CN114381421B - Lysozyme-induced mouse vascular smooth muscle cell inflammation model and establishment method and application thereof - Google Patents
Lysozyme-induced mouse vascular smooth muscle cell inflammation model and establishment method and application thereof Download PDFInfo
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Abstract
The invention relates to a lysozyme-induced mouse vascular smooth muscle cell inflammation model and an establishment method and application thereof, which comprises the following steps: (1) Continuously incubating lysozyme for 4 days by using PBS with the pH value of 7.35-7.45 at the temperature of 37+/-1 ℃ of an incubator; (2) Preparing a high sugar culture medium containing 1% FBS and 1% lysozyme solution after incubation as a stimulating solution; (3) The cell density was taken to be 2.3X10 6 2ml of mouse vascular smooth muscle cell suspension is placed on a 60mm culture dish, 2ml of 10% FBS-containing culture medium is added, uniformly mixed, adhered for 24 hours, starved for 24 hours, liquid is changed, old liquid is discarded, 4ml of stimulation liquid is taken and added into the 60mm dish, the mixture is placed in an incubator for 24 hours, and cells are collected, so that the model of mouse vascular smooth muscle cell inflammation is built by using lysozyme induction for the first time, the operation is easy, the cost is low, and a foundation is laid for researching the relation between cardiovascular diseases and inflammation.
Description
Technical Field
The invention belongs to the technical field of cell inflammation models, and particularly relates to a lysozyme-induced mouse vascular smooth muscle cell inflammation model, and an establishment method and application thereof.
Background
Phosphodiesterases (PDEs) are the only intracellular hydrolases of cyclic nucleotides (cAMP and cGMP). Phosphodiesterase 4 (PDE 4) is one of the important family members of phosphodiesterases, PDE4 has the function of specifically hydrolyzing the intracellular second messenger cyclic adenosine monophosphate (cAMP), and hydrolyzes intracellular cAMP to form inactive 5' nucleotides, thereby blocking its mediated physiological functions, and cAMP-PDEs are therefore recognized as new targets for the treatment of inflammation.
cAMP is an important second messenger involved in a variety of physiological processes in the body, such as inflammatory responses, immune responses, division and differentiation of cells, neurotransmitter delivery, and synaptic plasticity. cAMP is involved in inflammatory reactions, exudation and fibrosis; elevation of cAMP levels can inhibit and regulate inflammation. Inflammation is the basis of the pathogenesis of many vascular diseases. Thus, inhibition of intracellular PDE4 can raise cAMP levels and reduce the occurrence of inflammation.
Vascular smooth muscle cells are used as the main structure of the blood vessel wall medium membrane and have important functions for maintaining the normal physiological functions of blood vessels. A variety of factors such as abnormal lipid metabolism, endothelial injury, hemodynamic injury, and physicochemical injury may lead to chronic inflammation of blood vessels. Inflammatory response is the pathological basis and key link of numerous cardiovascular diseases. Vascular smooth muscle cells are involved in pathophysiological processes of various cardiovascular diseases including atherosclerosis, and prevention and treatment of vascular smooth muscle cell inflammation have become an important field of medical research. cAMP participates in inflammatory reaction, and can inhibit ERK and NF-KB channels, thereby further inhibiting expression of adhesion molecules and release of pro-inflammatory factors. PDE4 is one of the specific hydrolases of cAMP, thereby modulating various aspects of vascular smooth muscle. Elevation of cAMP levels can inhibit and regulate inflammation. Therefore, cAMP-PDEs become an important target for treating inflammation.
Lysozyme, also known as muramidase or N-acetylmuramidase, is a small monomeric protein widely distributed in nature, and the lysozyme with a normal structure has a protective effect on organisms. However, the secondary structure of lysozyme through in vitro incubation is changed compared with that of normal lysozyme, so that the normal biological function of lysozyme is changed.
At present, methods for inducing a mouse vascular smooth muscle inflammation model by using angiotensin II and Lipopolysaccharide (LPS) and inducing the mouse vascular smooth muscle inflammation model by using lysozyme are not reported.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a lysozyme-induced mouse vascular smooth muscle cell inflammation model, and simultaneously provides a method for establishing the same and application of lysozyme in establishing the mouse vascular smooth muscle cell inflammation model.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a method for establishing a lysozyme-induced mouse vascular smooth muscle cell inflammation model, which comprises the following steps:
(1) Continuously incubating lysozyme for 4 days by using PBS with pH of 7.35-7.45 at 37+/-1 ℃ of an incubator, wherein the concentration of the lysozyme is 1 multiplied by 10 -4 mol/L；
(2) Preparing a high sugar culture medium containing 1% FBS and 1% lysozyme solution after incubation as a stimulating solution (1% refers to volume ratio);
(3) The cell density was taken to be 2.3X10 6 2ml of mouse vascular smooth muscle cell suspension is placed on a 60mm culture dish, 2ml of 10% FBS medium is added, evenly mixed, adhered for 24 hours, starved for 24 hours, liquid is changed, old liquid is discarded, 4ml of stimulation liquid is taken and added into the 60mm dish, and the temperature is 37+/-1 ℃ and the concentration of CO is 5+/-1% 2 Placing in an incubator with 95% relative humidity for 24 hours, and collecting cells.
As some preferred embodiments of the present invention, the cell density of the mouse vascular smooth muscle cell suspension in the step (3) is 2.3X10 6 。
The invention also provides a lysozyme-induced mouse vascular smooth muscle cell inflammation model obtained by the establishment method.
In a further aspect, the invention provides the use of lysozyme in the establishment of a model of vascular smooth muscle cell inflammation in mice.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in:
the invention establishes the model of the mouse vascular smooth muscle cell inflammation by using lysozyme for the first time, has easy operation and low cost, and lays a foundation for researching the relationship between cardiovascular diseases and inflammations.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows PDE4 levels in a mouse vascular smooth muscle cell inflammation model constructed in accordance with the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be clearly and completely described in connection with the following specific embodiments.
Example 1
Lysozyme was incubated continuously with PBS at ph=7.4 at 37 ℃ in an incubator for 4 days at a concentration of 1×10 lysozyme -4 mol/L。
A high sugar medium containing 1% FBS and 1% post-incubation lysozyme solution was formulated as a stimulating solution.
Preparation of cell suspension: when the vascular smooth muscle cells of the mice are attached to more than 90%, 0.25% trypsin is digested, when the cells are slightly spherical, pancreatin is poured out, 4ml of 10% FBS culture medium is added to stop digestion, and the cells are blown to prepare cell suspension.
The cell density was taken to be 2.3X10 6 2ml of the vascular smooth muscle cell suspension of the mice is placed on a culture dish with the thickness of 60mm, 2ml of a culture medium containing 10% FBS is added, the mixture is uniformly mixed, the mixture is adhered to the wall for 24 hours, starvation is carried out for 24 hours, and liquid exchange and stimulation are carried out. 4ml of the stimulating solution was added to a 60mm dish and the incubator was left for 24 hours.
Effect example 1
The procedure was as per the instructions of the mouse enzyme-linked immunosorbent phosphodiesterase 4 (PDE 4) kit. The lysozyme after incubation significantly increased intracellular PDE4 levels compared to the placebo group, see in particular fig. 1.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (1)
1. A method for establishing a lysozyme-induced vascular smooth muscle cell inflammation model of a mouse, comprising the steps of:
(1) Continuously incubating lysozyme for 4 days by using PBS with pH of 7.35-7.45 at 37+/-1 ℃ of an incubator, wherein the concentration of the lysozyme is 1 multiplied by 10 -4 mol/L；
(2) Preparing a high-sugar culture medium containing 1% FBS and 1% lysozyme solution after incubation as a stimulating solution;
(3) Placing 2ml of mouse vascular smooth muscle cell suspension on a 60mm culture dish, adding 2ml of 10% FBS-containing culture medium, mixing, adhering to the wall for 24 hr, starving for 24 hr, changing liquid, discarding old liquid, adding 4ml of stimulating liquid into the 60mm dish, adding 5+ -1% CO at 37+ -1deg.C 2 Placing in an incubator with 95% relative humidity for 24 hours, and collecting cells.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1583167A (en) * | 2004-06-10 | 2005-02-23 | 张华� | Use of recommbined human lysozyme in preparation of medicine for bronchial asthma |
| CN104726393A (en) * | 2013-12-24 | 2015-06-24 | 江苏省农业科学院 | Preparation method of TNF-alpha-induced human umbilical vein endothelial cell HUVEC inflammatory reaction model |
| CN108210513A (en) * | 2017-12-12 | 2018-06-29 | 浙江海洋大学 | A kind of oligomeric glycosaminoglycan of selenizing influences ulcerative colitis mouse the method for building up of model |
| WO2019018441A1 (en) * | 2017-07-17 | 2019-01-24 | Massachusetts Institute Of Technology | Cell atlas of healthy and diseased barrier tissues |
| CN110327431A (en) * | 2019-07-30 | 2019-10-15 | 上海市农业科学院 | Application and drug of the Germinatus Phragmitis extract in the drug of preparation treatment inflammation |
| CN111789835A (en) * | 2020-06-05 | 2020-10-20 | 遵义医科大学 | Medicine for resisting atherosclerosis inflammation and anti-inflammatory action detection method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011526619A (en) * | 2008-06-30 | 2011-10-13 | サイレンシード リミテッド | Topical drug delivery system, method and composition thereof |
| AU2009271598A1 (en) * | 2008-07-15 | 2010-01-21 | Briannan Bintz | Wound healing |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1583167A (en) * | 2004-06-10 | 2005-02-23 | 张华� | Use of recommbined human lysozyme in preparation of medicine for bronchial asthma |
| CN104726393A (en) * | 2013-12-24 | 2015-06-24 | 江苏省农业科学院 | Preparation method of TNF-alpha-induced human umbilical vein endothelial cell HUVEC inflammatory reaction model |
| WO2019018441A1 (en) * | 2017-07-17 | 2019-01-24 | Massachusetts Institute Of Technology | Cell atlas of healthy and diseased barrier tissues |
| CN108210513A (en) * | 2017-12-12 | 2018-06-29 | 浙江海洋大学 | A kind of oligomeric glycosaminoglycan of selenizing influences ulcerative colitis mouse the method for building up of model |
| CN110327431A (en) * | 2019-07-30 | 2019-10-15 | 上海市农业科学院 | Application and drug of the Germinatus Phragmitis extract in the drug of preparation treatment inflammation |
| CN111789835A (en) * | 2020-06-05 | 2020-10-20 | 遵义医科大学 | Medicine for resisting atherosclerosis inflammation and anti-inflammatory action detection method |
Non-Patent Citations (1)
| Title |
|---|
| Yun Huang.Lysozyme Improves the Inhibitory Effects of Panax notoginseng Saponins on Phenotype Transformation of Vascular Smooth Muscle Cells by Binding to Ginsenoside Re.Front Nutr.2021,第8卷全文. * |
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