Use of a polypeptide in the preparation of a promoter of adipose mesenchymal stem cell proliferation

Abstract

This invention provides an application of a polypeptide in the preparation of an adipose-derived mesenchymal stem cell proliferation promoter, belonging to the field of stem cell technology. Through experiments, this invention has found that the Nigroain-E1 polypeptide can effectively promote the proliferation of adipose-derived mesenchymal stem cells and can effectively regulate cell cycle proteins. Therefore, the Nigroain-E1 polypeptide can be used to prepare a promoter that promotes the proliferation of adipose-derived mesenchymal stem cells.

Description

[0001] This case is a divisional application. The original application was entitled "Application of a Nigroain-E1 polypeptide in maintaining the stemness of adipose stem cells". The original application date was September 8, 2021, and the original application number was CN202111048277.8. Technical Field

[0002] This invention belongs to the field of stem cell technology, and particularly relates to the application of a polypeptide in the preparation of an adipose-derived mesenchymal stem cell proliferation promoter. Background Technology

[0003] Stem cells are a type of cell with multi-lineage differentiation capacity, capable of differentiating into osteoblasts, osteoclasts, adipocytes, and fibroblasts. Adipose-derived stem cells (ADSCs) are a type of cell with self-renewal capacity and multi-lineage differentiation potential. ADSCs are characterized by easy availability, wide distribution, and suitability for large-scale culture; therefore, they have become one of the most actively researched adult stem cell types in the field of tissue engineering.

[0004] Adipose-derived stem cells can differentiate into various cell lines, such as osteocytes, vascular endothelial cells, adipocytes, nerve cells, and smooth muscle cells. However, during continuous cell culture, their proliferative capacity decreases and stem cell characteristics are lost. Therefore, effectively improving the proliferative capacity of adipose-derived stem cells and maintaining their stem cell characteristics is a problem that needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Nigroain-E1 polypeptide in maintaining the stemness of adipose stem cells.

[0006] To achieve the above applications, the present invention provides the following technical solution:

[0007] This invention provides the application of Nigroain-E1 peptide in maintaining stem cell stemness, wherein the concentration of Nigroain-E1 peptide is greater than 25 ug / ml.

[0008] Preferably, the sequence of the Nigroain-E1 polypeptide is as follows:

[0009] Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp.

[0010] Preferably, the stem cells are adipose-derived mesenchymal stem cells.

[0011] Secondly, this invention provides the application of Nigroain-E1 polypeptide in promoting stem gene expression in stem cells.

[0012] Preferably, the stem genes include the OCT4 gene, the KLF4 gene, and the NANOG gene.

[0013] Preferably, the sequence of the Nigroain-E1 polypeptide is as follows:

[0014] Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp.

[0015] Secondly, this invention provides the application of Nigroain-E1 polypeptide in the preparation of adipose-derived mesenchymal stem cell stem cell maintenance agents.

[0016] Preferably, the sequence of the Nigroain-E1 polypeptide is as follows:

[0017] Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp.

[0018] Secondly, this invention provides the application of Nigroain-E1 polypeptide in the preparation of a stem gene expression promoter for adipose-derived mesenchymal stem cells.

[0019] Preferably, the stem genes include the OCT4 gene, the KLF4 gene, and the NANOG gene;

[0020] The sequence of the Nigroain-E1 polypeptide is as follows:

[0021] Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp.

[0022] The beneficial effects of this invention are:

[0023] This invention discovers that the Nigroain-E1 polypeptide can effectively promote the expression of the stemness genes OCT4, KLF4 and NANOG in adipose-derived mesenchymal stem cells, thereby effectively maintaining the stemness of adipose-derived mesenchymal stem cells. Attached Figure Description

[0024] Figure 1 Effects of different concentrations of Nigroain-E1 peptide on the proliferation of adipose-derived mesenchymal stem cells.

[0025] Figure 2 Effects of 25ug / ml Nigroain-E1 peptide on cell cycle-related proteins in adipose-derived mesenchymal stem cells;

[0026] Figure 3 Effects of 25ug / ml Nigroain-E1 peptide on stem cell-related genes in adipose-derived mesenchymal stem cells;

[0027] Figure 4 Effect of 25ug / ml Nigroain-E1 peptide on adipogenic differentiation of adipose-derived mesenchymal stem cells;

[0028] Figure 5 Effect of 25ug / ml Nigroain-E1 peptide on adipogenic differentiation-promoting protein of adipose-derived mesenchymal stem cells. Detailed Implementation

[0029] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0030] Example 1

[0031] Regulation of Nigroain-E1 peptide on the proliferation of adipose-derived mesenchymal stem cells

[0032] (1) P3 generation adipose mesenchymal stem cells were seeded into 96-well plates, with 1000 cells seeded per well and 100 μL per well, and one blank well containing no cells was set up.

[0033] (2) After the cells adhered, the cells were treated with 0ug / ml, 10ug / ml, 25ug / ml and 50ug / ml Nigroain-E1 peptide (using culture medium), and each group was set up with 3 replicates.

[0034] (3) Place the cell culture plate in a cell culture incubator and continue culturing for 72 hours;

[0035] (4) After the culture is completed, remove the culture medium, add 10 μL of CCK-8 to each well in the dark, incubate for 3 hours, and then measure the absorbance of each group at 450 nm.

[0036] Table 1 Absorbance after treatment with different concentrations of Nigroain-E1 peptide

[0037]

[0038]

[0039] The results obtained from the experiment are presented in Figure 1 As shown in Table 1, although the absorbance of the 10ug / ml Nigroain-E1 peptide group was higher than that of the 0ug / ml Nigroain-E1 peptide group, the P value was greater than 0.05, so the difference was not statistically significant.

[0040] The absorbance of the 25ug / ml Nigroain-E1 peptide group was significantly higher than that of the 0ug / ml Nigroain-E1 peptide group, and the P value was less than 0.05, so the difference was statistically significant.

[0041] Similarly, the absorbance of the 50 ug / ml Nigroain-E1 peptide group was significantly higher than that of the 0 ug / ml Nigroain-E1 peptide group, and the P value was less than 0.05, so the difference was statistically significant. However, the absorbance was not significantly improved compared to the 25 ug / ml Nigroain-E1 peptide group. Therefore, this invention considers 25 ug / ml Nigroain-E1 peptide to be the optimal treatment concentration.

[0042] Example 2

[0043] Detection of the regulation of cell cycle-related proteins in adipose-derived mesenchymal stem cells by 25 μg / ml Nigroain-E1 peptide.

[0044] (1) P3 generation adipose mesenchymal stem cells were seeded into 6-well plates. After the cells adhered, the control group was treated with 0 ug / ml Nigroain-E1 peptide for 72 h, and the experimental group was treated with 25 ug / ml Nigroain-E1 peptide for 72 h.

[0045] (2) After the treatment was completed, the culture medium was removed, the cells were washed three times with PBS, protein lysis buffer was added, the cells were lysed, and the cells were placed on ice for 30 min to lyse.

[0046] (3) After the treatment is completed, the cells are scraped off with a cell scraper and collected into a 1.5ml EP tube;

[0047] (4) Place the EP tube in a centrifuge, adjust the centrifuge parameters to 4℃, 12000rpm / min, and centrifuge for 18min;

[0048] (5) After centrifugation, collect the supernatant into a new EP tube, measure the protein concentration of each group using the BCA method, and then add 5×SDS loading buffer to obtain protein samples.

[0049] (6) Prepare separating gel and stacking gel to make gel plates. Load protein samples from the control group and experimental group into the gel plates. Set the power parameters to 80V. After 30 minutes, adjust the parameters to 120V until the bromophenol blue reaches the bottom, and then stop the electrophoresis.

[0050] (7) Install the electrophoresis clamp in the order of sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad, remove air bubbles, and adjust the electrophoresis parameters to 250mA;

[0051] (8) After electroporation for 90 minutes, remove the membrane, wash the membrane with TBST for 5 minutes, and then add the membrane to 5% skim milk powder and seal at room temperature for 1 hour.

[0052] (9) Remove the membrane and incubate it with Cyclin-D1, Cyclin-E1 and β-actin antibodies at 4°C overnight.

[0053] (10) After aspirating the primary antibody, wash the membrane three times with TBST for 5 minutes each time;

[0054] (11) Incubate the corresponding secondary antibody according to the primary antibody for 1 hour at room temperature;

[0055] (12) After incubation, wash the film with TBST 3 times, 5 minutes each time, and then add chemiluminescent solution for development and exposure.

[0056] The results obtained from the experiment are presented in Figure 2 The results show that the expression levels of Cyclin-D1 and Cyclin-E1 in adipose-derived mesenchymal stem cells treated with 25 μg / ml Nigroain-E1 peptide were significantly higher than those in the control group. This indicates that treatment with 25 μg / ml Nigroain-E1 peptide can effectively promote the protein expression of cyclins Cyclin-D1 and Cyclin-E1 in adipose-derived mesenchymal stem cells.

[0057] Example 3

[0058] Detecting the regulation of cell stemness-related genes by Nigroain-E1 in adipose-derived mesenchymal stem cells

[0059] Step 1: RNA extraction

[0060] (1) P3 generation adipose mesenchymal stem cells were seeded into 6-well plates. After the cells adhered, the control group was treated with 0 ug / ml Nigroain-E1 peptide for 72 h, and the experimental group was treated with 25 ug / ml Nigroain-E1 peptide for 96 h.

[0061] (2) After the treatment is completed, remove the culture medium, add 1 ml of Trizol to treat the cells, repeatedly pipette the cells, let stand for 5 min and then transfer the cells to an enzyme-free EP tube.

[0062] (3) Add 200 μL of chloroform, shake to mix, and let stand at room temperature for 5 minutes;

[0063] (4) Place the EP tube in a centrifuge and set the centrifuge parameters to 12000 r / min, 4℃, 10 min. After centrifugation, transfer the supernatant from the upper layer into a new EP tube.

[0064] (5) Add 500ul of pre-cooled isopropanol, mix well, and let stand on ice for 10 minutes;

[0065] (6) Place the EP tube in a centrifuge and set the centrifuge parameters to 12000 r / min, 4℃, 10min;

[0066] (7) Remove the supernatant, retain the white precipitate, and add 1 ml of 75% ethanol solution to mix the precipitate well;

[0067] (8) Place the EP in a centrifuge and set the centrifuge parameters to 8000 r / min, 4℃, and centrifuge for 5 min;

[0068] (9) After centrifugation, carefully remove the supernatant, let stand at room temperature for 10 minutes, and after the RNA dries, add 25 μL of DEPC water to dissolve the RNA.

[0069] The second step is the reverse transcription reaction.

[0070] (1) Removal of DNA from the genome

[0071] Prepare the reaction solution according to the following formula:

[0072]

[0073] Reaction conditions: 37℃ for 15 min, 85℃ for 5 s, 4℃ forever.

[0074] (2) Reverse transcription reaction

[0075] Prepare the reaction solution according to the following formula:

[0076]

[0077] Reaction conditions: 37℃ for 15 minutes, 85℃ for 5 seconds, 4℃.

[0078] Step 3: Real-time PCR reaction

[0079] (1) Primer design and synthesis

[0080]

[0081]

[0082] Real-time PCR reaction conditions: Stage 1 Reps 1 95℃ 15min; Stage 2 Reps 40 95℃ 15s 60℃ 30s 72℃ 15s.

[0083] Step 4 Application 2 -△△ct The relative expression level of mRNA was calculated and analyzed using this method.

[0084] The results obtained from the experiment are shown in Figure 3 From Figure 3 From A, we can see that the relative expression level of the OCT4 gene in the experimental group was 2.389±0.329, with a P value less than 0.05, indicating a statistically significant difference; from Figure 3 From B, the relative expression level of the NANOG gene was 2.481 ± 0.339, with a P value less than 0.05, indicating a statistically significant difference; from Figure 3 From C, we can see that the relative expression level of the KLF4 gene is 3.291±0.349, and the P value is less than 0.05, which is statistically significant.

[0085] The above results indicate that treatment of adipose-derived mesenchymal stem cells with 25 ug / ml Nigroain-E1 peptide resulted in high expression of stem cell stemness-related genes OCT4, NANOG, and KLF4, which effectively maintained stem cell stemness.

[0086] Example 4

[0087] Effects of Nigroain-E1 on Adipogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells

[0088] (1) Adipose-derived mesenchymal stem cells were seeded into 6-well culture plates. After the cell density reached 80%, the culture medium was removed and the cells were washed with PBS.

[0089] (2) Add adipogenic differentiation medium. The control group was given 0 ug / ml Nigroain-E1 peptide, and the experimental group was given 25 ug / ml Nigroain-E1 peptide. They were placed in an incubator and cultured. The medium was changed every 2-3 days.

[0090] (3) After culturing for 14 days, remove the culture medium, gently wash the cells 3 times with PBS, remove the PBS, add 4% paraformaldehyde, and fix for 30 min.

[0091] (4) After fixation, formaldehyde is fixed and Oil Red O staining solution is added for staining;

[0092] (5) After staining for 30 minutes, remove the staining solution and gently wash the cells with PBS;

[0093] (6) Observe and photograph the cells under a microscope.

[0094] The results obtained from the experiment are shown in Figure 4 As can be seen from the figure, the number of lipid droplets in the experimental group was significantly lower than that in the control group. This result indicates that adding 25ug / ml Nigroain-E1 peptide during the adipogenic differentiation of adipose-derived mesenchymal stem cells can effectively inhibit the adipogenic transformation of adipose-derived mesenchymal stem cells.

[0095] Example 5

[0096] Effects of Nigroain-E1 on Adipogenic Differentiation-Promoting Proteins in Adipose-Derived Mesenchymal Stem Cells

[0097] (1) Adipose-derived mesenchymal stem cells were seeded into 6-well culture plates. After the cell density reached 80%, the culture medium was removed and the cells were washed with PBS.

[0098] (2) Add adipogenic differentiation medium. The control group was given 0 ug / ml Nigroain-E1 peptide, and the experimental group was given 25 ug / ml Nigroain-E1 peptide. They were placed in an incubator and cultured. The medium was changed every 2-3 days.

[0099] (3) After culturing for 7 days, remove the culture medium, gently wash the cells 3 times with PBS, remove the PBS, add protein lysis buffer, lyse the cells, and place them on ice for 30 min to lyse.

[0100] (4) After the treatment is completed, scrape off the cells with a cell scraper and collect them into a 1.5ml EP tube;

[0101] (5) Place the EP tube in the centrifuge, adjust the centrifuge parameters to 4℃, 12000rpm / min, and centrifuge for 18min;

[0102] (6) After centrifugation, collect the supernatant into a new EP tube, measure the protein concentration of each group using the BCA method, and then add 5×SDS loading buffer to obtain protein samples.

[0103] (7) Prepare separating gel and stacking gel to make gel plates. Swim the protein samples of the control group and experimental group into the gel plates. Set the power parameters to 80V. After 30 minutes, adjust the parameters to 120V until the bromophenol blue reaches the bottom, and then end the electrophoresis.

[0104] (8) Install the electrophoresis clamp in the order of sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad, remove air bubbles, and adjust the electrophoresis parameters to 250mA;

[0105] (9) After electroporation for 90 minutes, remove the membrane, wash the membrane with TBST for 5 minutes, and then add the membrane to 5% skim milk powder and seal at room temperature for 1 hour.

[0106] (10) Remove the membrane and incubate it with PPARγ, C / EBPα, aP2 and β-actin antibodies at 4°C overnight.

[0107] (11) After aspirating the primary antibody, wash the membrane three times with TBST for 5 minutes each time;

[0108] (12) Incubate the corresponding secondary antibody according to the primary antibody for 1 hour at room temperature;

[0109] (13) After incubation, wash the film with TBST 3 times for 5 minutes each time, and then add chemiluminescent solution for development and exposure.

[0110] The results obtained from the experiment are shown in Figure 5 As shown in the figure, the expression levels of PPARγ, C / EBPα, and aP2 proteins in the experimental group were significantly lower than those in the control group. This result indicates that adding 25ug / ml Nigroain-E1 peptide during the adipogenic differentiation of adipose-derived mesenchymal stem cells can effectively inhibit the expression of adipogenic transformation-related proteins in adipose-derived mesenchymal stem cells.

Claims

1. Use of a Nigroain-E1 polypeptide for the preparation of a promoter of proliferation of adipose mesenchymal stem cells, characterized in that, The concentration of the Nigroain-E1 polypeptide is 25 μg / ml; The sequence of the Nigroain-E1 polypeptide is as follows: Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp.

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