Phenylacetylglutamine / phenylacetylglycine induced vascular smooth muscle cell senescence model as well as establishment method and application thereof

Abstract

The invention relates to a phenylacetylglutamine / phenylacetylglycine induced vascular smooth muscle cell senescence model and an establishment method and application thereof, and belongs to the technical field of cell models.The method comprises the following steps that vascular smooth muscle cell suspension is placed in a porous plate, 10% of FBS and 1% of mycillin are added into a culture medium, 24 h after cells adhere to the wall, the vascular smooth muscle cell senescence model is obtained, and the vascular smooth muscle cell senescence model is obtained. The method comprises the following steps of: adding a proper amount of phenylacetyl glutamine or phenylacetyl glycine dimethyl sulfoxide solution into a porous plate, changing a serum-free culture medium, continuously culturing for 24 hours, adding a proper amount of phenylacetyl glutamine or phenylacetyl glycine dimethyl sulfoxide solution into the porous plate, changing the culture medium added with phenylacetyl glutamine or phenylacetyl glycine once every 24 hours, and collecting cells after 72 hours. According to the invention, phenylacetylglutamine / phenylacetylglycine is used for inducing and establishing the vascular smooth muscle cell senescence model for the first time, the method is easy to operate and low in cost, and a foundation is laid for researching the relationship between cardiovascular diseases and senescence.

Description

Technical Field

[0001] The present invention belongs to the field of cell model construction, and specifically relates to a phenylacetylglutamine / phenylacetylglycine-induced vascular smooth muscle cell aging model, and a method for establishing and applying the model. Background Art

[0002] Although senescent cells participate in important physiological processes throughout life, such as tissue development, wound healing, and tissue repair, cellular senescence is crucial in tissue pathophysiology and a key driver of aging and age-related diseases. Vascular cell senescence and dysfunction are fundamental pathological hallmarks of cardiovascular disease. Vascular smooth muscle cells (VSMCs), the predominant cell type in the vascular wall, undergo phenotypic transformation. Aged vessels exhibit decreased compliance and elasticity, while increasing stiffness, leading to a senescence-associated secretory phenotype. Furthermore, senescent cells secrete a variety of inflammatory factors, contributing to atherosclerosis. To support clinical and preclinical research, reliable and convenient vascular cell-based cutting-edge research is essential to support the study of vascular aging.

[0003] Phenylacetylglutamine (PAGln) is a metabolite derived from gut microbiota. Dietary phenylalanine is absorbed in the small intestine and, in addition to participating in energy metabolism, is metabolized in the large intestine by gut microbiota to produce phenylpyruvate. Phenylpyruvate is converted to phenylacetate by ferredoxin oxidoreductase (PPFOR), phenylpyruvate decarboxylase (PPDC), or porA. Phenylacetate, under the action of liver and kidney enzymes, is primarily converted to PAGln in humans by combining with glutamine and to phenylacetylglycine (PAGly) in mice by combining with glycine. Recent studies have shown that PAGln plasma levels are positively correlated with major adverse cardiovascular events such as heart failure, atrial fibrillation, stroke, and death. AGln / PAGly promotes thrombosis by acting on α2A, α2B, and β2-adrenergic receptors on the platelet cell membrane surface, and is involved in the pathological progression of many cardiovascular events.

[0004] At present, most of the vascular smooth muscle cell aging models are induced by stress factors such as angiotensin II and hydrogen peroxide. There are no reports on the methods of inducing human umbilical artery vascular smooth muscle cell aging model using the intestinal flora-derived metabolite PAGln, and inducing mouse vascular smooth muscle cell aging model using PAGly. Summary of the Invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art by providing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine, and at the same time provide a method for establishing the same and the application of the phenylacetylglutamine / phenylacetylglycine-induced vascular smooth muscle cell aging model.

[0006] To achieve the purpose, the technical solution adopted by the present invention is: In one aspect, the present invention provides a method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine, comprising the following steps: S1: Prepare phenylacetylglutamine solution and phenylacetylglycine solution using dimethyl sulfoxide. S2: Place a vascular smooth muscle cell suspension in a multiwell plate. Add 10% FBS and 1% penicillin-streptomycin to the culture medium. After the cells have adhered for 24 hours, switch to serum-free medium and continue culturing for another 24 hours. Add an appropriate amount of phenylacetylglutamine or phenylacetylglycine solution to the multiwell plate. Change the medium with phenylacetylglutamine or phenylacetylglycine every 24 hours. Collect the cells after 72 hours.

[0007] As a further improvement of the present invention, the concentration of the phenylacetylglutamine solution and the phenylacetylglycine solution in step S1 is 6 mol / L.

[0008] As a further improvement of the present invention, the vascular smooth muscle cells in step S2 are human umbilical artery vascular smooth muscle cells or mouse vascular smooth muscle cells; when the vascular smooth muscle cells are human umbilical artery vascular smooth muscle cells, phenylacetylglutamine is used for induction; when the vascular smooth muscle cells are mouse vascular smooth muscle cells, phenylacetylglycine is used for induction.

[0009] As a further improvement of the present invention, the proportion of dimethyl sulfoxide in the culture medium does not exceed 1‰.

[0010] As a further improvement of the present invention, step S2 is to take the cell density of 1×10 6 2 mL of human umbilical artery vascular smooth muscle cell suspension was placed in a 6-well plate.

[0011] As a further improvement of the present invention, in step S2, an appropriate amount of phenylacetylglutamine or phenylacetylglycine solution is added to the multi-well plate, wherein the amount is 2 μL.

[0012] As a further improvement of the present invention, step S2 is to take the cell density of 1×10 62 mL of human umbilical artery vascular smooth muscle cell suspension was placed in a 6-well plate. 10% FBS and 1% penicillin-streptomycin were added to the primary smooth muscle cell culture medium. After the cells adhered for 24 hours, the serum-free medium was changed and cultured for another 24 hours. 2 μL of 6 mol / L phenylacetylglutamine solution was added to the 6-well plate. The serum-free medium was changed every 24 hours and 2 μL of 6 mol / L phenylacetylglutamine stock solution was added. The proportion of dimethyl sulfoxide in the culture medium should not exceed 1‰. The cells were collected after 72 hours.

[0013] As a further improvement of the present invention, step S2 is to take the cell density of 1×10 6 2 mL of mouse vascular smooth muscle cell suspension was placed in a 6-well plate, in which 10% FBS and 1% penicillin-streptomycin were added to DMEM high-glucose medium. After the cells adhered for 24 h, serum-free medium was changed and cultured for another 24 h. 2 μL of 6 mol / L phenylacetylglycine solution was added to the 6-well plate. The serum-free medium was changed every 24 h and 2 ul of 6 mol / L phenylacetylglycine was added. The proportion of dimethyl sulfoxide in the culture medium should not exceed 1‰. The cells were collected after 72 h.

[0014] In another aspect, the present invention provides a phenylacetylglutamine-induced human umbilical artery vascular smooth muscle cell aging model or a phenylacetylglycine-induced mouse vascular smooth muscle cell aging model obtained by the above method.

[0015] In another aspect, the present invention provides the use of phenylacetylglutamine or phenylacetylglycine in establishing a vascular smooth muscle cell aging model.

[0016] The beneficial effects of adopting the above technical solution are: The present invention is the first to use phenylacetylglutamine / phenylacetylglycine to induce the establishment of a vascular smooth muscle cell aging model. The method is easy to operate and low in cost, laying the foundation for studying the relationship between cardiovascular disease and aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figures are the staining intensity graphs of SA-β-gal staining in the human umbilical artery vascular smooth muscle cell aging model constructed by the present invention, wherein (A) is the staining graph; (B) is the staining intensity statistical graph; Figure 2 The diagram shows the proportion of SA-β-gal-stained positive cells in the mouse vascular smooth muscle cell aging model constructed in the present invention, wherein (A) is a staining diagram; and (B) is a statistical diagram. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0019] Example 1 Preparation of phenylacetylglutamine mother solution: Use dimethyl sulfoxide to prepare phenylacetylglutamine solution with a concentration of 6 mol / L, which is the mother solution.

[0020] Passaging and culture of primary human umbilical artery smooth muscle cells: Under normal circumstances, primary human umbilical artery smooth muscle cells are cultured in primary smooth muscle cell culture medium containing 10% FBS and 1% penicillin-streptomycin. When the cell density reaches above 80%, the old culture medium is discarded and 1 mL of 0.25% trypsin is added to the cell culture flask to wash the cells once to remove the old culture medium that was not discarded. Then, 1 mL of trypsin is added to the culture flask again and digested for an appropriate amount of time (until the cells are digested and separated from the cell culture flask). The trypsin is discarded and 3 mL of culture medium is added to mix the cells by pipetting to form a cell suspension. The cells are then passaged at a ratio of 1:3 or used for subsequent experiments.

[0021] Cell treatment before SA-β-gal staining: Take the cell suspension after cell digestion and resuspend it, count it with a cell counting plate, and then dilute the cell suspension to a cell density of 1×10 6 , take 2 mL and add it to a 6-well plate, wherein 10% FBS and 1% penicillin-streptomycin were added to the primary smooth muscle cell culture medium. After the cells adhered for 24 hours, the serum-free medium was changed and cultured for another 24 hours. 2 μL of 6 mol / L phenylacetylglutamine solution was added to the 6-well plate. The serum-free medium was changed every 24 hours, and 2 μL of 6 mol / L phenylacetylglutamine stock solution was added. The proportion of dimethyl sulfoxide in the culture medium should not exceed 1‰. After 72 hours, the cells were collected for SA-β-gal staining.

[0022] Effect Example 1 The operation was performed according to the instructions of the SA-β-gal staining kit. Compared with the control group, phenylacetylglutamine can significantly increase the staining intensity of SA-β-gal staining in aged human umbilical artery vascular smooth muscle cells. Figure 1 .

[0023] Example 2 Passaging and culture of mouse vascular smooth muscle cells: Under normal circumstances, mouse vascular smooth muscle cells are cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin. When the cell density reaches above 80%, the old medium is discarded and 1 mL of 0.25% trypsin is added to the cell culture flask to wash the cells once to remove the old medium that has not been discarded. Then, 1 mL of trypsin is added to the culture flask again and digested for an appropriate amount of time (until the cells are digested and separated from the cell culture flask). The trypsin is discarded and 3 mL of culture medium is added to mix the cells by pipetting to form a cell suspension. The cells are then passaged at a ratio of 1:4 or used for subsequent experiments.

[0024] Cell treatment before SA-β-gal staining: Take the cell suspension after cell digestion and resuspend it, count it with a cell counting plate, and then dilute the cell suspension to a cell density of 1×10 6 , take 2 mL and add it to a 6-well plate, wherein 10% FBS and 1% penicillin-streptomycin were added to the culture medium of mouse vascular smooth muscle cells. After the cells adhered for 24 hours, the serum-free medium was changed and cultured for another 24 hours. 2 μL of 6 mol / L phenylacetylglycine solution was added to the 6-well plate, and the serum-free medium was changed every 24 hours, and 2 ul of 6 mol / L phenylacetylglycine solution was added. The proportion of dimethyl sulfoxide in the culture medium should not exceed 1‰. After 72 hours, the cells were collected for SA-β-gal staining.

[0025] Effect Example 2 The operation was performed according to the instructions of the SA-β-gal staining kit. Compared with the control group, phenylacetylglycine can significantly increase the proportion of SA-β-gal positive cells in vascular smooth muscle cells of aged mice. Figure 2 .

[0026] The primary smooth muscle cell culture medium used in this example was purchased from Guangzhou Xinyuan Biotechnology Co., Ltd., Bios primary smooth muscle cell culture system PriMed-Bios-004; DMEM high glucose medium was purchased from Gibco, product number 12800-017 10*1L.

[0027] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine, characterized in that: It includes the following steps: S1: Prepare phenylacetylglutamine solution and phenylacetylglycine solution using dimethyl sulfoxide. S2: Place a vascular smooth muscle cell suspension in a multiwell plate. Add 10% FBS and 1% penicillin-streptomycin to the culture medium. After the cells have adhered for 24 hours, switch to serum-free medium and continue culturing for another 24 hours. Add an appropriate amount of phenylacetylglutamine or phenylacetylglycine solution to the multiwell plate. Change the medium with phenylacetylglutamine or phenylacetylglycine every 24 hours. Collect the cells after 72 hours.

2. The method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine according to claim 1, characterized in that: The concentration of the phenylacetylglutamine solution and the phenylacetylglycine solution in step S1 is 6 mol / L.

3. The method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine according to claim 1, characterized in that: The vascular smooth muscle cells in step S2 are human umbilical artery vascular smooth muscle cells or mouse vascular smooth muscle cells; when the vascular smooth muscle cells are human umbilical artery vascular smooth muscle cells, phenylacetylglutamine is used for induction; when the vascular smooth muscle cells are mouse vascular smooth muscle cells, phenylacetylglycine is used for induction.

4. The method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine according to claim 1, characterized in that: The proportion of dimethyl sulfoxide in the culture medium should not exceed 1‰.

5. The method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine according to claim 1, characterized in that: The step S2 is to take the cell density of 1×10 6 2 mL of human umbilical artery vascular smooth muscle cell suspension was placed in a 6-well plate.

6. The method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine according to claim 1, characterized in that: In step S2, an appropriate amount of phenylacetylglutamine or phenylacetylglycine solution is added to the multi-well plate, an appropriate amount being 2 µL.

7. The method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine according to claim 1, characterized in that: The step S2 is to take the cell density of 1×10 6 2 mL of human umbilical artery vascular smooth muscle cell suspension was placed in a 6-well plate. 10% FBS and 1% penicillin-streptomycin were added to the primary smooth muscle cell culture medium. After the cells adhered for 24 hours, the serum-free medium was changed and cultured for another 24 hours. 2 μL of 6 mol / L phenylacetylglutamine solution was added to the 6-well plate. The serum-free medium was changed every 24 hours and 2 μL of 6 mol / L phenylacetylglutamine stock solution was added. The proportion of dimethyl sulfoxide in the culture medium should not exceed 1‰. The cells were collected after 72 hours.

8. The method for establishing a vascular smooth muscle cell aging model induced by phenylacetylglutamine or phenylacetylglycine according to claim 1, characterized in that: The step S2 is to take the cell density of 1×10 6 2 mL of mouse vascular smooth muscle cell suspension was placed in a 6-well plate, in which 10% FBS and 1% penicillin-streptomycin were added to DMEM high-glucose medium. After the cells adhered for 24 h, serum-free medium was changed and cultured for another 24 h. 2 μL of 6 mol / L phenylacetylglycine solution was added to the 6-well plate. The serum-free medium was changed every 24 h and 2 ul of 6 mol / L phenylacetylglycine was added. The proportion of dimethyl sulfoxide in the culture medium should not exceed 1‰. The cells were collected after 72 h.

9. A phenylacetylglutamine-induced human umbilical artery vascular smooth muscle cell senescence model or a phenylacetylglycine-induced mouse vascular smooth muscle cell senescence model obtained by the method according to any one of claims 1 to 8.

10. Application of phenylacetylglutamine or phenylacetylglycine in establishing a vascular smooth muscle cell aging model.

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