A human mesenchymal stem cell preparation that effectively delays cell aging, and its preparation method and application

Abstract

The present invention discloses a human mesenchymal stem cell preparation that effectively delays cell aging. The human mesenchymal stem cell preparation is based on human mesenchymal stem cells and further includes any one or two of SIRT3 protein polypeptide and KAT7 protein single domain antibody. The amino acid sequence of the SIRT3 protein polypeptide of the present invention is shown in SEQ ID NO.2, and the nucleotide sequence of the SIRT3 protein polypeptide after codon optimization is shown in SEQ ID NO.3. The amino acid sequence of the KAT7 protein single domain antibody of the present invention is shown in SEQ ID NO.4, and the nucleotide sequence of the KAT7 protein single domain antibody after codon optimization is shown in SEQ ID NO.5. The human mesenchymal stem cells prepared by the present invention, combined with the SIRT3 protein polypeptide and KAT7 protein single domain antibody prepared by the present invention, have a good effect of delaying cell aging, and are better than the results of commercial SIRT3 protein and KAT7 antibody. This provides a good reference for the application of human mesenchymal stem cells in delaying cell aging.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a human mesenchymal stem cell preparation that can effectively delay cell aging, and a preparation method and application thereof. Background Art

[0002] Human mesenchymal stem cells (MSCs) are a type of adult stem cells with self-renewal and multidirectional differentiation potential. They are derived from the mesoderm and can differentiate into various types of cells, such as adipocytes, osteoblasts, chondrocytes, etc. under specific conditions. This type of stem cell has not only attracted much attention in regenerative medicine and cell therapy, but has also been used to treat a variety of diseases, including inflammatory and degenerative diseases. They can be obtained from a variety of tissues, such as bone marrow, adipose tissue, dental pulp and periodontal ligament, dermis and synovium. In addition, MSCs have many advantages in clinical applications, including low immunogenicity and good tissue repair ability.

[0003] In 1881, Weismann et al. found that organ performance is affected by the limited cell division of tissues. Subsequently, Hayflick et al. found in 1961 that normal human cells have limited cell replication and lifespan. When cells reach their maximum replication capacity, this phenomenon is called cell senescence. Aging is characterized by the cumulative loss of biological integrity at the cellular level, which affects the physiological functions of the whole body. The main biological hallmarks of the aging process include: (1) decreased tissue regeneration capacity and exhaustion or disorder of endogenous stem cell populations; (2) stable arrest of the cell cycle and DNA accumulation that impair protein homeostasis, cell function and communication, and normal organ physiology.

[0004] Aging is closely related to stem cell integrity. One of the main goals of stem cell biology and regenerative medicine is to use stem cells to reverse aging and its accompanying dysfunction. However, there is currently no better stem cell that can effectively delay cell aging. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a human mesenchymal stem cell preparation that effectively delays cell aging, and a preparation method and application thereof.

[0006] Therefore, the present invention discloses a human mesenchymal stem cell preparation that effectively delays cell aging. The human mesenchymal stem cell preparation is based on human mesenchymal stem cells and also includes any one or two of SIRT3 protein polypeptide and KAT7 protein single domain antibody.

[0007] Preferably, the amino acid sequence of the SIRT3 protein polypeptide of the present invention is shown as SEQ ID NO.2, and the nucleotide sequence of the SIRT3 protein polypeptide after codon optimization is shown as SEQ ID NO.3.

[0008] Preferably, the amino acid sequence of the KAT7 protein single domain antibody of the present invention is shown as SEQ ID NO.4, and the nucleotide sequence of the KAT7 protein single domain antibody after codon optimization is shown as SEQ ID NO.5.

[0009] Preferably, the human mesenchymal stem cells, SIRT3 protein polypeptide, and KAT7 protein single domain antibody of the present invention are diluted with physiological saline when used.

[0010] Preferably, the content of human mesenchymal stem cells in the human mesenchymal stem cell preparation of the present invention is 10 5 cells / 0.2ml.

[0011] Preferably, the SIRT3 protein polypeptide and the KAT7 protein single domain antibody in the human mesenchymal stem cell preparation of the present invention are used at a concentration of 5 mg / kg.

[0012] In another aspect, the present invention also discloses a use of the SIRT3 protein polypeptide in the preparation of a human mesenchymal stem cell preparation that is effective in delaying cell aging.

[0013] In another aspect, the present invention also discloses a use of the KAT7 protein single domain antibody in the preparation of a human mesenchymal stem cell preparation that effectively delays cell aging.

[0014] The human mesenchymal stem cells prepared by the present invention combined with the SIRT3 protein polypeptide and KAT7 protein single domain antibody prepared by the present invention have a good effect of delaying cell aging, and the results are better than those of the commercial SIRT3 protein (ab125586) and KAT7 antibody (MCEYA3027). This provides a good reference for the application of human mesenchymal stem cells in delaying cell aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Photo of cultured human mesenchymal stem cells.

[0016] Figure 2 SIRT3 protein peptide SDS-PAGE detection results. 1 and 2 are SIRT3 protein peptides.

[0017] Figure 3 KAT7 protein single domain antibody SDS-PAGE detection results. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] Example 1: Isolation and culture of human mesenchymal stem cells

[0021] 1. Isolation of human mesenchymal stem cells: Mesenchymal stem cells are isolated from a variety of tissues, including human bone marrow, adipose tissue, umbilical cord and dental pulp.

[0022] (1) Take an appropriate amount of fresh tissue aspirate collected under sterile conditions.

[0023] (2) Filter the cell suspension through a 70 mm filter to remove any cell clumps, then centrifuge at 500 g for 5 minutes and carefully discard the supernatant.

[0024] (3) The pellet was resuspended in 5 ml of complete MSC medium and counted. The cells were diluted to 25 × 10 6 The cells were then transferred to a T75 culture dish and placed in a 37°C, 5% CO 2 Incubate in the incubator for 3 hours. After the cells adhere to the wall, carefully remove the culture medium and add 10 ml of fresh complete culture medium.

[0025] (4) At 37°C and 5% CO 2 After 8 hours of static culture in the incubator, carefully remove the culture medium and add 10 ml of fresh complete culture medium. Thereafter, repeat this step every 8 hours until the initial culture of 72 hours is completed.

[0026] (5) The cultured cells were resuspended in MSC growth medium containing 10% DMSO (cell density was 2×10 6 / ml), gradually cooled and frozen in liquid nitrogen for later use.

[0027] 2. Expansion of human mesenchymal stem cells

[0028] (1) Add 10 mL of 0.1% gelatin solution to a T75 culture flask and incubate at room temperature for 30 min. Before use, remove the gelatin solution from the T75 culture flask.

[0029] (2) Remove the human mesenchymal stem cell vial from liquid nitrogen and immediately place it in a 37°C water bath to thaw. After the cells are completely thawed, immediately disinfect the outside of the vial with 70% ethanol. In a biosafety cabinet, use a 1 ml pipette to transfer the cells to a sterile 15 ml centrifuge tube. Slowly add 9 ml of expansion medium or a suitable substitute preheated to 37°C to the 15 ml centrifuge tube. Slowly pipette up and down twice to gently mix the cell suspension. Centrifuge at 500 rpm for 5 min and carefully remove the supernatant.

[0030] (3) Resuspend the cells in a total volume of 10 ml of expansion medium or a suitable substitute preheated to 37°C, containing freshly added 8 ng / ml FGF-2 (Merck), and then inoculate the cell suspension onto a T75 tissue culture flask or a T75 tissue culture flask. Place the cells at 37°C and 5% CO. 2 Culture in an incubator.

[0031] (4) On the next day, replace the culture medium with fresh expansion medium (pre-warmed to 37°C) containing 8 ng / ml FGF-2. Replace the culture medium containing FGF-2 every two to three days.

[0032] (5) When the cells reach approximately 80% confluence, they can be digested with trypsin-EDTA and further passaged or frozen for later use.

[0033] 3. Expansion of Mesenchymal Stem Cells

[0034] (1) When the cells reach about 80% confluence, cell subculture (e.g. Figure 1 ).

[0035] (2) Carefully remove the culture medium from the T75 culture flask containing human mesenchymal stem cells. Add 3-5 ml of trypsin-EDTA solution and incubate in a 37°C incubator for 3-5 minutes. After the cells are completely digested, add 5 ml of expansion medium to the culture flask. Gently rotate the culture plate to mix the cell suspension. Transfer the dissociated cells to a 15 ml offline tube, centrifuge at 500 rpm for 5 minutes, and carefully remove the supernatant.

[0036] (3) Add 2 ml of expansion medium (preheated to 37°C) containing 8 ng / ml FGF-2 to a 15 ml offline tube to resuspend the cells. Count the cells and, based on the count results, seed the cells at a density of 5,000-6,000 cells / cm2 into appropriate culture flasks containing expansion medium containing 8 ng / ml FGF-2.

[0037] (4) When the cells reach a confluence of about 80%, they are passaged, expanded, or frozen for future use.

[0038] Example 2: Screening and preparation of SIRT3 protein polypeptides

[0039] Studies have shown that in human mesenchymal stem cells, SIRT3 expression is reduced in aged human mesenchymal stem cells. Among them, SIRT3 interacts with the nuclear lamina and heterochromatin proteins to maintain the stability of heterochromatin and antagonize cell aging. Therefore, SIRT3 protein may be a potential target for delaying aging and intervening in aging-related diseases. [Zhiqing, Diao, et al. "SIRT3 consolidates heterochromatin and counteracts senescence." Nucleic Acids Research (2021).]

[0040] The amino acid sequence of SIRT3 protein (shown in SEQ ID NO.1) was analyzed, and the amino acids at positions aa145 to aa165 and aa228 to aa346 were selected to prepare SIRT3 protein polypeptide, whose amino acid sequence is shown in SEQ ID NO.2.

[0041] The nucleotide sequence of the codon-optimized SIRT3 protein polypeptide (shown in SEQ ID NO.3) is cloned into a eukaryotic expression vector (such as pCDNA3.1). After sequencing verification, the SIRT3 protein polypeptide is expressed and prepared (for specific methods, please refer to the embodiment of Chinese invention patent 201710551363.8). The SDS-PAGE purity of the prepared SIRT3 protein polypeptide can reach more than 90% ( Figure 2 The expression level after purification can reach 2g / L.

[0042] Example 3: Preparation of KAT7 protein single domain antibody

[0043] Other researchers have found that KAT7 expression is upregulated in both physiologically and pathologically aged human mesenchymal stem cells. Knocking out this gene can effectively delay cell senescence; overexpressing this gene will promote cell senescence. Related animal experiments have shown that knocking down KAT7 can reduce the proportion of senescent cells in the liver of aged mice, significantly reduce the level of pro-inflammatory factors in the blood, improve the health of mice, and prolong the lifespan of mice with physiological aging and progeria. [Wang, Wei, et al. "A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence." Science Translational Medicine 13.575(2021):eabd2655.]

[0044] The recombinant human KAT7 protein (ab268698) was diluted to 50 μg / ml with shark saline, and the diluted antigen was mixed with the adjuvant in a ratio of 1:1 for emulsification. Immunization was performed by intraperitoneal injection. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent immunization. A total of 3 to 4 immunizations were performed, with injections every two weeks. The immunized shark serum was used for transcriptome sequencing to analyze the antibody production in the shark, and the spleen and lymphocyte RNA of the immunized shark were extracted for RT-PCR to obtain a high-throughput database containing only shark VNAR. The obtained transcriptome high-throughput sequencing results were compared with the high-throughput database of VNAR to screen sequences that were contained in both databases and had a high content. The VNAR sequence obtained in combination with the screening results is shown in SEQ ID NO.4, which is a single-domain antibody of the KAT7 protein.

[0045] The codon of the KAT7 protein single domain antibody was optimized (shown in SEQ ID NO.5), and the optimized nucleotide was subcloned into the Nco I and Xho I sites of pET28a. The plasmid was transformed into BL21 (DE3) competent cells, spread on LB agar plates containing kanamycin, and cultured at 36-37°C at a constant temperature of 16-20 hours. A single colony was picked from the plate and inoculated into 3 ml of LB medium containing kanamycin, and cultured at 36-37°C and 220-240r / min with shaking until the OD600nm value reached 0.4-0.6, and the bacterial liquid was harvested. The bacterial liquid was amplified and cultured at 37°C, then cooled to 20°C, induced to express with IPTG, and the bacteria were collected; the bacteria were resuspended, crushed, centrifuged to collect the supernatant, purified by nickel column and dialyzed for liquid exchange, and filtered and sterilized through a 0.22μm low protein adsorption filter membrane to obtain the KAT7 protein single domain antibody, and its SDS-PAGE detection results are as follows Figure 3 As shown, its expression level is about 345 mg / L.

[0046] KAT7 antibody (MCEYA3027) and the above-prepared KAT7 protein single domain antibody were diluted to a concentration of 1 μg / ml with 50mM carbonate buffer (pH 9.6), 100 μl was plated per well, and the plate was coated overnight at 4°C. After washing 3 times with PBST, PBST solution containing 2% BSA was added to each well and incubated at 37°C for 2 hours. After washing 3 times with PBST, the recombinant human KAT7 protein was diluted to 1 μg / ml with PBST solution containing 1% BSA, 100 μl was added to each well, and incubated at 37°C for 1h. Recombinant HRPAnti-KAT7 antibody (ab70183) was used as a secondary antibody, diluted 1:5000 with PBST, incubated at 37°C for 1h, washed 3 times with PBST, and patted dry. 100 μl of TMB colorimetric solution was added to each well, and the reaction was protected from light for 10 minutes at room temperature. After the reaction, 50 μl of 2M H 2SO 4 The absorbance of the sample at 450 nm was measured by an ELISA instrument. The test results are shown in the table below. The single domain antibody can specifically bind to the KAT7 protein, indicating that the single domain antibody has good activity.

[0047]

[0048] Example 5: Human mesenchymal stem cell preparation treatment study

[0049] C57BL / 6 mice were fed with conventional feed until 18 months of age, and a total of 60 aged mice were obtained. They were screened based on the methods of coat color, physical activity, mental state, stool observation and weight detection. According to the standards of dark coat color, reduced gloss, appearance of white hair, poor mental state, reduced activity, increased frequency of dry or loose stools, and body weight in the range of 20±5g, the aged mice that met the standards were included in this experiment as a natural aging mouse model, a total of 40 mice, 20 males and 20 females.

[0050] Five young mice (6-7 weeks) were randomly selected as the young group. Aged mice were randomly divided into 8 groups (as shown in Table 1), with 5 mice in each group. All were fed conventionally. Different human mesenchymal stem cell preparations (as shown in Table 1) were injected into each group through the tail vein, and they were fed conventionally. The drugs were administered once every 2 weeks, and the effect was evaluated 1 month after treatment. The differences between each group and the young group of mice were observed by hair, spirit, activity, diet and body weight. At the end of the observation, the mice were killed by dislocation of the neck, and the heart, liver, spleen, lung, kidney and other tissues and organs were immediately removed, and the total SOD activity was detected using a kit (total SOD activity detection kit, Biyuntian, S0109).

[0051] Table 1 Treatment groups and treatment status

[0052]

[0053] Clinical observation results showed that in the group treated with human mesenchymal stem cell preparations, the mice had improved fur gloss, increased physical activity, better mental state, and lower rate of abnormal stool; and the differences between the groups were: young group > group 7 > group 4 ≈ group 5 ≈ group 6 > group 2 ≈ group 3 > group 1 > group 8.

[0054] The total SOD activity test results in each group are shown in Table 2. The results are consistent with the clinical observation results, and the order is: young group > group 7 > group 4 ≈ group 5 ≈ group 6 > group 2 ≈ group 3 > group 1 > group 8.

[0055] The above results show that the human mesenchymal stem cells prepared by the present invention combined with the SIRT3 protein polypeptide and KAT7 protein single domain antibody prepared by the present invention have a good effect of delaying cell aging, and the results are better than those of the commercial SIRT3 protein (ab125586) and KAT7 antibody (MCE YA3027). This provides a good reference for the application of human mesenchymal stem cells in delaying cell aging.

[0056] Table 2 Total SOD activity test results of each group

[0057] Group heart liver spleen lung kidney Group 1 8.91±0.65 6.28±0.35 9.78±0.65 9.35±0.54 6.92±0.52 Group 2 9.45±0.78 6.56±0.43 10.12±0.56 9.78±0.44 7.35±0.34 Group 3 9.41±0.65 6.67±0.47 10.09±0.67 9.89±0.55 7.44±0.43 Group 4 9.94±0.76 7.12±0.42 11.14±0.65 10.56±0.67 7.87±0.51 Group 5 10.01±0.67 7.15±0.44 11.09±0.67 10.54±0.67 7.81±0.46 Group 6 9.96±0.66 7.11±0.51 11.01±0.56 10.49±0.66 7.78±0.45 Group 7 13.76±1.01 7.98±0.35 12.43±0.89 13.34±0.78 8.42±0.56 Group 8 7.87±0.76 5.25±0.54 8.65±0.87 8.15±0.56 5.87±0.43 Young Group 14.67±1.24 8.56±0.76 13.87±1.12 14.77±0.98 8.75±0.87

[0058] Total SOD activity is an effective indicator for evaluating cell aging. SOD, or superoxide dismutase, is one of the important antioxidant enzymes in the body, which can remove superoxide anion free radicals and protect cells from oxidative damage. During cell aging, the increase in oxidative stress will lead to the accumulation of free radicals, thereby accelerating cell aging. Therefore, by measuring the total SOD activity, the antioxidant capacity of cells and the level of oxidative stress can be indirectly evaluated, and then the state of cell aging can be evaluated.

[0059] 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 human mesenchymal stem cell preparation that effectively delays cell aging, characterized in that: The human mesenchymal stem cell preparation not only includes human mesenchymal stem cells as a basis, but also includes SIRT3 protein polypeptide and KAT7 protein single domain antibody; the amino acid sequence of the SIRT3 protein polypeptide is shown in SEQ ID NO.2, and the nucleotide sequence of the SIRT3 protein polypeptide after codon optimization is shown in SEQ ID NO.3; the amino acid sequence of the KAT7 protein single domain antibody is shown in SEQ ID NO.4, and the nucleotide sequence of the KAT7 protein single domain antibody after codon optimization is shown in SEQ ID NO.

5.

2. The human mesenchymal stem cell preparation according to claim 1, characterized in that: The human mesenchymal stem cells, SIRT3 protein polypeptide, and KAT7 protein single domain antibody were diluted with physiological saline when used.

3. The human mesenchymal stem cell preparation according to claim 1, characterized in that: The content of human mesenchymal stem cells in the human mesenchymal stem cell preparation is 10 5 cells / 0.2ml.

4. The human mesenchymal stem cell preparation according to claim 1, characterized in that The SIRT3 protein polypeptide and the KAT7 protein single domain antibody in the human mesenchymal stem cell preparation are both used at a concentration of 5 mg / kg.

5. Use of a KAT7 protein single domain antibody in the preparation of a human mesenchymal stem cell preparation that effectively delays cell aging, characterized in that: The amino acid sequence of the KAT7 protein single domain antibody is shown in SEQ ID NO.4.

Images