Construction method of coronary microcirculation disturbance cell model

Abstract

The invention discloses a construction method of a coronary artery microcirculation disturbance cell model, and belongs to the technical field of biological medicine. According to the method, mouse heart microvascular endothelial cells (MCMEC) are taken as objects, tetrachlorohydroquinone (TCHQ) is used for treating for 6 hours at the concentration of 20-40 [mu] M, and CMD typical characteristics such as apoptosis, increase of reactive oxygen species (ROS), reduction of nitric oxide (NO) and release of inflammatory factors (TNF-alpha and IL-1beta) of the cells are induced. The TCHQ is applied to construction of the CMD model for the first time, and the model is stable, good in repeatability and capable of being used for CMD pathogenesis research and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for constructing a cell model of coronary microcirculation disorder. Background Technology

[0002] Coronary microcirculatory disturbance (CMD) is a significant cause of myocardial ischemia, with an incidence rate as high as 45%-60% in patients with normal coronary angiography. Current CMD research models primarily rely on animal models, but these suffer from high costs and ethical controversies. While cell models have some applications, a stable induction method that can mimic the multiple pathological features of CMD is lacking. Tetrachlorohydroquinone (TCHQ), a metabolite of pentachlorophenol, has been studied for its effects on DNA damage and neurotoxicity, but its application in cardiovascular disease models has not been observed. Therefore, developing a TCHQ-based CMD cell model is of significant value. Summary of the Invention

[0003] The purpose of this invention is to provide a method for constructing a cell model of coronary microcirculation disorder.

[0004] The objective of this invention is achieved by the following steps:

[0005] (a) Culture of mouse cardiac microvascular endothelial cells;

[0006] (b) The target model can be obtained by treating the cells with tetrachlorohydroquinone at a concentration of 20-40 μM for 6 hours.

[0007] The concentration of the tetrachlorohydroquinone was 40 μM.

[0008] The preparation method of tetrachlorohydroquinone used in step (b) is as follows: Under aseptic conditions, add 2 mL of dimethyl sulfoxide (DMSO) to 50 mg of tetrachlorohydroquinone, dissolve it completely, prepare a 100 mM tetrachlorohydroquinone stock solution, filter it with a 0.22 μm cell filter for sterilization, and then use it for later use.

[0009] In step (a), mouse cardiac microvascular endothelial cells were cultured in complete endothelial cell culture medium and placed in a 5% CO2, 37°C incubator.

[0010] Compared with the prior art, the present invention has the following technical effects:

[0011] Tetrachlorohydroquinone (TCHQ) is the main active metabolite of pentachlorophenol, a major component of persistent organic pollutants, in the body, and its effects on DNA damage and neurotoxicity have been studied. However, no reports have been found regarding TCHQ's effects on cardiovascular diseases. This invention is the first to use TCHQ to construct a cell model of coronary microcirculatory disturbance (CMD). The cells used were mouse cardiac microvascular endothelial cells (MCMECs). A stable CMD cell model was established by using TCHQ at a concentration of 20-40 μM and an action time of 6 hours. Attached Figure Description

[0012] Figure 1 Comparison of the effects of different concentrations of TCHQ on the viability of MCMEC cells;

[0013] Figure 2 Comparison of flow cytometry results of different concentrations of TCHQ on apoptosis in MCMEC cells;

[0014] Figure 3 A bar chart comparing the effects of different concentrations of TCHQ on apoptosis in MCMEC cells;

[0015] Figure 4 Comparison of flow cytometry results of different concentrations of TCHQ on ROS (reactive oxygen species) levels in MCMEC cells;

[0016] Figure 5 A bar chart comparing the effects of different concentrations of TCHQ on ROS (reactive oxygen species) levels in MCMEC cells;

[0017] Figure 6 A bar chart comparing the effects of different concentrations of TCHQ on NO (nitric oxide) release in MCMEC cells;

[0018] Figure 7 A bar chart comparing the effects of different concentrations of TCHQ on TNF-α (tumor necrosis factor) release in MCMEC cells;

[0019] Figure 8 A bar chart comparing the effects of different concentrations of TCHQ on IL-1β (interleukin) release in MCMEC cells. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0021] 1. Cell Information

[0022] Mouse cardiac microvascular endothelial cells (MCMEC) were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0023] 2. Main reagents and consumables

[0024] Reagent Name company Tetrachlorohydroquinone Mce Endothelial cell culture medium ScienCell Trypsin (0.25%) Gibco CCK-8 reagent kit Azure Sky Annexin V-FITC Apoptosis Detection Kit Azure Sky Reactive oxygen species detection kit Azure Sky Nitric Oxide (NO) Content Detection Kit Azure Sky Mouse Tumor Necrosis Factor α (TNF-α) ELISA Kit Jianglai Biotechnology Mouse interleukin-1β (IL-1β) ELISA kit Jianglai Biotechnology

[0025] 3. Main Instruments

[0026] Instrument Name company Cell incubator Thermo Fisher Scientific 3141 Clean bench Haier HCB-1300V Inverted microscope OLYMPUS BX53 Flow cytometer ACEA Novocyte 2060R Multifunctional ELISA reader Feyond-A300 microscope OLYMPUS BX53

[0027] 4. Preparation of reagents

[0028] Preparation of Tetrachlorohydroquinone (TCHQ): Under aseptic conditions, add 2 mL of dimethyl sulfoxide (DMSO) to 50 mg of TCHQ, dissolve thoroughly to prepare a 100 mM TCHQ stock solution, filter sterilize using a 0.22 μm cell filter and use for later use.

[0029] 5. Experimental Methods

[0030] 5.1 Cell Culture

[0031] MCMEC cells were cultured in complete endothelial cell culture medium and placed in a 5% CO2, 37°C incubator.

[0032] 5.2 TCHQ Concentration Screening

[0033] Take a suspension of MCMEC cells in the logarithmic growth phase and adjust the cell concentration to 5 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / mL into each well of a 96-well plate and incubated overnight at 37°C in a 5% CO2 incubator to allow cell adhesion. The old culture medium was discarded, and 100 μL of medium containing different concentrations of TCHQ (0, 3.125, 6.25, 12.5, 25, 50, 100 μM) was added to each well according to experimental requirements, with three replicates for each concentration. After 6 hours of incubation, the culture medium was removed, and 100 μL of basal medium containing 10 μL of CCK-8 reagent was added to each well. The plates were then incubated in the dark for another 2 hours. After incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader to calculate cell viability. Cell viability % = (OD of experimental wells - OD of blank wells) / (OD of control wells - OD of blank wells) × 100% to determine the appropriate TCHQ concentration.

[0034] 5.3 Cell grouping and processing methods

[0035] MCMEC cells were divided into 5 groups, as follows:

[0036] ①NC group: Normally growing cells

[0037] ②Vehicle group: The concentration of TCHQ solvent in the cell culture medium was the same as that in group ⑤.

[0038] ③ 10μM TCHQ group: The final concentration of TCHQ in the cell culture medium was 10μM

[0039] ④ 20μM TCHQ group: The final concentration of TCHQ in the cell culture medium was 20μM.

[0040] ⑤ 40μM TCHQ group: The final concentration of TCHQ in the cell culture medium was 40μM.

[0041] MCMEC cells in the logarithmic growth phase were seeded at a density of 50,000 cells per well in 6-well plates and placed in an incubator. When the cell confluence reached about 60%, the medium was changed according to the above grouping and cultured for another 6 hours.

[0042] 5.4 Flow cytometry detection of apoptosis

[0043] Cells from different treatment groups were collected after trypsin digestion, resuspended in PBS, and counted. 1×10⁶ cells were collected. 5 After resuspending the cells, centrifuge and discard the supernatant, add 200 μL of Annexin V-FITC binding solution to resuspend the cells, add 5 μL of Annexin V-FITC and mix gently, then add 10 μL of PI staining solution and mix well. Incubate at room temperature in the dark for 15 min, and then perform flow cytometry analysis.

[0044] 5.5 Detection of intracellular reactive oxygen species (ROS) content using DCFH-DA fluorescent probe

[0045] Dilute DCFH-DA to a final concentration of 10 μmol / L with serum-free cell culture medium. Collect cells from different treatment groups, suspend them in 1 mL of diluted DCFH-DA, and incubate in the cell culture incubator in the dark for 20 min. Wash three times with serum-free cell culture medium, centrifuge at 350 g for 5 min, remove the supernatant, resuspend in PBS, and detect intracellular ROS using flow cytometry.

[0046] 5.6 Detection of cellular NO levels using the nitrate reductase method

[0047] After the cells were processed according to the groups, the cell supernatant was collected. The NO detection kit was operated according to the instructions, and the absorbance (OD value) of the sample was detected at 530 nm using an ELISA reader. The concentration of NO in the cell supernatant of each group was calculated.

[0048] 5.7 Detection of TNF-α and IL-1β levels in cell supernatant using ELISA

[0049] After grouping and processing, cell supernatant was collected and tested according to the kit instructions. The absorbance (OD value) of each well was measured at 450 nm using an ELISA reader, and the contents of TNF-α and IL-1β were calculated based on the standard curve.

[0050] 5.8 Data Analysis

[0051] GraphPad Prism 9.0.0 software was used for statistical analysis of the data. All experimental data are expressed as mean ± standard deviation. The data indicates that one-way ANOVA was used for comparisons among multiple groups, and LSD test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0052] 6. Experimental Results

[0053] 6.1 Effects of TCHQ on MCMEC cell viability

[0054] TCHQ significantly inhibited the cell viability of MCMEC cells in a concentration-dependent manner, with the inhibitory effect gradually increasing with increasing TCHQ concentration. The IC50 of TCHQ on MCMEC cells was calculated using Graphpad Prism 9.0.0 software, and the result was IC50 = 39.69 μM. Therefore, the TCHQ concentrations used in subsequent studies were 10 μM, 20 μM, and 40 μM.

[0055] Table 1 Effects of different concentrations of TCHQ on the viability of MCMEC cells

[0056] Experimental groups (n=3) Cell viability (%) 0 μM 101.555±3.196 3.125 μM 97.657±1.694 6.25 μM 90.922±1.571 12.5 μM 77.949±2.289 25 μM 62.781±2.15 50 μM 41.867±3.829 100 μM 27.58±4.324

[0057] 6.2 Effects of TCHQ on MCMEC cell apoptosis

[0058] Flow cytometry was used to detect the effect of different concentrations of TCHQ on apoptosis in MCMEC cells. The results showed that the apoptosis rate increased after the addition of TCHQ. Compared with the NC group, the difference was statistically significant when the TCHQ concentration was ≥10 μM (P < 0.001).

[0059] Table 2. Effects of different concentrations of TCHQ on apoptosis in MCMEC cells.

[0060] Experimental groups (n=3) Apoptosis rate (%) NC 4.857±0.582 Vehicle 4.977±0.599 10μM TCHQ <![CDATA[9.49±0.884 *** ]]> 20μM TCHQ <![CDATA[16.943±1.967 **** ]]> 40μM TCHQ <![CDATA[25.827±1.462 **** ]]>

[0061] n = 3, Compared with the NC group, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001

[0062] 6.3 Effects of TCHQ on intracellular ROS levels in MCMEC cells

[0063] The effect of TCHQ on intracellular ROS levels in MCMEC cells was detected using the DCFH-DA fluorescent probe method. The results showed that TCHQ effectively induced the generation of intracellular ROS in MCMEC cells in a concentration-dependent manner within the concentration range of 10-40 μM, and the difference was statistically significant (P < 0.01).

[0064] Table 3. Effects of different concentrations of TCHQ on ROS in MCMEC cells

[0065] Experimental Groups ROS (%) NC 13.013±2.135 Vehicle 12.467±2.52 10μM TCHQ <![CDATA[23.62±3.332 ** ]]> 20μM TCHQ <![CDATA[42.4±2.411 **** ]]> 40μM TCHQ <![CDATA[67.84±4.064 **** ]]>

[0066] n = 3, Compared with the NC group, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001

[0067] 6.4 Effects of TCHQ on NO release in MCMEC cells

[0068] To assess the effect of TCHQ on NO production in MCMEC cells, the concentration of nitrite in the cell culture supernatant was detected using Griess reagent from a NO detection kit. The results showed that, compared with the NC group, 10 μM TCHQ had no significant effect on NO release from MCMEC cells; however, when the TCHQ concentration increased to 20 μM, NO release significantly decreased, and the difference was statistically significant (P < 0.0001).

[0069] Table 4. Effects of different concentrations of TCHQ on NO in MCMEC cells

[0070] Experimental Groups NO NC 14.265±1.693 Vehicle 14.581±1.995 10μM TCHQ 12.077±1.236 20μM TCHQ <![CDATA[6.496±1.476 **** ]]> 40μM TCHQ <![CDATA[2.761±0.557 **** ]]>

[0071] n = 3, Compared with the NC group, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001

[0072] 6.5 Effects of TCHQ on TNF-α and IL-1β in MCMEC cell supernatant

[0073] To assess the effects of TCHQ on inflammation-related factors in MCMEC cells, the levels of TNF-α and IL-1β in the supernatant of TCHQ-stimulated MCMEC cells were detected by ELISA. The results showed that, compared with the NC group, TCHQ stimulation induced increased levels of TNF-α and IL-1β in cells, and the differences were statistically significant when the TCHQ concentration was ≥20 μM (P < 0.0001).

[0074] Table 5. Effects of different concentrations of TCHQ on TNF-α and IL-1β in MCMEC cells.

[0075] Experimental Groups TNF-α IL-1β NC 88.977±13.81 138.379±23.35 Vehicle 93.161±21.34 136.556±30.913 10μM TCHQ 122.645±24.946 184.917±31.37 20μM TCHQ <![CDATA[236.106±31.438 **** ]]> <![CDATA[502.915±45.194 **** ]]> 40μM TCHQ <![CDATA[407.8±23.842 **** ]]> <![CDATA[695.553±65.925 **** ]]>

[0076] n = 3, Compared with the NC group, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Claims

1. A method for constructing a cell model of coronary microcirculation disorder, characterized in that, Includes the following steps: (a) Culture of mouse cardiac microvascular endothelial cells; (b) The target model can be obtained by treating the cells with tetrachlorohydroquinone at a concentration of 20-40 μM for 6 hours.

2. The method according to claim 1, characterized in that, The concentration of the tetrachlorohydroquinone was 40 μM.

3. The method according to claim 1, characterized in that, The preparation method of tetrachlorohydroquinone used in step (b) is as follows: Under aseptic conditions, add 2 mL of dimethyl sulfoxide to 50 mg of tetrachlorohydroquinone, dissolve it completely, prepare a 100 mM tetrachlorohydroquinone stock solution, filter it with a 0.22 μm cell filter for sterilization, and then use it for later use.

4. The method according to claim 1, characterized in that, In step (a), mouse cardiac microvascular endothelial cells were cultured in complete endothelial cell culture medium and placed in a 5% CO2, 37°C incubator.

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