CN113069548A - Reagent for cardiac revascularization - Google Patents
Reagent for cardiac revascularization Download PDFInfo
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- CN113069548A CN113069548A CN202110438953.6A CN202110438953A CN113069548A CN 113069548 A CN113069548 A CN 113069548A CN 202110438953 A CN202110438953 A CN 202110438953A CN 113069548 A CN113069548 A CN 113069548A
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Abstract
The invention provides a reagent for regenerating cardiac vessels, and belongs to the technical field of cardiovascular diseases. The gene LINC01910 differentially expressed in human heart microvascular endothelial cells is screened out through hypoxia induction, and meanwhile, experiments show that the inhibitor si-LINC01910 of LINC01910 can effectively promote the proliferation and migration of HCMECs cells and the generation of a tubule-like structure, so that the inhibitor of LINC01910 can be used for preparing a reagent for regenerating blood vessels, and a new treatment medicine is provided for patients with coronary heart disease.
Description
Technical Field
The invention belongs to the technical field of cardiovascular diseases, and particularly relates to a reagent for regenerating cardiac vessels.
Background
Coronary heart disease myocardial infarction and coronary heart disease myocardial ischemia are very common cardiovascular system diseases in clinic, and are the first causes of global pathogenesis and death. At present, the main treatment modes of coronary heart disease are coronary bypass surgery, western medicine treatment and stent implantation. Although these treatments have achieved magnificent results over the past decades, they also have been associated with high treatment costs, surgical risks, etc. Therefore, the search for new treatment modes is of great significance for better treatment of patients with coronary heart disease.
Angiogenesis (Angiogenesis) refers to the development of new blood vessels from existing capillaries or postcapillary veins, and mainly comprises: degradation of vascular basement membrane during activation; activation, proliferation and migration of vascular endothelial cells; remodeling forms new blood vessels and vascular networks, and thus it is a complex process involving a variety of molecules of a variety of cells. In recent years, researchers have made intensive progress on the basis of angiogenesis and have achieved desirable effects. However, at present, many problems still exist in clinic for cardiovascular angiogenesis, so deep research on angiogenesis mechanism has important significance for better treating coronary heart disease patients.
Disclosure of Invention
The purpose of the present invention is to provide an agent for cardiac revascularization.
The invention provides application of an inhibitor targeting LINC01910 in preparation of a cardiac revascularization agent.
Preferably, the sequence of the transcript of LINC01910 is shown as SEQ ID NO. 1.
Preferably, the inhibitor is siRNA, and the base sequence of the siRNA is as follows:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', SEQ ID NO.10;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3', SEQ ID NO. 11.
Secondly, the invention provides a siRNA for preparing a cardiac revascularization reagent, wherein the base sequence of the siRNA is as follows:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', SEQ ID NO.10;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3', SEQ ID NO. 11.
Thirdly, the invention provides application of the LINC 01910-targeted inhibitor in preparation of a human heart microvascular endothelial cell proliferation promoting reagent.
Preferably, the inhibitor is siRNA, and the base sequence of the siRNA is as follows:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', SEQ ID NO.10;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3', SEQ ID NO. 11.
Fourthly, the invention provides application of the LINC 01910-targeted inhibitor in preparation of a human heart microvascular endothelial cell migration promoting reagent.
Preferably, the base sequence of the siRNA is as follows:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', SEQ ID NO.10;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3', SEQ ID NO. 11.
The invention has the beneficial effects that:
the gene LINC01910 differentially expressed in human heart microvascular endothelial cells is screened out through hypoxia induction, and meanwhile, experiments show that the inhibitor si-LINC01910 of LINC01910 can effectively promote the proliferation and migration of HCMECs cells and the generation of a tubule-like structure, so that the inhibitor of LINC01910 can be used for preparing a reagent for regenerating blood vessels, and a new treatment medicine is provided for patients with coronary heart disease.
Drawings
FIG. 10. mu.M CoCl2And 150. mu.M CoCl2The expression of LINC1978, LINC01012, LINC01910 and LINC01854 in the group is different;
FIG. 2 shows the inhibitory effect of siRNA si-LINC01910 of LINC01910 provided by the present invention;
FIG. 3 CCK-8 measurements of si-NC and si-LINC01910 transfected HCMECs cells at 24h, 48h, 72h and 96 h;
FIG. 4 cell migration assay results for HCMECs cells transfected with si-NC and si-LINC 01910;
FIG. 5 tubuloid structure of HCMECs cells transfected with si-NC and si-LINC01910 generated assay results.
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
Detecting the effect of hypoxia treatment on non-coding RNA in human heart microvascular endothelial cells (HCMECs)
(1) HCMECs cells were seeded in 6-well plates using 0. mu.M CoCl2And 150 μM CoCl2HCMECs cells were treated for 24h, with 3 replicates per treatment set;
(2) after the treatment is finished, removing the culture medium, washing the cells for 2 times by using precooled PBS, adding 1ml of Trizol into each hole, repeatedly blowing and beating by using a pipettor to ensure that the Trizol fully cracks the cells, and transferring the liquid into an EP tube;
(3) adding 200ul chloroform into each tube, mixing uniformly, standing at room temperature for 5min, and centrifuging at 12000rpm at 4 deg.C for 15 min;
(4) sucking 400ul of supernatant liquid into a new RNase-free EP tube, adding 400ul of isopropanol, reversing, uniformly mixing, standing at room temperature for 10min, centrifuging at 12000rpm at 4 ℃ for 10min, removing supernatant, and retaining precipitate;
(5) adding 75% ethanol, resuspending, precipitating, reversing, mixing, and centrifuging at 12000rpm at 4 deg.C for 5 min;
(6) removing supernatant, air drying at room temperature, adding 40ul DEPC water to dissolve precipitate;
(7) carrying out reverse transcription reaction by using a TaKaRa reverse transcription kit to obtain cDNA;
(8) primers for synthesizing non-coding RNA LINC1978, LINC01012, LINC01910 and LINC01854 are designed, and specific primer sequences are as follows:
| name of Gene | Reference sequence | Primer sequences | Size of product |
| LINC1978 | NR_110851.1 | CAAGCCACACAGCTGGAAAC，SEQ ID NO.2 | 144 |
| GATCGTGTCACTGCACTCCA,SEQ ID NO.3 | |||
| LINC01012 | NR_038294.1 | ATGACTCTGCAGGGACTCCT,SEQ ID NO.4 | 115 |
| CTCCACCTTGTCCACCATCC,SEQ ID NO.5 | |||
| LINC01910 | NR_110764.1 | CAAAGACCAGGACGAGCAGT,SEQ ID NO.6 | 141 |
| TGGTGAAGCTGCTGGAACAA,SEQ ID NO.7 | |||
| LINC01854 | NR_122042.1 | GGCAGACTGGTGCAGTATCA,SEQ ID NO.8 | 117 |
| TAGGGATGTCAGCACAGGGA,SEQ ID NO.9 |
(9) Preparing a fluorescent quantitative PCR reaction solution according to the following reaction system:
10μl SYBR Green Premix Ex Taq（2×），
mu.l of the upstream primer was added at 0.4. mu.l,
mu.l of the downstream primer was added at 0.4. mu.l,
mu.l of the cDNA template was added,
7.2μl ddH2O；
(10) the fluorescent quantitative PCR reaction was carried out according to the following reaction conditions:
5min at 95 ℃; at 95 ℃ for 10s, at 60 ℃ for 30s, at 72 ℃ for 30s, for 35 cycles;
(10) use 2-△△CtThe data obtained were processed to calculate the relative expression levels of LINC1978, LINC01012, LINC01910, and LINC 01854.
The results are shown in FIG. 1, from which it can be seen that CoCl was present at 0. mu.M2And 150. mu.M CoCl2The P values of the expression differences of LINC01978, LINC01012 and LINC01854 in the group are all larger than 0.05, so the differences have no statistical significance. While the P value of the expression difference of LINC01910 was less than 0.05, so the difference was statistically significant at 150. mu.M CoCl2The relative expression level of LINC01910 in the group was 0.394. + -. 0.069, indicating CoCl2The treatment can reduce the expression level of LINC01910 in human heart microvascular endothelial cells, and because hypoxia can effectively promote angiogenesis of the endothelial cells, we guess that the reduction of LINC01910 can effectively promote angiogenesis of the endothelial cells.
Example 2 design of siRNA for synthesizing LINC01910 and detection
(1) The designed siRNA of LINC01910 has the following sequence:
sense strand: GGGAGAGACAGAUAUGUAAAC, SEQ ID NO.10;
antisense strand: UUACAUAUCUGUCUCUCCCAG, SEQ ID NO. 11.
(2) And (3) transfecting the siRNA and the si-NC into HCMECs cells, and after transfecting for 48h, extracting RNA and performing fluorescent quantitative PCR reaction.
The experimental result is shown in figure 2, and it can be seen from the figure that the siRNA of LINC01910 provided by the invention can effectively inhibit the expression level of LINC01910 (the relative expression level is 0.1857 +/-0.036).
Example 3 modulation of cell proliferation of HCMECs by si-LINC01910
(1) Inoculating HCMECs cells into a 96-well plate, setting an si-NC group for transfecting si-NC and an si-LINC01910 group for transfecting si-LINC01910, and setting 3 multiple wells in each group;
(2) the OD value of 450nm was measured at 24h, 48h, 72h and 96h using CCK-8.
As shown in the figure 2, the OD value of the si-NC is 0.771 +/-0.042 at 96h, the OD value of the si-LINC01910 group is 1.154 +/-0.047, the difference between the two values is statistically significant, and the OD value of the si-LINC01910 group is also obviously higher than that of the si-NC group at 48h and 72h, so the results can show that the si-LINC01910 provided by the invention can effectively promote the cell proliferation of HCMECs.
Example 3 modulation of cell migration of HCMECs by si-LINC01910
(1) Inoculating HCMECs cells into 6-well culture plate, transfecting si-NC and si-LINC01910 when the cell density reaches 90%, digesting the cells after transfecting for 48h, suspending the cells by serum-free medium, adjusting the cell density to 2 × 10 after counting5/ml；
(2) The Transwell chamber was placed in a 24-well plate, 600 medium containing 10% FBS was added to the lower chamber, and 100ul of cell suspension was added to the upper chamber of the Transwell chamber;
(3) placing the 24-hole plate in a cell culture box for culturing for 24h, taking out the Transwell chamber by using a pair of tweezers after the culture is finished, transferring the upper chamber liquid to a hole with methanol added, and fixing for 30min at room temperature;
(4) after fixation, the fixative solution was blotted dry, the Transwell chamber was transferred to a well containing crystal violet and stained for 20min at room temperature;
(5) after dyeing is finished, lightly washing the cells for 3 times by using clear water, and wiping off the cells on the surface of the upper chamber membrane of the Transell chamber by using a cotton swab;
(6) and (5) placing the glass sample under an inverted microscope, and photographing and counting.
The experimental results are shown in FIG. 4, and it can be seen that the number of cells penetrated by the si-LINC01910 group is significantly greater than that of the si-NC group, and therefore, the results can indicate that the si-LINC01910 provided by the invention can effectively promote the migration of HCMECs.
Example 4 Regulation of production of tubuloid structures in HCMECs cells by si-LINC01910
(1) Placing 500ul of Matrigel gel in a refrigerator at 4 ℃ overnight to be fully melted;
(2) coating with 200ul Matrigel gel per well in 24-well plate at 37 deg.C for 30 min;
(3) digestion of transfected si-NC and si-LINC01910 to cell suspensions, 1X 10 inoculate each well5And (4) continuously culturing the cells for 6 hours, taking a picture under an inverted microscope, randomly selecting 5 visual fields, and calculating the length of the capillary vessel lumen structure.
The results of the experiment are shown in FIG. 5, from which it can be seen that the length of the tubule-like structures formed on the Matrigel gel by si-LINC01910 group is significantly increased compared to the si-NC group.
In conclusion, the inhibitor si-LINC01910 of LINC01910 can effectively promote the proliferation, migration and tubule-like structure generation of HCMECs cells, so that the inhibitor of LINC01910 can be used for preparing an agent for angiogenesis.
Sequence listing
<110> Qingdao city Chinese medical Hospital (Qingdao city Haikehospital, Qingdao city rehabilitation research institute)
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<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tggtgaagct gctggaacaa 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggcagactgg tgcagtatca 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tagggatgtc agcacaggga 20
<210> 10
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gggagagaca gauauguaaa c 21
<210> 11
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
uuacauaucu gucucuccca g 21
Claims (8)
1. Application of an inhibitor targeting LINC01910 in preparing a cardiac revascularization agent.
2. The use according to claim 1, wherein the transcript sequence of LINC01910 is shown in SEQ ID No. 1.
3. The use according to claim 1, wherein the inhibitor is an siRNA having the following base sequence:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', respectively;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3' are provided.
4. An siRNA for preparing a heart blood vessel regeneration reagent, wherein the base sequence of the siRNA is as follows:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', respectively;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3' are provided.
5. The application of the inhibitor of the targeting LINC01910 in preparing a human heart microvascular endothelial cell proliferation promoting reagent.
6. The use according to claim 5, wherein the inhibitor is siRNA, and the base sequence of the siRNA is as follows:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', respectively;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3' are provided.
7. Application of the LINC 01910-targeted inhibitor in preparation of a human heart microvascular endothelial cell migration promoting agent.
8. The use according to claim 7, wherein the siRNA has the following base sequence:
sense strand: 5'-GGGAGAGACAGAUAUGUAAAC-3', respectively;
antisense strand: 5'-UUACAUAUCUGUCUCUCCCAG-3' are provided.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921462A (en) * | 2021-08-18 | 2022-08-19 | 济南生发堂生物科技股份有限公司 | Application of non-coding gene in promoting growth of hair follicle stem cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190256846A1 (en) * | 2016-09-14 | 2019-08-22 | Agency For Science, Technology And Research | Modulation of tjp1 expression to regulate regeneration of heart cells |
| CN111032093A (en) * | 2017-06-19 | 2020-04-17 | 洛桑大学 | Methods and compositions for controlling myocardial fibrosis and remodeling |
| CN112656947A (en) * | 2021-01-27 | 2021-04-16 | 青岛市中心医院 | Endothelial cell injury inhibitor |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190256846A1 (en) * | 2016-09-14 | 2019-08-22 | Agency For Science, Technology And Research | Modulation of tjp1 expression to regulate regeneration of heart cells |
| CN111032093A (en) * | 2017-06-19 | 2020-04-17 | 洛桑大学 | Methods and compositions for controlling myocardial fibrosis and remodeling |
| CN112656947A (en) * | 2021-01-27 | 2021-04-16 | 青岛市中心医院 | Endothelial cell injury inhibitor |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921462A (en) * | 2021-08-18 | 2022-08-19 | 济南生发堂生物科技股份有限公司 | Application of non-coding gene in promoting growth of hair follicle stem cells |
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