CN116271049B - Biological agent for inhibiting adipose-derived mesenchymal stem cells from being converted into adipocytes - Google Patents
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Abstract
The invention provides a biological agent for inhibiting adipose-derived mesenchymal stem cells from being converted into adipocytes, belonging to the technical field of cell biology. The biological agent provided by the invention comprises a gene inhibitor for inhibiting LINC02516 gene expression and also comprises a pharmaceutically acceptable carrier. The biological agent provided by the invention can effectively inhibit the differentiation of adipose-derived mesenchymal stem cells into adipocytes.
Description
The application is a divisional application, the name of the original application is that a gene expression inhibitor is applied to treating obesity, the application date of the original application is 2022-05-18, and the application number of the original application is CN202210548230.6.
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a biological agent for inhibiting adipose-derived mesenchymal stem cells from being converted into adipocytes.
Background
With the improvement of the living standard of people, overweight and obesity of residents in China have a rapid rising trend, and have created a heavy disease burden and economic burden for the countries and society. Studies have shown that overweight and obese people are high risk groups for hypertension, coronary heart disease, dyslipidemia, abnormal insulin function, type 2 diabetes and fatty liver.
Obesity is due to adipogenesis and enlargement, which is mainly dependent on proliferation and differentiation of adipose stem cells. Therefore, from the viewpoint of cell proliferation and differentiation, if proliferation and differentiation of adipose stem cells are effectively inhibited, the generation of adipose cells can be effectively reduced, thereby effectively inhibiting obesity.
Disclosure of Invention
The invention aims to provide an application of a gene expression inhibitor in treating obesity.
In order to achieve the above object, the present invention provides the following technical solutions:
firstly, the invention provides application of a gene expression inhibitor in preparing an obesity treatment drug, wherein the gene expression inhibitor is a LINC02516 gene expression inhibitor, and a transcript sequence of LINC02516 is shown as SEQ ID NO. 1.
Preferably, the LINC02516 gene expression inhibitor is an siRNA that inhibits expression of the LINC02516 gene.
Preferably, the siRNA comprises a sense strand and an antisense strand, the sense strand of the siRNA is shown as SEQ ID NO.4, and the antisense strand of the siRNA is shown as SEQ ID NO. 5.
Secondly, the invention provides a therapeutic drug for treating obesity, which comprises a LINC02516 gene expression inhibitor, wherein the transcript sequence of the LINC02516 gene is shown as SEQ ID NO.1, and the LINC02516 gene expression inhibitor is siRNA for inhibiting the expression of the LINC02516 gene.
Preferably, the siRNA comprises a sense strand and an antisense strand, the sense strand of the siRNA is shown as SEQ ID NO.4, and the antisense strand of the siRNA is shown as SEQ ID NO. 5.
Preferably, the medicament further comprises a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier being one of a solvent, a liposome, or a viral vector.
Secondly, the invention provides application of a LINC02516 gene expression inhibitor in preparing a medicament for inhibiting proliferation of adipose-derived mesenchymal stem cells, which is characterized in that a transcript sequence of the LINC02516 gene is shown as SEQ ID NO.1, and the LINC02516 gene expression inhibitor is siRNA for inhibiting expression of a LINC02516 gene.
Preferably, the siRNA comprises a sense strand and an antisense strand, the sense strand of the siRNA is shown as SEQ ID NO.4, and the antisense strand of the siRNA is shown as SEQ ID NO. 5.
Finally, the invention provides application of the siRNA of the LINC02516 gene in preparing inhibitors for inhibiting the expression of the mesenchymal stem cell Cyclin-D1 and CDK1 gene proteins, wherein the transcript sequence of the LINC02516 gene is shown as SEQ ID NO.1, the siRNA comprises a sense strand and an antisense strand, the sense strand of the siRNA is shown as SEQ ID NO.4, and the antisense strand of the siRNA is shown as SEQ ID NO. 5.
The beneficial effects of the invention are as follows:
The invention discovers that the expression quantity of LINC02516 is obviously increased in the adipogenic differentiation process of the adipose-derived mesenchymal stem cells, and the inhibition of LINC02516 can effectively inhibit the adipogenic differentiation of the adipose-derived mesenchymal stem cells and can effectively inhibit the proliferation of the adipose-derived mesenchymal stem cells, so that the invention can effectively inhibit the generation of the adipose-derived mesenchymal stem cells, thereby being used for treating obesity.
Drawings
FIG. 1 changes in expression levels of LINC02516 during adipogenic transformation of adipose-derived mesenchymal stem cells;
FIG. 2 shows the inhibition effect of siRNA provided by the present invention;
FIG. 3 results of detection of oil red O staining;
FIG. 4 shows the protein expression of Cheng Zhiji due to PPARgamma and aP2 after siRNA transfection;
FIG. 5 protein expression of cell cycle genes Cyclin-D1 and CDK1 after siRNA transfection.
Detailed Description
In order to clearly illustrate the technical characteristics of the scheme, the scheme is explained below through a specific embodiment.
Example 1
Detection of expression level difference of LINC02516 during adipogenic differentiation of adipose-derived mesenchymal stem cells
(1) Inoculating adipose-derived mesenchymal stem cells into a 6-hole cell culture plate, and adding a complete culture medium for culture;
(2) When the cell density of the adipose-derived mesenchymal stem cells reaches about 90%, removing the complete medium, adding a adipogenic induction medium, and performing adipogenic induction, and extracting RNA on the 0 th day, the 3 rd day, the 7 th day, the 10 th day and the 14 th day respectively;
(3) After extracting the RNA, reverse transcribing the RNA into cDNA using a reverse transcription kit;
(4) The primer of gene LINC02516 was designed based on the transcript sequence of LINC02516 (SEQ ID NO. 1), and the primer sequence was as follows:
an upstream primer GTGTACTCCTGGGAGCTCCT (SEQ ID NO. 2);
A downstream primer GGCAGGAAAGGACATTCTCCA (SEQ ID NO. 3);
(5) The relative expression level of LINC02516 was detected using a fluorescent quantitative PCR kit, GAPDH was used as an internal control, and the results were analyzed by a2 -△△CT method, and the experimental results are shown in FIG. 1.
Wherein, when the culture is carried out for 3 days, the relative expression quantity of LINC02516 is 2.02+/-0.18, P is less than 0.05, and the difference has statistical significance;
When the culture is carried out for 7 days, the relative expression quantity of LINC02516 is 3.26+/-0.14, P is less than 0.05, and the difference has statistical significance;
When the culture is carried out for 10 days, the relative expression quantity of LINC02516 is 4.14+/-0.10, P is less than 0.05, and the difference has statistical significance;
When the culture is carried out for 14 days, the relative expression quantity of LINC02516 is 4.62+/-0.21, P is less than 0.05, and the difference has statistical significance;
From the above results, it can be seen that the increase in the expression level of LINC02516 during adipogenic differentiation suggests that the increase in the expression level of LINC02516 may be related to promotion of adipogenic differentiation of adipose mesenchymal stem cells, and therefore, it is considered that adipogenic differentiation of adipose mesenchymal stem cells can be inhibited by inhibiting the expression level of LINC02516 during adipogenic differentiation.
Example 2
Inhibiting LINC02516 expression quantity and detecting fat mesenchymal stem cell adipogenic differentiation change condition
1. Detection of inhibition effect of siRNA of LINC02516
(1) The siRNA was designed based on the transcript sequence of the gene LINC02516, and the sequence of this siRNA was as follows:
GAGAGAGAGUGCAAAGCAA (SEQ ID NO. 4) for siRNA sense strand;
UUGCUUUGCACUCUCUCUC (SEQ ID NO. 5) as siRNA antisense strand;
siNC supplied by siRNA synthesis;
(2) Inoculating adipose-derived mesenchymal stem cells into a 6-hole cell culture plate, and respectively transfecting siNC and siRNA into the adipose-derived mesenchymal stem cells;
(3) After 48h of transfection, RNA is extracted, reverse transcription and fluorescence quantitative PCR reaction are carried out, the relative expression quantity of LINC02516 is detected, and the obtained result is shown in figure 2;
After siRNA is transfected, the relative expression quantity of cells is 0.16+/-0.06, and the inhibition rate is about 84 percent, which proves that the siRNA designed by the invention can effectively inhibit the expression of LINC 02516.
2. Oil red O experiment detects changes in adipogenic differentiation of adipose-derived mesenchymal stem cells after transfection of siRNA
(1) Adipose-derived mesenchymal stem cells are inoculated into a 6-hole cell culture plate, and siNC and siRNA are respectively transfected;
(2) After 48 hours of transfection, changing into a lipid-forming induction culture medium for culture;
(3) After 7 days of culture, the culture medium is removed, the culture wells are washed with PBS, after 3 times of washing, PBS is removed, and 10% paraformaldehyde is added to fix the cells for 30min;
(4) After the fixation is finished, removing paraformaldehyde, adding the prepared oil red O staining solution, covering cells, and staining the cells at normal temperature;
(5) Staining for 30min, sucking the staining solution, gently washing the cells with PBS for 2-3 times, observing under an inverted microscope, and photographing, and the obtained results are shown in FIG. 3.
From the results, the cells transfected with LINC02516 siRNA were significantly less stained with cell oil red O, indicating that the lipid-forming differentiation of adipose-derived mesenchymal stem cells can be effectively inhibited after the expression of LINC02516 is inhibited.
Example 3
Protein expression of adipogenic genes PPARgamma and aP2 of adipose-derived mesenchymal stem cells after inhibition of LINC02516
(1) Adipose-derived mesenchymal stem cells are inoculated into a 6-hole cell culture plate, and siNC and siRNA are respectively transfected;
(2) After 48 hours of transfection, changing into a lipid-forming induction culture medium for culture;
(3) After 7 days of culture, the culture medium is removed, protein lysate is added, after complete lysis, the cells are scraped off and transferred to a 1.5ml centrifuge tube;
(4) After 30min of ice lysis, the centrifuge tube was placed in a centrifuge, centrifuged for 15min, the obtained supernatant was transferred to a new 1.5ml centrifuge tube, and the concentration of the protein sample was measured,
(5) Adding a sample buffer solution to adjust the protein sample to be uniform protein concentration, and heating the centrifuge tube in boiling water for 5min;
(6) Preparing protein electrophoresis gel, loading sample, and performing electrophoresis under the initial condition of constant pressure 90V, and adjusting to constant pressure 120V when bromophenol blue reaches the separation gel until bromophenol blue runs out of the bottom of the electrophoresis gel;
(7) After electrophoresis, installing an electric rotating clamp, setting electric rotating conditions, and carrying out electric rotating;
(8) After the electrotransformation is finished, taking out the membrane, placing the membrane into a sealing liquid for sealing, and after sealing for 1h, incubating PPARgamma, aP2 and GAPDH primary antibodies;
(9) After the incubation is finished, washing the membrane, and incubating the secondary antibody after washing the membrane;
(10) After the secondary antibody incubation was completed, development exposure was performed, and the experimental results obtained are shown in fig. 4.
From the figure, after the expression of LINC02516 is inhibited, the protein expression of the adipogenic genes PPARgamma and aP2 of the adipose-derived mesenchymal stem cells can be effectively inhibited.
Example 4
Proliferation of adipose-derived mesenchymal stem cells after inhibition of LINC02516
(1) Adipose-derived mesenchymal stem cells transfected with siNC and siRNA were inoculated into 96-well cell culture plates, 100ul was added per well, and 2000 cells were contained per 100 ul;
(2) After 96-well plates were placed in a cell incubator for 72 hours, the cells were examined by referring to the instructions of cck-8.
As a result of the detection, the OD value of the transfected siNC cells was 0.59.+ -. 0.04, the OD value of the transfected siRNA cells was 0.48.+ -. 0.05, the P value was less than 0.05, and the difference was statistically significant. The above results demonstrate that inhibition of expression of LINC02516 in adipose-derived mesenchymal stem cells can effectively inhibit proliferation of adipose-derived mesenchymal stem cells.
Example 5
Protein expression of mesenchymal stem cell cycle genes Cyclin-D1 and CDK1 after inhibition of LINC02516
(1) Adipose-derived mesenchymal stem cells are inoculated into a 6-hole cell culture plate, and siNC and siRNA are respectively transfected;
(2) After 48 hours of transfection, changing into a lipid-forming induction culture medium for culture;
(3) After 7 days of culture, the culture medium is removed, protein lysate is added, after complete lysis, the cells are scraped off and transferred to a 1.5ml centrifuge tube;
(4) After 30min of ice lysis, the centrifuge tube was placed in a centrifuge, centrifuged for 15min, the obtained supernatant was transferred to a new 1.5ml centrifuge tube, and the concentration of the protein sample was measured,
(5) Adding a sample buffer solution to adjust the protein sample to be uniform protein concentration, and heating the centrifuge tube in boiling water for 5min;
(6) Preparing protein electrophoresis gel, loading sample, and performing electrophoresis under the initial condition of constant pressure 90V, and adjusting to constant pressure 120V when bromophenol blue reaches the separation gel until bromophenol blue runs out of the bottom of the electrophoresis gel;
(7) After electrophoresis, installing an electric rotating clamp, setting electric rotating conditions, and carrying out electric rotating;
(8) After the electrotransformation is finished, the membrane is taken out and placed in a sealing liquid for sealing, and after sealing for 1h, primary incubation of Cyclin-D1, CDK1 and GAPDH is carried out;
(9) After the incubation is finished, washing the membrane, and incubating the secondary antibody after washing the membrane;
(10) After the secondary antibody incubation was completed, development exposure was performed, and the experimental results obtained are shown in fig. 5.
The technical features of the present invention that are not described in the present invention may be implemented by or using the prior art, and are not described in detail herein, but the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, but is also intended to be within the scope of the present invention by those skilled in the art.
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| Reference Sequence: NR_110838.1 Homo sapiens long intergenic non-protein coding RNA 2516 (LINC02516), long non-coding RNA.;NCBI;GENBANK;20220419;1-3 * |
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