A kind of stem cell freeze-dried powder and its use in preparing medicine or cosmetics

Abstract

The present invention discloses an IL-11 protein monoclonal antibody, the monoclonal antibody is monoclonal antibody 4D3, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4D3 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The present invention also discloses a stem cell freeze-dried powder, the stem cell freeze-dried powder contains an effective amount of human mesenchymal stem cells and an effective amount of IL-11 protein monoclonal antibody 4D3. The stem cell freeze-dried powder prepared by the present invention has good stability, and can inhibit the inflammatory response in the skin caused by long-term ultraviolet light, thereby treating the skin for ultraviolet damage repair. Therefore, it has a relatively broad application prospect in cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a stem cell freeze-dried powder and use thereof in preparing medicines or cosmetics. Background Art

[0002] Human mesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multidirectional differentiation potentials, which have broad application prospects in the fields of regenerative medicine and cosmetology. However, the preservation and transportation of human mesenchymal stem cells are a major problem in their application. Freeze-drying technology, as a method that can preserve biological materials for a long time at room temperature, provides a new idea for the preservation and transportation of human mesenchymal stem cells.

[0003] Skin photoaging is an exogenous aging phenomenon of the skin caused by long-term and repeated ultraviolet radiation. It is different from natural aging (endogenous aging), which is mainly caused by endogenous factors of the body. Photoaging can cause symptoms such as roughness, wrinkles, dilated capillaries, pigmentation spots, enlarged pores and dull skin. Ultraviolet rays (UV) are the main factors causing skin photoaging, among which UVA and UVB have the greatest impact on the skin. UVA can penetrate deep into the basal layer and dermis of the skin, while UVB acts on the epidermis of the skin. Both can cause skin damage and accelerate the aging process. Therefore, studying and treating them can help delay cell aging. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a stem cell freeze-dried powder and its use in the preparation of medicines or cosmetics.

[0005] Therefore, the present invention discloses a monoclonal antibody to IL-11 protein, wherein the monoclonal antibody is monoclonal antibody 4D3, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4D3 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0006] In another aspect, the present invention further discloses a stem cell freeze-dried powder, wherein the stem cell freeze-dried powder contains an effective amount of human mesenchymal stem cells and an effective amount of the IL-11 protein monoclonal antibody 4D3 according to claim 1.

[0007] Preferably, the content of human mesenchymal stem cells in the present invention is 10 6 Pieces / ml.

[0008] Preferably, the content of the IL-11 protein monoclonal antibody 4D3 of the present invention is 1 mg / ml.

[0009] In another aspect, the present invention further discloses a method for preparing the above-mentioned freeze-dried stem cell powder, the method comprising the following steps:

[0010] (1) first dialyzing the monoclonal antibody 4D3 with a lyophilization buffer to adjust the concentration to 1 mg / ml;

[0011] (2) The concentration of human mesenchymal stem cells was adjusted to 10 using a freeze-dried buffer containing monoclonal antibody 4D3. 6 / ml, mix well, and pipette 1ml / tube, and cool from room temperature to -80℃ at 10℃ / min;

[0012] (3) The packaged liquid is placed in a freeze dryer and freeze-dried for 24 hours according to the set freeze-drying program to obtain stem cell freeze-dried powder.

[0013] Preferably, the formula of the freeze-drying buffer in step (1) of the present invention is: add 20 ml of fetal bovine serum, 20 g of sucrose, and 100 ml of glycerol to 100 ml of DMEM culture medium, stir and mix, and sterilize through a 0.22 μm filter membrane.

[0014] In another aspect, the present invention also discloses a use of the monoclonal antibody 4D3 in preparing freeze-dried stem cell powder.

[0015] The prepared stem cell freeze-dried powder of the present invention has good stability, and can effectively inhibit the expression of inflammatory factors, increase the expression of SOD on the surface of the skin, improve the ability to scavenge free radicals, and inhibit the inflammatory reaction in the skin caused by long-term ultraviolet light, thereby treating the skin damage and repairing it against ultraviolet light. Therefore, it has a relatively broad application prospect in cosmetics. DETAILED DESCRIPTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0017] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The culture medium used for the isolation and culture of human mesenchymal stem cells was purchased from Merck. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0018] Example 1: Isolation and culture of human mesenchymal stem cells

[0019] The umbilical cord was removed and placed in Hank's solution containing antibiotics, the blood was washed away, the blood vessels were removed and the cord was cut into small pieces of 1.0 to 2.0 mm.

[0020] The tissue blocks were inoculated on culture plates at an appropriate density and cultured in DMEM containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Fresh culture medium was added every 3 days. When fibroblasts growing in colonies were observed around the tissue blocks under the microscope, the tissue blocks were removed and culture medium was added to continue the culture. After about 20 days, when the cell growth reached about 80%, the cells were digested with trypsin-EDTA solution and cultured at 10 °C. 4 / cm 2 Subculture or resuspend in DMEM containing 10% DMSO and 40% fetal bovine serum (cell density of 2×10 6 / ml), gradually cooled and frozen in liquid nitrogen for later use.

[0021] When the cells reach about 80% confluence, perform cell passage. Discard the culture medium, add 3-5ml trypsin-EDTA solution, and incubate in a 37℃ incubator for 3-5 minutes. After the cells are completely digested, add 5ml culture medium to the culture flask. Gently rotate the culture plate to mix the cell suspension. Transfer the dissociated cells to a 15ml offline tube, centrifuge at 500rpm for 5min, and carefully remove the supernatant. Add culture medium to the 15ml offline tube to resuspend the cells. Count the cells and divide the cells into 10 according to the count results. 4 / cm 2 The cells are inoculated at a density of 100 μg / mL into a suitable culture flask containing culture medium, and when the cells reach a confluence of about 80%, the cells are passaged, expanded, or frozen for future use.

[0022] Example 2: Preparation of IL-11 protein monoclonal antibody

[0023] Monoclonal antibodies were prepared using IL-11 protein (MCE, HY-P7031). The specific method was conventional mouse hybridoma cell technology, which is briefly described as follows:

[0024] Select SPF BALB / c mice of about 7 weeks of age. For the first immunization, emulsify the IL-11 protein with Freund's complete adjuvant in equal volumes and inoculate the mice intraperitoneally, 0.2 ml / mouse (the vaccine contains 100 μg / ml IL-11 protein); 2 weeks later, emulsify the IL-11 protein with Freund's incomplete adjuvant in equal volumes and inoculate the mice intraperitoneally for the second time, 0.2 ml / mouse (the vaccine contains 100 μg / ml IL-11 protein); 2 weeks later, the mice are directly immunized with the recombinant protein via the third intraperitoneal route, 0.2 ml / mouse (the vaccine contains 100 μg / ml IL-11 protein); on the 7th day after immunization, mouse spleen cells are taken and fused with mouse myeloma cells SP2 / 0 and cultured in HAT selective medium; about 7 days later, the IL-11 protein is used as the coating antigen, and the cell supernatant is detected by indirect ELISA to screen positive hybridoma cells.

[0025] SPF Balb / c mice of about 7 weeks old were intraperitoneally injected with 0.5 ml of pristane per mouse. Seven days later, each mouse was injected with 1×10 hybridoma cells. 6 After 7 days, the mouse ascites was extracted and centrifuged at 1000r / min for 10 minutes at 2-8°C to collect the supernatant. The monoclonal antibody was purified using a Protein G affinity chromatography column, and the quantitative aliquots were stored below -70°C for later use.

[0026] After the above steps, a better hybridoma cell was screened, and on this basis, the corresponding monoclonal antibody was prepared and marked as monoclonal antibody 4D3.

[0027] The commercial mouse monoclonal antibody subtype identification ELISA kit (Shanghai Kanglang Biotechnology Co., Ltd.) was used to identify the subtypes of the heavy chain and light chain of the monoclonal antibody according to the instructions. After testing, the heavy chain subtype of the monoclonal antibody 4D3 was IgG1, and the light chain was Kappa.

[0028] According to the antibody subtype results, the antibody gene sequence was cloned using a method based on the RACE technology route. Hybridoma cells with good growth status were collected, and the total RNA of the hybridoma cells was obtained using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the operating method of Takara's SMARTer RACE instructions, and the full-length sequence of the target antibody was amplified. After sequencing and analysis, the sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody 4D3 were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0029] Example 3: Lyophilization of human mesenchymal stem cells and preparation of drugs

[0030] Preparation of freeze-dried buffer: Add 20 ml fetal bovine serum, 20 g sucrose, and 100 ml glycerol to 100 ml DMEM medium, stir and mix, and sterilize through a 0.22 μm filter. Store at 2-8°C for later use.

[0031] The human mesenchymal stem cells cultured in Example 1 were added to the freeze-drying buffer (after centrifugation, the cell concentration was adjusted to 10 using the freeze-drying buffer). 6 / ml), mix well, and pipette 1ml / tube, and cool from room temperature to -80℃ at 10℃ / min. Recorded as lyophilized solution 1.

[0032] The monoclonal antibody 4D3 prepared in Example 2 was first dialyzed using a lyophilization buffer to adjust the concentration to 1 mg / ml, and then the human mesenchymal stem cells cultured in Example 1 were added to the lyophilization buffer containing the monoclonal antibody 4D3 (after centrifugation, the cell concentration was adjusted to 10 using a lyophilization buffer containing the monoclonal antibody 4D3). 6 1 ml / ml), mix well, and pipette 1 ml / tube, and cool from room temperature to -80°C at 10°C / min. This is called lyophilized solution 2.

[0033] Freeze-dried liquid 1 and freeze-dried liquid 2 were placed in freeze dryers respectively, and freeze-dried for 24 hours according to the freeze-drying program set in Table 1. Freeze-dried powder 1 (prepared from freeze-dried liquid 1) and freeze-dried powder 2 (prepared from freeze-dried liquid 2) were obtained.

[0034] Table 1 Freeze-drying procedure

[0035]

[0036]

[0037] The freeze-dried powder 1 and freeze-dried powder 2 were dissolved in PBS, respectively, and after trypan blue staining, the number of cells stained with trypan blue was counted, and the cell survival rate was calculated as [(total number of cells-number of cells stained with trypan blue) / total number of counted cells*100%. The results showed (Table 2) that the cell survival rate after freeze-drying by this method was higher.

[0038] Table 2 Lyophilized powder viable cell count results

[0039] Group Freeze-dried powder 1 Freeze-dried powder 2 Control (not freeze-dried) Cell survival rate (%) 92.5±4.3 93.2±3.8 95.5±6.7

[0040] The freeze-dried powder 1 and freeze-dried powder 2 were stored at -20°C for 6 months, respectively, and dissolved with PBS. The cells were counted and calculated according to the above method. The results showed (Table 3) that the cells freeze-dried by this method were stored for 6 months and the survival rate was still high.

[0041] Table 3 Lyophilized powder viable cell count results

[0042] Group Freeze-dried powder 1 Freeze-dried powder 2 Control (not lyophilized, stored in liquid nitrogen) Cell survival rate (%) 81.6±3.3 80.8±4.2 96.3±5.9

[0043] Example 4: Protective effect of human mesenchymal stem cell freeze-dried powder on skin photoaging model mice

[0044] The experiment was conducted with reference to Example 5 of patent CN 115141281 A, which is briefly described as follows:

[0045] ICR mice, half male and half female, clean grade, weighing 18-22 g. After being fed normally for 1 week, the mice were randomly divided into 6 groups, namely, blank group, model group, positive control group, freeze-dried powder group 1, freeze-dried powder group 2, and freeze-dried powder group 3 (using commercial IL-11 monoclonal antibody (abcam, ab89887) prepared according to the method of freeze-dried powder 2 in Example 3).

[0046] The back hair of the mice was shaved with a razor. The blank group was not irradiated with UV, and the remaining groups were shaved repeatedly before UV irradiation. The modeling method was a conventional method in the art for preparing a photoaging mouse model using UV, irradiating for 2 hours per day, accumulating irradiation for 14 days, and the cumulative UVB light dose was 250mJ / cm2. On the first day of irradiation, the drugs were administered according to each group, as follows:

[0047] Blank group: intraperitoneal injection of 0.5 ml PBS buffer;

[0048] Model group: after UV irradiation, 0.5 ml PBS buffer was injected intraperitoneally;

[0049] Lyophilized powder group 1: intraperitoneal injection of 0.5 ml / head of prepared lyophilized powder 1 (dissolved in 1 ml PBS buffer in each tube), once a week for 3 consecutive weeks.

[0050] Lyophilized powder group 2: intraperitoneal injection of 0.5 ml / head of prepared lyophilized powder 2 (dissolved in 1 ml PBS buffer in each tube), once a week for 3 consecutive weeks.

[0051] Lyophilized powder 3 group: intraperitoneal injection of 0.5 ml / head of prepared lyophilized powder 3 (dissolved in 1 ml PBS buffer in each tube), once a week for 3 consecutive weeks.

[0052] Positive control group: dipotassium glycyrrhizinate 150 μg, once a week, for 3 consecutive weeks.

[0053] One week after the end of the administration, the mice were killed, and the skin of the treated area of ​​the mice was taken and homogenized to determine the content of SOD, IL-6 and TNF-α in the skin. The detection was performed according to the instructions of the kit. The results (Table 4) showed that the effect of freeze-dried powder group 2 was the best, indicating that the combination of human mesenchymal stem cell freeze-dried powder prepared by the present invention and monoclonal antibody 4D3 had a better effect (increased SOD activity and decreased inflammatory factors), and was better than the commercial monoclonal antibody.

[0054] Table 4 Expression results of SOD, IL-6 and TNF-α in each group

[0055] Group SOD (U / mgprot) IL-6 (pg / L) TNF-α(ng / L) Blank Group 151.36±12.68 22.37±5.45 101.36±8.15 Model Group 35.69±3.65 100.25±6.87 470.36±15.57 Positive control group 93.56±5.25 65.65±4.12 289.67±10.36 Freeze-dried powder 1 set 116.25±5.68 49.36±3.56 172.36±8.45 Freeze-dried powder 2 groups 149.25±10.36 25.68±2.24 103.36±7.28 Freeze-dried powder 3 groups 126.01±10.87 36.27±4.36 154.36±8.15

[0056] The above research results of monoclonal antibody 4D3 are consistent with the conclusion of a research paper published by the research team of Duke-NUS Medical School in Nature, titled: Inhibition of IL-11 signaling extends mammalian healthspan and lifespan. That is, inhibiting IL-11 can significantly improve the health lifespan of elderly mice and delay aging.

[0057] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A monoclonal antibody against IL-11 protein, characterized in that: The monoclonal antibody is monoclonal antibody 4D3, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4D3 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. A freeze-dried stem cell powder, characterized in that: The stem cell freeze-dried powder contains an effective amount of human mesenchymal stem cells and an effective amount of the IL-11 protein monoclonal antibody 4D3 according to claim 1.

3. The freeze-dried stem cell powder according to claim 2, characterized in that: The content of human mesenchymal stem cells in the stem cell suspension before freeze-drying is 10 6 Pieces / ml.

4. The freeze-dried stem cell powder according to claim 2, characterized in that: The content of the IL-11 protein monoclonal antibody 4D3 in the stem cell suspension before freeze-drying is 1 mg / ml.

5. A method for preparing the freeze-dried stem cell powder according to claim 2, characterized in that: The method comprises the following steps: (1) First, dialyze the monoclonal antibody 4D3 described in claim 1 using a lyophilization buffer to adjust the concentration to 1 mg / ml; (2) Use the freeze-dried buffer containing monoclonal antibody 4D3 to adjust the concentration of human mesenchymal stem cells to 10 6 / ml, mix well, and pipette 1ml / tube, and cool from room temperature to -80℃ at 10℃ / min; (3) Place the packaged liquid into a freeze dryer and freeze dry for 24 hours according to the set freeze drying program to obtain stem cell freeze-dried powder.

6. The method according to claim 5, characterized in that The formula of the freeze-drying buffer in step (1) is as follows: add 20 ml of fetal bovine serum, 20 g of sucrose, and 100 ml of glycerol to 100 ml of DMEM culture medium, stir and mix, and sterilize through a 0.22 μm filter membrane.

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