Use of a polypeptide in adipose mesenchymal stem cells and related products

Abstract

The application provides use of Nigroain-E1 polypeptide in adipose mesenchymal stem cells and related products, and belongs to the technical field of stem cells. It is found that adding Nigroain-E1 polypeptide in the process of adipogenic differentiation of adipose mesenchymal stem cells can effectively inhibit the adipogenic differentiation of adipose mesenchymal stem cells, and can effectively inhibit the protein expression of adipogenic differentiation promoting proteins PPAR gamma, C / EBP alpha and aP2.

Description

Technical Field

[0001] This invention belongs to the field of mesenchymal stem cell technology, and particularly relates to the use of a polypeptide in adipose-derived mesenchymal stem cells and related products. Background Technology

[0002] Stem cells are a type of cell with multi-lineage differentiation capacity, capable of differentiating into osteoblasts, osteoclasts, adipocytes, and fibroblasts. Adipose-derived mesenchymal stem cells (ADMs) are a type of stem cell with multi-lineage differentiation potential and self-renewal capabilities. ADMs are characterized by their ease of acquisition, wide availability, and suitability for large-scale culture; therefore, they have become one of the most actively researched adult stem cells in the field of tissue engineering.

[0003] Adipose-derived mesenchymal stem cells (ADMs) can differentiate into various cell lines, such as osteocytes, vascular endothelial cells, adipocytes, nerve cells, and smooth muscle cells. However, during continuous cell culture, ADMs exhibit a certain degree of self-differentiation towards adipocytes, rendering them unsuitable for subsequent directed differentiation culture. Therefore, effectively inhibiting adipogenic differentiation of ADMs is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide the use of Nigroain-E1 polypeptide in adipose-derived mesenchymal stem cells and related products.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of Nigroain-E1 peptide in the preparation of an adipogenic differentiation inhibitor for adipose-derived mesenchymal stem cells, wherein the concentration of Nigroain-E1 peptide is greater than 25 ug / ml.

[0006] Preferably, 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.

[0007] Secondly, this invention provides the application of Nigroain-E1 polypeptide in the preparation of an inhibitor of adipogenic differentiation-promoting protein expression in adipose-derived mesenchymal stem cells, wherein the concentration of Nigroain-E1 polypeptide is greater than 25 ug / ml.

[0008] Preferably, the adipogenic differentiation promoting protein is PPARγ, C / EBPα, and aP2.

[0009] Preferably, 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.

[0010] Secondly, the present invention provides an inhibitor of adipogenic differentiation of adipose-derived mesenchymal stem cells, wherein the inhibitor contains a Nigroain-E1 polypeptide, and the concentration of the Nigroain-E1 polypeptide is greater than 25 ug / ml.

[0011] Preferably, 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.

[0012] Finally, this invention provides the application of Nigroain-E1 peptide in the preparation of adipose-derived mesenchymal stem cells as a promoter of cyclin-D1 and Cyclin-E1 protein expression, wherein the concentration of Nigroain-E1 peptide is greater than 25 ug / ml.

[0013] Preferably, 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.

[0014] The beneficial effects of this invention are: This invention reveals that adding Nigroain-E1 peptide during the adipogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs) can effectively inhibit ADMSCs and effectively suppress the protein expression of adipogenic differentiation-promoting proteins PPARγ, C / EBPα, and aP2. Furthermore, this invention also finds that adding Nigroain-E1 peptide during ADMSC culture can effectively promote the protein expression of cyclins Cyclin-D1 and Cyclin-E1. Attached Figure Description

[0015] Figure 1 Effects of different concentrations of Nigroain-E1 peptide on the proliferation of adipose-derived mesenchymal stem cells.

[0016] Figure 2 Effects of 25 ug / ml Nigroain-E1 peptide on cell cycle-related proteins in adipose-derived mesenchymal stem cells; Figure 3Effects of 25 ug / ml Nigroain-E1 peptide on stem cell-related genes in adipose-derived mesenchymal stem cells; Figure 4 Effect of 25 ug / ml Nigroain-E1 peptide on adipogenic differentiation of adipose-derived mesenchymal stem cells; Figure 5 Effect of 25 ug / ml Nigroain-E1 peptide on adipogenic differentiation-promoting protein of adipose-derived mesenchymal stem cells. Detailed Implementation

[0017] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0018] Example 1 Regulation of Nigroain-E1 peptide on the proliferation of adipose-derived mesenchymal stem cells (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. (2) After the cells adhered, the cells were treated with 0 ug / ml, 10 ug / ml, 25 ug / ml and 50 ug / ml Nigroain-E1 peptide (prepared with culture medium), and each group was set up with 3 replicates; (3) Place the cell culture plate in a cell culture incubator and continue culturing for 72 hours; (4) After the cell culture is completed, the culture medium is removed, and 10 μL of CCK-8 is added to each well under the dark. After incubation for 3 hours, the absorbance of each group under different treatments is detected at 450 nm.

[0019] Table 1. Absorbance after treatment with different concentrations of Nigroain-E1 peptide Treatment concentration Average absorbance Standard deviation of absorbance 0ug / ml Nigroain-E1 peptide 0.869 0.059 10ug / ml Nigroain-E1 peptide 0.912 0.060 25 ug / ml Nigroain-E1 peptide 1.119 0.093 50 ug / ml Nigroain-E1 peptide 1.146 0.086 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. The absorbance of the 25 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. 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.

[0020] Example 2 Detection of the regulation of cell cycle proteins in adipose-derived mesenchymal stem cells by 25 ug / ml Nigroain-E1 peptide (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. (2) After the treatment was completed, the culture medium was removed, the cells were washed three times with PBS, protein lysis buffer was added to lyse the cells, and the cells were placed on ice for 30 min to lyse. (3) After the treatment is completed, scrape off the cells with a cell scraper and collect them into a 1.5ml EP tube; (4) Place the EP tube in a centrifuge, adjust the centrifuge parameters to 4℃, 12000rpm / min, and centrifuge for 18min; (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. (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 electrophoresis. (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; (8) After electroporation for 90 minutes, remove the membrane, wash it with TBST for 5 minutes, and then add the membrane to 5% skim milk powder and seal it at room temperature for 1 hour. (9) Remove the membrane and incubate it with Cyclin-D1, Cyclin-E1 and β-actin antibodies at 4°C overnight; (10) After aspirating the primary antibody, wash the membrane three times with TBST for 5 minutes each time; (11) Incubate the corresponding secondary antibody according to the primary antibody at room temperature for 1 hour; (12) After incubation, wash the film with TBST 3 times, 5 minutes each time, and then add chemiluminescent solution for development and exposure.

[0021] 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 ug / ml Nigroain-E1 peptide were significantly higher than those in the control group. This indicates that treatment with 25 ug / ml Nigroain-E1 peptide can effectively promote the protein expression of cyclins Cyclin-D1 and Cyclin-E1 in adipose-derived mesenchymal stem cells.

[0022] Example 3 Detecting the regulation of cell stemness-related genes by Nigroain-E1 in adipose-derived mesenchymal stem cells Step 1: RNA extraction (1) P3 generation adipose mesenchymal stem cells were seeded in 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. (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; (3) Add 200 μL of chloroform, shake to mix, and let stand at room temperature for 5 minutes; (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 to a new EP tube. (5) Add 500 μL of pre-cooled isopropanol, mix well, and let stand on ice for 10 min; (6) Place the EP tube in a centrifuge and set the centrifuge parameters to 12000 r / min, 4℃, 10 min; (7) Remove the supernatant, retain the white precipitate, and add 1 ml of 75% ethanol solution to mix the precipitate well; (8) Place the EP in a centrifuge and set the centrifuge parameters to 8000 r / min, 4℃, and centrifuge for 5 min; (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.

[0023] The second step is the reverse transcription reaction. (1) Removal of DNA from the genome Prepare the reaction solution according to the following formula: Reaction reagents Added amount 5×gDNA Eraser Buffer 2.0μl gDNA Eraser 1.0μl Total RNA 1.0μg <![CDATA[RNase Free dH2O]]> up to 10.0μl Reaction conditions: 37℃ for 15 min, 85℃ for 5 s, 4℃ forever.

[0024] (2) Reverse transcription reaction Prepare the reaction solution according to the following formula: Reaction reagents Added amount The reaction solution in step (1) 10.0μl PrimeScript RT Enzyme Mix I 1.0μl RT Primer Mix 1.0 μl 5×PrimeScript Buffer 2 4.0 μl <![CDATA[RNase Free dH2O]]> 4.0 μl Reaction conditions: 37℃ for 15 minutes, 85℃ for 5 seconds, 4℃.

[0025] Step 3: Real-time PCR reaction (1) Primer design and synthesis Real-time PCR reaction conditions: Stage 1 Reps 1 95℃ 15min; Stage 2 Reps 40 95℃ 15s 60℃ 30s 72℃ 15s.

[0026] Step 4 Application 2 -△△ ct The relative expression level of mRNA was calculated and analyzed using this method.

[0027] 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.

[0028] The above results indicate that after treating adipose-derived mesenchymal stem cells with 25 ug / ml Nigroain-E1 peptide, the cells highly express stem cell-related genes OCT4, NANOG, and KLF4, thereby effectively maintaining stem cell stemness.

[0029] Example 4 Effects of Nigroain-E1 on Adipogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells (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. (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. The medium was placed in an incubator and cultured. The medium was changed every 2-3 days. (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. (4) After fixation, fix the formaldehyde and add Oil Red O staining solution for staining; (5) After staining for 30 minutes, remove the staining solution and gently wash the cells with PBS; (6) Observe and photograph the cells under a microscope.

[0030] 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 25 ug / 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.

[0031] Example 5 Effects of Nigroain-E1 on Adipogenic Differentiation-Promoting Proteins in Adipose-Derived Mesenchymal Stem Cells (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. (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. The medium was placed in an incubator and cultured. The medium was changed every 2-3 days. (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. (4) After the treatment is completed, scrape off the cells with a cell scraper and collect them into a 1.5ml EP tube; (5) Place the EP tube in a centrifuge, adjust the centrifuge parameters to 4℃, 12000rpm / min, and centrifuge for 18min; (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. (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 stop the electrophoresis. (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; (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; (10) Remove the membrane and incubate it with PPARγ, C / EBPα, aP2 and β-actin antibodies overnight at 4°C; (11) After aspirating the primary antibody, wash the membrane three times with TBST for 5 minutes each time; (12) Incubate the corresponding secondary antibody according to the primary antibody at room temperature for 1 hour; (13) After incubation, wash the film with TBST 3 times for 5 minutes each time, and then add chemiluminescent solution for development and exposure.

[0032] 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 25 ug / 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. The application of Nigroain-E1 polypeptide in the preparation of an inhibitor of adipogenic differentiation of adipose-derived mesenchymal stem cells, characterized in that, The concentration of the Nigroain-E1 polypeptide is 25-50 ug / 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.

2. The application according to claim 1, characterized in that, The polypeptide inhibits the expression of adipogenic differentiation-promoting proteins, namely PPARγ, C / EBPα, and aP2.

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