CN113717935B - Application of Nigroain-E1 polypeptide in maintaining adipose-derived stem cell (Stem) stem property - Google Patents
Application of Nigroain-E1 polypeptide in maintaining adipose-derived stem cell (Stem) stem property Download PDFInfo
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention provides application of Nigroain-E1 polypeptide in maintaining the stem property of adipose-derived stem cells, and belongs to the technical field of stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to application of a Nigroain-E1 polypeptide in maintaining the stem property of adipose-derived stem cells.
Background
Stem cells are a type of cells with the capacity of multidirectional differentiation, and can be differentiated into osteoblasts, osteoclasts, adipocytes and fibroblasts. The adipose-derived stem cells are cells with the multi-directional differentiation potential and have the characteristics of easy material acquisition, wide sources, suitability for large-scale culture and the like, so that the adipose-derived stem cells become the adult stem cells which are most actively researched in the field of tissue engineering.
Adipose stem cells are capable of differentiating into various cell lines such as bone cells, vascular endothelial cells, adipocytes, neural cells, smooth muscle cells, etc., however, there are cases where proliferation capacity is decreased and cell stem is lost during the continuous culture of cells. Therefore, how to effectively improve the proliferation capacity of adipose stem cells and maintain the cell stem properties thereof is a problem that needs to be solved at present.
Disclosure of Invention
The invention aims to provide an application of Nigroain-E1 polypeptide in maintaining the stem cell dryness.
In order to realize the application, the invention provides the following technical scheme:
the invention provides an application of Nigroain-E1 polypeptide in maintaining stem cell dryness, wherein the concentration of the Nigroain-E1 polypeptide is more than 25 ug/ml.
Preferably, the sequence of the Nigroain-E1 polypeptide is:
Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp。
preferably, the stem cells are adipose mesenchymal stem cells.
Second, the invention provides the use of a Nigroain-E1 polypeptide in promoting stem cell expression of a stem gene.
Preferably, the dry basis comprises OCT4 gene, KLF4 gene and NANOG gene.
Preferably, the sequence of the Nigroain-E1 polypeptide is:
Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp。
secondly, the invention provides application of the Nigroain-E1 polypeptide in preparation of adipose-derived mesenchymal stem cell stem property maintenance agents.
Preferably, the sequence of the Nigroain-E1 polypeptide is:
Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp。
secondly, the invention provides application of the Nigroain-E1 polypeptide in preparing a stem gene expression promoter of adipose-derived mesenchymal stem cells.
Preferably, the dry gene comprises OCT4 gene, KLF4 gene and NANOG gene;
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。
the beneficial effects of the invention are as follows:
the invention discovers that the Nigroain-E1 polypeptide can effectively promote the expression of the stem genes OCT4, KLF4 and NANOG genes of the adipose-derived mesenchymal stem cells, thereby effectively maintaining the stem property of the adipose-derived mesenchymal stem cells.
Drawings
FIG. 1 effect of different concentrations of Nigroain-E1 polypeptide on proliferation of adipose mesenchymal stem cells.
FIG. 2 effect of 25 ug/ml Nigroain-E1 polypeptide on the cell cycle associated protein of adipose mesenchymal stem cells;
FIG. 3 effect of 25 ug/ml Nigroain-E1 polypeptide on cell stem-related genes of adipose mesenchymal stem cells;
FIG. 4 effect of 25 ug/ml Nigroain-E1 polypeptide on adipogenic differentiation of adipose mesenchymal stem cells;
FIG. 5 effect of 25 ug/ml Nigroain-E1 polypeptide on adipogenic differentiation promoting protein of adipose mesenchymal stem cells.
Detailed Description
In order to clearly illustrate the technical characteristics of the scheme, the scheme is explained below through a specific embodiment.
Example 1
Regulation of adipose mesenchymal stem cell proliferation by Nigroain-E1 polypeptides
(1) Inoculating P3 generation adipose-derived mesenchymal stem cells into a 96-well plate, inoculating 1000 cells per well, 100ul per well, and setting 1 blank well without cells;
(2) After cell attachment, cells were treated with 0ug/ml,10 ug/ml,25 ug/ml,50 ug/ml of the Nigroain-E1 polypeptide (configured with medium), each set of 3 duplicate wells;
(3) Placing the cell culture plate in a cell culture box for continuous culture for 72 hours;
(4) After the end of the incubation, the medium was removed and 10ul CCK-8 was added to each well in the absence of light, incubated for 3 hours, and the absorbance of each group was measured at 450 nm.
TABLE 1 absorbance after treatment with different concentrations of Nigroain-E1 polypeptide
| Treatment concentration | Average of absorbance | Standard deviation of absorbance |
| 0ug/ml Nigroain-E1 polypeptide | 0.869 | 0.059 |
| 10ug/ml Nigroain-E1 polypeptide | 0.912 | 0.060 |
| 25 ug/ml Nigroain-E1 polypeptide | 1.119 | 0.093 |
| 50 ug/ml Nigroain-E1 polypeptide | 1.146 | 0.086 |
The results obtained from the experiments are shown in FIG. 1 and Table 1, from which it can be seen that the absorbance of the 10ug/ml Nigroain-E1 polypeptide group is higher than that of the 0ug/ml Nigroain-E1 polypeptide group, but the P value is greater than 0.05, so that the difference is not statistically significant;
25 The absorbance of the ug/ml Nigroain-E1 polypeptide group is obviously higher than that of the 0ug/ml Nigroain-E1 polypeptide group, and the P value is smaller than 0.05, so that the difference has statistical significance;
similarly, the absorbance of the 50 ug/ml Nigroain-E1 polypeptide group is also significantly higher than that of the 0ug/ml Nigroain-E1 polypeptide group, and the P value is less than 0.05, so that the difference is statistically significant, but the absorbance of the 50 ug/ml Nigroain-E1 polypeptide group is not significantly improved compared with that of the 25 ug/ml Nigroain-E1 polypeptide group, so that the 25 ug/ml Nigroain-E1 polypeptide is considered to be the optimal treatment concentration.
Example 2
Detection of regulation of adipose mesenchymal Stem cell cycle-related proteins by 25 ug/ml Nigroain-E1 polypeptide
(1) Adipose-derived mesenchymal stem cells of the generation P3 are inoculated into a 6-well plate, after the cells are attached, a control group is treated by 0ug/ml of Nigroain-E1 polypeptide for 72 hours, and an experimental group is treated by 25 ug/ml of Nigroain-E1 polypeptide for 72 hours;
(2) After the treatment is finished, removing the culture medium, cleaning the cells for 3 times by using PBS, adding protein lysate, lysing the cells, and putting the cells on ice for lysis for 30min;
(3) After the treatment was completed, the cells were scraped off using a cell scraper and collected into 1.5ml ep tubes;
(4) Placing the EP tube in a centrifuge, adjusting the parameters of the centrifuge to 4 ℃,12000rpm/min, and centrifuging for 18min;
(5) After centrifugation, collecting the supernatant into a new EP tube, measuring the protein concentration of each group by using a BCA method, and adding a 5 XSDS loading buffer to obtain protein samples;
(6) Preparing separation gel and concentrated gel into gel plates, loading protein samples of a control group and an experimental group into the gel plates, setting the power supply parameter to 80V, adjusting the parameter to 120V after 30min, and ending electrophoresis until bromophenol blue reaches the bottom;
(7) Installing an electric rotating clamp according to the sequence of the foam cushion, the filter paper, the gel, the PVDF film, the filter paper and the foam cushion, removing bubbles, and adjusting the electrophoresis parameter to 250mA;
(8) After 90min of electric rotation, the membrane was taken out, washed with TBST for 5min, then added into 5% skim milk powder, and sealed at room temperature for 1h;
(9) Taking out the membrane, incubating the Cyclin-D1, the Cyclin-E1 and the beta-actin antibody, and standing at 4 ℃ for overnight incubation;
(10) After primary antibody is absorbed, the membrane is washed 3 times by TBST for 5min each time;
(11) Incubating the corresponding secondary antibody according to the primary antibody, and incubating for 1h at room temperature;
(12) After the incubation, the membrane was washed 3 times with TBST for 5min each, and then developed and exposed by adding a chemiluminescent solution.
The results of the experiment are shown in FIG. 2, from which it can be seen that the expression levels of Cyclin-D1 and Cyclin-E1 in adipose-derived mesenchymal stem cells treated with 25 ug/ml of Nigroain-E1 polypeptide are significantly higher than those of the control group, and this result demonstrates that the protein expression of Cyclin-D1 and Cyclin-E1 in adipose-derived mesenchymal stem cells can be effectively promoted after the adipose-derived mesenchymal stem cells are treated with 25 ug/ml of Nigroain-E1 polypeptide.
Example 3
Detection of regulation of cell-stem-related genes of Nigroain-E1 on adipose-derived mesenchymal stem cells
First step RNA extraction
(1) Adipose-derived mesenchymal stem cells of the generation P3 are inoculated into a 6-well plate, after the cells are attached, a control group is treated by 0ug/ml of Nigroain-E1 polypeptide for 72 hours, and an experimental group is treated by 25 ug/ml of Nigroain-E1 polypeptide for 96 hours;
(2) After the treatment is finished, removing the culture medium, adding 1ml of Trizol to treat the cells, repeatedly blowing the cells by using a liquid transfer device, standing for 5min, and transferring the cells into an enzyme-free EP tube;
(3) Adding 200ul of chloroform, shaking and uniformly mixing, and standing at room temperature for 5min;
(4) Placing the EP tube in a centrifuge, setting the parameters of the centrifuge to 12000r/min,4 ℃ and 10min, and sucking the supernatant of the upper layer into a new EP tube after the centrifugation is finished;
(5) Adding 500ul of precooled isopropanol, mixing, standing on ice for 10min;
(6) Placing the EP pipe in a centrifuge, setting the parameters of the centrifuge to 12000r/min, and setting the temperature to 4 ℃ for 10min;
(7) Removing the supernatant, retaining the white precipitate, and adding 1ml of 75% ethanol solution to uniformly mix the precipitate;
(8) Placing the EP in a centrifuge, setting the parameters of the centrifuge to 8000r/min, and centrifuging at 4 ℃ for 5min;
(9) After centrifugation, the supernatant was carefully removed, allowed to stand at room temperature for 10min, and after drying the RNA, 25ul of DEPC water was added to dissolve the RNA.
Second step reverse transcription reaction
(1) Removal of DNA from the genome
The reaction solution was prepared according to the following formulation:
| reactive reagent | Additive amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1.0μg |
| RNase Free dH 2 O | up to 10.0μl |
Reaction conditions: 15min at 37 ℃, 5s at 85 ℃, and forever at 4 ℃.
(2) Reverse transcription reaction
The reaction solution was prepared according to the following formulation:
| reactive reagent | Additive amount |
| Reaction liquid of step (1) | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0 μl |
| 5×PrimeScript Buffer 2 | 4.0 μl |
| RNase Free dH 2 O | 4.0 μl |
The reaction conditions were 37℃for 15 minutes, 85℃for 5 seconds and 4 ℃.
Third step fluorescent quantitative PCR reaction
(1) Primer design and Synthesis
Fluorescent quantitative PCR reaction conditions: stage1 Reps 1℃for 15min at 95 ℃; stage2 Reps 40℃15s 60℃30s 72℃15s.
Fourth step application 2 -△△ ct The relative expression amount of mRNA was calculated and analyzed by the method.
The results obtained by the experiment are shown in FIG. 3, and as can be obtained from FIG. 3A, the relative expression amount of OCT4 gene in the experimental group is 2.389 +/-0.329, the P value is less than 0.05, and the difference has statistical significance; as can be seen from FIG. 3B, the relative expression level of the NANOG gene is 2.481 + -0.339, the P value is less than 0.05, and the difference is statistically significant; as can be seen from FIG. 3C, the relative expression level of KLF4 gene was 3.291.+ -. 0.349, the P value was less than 0.05, and the difference was statistically significant.
The above results demonstrate that the cells highly express stem cell stem-related genes OCT4, NANOG and KLF4 genes after treating adipose mesenchymal stem cells with 25 ug/ml Nigroain-E1 polypeptide, thereby effectively maintaining the stem cell stem.
Example 4
Effect of Nigroain-E1 on adipogenic differentiation of adipose-derived mesenchymal Stem cells
(1) Inoculating adipose-derived mesenchymal stem cells into a 6-hole culture plate, removing the culture medium after the cell density reaches 80%, and cleaning the cells by using PBS;
(2) Adding lipid-forming differentiation medium, adding 0ug/ml Nigroain-E1 polypeptide into control group, adding 25 ug/ml Nigroain-E1 polypeptide into experimental group, culturing in incubator, and changing liquid every 2-3 days;
(3) After 14 days of incubation, the medium was removed, the cells were gently washed 3 times with PBS, PBS was removed, 4% paraformaldehyde was added, and fixation was performed for 30min;
(4) After the fixation is finished, formaldehyde is fixed, and oil red O staining solution is added for staining;
(5) After staining for 30min, the staining solution was removed and the cells were gently washed with PBS;
(6) The cells were observed under a microscope and photographed.
The results of the experiment are shown in fig. 4, and it can be seen from the graph that the number of lipid droplets in the experimental group is significantly lower than that in the control group, and the results indicate that the addition of 25 ug/ml of the Nigroain-E1 polypeptide during adipogenic differentiation of the adipose mesenchymal stem cells can effectively inhibit adipogenic transformation of the adipose mesenchymal stem cells.
Example 5
Effect of Nigroain-E1 on adipose mesenchymal Stem cell adipogenic differentiation-promoting protein
(1) Inoculating adipose-derived mesenchymal stem cells into a 6-hole culture plate, removing the culture medium after the cell density reaches 80%, and cleaning the cells by using PBS;
(2) Adding lipid-forming differentiation medium, adding 0ug/ml Nigroain-E1 polypeptide into control group, adding 25 ug/ml Nigroain-E1 polypeptide into experimental group, culturing in incubator, and changing liquid every 2-3 days;
(3) After 7 days of incubation, the medium was removed, the cells were gently washed 3 times with PBS, PBS was removed, protein lysate was added, the cells were lysed, and the cells were lysed on ice for 30min;
(4) After the treatment was completed, the cells were scraped off using a cell scraper and collected into 1.5ml ep tubes;
(5) Placing the EP tube in a centrifuge, adjusting the parameters of the centrifuge to 4 ℃,12000rpm/min, and centrifuging for 18min;
(6) After centrifugation, collecting the supernatant into a new EP tube, measuring the protein concentration of each group by using a BCA method, and adding a 5 XSDS loading buffer to obtain protein samples;
(7) Preparing a gel plate by preparing separation gel and concentrated gel, arranging protein samples of a control group and an experimental group upstream in the gel plate, setting a power parameter to 80V, adjusting the parameter to 120V after 30min, and ending electrophoresis until bromophenol blue is obtained at the bottom;
(8) Installing an electric rotating clamp according to the sequence of the foam cushion, the filter paper, the gel, the PVDF film, the filter paper and the foam cushion, removing bubbles, and adjusting the electrophoresis parameter to 250mA;
(9) After 90min of electric rotation, the membrane was taken out, washed with TBST for 5min, then added into 5% skim milk powder, and sealed at room temperature for 1h;
(10) The membranes were removed, incubated with PPARgamma, C/EBPalpha, aP2 and beta-actin antibodies, and incubated overnight at 4 ℃;
(11) After primary antibody is absorbed, the membrane is washed 3 times by TBST for 5min each time;
(12) Incubating the corresponding secondary antibody according to the primary antibody, and incubating for 1h at room temperature;
(13) After the incubation, the membrane was washed 3 times with TBST for 5min each, and then developed and exposed by adding a chemiluminescent solution.
The results obtained by the experiment are shown in fig. 5, and it can be seen from the graph that the expression amounts of pparγ, C/ebpα and aP2 proteins in the experimental group are significantly lower than those in the control group, and the results indicate that the addition of 25 ug/ml of the Nigroain-E1 polypeptide in the adipogenic differentiation process of adipose-derived mesenchymal stem cells can effectively inhibit the expression of adipogenic transformation-related proteins of adipose-derived mesenchymal stem cells.
Claims (3)
- Use of a Nigroain-E1 polypeptide for maintaining stem cell stem properties, wherein the concentration of said Nigroain-E1 polypeptide is greater than 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; the stem cells are adipose mesenchymal stem cells.
- Use of a Nigroain-E1 polypeptide in the preparation of a cell stem maintenance agent for adipose-derived mesenchymal stem cells, characterized in that the concentration of said Nigroain-E1 polypeptide is greater than 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.
- 3. The use according to claim 2, wherein the cell stem maintenance agent is used to promote expression of stem genes in adipose mesenchymal stem cells, the stem genes being OCT4 gene, KLF4 gene and NANOG gene.
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| CN110592009A (en) * | 2019-10-14 | 2019-12-20 | 杨姣姣 | Application of asiatic acid derivatives in promoting in-vitro proliferation and maintaining dryness of human adipose mesenchymal stem cells |
| WO2020051141A1 (en) * | 2018-09-04 | 2020-03-12 | Academia Sinica | A medium composition and method for culturing mesenchymal stem cells |
| WO2020233119A1 (en) * | 2019-05-23 | 2020-11-26 | 广州赛莱拉干细胞科技股份有限公司 | Serum-free polypeptide composition for promoting proliferation of mesenchymal stem cells |
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| WO2020051141A1 (en) * | 2018-09-04 | 2020-03-12 | Academia Sinica | A medium composition and method for culturing mesenchymal stem cells |
| WO2020233119A1 (en) * | 2019-05-23 | 2020-11-26 | 广州赛莱拉干细胞科技股份有限公司 | Serum-free polypeptide composition for promoting proliferation of mesenchymal stem cells |
| CN110592009A (en) * | 2019-10-14 | 2019-12-20 | 杨姣姣 | Application of asiatic acid derivatives in promoting in-vitro proliferation and maintaining dryness of human adipose mesenchymal stem cells |
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