Early atherosclerosis cell model as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biomedical experimental model construction, and particularly relates to an early atherosclerosis cell model and a construction method and application thereof, and the construction method comprises the following steps: using low density lipoprotein (LDL) to synthesize enzyme modified low density lipoprotein (ELDL) under the treatment of trypsin and cholesterol esterase; the ELDL and a tumor necrosis factor alpha (TNF-alpha) are mixed into a culture medium to culture cells, so that the early atherosclerosis model is constructed. The early atherosclerosis blood vessel model constructed by the method is very close to real pathological development and environment in vivo.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical experimental model construction, and in particular relates to an early atherosclerosis cell model and a construction method and application thereof. Background Art

[0002] Cardiovascular disease has always been the disease category with the highest morbidity and mortality worldwide. It is estimated that more than 17 million people die from cardiovascular disease each year worldwide. Atherosclerosis (AS) is the main pathological basis of cardiovascular disease, covering ischemic heart disease, coronary heart disease, acute myocardial infarction and cerebrovascular disease, etc., which seriously threatens human health. Therefore, in-depth research on the pathogenesis of atherosclerosis, the development of drug treatment plans to prevent its early occurrence, and the establishment of new accurate and efficient drug screening methods are of vital importance for clinical treatment.

[0003] The causes and pathogenesis of AS are extremely complex, involving multiple aspects such as inflammatory response, immune system function, and lipid metabolism disorders. Its pathological characteristics include lipid accumulation, thickening and loss of elasticity of the arterial wall, and increased inflammatory factors. In the early stage, due to the thickening of the arterial intima and the disorder of lipid metabolism in the body, LDL will be retained in the arterial intima and modified by enzymes to produce ELDL. ELDL can activate the complement system in the early pathological stage, promote inflammatory response, and trigger cell uptake and metabolism through various pathways, thereby accelerating the formation of foam cells. Vascular endothelial cells (ECs) are a barrier between blood and vascular walls, responsible for material exchange, and regulate vascular function such as vascular tension, inflammatory response, blood pressure, and coagulation and anticoagulation balance by secreting a variety of bioactive substances. When ECs are stimulated by ELDL, they express adhesion molecules such as ICAM-1, PECAM-1, P-selectin, and E-selectin, promote the adhesion of monocytes and lymphocytes to ECs, and enhance the inflammatory response. In addition, after smooth muscle cells (SMCs) take up ELDL, lipids accumulate in the cells to form lipid droplets mainly composed of cholesterol and fatty acids, which is one of the hallmarks of foam cell formation. Existing research mainly focuses on the late AS stage, and there are few methods for constructing early AS models. Therefore, the present invention provides a method for constructing an early atherosclerosis cell model, and provides an in vitro simulation test method for preventive treatment drugs and detection. Summary of the invention

[0004] The purpose of the present invention is to provide an early atherosclerosis cell model and a construction method and application thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for constructing an early atherosclerosis cell model comprises the following steps:

[0007] The enzymatically modified low-density lipoprotein (ELDL) was synthesized using low-density lipoprotein (LDL) under the treatment of trypsin and cholesterol esterase. The synthesis and verification of ELDL were obtained through the following steps:

[0008] S1. Synthesis of enzymatically modified low-density lipoprotein ELDL: Add 7 μg of trypsin to 1 mg of LDL protein and incubate at 37 °C for 6 h, then add 12 μg of cholesterol esterase and incubate at 37 °C for 14 - 18 h; then, add 24 μg of trypsin and incubate at 37 °C for 6 h, and then add 24 μg of cholesterol esterase and incubate at 37 °C for 46 - 50 h. After that, dialyze with phosphate buffer for 24 h, filter and sterilize, and store at 4 °C for later use, where the molecular weight of the dialysis bag is 100 kDa;

[0009] S2. Construction of an early atherosclerotic cell model: Mix ELDL and TNF-α into the cell culture medium, and place normal cells (i.e., endothelial cells, smooth muscle cells, and macrophages) in the culture medium and treat for 24 h to obtain an early atherosclerotic cell model.

[0010] Furthermore, the concentrations of ELDL and TNF-α in the culture medium are 10 - 55 μg / ml and 1.5 - 10.2 ng / mL, respectively.

[0011] Furthermore, the particle size of the synthesized ELDL is 100 - 200 μm.

[0012] Furthermore, the concentration of trypsin is 4 - 15 μg / ml.

[0013] Furthermore, the concentration of cholesterol esterase is 20 - 40 μg / ml.

[0014] An early atherosclerotic cell model is constructed by the above construction method.

[0015] An application of an early atherosclerotic cell model, which is used in the preventive treatment drugs and detection of early atherosclerosis.

[0016] The advantages of the present invention are: The early atherosclerotic cell model constructed by the present invention conforms to the law of lesion occurrence and can effectively screen and test candidate drugs for early atherosclerosis in vitro. Description of the Drawings

[0017] Figure 1 It is the preparation and characterization diagram of ELDL in Example 1 of the present invention.

[0018] Figure 2Oil Red O staining results of smooth muscle cells and macrophages under different treatment conditions in Example 1 of the present invention.

[0019] Figure 3 Adhesion results of ECs to THP-1 and MCs under different treatment conditions in Example 1 of the present invention.

[0020] Figure 4 Results of the ELDL+TNF-α combination on endothelial cell modeling in Example 2 of the present invention.

[0021] Figure 5 Results of the ELDL+TNF-α combination on smooth muscle cell modeling in Example 2 of the present invention.

[0022] Figure 6 Results of the ELDL+TNF-α combination on macrophage modeling in Example 2 of the present invention. Detailed implementation manners

[0023] The technical solutions of the present invention will be further described and illustrated below through specific implementation manners in conjunction with the accompanying drawings.

[0024] The experimental materials and reagents in the following examples are specifically as follows:

[0025]

[0026]

[0027] Example 1 Preparation and characterization of ELDL

[0028] Preparation of ELDL: Add 7 μg of trypsin to every 1 mg of LDL protein and incubate at 37 °C for 6 h, then add 12 μg of cholesterol esterase and incubate at 37 °C for 16 h; then add 24 μg of trypsin and incubate at 37 °C for 6 h, and then add 24 μg of cholesterol esterase and incubate for 48 h. After incubation, the prepared ELDL appears turbid, and then it is fully dialyzed (cut-off molecular weight is 100 KDA) with phosphate buffer (PBS) for 24 h. The ELDL is filtered and sterilized, and its final protein concentration is measured using a BCA protein detection kit / Lowry method, and then it is stored in an environment at 4 °C and can be stored for up to 3 months.

[0029] B. Morphological and functional verification of ELDL: First, morphologically, the solution of ELDL becomes turbid. Meanwhile, the particle size is characterized by a nano particle size and Zeta potential analyzer, and the particle diameter increases from 10 - 20 μm to 100 - 200 μm. Since ELDL has a strong ability to promote foam cell formation, to test this, bovine serum albumin (BSA, as a control), LDL, ox - LDL, and ELDL were added to smooth muscle cells (SMCs) and macrophages (MCs) at specified doses (20, 50, 100, 200 μg / ml) and incubated at 37 °C for 24 h. The formation of foam cells was visually evaluated using an Oil Red O staining kit. Figure 2 It can be visually seen that at the same concentration, ELDL has a stronger ability to promote foam cell generation. In addition, ECs treated with ELDL will express ICAM - 1 and VCAM - 1, making it easier for THP - 1 and MCs to adhere to the surface of damaged ECs. To verify this, ECs were seeded in 12 - well plates at a density of 75,000 cells per well, cultured for 2 days, and then treated with 50 μg / ml ELDL for 24 h. Subsequently, THP - 1 cells were labeled with Calcein - AM and added to the 12 - well plates at a density of 3×10 8 cells per well. After 1 h, bright - field and fluorescence images were obtained before and after washing with growth medium, and the number of cells was analyzed and counted. Figure 3 The results in [[]] show that more THP - 1 and MCs can adhere to the surface of ECs treated with ELDL alone. In summary, the successful synthesis of ELDL was verified.

[0030] Example 2 Construction of an early atherosclerotic endothelial cell model

[0031] ECs were treated with a medium containing 50 μg / ml ELDL and 10 ng / ml TNF - α for 24 h, and the cells were subjected to corresponding verification ( Figure 4 ).

[0032] ECs were seeded at a density of 3×10 5 cells per well into 24 - well plates pre - coated with coverslips. After 1 day of adherent cell culture, 50 μg / ml ELDL and 10 ng / ml TNF - α were added and treated for 24 h. Subsequently, a reactive oxygen species (ROS) detection kit was used to confirm that the ECs were damaged. Finally, images were collected using a confocal laser scanning microscope (CLSM) and analyzed using ImageJ software. The results showed that there was a significant difference in ROS between the ECs group induced by ELDL + TNF - α and the control group, proving that ECs damage was successfully induced.

[0033] Construction of an early atherosclerotic smooth muscle cell model

[0034] Treat SMCs with a medium containing 50 μg / ml ELDL and 10 ng / ml TNF-α for 24 h, and perform corresponding verification on the cells ( Figure 5 ).

[0035] In AS, SMCs will undergo phenotypic changes, transforming from a proliferative type to a migratory type, specifically manifested as enhanced migratory ability. Take 500 μl of SMC medium containing 10% FBS (50% 1640 medium + 40% SMCM medium + 10% FBS) and place it in a 24-well plate. Then gently place a Transwell chamber (PET material) with a pore size of 8 μm on it. Resuspend SMCs with SMC medium without FBS (50% 1640 medium + 40% SMCM medium), and take 200 μl of cells with a density of 2.5×10 5 cells / ml and place them in the upper chamber of the Transwell. Incubate for 4 hours. Subsequently, gently scrape off the cells on one side of the upper chamber of the Transwell membrane with a cotton swab, and place the Transwell chamber (with cells on the lower layer of the membrane) in 10% formalin for fixation for 10 minutes. Gently rinse the fixed Transwell chamber twice with PBS, and then incubate it in 0.5% crystal violet solution for 10 minutes. Gently wash the cells with PBS several times until the background on the membrane is clean. Completely dry the Transwell chamber, cut the membrane from the Transwell chamber with a blade, and place it on a glass slide for microscopic observation. Use ImageJ software to collect images to count the number of SMCs, and use GraphPad for analysis. The results show that the number of migratory cells in the SMCs group induced by ELDL + TNF-α is more than that in the control group, proving the successful induction of SMC phenotypic transformation.

[0036] Construction of an early atherosclerotic macrophage model

[0037] Treat MCs with a medium containing 50 μg / ml ELDL and 10 ng / ml TNF-α for 24 h, and perform corresponding verification on the cells ( Figure 6 ).

[0038] In AS, M1 macrophages are pro-inflammatory and express CD86; M2 macrophages are anti-inflammatory and express CD206. First, THP-1 cells were treated with M0 medium (95% 1640 medium + 5% FBS) containing 100 ng / ml phorbol myristate acetate (PMA) for 48 h. THP-1 cells were seeded at 200,000 cells per well, 1 ml per well, in 12-well plates. Under the microscope, it was observed that the cells changed from suspension cells to adherent cells. Then, after changing to M0 medium (95% 1640 medium + 5% FBS) and culturing for 24 h, M0 medium containing 50 μg / ml ELDL and 10 ng / ml TNF-α was added and cultured for 24 h. The induced M0 cells were fixed in 10% formalin for 10 minutes. Gently rinsed twice with PBS, then blocked with 5% BSA solution, and gently rinsed twice with PBS again; 200 μl of the primary antibody mixture was added to the wells, which contained 1 μg / ml Anti-CD86 antibody and 1 μg / ml Anti-Mannose Receptor antibody, and incubated overnight at 4 °C in a refrigerator. Then, the secondary antibody Alexa Fluor® 488 for detecting CD86 and the secondary antibody Alexa Fluor® 647 for detecting CD206 were added. After incubation for 2 h, DAPI was added for final nuclear staining. Confocal microscopy was used to take pictures, and ImageJ software was used for statistical analysis. The results showed that the MCs group induced by ELDL + TNF-α expressed CD86, indicating the transformation of M0 macrophages into M1 macrophages.

Claims

1. A method for constructing an early atherosclerosis cell model, characterized in that, It includes the following steps: S1. Synthesis of enzyme-modified low-density lipoprotein (ELDL): Add 7 μg of trypsin to 1 mg of LDL protein, incubate at 37°C for 6 h, then add 12 μg of cholesterol esterase and incubate at 37°C for 14 - 18 h; then, add 24 μg of trypsin and incubate at 37°C, after 6 h, add 29 μg of cholesterol esterase and incubate at 37°C for 46 - 50 h. After that, dialyze with phosphate buffer for 24 h, filter and sterilize, and store for standby in an environment at 4°C. The molecular weight of the dialysis bag is 100 kDa; S2. Construction of an early atherosclerosis cell model: Mix ELDL and TNF-α into the cell culture medium, and place normal cells in the culture medium for treatment for 24 h to obtain an early atherosclerosis cell model.

2. The method for constructing an early atherosclerosis cell model according to claim 1, characterized in that: The concentrations of ELDL and TNF-α in the culture medium are 10 - 55 μg / ml and 1.5 - 10.2 ng / mL in sequence.

3. The method for constructing an early atherosclerosis cell model according to claim 1, wherein: The particle size of the synthesized ELDL is 100 - 200 μm.

4. The method for constructing an early atherosclerosis cell model according to claim 1, characterized in that: The concentration of the trypsin is 4 - 15 μg / ml.

5. The method for constructing an early atherosclerosis cell model according to claim 1, characterized in that: The concentration of the cholesterol esterase is 20 - 40 μg / ml.

6. An early atherosclerosis cell model, characterized in that: It is constructed by using the construction method described in any one of claims 1 - 5.

7. The application of the cell model according to claim 6, wherein: It is used in the preventive treatment drugs and detection of early atherosclerosis.

Images