CN113930384B - Rat and modeling method for in-vitro vascular smooth muscle cell vascular remodeling of rat - Google Patents
Rat and modeling method for in-vitro vascular smooth muscle cell vascular remodeling of rat Download PDFInfo
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Abstract
The invention relates to the field of medical biology, in particular to a rat and a modeling method for in-vitro vascular smooth muscle cell vascular remodeling thereof. The invention uses glucagon peptide GLP-1, liraglutide or exenatide as an inducing factor to treat rat or rat in vitro vascular smooth muscle cells to construct a vascular remodeling rat model or vascular remodeling in vitro cell model. The invention firstly uses GLP-1 and GLP-1 analogues with the concentration of 100nM and above to construct the vascular remodeling model, the construction methods of the animal model and the in vitro cell model are simple, the operation step time is short, the time cost and the economic cost are saved, the support is provided for the multifactor induced pathogenesis of the cardiovascular disease, and the important theoretical basis is provided for developing a new pharmaceutical preparation for effectively treating the cardiovascular disease.
Description
Technical Field
The invention relates to the field of medical biology, in particular to a rat and a modeling method for in-vitro vascular smooth muscle cell vascular remodeling thereof.
Background
GLP-1 is a long insulinotropic polypeptide formed by processing a glucagon gene, the gene is successfully cloned for the first time in 1983, and a protein product of the gene is approved as a therapeutic drug for type II diabetes in 2005. GLP-1 has been found to have protective effects on the cardiovascular system during treatment of type II diabetes, such as improving endothelial dysfunction, increasing cardiomyocyte survival, improving local and global cardiac output following myocardial injury and heart failure. In addition, GLP-1 can reduce blood pressure of hypertension patients and improve cardiovascular prognosis effect of diabetes patients.
The function of GLP-1 is mediated by GLP-1R. GLP-1R is expressed in pancreas, lung, kidney, brain, adipose tissue, gastrointestinal tract, blood vessel, heart, liver, etc. Like most GPCRs, GLP-1R has long been recognized as a classical membrane receptor with subcellular localization focused primarily on the cytoplasm and cell membrane. Ligand-induced GLP-1R internalization and circulation has been demonstrated in Chinese hamster lung fibroblasts, rat insulinoma cells, and rat islet cells. The expression of GLP-1R can play a protective role in the treatment of cardiovascular diseases, the expression of GLP-1R in renal arteries of hypertensive rats can be reduced by the up-regulation of the expression of beta-type subunit of protein kinase C, and the reduction of the expression of GLP-1R causes the vasomotor function of hypertensive patients to be damaged. In chronic kidney disease and myocardial infarction, inhibition of dipeptidyl IV can increase GLP-1R expression in the renal tubules and myocardium. GLP-1 (10-20 nmol/L) in a physiological concentration range can improve the effect of cardiovascular diseases, however, the effect of GLP-1 in high concentration on vascular remodeling is not reported in the literature. We have established a method for using high concentrations of GLP-1 and its analogs to intervene in rats, and to intervene in vitro in rat aortic vascular smooth muscle cells, which can cause the vascular remodeling phenomenon to occur. The method for establishing the blood vessel reconstruction model provides a good model and theoretical basis for future treatment and prevention of cardiovascular diseases.
Disclosure of Invention
The invention aims to solve the technical problem of providing a rat and a modeling method for in-vitro vascular smooth muscle cell vascular remodeling thereof, which utilize high-concentration GLP-1 and analogues thereof to promote abnormal proliferation, migration and extracellular matrix remodeling of in-vivo and in-vitro vascular smooth muscle cells of the rat and establish a vascular remodeling rat model or a vascular remodeling in-vitro cell model.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention provides a rat and a modeling method for in vitro vascular smooth muscle cell vascular remodeling of the rat, and the method takes glucagon-like peptide GLP-1, liraglutide or exenatide as an induction factor to treat rat or rat in vitro vascular smooth muscle cells to construct a vascular remodeling rat model or a vascular remodeling in vitro cell model.
The construction method of the rat model with vascular remodeling comprises the following steps: injecting GLP-1, liraglutide or exenatide into abdominal cavity of a rat, wherein the dosage of each time is 3mg/kg of the rat, and the injection is performed 2 times a day for 8 weeks continuously to obtain a rat vascular remodeling model.
Further, the rat is a Wistar rat.
The construction method of the vascular reconstruction in-vitro cell model comprises the following steps: GLP-1, liraglutide or exenatide with the concentration of 100nM or more is added into a complete culture medium of rat aortic vascular smooth muscle cells, and the rat aortic vascular smooth muscle cells are cultured to obtain a vascular remodeling in-vitro cell model.
Further, the complete medium contains L-glutamic acid, hydroxyethyl piperazine ethanesulfonic acid, glucose, sodium bicarbonate, sodium pyruvate and fetal calf serum.
Further, in the complete culture medium, the content of L-glutamic acid is 4mM, the content of hydroxyethyl piperazine ethanesulfonic acid is 6.0g/L, the content of glucose is 4.5g/L, the content of sodium bicarbonate is 3.7g/L, the content of sodium pyruvate is 0.11g/L, and the content of fetal calf serum is 10%.
Further, the culture time is 24-48 h.
The invention also provides application of the vascular remodeling rat model or the vascular remodeling in-vitro cell model in screening or preparing a medicament for treating cardiovascular diseases.
Compared with the prior art, the invention has the following beneficial effects:
(1) GLP-1 in a normal physiological concentration range plays a beneficial role in preventing and treating diseases related to vascular remodeling, a vascular remodeling model is constructed by GLP-1 and GLP-1 analogues with the concentration of 100nM and above for the first time, the construction method of an animal model and an in-vitro cell model is simple, the operation step time is short, and the time cost and the economic cost are saved.
(2) The vascular remodeling rat model or the vascular remodeling in-vitro cell model is a vascular remodeling model induced by GLP-1 and analogues thereof, provides support for a multi-factor induced pathogenesis of cardiovascular diseases, and provides an important theoretical basis for developing a new pharmaceutical preparation for effectively treating the cardiovascular diseases.
Drawings
FIG. 1 is a graph comparing the vascular wall thickness and collagen content of rats in the liraglutide intervention group and the control group according to example 1 of the present invention;
FIG. 2 is a graph comparing the extracellular matrix remodeling of rat blood vessels in the liraglutide intervention group and the control group in example 1 of the present invention;
FIG. 3 is a graph comparing the proliferation of aortic vascular smooth muscle cells in rats in GLP-1 intervention group, liraglutide intervention group and exenatide intervention group in example 2 of the present invention;
FIG. 4 is a graph comparing the migration of rat aortic vascular smooth muscle cells in GLP-1 intervention group, liraglutide intervention group and exenatide intervention group of example 3 of the present invention;
FIG. 5 is a graph comparing the rat aortic vascular smooth muscle extracellular matrix reconstructions in GLP-1 intervention group, liraglutide intervention group, and exenatide intervention group of example 4 of the present invention.
Detailed Description
The following examples are given in the detailed description and the specific operation on the premise of the technical solutions of the present invention, but do not limit the protection scope of the patent of the present invention, and all technical solutions obtained by using equivalent alternatives or equivalent variations should fall within the protection scope of the present invention.
Example 1
Construction of vascular remodeling rat model: 12 WT rats at 8 weeks of age were weighed on the day of the start of the experiment, which was recorded as the start of the day 0, and the WT rats were randomly divided into 2 groups according to body weight: liraglutide intervention group and control group, 6 each. The liraglutide or the normal saline is given at the beginning of day 1, the liraglutide intervention group is injected into the abdominal cavity of the liraglutide Lirglutide, the single dosage is 3mg/kg of rats, and the control group is given with the same amount of normal saline at the same time and is injected for 2 times a day for 8 weeks continuously.
Identification of rat model for vascular remodeling: the rats were sacrificed and tissue samples were collected. The blood vessel part is embedded and sliced, and then the change of the thickness of the blood vessel wall and the content of collagen fibers are detected by HE staining and Masson staining respectively.
And (3) an HE dyeing method: the extracted vascular tissue was fixed in 10% neutral formalin for 24h. Performing conventional paraffin embedding, cutting into 2 μm thick slices, dewaxing with xylene, dehydrating with ethanol with concentration gradient, staining with hematoxylin for 10min, washing with distilled water and ethanol, staining with 0.5% eosin for 1-3min, performing gradient dehydration according to conventional method, transparentizing, and sealing.
Masson staining method: dewaxing the blood vessel section to water by a conventional method, and staining the blood vessel section for 5-10min by hematoxylin; differentiating by acidic ethanol solution for 5-10s, washing with distilled water for 5min; dyeing with acid fuchsin solution for 5min, dyeing with 1% phosphomolybdic acid for 1-2min, washing with 1% acetic acid solution for 1min; treating with aniline acetate blue dye solution for 1min, and treating with 1% acetic acid for 1min; and (5) performing gradient dehydration by using ethanol, and sealing the slices by using neutral resin.
Referring to fig. 1 and fig. 2, the results show that compared with the control group, the vascular wall thickness of vascular smooth muscle cells in rats in the liraglutide intervention group is increased, which in turn causes vascular stenosis, the expression of collagen fibers in blood vessels is increased, and the expression of col1a1, col3a1, MMP2 and MMP9 proteins is increased, which indicates that the construction of the liraglutide group vascular remodeling rat model is successful.
Example 2
Construction of in vitro cell model of vascular remodeling: after the cells of rat aorta vascular smooth muscle cells in a good state are counted, the cells are inoculated in a 96-well plate at the density of 5000 cells/well; after culturing for 24h in complete medium containing fetal calf serum (4 mM L-glutamic acid, 6.0 g/L4-hydroxyethylpiperazine ethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate, 0.11g/L sodium pyruvate, 10% fetal calf serum) and after the cells adhere to the wall, they were divided into four groups: the control group is intervened by a serum-free culture medium (4 mM L-glutamic acid, 6.0 g/L4-hydroxyethyl piperazine ethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate and 0.11g/L sodium pyruvate), and the GLP-1 dry pre-group, the liraglutide dry pre-group and the exenatide dry pre-group are intervened by 100nM GLP-1, liraglutide dry pre-group and exenatide prepared by the serum-free culture medium (the components are the same as those of the control group) respectively and then are placed in a carbon dioxide constant temperature culture box for 24 hours.
And (3) detecting cell proliferation activity: adding 20 mu L of MTT into each hole in the different treatment groups, reacting for 3-4h in an incubator, discarding the solution, adding 150 mu L of dimethyl sulfoxide, mixing uniformly, and detecting the light absorption value at 490 nm.
And (3) detecting the cyclin: (1) collecting the cells of the different treatment groups, placing the cells in a cell lysis solution prepared by protease inhibitor, phosphatase inhibitor and phenylmethylsulfonyl fluoride in advance, and lysing for 20min on ice; (2) centrifuging at 12000rpm and 4 ℃ for 15min, taking out, and detecting the protein concentration by using a BCA quantitative kit; (3) ensuring the same sample loading amount of each group of samples, and performing SDS-PAGE; (4) after electrophoresis is finished, putting the PVDF membrane in methanol for 5s to activate the PVDF membrane, preparing a membrane transfer sandwich from the anode to the cathode of a membrane transfer instrument according to the sequence of filter paper-PVDF membrane-SDS gel-filter paper, adding a prepared electrotransformation liquid in advance, and setting corresponding membrane transfer conditions according to the molecular weight of the required target protein; (5) after the wet conversion is finished, taking out the PVDF membrane from the middle of the filter paper, washing the PVDF membrane by using a membrane washing buffer TBST, and finally sealing the PVDF membrane for 2 hours at room temperature by using 5% skimmed milk powder prepared by TBST; (6) washing with membrane washing buffer TBST for 3 times (5 min each time), and incubating the primary antibody at 4 deg.C overnight; (7) washing with membrane washing buffer TBST for 3 times (5 min each time), and incubating the secondary antibody at 4 deg.C overnight; (8) and performing X-ray film development and fixation by ECL in a dark room.
Referring to FIG. 3, the results show that treatment of 100nM GLP-1 or GLP-1 analogs liraglutide and exenatide increases cell viability; the western blotting result shows that the intervention of 100nM GLP-1 or liraglutide and exenatide can obviously up-regulate the expression of cyclin PCNA and cyclin D1, and the result shows that 100nM GLP-1 or liraglutide and exenatide can cause the abnormal proliferation of rat aortic vascular smooth muscle cells.
Example 3
Construction of in vitro cell model of vascular remodeling: rat aortic vascular smooth muscle cells in good state are inoculated in a 6-well plate, and the number of each well is ensured to be 1 multiplied by 10 5 (ii) individual cells; after culturing for 24 hours in complete medium containing fetal calf serum (4 mM L-glutamic acid, 6.0 g/L4-hydroxyethylpiperazineethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate, 0.11g/L sodium pyruvate, 10% fetal calf serum) and cell density reached 90% or more, the wells were scored at the center with a 200. Mu.L tip and divided into four groups: control group, GLP-1 intervention group, liraglutide intervention group and exenatideThe peptide dry pre-group and the control group are intervened by a serum-free culture medium (4 mM L-glutamic acid, 6.0 g/L4-hydroxyethyl piperazine ethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate and 0.11g/L sodium pyruvate), and the GLP-1 dry pre-group, the liraglutide dry pre-group and the exenatide dry pre-group are intervened by 100nM GLP-1, liraglutide dry pre-group and exenatide which are prepared by the serum-free culture medium (the components are the same as the control group) respectively and then are placed in a carbon dioxide constant temperature incubator for 24 hours.
Cell migration assay: the extent of rat aortic vascular smooth muscle cell migration was evaluated by taking pictures of the width of the scratch using an inverted microscope (× 10 times mirror), capturing images of 0h and 24h of each sample, and measuring the width of the scratch measured at 2 time points using Image-ProPlus 6 software, cell migration = (control-experimental)/control 100%.
Referring to fig. 4, the results, which show that the treatment of 100nM of GLP-1 or liraglutide and exenatide induces a significant increase in the migration ability of RASMC cells, indicate that 100nM of GLP-1 or liraglutide and exenatide can promote abnormal migration of rat aortic vascular smooth muscle cells.
Example 4
Construction of in vitro cell model of vascular remodeling: well-conditioned rat aortic vascular smooth muscle cells were placed in complete medium (4 mM L-glutamic acid, 6.0 g/L4-hydroxyethylpiperazine ethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate, 0.11g/L sodium pyruvate, 10% fetal bovine serum), and then placed at 37 ℃ with a volume concentration of 5% CO 2 Culturing in a constant-temperature incubator; when the cell density reaches more than 95%, discarding the old culture medium, washing with Phosphate Buffered Saline (PBS) for 3 times, adding 0.25% pancreatin to eliminate the adherent cells in the incubator for 1-2min; then adding fresh complete culture medium (the components are the same as above) containing fetal calf serum to stop digestion, centrifuging at 1100rpm for 5min, and removing supernatant; the cells were resuspended in fresh complete medium containing fetal bovine serum (as above) and the cell suspension was seeded into 6-well plates and divided into four groups: a control group, a GLP-1 intervention group, a liraglutide intervention group and an exenatide intervention group; culturing with serum-free medium when cell density reaches above 90%100nM GLP-1, liraglutide and exenatide are respectively prepared by replacing a complete culture medium with a nutrient medium (4 mM L-glutamic acid, 6.0 g/L4-hydroxyethylpiperazine ethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate and 0.11g/L sodium pyruvate), and are respectively added into cell suspensions of corresponding groups after preparation, cells are continuously intervened for 24h, and a control group is intervened for the same time by using an equal-volume serum-free culture medium.
Detection of protein expression in the extracellular matrix: (1) collecting the cells of the different treatment groups, placing the cells in a cell lysis solution prepared by protease inhibitor, phosphatase inhibitor and phenylmethylsulfonyl fluoride in advance, and lysing for 20min on ice; (2) centrifuging at 12000rpm and 4 ℃ for 15min, taking out, and detecting the protein concentration by using a BCA quantitative kit; (3) ensuring the same sample loading amount of each group of samples, and performing SDS-PAGE; (4) after electrophoresis is finished, putting the PVDF membrane in methanol for 5s to activate the PVDF membrane, preparing a membrane transfer sandwich from the anode to the cathode of a membrane transfer instrument according to the sequence of filter paper-PVDF membrane-SDS gel-filter paper, adding a prepared electrotransformation liquid in advance, and setting corresponding membrane transfer conditions according to the molecular weight of the required target protein; (5) after the wet conversion is finished, taking out the PVDF membrane from the middle of the filter paper, washing the PVDF membrane by using a membrane washing buffer TBST, and finally sealing the PVDF membrane for 2 hours at room temperature by using 5% skimmed milk powder prepared by TBST; (6) washing with membrane washing buffer TBST for 3 times (5 min each time), and incubating the primary antibody at 4 deg.C overnight; (7) washing with membrane washing buffer TBST for 3 times, 5min each time, and incubating the secondary antibody overnight at 4 deg.C; (8) and performing X-ray film development and fixation by ECL in a dark room.
Referring to fig. 5, the results show that 100nM of GLP-1, liraglutide or exenatide can significantly up-regulate the expression of col1a1, col3a1, MMP9, MMP2 and MMP1 proteins, and the proteins in the extracellular matrix are reconstructed, which indicates that the GLP-1 vascular reconstruction in vitro cell models in the GLP-1 stem cell group, the liraglutide stem cell group and the exenatide stem cell group are successfully constructed.
Example 5
Rat aortic vascular smooth muscle cells in good state are inoculated in a 6-well plate, and the number of each well is ensured to be 1 multiplied by 10 5 (ii) individual cells; the whole culture medium containing fetal bovine serum (4 mM L-glutamic acid,6.0 g/L4-hydroxyethylpiperazine ethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate, 0.11g/L sodium pyruvate, 10% fetal bovine serum), and when the cell density reached 90% or more, the center of each well was scratched with a 200. Mu.L tip to divide it into four groups: the control group is intervened by a serum-free culture medium (4 mM L-glutamic acid, 6.0 g/L4-hydroxyethyl piperazine ethanesulfonic acid, 4.5g/L glucose, 3.7g/L sodium bicarbonate and 0.11g/L sodium pyruvate), and the GLP-1 dry pre-group, the liraglutide dry pre-group and the exenatide dry pre-group are intervened by 200nM GLP-1, liraglutide and exenatide which are prepared by the serum-free culture medium (the components are the same as the control group) respectively and then are placed in a carbon dioxide constant-temperature culture box for 24h.
And (3) detecting cell proliferation activity: the detection method is shown in example 2, and MTT detection results show that 200nM GLP-1, liraglutide intervention group and exenatide obviously improve cell viability.
Cyclin detection: the detection method is shown in example 2, and the western blotting result shows that the intervention of 200nM GLP-1, liraglutide and exenatide can obviously up-regulate the expression of PCNA and cyclinD1, and the result shows that the 200nM GLP-1, liraglutide and exenatide can cause the abnormal proliferation of rat aortic vascular smooth muscle cells.
Detecting extracellular matrix proteins: the detection method is shown in example 2, and the result shows that 200nM GLP-1, liraglutide and exenatide can obviously up-regulate the expression of col1a1, col3a1, MMP9, MMP2 and MMP1 proteins, and the proteins in the extracellular matrix are reconstructed.
Cell migration detection: the results of the 200nM GLP-1, liraglutide and exenatide treatment, which show that the migration ability of RASMC cells is significantly increased, show that 200nM GLP-1, liraglutide and exenatide can promote abnormal migration of rat aortic vascular smooth muscle cells.
In conclusion, 200nM GLP-1, liraglutide and exenatide can also induce vascular remodeling of smooth muscle cells in vitro, and GLP-1 vascular remodeling in vitro cell models in the GLP-1 intervention group, the liraglutide intervention group and the exenatide intervention group are successfully constructed.
Claims (6)
1. A model making method for rat vascular remodeling is characterized in that a glucagon-like peptide GLP-1, liraglutide or exenatide is used as an induction factor to process a rat to construct a vascular remodeling rat model;
the construction method of the rat model with vascular remodeling comprises the following steps: injecting GLP-1, liraglutide or exenatide into abdominal cavity of a rat, wherein the dosage of a single time is 3mg/kg of rat, and injecting for 2 times a day for 8 weeks continuously to obtain a rat vascular remodeling model.
2. The modeling method for rat vascular remodeling of claim 1, wherein the rat is a Wistar rat.
3. A model building method for rat in vitro vascular smooth muscle cell vascular remodeling is characterized in that glucagon-like peptide GLP-1, liraglutide or exenatide is used as an induction factor to process rat in vitro vascular smooth muscle cells to construct a vascular remodeling in vitro cell model;
the construction method of the blood vessel reconstruction in-vitro cell model comprises the following steps: adding 100-200nM GLP-1, liraglutide or exenatide into a complete culture medium of rat aortic vascular smooth muscle cells, and culturing the rat aortic vascular smooth muscle cells to obtain a vascular remodeling in-vitro cell model.
4. The method of claim 3, wherein the complete medium comprises L-glutamic acid, hydroxyethylpiperazine ethanesulfonic acid, glucose, sodium bicarbonate, sodium pyruvate, and fetal calf serum.
5. The modeling method for rat in-vitro vascular smooth muscle cell vascular remodeling of claim 3, wherein in the complete medium, the content of L-glutamic acid is 4mM, the content of hydroxyethylpiperazine ethanesulfonic acid is 6.0g/L, the content of glucose is 4.5g/L, the content of sodium bicarbonate is 3.7g/L, the content of sodium pyruvate is 0.11g/L, and the content of fetal bovine serum is 10%.
6. The modeling method for rat in-vitro vascular smooth muscle cell vascular remodeling of claim 3, wherein the culture time is 24 to 48h.
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| WO2014160524A1 (en) * | 2013-03-14 | 2014-10-02 | The Trustees Of The University Of Pennsylvania | Cardio- metabolic and cascular effects of glp-1 metabolites |
| CN105999272A (en) * | 2015-07-20 | 2016-10-12 | 汪蕾 | Application of glucagon-like peptde-1 receptor agonist to preparation of pulmonary hypertension treating medicine |
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| Glucagon-like peptide-1 inhibits vascular smooth muscle cell dedifferentiation through mitochondrial dynamics regulation;Torres G等;《Biochem Pharmacol》;20160315;第104卷;第52-61页 * |
| LNK在高血压血管重构中的作用研究;邵明明等;《中国心血管病研究》;20200531;第18卷(第5期);第441-445页 * |
| 胰高血糖素样肽1与心血管疾病的研究进展;范韶华等;《生命的化学》;20161231;第36卷(第6期);第940-944页 * |
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