Biomarker genetic toxicity detection method based on gamma-H2AX
阅读说明：本技术 一种基于γ-H2AX的生物标志物遗传毒性检测方法 (Biomarker genetic toxicity detection method based on gamma-H2AX ) 是由 黄鹏程 李若婉 周长慧 常艳 于 2018-08-16 设计创作，主要内容包括：本发明公开了一种基于γ-H2AX的生物标志物遗传毒性检测方法。该检测方法包括下述步骤：(1)将与计数微珠混合后的待检测细胞与抗体、以及通透液和封闭液混合、孵育后使用流式细胞仪进行荧光检测；所述的抗体包括抗γ-H2AX抗体、p53蛋白抗体、磷酸化组蛋白H3抗体和抗Cleaved PARP抗体；(2)结果分析：排除死亡细胞,并通过Cleaved-PARP阳性排除凋亡细胞后,根据活细胞的γ-H2AX的表达分析细胞毒性信息。该检测方法检测体外细胞遗传毒性的方法简便、快速、准确并且可以提供多种机制信息的体外遗传毒性检测方法。(The invention discloses a biomarker genetic toxicity detection method based on gamma-H2 AX. The detection method comprises the following steps: (1) mixing the cells to be detected mixed with the counting microbeads with the antibody, the permeable liquid and the confining liquid, incubating and then carrying out fluorescence detection by using a flow cytometer; the antibody comprises an anti-gamma-H2 AX antibody, a p53 protein antibody, a phosphorylated histone H3 antibody and an anti-cleared PARP antibody; (2) and (4) analyzing results: after elimination of dead cells and positive elimination of apoptotic cells by clear-PARP, cytotoxicity information was analyzed based on the expression of gamma-H2AX in living cells. The method for detecting the in vitro cell genotoxicity is simple, convenient, rapid and accurate, and can provide a plurality of mechanism information.)
1. A method for detecting genetic toxicity of a biomarker based on gamma-H2AX, which is characterized by comprising the following steps:
(1) mixing the cells to be detected mixed with the counting microbeads with the antibody, the permeable liquid and the confining liquid, incubating and then carrying out fluorescence detection by using a flow cytometer; the antibodies include anti-gamma-H2 AX antibody and anti-cleared-PARP antibody;
(2) and (4) analyzing results: after elimination of dead cells and positive elimination of apoptotic cells by clear-PARP, cytotoxicity information was analyzed based on the expression of gamma-H2AX in living cells.
2. The method for detecting genetic toxicity of a biomarker according to claim 1, wherein the genetic toxicity is the genetic toxicity of a chemical substance; and/or, the cells to be detected in the step (1) are human lymphoblastic TK6 cells.
3. The method for detecting the genetic toxicity of a biomarker according to claim 1, wherein the method for detecting the genetic toxicity of a biomarker further comprises using p53 gene as the biomarker; preferably, the antibody in step (1) further comprises an anti-p53 protein antibody;
and/or, the biomarker genetic toxicity detection method further comprises the step of taking histone H3 as a biomarker, mixing a part of the cells to be detected mixed with the counting microbeads in the step (1) with an anti-histone H3 antibody, a permeable solution and a blocking solution, incubating, and then carrying out fluorescence detection by using a flow cytometer.
4. The method for detecting genetic toxicity of a biomarker according to claim 1, wherein the blocking solution in step (1) is 1% bovine albumin, and the permeation solution is 1 x Perm/Wash buffer.
5. The method for detecting genetic toxicity of biomarkers according to claim 1, wherein the cells to be detected in step (1) are obtained by immobilizing ① the cells to be detected, eluting ② the immobilized cells to be detected with a reagent containing counting beads, or eluting the immobilized cells to be detected and then adding counting beads, preferably:
the reagent used for fixing in the step ① is 70% ethanol, preferably 70% ethanol pre-cooled at-20 ℃, and the fixing time is 14-18 hours, preferably 16 hours;
and/or the reagent used for elution in the step ② is a PBS solution, and the concentration of the counting microbeads is preferably (0.8-1.2) multiplied by 104one/mL PBS solution, more preferably 1X 10 counting bead concentration4pieces/mL in PBS.
6. The method for detecting genetic toxicity of a biomarker according to any one of claims 1 to 5, wherein the method for detecting genetic toxicity of a biomarker comprises the following steps:
(1) fixing the cells to be detected;
(2) eluting the fixed cell to be detected by using a reagent with counting microbeads, or eluting the fixed cell to be detected and then adding the counting microbeads
(3) Dividing the cells to be detected treated in the step (2) into two parts, mixing and incubating one part with anti-gamma-H2 AX antibody, anti-cleared PARP antibody and anti-p53 protein antibody, as well as the penetration solution and the confining solution, and mixing and incubating the other part with histone H3, as well as the penetration solution and the confining solution; then, carrying out fluorescence detection by using a flow cytometer;
(4) and (4) analyzing results: after dead cells are eliminated and apoptotic cells are positively eliminated through cleaned-PARP, cytotoxicity information is analyzed according to the expression of gamma-H2AX, p53 protein and histone H3 of living cells, and the positive expression of gamma-H2AX and p53 protein can indicate that a genotoxicity mechanism is a breaking agent; positive expression of histone H3 and p53 proteins may suggest that the genotoxic mechanism is an aneuploid inducer; negative expression of gamma-H2AX, histone H3 and p53 proteins suggested no genotoxic effect.
7. A biomarker genotoxicity detection kit based on gamma-H2AX, which is characterized by comprising an anti-cleared PARP antibody and an anti-gamma-H2 AX antibody.
8. The biomarker genotoxicity test kit of claim 7, wherein the kit further comprises an anti-p53 protein antibody and/or an anti-histone H3 antibody.
9. The biomarker genotoxicity detection kit of claim 7 or 8, further comprising PBS, counting beads, blocking fluid, and/or permeabilizing fluid; the confining liquid is preferably 1% bovine albumin, and the penetrating liquid is preferably 1 XPerm/Wash buffer.
Use of cleaned-PARP in a method of genotoxicity detection based on a biomarker of γ -H2 AX.
Technical Field
The invention belongs to the field of genotoxicity detection, and particularly relates to a method for detecting genotoxicity of a biomarker based on gamma-H2 AX.
Background
Among the different DNA damage, DNA double strand breaks are the most serious one, and DNA damage is closely related to the genotoxicity of chemicals. Chemicals with spindle toxicity can affect the synthesis of cellular spindle threads or the formation of spindles, thereby causing cell cycle arrest and apoptosis, and in severe cases, causing aneuploid changes in the chromosome set of cells. Aneuploidy of cellular chromosomes can lead to various diseases, such as trisomy 21 syndrome, etc., and cancer formation is also closely related to aneuploidy of cells. Detecting compound-induced DNA damage and aneuploidy changes in cells is an important marker for assessing compound genotoxicity.
The in vitro test commonly used in the current stage of genotoxicity detection mainly comprises a bacterial back mutation test (Ames test), a mammalian cell chromosome aberration test, a mammalian cell micronucleus test, a mammalian cell comet test and the like. These in vitro detection methods play an important role in the safety evaluation of genetic toxicity of drugs and the like, and are also detection methods mainly used at present at home and abroad. However, these in vitro methods have many limitations: (1) the false positive result rate is higher; (2) the currently used cell lines (such as CHL, CHO-K1 cell lines and the like) for in vitro genotoxicity test are mostly cells of rodent origin, and the test results are extrapolated to the poor compliance of human body tests (Huangpeng, Zhongchang, Changbai. the research progress of the DNA genotoxicity test method based on the gamma-H2AX biomarker [ J ]. Chinese New drug journal, 2016(4):418 and 424.) (3) the cell lines used in the currently used genotoxicity test lack p53 gene, and the DNA damage repair mechanism is incomplete (Erythium prunifolia, Zhongchang, Mega, and Changbai, Changbai. the P53 gene state has influence on the in vitro micronucleus test results [ J ]. Chinese pharmacology and toxicology journal, 2015,29(01):170 and 173.). The existing genotoxicity evaluation of chemicals such as drugs and the like usually uses genotoxicity in vitro experiments and animal models to cause great uncertainty on evaluation results, particularly when the results are extrapolated to humans, and the chemicals can be stopped from being continuously developed due to false positive results. Meanwhile, the conventional in vitro genotoxicity evaluation methods cannot directly provide evaluation indexes of the aneuploidy effect induced by the compound. Aiming at the limitation of the conventional in vitro genotoxicity evaluation method, the humanized in vitro cultured cell strain is applied to genotoxicity evaluation, and a plurality of biomarkers are integrated to detect a DNA double-strand break agent and an aneuploid inducer, so that a good in vitro genotoxicity evaluation method is expected to be established.
Disclosure of Invention
The invention aims to solve the technical problems of high false positive rate, low sensitivity, few detection indexes and the like of a method for detecting in vitro cell genetic toxicity in the prior art, and provides a genetic toxicity detection method of a biomarker based on gamma-H2 AX. The method for detecting the in vitro cell genotoxicity is simple, convenient, rapid and accurate and can provide a plurality of mechanism information.
The main reason for the high false positive rate affecting the γ -H2AX biomarker DNA genotoxicity detection method is the presence of normal dead cells (i.e. apoptotic cells) (Maria Tsamou, Danyel g.j. jennen, sandra m.h.claisen, christina magkoufopoulou, Jos c.s.kleinjans and Joost h.m.vandelft.performance of in vitro γ H2AXassay in HepG2cells to predict in vivo genotoxicity [ J ] Mutagenesis,2012,27(6), 645-; however, how to improve the specificity of detection, reduce the false positive rate and ensure the sensitivity of detection is always an urgent problem to be solved in the field. The inventor creatively selects the cleavage-PARP from a plurality of apoptosis markers such as PI cell nucleus single stain, cell membrane surface phosphatidylserine (Annexin V), Caspase 3(Caspase-3), cleavage substrate cleavage-PARP of DNA repair enzyme PARP and the like for reducing the false positive rate, solves the technical problem in the field, and promotes the development of the detection method of genetic toxicity in the field.
The invention mainly solves the technical problems by the following technical means:
the invention provides a biomarker genetic toxicity detection method based on gamma-H2AX, which comprises the following steps:
(1) mixing the cells to be detected mixed with the counting microbeads with the antibody, the permeable liquid and the confining liquid, incubating and then carrying out fluorescence detection by using a flow cytometer; the antibodies include anti-gamma-H2 AX antibody and anti-cleared PARP antibody;
(2) and (4) analyzing results: after elimination of dead cells and positive elimination of apoptotic cells by clear-PARP, cytotoxicity information was analyzed based on the expression of gamma-H2AX in living cells.
The genetic toxicity is conventional in the art and refers to a toxic effect caused by physical and chemical factors in the environment acting on an organism to cause various damages to genetic materials at the chromosome level, the molecular level and the base level, and the genetic toxicity in the invention is preferably the genetic toxicity of chemical substances.
In order to reduce the false positive rate of the in vitro mammalian cell assay system caused by the species difference, the cells to be detected in the above steps (1) to (2) are preferably human lymphoblastic TK6 cells.
The blocking solution described in step (1) above may be conventional in the art, for example: the confining liquid can be skimmed milk, fetal calf serum and the like, and the penetrating liquid can be Triton X-100, Tween-20, 1 XPerm/Wash buffer and the like. In the invention, the blocking liquid is preferably 1% bovine albumin, and the penetrating liquid is preferably 1 XPerm/Wash buffer. The technical personnel in the field generally add the confining liquid and the permeation liquid in sequence according to the instructions of the confining liquid, however, the inventor tries to mix the confining liquid and the permeation liquid with the cells to be detected and the antibody simultaneously, and unexpectedly finds that the experimental effect is not affected, the operation time can be greatly shortened, and the time cost can be saved. Therefore, in the present invention, the permeation solution and the blocking solution are preferably mixed with the cell to be detected and the antibody simultaneously in a mixed state or in an immiscible state.
Preferably, the method for detecting the genetic toxicity of the biomarkers also takes the p53 gene as the biomarker, and specifically comprises the following steps: in the step (1), the antibody further comprises an anti-p53 protein antibody.
The biomarker genetic toxicity detection method can also use histone H3 as a biomarker, and specifically comprises the steps of mixing a part of cells to be detected mixed with counting microbeads in the step (1) with an anti-histone H3 antibody, a permeable solution and a confining solution, incubating and then carrying out fluorescence detection by using a flow cytometer; the cells were treated with the permeation and blocking solutions containing anti-gamma-H2 AX antibody and anti-cleared PARP antibody (or also anti-p53 protein antibody), respectively.
Preferably, in order to enhance the binding efficiency of the antibody, the cells to be detected in step (1) are obtained by fixing ① the cells to be detected, eluting ② the fixed cells to be detected with a reagent with counting beads, or eluting the fixed cells to be detected and then adding the counting beads, wherein ① to ② are conventional in the art, for example, the fixed reagent in
In a preferred embodiment of the present invention, the method for detecting the genetic toxicity of the biomarker comprises the following steps:
(1) fixing the cells to be detected;
(2) adding counting microbeads after elution or eluting by using a reagent with the counting microbeads;
(3) dividing the cells to be detected treated in the step (2) into two parts, mixing and incubating one part with anti-gamma-H2 AX antibody, anti-CleavedPARP antibody and anti-p53 protein antibody, and the penetration liquid and the confining liquid, and mixing and incubating the other part with histone H3, and the penetration liquid and the confining liquid; then, carrying out fluorescence detection by using a flow cytometer;
(4) and (4) analyzing results: after dead cells are eliminated and apoptotic cells are positively eliminated through cleaned-PARP, cytotoxicity information is analyzed according to the expression of gamma-H2AX, p53 protein and histone H3 of living cells, and the positive expression of gamma-H2AX and p53 protein can indicate that a genotoxicity mechanism is a breaking agent; positive expression of histone H3 and p53 proteins may suggest that the genotoxic mechanism is an aneuploid inducer; negative expression of gamma-H2AX, histone H3 and p53 proteins suggested no genotoxic effect.
The invention also provides a biomarker genetic toxicity detection kit based on gamma-H2AX, wherein the kit comprises an anti-cleared PARP antibody and an anti-gamma-H2 AX antibody; preferably, the biomarker genotoxicity detection kit further comprises an anti-p53 protein antibody and/or an anti-histone H3 antibody. More preferably, the biomarker genotoxicity detection kit further comprises PBS, counting microbeads, a confining liquid and/or a penetrating liquid; the confining liquid is preferably 1% bovine albumin, and the penetrating liquid is preferably 1 XPerm/Wash buffer.
The invention also provides application of the cleaned-PARP in a method for detecting the genetic toxicity of the biomarker based on the gamma-H2 AX.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the invention provides an in vitro genotoxicity detection method based on a flow cytometry analysis method, which is simple, convenient, rapid and accurate and provides a plurality of mechanism information; and the clear-PARP is taken as a biomarker to eliminate apoptotic cells, and counting microbeads are added to calculate cytotoxicity, so that false positive results caused by cytotoxicity are reduced. And multiple genotoxicity detection indexes can be integrated in the same test, so that the method for detecting the potential genotoxicity of the predicted chemicals is low in false positive rate, high in sensitivity, multiple in detection indexes and capable of providing action mechanism information. The method can use human lymphoblastic TK6 cells with wild-type p53 gene as genotoxicity detection materials, and avoids the problem that experimental data cannot be effectively extrapolated due to species difference when microorganisms or animal cell strains are used for genotoxicity detection.
Drawings
FIGS. 1A-F are flow charts for detecting histone H3 phosphorylation.
FIGS. 2A-H are flow charts illustrating the establishment of multiple endpoint detection.
FIGS. 3A-C show the ratios of histone H3 phosphorylation positive cells of ETO, CP and COL.
FIGS. 4A-C are the fold increase in cytotoxicity, apoptosis ratio, and fluorescence intensity of γ -H2AX, p53 protein expression relative to solvent control for ETO, CP, and COL.
FIGS. 5A-C are statistical plots of mean fluorescence intensity of γ -H2AX, mean fluorescence intensity of p53 protein, and phosphorylation ratio of histone H3 after TK6 cells were treated with MMC under +/-S9 conditions, respectively. Compared with the solvent control group, P is less than or equal to 0.05, and P is less than or equal to 0.01.
FIGS. 6A-C are statistical plots of mean fluorescence intensity of γ -H2AX, mean fluorescence intensity of P53 protein, and phosphorylation ratio of histone H3 after TK6 cells were treated with B [ α ] P under +/-S9 conditions, respectively.
FIGS. 7A-C are statistical plots of the mean fluorescence intensity of gamma-H2AX, the mean fluorescence intensity of p53 protein, and the phosphorylation ratio of histone H3 after TK6 cells were treated individually with VCR under +/-S9 conditions. Compared with the solvent control group, P is less than or equal to 0.05, and P is less than or equal to 0.01.
FIGS. 8A-C are statistical plots of mean fluorescence intensity of γ -H2AX, mean fluorescence intensity of p53 protein, and phosphorylation ratio of histone H3 after Amp G has treated TK6 cells under +/-S9 conditions, respectively. Compared with the solvent control group, P is less than or equal to 0.05, and P is less than or equal to 0.01.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Etoposide (cat # E1383), cyclophosphamide (cat # C0768) and RNase (RNaseA) (cat # R6513) were purchased from Sigma; human lymphoblasts TK6 cells were purchased from American Type Culture Collection (ATCC) (Cat. No.: CRL-8015); RPMI-1640 medium (cat # 72400-; cell permeation/washing buffer (BD Perm/Wash)TMBuffer) (cargo number: 51-2091KZ), anti-H2AX (pS139) Alexa
Statistical analysis method
Results of flow cytometry measurements Using BD Accuri C6TMstatistical analysis was performed using IBM SPSS staticiscs 21(Version 21) software, and the phosphorylation ratio of H3 histone between different concentrations of compound and solvent control was determined using Fisher's exact probability method, the four-grid chi-square test, at α ═ 0.05 test level, the fluorescence intensity values for γ -H2AX and p53 protein expression are expressed in fold relation.