CN112656947A - Endothelial cell injury inhibitor - Google Patents
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Abstract
The invention provides an endothelial cell injury inhibitor, belonging to the technical field of cardiovascular diseases. The invention discovers that the gene AL592494.4 is obviously highly expressed in ox-LDL induced coronary artery endothelial cell injury through fluorescent quantitative PCR detection. Secondly, the invention discovers that the knocking-down of AL592494.4 can effectively reduce the decrease of the survival rate of HCAECs cells caused by ox-LDL, the increase of apoptosis and the increase of Caspase3 protein expression, so that the AL592494.4 inhibitor can be used for preparing a coronary artery endothelial cell damage inhibitor, thereby providing a new treatment mode for treating coronary atherosclerotic heart disease.
Description
Technical Field
The invention belongs to the technical field of cardiovascular diseases, and particularly relates to an endothelial cell injury inhibitor.
Background
Currently, cardiovascular-related diseases remain the leading cause of human health threats worldwide. Coronary atherosclerotic heart disease is also known as ischemic heart disease, which refers to heart disease in which atherosclerosis of the coronary arteries causes stenosis or occlusion of the lumen, resulting in hypoxia or necrosis of the heart muscle. The pathogenesis of atherosclerosis is not yet fully studied. Currently, the accepted theories relating to pathogenesis include the endothelial injury-response theory, the lipid infiltration theory, the inflammation theory, platelet aggregation and the thrombosis hypothesis. Among them, the endothelial injury-response theory is currently the most recognized theory. The theory of endothelial injury-response refers to the fact that oxidized low-density lipoprotein is accumulated on the vascular wall in a large quantity, adhesion molecules and chemotactic protein are triggered to be produced on the inner side of the lumen of the vascular wall, and finally endothelial cell injury is caused, and the formation and the progression of atherosclerotic lesions and the occurrence of cardiovascular events can be caused after the endothelial cell injury. Therefore, a detailed study of the mechanisms of endothelial cell damage, effective reduction of coronary endothelial cell damage is an effective way to treat coronary atherosclerotic heart disease.
Disclosure of Invention
The invention aims to provide an inhibitor which can effectively reduce damage of endothelial cells of coronary arteries, thereby providing a novel treatment mode for coronary atherosclerotic heart disease.
In order to achieve the purpose, the invention provides the following technical scheme:
in one aspect, the invention provides the use of a gene AL592494.4 as a target molecule in inhibiting damage to endothelial cells of coronary arteries, wherein the External Transcript ID of the gene AL592494.4 is ENST 00000437523.1.
Secondly, the invention provides application of an inhibitor of gene AL592494.4 in preparation of drugs for treating coronary atherosclerotic heart disease.
Preferably, the inhibitor is siRNA.
Preferably, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.13, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO. 14.
Thirdly, the invention provides an application of an inhibitor of gene AL592494.4 in preparing a coronary artery endothelial cell injury inhibitor.
Preferably, the inhibitor is siRNA, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.13, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO. 14.
The invention also provides a siRNA for preparing the coronary artery endothelial cell injury inhibitor, wherein the sense strand sequence of the siRNA is shown as SEQ ID NO.13, and the antisense strand sequence of the siRNA is shown as SEQ ID NO. 14.
The invention also provides a medicament for treating coronary atherosclerotic heart disease, which comprises an inhibitor of gene AL592494.4, wherein the inhibitor is siRNA, the sequence of the sense strand of the siRNA is shown as SEQ ID NO.13, and the sequence of the antisense strand of the siRNA is shown as SEQ ID NO. 14.
The invention has the beneficial effects that:
experiments show that the gene AL592494.4 is obviously highly expressed in ox-LDL induced coronary artery endothelial cell injury;
secondly, the invention provides si-AL592494.4 which can obviously inhibit the expression of AL 592494.4;
secondly, the invention discovers that the knocking-down of AL592494.4 can effectively reduce the decrease of the survival rate of HCAECs cells caused by ox-LDL, the increase of apoptosis and the increase of Caspase3 protein expression, so that the AL592494.4 inhibitor can be used for preparing a coronary artery endothelial cell damage inhibitor, thereby providing a new treatment mode for treating coronary atherosclerotic heart disease.
Drawings
FIG. 1 genes differentially expressed in coronary endothelial cells HCAECs following ox-LDL induction;
FIG. 2 is a graph of the results of inhibitor effect for si-AL 592494.4;
FIG. 3 is a graph of the results of knocking down the effect of AL592494.4 on the decrease in survival of HCAECs cells by ox-LDL;
FIG. 4 is a graph of the results of knocking down the effect of AL592494.4 on ox-LDL induced apoptosis of HCAECs;
FIG. 5 is a graph showing the effect of knocking down AL592494.4 on ox-LDL induced upregulation of Caspase3 protein expression in HCAECs cells.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1
Detection of differentially expressed genes in ox-LDL induced coronary endothelial cells HCAECs
1. Extraction of cellular RNA from HCAECs after ox-LDL treatment
(1) Inoculating HCAECs cells into a 6-well cell culture plate, adding 2ml of 0 mu g/ml ox-LDL and 100 mu g/ml ox-LDL respectively, putting into a cell culture box, and culturing for 24 h;
(2) after 24h of culture, adding 1ml of Trizol into each hole, uniformly blowing and stirring, and standing for 5min at room temperature;
(3) transferring the cells to a new EP tube, adding 200 μ l of chloroform, mixing well, standing at room temperature for 5 min;
(4) adjusting the parameters of the centrifuge to 4 ℃, 12000rpm/min, centrifuging for 15min, and carefully absorbing the upper aqueous phase into a new EP tube after centrifuging;
(5) adding isovoluminal precooled isopropanol, uniformly mixing, standing at room temperature for 10min, and centrifuging at 4 ℃ at 12000rpm/min for 10 min;
(6) removing supernatant, adding 75% ethanol prepared from DEPC water, washing precipitate, adjusting centrifuge parameter to 4 deg.C, 8000rpm/min, and centrifuging for 5 min;
(7) removing supernatant, placing in a superclean bench, drying until ethanol is completely evaporated, and adding 30 μ l DEPC water;
2. reverse transcription to obtain cDNA
(1) Removal of genomic DNA
The reaction system is as follows:
the reaction conditions were as follows: stored at 42 ℃ for 2 minutes and 4 ℃.
(2) Reverse transcription reaction
The reaction system is as follows:
the reaction conditions were as follows: 15min at 37 ℃; 5s at 85 ℃; storing at 4 ℃.
3. Experiment fluorescent quantitative PCR detection of differentially expressed genes
(1) Primers of RP11-776H12.1, AL592494.4, AL162400.1, LINC01927 and LINC01618 are designed, and specific sequences are as follows:
(2) preparing a fluorescent quantitative PCR reaction solution, which comprises the following specific steps:
(3) the fluorescent quantitative PCR reaction was carried out according to the following reaction conditions:
1min at 95 ℃; 5s at 95 ℃, 40s at 60 ℃, 60s at 72 ℃ and 35 cycles;
(4) use 2-ΔΔCtThe method performs data analysis, and the relative expression result is shown in FIG. 1.
As can be seen from the figure, the relative expression level of the gene RP11-776H12.1 is 0.959 +/-0.041, and the difference has no statistical significance; the relative expression level of the gene AL592494.4 is 3.381 +/-0.279, and the difference has statistical significance; the relative expression quantity of the gene AL162400.1 is 0.737 +/-0.074, and the difference has statistical significance; the relative expression quantity of the gene LINC01927 is 1.105 +/-0.045, and the difference has no statistical significance; the relative expression level of the gene LINC01618 is 1.226 +/-0.113, and the difference has no statistical significance.
From the above results, it can be seen that the gene AL592494.4 was highly expressed and the difference was statistically significant in HCAECs cells after ox-LDL treatment.
Example 2
Design of interfering RNA for Gene AL592494.4
(1) siRNA is designed according to the sequence of the gene AL592494.4, and the specific sequence of si-AL592494.4 is as follows:
sense strand: AGUUUCUUGAUAAGUCAUCCU, SEQ ID NO. 13;
antisense strand: GAUGACUUAUCAAGAAACUUU, SEQ ID NO. 14;
(2) AL592494.4 was transfected into HCAECs according to the protocol for lip2000 lipofection, 48h after transfection, the knockdown effect of si-AL592494.4 was detected using fluorescent quantitative PCR, the specific steps of which refer to example 1;
(3) the expression level of AL592494.4 after transfection of si-AL592494.4 is shown in FIG. 2.
It can be seen that si-AL592494.4 was able to efficiently knock down AL592494.4 (the relative expression of AL592494.4 in the si-AL592494.4 group was 0.251 ± 0.026).
Example 3
Effect of knockdown of AL592494.4 on ox-LDL induced reduction in survival of HCAECs cells
(1) Inoculating HCAECs cells into a 96-well cell culture plate, and putting the cell culture plate into a cell culture box to incubate overnight;
(2) cells were treated in cell groups as follows:
group A: 0 μ g/ml ox-LDL; group B: 100 μ g/ml ox-LDL, group C: 0 μ g/ml ox-LDL + si-NC; group D: 100 μ g/ml ox-LDL + si-AL592494.4, 3 replicates per group set-up;
(3) after 24 times of treatment, 100. mu.l of serum-free medium containing 10. mu.l of CCK-8 solution was added to each well, and the plates were placed in an incubator for further 3-4 hours;
(4) the absorbance at 450nm was measured using a microplate reader, and the results are shown in FIG. 3.
The cell survival rate of the B group is 76.61 +/-1.28%, and the cell survival rate of the C group is 72.16 +/-1.21%; the cell survival rate of group D was 92.24. + -. 1.57%, and from the results, it can be seen that the knock-down of AL592494.4 by si-AL592494.4 was effective in reducing the decrease in cell survival rate of HCAECs cells caused by ox-LDL.
Example 4
Effect of knockdown of AL592494.4 on ox-LDL induced apoptosis of HCAECs
(1) Inoculating HCAECs on a square glass slide of a 24-pore plate, and placing the square glass slide in a cell culture box to adhere to the wall for 12 hours;
(2) cells were treated in cell groups as follows:
group A: 0 μ g/ml ox-LDL; group B: 100 μ g/ml ox-LDL, group C: 0 μ g/ml ox-LDL + si-NC; group D: 100 μ g/ml ox-LDL + si-AL592494.4, 3 replicates per group set-up;
(3) removing the culture medium, washing the cells with PBS 2 times, and fixing the cells with 4% paraformaldehyde for 30 min;
(4) discarding the stationary liquid, gently washing with PBS for 2 times, adding PBS containing 0.1% TritonX-100, and incubating in ice bath for 2 min;
(5) preparing TUNEL detection solution according to TUNEL detection kit instructions, incubating at 37 deg.C in dark for 1h, adding prepared DAPI solution, and incubating at room temperature in dark for 5 min;
(6) after washing 1-2 times with PBS, mounting was performed using an anti-fluorescence quenching mounting solution, followed by observation and technique under a microscope, and the results are shown in FIG. 4.
From the experimental results, it can be seen that the use of si-AL592494.4 to knock down AL592494.4 can effectively reduce ox-LDL induced apoptosis of HCAECs cells.
Example 5
Effect of knock-down of AL592494.4 on ox-LDL induced upregulation of Caspase3 protein expression in HCAECs cells
(1) Inoculating HCAECs cells into a 6-well cell culture plate, and putting the cell culture plate into a cell culture box for overnight incubation;
(2) cells were treated in cell groups as follows:
group A: 0 μ g/ml ox-LDL; group B: 100 μ g/ml ox-LDL, group C: 0 μ g/ml ox-LDL + si-NC; group D: 100 μ g/ml ox-LDL + si-AL592494.4, 3 replicates per group set-up;
(3) after 24h of treatment, the cells were washed with experimental PBS, 100 μ l RIPA cell lysate was added to each well, and the cells were scraped off using a cell scraper and transferred to an EP tube;
(4) after cell lysis for 30min, placing in a centrifuge at 12000rpm/min, centrifuging for 10min, and removing supernatant to a new EP tube;
(5) quantifying protein by using a BCA method, adding 5 Xloading buffer solution, and boiling for 5min with boiling water to obtain a protein sample;
(6) preparing 5% of upper layer glue and 12% of lower layer glue, organizing an electrophoresis tank, adding 20 mug of protein sample and Marker indicator, and adding electrophoresis liquid for electrophoresis;
(7) after electrophoresis is finished, assembling an electric transfer clamp, transferring the PVDF membrane into 5% of skimmed milk powder for 1.5h at 200mA, and sealing at room temperature for 1 h;
(8) after membrane washing, Caspase3 and GAPDH primary antibody are incubated, and the membrane is sealed for 1h at 4 ℃;
(9) after washing the membrane, incubating corresponding secondary antibody, and incubating for 1h in a shaking table at room temperature;
(10) in the dark, development exposure was performed, and the experimental results are shown in fig. 5.
As can be seen from the figure, the knock-down of AL592494.4 by si-AL592494.4 can effectively reduce ox-LDL induced Caspase3 protein up-regulation of HCAECs cells.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.
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Claims (8)
1. The application of the gene AL592494.4 as a target molecule in inhibiting the damage of endothelial cells of coronary arteries is characterized in that the External Transcript ID of the gene AL592494.4 is ENST 00000437523.1.
2. Application of an inhibitor of gene AL592494.4 in preparation of drugs for treating coronary atherosclerotic heart disease.
3. The use of claim 2, wherein the inhibitor is an siRNA.
4. The use of claim 3, wherein the sense strand of the siRNA is represented by SEQ ID No.13 and the antisense strand of the siRNA is represented by SEQ ID No. 14.
5. Application of an inhibitor of gene AL592494.4 in preparing a coronary artery endothelial cell injury inhibitor.
6. The use of claim 5, wherein the inhibitor is an siRNA, wherein the sequence of the sense strand of the siRNA is shown in SEQ ID No.13, and the sequence of the antisense strand of the siRNA is shown in SEQ ID No. 14.
7. The siRNA for preparing the coronary artery endothelial cell injury inhibitor is characterized in that the sense strand sequence of the siRNA is shown as SEQ ID NO.13, and the antisense strand sequence of the siRNA is shown as SEQ ID NO. 14.
8. The medicine for treating the coronary atherosclerotic heart disease is characterized by comprising an inhibitor of a gene AL592494.4, wherein the inhibitor is siRNA, the sequence of a sense strand of the siRNA is shown as SEQ ID NO.13, and the sequence of an antisense strand of the siRNA is shown as SEQ ID NO. 14.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113069548A (en) * | 2021-04-23 | 2021-07-06 | 青岛市中医医院(青岛市海慈医院、青岛市康复医学研究所) | Reagent for cardiac revascularization |
| CN115804827A (en) * | 2022-03-18 | 2023-03-17 | 山东大学齐鲁医院(青岛) | Application of polypeptide in preparation of protective agent for reducing vascular endothelial function damage |
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| CN112159842A (en) * | 2020-10-11 | 2021-01-01 | 宁夏医科大学 | Molecular marker for early warning of atherosclerosis and application thereof |
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| CN113069548A (en) * | 2021-04-23 | 2021-07-06 | 青岛市中医医院(青岛市海慈医院、青岛市康复医学研究所) | Reagent for cardiac revascularization |
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| CN115804827A (en) * | 2022-03-18 | 2023-03-17 | 山东大学齐鲁医院(青岛) | Application of polypeptide in preparation of protective agent for reducing vascular endothelial function damage |
| CN115804827B (en) * | 2022-03-18 | 2023-06-30 | 山东大学齐鲁医院(青岛) | Application of polypeptide in preparation of protective agent for reducing vascular endothelial function injury |
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