Application of Haematococcus pluvialis active factors and stem cells in skin repair

Abstract

The present invention relates to the application of active factors of Haematococcus algae and stem cells in skin repair. The present invention provides umbilical cord mesenchymal stem cell cytokines and Haematococcus algae active factors, both of which have the effect of inhibiting ultraviolet damage to the skin. At the same time, based on the COX-2 protein target, a monoclonal antibody that specifically inhibits COX-2 is developed, and the antibody can effectively inhibit skin damage by ultraviolet rays. At the same time, when used in combination with umbilical cord mesenchymal stem cell cytokines and Haematococcus algae active factors, it can effectively inhibit the apoptosis of skin cells induced by ultraviolet rays, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine, and more specifically to the application of active factors of Haematococcus pluvialis and stem cells in skin repair. Background Art

[0002] As people's living standards gradually improve, people's demand for delaying skin aging becomes stronger, and the awareness of protecting the skin is gradually increasing. The skin is the first barrier that isolates the human body from the external environment. Outdoors, the skin is exposed to ultraviolet (UV) radiation all the time. Epidemiological surveys and laboratory studies have confirmed that continuous exposure to UV (including UVA and UVB) can cause acute or chronic skin disorders, injuries, skin cancer, skin aging and other skin lesions. UV radiation is the main factor causing exogenous skin aging, so how to prevent skin exposure to UV or alleviate skin UV damage, thereby delaying skin photoaging, is receiving increasing attention.

[0003] Skin aging is a complex biological process, which is divided into two types: endogenous aging and exogenous aging. Endogenous aging is skin aging that occurs over time and is gene-dependent. Exogenous aging is caused by external environmental factors, among which UV radiation is the main factor, accounting for 80% of external influences, so exogenous aging is also called photoaging. According to the wavelength, UV is divided into UVA (320-400nm), UVB (280-320nm) and UVC (100-280nm). When UV passes through the earth's atmosphere, UVC is filtered out. UVB has a shorter wavelength and is 30-40 times more energetic than UVA. Most of UVB can be absorbed by the epidermis, mainly causing skin erythema, burns, skin cancer and immunosuppression. UVA is a longer wavelength ultraviolet light that can penetrate into the deep dermis of the skin. UVA is the main component (95%) of ultraviolet rays to which the skin is exposed. It is generally believed that UVA has weak carcinogenicity, but it is the main cause of skin photoaging. At present, the focus of anti-UV research is on UVB. For example, SPF is a sun protection factor based on the protection against UVB, but some studies have gradually turned to protecting the skin from UVA damage. The most significant clinical features of photoaging are dry skin, loose nodules, increased fine lines, decreased elasticity, and pigmentation. Histologically, photoaged skin has an increase in melanocytes, excessive pigmentation, a significant decrease in Langerhans cells, thickening of the dermis, a significant decrease in collagen fibers, but a large increase in elastic fibers and degeneration. The characteristics of photoaging at the cellular level are mainly manifested in the excessive accumulation of reactive oxygen species (ROS) and changes in the components of the extracellular matrix. UV irradiation causes epidermal sunburn and erythema, which is related to the production of inflammatory mediators such as prostaglandins (PGs) and leukotrienes. COX has two enzyme functions: COX catalyzes arachidonic acid (AA) to generate prostaglandin G2 (PGG2) and O; peroxidase catalyzes PGG2 to generate prostaglandin H2 (PGH2) and 2 unpaired electrons, thereby generating oxygen free radicals J. Recent studies have confirmed that UV irradiation can induce COX-2 expression in HaCaT, and the signal transduction mechanism shows that tyrosine kinase and reactive oxygen intermediates promote the expression of COX-2 mRNA. This suggests that the increase in COX-2 expression plays an important role in UV-induced inflammation and tumorigenesis. By inhibiting the activity of COX-, it can be used to treat UV-induced inflammation.

[0004] Astaxanthin belongs to a class of carotenoids that are naturally present in organisms such as microalgae, crustaceans, fish and some birds, and is classified as a xanthophyll because of its strong antioxidant capacity. Haematococcus pluvialis accumulates astaxanthin under stressful environmental conditions and is the richest source of astaxanthin in nature, with content up to %. Carotenoids, including astaxanthin and fucoxanthin, are known as singlet oxygen quenchers. These photosynthetic pigments mitigate the harmful effects associated with UV exposure by dissipating excess energy as heat and returning to the initial ground state. Many studies have shown that fucoxanthin has better antioxidant and photoprotective properties than the most common terrestrial antioxidants and carotenoids. Initially, it was believed that MAAs worked only by absorbing UV before reaching key cellular targets, but studies have found that different MAAs from various species also have antioxidant properties, which is a very desirable property for photoprotective molecules.

[0005] In recent years, regenerative medicine has become the research focus of many medical researchers. Regenerative medicine is the use of one's own stem cells or growth factors to repair one's own injured tissue. Adipose-derived mesenchymal stem cells are used because of their multidirectional differentiation properties. The growth factors they secrete, including vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF) and transforming growth factor, can control or maintain injured tissue cells and guide their own repair. At present, there are few studies on the antioxidant effect of ADSC, but some studies have shown that cytokines secreted by stem cells have a protective effect on oxidative damage. IGF can reduce oxidative damage to fibroblasts and intestinal epithelial cells; HGF has a protective effect on oxidative damage to retinal pigment epithelial cells caused by glutathione deficiency. Interleukin-6 (IL-6) can reduce the mortality of epithelial cells caused by hydrogen peroxide. These studies all show that ADSC has an antioxidant effect. Studies have shown that adipose-derived mesenchymal stem cells and their conditioned medium (ADSC-CM) can promote the synthesis and transfer of collagen in fibroblasts in injured tissues. ADSC-CM reduces the formation of melanin by downregulating the expression of tyrosine and tyrosine-related proteins in B16 melanoma cells. ADSC and its secreted growth factors also have a protective effect on UVB-induced fibroblasts. Clinical studies have shown that injection of autologous adipose-derived mesenchymal stem cells can increase dermal thickness by 20-30% and reduce wrinkle formation. These studies have shown that adipose-derived mesenchymal stem cells and their conditioned medium have an anti-skin photoaging effect.

[0006] Although the existing technology has proven that inhibiting COX-2 activity or using astaxanthin or stem cells can be used to treat UV-induced diseases, there are not many studies on the combined use of these drugs, and the therapeutic effect is not well predicted. Summary of the invention

[0007] In one aspect, the present invention provides a COX-2 monoclonal antibody.

[0008] The COX-2 monoclonal antibody of the present invention has been found to have good specificity through identification, and can only specifically bind to human COX-2, but not to mouse COX-2 or other proteins, and has excellent specificity.

[0009] The COX-2 monoclonal antibody C6A4 has a light chain variable region sequence as shown in SEQ ID NO: 1, and a heavy chain variable region sequence as shown in SEQ ID NO: 2.

[0010] In some embodiments, any of the isolated anti-COX-2 antibodies described above, the isolated anti-COX-2 antibody comprises: H , which comprises the amino acid sequence shown in SEQ ID NO:1 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1; and VL, which comprises the amino acid sequence shown in SEQ ID NO:2 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0011] In some embodiments, any of the anti-COX-2 antibodies isolated as described above, the isolated anti-COX-2 antibody comprises an Fc fragment. In some embodiments, the isolated anti-COX-2 antibody is a full-length IgG antibody. In some embodiments, the isolated anti-COX-2 antibody is a full-length IgG1, IgG2, IgG3 or IgG4 antibody. In some embodiments, the isolated anti-COX-2 antibody is chimeric, fully human or humanized. In some embodiments, the isolated anti-COX-2 antibody is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, double-chain antibody (diabody) and linear antibody.

[0012] The "percent (%) amino acid sequence identity" or "homology" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues in the candidate sequence and the polypeptide sequence to be compared when the sequence is compared, assuming that conservative substitutions are part of the sequence identity. The percentage of amino acid sequence identity can be determined by a variety of alignment methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to maximize alignment over the full length of the compared sequences. Further, the present invention also provides the use of COX-2 monoclonal antibodies in the preparation of skin repair drugs for treating UV-damage.

[0013] In some embodiments, the anti-COX-2 antibody has a Kd value of 10 for binding to a non-target -1 M to 10 -6 M (e.g. 10 - 1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M). In some embodiments, the non-target is an antigen other than COX-2. Thus, in some embodiments, the Kd value of the binding between the anti-COX-2 antibody and the non-COX-2 target is 10 -1 M to 10 -6 M, 1×10 -1 M to 5×10 -6 M, 10 -1 M to 10 -5 M, 1×10 -1 M to 5×10 -5 M, 10 -1 M to 10 -4 M, 1×10 -1 M to 5×10 -4 M, 10 -1 M to 10 -3 M, 1×10 -1 M to 5×10 -3 M, 10 -1 M to 10 -2 M, 10 -2 M to 10 -6 M, 1×10 -2 M to 5×10 -6 M, 10 -2 M to 10 -5 M, 1×10 -2 M to 5×10 -5 M, 10 -2 M to 10 -4 M, 1×10 -2 M to 5×10 -4 M, 10 -2 M to 10 -3 M, 10 -3 M to 10 -6 M, 1×10 -3 M to 5×10 -6 M, 10 -3 M to 10 -5 M, 1×10 -3 M to 5×10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -6 M, 1×10 -4 M to 5×10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -6 M.

[0014] In some embodiments, an isolated nucleic acid molecule is provided, the nucleic acid molecule encoding any of the anti-COX-2 antibodies described above. In some embodiments, a vector is provided, the vector comprising any of the nucleic acid molecules described above. In some embodiments, a host cell is provided, the host cell comprising any of the anti-COX-2 antibodies described above, any of the nucleic acid molecules described above, or any of the vectors described above. In some embodiments, a method for preparing an anti-COX-2 antibody is provided, comprising: a) culturing any of the host cells described above under conditions that effectively express the anti-COX-2 antibody; and b) obtaining the expressed anti-COX-2 antibody from the host cell.

[0015] Furthermore, the present invention also provides the use of COX-2 monoclonal antibody in the preparation of a drug for treating ultraviolet damage-based reduction of HaCaT cell apoptosis.

[0016] Furthermore, the drug contains an acceptable carrier.

[0017] Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM Or polyethylene glycol (PEG); exemplary formulations are described in WO98 / 56418 and are expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted into a high protein concentration formulation by a suitable diluent, and the reconstituted formulation can be administered to the subject to be treated herein by subcutaneous administration. Cationic liposomes or liposomes can be used to deliver the anti-COX-2 antibodies in the present application to cells.

[0018] Mesenchymal stem cells can promote the secretion of cytokines under certain specific environmental stimulations, and these cytokines can promote the repair of skin damage.

[0019] The mesenchymal stem cells may be umbilical cord mesenchymal stem cells, which are purchased commercially or obtained by separation from umbilical cord using a conventional separation method in the art.

[0020] Furthermore, the present invention also provides Haematococcus algae active factors.

[0021] Furthermore, the present invention also provides a method for preparing active factors of Haematococcus pluvialis, which specifically includes preparing Haematococcus pluvialis and then extracting the active factors therein, wherein the main component of the active factors is astaxanthin, which has the effect of inhibiting ultraviolet damage to the skin in the art.

[0022] Furthermore, the present invention also provides the use of COX-2 monoclonal antibody, umbilical cord mesenchymal stem cell cytokine and Haematococcus active factor in the preparation of a drug for treating ultraviolet damage-based reduction of HaCaT cell apoptosis.

[0023] Furthermore, the present invention also provides the use of COX-2 monoclonal antibody, umbilical cord mesenchymal stem cell cytokine and Haematococcus active factor in the preparation of a drug for treating skin damage caused by ultraviolet damage.

[0024] Furthermore, the drug contains an acceptable carrier.

[0025] Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM Or polyethylene glycol (PEG); exemplary formulations are described in WO98 / 56418 and are expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted into a high protein concentration formulation by a suitable diluent, and the reconstituted formulation can be administered to the subject to be treated herein by subcutaneous administration. Cationic liposomes or liposomes can be used to deliver the anti-COX-2 antibodies in the present application to cells.

[0026] Beneficial Effects

[0027] The present invention provides umbilical cord mesenchymal stem cell cytokines and Haematococcus algae active factors, both of which have the effect of inhibiting ultraviolet damage to the skin. At the same time, based on the COX-2 protein target, a monoclonal antibody that specifically inhibits COX-2 is developed. The antibody can effectively inhibit skin damage by ultraviolet rays. At the same time, when used in combination with the umbilical cord mesenchymal stem cell cytokines and the Haematococcus algae active factors, it can effectively inhibit the apoptosis of skin cells induced by ultraviolet rays, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Effects of monoclonal antibodies on apoptosis of HaCaT cells induced by ultraviolet irradiation

[0029] Figure 2 Effects of active factors from umbilical cord mesenchymal stem cells on the antioxidant capacity of human skin cells

[0030] Figure 3 The synergistic effect of each component on reducing the apoptosis of HaCaT cells induced by ultraviolet irradiation DETAILED DESCRIPTION

[0031] Those skilled in the art will recognize that several embodiments are possible within the scope and purpose of the present application. The present application will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present application, but should not be construed as limiting its scope in any way.

[0032] Example 1 Preparation of COX-2 Monoclonal Antibody Inhibitor

[0033] Recombinant COX-2 was used as the immunogen and was purchased from Xinbosheng Biotechnology, with the catalog number: 60122.

[0034] The antigen was emulsified with Freund's complete adjuvant and immunized into female Balb / c mice aged 18 weeks. The dose was 120 μg / mouse and injected subcutaneously at 6 points per mouse. The antigen was emulsified with Freund's incomplete adjuvant and the dose was 60 μg / mouse. Seven days after the third booster immunization, the polyantibody titer against the immunogen in the mouse serum was detected by indirect ELISA. The mouse No. 4 with the highest titer was immunized by tail vein injection. The antigen was mixed with physiological saline and the dose was 60 μg / mouse. One week after the shock immunization, the spleen was taken to prepare a cell suspension, and the cell fusion was performed with mouse myeloma cells SP2 / 0 at a ratio of 10:1. The fused cells were gently suspended with HAT selection medium, and the fused cells were dispersed into a 96-well cell culture plate, 220μl / well, and cultured in a 37°C, 5% CO2 incubator. Small cell clusters can be observed under a microscope after 3-4 days of culture. After fusion, HT culture medium was replaced with half the medium on the 7th day, and 25μl of cell culture supernatant was aspirated on the 10th day for initial screening by ELISA. The positive antibodies obtained by screening were diluted until a single subclone cell was obtained. Through ELISA screening, 6 monoclonal clones with good positive results were obtained, among which C6A4 and C8H5 had the strongest resistance. The two clones were expanded and cultured for detection and determination.

[0035] Preparation of C6A4 and C8H5 monoclonal antibodies by ascites induction method: C6A4 and C8H5 cells in logarithmic growth phase were washed and suspended in serum-free medium, and 5×10 5 / mL, 1mL of suspended cells was intraperitoneally injected into mice sensitized with paraffin oil in advance. After the mouse abdomen was swollen, ascites was collected and collected multiple times. The ascites was centrifuged at 4000rpm at 4°C for 10 minutes. Carefully aspirate the ascites in the middle and collect it in a centrifuge tube. Purify the antibody from the ascites using Protein G (GE) affinity chromatography according to the instructions. The concentration was determined using the Bradford method, and the concentration of the two antibodies was adjusted to 5mg / mL and stored at -20°C.

[0036] Identification of monoclonal antibody characteristics: Subtype detection: Mouse Immunoglobulin Isotyping ELISA kit (BD) was used, and the goat anti-mouse IgG plate was coated and blocked according to the operating instructions. The hybridoma supernatant was taken for detection. The results showed that both C6A4 and C8H5 monoclonal antibodies were IgG1, λ type mouse monoclonal antibodies.

[0037] Antibody titer detection after purification: Dilute the recombinant COX-2 coated with PBS (pH 7.2) at 1 μg / mL, add 100 μL to each well, 4°C, overnight. Pour off the liquid, wash 3 times with PBS containing 0.05% Tween, add 200 μL of blocking solution (PBS containing 3% BSA) to each well, incubate at 37°C for 1 hour, pour off the liquid, and set aside. Add the diluted antibody solution to each well, make a gradient dilution of 1:3, 100 μL of reaction solution to each well, incubate at 37°C for 1 hour, pour off the liquid, and wash 3 times with PBST. Dilute HRP-labeled goat anti-mouse antibody Anti-Mouse IgG (Fc specific) (Sigma) with blocking solution at 1:10000, add 100 μL to each well, incubate at 37°C for 1 hour, pour off the liquid, and wash 3 times with PBST. Add 100 μL of TMB color development solution to each well for color development, and measure the OD value at a wavelength of 450 nm. The results are shown in Table 1 below.

[0038] Table 1 Monoclonal antibody titer

[0039]

[0040]

[0041] From the results in Table 1, it can be seen that the titers of C6A4 and C8H5 monoclonal antibodies both reached 1:5.12×10 5 It has good binding activity with COX-2.

[0042] Example 2 Affinity determination and variable region sequence determination of COX-2 monoclonal antibody C6A4

[0043] The COX-2 protein was diluted with CBS solution to a coating solution of 1 μg / mL and 2 μg / mL, and the ELISA plate was coated respectively. The titer of the monoclonal antibody ascites was determined by indirect ELISA. The monoclonal antibody concentration was used as the horizontal axis and the OD450 value was used as the vertical axis to draw the corresponding two indirect ELISA reaction curves. The OD450 value of the flat section on each curve was taken as 100%, and the antibody concentration corresponding to the 50% OD450 value was calculated on the curve. The affinity constant of the monoclonal antibody was calculated according to the formula Kaff = (n-1) / 2(n[Ab′]t-[Ab]t), where n = [Ag]t / [Ag′]t, [Ag]t, [Ag′]t are two different coating original concentrations, and [Ab]t, [Ab′]t are the antibody concentrations corresponding to the 50% OD450 value at each coating original concentration. According to the affinity determination results, the dissociation constant Kd value of the C6A4 monoclonal antibody was calculated to be 6.23×10 -9 mol / L.

[0044] The cultured hybridoma cells were centrifuged to obtain the supernatant, and the cell pellet was entrusted to Shanghai Bioengineering to determine the antibody heavy chain and light chain variable region sequences. The results showed that the antibody light chain variable region sequence was as shown in SEQ ID NO: 1, and the heavy chain variable region sequence was as shown in SEQ ID NO: 2.

[0045] Example 3 Effect of C6A4 monoclonal antibody on ultraviolet irradiation-induced apoptosis of HaCaT cells

[0046] HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and subcultured at 37°C in a 5% CO2 saturated humidity environment. The cells were digested with 0.25% trypsin and 0.02% EDTA and the cell density was adjusted to 1×10 cells / mL in DMEM containing 10% fetal bovine serum. 4 / ml, 100μl / well was quantitatively inoculated in a 96-well culture plate and divided into the following groups: Group 1 was a blank control group; Groups 2 to 5 were added with C6A4 monoclonal antibody 1 / 10 / 50 / 200μg / mL, and the concentration of NS398 positive control group was 10μg / mL, with 4 replicates for each concentration. After incubation for 4 hours, long-wave ultraviolet / medium-wave ultraviolet (UVA / UVB) irradiation was performed. The 96-well culture plate was treated with aluminum foil paper to avoid light, and the cell wells to be treated were untied. The SS-05 ultraviolet light therapy device was used for light irradiation. The distance between the ultraviolet light and the cells was 20cm, and the irradiation dose was set as follows:

[0047] UVA / UVB(1.00 / 0.02J / cm 2 ), and a normal control group (no illumination group) was set up at the same time. Each group had 4 replicate wells. After illumination, the culture was continued for 12 hours. The adherent cells were digested and prepared into a single cell suspension with DMEM culture medium containing 10% fetal bovine serum. The cell density was adjusted to about 1×10 6 / ml, centrifuge at 1000r / min for 5min, collect cells and discard culture medium; wash once with PBS, centrifuge to remove supernatant; add ice-cold 70% ethanol to fix single cell suspension, 4℃, 2h; centrifuge to discard fixative, resuspend in 3ml PBS for 5min, centrifuge to discard supernatant, add RNaseA (final concentration 1mg / m1) 80μl / tube, oscillate to mix, and bathe in 37℃ water for 30min; put on ice for 2min to terminate RNaseA action; add P1 comprehensive dye 1ml / tube, oscillate to mix, stand at 4℃ in the dark for 30min, filter with 300-mesh nylon mesh, and detect cell apoptosis rate on the machine.

[0048] The results are as follows Figure 1 As shown, from Figure 1 From the results, it can be seen that the apoptosis rate of the normal control group (i.e., cells that were not given UV irradiation and experimental treatment) was about 5.00%, and the apoptosis rate of the blank-administered control group cells that were not given experimental treatment after UV irradiation reached 37.12%, which also shows that UV irradiation can induce apoptosis in HaCaT cells. After monoclonal antibody treatment and then UV irradiation, the cell apoptosis rate gradually decreased with the increase of antibody concentration, and had a good concentration dependence. Under the condition of 200μg / mL C6A4 monoclonal antibody treatment, the cell apoptosis rate was only 6.83%, which was extremely significant compared with no antibody treatment, P<0.01. The results show that the antibody of the present invention can more effectively resist UV irradiation-induced cell apoptosis than the positive control NS398 at the same concentration.

[0049] Example 4 Preparation method of active factors of Haematococcus

[0050] Use GY-D2 Haematococcus pluvialis (purchased from Shanghai Guangyu Biotechnology Co., Ltd.) with BBM as the basic culture medium. The components of BBM culture medium are as follows: NaNO3 250mg, KH2PO4 175mg, K2HPO4 75mg, MgSO4·7H2O 75mg, CaCl2·2H2O 25mg, NaCl 25mg, EDTA 50mg, KOH 31mg, FeSO4·7H2O4.98mg, H3BO3 11.42mg, ZnSO4·7H2O 8.82mg, MnCl2 1.44mg, MoO3 0.71mg, CuSO4·5H2O 1.57mg, Co(NO3)2·6H2O 0.49mg, and distilled water to make up to 1000ml.

[0051] The algae were cultured in BBM medium at 23°C for 10 days, precipitated by low-speed centrifugation, the supernatant was discarded, nitrogen-deficient medium (nitrogen was removed from BBM medium) was added, and cultured for 7 days under 5000lx light intensity. The algae were collected by centrifugation, dried and stored.

[0052] Take 5.0g of the above-mentioned cultured Haematococcus algae, add 200mL of cellulase solution (0.6mg / mL), break the algae wall at 50℃ for 30min, then centrifuge at 5000rpm for 5min, discard the supernatant, add 500mL of acetone to the algae powder, extract for 40min, centrifuge again, discard the lower precipitate to obtain a crude extract. The crude extract is concentrated and evaporated to dryness to obtain the active factor of Haematococcus algae.

[0053] Calculation of astaxanthin content in Haematococcus algae active factor: Accurately weigh 2.5 mg of pure astaxanthin sample, dissolve it with chloroform, and make up to 25 mL. Accurately transfer 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL respectively, dilute with chloroform and make up to 50 mL, and measure the absorbance at 490 nm. According to the standard curve drawn with astaxanthin samples, the astaxanthin content in the entire sample of Haematococcus algae active factor was calculated to be 146 mg, and the specific extraction efficiency was 29.2 mg astaxanthin / g (haematococcus algae dry weight).

[0054] Example 5 Preparation of Umbilical Cord Mesenchymal Stem Cell Active Factors

[0055] Under sterile conditions, 10 cm of umbilical cord from a full-term infant was obtained, washed with D-Hanks solution until the tissue was bloodless, the epidermis, two arteries and one vein were removed, the tissue was cut into pieces of about 1 mm3 / piece, and DMEM / F12 medium (containing double antibodies) containing 10% FBS was added, and cultured at 37°C, saturated humidity, and 5% CO2 for about a week to obtain primary human umbilical cord mesenchymal stem cells. When the cell confluence was 80%, 0.25% trypsin (containing 0.02% EDTA) was used to digest the cells for subculture, and the third generation of cells was used for experiment.

[0056] The third generation of stem cells were cultured in 10% FBS DMEM / F12 medium. When the cell confluence reached 80%, the supernatant of the medium containing 10% FBS was discarded, the cells were washed three times with PBS (pH=7.0), and the cells were incubated with PBS (pH=7.0). Sterilized Tips were used to rub the cell surface back and forth to damage the cells. The cells were treated at 10 degrees Celsius for 1 hour, and then 20 μW / cm 2 / s UVA irradiated cells for 2h, discarded PBS (pH=7.0), added serum-free supernatant, cultured for 72h, and collected serum-free supernatant. After concentration and desalting with a membrane package with a molecular cutoff of 3KD, concentrated to one tenth of the original volume, filtered with a 0.22μm membrane, and obtained sterile stem cell secretion supernatant, which is the stem cell active factor.

[0057] When human dermal fibroblasts grow to 70% confluency, USFM is diluted to the final concentration (20 μg / ml for high-dose group and 10 μg / ml for low-dose group) with DMEM / F12 medium containing 10% FBS, and human dermal fibroblasts are added for co-culture. VitC 100 μg / ml is used as a positive drug control. At the same time, normal cells without treatment are the normal group, and cells treated with UV without adding drugs are the model group. After 12 hours of culture, the UVA radiation intensity is 200 μW / cm 2 After UV damage for 36 h, the cells were cultured for 48 h, and the cells were collected to measure the intracellular SOD enzyme activity.

[0058] from Figure 2 The results show that both high and low doses of stem cell active factor can increase the activity of intracellular SOD enzyme to varying degrees. The intracellular SOD enzyme activity of the pretreatment group of the high dose group (20 μg / ml) of stem cell active factor is 46.88±1.21U / ml; the low dose group (10 μg / ml) of stem cell active factor is 43.26±2.03U / ml. The results show that stem cell active factor can increase the intracellular SOD enzyme activity, thereby reducing the attack and damage of oxygen free radicals induced by ultraviolet radiation on cells and reducing the apoptosis rate of cells.

[0059] Example 6 The components act synergistically to reduce the effect of ultraviolet irradiation-induced apoptosis in HaCaT cells

[0060] HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and subcultured at 37°C in a 5% CO2 saturated humidity environment. The cells were digested with 0.25% trypsin and 0.02% EDTA and the cell density was adjusted to 1×10 cells / mL in DMEM containing 10% fetal bovine serum. 4 / ml, 100μl / well was quantitatively inoculated in a 96-well culture plate and grouped as follows:

[0061] Group 1 was the blank medication control group;

[0062] Group 2 was added with 200 μg / mL of C6A4 monoclonal antibody + 200 μg / mL of Haematococcus active factor prepared in Example 4 + 20 μg / mL of stem cell active factor;

[0063] Group 3 was added with 200 μg / mL of Haematococcus active factor and 20 μg / mL of stem cell active factor prepared in Example 4;

[0064] Group 4: 200 μg / mL of the Haematococcus active factor prepared in Example 4;

[0065] Four replicate wells were set for each concentration. After incubation for 4 hours, the cells were exposed to long-wave ultraviolet / medium-wave ultraviolet (UVA / UVB). The 96-well culture plate was treated with aluminum foil to avoid light, and the wells with cells to be treated were untied. The cells were irradiated with light using an SS-05 ultraviolet light therapy device. The distance between the ultraviolet light and the cells was 20 cm, and the irradiation doses were set as: UVA / UVB (1.00 / 0.02 J / cm2). A normal control group (no light group) was also set up. Each group had four replicate wells. After the light exposure was completed, the cells were cultured for 12 hours. The adherent cells were digested and made into a single-cell suspension using DMEM culture medium containing 10% fetal bovine serum. The cell density was adjusted to about 1×10 6 / ml, centrifuge at 1000r / min for 5min, collect cells and discard culture medium; wash once with PBS, centrifuge to remove supernatant; add ice-cold 70% ethanol to fix single cell suspension, 4℃, 2h; centrifuge to discard fixative, resuspend in 3ml PBS for 5min, centrifuge to discard supernatant, add RNaseA (final concentration 1mg / m1) 80μl / tube, oscillate to mix, and bathe in 37℃ water for 30min; put on ice for 2min to terminate RNaseA action; add P1 comprehensive dye 1ml / tube, oscillate to mix, stand at 4℃ in the dark for 30min, filter with 300-mesh nylon mesh, and detect cell apoptosis rate on the machine.

[0066] from Figure 3 The results show that the combined use of the three components of C6A4 monoclonal antibody 200μg / mL + Haematococcus active factor 200μg / mL prepared in Example 4 + stem cell active factor 20μg / ml can significantly reduce the apoptosis rate of cells, and the apoptosis rate is only (2.03±0.38)%, which is significantly different from that of group 1 (P<0.01). The inhibitory effect of the apoptosis rate of groups 3 and 4 using two components or one component respectively is not as good as that of group 1, which fully demonstrates that the use of the three components together can significantly inhibit cell apoptosis and has excellent application prospects.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-COX-2 monoclonal antibody, named C6A4, wherein the light chain variable region sequence of the antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO:

2.

2. Use of the monoclonal antibody according to claim 1 in the preparation of a drug for inhibiting ultraviolet damage to skin cells.

3. Use of anti-COX-2 monoclonal antibodies, umbilical cord mesenchymal stem cell cytokines and Haematococcus active factors in the preparation of drugs for inhibiting ultraviolet damage to skin cells; wherein: The anti-COX-2 monoclonal antibody is named C6A4, and the sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the active factor of Haematococcus pluvialis is prepared by adding cellulase solution to the algae body of Haematococcus pluvialis, breaking the wall at 50°C, centrifuging at 5000 rpm for 5 min, discarding the supernatant, adding 500 mL of acetone to the algae powder, extracting for 40 min, centrifuging again, discarding the lower layer of precipitation to obtain a crude extract, and concentrating and evaporating the crude extract to obtain the active factor of Haematococcus pluvialis; the umbilical cord mesenchymal stem cell cytokine is prepared by inoculating the stem cells into 10% FBS DMEM / F12 culture medium to culture the cells, discarding the culture medium supernatant containing 10% FBS when the cell confluence is 80%, washing the cells three times with PBS pH=7.0, incubating the cells with PBS pH=7.0, rubbing the cell surface with sterilized Tips to damage the cells, treating at 10 degrees Celsius for 1 hour, and then 20μW / cm 2 / s UVA irradiates the cells for 2 hours, discards PBS pH=7.0, adds serum-free supernatant, and cultures for 72 hours, collects serum-free supernatant, concentrates and desalts through a membrane package with a molecular cutoff of 3KD, concentrates to one tenth of the original volume, and filters through a 0.22μm membrane to obtain a sterile stem cell secretion supernatant, which is the umbilical cord mesenchymal stem cell cytokine.

4. The use according to claim 3, characterized in that The medicine also contains a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that The pharmaceutically acceptable carrier includes phosphate or citric acid.

6. The use according to claim 4, characterized in that The pharmaceutically acceptable carrier includes ascorbic acid or methionine.

7. The use according to claim 4, characterized in that The pharmaceutically acceptable carrier includes the preservative octadecyldimethylbenzyl ammonium chloride.

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