Molecular index screening of MDM2 gene in occupational irradiation-induced liver injury
Through MDM2 gene-specific primer amplification and qRT-PCR analysis, molecular markers of hepatocyte injury induced by the mixture of α particles and alcohol were screened, solving the problem of early warning of liver injury in occupational irradiation, and achieving early warning and accurate assessment of liver injury in occupational personnel.
Patent Information
- Application Number
- CN202510392184.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art cannot accurately evaluate the liver damage caused by intra-occupational radiation, especially the effects of decayed alpha particles radiation of transuronic nuclides such as plutonium on the liver, and whether drinking habits increase health risks lack data support, resulting in difficulty in early tumor detection.
qRT-PCR analysis was performed using MDM2 gene-specific primers. By amplifying the cDNA template of hepatocytes after intra-occupation irradiation, the relative expression of the MDM2 gene was analyzed using fluorescence quantitative data, and molecular markers of hepatocyte damage induced by α particles and alcohol mixture were screened out.
It provides the MDM2 gene as an early warning molecular marker for liver injury after intra-occupational irradiation, which can accurately predict early radiation damage to hepatocytes and support early warning for occupational health monitoring.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of radiation medicine and relates to the screening of molecular indicators of MDM2 gene for occupational internal irradiation-induced liver injury. Background Art
[0002] Exposure to radioactive substances such as plutonium, americium, neptunium, and curium is usually the direct cause of pollution in spent fuel reprocessing plants. It mainly enters the body through occupational inhalation, skin wounds, and gastrointestinal absorption, and deposits in target organs and is difficult to excrete. Transuranic nuclides and their decay products are listed as Class I carcinogens by the International Agency for Research on Cancer (IARC), and their hazards are mainly caused by the alpha particle radiation of transuranic nuclide decay.
[0003] In the process of occupational health surveillance, regular individual dose monitoring and occupational health examinations are the main means of health surveillance to achieve early detection and treatment of diseases. However, due to the extremely complex biokinetics of the metabolism of extremely toxic transuranics such as plutonium in the human body, individual dose estimation often cannot accurately reflect the damage of individuals. At present, the occupational health examinations for workers mainly rely on eye lens examinations, blood routine, urine routine examinations of hematopoiesis, thyroid, liver and kidney functions. When positive lesions in the target organs are found, tumors have already developed irreversibly.
[0004] Whether it is the occurrence of tumors in human tissues or the tumor models established in experimental studies, they are all the results of the interaction between environmental factors and the genetic material of the body, ultimately leading to genetic instability of cells themselves, mutations, and disorders of cell growth behavior. Whether the drinking habits of workers exposed to occupational internal irradiation will increase the health risks of occupational personnel has always been an occupational health issue of concern in radiation protection. At present, there is not enough population epidemiological investigation and data at the experimental animal and cell levels to support it.
[0005] This patent separately conducts experiments on alpha particle internal irradiation and alcohol-induced hepatocyte injury, and uses multi-chip technology to screen out the differential genes induced by the combined factors of cumulative alpha particle irradiation and alcohol, in order to be used as molecular indicators in the process of liver injury after occupational internal irradiation with alpha particles, and to provide molecular markers for the health surveillance of occupational personnel exposed to internal irradiation and the early warning of liver injury in target organs. Summary of the Invention
[0006] The purpose of the present invention is to provide a method for screening molecular indicators of MDM2 gene for occupational internal irradiation-induced liver injury, and to provide molecular markers for the health surveillance of occupational personnel exposed to internal irradiation and the early warning of liver injury in target organs.
[0007] The screening of molecular indicators of MDM2 gene for occupational internal irradiation-induced liver injury in the present invention uses the specific primers of MDM2 gene as:
[0008] Forward primer: 5′-ATCTACAGGGACGCCATCGAA-3′
[0009] Reverse primer: 5′-TGAAACTGAATCCTGATCCAACCA-3′
[0010] Amplify the cDNA template of hepatocytes from irradiated personnel in the occupation to be tested. Analyze the 2-ΔΔCt results of qRT-PCR fluorescence quantification data to measure the relative expression levels of the MDM2 gene in hepatocytes and the relative expression levels of the MDM2 gene in hepatocytes of the control group. If the mRNA expression level of the MDM2 gene in the hepatocytes of the group to be tested increases compared with the relative expression level of the MDM2 gene in the hepatocytes of the control group, and the difference reaches a significant level of p < 0.05, it indicates early radiation damage to hepatocytes after internal irradiation with α particles in the occupation to be tested.
[0011] The MDM2 gene described in the present invention is used for screening molecular indicators of liver injury induced by internal irradiation in the occupation. The specific method is as follows:
[0012] 1) Extraction of total RNA from hepatocytes: Extract the total RNA of hepatocytes to be tested and hepatocytes of the control group;
[0013] 2) Synthesis of the first strand of cDNA:
[0014] (1) Add the following solutions to an EP tube, mix well, and incubate at 42 °C for 3 min:
[0015] 5×gDNA Buffer 2 μL
[0016] Total RNA of hepatocytes 1 μL
[0017] Make up to 10 μL with RNase-Free ddH2O
[0018] (2) Prepare a premixed solution according to the following solutions:
[0019]
[0020] Incubate at 42 °C for 15 min; incubate at 95 °C for 3 min, then place on ice, and then store at -20 °C for subsequent experiments;
[0021] 3) q-PCR amplification of the target gene:
[0022] Primers for the MDM2 differential gene:
[0023] Forward primer: 5′-ATCTACAGGGACGCCATCGAA-3′
[0024] Reverse primer: 5′-TGAAACTGAATCCTGATCCAACCA-3′
[0025] The reaction system and reaction conditions are as follows:
[0026]
[0027] Pre-denaturation is carried out at 95°C for 10 min; denaturation at 95°C for 15 s, annealing / extension at 72°C for 45 s, and the number of cycles is 40;
[0028] For qRT-PCR fluorescence quantitative data analysis of the 2-ΔΔCt results, the relative expression levels of MDM2 in hepatocytes of the test group and the control group of hepatocytes after 24 h of hepatocyte culture are measured. If the mRNA expression level of the MDM2 gene in hepatocytes of the test group increases compared with the relative expression level of MDM2 in hepatocytes of the control group, and the difference reaches a significant level of p < 0.05, it indicates that the hepatocytes of the tested occupational personnel have early radiation damage after internal irradiation with α-particles.
[0029] The control group hepatocytes mentioned above are L-02 human hepatocytes.
[0030] Beneficial effects
[0031] This patent belongs to the screening of sensitive molecules during the process of occupational personnel ingesting α-radionuclides and inducing liver injury. By conducting experiments on α-particle irradiation and alcohol-induced hepatocyte injury, and using multi-chip technology to screen out the differential genes induced by the combined factors of cumulative α-particle irradiation and alcohol in hepatocyte injury, it provides the MDM2 gene as a molecular marker for molecular flow adjustment of health monitoring of occupational personnel exposed to internal irradiation and early warning of liver injury in the target organ.
[0032] The cDNA template of early hepatocyte injury after internal irradiation of α-particles in the tested occupational personnel is amplified with MDM2 gene-specific primers. For qRT-PCR fluorescence quantitative data analysis of the 2-ΔΔCt results, it is measured that the relative expression level of the MDM2 gene in hepatocytes of the test group has a significant difference after radiation, so as to predict the early radiation injury of hepatocytes in the tested occupational personnel after internal irradiation. Specific implementation manners
[0033] ① Establish an alcohol-induced cell injury model
[0034] The liver is the largest metabolic organ in the human body. Alcohol has been clearly listed as a Class I carcinogen by the International Agency for Research on Cancer (IARC). Whether the drinking habits of workers exposed to occupational internal irradiation will increase the health risks of occupational personnel. This patent first establishes an alcohol-induced cell injury model.
[0035] According to the literature research, referring to the body weight, blood content and drinking habits of Chinese people, calculate the alcohol metabolism concentration in the blood. Select alcohol concentrations of 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% by volume for cell poisoning. Use human normal liver epithelial cells LO2 and perform long-term multiple hepatocyte poisoning treatments with 1640 medium containing different alcohol concentrations. Conduct an alcohol poisoning experiment treatment once per generation, and passage the cells every three days. The cells are treated with alcohol (1% each time) 10 times and passaged and cultured for 10 generations, denoted as 1%*10-10. 0% is the control group of cells that are not poisoned and synchronously cultured for the same number of generations.
[0036] ② Establish an α-particle cell damage model
[0037] The liver is one of the important target organs for transuranic nuclides. Use human normal liver epithelial cells LO2 cultured in vitro, irradiate the cells with α-particles, and establish an α-particle cell damage model. The irradiation dose rate is 0.138 Gy / min, and the irradiation dose each time is 0, 0.5, 1, 2 Gy. After each irradiation, continue to passage and culture. 0 Gy is the control group of cells that are not irradiated and synchronously cultured for the same number of generations. The hepatocytes are irradiated with α-particles (1 Gy each time) for a cumulative of 2 times, and after passage and culture for 10 generations, they are irradiated and cultured again until 20 generations, denoted as 1 Gy*2-20.
[0038] ③ Establish an α-particle and alcohol combined action cell damage model
[0039] According to the alcohol-induced cell damage model established in ①, select the lowest alcohol concentration that does not affect cell growth and proliferation. According to the α-particle-induced cell damage model established in ②, select the lowest α-particle irradiation dose that does not affect normal cell growth and proliferation, and establish an α-particle and alcohol combined action cell damage model. First, perform alcohol poisoning treatment and then carry out α-particle irradiation.
[0040] For the cells with combined factors, first use the CCK8 method to detect the proliferation viability of the cells. Take the cells in the logarithmic growth phase of each experimental group and the control group, inoculate them in a 96-well culture plate according to a certain cell concentration, set 4 replicates for each sample, and after incubating at 37°C and 5% CO2 for 0 h, 12 h, 24 h, 48 h, 72 h, add reagents according to the CCK8 kit instructions. After reacting for 2 - 4 h, measure the absorbance value at 450 nm of the 96-well plate and draw a growth curve.
[0041] Use the Oil Red O staining method to detect the lipid metabolism function of the cells. Stain the hepatocytes in each experimental group to specifically stain neutral fats such as intracellular triglycerides, and microscopically observe the cell morphology and growth status dynamically. It can be seen that the lipid droplets in normal hepatocytes and experimental group hepatocytes are red granules, and the cell nuclei are blue.
[0042] The Annexin-V-PI double staining method was used to detect cell apoptosis. Cells in the logarithmic growth phase of each experimental group and the control group were digested with 0.25% trypsin without EDTA, and then the cells were collected by centrifugation and washed twice with PBS. Annexin V-FITC and PI dyes were added respectively, and after reacting for 15 min at room temperature in the dark, the apoptosis rate of hepatocytes was detected by flow cytometry. The PI single staining method was used to detect the cell cycle. Cells in the logarithmic growth phase of the experimental group and the control group were digested with 0.25% trypsin without EDTA, and then the cells were collected by centrifugation and fixed with 70% cold ethanol, and the cell cycle was detected by flow cytometry.
[0043] The Transwell assay was used to determine the migration and invasion abilities of hepatocytes. Cells in the logarithmic growth phase were made into a single cell suspension, and were inoculated onto the upper chamber of a 24-well plate with an 8-μm pore size filter membrane at a certain cell concentration. A certain amount of medium containing fetal bovine serum was added to the lower chamber, making the liquid level just cover the cells and avoiding the generation of bubbles. After culturing for 1-2 days, the cells were stained with 0.1% crystal violet, and the cells that passed through the Transwell chamber membrane were observed under an inverted microscope. Five to ten fields of view were randomly selected to collect images.
[0044] ④ Screening and verification of molecular indicators for cell malignant transformation induced by the combined action of α-particles and alcohol
[0045] According to the results of the Transwell assay in ①②③, cells with migration / invasion ability were selected and hybridized and detected using Agilent Human miRNA microarray, methylation gene microarray and lncRNA microarray respectively. Differentially expressed genes in the control group and those causing malignant transformation of hepatocytes by alcohol, α-particles and alcohol / α-particles combined factors were screened out respectively. Then, according to the sequences published in Genebank, primers were designed for real-time quantitative fluorescence PCR verification of cells in each experimental group.
[0046] Take hepatocytes from the radiation group at 1 Gy * 1 - 10, the alcohol group at 1% * 1 - 10, and the combined exposure group at 1 Gy * 1 - 10 + 1%, as well as hepatocytes from the control group. Remove the supernatant, wash twice with PBS at 37°C, and then directly add Trizol reagent or DNA lysis solution from QIAGEN. Extract total RNA and DNA according to the instructions. The total RNA is purified and reverse-transcribed, and the DNA is treated with bisulfite. Hybridization reactions are performed using Agilent Human miRNA chips (8 × 60K, Design ID: 070156), Illumina 850K methylation gene chips, and Agilent Human lncRNA V6 chips (4 × 180K, Design ID: 084410). After washing, the Agilent scanner automatically scans once at 100% and once at 10% photomultiplier tube (PMT) signal intensity, and the two scan results are automatically merged. The Feature Extraction 10.7.1.1 software is used to process the original images and extract the original data. The Genespring 12.5 software is used for quantile normalization and subsequent processing. Probes with at least one set marked as Detected in each group of samples are used for subsequent analysis. Differentially expressed genes are screened using the fold change value, and the screening criterion is that the upregulated or downregulated fold change value ≥ 2.0. Differentially expressed LncRNA, mRNA, circRNA, miRNA, and methylated DNA in hepatocytes of each cumulative irradiation group are obtained, and common differentially expressed RNA molecules are screened: AGBL3, DST, ERCC6, HNRNPR, LOC441666, MAP4K4, MDM2, MIR764, PPIG, ROCK2, SHARPIN, and XLOC_l2_007395.
[0047] The screened differentially expressed genes are verified by Real-time PCR. For the extraction of total RNA from hepatocytes, take hepatocytes from the radiation group at 1 Gy * 1 - 10, the alcohol group at 1% * 1 - 10, and the combined exposure group at 1 Gy * 1 - 10 + 1%, as well as hepatocytes from the control group. 6 cells are seeded into 25 cm 2In a cell culture flask, culture overnight at 5% CO2 and 37°C to allow the cells to adhere. When the cells grow normally to the logarithmic phase, wash the cells once with PBS and discard the supernatant. Add 2.5 ml of Trizol solution to each culture flask, mix well with a cell scraper, and let stand at room temperature for 5 min to fully lyse the cells. Collect the solution in a 5 ml centrifuge tube, add 0.5 ml of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and then centrifuge at 4°C for 15 minutes. Transfer the upper aqueous phase to a new centrifuge tube, add 1.25 ml of isopropanol, let stand at room temperature for 10 minutes, and then centrifuge at 4°C for 10 minutes. Carefully discard the supernatant, add 1 ml of 75% ethanol (dilute absolute ethanol with DEPC water), gently invert to mix well, wash the RNA, and the RNA precipitate can be seen floating. Then let stand at room temperature for 3 - 5 min to sediment the RNA precipitate. Centrifuge at 4°C and 7500 rpm for 5 min, carefully discard the supernatant, and let the RNA precipitate dry naturally in the fume hood. Depending on the amount of the precipitate, add 20 - 50 μl of RNase-free water to dissolve the RNA. Gently pipette to dissolve the precipitate and place on ice. Measure the sample concentration with a Nano-2000 micro nucleic acid and protein detector and calculate OD260 / OD280. If it is between 1.8 - 2.2, the sample purity is good. Use RNA gel electrophoresis method and observe the integrity of the RNA sample with a UV gel imaging system. If the electrophoresis result shows three bright bands, it indicates less RNA degradation and successful extraction, and it can be used for subsequent experiments.
[0048] For PCR amplification, according to the sequences published in Genebank, design primers for the screened differential genes: MDM2, MAP4K4, and CDKN1A (primers for these 3 target genes, as well as the internal reference gene GADPH. Entrust Invitrogen to synthesize Real-time PCR primers according to the following table.
[0049] (1) MDM2
[0050] Forward primer: 5′-ATCTACAGGGACGCCATCGAA-3′
[0051] Reverse primer: 5′-TGAAACTGAATCCTGATCCAACCA-3′
[0052] (2) MAP4K4
[0053] Forward primer: 5′-AGATCCTGCAGCAGCAGCTG-3′
[0054] Reverse primer: 5′-CAGGGACGACAGGTCGATGTCCAC-3′
[0055] (3) CDKN1A
[0056] Forward primer: 5′-AGGCACTCAGAGGAGGCGCCA-3′
[0057] Reverse primer: 5′-GGT GAC AAAGTC GAAGTT CCA-3′
[0058] (4) GADPH
[0059] Forward primer: 5′-GTCAGTGGTGGACCTGACCT-3′’
[0060] Reverse primer: 5′-CCCTGTTGCTGTAGCCAAAT-3′
[0061] Synthesis of the first strand of cDNA: The first strand of cDNA was synthesized according to the FastKing cDNA First Strand Synthesis Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. The steps are as follows:
[0062] (1) Add the following solutions to an EP tube, mix well, and incubate at 42 °C for 3 min.
[0063] 5×gDNA Buffer 2 μL
[0064] Total RNA of hepatocytes 1 μL
[0065] RNase-Free ddH2O was added to make up to 10 μL
[0066] (2) Prepare the following mixed solution.
[0067]
[0068] Add the premixed solution of (2) to the reaction solution of (1).
[0069] Incubate at 42 °C for 15 min. After incubating at 95 °C for 3 min, place it on ice and then store it at -20 °C for subsequent experiments.
[0070] q-PCR amplification of the target gene: According to the instructions of the GoTaq@PCR Master Mix kit, the reaction system and reaction conditions are as follows:
[0071] cDNA template 2 μL
[0072] Forward primer 0.4 μL (final concentration 0.2 μmol / L)
[0073] Reverse primer 0.4 μL (final concentration 0.2 μmol / L)
[0074] GoTaq@PCR Master Mix, 2X 10 μL (final concentration 1×)
[0075] Make up to 20 μL with Nuclease-Free Water
[0076] Place the above solution in a PCR instrument and perform pre-denaturation at 95 °C for 10 min.
[0077] Set the PCR amplification cycle parameters: denaturation (95 °C, 15 s), annealing / extension (72 °C, 45 s), number of cycles (40).
[0078] Table 1: Relative expression levels of MDM2, MAP4K4, and CDKN1A in hepatocytes after 24 h of culture in different experimental groups
[0079] 2MDM2 MAP4K4 CDKN1A Control group 1.004±0.105 1.004±0.295 1.004±0.295 Irradiation group 1.860±0.249 1.428±0.350 1.428±0.796 Alcohol group 2.563±0.504 0.631±0.109 2.203±0.910 Irradiation + alcohol group 3.263±0.352 2.009±0.808 3.082±1.019
[0080] Analysis of the 2-ΔΔCt results of qRT-PCR fluorescence quantitative data (Table 1) showed that the mRNA expression levels of the MDM2 gene in hepatocytes of the radiation group (1 Gy * 1 - 10), alcohol group (1% * 1 - 10), and radiation combined with alcohol group (1 Gy * 1 - 10 + 1%) increased at the 10th generation, and the differential expression was statistically significant (p < 0.05). MDM2 is expected to become a molecular indicator of a-particle or alcohol-induced liver injury and be used for molecular epidemiological investigation of liver injury in occupational workers.
Claims
1. Screening of molecular indicators for MDM2 gene in occupational internal irradiation-induced liver injury.
2. The use of the MDM2 gene according to claim 1 for screening molecular indicators of occupational internal irradiation-induced liver injury, characterized in that, The specific primers for the MDM2 gene are: Forward primer: 5′-ATCTACAGGGACGCCATCGAA-3′ Reverse primer: 5′-TGAAACTGAATCCTGATCCAACCA-3′ Amplify the cDNA template of hepatocytes from irradiated occupational personnel to be tested. Analyze the 2-ΔΔCt results of qRT-PCR fluorescence quantitative data to measure the relative expression levels of the MDM2 gene in hepatocytes of the tested group and the control group. If the mRNA expression level of the MDM2 gene in hepatocytes of the tested group increases compared with the relative expression level of the MDM2 gene in hepatocytes of the control group, and the difference reaches a significant level of p < 0.05, it indicates early radiation injury of hepatocytes in the tested occupational personnel after internal irradiation with α-particles.
3. The use of the MDM2 gene according to claim 1 or 2 for screening molecular indicators of occupational internal irradiation-induced liver injury, characterized in that, The specific method is as follows: 1) Total RNA extraction from hepatocytes: Extract total RNA from the tested hepatocytes and hepatocytes of the control group. 2) First-strand cDNA synthesis: (1) Add the following solutions to an EP tube, mix well, and incubate at 42°C for 3 min: 5×gDNA Buffer 2 μL Total RNA of hepatocytes 1 μL RNase-Free ddH2O to make up to 10 μL (2) Prepare a premix according to the following solutions: Add the premix in (2) to the reaction solution in (1); incubate at 42°C for 15 min; incubate at 95°C for 3 min, then place on ice, and then store at -20°C for subsequent experiments. 3) q-PCR amplification of the target gene: Primers for the MDM2 differential gene: Forward primer: 5′-ATCTACAGGGACGCCATCGAA-3′ Reverse primer: 5′-TGAAACTGAATCCTGATCCAACCA-3′ The reaction system and reaction conditions are as follows: cDNA template 2 μL Forward primer 0.4 μL Reverse primer 0.4 μL GoTaq@PCR Master Mix, 2X 10 μL Nuclease-Free Water to make up to 20 μL Perform pre-denaturation at 95°C for 10 min; denature at 95°C for 15 s, anneal / extend at 72°C for 45 s, with 40 cycles. Analyze the 2-ΔΔCt results of qRT-PCR fluorescence quantitative data to measure the relative expression levels of the MDM2 gene in hepatocytes of the tested group and the control group after 24 h of hepatocyte culture. If the mRNA expression level of the MDM2 gene in hepatocytes of the tested group increases compared with the relative expression level of the MDM2 gene in hepatocytes of the control group, and the difference reaches a significant level of p < 0.05, it indicates damage to hepatocytes of the irradiated occupational personnel in the tested group due to the combined effect of irradiation and alcohol.
4. The use of the MDM2 gene according to claim 1 or 2 for screening molecular indicators of occupational internal irradiation-induced liver injury, characterized in that, The control group of hepatocytes is L-02 human hepatocytes.
5. The use of the MDM2 gene according to claim 3 for screening molecular indicators of occupational internal irradiation-induced liver injury, characterized in that, The control group of hepatocytes is L-02 human hepatocytes.