CN116338187B - Application of WTAP protein as acute ionizing radiation injury marker - Google Patents
Application of WTAP protein as acute ionizing radiation injury marker Download PDFInfo
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- CN116338187B CN116338187B CN202310531371.1A CN202310531371A CN116338187B CN 116338187 B CN116338187 B CN 116338187B CN 202310531371 A CN202310531371 A CN 202310531371A CN 116338187 B CN116338187 B CN 116338187B
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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Abstract
The application discloses application of WTAP protein as an acute ionizing radiation injury marker. The application provides application of WTAP protein nucleoplasm shuttle as a marker in preparation of a product for identifying or assisting in identifying acute ionizing radiation injury. The research of the application discovers that WTAP protein changes nuclear shuttle after the stimulation of acute ionizing radiation, the WTAP protein enters the nucleus from cytoplasm, the WTAP protein in the nucleus is increased, and the WTAP protein in the cytoplasm is reduced. The application can be used for assisting in judging whether the object suffers from ionizing radiation, is beneficial to improving the rapid and accurate evaluation of the irradiated personnel during nuclear leakage accidents, and has important significance for the subsequent effective classification diagnosis and the determination of clinical treatment schemes.
Description
Technical Field
The application relates to the technical field of biology, in particular to application of WTAP protein as an acute ionizing radiation injury marker.
Background
In recent years, nuclear technology has been widely used in aspects of human life, such as radiation therapy and radiodiagnosis in the medical field, food sterilization in the food processing field, and the like. But the nuclear technology brings benefits and increases the possibility of nuclear accidents. Once a large-scale nuclear accident occurs, the suspected irradiated personnel can be rapidly and accurately judged in the early stage of the accident, so that the method is beneficial to the subsequent effective classification diagnosis and the determination of clinical treatment schemes, and has very important significance.
Currently, common radiation biomarkers mainly include: chromosome aberration analysis, peripheral blood lymphocyte count, gamma-H2 AX analysis, and the like. However, existing radiation biomarkers cannot make a rapid and efficient judgment for suspected persons to be irradiated in the early stage of a large-scale radiation accident, and therefore, searching for new radiation biomarkers is a goal of continuous efforts of radiation biologists in recent years. The field of research for the irradiation molecular markers can be divided into four histologic directions: genomics, transcriptomics, metabolomics, and proteomics. Genomic studies mainly include gene mutation analysis, DNA modification analysis, and the like. By genetic mutation analysis of atomic bomb explosion survivors, researchers found that the higher the dose irradiated, the greater the GPA mutation index. Meanwhile, through DNA modification analysis, researchers find that radiation can cause hypomethylation of whole genome DNA, and has dose dependence, sex variability and tissue specificity. However, the genomics analysis method still has the problems of large individual difference, high spontaneous rate and the like. With the increasing research approaches, transcriptomics is also increasingly being applied to the study of irradiated molecular markers. The multi-molecular expression profile analysis realizes high-flux detection of mRNA level change, and promotes research and study of the radiation sensitive gene mRNA and miRNA as a radiation molecular marker. Metabonomics studies are mainly based on animal models and serum or urine of clinical patients to identify various differential metabolites that are subject to radiation modulation, exploring potential radiation damage markers. The process is susceptible to dietary, individual differences, gender and environmental factors. Proteomics research mainly uses stress response molecules, inflammatory factors, apoptosis and DNA damage repair related molecules, wherein the research using gamma H2AX aggregation point analysis as a radiation damage marker is more systematic. But has the disadvantages of complicated microscopic examination and low flux.
m 6 The A modification is methylation modification of adenine sixth N in nucleotide, and is a dynamic reversible modification mode. With respect to m 6 On continuing research on modification A, scientists found m 6 The A modification and related enzymes are involved in a variety of biological processes. Through m 6 A modified methylase and demethylase, and the action of binding proteins, m 6 The A modification plays a regulatory role in the processes of RNA processing, nuclear export, translation, degradation, RNA-protein interaction and the like. There are also continuous studies showing that m 6 The A modification plays an important role in various biological processes such as cancers, fat metabolism, biological rhythms, reproductive development, immune regulation, virus replication, myocardial cell remodeling, stress response and the like. It is worth mentioning that m 6 The A modification is the most common modification mode in eukaryotic RNA modification, accounting for about 60% of all RNA modifications, and is a research hotspot in epigenetic modification. In recent years, studies have been carried out to show that RNA m 6 The a modification is responsive to a variety of stimuli, with significant changes in the level of RNA m6A modification in eukaryotic cells under the influence of UV, thermal stimuli, strong oxidants, and ionizing radiation. Nephroblastoma 1-binding protein (WTAP) as m 6 The important methyltransferase related to A modification is expressed in the nucleus and the outside. In the catalysis of m 6 During the A modification, METLl 4 forms heterodimerization with METTL3, is recruited by WTAP in the nucleus and binds to mRNA upper body to play a role in catalysis, and regulates dynamic change of m6A modification level. From RNA m 6 Starting from the A modification angle, RNA m was explored in response to ionizing radiation 6 A modification of methylation modification enzyme or demethylase, m was found 6 No studies of a modification related radiation biomarkers have been reported.
Disclosure of Invention
The object of the present application is to provide the use of a wilt's tumor 1-binding protein (WTAP) as a marker of acute ionizing radiation injury.
In a first aspect, the application claims the use of WTAP protein nucleoplasm shuttle as a marker in any of the following:
(A1) Preparing a product for identifying or aiding in identifying acute ionizing radiation injury;
(A2) Preparing a product for detecting or assisting in detecting whether a cell to be detected or an organism to be detected suffers from acute ionizing radiation injury;
(A3) Preparing a product for distinguishing or assisting in distinguishing an acute ionizing radiation injury group from a normal control group; the acute ionizing radiation injury group is a cell or organism suffering from acute ionizing radiation injury; the normal control group is normal cells or healthy organisms which are not treated by radiation.
In a second aspect, the application claims the use of a substance for detecting a WTAP protein nucleoplasmic shuttle in any of the following:
(A1) Preparing a product for identifying or aiding in identifying acute ionizing radiation injury;
(A2) Preparing a product for detecting or assisting in detecting whether a cell to be detected or an organism to be detected suffers from acute ionizing radiation injury;
(A3) Preparing a product for distinguishing or assisting in distinguishing an acute ionizing radiation injury group from a normal control group; the acute ionizing radiation injury group is a cell or organism suffering from acute ionizing radiation injury; the normal control group is normal cells or healthy organisms which are not treated by radiation.
In the first and second aspects, the substance for detecting a nuclear shuttle of the WTAP protein may be a substance capable of detecting a ratio of an expression amount of the WTAP protein in a nucleus to an expression amount in a cytoplasm.
Further, the substance for detecting the WTAP protein nucleoplasmic shuttle may consist of (B1) and (B2) as follows, or may be (B3) as follows:
(B1) Reagents and/or instrumentation for nuclear mass separation;
(B2) Reagents and/or instruments for detecting WTAP protein expression levels;
(B3) Reagents and/or instruments capable of performing in situ quantitative detection of WTAP proteins.
Wherein (B1) and (B2) correspond to nuclear mass separation techniques; (B3) Can correspond to, for example, immunofluorescence techniques, wherein the instrument can be, for example, a laser scanning confocal microscope.
In the first and second aspects, the ionizing radiation may be gamma-radiation (e.g., cobalt 60-gamma radiation).
In the first and second aspects, the ionizing radiation is gamma-ray irradiated for 5 to 10Gy for 0.5 to 24 hours.
In a specific embodiment of the application, the ionizing radiation irradiates the human cells with a treatment mode of 10Gy of gamma-rays (cobalt 60-gamma rays) for 0.5-4 hours (such as 1 hour), and irradiates the mice with a treatment mode of 6.5Gy of gamma-rays (cobalt 60-gamma rays) for 1-24 hours (such as 6 hours).
In a third aspect, the application claims a system for identifying or aiding in the identification of acute ionizing radiation damage.
The system for identifying or aiding in the identification of acute ionizing radiation injury claimed in the present application may comprise:
(C1) Kit and/or instrument;
the kit and/or instrument has the following functions: detecting the ratio of the expression quantity of WTAP protein in cell nucleus to the expression quantity in cytoplasm;
(C2) A device;
the device comprises a data input module, a threshold storage module, a data comparison module and a judgment module;
the data input module is configured to input (C1) the detected following values: the ratio of the expression level of WTAP protein in cell nucleus to the expression level in cytoplasm from the cell to be tested or the body to be tested;
the threshold storage module is configured to store a threshold; the threshold value is the ratio of the expression amount of WTAP protein in cell nucleus to the expression amount in cytoplasm of normal cells or healthy organisms which are not treated by radiation;
the data comparison module is configured to receive the value detected by the (C1) sent by the data input module, call the threshold value in the threshold value storage module, and compare the ratio of the expression amount of the WTAP protein in the cell nucleus and the expression amount in the cytoplasm of the cell or the organism to be tested with the threshold value;
the judging module is configured to receive the comparison result sent by the data comparing module, and then to judge the result according to the following steps: if the ratio of the expression level of the WTAP protein in the cell nucleus to the expression level of the WTAP protein in the cytoplasm of the cell or the organism to be tested is larger than the threshold value, the cell or the organism to be tested is or is candidate to suffer from acute ionizing radiation damage; otherwise, the cell or the body to be tested is or is candidate to be not damaged by the acute ionizing radiation.
Further, the kit and/or instrument is composed of (B1) and (B2) as follows, or (B3) as follows:
(B1) Reagents and/or instrumentation for nuclear mass separation;
(B2) Reagents and/or instruments for detecting WTAP protein expression levels;
(B3) Reagents and/or instruments capable of performing in situ quantitative detection of WTAP proteins.
Wherein (B1) and (B2) correspond to nuclear mass separation techniques; (B3) Can correspond to, for example, immunofluorescence techniques, wherein the instrument can be, for example, a laser scanning confocal microscope.
Further, the ionizing radiation may be gamma-radiation (e.g., cobalt 60-gamma radiation).
Further, the ionizing radiation is gamma-ray irradiation of 5-10Gy for 0.5-24h.
In a specific embodiment of the application, the ionizing radiation irradiates the human cells with a treatment mode of 10Gy of gamma-rays (cobalt 60-gamma rays) for 0.5-4 hours (such as 1 hour), and irradiates the mice with a treatment mode of 6.5Gy of gamma-rays (cobalt 60-gamma rays) for 1-24 hours (such as 6 hours).
In a fourth aspect, the application claims the use of a system as described in the third aspect hereinbefore in any of the following:
(D1) Preparing a product for classifying and diagnosing nuclear leakage irradiated personnel;
(D2) A product is prepared for use in determining a clinical treatment regimen for a nuclear leak radiation subject.
In each of the above aspects, the body is an animal (e.g., a mouse) or a human.
In a specific embodiment of the application, the cell is a Hela cell or a 293T cell.
In the aspects, the amino acid sequence of the WTAP protein is shown as SEQ ID No. 1.
The application discovers for the first time that WTAP protein changes nuclear shuttle after the stimulation of acute ionizing radiation, WTAP protein enters the nucleus from cytoplasm, WTAP protein in the nucleus is increased, and WTAP protein in the cytoplasm is reduced. The application can be used for assisting in judging whether the object is subjected to ionizing radiation (such as gamma-ray irradiation), is beneficial to improving the rapid and accurate evaluation of the irradiated personnel during nuclear leakage accidents, and has important significance for the subsequent effective classification diagnosis and the determination of clinical treatment schemes.
Drawings
FIG. 1 shows that acute ionizing radiation stimulation does not affect intracellular multiple RNAs m 6 A modifies the mRNA level of a methylation modifying enzyme or a demethylase. A is m of RNA in Hela cells of the non-irradiated and acute ionizing radiation damaged groups analyzed by transcriptome sequencing 6 A modification of the expression difference of a methylation modification enzyme or a demethylase; b is a plurality of RNA m in Hela cells after acute ionizing radiation injury compared with non-irradiated cells 6 The mRNA levels of the A modified methylation modification enzyme or the demethylase are not significantly different; c is the multiple RNA m in 293T cells after acute ionizing radiation injury as compared to non-irradiated cells 6 There was no significant difference in mRNA levels of the a-modified methylation-modified or demethylase enzymes. ns indicates no significant difference.
FIG. 2 shows that acute ionizing radiation stimulation does not affect intracellular multiple RNAs m 6 A modifies the protein level of a methylation modifying enzyme or a demethylase. A is a plurality of RNA m in Hela cells after acute ionizing radiation injury compared with non-irradiated cells 6 The protein level of the A modified methylation modification enzyme or the demethylase is not significantly different; b is a plurality of RNA m in 293T cells after acute ionizing radiation injury compared with non-irradiated cells 6 There was no significant difference in protein levels of the a-modified methylation-modified or demethylase enzymes.
FIG. 3 shows that WTAP protein levels in the nucleus and in the cytoplasm decreased after stimulation with acute ionizing radiation. A is a nuclear cytoplasm separation experiment, and compared with an unirradiated cell, after acute ionizing radiation injury, the WTAP protein level in the nucleus of the Hela cell is increased, and the WTAP protein level in the cytoplasm is reduced; b is a nucleoplasm separation experiment, and compared with the non-irradiated cells, the WTAP protein level in the nucleus of the 293T cell is increased after acute ionizing radiation injury, and the WTAP protein is in cytoplasmReduced WTAP protein levels; c is immunofluorescence experiment, compared with the non-irradiated cells, after acute ionizing radiation injury, the WTAP protein level in the nucleus of the Hela cells is increased, and the WTAP protein level in cytoplasm is reduced; d is immunofluorescence experiment, and compared with the non-irradiated cells, after acute ionizing radiation injury, the WTAP protein level in the nucleus of 293T cells is increased, and the WTAP protein level in cytoplasm is reduced; e is a WTAP protein fluorescence signal ratio statistical chart of cell nucleus and cytoplasm in a Hela cell immunofluorescence experiment; f is a statistical diagram of WTAP protein fluorescence signal ratio of cell nucleus and cytoplasm in 293T cell immunofluorescence experiment. *p＜0. 1，**p＜0.01，***p＜0.001。
FIG. 4 is a blood image test of mice after acute ionizing radiation stimulation. A is the analysis of hemogram, compared with the non-irradiated mice, after acute ionizing radiation injury, lymphocytes in the peripheral blood of the mice gradually decrease at 1h, 6h and 24h after irradiation; b is a blood image analysis, and compared with a non-irradiated mouse, white blood cells in peripheral blood of the mouse are increased 1h and 6h after irradiation and are sharply reduced 24h after irradiation after acute ionizing radiation injury; c is the analysis of hemogram, compared with the non-irradiated mice, the mononuclear cells in the peripheral blood of the mice are increased after irradiation for 6 hours and are sharply reduced after irradiation for 24 hours after acute ionizing radiation injury; d is the analysis of hemogram, compared with the non-irradiated mice, after acute ionizing radiation injury, the red blood cells in the peripheral blood of the mice have no obvious difference between 1h, 6h and 24h after irradiation; e is a hemogram analysis, and compared with an unirradiated mouse, after acute ionizing radiation injury, hemoglobin in peripheral blood of the mouse has no obvious difference between 1h, 6h and 24h after irradiation; f is a hemogram analysis that shows that platelets in the peripheral blood of mice have no significant difference between 1h, 6h and 24h after irradiation, compared to non-irradiated mice, after acute ionizing radiation injury. ns represents no significant difference, xp＜0. 1，**p＜0.01，***p＜0.001。
Fig. 5 shows that WTAP protein levels in the nuclei of mouse Peripheral Blood Mononuclear Cells (PBMCs) were increased and WTAP protein levels in the cytoplasm were decreased after acute ionizing radiation stimulation. A is immunofluorescence experiment, compared with non-irradiated mice, after acute ionizing radiation injury, the WTAP protein level in the cell nucleus of the PBMC of the mice is increased, and the WTAP protein level in cytoplasm is reduced; compared with the non-irradiated mice, the foci of gamma H2AX is increased 1H after acute ionizing radiation injury, and the foci is gradually decreased 6H and 24H after irradiation; b is a statistical chart of WTAP protein fluorescence signal ratio of cell nuclei and cytoplasm in mouse PBMC cell immunofluorescence experiment.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The culturing methods for Hela and 293T cell lines as described in the examples below were as follows: hela and 293T cell lines were inoculated in DMEM medium containing 10% (volume percent) fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin, 37℃and 5% CO 2 Culturing.
Male C57BL/6 mice of 6 to 8 weeks of age referred to in the examples below were purchased from Beijing Veitz Lihua. All mice were housed in the military medical institute 27 animal house and were acclimatized for at least 3 days prior to the development of the particular experiment. 5 mice were housed in the same cage. All mice in the animal house were cycled daily in a 12 hour light and 12 hour dark environment with ad libitum feeding and drinking conditions.
The sources of materials, reagents and test techniques used in the examples below are shown in table 1.
MonScript TM RTIII AII-The in-One Mix reverse transcription kit is a product of monarch, and the product number is: MR05101. The fluorescent quantitative detection kit (KAPA SYBR cube FAST qRT-PCR kit) is manufactured by KAPA Biosystems, inc. of America, and has the product number of: KK4601.
In the following examples, the irradiation conditions for Hela cells and 293T cells were as follows:
hela (or 293T) cells with good growth status were irradiated with cobalt 60-gamma rays. Wherein the irradiation condition of the cobalt 60-gamma ray irradiation is room temperature, the irradiation dose is 10Gy, the irradiation distance is 3 meters, and the dose rate is 69.1cGy/min. Non-irradiated cells were harvested as control, labeled IR-, and cells 1 hour after 10Gy irradiation were harvested as acute ionizing radiation injury, labeled ir+.
In the following examples, the irradiation conditions of the mice were as follows:
cobalt 60-gamma irradiation was given to 5 male C57BL/6 mice of 6-8 weeks of age. Wherein the irradiation condition of the cobalt 60-gamma ray irradiation is room temperature, the irradiation dose is 6.5Gy, the irradiation distance is 3 meters, and the dose rate is 69.1cGy/min. Tail vein blood collection peripheral blood from mice 1 hour before irradiation, 1, 6 and 24 hours after irradiation. About 100 mu L of peripheral blood is collected from each mouse, 20 mu L of peripheral blood is taken for hemogram detection, and the rest samples are subjected to PBMC separation and immunofluorescence experiments.
Example 1 multiple RNAs m in cells after acute ionizing radiation injury 6 mRNA levels of A-modified methylation-modified or demethylase are not significantly different
1. Bioinformatics analysis
The unirradiated Hela cells and Hela cells after 1 hour of irradiation with 10Gy of cobalt 60-gamma rays were subjected to RNA-seq extraction of RNA samples.
As shown in FIG. 1A, based on the results of RNA-seq, it was found that 12 RNA m were found after acute ionizing radiation compared with untreated Hela cells 6 A modified methylases METTL3, METTL14, WTAP, KIAA1429, RBM15B, METTL16, ZC3H13, CBLL1, METTL5, TRMT112, ZCCHC4 and 1 RNA m 6 There was no significant difference in mRNA levels of the a-modified demethylase FTO, another RNA m 6 A repairThe trim demethylase ALKBH5 may not be detected due to low abundance.
2. Acute ionizing radiation does not affect multiple RNAs m 6 Verification of mRNA level of A-modified methylation modification enzyme or demethylase
Total RNA was isolated from Hela and 293T cells 1 hour after non-and 10Gy irradiation by TRIzol. Using MonScript TM The RTIII AII-in-One Mix reverse transcription kit reverse transcribes RNA into cDNA, and the specific steps are as follows:
taking out the components from the kit, putting the components on ice for dissolving, uniformly mixing the dissolved components, centrifuging for a short time, and putting the components on ice for standby; reverse transcription systems were formulated in 200 μl of RNase-free inlet PCR tubes as shown in table 2:
mixing, placing into a PCR instrument, and performing reverse transcription reaction at first step 55deg.C for 15min, second step 85deg.C for 5min, and third step 4deg.C for 10min; taking out the cDNA product after reverse transcription, and adding 200 mu L of RNase-free water for dilution; for RNA m 6 A modification of the gene sequence of the methylation modification enzyme or the demethylase specific primers were designed, and for ease of analysis, the expression of the target gene was normalized to GAPDH (GAPDH is a reference gene), and the primer sequences were as shown in Table 3:
quantitative PCR analysis was performed using a real-time quantitative PCR kit KAPA FAST qRT-PCR, and samples were loaded according to the system shown in Table 4 and packed into 96-well plates, and three repeated reaction systems were performed for each sample, i.e., three sets of parallel experiments.
Centrifuging the 96-well plate at 3000rpm for 5min, putting the mixture into a qPCR instrument for PCR reaction, and setting a qPCR program as follows: the first step is 95 ℃ for 5min;the second step is 95 ℃ for 5s; third, 60 ℃ for 30s; the second step to the third step are repeated for 40 times; fourth step: dissolution profile (i.e., 95 ℃,15s;60 ℃,1min 20s;95 ℃,30 s); fifth step: preserving at 4 ℃. Analysis of the data, normalized to the reference gene GAPDH, at 2 -△△CT The value shows the relative expression level of the target genes, and the difference in expression of each target gene is determined. Calculation using an unpaired t-testPValues.
As shown in FIG. 1B, to encode RNA m 6 Specific primers for A-modified methylase and demethylase genes detected mRNA levels in HeLa cells before and after irradiation, and the results showed that 12 RNA m in HeLa cells after acute ionizing radiation injury compared with non-irradiated cells 6 mRNA levels of the A-modified methylases (METTL 3, METTL14, WTAP, KIAA1429, RBM15B, METTL, ZC3H13, CBLL1, METTL5, TRMT112, ZCCHC 4) and 2 demethylases (FTO, ALKBH 5) were not significantly different. Similarly, as shown in FIG. 1C, in 293T cells, 12 RNA m in the cells after acute ionizing radiation injury compared to the non-irradiated group 6 mRNA levels of the A-modified methylases (METTL 3, METTL14, WTAP, KIAA1429, RBM15B, METTL, ZC3H13, CBLL1, METTL5, TRMT112, ZCCHC 4) and 2 demethylases (FTO, ALKBH 5) were not significantly different.
Example 2 acute ionizing radiation injury on multiple RNAs m in cells 6 Effects of A modification on protein levels of methylation-modified or demethylase enzymes
Hela and 293T cells are divided into two groups, namely an unirradiated group and an acute ionizing radiation injury group (gamma rays of 10 Gy), proteins in the cells are extracted 1 hour after irradiation, and a plurality of RNA m in the cells are detected by using western blot 6 The effect of A on protein level of modified methylation modification enzyme or demethylase is as follows:
(1) The protein is extracted by the following steps: after cells were lysed on ice with RIPA lysate for 15min, centrifuged at 4℃for 5min at 12000g, the supernatant was transferred to another new EP tube, labeled as total protein sample of cells, added with the same volume of 2 Xprotein loading buffer, mixed well and heated in boiling water for 5min to denature the protein, and the protein sample required for the western blot test was prepared.
(2) SDS-PAGE gels were prepared, comprising the following steps: and cleaning the two sides of the glue-making glass plate, and then placing the glue-making glass plate on a plate frame for airing. After the glass plate is dried, the bottom end is aligned with the glass plate, the glass plate is clamped, and the glass plate is fixed on a glue making frame. The separation gel was prepared according to actual requirements using deionized water, 30% polyacrylamide, 4 x separation gel, 10% aps and coagulant, the mixture was slowly added between the two glass plates with a pipette to a position of 1 cm remaining, then isopropyl alcohol was added to compress the gel, and left at room temperature for about 20 minutes. When significant stratification of the liquid surface had occurred, the isopropanol was decanted and the residual liquid was sucked dry with filter paper. Preparing concentrated glue, adding the concentrated glue into a glass plate to separate the glue after the isopropanol is volatilized cleanly, inserting a comb for airing, and standing at room temperature for about 20 minutes.
(3) The protein sample is subjected to SDS-PAGE gel electrophoresis, and the specific steps are as follows: the comb was pulled out, the gel was set into the electrophoresis tank, and 1×tgs was added into the electrophoresis tank. The formula of the 10 xTGS running buffer is as follows: 144g glycine, 30.3g Tris-base and 10g SDS were dissolved thoroughly in 1L deionized water and stored at room temperature. In use, the gel is diluted with deionized water to 1 xTGS running buffer. According to the actual demand, a pipetting gun is used for loading protein samples. And (5) plugging in a power supply, and setting 80V for electrophoresis. And when obvious layering occurs in the Marker protein, adjusting the parameters of the electrophoresis apparatus to 120V, and determining the electrophoresis position according to the molecular weight of the target protein.
(4) After SDS-PAGE is finished, a semi-dry transfer method is adopted for transferring membranes, and the specific steps are as follows: and transferring the PVDF film, and cutting the PVDF film with a proper size according to actual requirements. The cut PVDF film was placed in a clean tray, and methanol was added thereto for activation for 1 minute. Methanol is recovered and a proper amount of transfer solution is poured. The transfer buffer solution comprises the following components: 14.4g glycine, 2.9g Tris-base and 200 mL methanol were dissolved thoroughly in deionized water and the volume was set to 1L. And taking out the separation gel, placing the separation gel on the activated PVDF membrane, and transferring the separation gel to filter paper infiltrated by the membrane transferring liquid to avoid generating bubbles. And then a layer of filter paper soaked by the transfer film liquid is filled. And (3) gently pressing a glass rod to remove bubbles possibly existing between the filter paper and the glue, pouring a proper amount of film transfer liquid into the upper filter paper, and preventing the film transfer liquid from volatilizing to cause drying between the film and the gel in the film transfer process. Transfer film was performed using a semi-dry transfer machine, and corresponding voltages (15-20V) were set according to the number of gels for 40 minutes.
(5) After the transfer was completed, the membrane was washed 3 times with 1 XTBST buffer, 5min each time, and 5% skim milk was blocked for 1h. Using the corresponding RNA m 6 Antibodies to A-modified methylation-modified or demethylase (Table 1) were incubated overnight at 4℃and the primary antibodies were recovered and washed 3 times with 1 XTBST buffer for 5min each. The membrane was washed 3 times with 1 XTBST buffer 5min each time after incubation with the appropriate HRP-labeled secondary antibody (Table 1) for 1 hour at room temperature.
(6) Protein signals were detected using a Thermo ELC luminescence kit, the detection instrument being a Bio-Rad chemiluminescent gel imaging system.
As shown in FIG. 2A, RNA m 6 The specific antibodies of A modified methylase and demethylase genes detect the total protein level in Hela cells before and after irradiation, and the result shows that 12 RNA m in Hela cells after acute ionizing radiation injury compared with non-irradiated cells 6 There was no significant difference in total protein levels of a modified methylases (METTL 3, METTL14, WTAP, KIAA1429, RBM15B, METTL, ZC3H13, CBLL1, METTL5, TRMT112, zchc 4) and 2 demethylases (FTO, albh 5). Similarly, as shown in FIG. 2B, in 293T cells, 12 RNA m in the cells after acute ionizing radiation injury compared to non-irradiated cells 6 There was no significant difference in total protein levels of a modified methylases (METTL 3, METTL14, WTAP, KIAA1429, RBM15B, METTL, ZC3H13, CBLL1, METTL5, TRMT112, zchc 4) and 2 demethylases (FTO, albh 5).
Example 3 acute ionizing radiation injury to multiple RNAs m 6 Effects of A-modified methylation modification enzymes or demethylases on protein levels in the nucleus and cytoplasm
Hela and 293T cells were divided into two groups, an unirradiated group and an acute ionizing radiation damaged group (gamma rays of 10 Gy), and proteins in cytoplasm and nucleus were extracted by nuclear mass separation method 1 hour after irradiation, specifically, the steps were as follows:
(1) Cells were lysed on ice with NETN lysate (formulation: 100mM NaCl, 1mM EDTA, 10mM Tris-HCl (pH=7.4), 0.1% (volume percent) NP-40) for 15min. Centrifuging at 4deg.C for 5min at 12000g, transferring the supernatant to another new EP tube, adding 2×protein loading buffer solution of the same volume, mixing, and heating in boiling water for 5min to obtain cytoplasmic protein sample.
(2) Washing the centrifuged precipitate twice with NETN lysate, adding 0.2M HCl on ice for 15min, adding Tris-HCl with the same volume of pH=8.0 for neutralization for 15min, centrifuging at 4 ℃ for 5min and 12000g, transferring the supernatant to another new EP tube, adding 2 x protein loading buffer with the same volume, mixing uniformly, heating in boiling water for 5min, and preparing a nuclear protein sample.
(3) The obtained cytoplasm and the obtained multiple RNA m in the nuclear protein sample are subjected to a Western blot method 6 A modification of methylation modification enzyme or demethylase was analyzed (see example 2 for specific procedures). GAPDH is used as a cytoplasmic reference, and histone 3 (H3) is used as a nuclear reference.
As shown in FIG. 3A, RNA m 6 Specific antibodies to A-modified methylase and demethylase genes detected protein levels in the nuclei and cytoplasm of HeLa cells before and after irradiation, and the results showed that, compared with non-irradiated cells, after acute ionizing radiation injury, the level of WTAP protein in the nuclei of HeLa cells was increased, the level of WTAP protein in the cytoplasm was decreased, and the remaining 8 RNAs m 6 Protein levels of a modified methylases (METTL 3, METTL14, KIAA1429, RBM15B, METTL, ZC3H13, CBLL 1) and 2 demethylases (FTO, albh 5) were not significantly different in the nucleus and cytoplasm. Similarly, as shown in FIG. 3B, in 293T cells, the level of WTAP protein in the nucleus was increased, the level of WTAP protein in the cytoplasm was decreased, and the remaining RNA m was increased after acute ionizing radiation injury as compared with non-irradiated cells 6 The protein levels of the a-modified methylase and demethylase in the nucleus and cytoplasm were not significantly different.
Example 4 immunofluorescence assay detectionAcute ionizing radiation injury to RNA m 6 Influence of A-modified methylation modification enzyme WTAP on protein level in nucleus and cytoplasm
Hela cells were divided into two groups, an unirradiated group and an acute ionizing radiation damaged group (gamma rays of 10 Gy), inoculated on slides in 12-well plate dishes 24 hours in advance, and cells were harvested 0, 0.5, 1, 2, 4 hours after irradiation, and immunofluorescence experiments were performed, specifically as follows:
(1) Cells were washed once with PBS and fixed for 10min with the addition of 4% paraformaldehyde pre-chilled at 4 ℃.
(2) The fixed cells were washed once with PBS and punched for 5min with 0.5% Triton X-100 buffer pre-chilled at 4 ℃.
(3) The punched cells were washed once with PBS, and the primary antibody was diluted in PBS containing 10% horse serum (Anti-WTAP, proteintech; cat# 60188-1-Ig; anti-gamma H2AX, NOVUS; cat# NB 100-384) in proportion and incubated at room temperature for 20min.
(4) The cells after incubation of the primary antibody were washed once with PBS, and the fluorescent conjugated secondary antibody was diluted in proportion with PBS containing 10% horse serum (FITC Goat anti Mouse IgG, jackson Immuno Research, cat# 115-095-146;Rhodamine Red Goat anti Rabbit IgG,Jackson Immuno Research, cat# 111-295-144), and incubated at room temperature in the absence of light for 20min.
(5) Cells incubated with the secondary antibody were washed once with PBS, stained for nuclei with DAPI, stained for appropriate amounts of anti-quencher on slides, covered with coverslips, sealed with clear nail polish, and examined under a laser scanning confocal microscope.
As shown in FIG. 3C, RNA m 6 The specific antibody of the A modified methylase WTAP detects the distribution of WTAP proteins in cytoplasm and nucleus in Hela cells at different time points after irradiation, and the result shows that the WTAP proteins in the non-irradiated Hela cells are distributed in cytoplasm and nucleus, the level of WTAP proteins in the nuclei of the Hela cells is increased and the level of WTAP proteins in the cytoplasm is reduced after acute ionizing radiation injury. Similarly, as shown in FIG. 3D, following acute ionizing radiation injury, 293T cells were nuclear compared to non-irradiated 293T cellsIncreased levels of WTAP protein and decreased levels of WTAP protein in the cytoplasm. In fig. 3, E is a statistical chart of WTAP protein fluorescent signal ratio of nuclei and cytoplasm in Hela immunofluorescence experiment. F in FIG. 3 is a statistical chart of the ratio of WTAP protein fluorescence signals of nuclei and cytoplasm in 293T cell immunofluorescence experiments.
EXAMPLE 5 blood image detection of acute ionizing radiation injured mice and analysis of the influence of ionizing radiation on blood image
Male 6-8 week old C57BL/6N mice are from Beijing Vetong Lihua laboratory animal technology Co., ltd, after the C57BL/6N mice with good health status are irradiated with 6.5Gy cobalt 60-gamma rays, blood image detection analysis is carried out at different time points (0 h, 1h, 6h and 24 h) by utilizing tail vein blood sampling and five-class full-automatic blood cell analyzers, and the specific steps are as follows:
(1) The EP tube, labeled 1.5mL, was pre-labeled and 480. Mu.L of animal blood cell assay diluent was added.
(2) The mice were fixed in tail vein blood collection devices with the tail cut approximately 1 to 2 mm.
(3) The capillaries aspirate 20. Mu.L of the shed mouse peripheral blood.
(4) The ear-washing ball is used to blow the blood into the animal blood cell analysis diluent, and the mixture is mixed up and down for 3 times.
(5) The five-class full-automatic blood cell analyzer was used to perform the mouse whole blood cell count using the pre-dilution mode.
(6) Mice were analyzed for the number of White Blood Cells (WBC), lymphocytes (Lymphocyte, lym), monocytes (monocytote, mono), red Blood Cells (RBC), hemoglobin (HGB), and Platelets (PLT) at various time points before and after irradiation.
As shown in fig. 4 a, the lymphocyte numbers in the blood of mice were gradually decreased 1h, 6h, and 24h after acute ionizing radiation injury, as compared with the lymphocyte numbers in the blood of non-irradiated mice (group 0 h). As shown in fig. 4B, the number of white blood cells in the blood of mice was gradually increased 1h and 6h after the acute ionizing radiation injury, compared to the number of white blood cells in the blood of non-irradiated mice (group 0 h), and the number of white blood cells in the blood of mice was significantly lower than that of non-irradiated mice 24h after the acute ionizing radiation injury. As shown in fig. 4C, the number of monocytes in the blood of the mice was not significantly different from the number of monocytes in the blood of the non-irradiated mice (group 0 h), 1h after the acute ionizing radiation injury, 6h after the acute ionizing radiation injury, the number of monocytes in the blood of the mice was significantly increased, and 24h after the acute ionizing radiation injury, the number of monocytes in the blood of the mice was significantly lower than that of the non-irradiated mice. As shown in fig. 4D, the number of red blood cells in the blood of mice was not significantly changed 1h, 6h, and 24h after acute ionizing radiation injury, compared to the number of red blood cells in the blood of non-irradiated mice (group 0 h). As shown in fig. 4E, the amount of hemoglobin in the blood of mice was not significantly changed 1h, 6h, and 24h after acute ionizing radiation injury, as compared to the amount of hemoglobin in the blood of non-irradiated mice (group 0 h). As shown in F in fig. 4, there was no significant change in the number of platelets in the blood of mice 1h, 6h, and 24h after acute ionizing radiation injury, compared to the number of platelets in the blood of non-irradiated mice (group 0 h).
Example 6 immunofluorescence assay of PBMC cells of acute ionizing radiation injured mice and analysis of the effect of acute ionizing radiation injury on protein levels of WTAP in the nucleus and cytoplasm
Male 6-8 week old C57BL/6N mice are from Beijing Veitz Lihua laboratory animal technology Co., ltd, after the C57BL/6N mice with good health status are irradiated with 6.5Gy cobalt 60-gamma rays, orbital blood collection is carried out at different time points (0 h, 1h, 6h and 24 h), and peripheral blood mononuclear cells are separated by adopting a mouse peripheral blood lymphocyte separation liquid KIT (brand: TBD; product number: LTS 1092-KIT) and immunofluorescence detection is carried out. The method comprises the following specific steps:
(1) 0.5mL of anticoagulation (blood is placed for a long time and is easy to separate, and one mixture is needed), and 0.5mL of diluent is added and mixed uniformly. 3mL of the separation liquid is taken and added into a 15mL centrifuge tube, and after the centrifuge tube is inclined at an angle of 45 degrees, diluted peripheral blood is taken and slowly added into the centrifuge tube containing the separation liquid along the tube wall.
(2) The tube was placed in a centrifuge and centrifuged at 500g for 20min. Blood cells were separated into 4 layers in a centrifuge tube, the white lymphocyte layer was carefully aspirated, transferred into a fresh centrifuge tube, added with wash solution or PBS to 10mL, and mixed well with a dropper.
(3) Centrifugation at 1100rpm for 15min, discarding the supernatant, resuspension with 1mL of wash or PBS, cell counting 20. Mu.L, adding the remaining cells to a fresh 1.5mL EP tube, and diluting the sample with 1 XPBS to a cell number of 5000 cells/mL.
(4) Sucking 20 mu L of sample to the glass slide, and placing the glass slide in a metal bath with constant temperature of 37 ℃ for about 5min until the liquid is dried. The sample was covered by dropping 1mL of 4% paraformaldehyde solution, and the sample was fixed for 10 minutes and washed 2 times with 1 XPBS.
(5) A0.5% Triton X-100 solution of 1mL was added dropwise to cover the sample, and the sample was punched out for 5min and washed 2 times with 1 XPBS.
(6) A primary antibody (Anti-WTAP, proteintech; cat# 60188-1-Ig; anti-gamma H2AX, NOVUS; cat# NB 100-384) was prepared by proportional dilution with a1 XPBS buffer containing 5% horse serum.
(7) The surrounding PBS of the sample was blotted with a piece of absorbent paper, 200. Mu.L of primary antibody was added dropwise, incubated for 15min, and washed 2 times with 1 XPBS.
(8) Secondary antibodies (FITC Goat anti Mouse IgG, jackson Immuno Research, cat# 115-095-146;Rhodamine Red Goat anti Rabbit IgG,Jackson Immuno Research, cat# 111-295-144) were formulated by proportional dilution with 1 XPBS buffer containing 5% horse serum. 200. Mu.L of secondary antibody was added and incubated for 15min in the dark and washed 2 times with 1 XPBS.
(9) 200. Mu.L of DAPI was added, covered with coverslips and fixed with nail polish.
(10) The results were observed using a confocal microscope.
As shown in fig. 5 a, the levels of WTAP protein in the nuclei of mice Peripheral Blood Mononuclear Cells (PBMCs) were increased and the levels of WTAP protein in the cytoplasm were decreased 1h, 6h, and 24h after acute ionizing radiation injury, as compared to non-irradiated mice (group 0 h). In PBMCs of acute ionizing radiation injured mice (1H, 6H, and 24H), DNA damage marker protein γh2ax forms a DNA damage generating focal spot (foci), γh2ax foci gradually decreases with the time after irradiation. FIG. 5B is a statistical plot of the ratio of WTAP protein fluorescent signals of nuclei and cytoplasm in mouse PBMC immunofluorescence experiments.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (8)
1. Use of WTAP protein nuclear shuttles as markers in any of the following:
(A1) Preparing a product for detecting or assisting in detecting whether a cell to be detected or an organism to be detected suffers from acute ionizing radiation injury; in the application, if the ratio of the expression level of the WTAP protein in the nucleus to the expression level of the WTAP protein in the cytoplasm of the test cell or the test organism is greater than a threshold value, the test cell or the test organism is or is candidate to suffer from acute ionizing radiation damage; otherwise, the cell to be detected or the organism to be detected is or is candidate to be not suffered from acute ionizing radiation injury; the threshold value is the ratio of the expression amount of WTAP protein in cell nucleus to the expression amount in cytoplasm of normal cells or healthy organisms which are not treated by radiation;
(A2) Preparing a product for distinguishing or assisting in distinguishing an acute ionizing radiation injury group from a normal control group; the acute ionizing radiation injury group is a cell or organism suffering from acute ionizing radiation injury; the normal control group is normal cells or healthy organisms which are not subjected to radiation treatment; in the application, a group with relatively high ratio of the expression amount of WTAP protein in the cell nucleus to the expression amount in the cytoplasm is or is candidate as an acute ionizing radiation injury group; a group with relatively low ratio of the expression level of WTAP protein in the cell nucleus to the expression level in the cytoplasm is or is candidate as a normal control group;
in (A1) and (A2), the body is a mouse.
2. Use of a substance for detecting a WTAP protein nucleoplasmic shuttle in any of the following:
(A1) Preparing a product for detecting or assisting in detecting whether a cell to be detected or an organism to be detected suffers from acute ionizing radiation injury; in the application, if the ratio of the expression level of the WTAP protein in the nucleus to the expression level of the WTAP protein in the cytoplasm of the test cell or the test organism is greater than a threshold value, the test cell or the test organism is or is candidate to suffer from acute ionizing radiation damage; otherwise, the cell to be detected or the organism to be detected is or is candidate to be not suffered from acute ionizing radiation injury; the threshold value is the ratio of the expression amount of WTAP protein in cell nucleus to the expression amount in cytoplasm of normal cells or healthy organisms which are not treated by radiation;
(A2) Preparing a product for distinguishing or assisting in distinguishing an acute ionizing radiation injury group from a normal control group; the acute ionizing radiation injury group is a cell or organism suffering from acute ionizing radiation injury; the normal control group is normal cells or healthy organisms which are not subjected to radiation treatment; in the application, a group with relatively high ratio of the expression amount of WTAP protein in the cell nucleus to the expression amount in the cytoplasm is or is candidate as an acute ionizing radiation injury group; a group with relatively low ratio of the expression level of WTAP protein in the cell nucleus to the expression level in the cytoplasm is or is candidate as a normal control group;
in (A1) and (A2), the body is a mouse.
3. The use according to claim 2, characterized in that: the substance for detecting the nuclear shuttle of the WTAP protein is a substance capable of detecting the ratio of the expression quantity of the WTAP protein in the cell nucleus to the expression quantity in the cytoplasm.
4. A use according to claim 3, characterized in that: the substance for detecting the WTAP protein nuclear shuttling consists of the following (B1) and (B2) or is the following (B3):
(B1) Reagents and/or instrumentation for nuclear mass separation;
(B2) Reagents and/or instruments for detecting WTAP protein expression levels;
(B3) Reagents and/or instruments capable of performing in situ quantitative detection of WTAP proteins.
5. Use according to any one of claims 1-4, characterized in that: the ionizing radiation is gamma-ray irradiation of 5-10Gy for 0.5-24h.
6. A system for or aiding in the identification of acute ionizing radiation damage comprising:
(C1) Kit and/or instrument;
the kit and/or instrument has the following functions: detecting the ratio of the expression quantity of WTAP protein in cell nucleus to the expression quantity in cytoplasm;
(C2) A device;
the device comprises a data input module, a threshold storage module, a data comparison module and a judgment module;
the data input module is configured to input (C1) the detected following values: the ratio of the expression level of WTAP protein in cell nucleus to the expression level in cytoplasm from the cell to be tested or the body to be tested;
the threshold storage module is configured to store a threshold; the threshold value is the ratio of the expression amount of WTAP protein in cell nucleus to the expression amount in cytoplasm of normal cells or healthy organisms which are not treated by radiation;
the data comparison module is configured to receive the value detected by the (C1) sent by the data input module, call the threshold value in the threshold value storage module, and compare the ratio of the expression amount of the WTAP protein in the cell nucleus and the expression amount in the cytoplasm of the cell or the organism to be tested with the threshold value;
the judging module is configured to receive the comparison result sent by the data comparing module, and then to judge the result according to the following steps: if the ratio of the expression level of the WTAP protein in the cell nucleus to the expression level of the WTAP protein in the cytoplasm of the cell or the organism to be tested is larger than the threshold value, the cell or the organism to be tested is or is candidate to suffer from acute ionizing radiation damage; otherwise, the cell to be detected or the organism to be detected is or is candidate to be not suffered from acute ionizing radiation injury;
the organism is a mouse.
7. The system according to claim 6, wherein: the kit and/or the instrument consists of the following (B1) and (B2), or is the following (B3):
(B1) Reagents and/or instrumentation for nuclear mass separation;
(B2) Reagents and/or instruments for detecting WTAP protein expression levels;
(B3) Reagents and/or instruments capable of performing in situ quantitative detection of WTAP proteins.
8. The system according to claim 6 or 7, characterized in that: the ionizing radiation is gamma-ray irradiation of 5-10Gy for 0.5-24h.
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| CN113573719A (en) * | 2018-11-08 | 2021-10-29 | 朱诺治疗学股份有限公司 | Methods and combinations of therapy and T cell modulation |
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