing recombinant human tyrosinase after lysis showed that the target protein was obtained and was purified and formulated to a concentration of 1mg/mL for use.

(2) Preparation of hybridomas

Taking the recombinant human tyrosinase protein in the step (1) as an immunogen, adding Freund's Complete Adjuvant (FCA) for emulsification, and then immunizing Balb/c mice (15 mu g/mouse) by adopting a back multipoint subcutaneous injection mode; booster immunizations were performed at 5 and 9 weeks after the primary immunization, respectively, and the immunogen protein (30 μ g/mouse) emulsified by Freund's Incomplete Adjuvant (FIA) was intraperitoneally injected; after 1 week, the tail of the immunized mouse is cut off and blood is taken, the serum antibody titer is detected by an ELISA method, and the antibody titer is selected>10 ⁵ Performing a cell fusion experiment; the cells were boosted again 5 days prior to fusion and were injected intraperitoneally with immunogen protein without adjuvant (30. mu.g/cell). Successfully immunized mouse spleen lymphocytes are isolated and fused with mouse myeloma cells SP2/0 in the logarithmic growth phase in a ratio of 5: 1. Selectively culturing hybridoma cells by using a DMEM complete culture medium containing 10% fetal calf serum, a HAT culture medium and an HT culture medium, taking immunogen recombinant human tyrosinase protein as a coating antigen, and detecting the secretion level of a tyrosinase monoclonal antibody in a hybridoma cell culture solution by an ELISA method; hybridoma cells in culture wells with positive ELISA detection results are collected, and cloning culture is continued by adopting a limiting dilution method. After continuous multiple cloning culture, two hybridoma cell strains of clone type hybridoma cells LYSM-3 and LYSM-7 with ELISA detection positive rate of 100% are finally selected for preservation.

(3) Large-scale preparation of monoclonal antibodies

Hybridoma cells LYSM-3 and LYSM-7 were washed with sterile PBS solution and resuspended at 2X 10 ⁶ Perml, the resuspended hybridoma cells were injected intraperitoneally into pristine-primed Balb/c mice (0.5 ml/mouse). After 10 days, the abdomen is collected after ascites is formedWater; centrifuging at room temperature at 3000r/min for 10min, collecting ascites supernatant (containing tyrosinase monoclonal antibody), purifying the tyrosinase monoclonal antibody in the ascites supernatant by ammonium sulfate precipitation, and storing for later use.

Example 2 determination of equilibrium dissociation constant (KD) of LYSM-3 monoclonal antibody

The affinity of LYSM-3 monoclonal antibody to tyrosinase was determined using Biacore T200, all at 25 ℃. A commercial ProteinA chip is adopted, and a proper amount of antibody is fixed by a capture method, so that Rmax is about 50RU, and the capture flow rate is 10 mul/min. The antigen is subjected to gradient dilution, the flow rate of the instrument is switched to 30 mul/min, the antigen sequentially flows through a reference channel and a channel for fixing the antibody according to the sequence of the concentration from low to high, and the antigen flows through a buffer solution to serve as a negative control. After each binding and dissociation, the chip was regenerated with glycine of pH 1.5. And selecting a 1:1 binding model in a Kinetics option for fitting by using an instrumental self-contained analysis software, and calculating the dissociation equilibrium constant KD value of the antibody. The results are shown in Table 1.

TABLE 1 affinity of antibodies for binding human tyrosinase

Sample name	KD(nM)
		LYSM-3 monoclonal antibodies	0.47±0.02

As shown in the results in Table 1, the LYSM-3 monoclonal antibody prepared by the invention has a dissociation equilibrium constant of (0.47 +/-0.02) nM and a good binding effect.

EXAMPLE 3 sequence identification of LYSM-3 monoclonal antibodies

LYSM-3 hybridoma cells are subjected to RNA extraction and reverse transcription to form cDNA, VL and VH genes are amplified by adopting a PCR method, an amplification kit by the PCR method is purchased from Takara company, an amplification product is subjected to sequencing identification, and a light chain variable region sequence and a heavy chain variable region sequence of an obtained antibody are identified as follows.

The amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2:

DLVMTQTAPSVPVTPGESVSISCRSTGDYLDLRQAPCLYWFLQRPGQSPQLLIYFSWNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCEQVAASVPGFGSGTKLEIK

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3:

VKPGGSLKLSCAASMQRNHQETMSWVRQTPDKRLEWVAIIPQIDEYSYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCDICPTDGLGMDWGQGTTVTVS。

example 4LYSM-3 monoclonal antibody inhibits melanoma cell proliferation and melanin synthesis assays

The melanoma cell line B16 is prepared by inoculating cells in logarithmic growth phase into a 96-well plate (1000 cells), and culturing the cells for 24H by using DMEM-H10% FBS; changing the culture medium, adding LYSM-3 monoclonal antibodies (1O, 100, 200 and 500 mu g/mL) with different mass concentrations into each well, adding imatinib mesylate with 100 mu g/mL into a positive control group, adding PBS with the same volume into a negative control group, setting 6 multiple wells for the experimental group and the control group, and respectively incubating for 96 h; adding 20 mu l of MTT (5mg/ml) into each well, and incubating for 4h in a dark place; carefully discard the supernatant, add 150. mu.L of dimethyl sulfoxide into each well, and shake for 5 min; the absorbance (A) was measured at a wavelength of 570nm using an enzyme-linked immunosorbent assay and the inhibition rate was calculated. The inhibition ratio (%) - (test group average absorbance-control group average absorbance)/control group average absorbance × l 00%.

The results are shown in MTT statistics of FIG. 1, and compared with the control group, each administration group has proliferation inhibition effect (P < 0.05), the difference between each group and the control group has statistical significance, and LYSM-3 monoclonal antibody has certain concentration and time dependence on the proliferation inhibition effect of cells (FIG. 1), and the inhibition rate reaches (95.3 +/-1.9)%, when the concentration is 500 mug/mL.

The cell suspension cultured for 96 hours was centrifuged, washed twice with PBS (pH 7.2), added with 1mol/L NaOH, placed in a water bath at 80 ℃ for 30min to completely dissolve the cell pellet, and diluted to a final concentration of 0.2mol/L with ultrapure water. Mixing well, placing each group of solution into 96-well plate, setting up 8 heavy wells with each well at 100 μ L. OD was measured at 492nm with a microplate reader. Melanin content (%) × 100% (OD experimental group/OD control group).

From the results shown in fig. 2, it can be seen that the monoclonal antibody of the present invention has a significant inhibitory effect on melanin synthesis in B16 cells, and the inhibitory rate reached (3.5 ± 0.8)% (fig. 2) at a concentration of 500 μ g/mL, compared to the control group.

Example 5LYSM-3 monoclonal antibody inhibition of melanoma cell migration assay

Inoculating cells in logarithmic phase into the plate, after the cells are converged into a single layer, scratching a scratch with the width of about 1mm on a culture dish by using a 10-microliter gun head, setting 6 observation points in each group, and photographing and recording a scratch area; grouping and giving LYSM-3 monoclonal antibody treatment of 1O, 100, 200 and 500 μ g/mL, positive control group is 100 μ g/mL imatinib mesylate, negative control group is PBS, and the serum concentration of the culture medium is changed to 2%; after culturing for 48h, photographing and recording; the relative distance of the scratched area was measured and the cell migration rate was calculated using Image-ProPlus software. Mobility (%) (relative distance of scratched area before drug treatment-relative distance of scratched area after drug treatment)/relative distance of scratched area before drug treatment × 100%.

The scratch test result in fig. 3 shows that the migration ability of the cells after the monoclonal antibody is acted is significantly reduced (P < 0.05) compared with the control group, wherein the inhibition effect is most significant (P < 0.01) when the mass concentration of the monoclonal antibody is 500 μ g/mL, and the inhibition effect is dependent on a certain concentration. At a concentration of 500. mu.g/mL, the mobility was only (9.8. + -. 1.8)% (FIG. 3).

EXAMPLE 6 preparation of Spirulina polysaccharides

Pulverizing 10g of dried spirulina, sieving with a 10-mesh sieve, hydrolyzing the sieved spirulina powder with 0.5% trypsin at 36 ℃ for 2h, and adding 0.5% papain to hydrolyze for 2 h; then ultrasonic extracting with 1% calcium chloride water solution for 2 times, wherein the power of ultrasonic extraction is 400W for 20 minutes each time. Mixing the two extractive solutions, and filtering with 5 μm polypropylene membrane to obtain filtrate; adding polyamide and snake cage resin into the filtrate, stirring for 1 hour at room temperature, and filtering with 0.8 μm polypropylene membrane to obtain filtrate; concentrating the filtrate at 55 deg.C under reduced pressure to 300ml, filtering with 0.8 μm polypropylene membrane, collecting filtrate, adding ethanol to the filtrate to make ethanol concentration (volume percentage) at 75%, standing at 4 deg.C for 5 hr, centrifuging, collecting solid, and drying to obtain polysaccharide extract. Dissolving the extract at constant volume, taking 0.2mL, determining polysaccharide content by sulfuric acid-anthrone method, and calculating spirulina polysaccharide extraction rate to be 27.53%.

Example 7 in vivo antitumor assay of monoclonal antibody and combination thereof

Removing the culture medium in the culture bottle from B16 cells growing at 80% of the bottom of the culture bottle, flushing the culture bottle with phosphate buffer solution for 2 times, adding 2.5g/l trypsin for several drops for digestion, sucking the digested cells into a centrifugal tube, centrifuging for 5min at 1000rpm, removing the supernatant, adding 3ml of RPMI1640 medium without calf serum, and uniformly blowing and beating the tube (ice bath operation). Cell counting: dissolving in PBS, diluting 100 times (10ul cell suspension +990ul PBS), staining with trypan blue, and making cell density at 20-30 × 10 ⁴ And/ml. Centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell concentration to 1 × 10 ⁶ And/ml. Each C57BL/6J mouse was inoculated with 0.5ml of the vaccine subcutaneously in the right forelimb axilla. On day 15 after inoculation, tumor-bearing mice were sacrificed by dislocation of the cervical vertebrae and melanoma was removed. Under aseptic condition, removing envelope and necrotic part from melanoma in tumor mouse, selecting intact tumor mass, placing in glass homogenizer, adding appropriate amount of normal saline, preparing into tumor cell suspension, filtering with filter screen, centrifuging at 1000rpm for 6min, staining with trypan blue, counting, and counting to obtain cell number of 1 × 10 ⁷ The vaccine is inoculated under the axilla of the right forelimb of a C57 mouse, and each mouse is injected with 0.5 ml.

Mice were weighed and randomly divided into the following groups, and intraperitoneal administration was started 24h after inoculation: saline group (200 μ L/time, days 0, 7, 14, intraperitoneal injection), stem cell factor group (HY StemCell HZ0301C lyophilized preparation of retentate obtained by ultrafiltering human umbilical cord mesenchymal stem cell lysate with 1KD ultrafiltration membrane), 200 μ g/dose, days 0, 7, 14, intraperitoneal injection), Spirulina polysaccharide group (200 μ g/dose, days 0, 7, intraperitoneal injection),14 days i.p.), imatinib mesylate control group (1mg/kg/d, day 0, 7, 14, i.p.), LYSM-3 monoclonal antibody group (1 mg/kg/time, day 0, 7, 14, i.p.), LYSM-3 monoclonal antibody in combination with stem cell factor and spirulina polysaccharide group (100 μ g/spirulina polysaccharide, 100 μ g/stem cell factor (origin same) 100 μ g/LYSM, 1 mg/kg/time LYSM-3 monoclonal antibody, 1h interval administration, day 0, 7, 14, i.p.). Tumor volume was measured at day 0 and day 21. Tumor volume (V) ═ a × b ² And/2 (a is the major diameter and b is the minor diameter). Using a conventional tumor inhibition calculation method in the art, the tumor inhibition was calculated as (1-experimental group tumor volume/control group tumor volume) × 100%, and the results are shown in fig. 4.

From the results in fig. 4, it can be seen that the tumor inhibition effect of each group of treatments was significant (P < 0.05), and the tumor inhibition effect of both the LYDM-3 monoclonal antibody group and the monoclonal antibody combination treatment group was better than that of the positive control group, especially the tumor inhibition rate of the combination treatment group reached (97.2 ± 2.0)%.

And meanwhile, the life activity state of each group of mice is detected, and the result shows that each treatment group of mice has no obvious symptom of toxic reaction.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred embodiments of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims. We therefore claim as our invention all those inventions that come within the scope and spirit of these claims.

Sequence listing

<110> Guangzhou Taishu Biotechnology Ltd

<120> use of stem cell lysate in combination with active polysaccharide and tyrosinase inhibitor for preparing medicine or cosmetics

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ggcaactgca agttcggttt ctggggtccc aactgcaccg agcgtcgtct gctcgtgcgt 360

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cctggcaacc acgacaagtc ccgtaccccc cgtctgccat cctccgctga cgtcgagttc 960

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tcctctaagg acctgggtta cgactacagc tacctgcagg actccgaccc agactccttc 1380

caggactaca tcaagtccta cctggaacag gcttcc 1416

<210> 2

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Leu Val Met Thr Gln Thr Ala Pro Ser Val Pro Val Thr Pro Gly

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Thr Gly Asp Tyr Leu Asp Leu

20 25 30

Arg Gln Ala Pro Cys Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Phe Ser Trp Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Glu Gln Val

85 90 95

Ala Ala Ser Val Pro Gly Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 3

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Met Gln

1 5 10 15

Arg Asn His Gln Glu Thr Met Ser Trp Val Arg Gln Thr Pro Asp Lys

20 25 30

Arg Leu Glu Trp Val Ala Ile Ile Pro Gln Ile Asp Glu Tyr Ser Tyr

35 40 45

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gln Asp

50 55 60

Lys Gln Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr

65 70 75 80

Ala Met Tyr Tyr Cys Asp Ile Cys Pro Thr Asp Gly Leu Gly Met Asp

85 90 95

Trp Gly Gln Gly Thr Thr Val Thr Val Ser

100 105

Claims (6)

1. The use of the tyrosinase monoclonal antibody LYSM-3 in the preparation of a pharmaceutical composition for inhibiting melanoma; wherein the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 3.

2. The use of tyrosinase monoclonal antibody LYSM-3 in combination with spirulina polysaccharide and stem cell factor in the preparation of a pharmaceutical composition for inhibiting melanoma; wherein the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3; wherein, the stem cell factor is HY StemCell HZ0301C human umbilical cord mesenchymal stem cells, the stem cell factor is cultured, the cells are collected, the lysate obtained after the cells are cracked through ultrasonic disruption is centrifuged to remove the bacterial debris, the supernatant is ultrafiltered by a 1KD ultrafiltration membrane to obtain the trapped fluid which is the target product containing the stem cell factor, and the trapped fluid is freeze-dried to obtain the stem cell factor.

3. The application of the tyrosinase monoclonal antibody LYSM-3 in preparing whitening cosmetics for inhibiting melanin generation; wherein the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 3.

4. Use according to claim 1 or 2, characterized in that the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

5. The use of claim 4, wherein the antibody is prepared as a formulation in a pharmaceutically acceptable diluent comprising saline or glucose.

6. Use according to claim 5, characterized in that the pharmaceutical composition further comprises a preservative to retard the growth of microorganisms and the preservative is added in an amount of 0.2-1% (w/v).

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