CN112375802A - Reagent or kit for evaluating toxicity effect of resveratrol interfering with pulmonary epithelial cell polycyclic aromatic hydrocarbon and application and detection thereof - Google Patents
Reagent or kit for evaluating toxicity effect of resveratrol interfering with pulmonary epithelial cell polycyclic aromatic hydrocarbon and application and detection thereof Download PDFInfo
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- CN112375802A CN112375802A CN202011230199.9A CN202011230199A CN112375802A CN 112375802 A CN112375802 A CN 112375802A CN 202011230199 A CN202011230199 A CN 202011230199A CN 112375802 A CN112375802 A CN 112375802A
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Abstract
本发明涉及β‑catenin和Fibronectin mRNA在制备用于评价白藜芦醇干预肺上皮细胞多环芳烃毒性效应的试剂盒和检测方法的新应用。应用本发明的试剂和方法可测定肺上皮细胞β‑catenin和Fibronectin mRNA的表达量。本发明进一步提供了利用定量聚合酶链式反应检测肺上皮细胞β‑catenin和Fibronectin mRNA表达量的方法，可用于评价白藜芦醇抑制和预防肺上皮细胞多环芳烃毒性效应，并分析相关诊断性能。本发明实验条件温和、操作简单、成本低，可高灵敏、高特异性地评价肺上皮细胞多环芳烃毒性效应及其白藜芦醇抑制和预防效果，应用前景广。
The invention relates to the new application of β-catenin and Fibronectin mRNA in the preparation of a kit and a detection method for evaluating the toxic effect of resveratrol intervening polycyclic aromatic hydrocarbons in lung epithelial cells. Using the reagent and method of the present invention, the expression levels of β-catenin and Fibronectin mRNA in lung epithelial cells can be determined. The present invention further provides a method for detecting the mRNA expression levels of β-catenin and Fibronectin in lung epithelial cells by using quantitative polymerase chain reaction, which can be used to evaluate resveratrol's inhibition and prevention of the toxic effects of polycyclic aromatic hydrocarbons in lung epithelial cells, and to analyze related diagnosis performance. The invention has mild experimental conditions, simple operation and low cost, can evaluate the toxicity effect of polycyclic aromatic hydrocarbons in lung epithelial cells and the inhibitory and preventive effects of resveratrol with high sensitivity and high specificity, and has wide application prospects.
Description
Technical Field
The invention relates to a reagent or a kit for evaluating toxicity effect of Resveratrol (RSV) on pulmonary epithelial cell polycyclic aromatic hydrocarbon in mRNA (messenger ribonucleic acid) preparation and application thereof and new application of detection, belonging to the fields of molecular nutrition and science, environmental exposure and toxicology.
Technical Field
The incidence and mortality of malignant tumors are rapidly increasing globally, wherein the incidence and mortality of lung cancer are the first of malignant tumors, the incidence and mortality of lung cancer account for 11.6% of the total number of malignant tumors in 2018 of new lung cancer cases, and the incidence and mortality of lung cancer accounts for 18.4% of the total mortality of malignant tumors. In China, lung cancer is also the most common malignant tumor, the morbidity and mortality of the lung cancer are the first of the malignant tumors, new lung cancer cases account for 18.1 percent of the total number of the malignant tumors in 2018, and death cases account for 24.1 percent of the total death number of the malignant tumors. Therefore, the incidence and mortality of lung cancer in China are far higher than the average level of the whole world, and the prevention and control situation of lung cancer in China is quite severe at present.
Polycyclic aromatic hydrocarbon as a product of incomplete combustion of fuel exists in a large amount in smoke and aerosol generated by combustion of fuel (such as coal, gasoline, diesel oil and the like), and is a primary factor for occurrence and development of lung cancer. 75% of lung cancer deaths in China are related to smoking and solid fuel combustion emission, and lung cancer deaths can be reduced by reducing polycyclic aromatic hydrocarbon emission. PM Beijing10And PM2.5Data on lung cancer risk from exposure to attached polycyclic aromatic hydrocarbons indicate that residents in Beijing and other areas with higher levels of associated pollution than Beijing are often exposed to a high risk of life-long lung cancer, even in summer months. Therefore, the research on the molecular toxicology and prevention and control of lung cancer caused by exposure of polycyclic aromatic hydrocarbon is not slow.
Under the exposure of polycyclic aromatic hydrocarbons such as benzo [ a ] pyrene (BaP) and the like, cytoplasm AhR is combined with a ligand and then enters a cell nucleus to form a heterodimer with ARNT, and is combined to an aromatic response element sequence to induce the transcription of target genes (such as polycyclic aromatic hydrocarbon metabolizing enzyme genes, NRF2 and RAS); in addition, under the action of AhR activated phase I metabolic enzyme, the generated active metabolite can form a compound with DNA or protein to regulate and control the transcription of a target gene; secondly, AhR can activate the cell Epithelial Mesenchymal Transition (EMT) regulated and controlled by beta-Catenin through biochemical processes such as downstream factor regulation phosphorylation and the like, and the occurrence and development of lung cancer are promoted.
RSV, a natural plant polyphenol, is an AhR inhibitor that inhibits the development of lung cancer by AhR, NRF2, NFkB, MAPK, PPAR, and other signals. In addition, EMT is an early molecular event in the development of lung cancer. Therefore, the lung epithelial cells BEAS-2B are taken as a model, the lung epithelial cells are treated by a polycyclic aromatic hydrocarbon representative component BaP to generate EMT, RSV is used for treatment during and before BaP exposure, and the polycyclic aromatic hydrocarbon toxicity effect and the RSV intervention effect of the lung epithelial cells are evaluated by a binary logistic regression model based on the mRNA expression information of cell EMT related factors beta-catenin and fibrinectin. At present, no relevant report is found. The invention can provide references for polycyclic aromatic hydrocarbon molecular toxicology research, health risk evaluation, RSV prevention and control of tumorigenesis and development, tumor (such as lung cancer, colon cancer, ovarian cancer and the like) prevention and control and the like.
Disclosure of Invention
The invention aims to provide a reagent or a kit for evaluating a pulmonary epithelial cell polycyclic aromatic hydrocarbon toxicity effect and an RSV intervention effect thereof with high sensitivity and high specificity and a detection method thereof.
Beta-catenin and fibrinectin mRNA are used for preparing a reagent or a kit for evaluating toxicity effect of resveratrol intervening polycyclic aromatic hydrocarbon in lung epithelial cells, and application and detection thereof.
The reagent or the kit is used for detecting the expression quantity of lung epithelial cell beta-catenin and fibrinectin mRNA.
The reagent or the kit adopts quantitative polymerase chain reaction (Q-PCR) to detect the expression quantity of the lung epithelial cell beta-catenin and fibrinectin mRNA.
An agent or kit for evaluating toxicity effect of resveratrol interfering polycyclic aromatic hydrocarbon in lung epithelial cells, comprising:
cell lysate: TRIzol reagent, the lysate is used to lyse cells;
② reagent for extracting and purifying total RNA: chloroform, isopropanol and 70-80% (V/V) ethanol, said reagents being used for total RNA extraction and purification;
③ mRNA reverse transcription reagent: PrimeScirpt TM RT Master Mix (Perfect Real Time, Takara Inc.), which is a reagent for reverse transcription of mRNA into cDNA;
beta-catenin, Fibronectin and beta-Actin mRNA primers: primer sequences are shown in Table 1;
polymerase chain reaction system: mu.L of cDNA template, 1.6. mu.L of forward primer (5. mu.M), 1.6. mu.L of reverse primer (5. mu.M), 5.8. mu.L of RNase Free dH2O and 10. mu.L of TB Green TM Premix Ex Taq TM II, the reaction system was used for cDNA amplification.
In order to realize the purpose, the invention takes lung epithelial BEAS-2B cells as a model and takes a polycyclic aromatic hydrocarbon representative component BaP as an example, and adopts the following technical scheme:
after exposing BEAS-2B cells through BaP, extracting total RNA of the cells, detecting mRNA expression of beta-catenin and fibrinectin of the cells by Q-PCR, jointly using the two indexes, predicting the classification probability of the samples by using a binary logistic regression model, setting the critical value of the classification probability as 0.5, and if the classification prediction probability value of the samples is less than 0.5, not inducing the BEAS-2B cytotoxic effect by the BaP; if the sample classification prediction probability value is more than or equal to 0.5, the BaP induces the BEAS-2B cytotoxic effect; the control group was 1 ‰ (V/V) dimethyl sulfoxide treated BEAS-2B cells.
When the BEAS-2B cells are exposed in BaP, simultaneously intervening with RSV (respiratory syncytial virus), extracting total RNA (ribonucleic acid) of the cells, detecting mRNA expression of beta-catenin and fibrinectin of the cells by Q-PCR (polymerase chain reaction), jointly using the two indexes, predicting the classification probability of the samples by using a binary logistic regression model, setting the critical value of the classification probability to be 0.5, and if the prediction probability value of the classification of the samples is less than 0.5, the RSV can not inhibit BaP toxic effect of the BEAS-2B cells; the classification prediction probability value of a plurality of pre-group samples is more than or equal to 0.5, and then the RSV can inhibit the BaP toxic effect of BEAS-2B cells; the control group was BaP-exposed BEAS-2B cells.
The method comprises the following steps that (1) BEAS-2B cells are treated by RSV, then BaP exposure is carried out, total RNA of the cells is extracted, Q-PCR is used for detecting mRNA expression of beta-catenin and fibrinectin of the cells, the two indexes are jointly used, a binary logistic regression model is used for predicting the classification probability of a sample, the critical value of the classification probability is set to be 0.5, and if the prediction probability value of the classification of the sample is less than 0.5, the RSV can not prevent BaP toxic effect of the BEAS-2B cells; if the sample classification prediction probability value is more than or equal to 0.5, the RSV can prevent the BEAS-2B cell BaP toxic effect; the control group was BaP-exposed BEAS-2B cells.
The specific method comprises the following steps:
BEAS-2B cell culture and treatment
BEAS-2B cells were cultured in DMEM medium (high sugar) and supplemented with 10% fetal bovine serum. The cells were treated as follows: processing BaP for 48 hours, and evaluating whether the BaP induces a lung epithelial cell toxicity effect; processing BaP and RSV for 48 hours at the same time, and evaluating whether the RSV can inhibit the BaP toxic effect of lung epithelial cells; ③ 48h after RSV treatment and 48h after BaP treatment, whether RSV can prevent BaP toxic effect of lung epithelial cells is evaluated. Treatment concentration of BaP: 5-800 mu g/L. The concentration of RSV treatment was 0.1. mu.g/mL.
RNA extraction and Q-PCR analysis
The BEAS-2B cells were lysed with TRIzol reagent, extracted with chloroform, precipitated with isopropanol, washed with 75% (V/V) ethanol to remove impurities, dried at room temperature, and then applied with PrimeScriptTMThe RT master mix premix reverse transcribes RNA to cDNA for real-time RT-PCR analysis. Correcting mRNA expression by taking beta-Actin as an internal reference and regulating the expression level by 2–ΔΔCtThe method calculates the relative expression quantity of mRNA. In addition, primer sequences were designed for Q-PCR analysis based on the NCBI database. The sequences of the beta-catenin and Fibronectin primers are shown in Table 1.
Evaluation method of BaP toxic Effect of BEAS-2B cells: after the BEAS-2B cells are exposed by BaP, the mRNA expression of cell beta-catenin and Fibronectin is obviously increased (p is less than 0.05, and two-tailed independent sample t-test); substituting the relative expression quantities of the beta-catenin and the fibrinectin mRNA into a binary logistic regression model for analysis to obtain a constant term and coefficients of the beta-catenin and the fibrinectin mRNA in a regression equation, and establishing an equation 1; the classification probability of each sample is predicted by equation 1. The established binary logistic regression model and equation 1 are as follows:
binary logistic regression model: sample classification prediction probability ═ 1/[1+ e--(c+K*a+L*b)]
Wherein c is a constant term; a is the relative expression quantity of beta-catenin mRNA; b is the relative expression quantity of the mRNA of the Fibronectin; k is the coefficient of beta-catenin mRNA in the equation; l is the coefficient of Fibronectin mRNA in the equation.
Binary logistic regression equation 1: sample classification prediction probability ═ 1/[1+ e--(-26.243+11.372*a+3.498*b)]
Wherein a and b represent the relative expression amounts of mRNA of beta-catenin and fibrinectin respectively.
Setting the critical value of the sample classification prediction probability as 0.5, and if the sample classification prediction probability value is less than 0.5, not inducing the BEAS-2B cytotoxic effect by the BaP; if the sample classification prediction probability value is more than or equal to 0.5, the BaP induces the BEAS-2B cytotoxic effect; the control group was 1 ‰ (V/V) dimethyl sulfoxide treated BEAS-2B cells.
Evaluation method of RSV inhibition of the BaP toxic Effect of BEAS-2B cells: when the BEAS-2B cells are exposed to BaP and are subjected to dry prediction by RSV, the mRNA expression of the beta-catenin and the fibrinectin of the cells is obviously reduced (p is less than 0.05, and two-tailed independent sample t-test); substituting the relative expression quantities of the beta-catenin and the fibrinectin mRNA into a binary logistic regression model for analysis to obtain a constant term and coefficients of the beta-catenin and the fibrinectin mRNA in a regression equation, and establishing an equation 2; the classification probability of each sample is predicted by equation 2. The established binary logistic regression equation 2 is as follows:
binary logistic regression equation 2: sample classification prediction probability ═ 1/[1+ e--(31.435-11.725*a-5.527*b)]
Wherein a and b represent the relative expression amounts of mRNA of beta-catenin and fibrinectin respectively.
Setting the critical value of the sample classification prediction probability as 0.5, and if the sample classification prediction probability value is less than 0.5, the RSV can not inhibit the BaP toxic effect of BEAS-2B cells; if the sample classification prediction probability value is more than or equal to 0.5, the RSV can inhibit the BaP toxic effect of BEAS-2B cells; the control group was BaP-exposed BEAS-2B cells.
Method for evaluating RSV to prevent the toxic effect of BEAS-2B cells: after the BEAS-2B cells are treated by RSV and then are exposed by BaP, the mRNA expression of the beta-catenin and the fibrinectin of the cells is obviously reduced (p is less than 0.05, and two-branched independent sample t-test); substituting the relative expression quantities of the beta-catenin and the fibrinectin mRNA into a binary logistic regression model for analysis to obtain a constant term and coefficients of the beta-catenin and the fibrinectin mRNA in a regression equation, and establishing an equation 3; the classification probability of each sample is predicted by equation 3. The established binary logistic regression equation 3 is as follows:
binary logistic regression equation 3: sample classification prediction probability ═ 1/[1+ e--(33.74-13.371*a-4.548*b)]
Wherein a and b represent the relative expression amounts of mRNA of beta-catenin and fibrinectin respectively.
Setting the critical value of the sample classification prediction probability as 0.5, and if the sample classification prediction probability value is less than 0.5, the RSV can not prevent the BEAS-2B cell BaP toxic effect; if the sample classification prediction probability value is more than or equal to 0.5, the RSV can prevent the BEAS-2B cell BaP toxic effect; the control group was BaP-exposed BEAS-2B cells.
6. Evaluation of BaP toxic effects of lung epithelial cells and their RSV inhibitory and prophylactic effects: the probability value is predicted by sample classification, and the classification of each sample is judged, so that the accuracy of sample classification can be obtained, and the BaP toxic effect of the lung epithelial cells and the RSV inhibition and prevention effect of the lung epithelial cells can be evaluated; and (3) performing Receiver Operating Characteristic (ROC) curve analysis by taking the sample classification prediction probability as a variable, and evaluating the diagnosis performance of the BaP toxicity effect of the lung epithelial cells and the RSV inhibition and prevention effect thereof, wherein evaluation indexes are the area under the ROC curve (AUC), the sensitivity and the specificity.
Researchers of the invention find that BaP exposure activates AhR signals and EMT process regulated by the AhR signals, and RSV can inhibit or even eliminate the effects; among changes in AhR signaling and EMT-related factors induced by BaP exposure, β -catenin and fibrinectin mRNA expression changed the most. Therefore, the invention uses EMT related factors beta-catenin and fibrinectin mRNA as combined indexes to evaluate the BaP toxicity effect of the lung epithelial cells and the RSV inhibition and prevention effect thereof. At present, no relevant report is found.
According to the invention, the combination of beta-catenin and fibrinectin mRNA in lung epithelial cells is used as an index, and a binary logistic regression model is adopted, so that the BaP toxic effect and the RSV inhibition and prevention effect of the lung epithelial cells can be evaluated with high sensitivity and high specificity; the experimental conditions are mild, the operation is simple, and the cost is low; can provide reference for polycyclic aromatic hydrocarbon molecular toxicology research and health risk evaluation thereof, RSV (respiratory syncytial virus) reduction and even elimination of the health hazard of polycyclic aromatic hydrocarbon in the environment, RSV prevention and control of malignant tumor transformation, tumor (such as lung cancer, colon cancer, ovarian cancer and the like) prevention and control and the like.
Drawings
FIG. 1: RSV inhibits and prevents BaP exposure-induced lung epithelial cell invasion and migration. Control (Control group): treating with 1 ‰ (V/V) dimethyl sulfoxide; and (3) BaP: 5 μ g/L of BaP exposed group; BaP + RSV: 5 μ g/L BaP exposure with simultaneous intervention with 0.1 μ g/mL RSV; Pre-RSV: 0.1. mu.g/mL RSV was pretreated and subjected to 5. mu.g/L BaP exposure. (A) Crystal violet staining evaluates BaP exposure-induced changes in lung epithelial cell invasion and migration behavior. (B) Cell counts assessed BaP exposure-induced changes in lung epithelial cell invasion and number of migrating cells. The histogram represents mean + standard deviation. **: p < 0.01, two-tailed independent sample t-test. Each experimental group had 5 replicates.
FIG. 2: the heatmap shows that RSV inhibits and prevents BaP exposure-induced changes in AhR signaling and EMT-related factors in lung epithelial cells. Control (Control group): treating with 1 ‰ (V/V) dimethyl sulfoxide; and (3) BaP: 5 μ g/L of BaP exposed group; BaP + RSV: 5 μ g/L BaP exposure with simultaneous intervention with 0.1 μ g/mL RSV; Pre-RSV: 0.1. mu.g/mL RSV was pretreated and subjected to 5. mu.g/L BaP exposure. The listed indices varied significantly (p < 0.05, two-tailed independent sample t-test) in all three comparisons (Control vs. BaP, BaP vs. BaP + RSV, BaP vs. Pre-RSV). 4 replicates per experimental group.
FIG. 3: the Z-value plot shows changes in AhR signaling and EMT-related factors in lung epithelial cells induced by BaP exposure. 4 replicates per experimental group.
FIG. 4: RSV inhibition and prevention of the toxic effect of BaP on lung epithelial cells. Control (Control group): treating with 1 ‰ (V/V) dimethyl sulfoxide; and (3) BaP: 5 μ g/L of BaP exposed group; BaP + RSV: 5 μ g/L BaP exposure with simultaneous intervention with 0.1 μ g/mL RSV; Pre-RSV: 0.1. mu.g/mL RSV was pretreated and subjected to 5. mu.g/L BaP exposure. (A) Histograms show RSV intervention in BaP exposure induced disorders of pulmonary epithelial cell β -catenin and fibrinectin mRNA expression. **: p < 0.01, two-tailed independent sample t-test. (B) Beta-catenin and fibrinectin mRNA were used as combined indicators to evaluate the BaP toxic effects of lung epithelial cells and their RSV intervention effects. (C) Beta-catenin and fibrinectin mRNA as a combined index identifies the diagnostic properties of the BaP toxic effects of lung epithelial cells and their RSV intervention effects. 4 replicates per experimental group.
Table 1: Q-PCR analysis of the relevant primer sequences.
Detailed Description
Examples
The invention is further described in the following examples, which are intended to be illustrative only and not limiting.
BEAS-2B cell culture and treatment
BEAS-2B cells were cultured in high-sugar DMEM medium (HyClone, USA) to which fetal bovine serum (HyClone, USA) was added at a final volume concentration of 10%. Incubator temperature 37 deg.C, CO2Air at a concentration of 5%. After 48h exposure of BEAS-2B cells from 5. mu.g/L BaP (added to a final concentration of 5. mu.g/L in cell culture broth), cells were harvested for evaluation of the BaP toxic effects of lung epithelial cells. Furthermore, BEAS-2B cells were treated simultaneously (added to corresponding final concentrations in cell culture broth) with 5. mu.g/L BaP and 0.1. mu.g/mL RSV for 48h before harvesting for evaluation of whether RSV could inhibit BaP toxic effects in lung epithelial cells. Second, BEAS-2B cells were treated with 0.1. mu.g/mL RSV (added to the corresponding final concentration in cell culture) for 48h, and then exposed with 5. mu.g/L BaP (added to the corresponding final concentration in cell culture) for 48h before cells were harvested for evaluation of whether RSV could prevent BaP toxic effects in lung epithelial cells.
Transwell experiment
Fine preparation was carried out in a 24-well Transwell chamber (Corning, USA)Cell invasion and migration experiments. In the invasion experiment, the upper chamber of the Transwell was coated with an artificial basement membrane (BD Biosciences, USA) with 8 μm pores in advance, 200 μ L of serum-free high-sugar DMEM medium was added, and then BaP and RSV-treated cells were cultured at 5X 104The density is seeded in the upper chamber. Add 600. mu.L of high glucose DMEM medium containing fetal bovine serum at a final volume concentration of 10% to the lower chamber. After incubation of the cells at 37 ℃ for 6h, the upper chamber was washed twice with phosphate buffer and air dried. Cells adhering to the underside of the artificial basement membrane were fixed, stained and counted. In migration experiments, after 200. mu.L of serum-free high-glucose DMEM medium was added to the upper chamber of the Transwell, BaP and RSV treated cells were cultured at 2.5X 104The density is seeded in the upper chamber. After incubation of the cells at 37 ℃ for 4h, the cells on the underside of the upper chamber were fixed, stained and counted.
RNA extraction and Q-PCR analysis
BEAS-2B cells were lysed with TRIzol reagent (Thermo Fisher Scientific, MA, US), extracted with chloroform, precipitated with isopropanol, washed with 75% (V/V) ethanol to remove impurities, dried at room temperature, and then used PrimeScriptTMRT master mix premix (Takara, Dalian, China) reverse transcribes RNA to cDNA. By
Premix Ex TaqTMII (Takara, Dalian, China) performed real-time Q-PCR analysis with the following reaction program set: pre-incubation: reacting at 95 ℃ for 30s for 1 cycle; amplification: firstly reacting at 95 ℃ for 5s, then annealing at 60 ℃ for 20s, and performing 45 cycles; analysis of melting curve: running at 95 ℃ for 1s, then running at 65 ℃ for 15s, and finally rising to 97 ℃ for 1 cycle; cooling to 40 ℃. Correcting mRNA expression with beta-Actin as an internal reference, and using 2–ΔΔCtThe method calculates the relative expression quantity of mRNA. In addition, primer sequences for Q-PCR analysis (see Table 1) were designed according to the NCBI database and synthesized by Shanghai.
4. Statistical analysis
The mRNA relative expression value is introduced into PASW Statistics 18.0 software to perform two-dimensional exponential sample t-test and binary logistic regression analysis. ROC curve analysis was performed in PASW Statistics 18.0 software using sample predicted probability values obtained from binary logistic regression analysis. mRNA relative expression values were introduced into MeV 4.9.0 and MATLAB 2010b software for heatmap and Z-value analysis, respectively.
RSV intervention in BaP Exposure induced Lung epithelial cell invasion and migration
The crystal violet staining result shows that the BaP exposure obviously enhances the invasion and migration capacity of BEAS-2B cells, and RSV can inhibit the change; in addition, RSV pretreatment also reduced BaP exposure-induced bias-2B cell invasion and migration (fig. 1A). Cell counting results show that BaP exposure obviously improves the number of BEAS-2B cells which invade and migrate, and RSV can inhibit the change; in addition, RSV pretreatment also reduced the number of BaP exposure-induced invasive and migratory BEAS-2B cells (fig. 1B). This section of the data demonstrates that RSV inhibits and prevents BaP exposure-induced lung epithelial cell invasion and migration.
RSV intervention on BaP Exposure induced changes in AhR Signaling and EMT associated in Lung epithelial cells
PCR results show that BaP exposure activates the expression of AhR signals (such as AhR, ARNT, HSP90AA1, CYP1A1 and the like) and EMT related factors (such as Twist1/2, Snail2, beta-catenin, Fibronectin, Vimentin and the like), and RSV can inhibit or even eliminate the changes; furthermore, RSV pretreatment may also suppress or even eliminate the above-described effects induced by BaP exposure (fig. 2). This data shows that RSV inhibits and prevents BaP exposure-induced AhR signaling and EMT activation in lung epithelial cells.
Evaluation of the toxic Effect of RSV intervention on BaP in Lung epithelial cells
The changes of the EMT related factors beta-catenin and fibrinectin mRNA are the largest in the changes of the AhR signal and the EMT related factors induced by the BaP exposure, so that the invention further evaluates the BaP toxicity effect of the lung epithelial cells and the RSV intervention effect of the lung epithelial cells by taking the beta-catenin and the fibrinectin mRNA as combined indexes (figures 3 and 4).
Substituting the relative expression quantities of beta-catenin and fibrinectin mRNA into a binary logistic regression model to obtain a binary logistic regression equation 1 for evaluating the BaP toxicity effect of the lung epithelial cells: sample classification prediction probability ═ 1/[1+ e--(-26.243+11.372*a+3.498*b)]Wherein a and b each representThe relative expression quantity of mRNA of beta-catenin and fibrinectin; binary logistic regression equation 2 to evaluate the toxic effect of RSV on BEAS-2B cell BaP: sample classification prediction probability ═ 1/[1+ e--(31.435-11.725*a-5.527*b)]Wherein a and b respectively represent the relative expression amounts of mRNA of beta-catenin and fibrinectin; binary logistic regression equation 3 to evaluate the effect of RSV on preventing BEAS-2B cell BaP toxicity: sample classification prediction probability ═ 1/[1+ e--(33.74-13.371*a-4.548*b)]Wherein a and b represent the relative expression amounts of mRNA of beta-catenin and fibrinectin respectively. The result shows that the prediction probabilities of the BaP exposure samples are close to 1 (more than 0.5), and the prediction probabilities of the control samples are close to 0 (less than 0.5), so that the method can correctly identify the BaP toxic effect of the lung epithelial cells, and the correct rate is 100.0%; similarly, the invention can also correctly identify whether the RSV can inhibit the BaP toxic effect of the lung epithelial cells, and the correct rate is 100.0%; secondly, after the cells are pretreated by RSV and then are exposed by BaP, whether the RSV can prevent the BaP toxic effect of the lung epithelial cells can be correctly identified by applying the method of the invention, and the correct rate is 100.0%. The results show that the method provided by the invention can accurately evaluate the BaP toxic effect of the lung epithelial cells and the RSV intervention effect of the lung epithelial cells. Finally, ROC analysis is used for evaluating the diagnostic performance of the invention, and the result shows that the invention has excellent diagnostic performance on the BaP toxic effect of the lung epithelial cells and the RSV intervention effect thereof, the related optimal sensitivity and specificity can reach 100.0 percent, and the AUC is 1.0.
8. And (4) conclusion: the reagent and the method provided by the invention can be used for detecting the expression quantity of the lung epithelial cell beta-catenin and fibrinectin mRNA, and can realize the evaluation of the BaP toxic effect and the RSV intervention effect of the lung epithelial cell with high sensitivity and high specificity based on a binary logistic regression model according to the expression quantity of the beta-catenin and the fibrinectin mRNA; secondly, the method has mild experimental conditions, simple operation and low cost, and is suitable for popularization and application; in addition, the invention can provide references for polycyclic aromatic hydrocarbon molecular toxicology research and health risk evaluation thereof, RSV (respiratory syncytial virus) reduction and even elimination of polycyclic aromatic hydrocarbon health hazard in the environment, RSV prevention and intervention of tumor occurrence and development, tumor (such as lung cancer, colon cancer, ovarian cancer and the like) prevention and control and the like.
Claims (10)
1. Application of beta-catenin and fibrinectin mRNA in preparing a reagent or a kit for evaluating toxicity effect of resveratrol interfering with polycyclic aromatic hydrocarbon in lung epithelial cells.
2. The application of the reagent or the kit as claimed in claim 1, wherein the reagent or the kit is used for detecting the expression quantity of the beta-catenin and the fibrinectin mRNA of the lung epithelial cells by quantitative polymerase chain reaction, and evaluating the polycyclic aromatic hydrocarbon toxicity effect of resveratrol interfering in the lung epithelial cells according to the expression quantity of the beta-catenin and the fibrinectin mRNA;
the intervention refers to inhibition and/or prevention.
3. A reagent or kit for evaluating or detecting toxicity effect of resveratrol interfering with polycyclic aromatic hydrocarbon in lung epithelial cells comprises:
cell lysate: TRIzol reagent, the lysate is used to lyse cells;
② reagent for extracting and purifying total RNA: chloroform, isopropanol and 70-80% (V/V) ethanol, said reagents being used for total RNA extraction and purification;
③ mRNA reverse transcription reagent: such as: PrimeScirpt TM RT Master Mix (Perfect Real Time, Takara Inc.), which is a reagent for reverse transcription of mRNA into cDNA;
beta-catenin, Fibronectin and beta-Actin mRNA primers: the primer sequences are shown in the corresponding sequences shown in Table 1;
polymerase chain reaction system: in the case of a 20. mu.L reaction system for cDNA amplification, 1. mu.L of cDNA template, forward and reverse primers (final concentrations of 0.2-1.0. mu.M, respectively), and 10. mu.L of TB Green TM Premix Ex Taq TMII (2X) were added.
4. A method for detecting the mRNA expression level of lung epithelial cells beta-catenin and fibrinectin comprises the following steps: firstly, collecting lung epithelial cells, cracking by a TRIzol method and extracting total RNA of the cells; reverse transcribing the extracted total RNA into cDNA; amplifying cDNA by real-time quantitative fluorescent polymerase chain reaction; fourthlyAccording to the amplification curve and the melting curve, confirming that only one unique target product is successfully amplified and detected; beta-Actin is used as internal reference to correct the expression of beta-catenin and fibrinectin mRNA and the expression is expressed by 2–ΔΔCtThe method calculates the relative expression quantity of mRNA.
5. The detection method according to claim 4, characterized in that: the real-time quantitative fluorescent polymerase chain reaction program is set as follows: pre-incubation: reacting at 95 ℃ for 30s for 1 cycle; amplification: firstly reacting at 95 ℃ for 5s, then annealing at 60 ℃ for 20s, and performing 45 cycles; analysis of melting curve: running at 95 ℃ for 1s, then running at 65 ℃ for 15s, and finally rising to 97 ℃ for 1 cycle; cooling to 40 ℃.
6. The detection method according to claims 1-5, characterized in that it comprises
The method for evaluating the toxicity effect of polycyclic aromatic hydrocarbon of lung epithelial cells comprises the following steps: after the lung epithelial cells are exposed by polycyclic aromatic hydrocarbons, extracting total RNA of the cells, detecting mRNA expression of beta-catenin and fibrinectin of the cells, using the two indexes in a combined manner, calculating the classification probability of the samples by using a binary logistic regression model, setting the critical value of the classification probability to be 0.5, and if the classification prediction probability value of the samples is less than 0.5, not inducing the toxicity effect of the lung epithelial cells by the polycyclic aromatic hydrocarbons; if the sample classification prediction probability value is more than or equal to 0.5, the polycyclic aromatic hydrocarbon induces the pulmonary epithelial cytotoxicity effect; the control group is lung epithelial cells treated by 1 per thousand (V/V) dimethyl sulfoxide;
or, the method for evaluating the toxicity effect of resveratrol on inhibiting the polycyclic aromatic hydrocarbon of the lung epithelial cells comprises the following steps: when the pulmonary epithelial cells are exposed to polycyclic aromatic hydrocarbons, simultaneously using resveratrol for intervention, extracting total RNA of the cells, detecting mRNA expression of beta-catenin and fibrinectin of the cells, using the two indexes in a combined manner, using a binary logistic regression model to calculate the classification probability of the samples, setting the critical value of the classification probability as 0.5, and if the classification prediction probability value of the samples is less than 0.5, the resveratrol cannot inhibit the polycyclic aromatic hydrocarbon toxicity effect of the pulmonary epithelial cells; if the sample classification prediction probability value is more than or equal to 0.5, the resveratrol can inhibit the polycyclic aromatic hydrocarbon toxicity effect of the lung epithelial cells; the control group was polycyclic aromatic hydrocarbon-exposed lung epithelial cells;
or, the resveratrol evaluation method for preventing the polycyclic aromatic hydrocarbon toxicity effect of the lung epithelial cells comprises the following steps: treating lung epithelial cells with resveratrol, then exposing with polycyclic aromatic hydrocarbons, extracting total RNA of the cells, detecting mRNA expression of beta-catenin and fibrinectin of the cells, using the two indexes in a combined manner, utilizing a binary logistic regression model to calculate the classification probability of a sample, setting the critical value of the classification probability to be 0.5, and if the classification prediction probability value of the sample is less than 0.5, the resveratrol cannot prevent the polycyclic aromatic hydrocarbon toxic effect of the lung epithelial cells; if the sample classification prediction probability value is more than or equal to 0.5, the resveratrol can prevent the polycyclic aromatic hydrocarbon toxicity effect of the lung epithelial cells; the control group was polycyclic aromatic hydrocarbon-exposed lung epithelial cells.
7. The method of claims 1-5, wherein the diagnostic performance analysis of the evaluation results of pulmonary epithelial cell polycyclic aromatic hydrocarbon toxicity effect, resveratrol inhibition and pulmonary epithelial cell polycyclic aromatic hydrocarbon toxicity effect prevention comprises: and (3) analyzing the working characteristic curve of the testee by taking the sample classification prediction probability as a variable, wherein if the optimal sensitivity and specificity of the evaluation result of the pulmonary epithelial cell polycyclic aromatic hydrocarbon toxicity effect and resveratrol inhibition and pulmonary epithelial cell polycyclic aromatic hydrocarbon toxicity effect prevention evaluation result and the area under the working characteristic curve of the testee are respectively 80%, 80% and 0.8 or more, the sensitivity and specificity of the related evaluation result are high, and the diagnosis performance is excellent.
8. The method of claims 1-7, wherein: the lung epithelial cells comprise one or more than two of various human-derived and animal-derived lung epithelial cells with significant changes (p is less than 0.05 and the expression of beta-catenin and/or fibrinectin mRNA is t-test) after polycyclic aromatic hydrocarbon exposure.
9. The method of claims 1-8, wherein polycyclic aromatic hydrocarbon exposure is performed by exposing the cells to an environment comprising polycyclic aromatic hydrocarbons, such as, preferably: polycyclic aromatic hydrocarbons are added to the cell culture fluid.
10. The method according to claims 1-9, wherein polycyclic aromatic hydrocarbon refers to aromatic hydrocarbon containing two or more benzene rings, such as: one or more of benzo [ a ] pyrene, anthracene, benzo [ a ] anthracene and benzo [ b ] fluoranthene.
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| US20030069199A1 (en) * | 2000-12-07 | 2003-04-10 | Hanan Polansky | Treatment methods based on microcompetition for a limiting GABP complex |
| CN103088105A (en) * | 2013-01-15 | 2013-05-08 | 中国烟草总公司郑州烟草研究院 | MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke |
| CN109803664A (en) * | 2016-06-15 | 2019-05-24 | 尚特·德扎尔基西安 | Reagents, compositions and methods for improving viability and function of cells, tissues and organs |
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