CN113355294A - System for detecting eukaryotic cell genetic toxicity and method for detecting eukaryotic cell genetic toxicity - Google Patents
System for detecting eukaryotic cell genetic toxicity and method for detecting eukaryotic cell genetic toxicity Download PDFInfo
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Abstract
The invention discloses a system for detecting the genetic toxicity of eukaryotic cells and a method for detecting the genetic toxicity of the eukaryotic cells. The system for detecting the genetic toxicity of the eukaryotic cell comprises a first eukaryotic cell, a recombinant virus and a second eukaryotic cell; said first eukaryotic cell being reactive to infection by said recombinant virus and comprising a first reporter gene; the first reporter gene is expressed after infection with the recombinant virus; said recombinant virus is capable of replication in said second eukaryotic cell; the recombinant virus contains a second reporter gene, which has reduced or eliminated activity after mutation induction, and a nuclease that causes gene recombination. The invention provides a system and a method for detecting the genetic toxicity of eukaryotic cells by directly adopting the eukaryotic cells, which can effectively judge the genetic toxicity of an object to be detected, avoid the inaccuracy of detecting the compound/medicine eukaryotic genetic toxicity by using prokaryotic bacteria, and improve the accuracy and the practicability of detecting the genetic toxicity of the eukaryotic cells.
Description
Technical Field
The invention relates to the field of medicine and health, in particular to a system for detecting eukaryotic cell genotoxicity, a method for detecting eukaryotic cell genotoxicity and application thereof.
Background
Genotoxicity refers to the toxic effect caused by physical and chemical factors in the environment acting on the organism, causing various damages to its genetic material at the chromosome level, molecular level and base level.
After ten years of efforts, Ames and the like establish and continuously develop and perfect salmonella reverse mutation test (also called Ames test) in 1975, the test is a method for rapidly detecting mutagenicity and potential carcinogenicity of compounds, is a method which is most widely applied in mutation detection worldwide and is also an essential item in the standard test combination of national drug genotoxicity.
For example, chinese patent publication No. CN107478795A, a method for detecting water genotoxicity of city drinking water, discloses a method for detecting water genotoxicity of city drinking water, which comprises: treating a water sample by adopting a reverse osmosis technology; carrying out solid phase extraction on the concentrated water sample subjected to reverse osmosis treatment; respectively carrying out an Ames experiment, a recombinant Escherichia coli SOS effect and a comet experiment to obtain the equivalent concentration of the genotoxicity effect. The method for determining the water body genotoxicity by adopting reverse osmosis, solid-phase extraction and grouped genotoxicity experiments can comprehensively evaluate the genotoxicity of the water body, and solves the problems of complicated pretreatment process, large organic reagent usage amount and the like in the water body genotoxicity determination. The technical scheme provided by the patent is to detect the water body genetic toxicity by using an Ames experiment.
With the development of technology, researchers have also proposed new genotoxicity detection methods based on the Ames assay. For example, chinese patent publication No. CN106636164A, a genotoxic substance detection vector and detection method, discloses a genotoxic substance detection vector and detection method, wherein the vector is an escherichia coli expression vector having a genotoxic response promoter, a phage lytic gene and an escherichia coli terminator connected in sequence from 5 'to 3' end. The detection method comprises the steps of introducing the genetic toxic substance response vector into escherichia coli to obtain recombinant bacteria, incubating the recombinant bacteria and the genetic toxic substance, and cracking escherichia coli cells. The Escherichia coli recombinant strain carries a genotoxic response carrier, the recombinant strain can initiate self cell lysis when contacting genotoxic chemical substances, and the genotoxic substance is quantitatively detected through the lysis efficiency.
However, the methods provided by the prior art have the following commonalities: bacteria from prokaryotic cells are used for detection. The human body or animal body is eukaryotic cell, the result of prokaryotic cell detection is not suitable for the human body or animal body, and the prokaryotic cell detection result has more false positive and false negative. Many scientists have come to appreciate the problem of Ames testing for false positives and false negatives, and even some scientists have studied the methods of Ames testing for false positive and false negative determinations.
Therefore, it is desirable to provide a system and a method for detecting the genetic toxicity of eukaryotic cells, which can detect the genetic toxicity of an object to be detected comprehensively and accurately by using the eukaryotic cells.
Disclosure of Invention
Aiming at the technical problems, the invention provides a system and a method for detecting the genetic toxicity of eukaryotic cells, which adopt the eukaryotic cells to detect the genetic toxicity, reduce the false positive and the false negative of the detection result, and particularly have higher accuracy and practicability for the detection of the drug genetic toxicity.
The invention provides a system for detecting the genetic toxicity of eukaryotic cells, which comprises a first eukaryotic cell, a second eukaryotic cell and a recombinant virus, wherein the recombinant virus can replicate in the second eukaryotic cell;
the recombinant virus contains a second reporter gene and a nuclease that causes gene recombination.
In another preferred embodiment, said first eukaryotic cell is reactive to infection by said recombinant virus.
In another preferred embodiment, the genome of the first eukaryotic cell comprises a first reporter gene; the first reporter gene is expressed only under conditions of infection by the recombinant virus.
In another preferred embodiment, the first reporter gene is selected from at least one of a fluorescent protein, a chromogenic protein with enzymatic activity and a protein that indirectly activates a detectable signal.
In another preferred example, the first reporter gene may be a tomato fluorescent protein.
In another preferred example, the color-developing protein having enzymatic activity is at least one of peroxidase, alkaline phosphatase, and luciferase. Specifically, the color-developing protein having enzymatic activity is an enzyme capable of developing color by acting on a substrate.
In another preferred example, the nuclease is at least one of cre Enzyme (Cyclization Enzyme), Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas system.
In another preferred embodiment, said second reporter gene is mutagenized by a mutagenic agent during replication of said recombinant virus in said second eukaryotic cell. If the second reporter gene is mutated, the activity of the second reporter gene is reduced or eliminated.
In another preferred embodiment, the second reporter gene is selected from at least one of a fluorescent protein, a chromogenic protein having enzymatic activity, and a protein that indirectly activates a detectable signal; and the second reporter gene is not the same as the first reporter gene.
In another preferred embodiment, the recombinant virus is one of adenovirus, adeno-associated virus, lentivirus and retrovirus. The recombinant virus is a virus capable of replicating in eukaryotic cells.
In another preferred embodiment, the first eukaryotic cell is one of a mammalian cell, an insect and plant cell, a fungal cell.
In another preferred embodiment, the second eukaryotic cell is one of a mammalian cell, an insect and plant cell, a fungal cell.
In another preferred embodiment, the first eukaryotic cell and/or the second eukaryotic cell may be a neural stem cell.
The invention also provides an application of the system for detecting the eukaryotic cell genotoxicity in the eukaryotic cell genotoxicity detection.
The invention also provides a detection method of the eukaryotic cell genetic toxicity, which adopts the system for detecting the eukaryotic cell genetic toxicity and comprises the following steps:
step one, infecting a second eukaryotic cell by adopting a recombinant virus;
step two, placing the infected second eukaryotic cell in the step one into a solution of an object to be detected, and replicating the recombinant virus;
step three, placing the infected second eukaryotic cell in the step one into a negative control solution, and replicating the recombinant virus; wherein the negative control solution does not contain a factor that induces a mutation in a second reporter gene;
step four, infecting the recombinant viruses obtained in the step two and the step three with first eukaryotic cells respectively to ensure that a second reporter gene in the recombinant viruses has sufficient expression;
step five, activating the second reporter gene, and observing the activity of the second reporter gene obtained in the step four; and respectively obtaining the activity of the second reporter genes after the recombinant viruses of the object solution group to be detected and the negative control solution group infect the first eukaryotic cell, wherein the activity ratio of the two groups of second reporter genes represents the genetic toxicity of the eukaryotic cells of the object solution to be detected.
In another preferred example, the detection method further includes:
infecting the recombinant viruses obtained in the second step and the third step with first eukaryotic cells containing a first reporter gene respectively;
activating the first reporter gene and observing its activity; activating the second reporter gene and observing its activity; obtaining the activity of a first reporter gene and the activity of a second reporter gene after the recombinant viruses of the solution group of the substance to be detected and the negative control solution group infect a first eukaryotic cell containing the first reporter gene;
and taking the ratio of (the activity of the second reporter gene of the solution group to be detected/the activity of the first reporter gene of the solution group to be detected)/(the activity of the second reporter gene of the negative control solution group/the activity of the first reporter gene of the negative control solution group) as the eukaryotic cell genotoxicity index of the solution to be detected. Thus, the eukaryotic cell genetic toxicity of the object to be detected can be judged more comprehensively and accurately.
The invention also provides a kit for detecting the genetic toxicity, and the kit comprises the system for detecting the genetic toxicity of the eukaryotic cells. Specifically, the kit for detecting the genetic toxicity comprises a first eukaryotic cell, a second eukaryotic cell and a recombinant virus in the system for detecting the genetic toxicity of the eukaryotic cell, and can realize the detection of the genetic toxicity of the eukaryotic cell of the solution to be detected.
The kit can be used for detecting the genetic toxicity of the eukaryotic cells.
In the present invention, the activity of the first reporter gene is detected, i.e., the recombinant virus transfection is successful.
In the present invention, if the activity of the second reporter gene in the test substance solution group is lower than the activity of the second reporter gene in the negative control solution group, the test substance solution induces the mutation of the second reporter gene.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention provides a brand-new system for detecting the genetic toxicity of eukaryotic cells, which can detect the genetic toxicity by utilizing the eukaryotic cells.
The recombinant virus contains nuclease (cre enzyme) causing gene recombination and a second reporter gene, so that whether eukaryotic cells are infected by the recombinant virus can be judged under proper conditions, and the condition of cell mutation can be seen by the second reporter gene, so that the combination of the two data can detect the genetic toxicity more comprehensively and accurately. Furthermore, the invention can combine the data of the control group (negative control solution) and avoid the inaccurate detection result caused by silent mutation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Fig. 1 is a first schematic view of a light emitting principle in embodiment 5 of the present invention;
FIG. 2 is a second schematic view of the light-emitting principle in embodiment 5 of the present invention;
FIG. 3 is a graph showing the results of luminescence of fluorescent proteins in 6 experiments of example 6 of the present invention under excitation light;
FIG. 4 is a graph showing the results of luminescence of fluorescent proteins in 3 experiments of example 7 of the present invention under excitation light;
FIG. 5 is a graph of mean mutation results of 3 experiments in example 7 of the present invention.
Examples of the embodiments
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention. In the description of the present invention, the term "including" means "including but not limited to". The terms first, second, third and the like are used merely as labels, and do not impose numerical requirements or an established order.
Example 1
The embodiment of the invention provides a system for detecting the genetic toxicity of eukaryotic cells, which comprises a first eukaryotic cell, a second eukaryotic cell and a recombinant virus, wherein the recombinant virus can replicate in the second eukaryotic cell;
the recombinant virus contains a second reporter gene and a nuclease that causes gene recombination.
The first eukaryotic cell is reactive to infection by the recombinant virus.
The nuclease is at least one of cre Enzyme (Cyclization Enzyme, namely Cyclization recombinase), Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas system.
The second reporter gene may be mutagenized by a mutagen/mutagen during replication of the recombinant virus in the second eukaryotic cell.
The recombinant virus is a virus capable of replicating in eukaryotic cells.
The recombinant virus can be any one of adenovirus, adeno-associated virus, lentivirus and retrovirus. The first eukaryotic cell may be any one of mammalian cells, insect and plant cells, fungal cells. The second eukaryotic cell may be any one of a mammalian cell, an insect, a plant cell, and a fungal cell.
Example 2
The embodiment of the invention provides a system for detecting the genetic toxicity of eukaryotic cells, which comprises a first eukaryotic cell, a second eukaryotic cell and a recombinant virus, wherein the recombinant virus can replicate in the second eukaryotic cell;
the recombinant virus comprises a second reporter gene and a nuclease that causes gene recombination.
The first eukaryotic cell is reactive to infection by the recombinant virus.
The genome of the first eukaryotic cell contains a first reporter gene; the first reporter gene is expressed only under conditions of infection by the recombinant virus.
The first reporter gene is at least one selected from the group consisting of a fluorescent protein, a chromogenic protein having enzymatic activity, and a protein that indirectly activates a detectable signal.
The chromogenic protein with enzyme activity is at least one selected from peroxidase, alkaline phosphatase and luciferase. The enzyme having an enzymatic activity is an enzyme capable of developing a color by acting on a substrate.
The nuclease is selected from at least one of cre Enzyme (Cyclization Enzyme, i.e. Cyclization recombinase), Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas system.
The second reporter gene may be mutagenized by a mutagen/mutagen during replication of the recombinant virus in the second eukaryotic cell.
The recombinant virus is a virus capable of replicating in eukaryotic cells.
The recombinant virus can be any one of adenovirus, adeno-associated virus, lentivirus and retrovirus. The first eukaryotic cell may be any one of mammalian cells, insect and plant cells, fungal cells. The second eukaryotic cell may be any one of a mammalian cell, an insect, a plant cell, and a fungal cell.
Example 3
The invention provides a method for detecting the genetic toxicity of eukaryotic cells, which adopts the system for detecting the genetic toxicity of the eukaryotic cells in embodiment 1, and comprises the following steps:
step one, infecting a second eukaryotic cell by adopting a recombinant virus;
step two, placing the infected second eukaryotic cell in the step one into a solution of an object to be detected, and replicating the recombinant virus;
step three, placing the infected second eukaryotic cell in the step one into a negative control solution, and replicating the recombinant virus; wherein the negative control solution does not contain a factor that induces a mutation in a second reporter gene;
step four, infecting the recombinant viruses obtained in the step two and the step three with first eukaryotic cells respectively to ensure that a second reporter gene in the recombinant viruses has sufficient expression;
step five, activating the second reporter gene, and observing the activity of the second reporter gene obtained in the step four; and respectively obtaining the activity of the second reporter genes after the recombinant viruses of the object solution group to be detected and the negative control solution group infect the first eukaryotic cell, wherein the activity ratio of the two groups of second reporter genes represents the genetic toxicity of the eukaryotic cells of the object solution to be detected.
Example 4
The invention provides a method for detecting the genetic toxicity of eukaryotic cells, which adopts the system for detecting the genetic toxicity of the eukaryotic cells of the embodiment 2, and comprises the following steps:
step one, infecting a second eukaryotic cell by adopting a recombinant virus;
step two, placing the infected second eukaryotic cell in the step one into a solution of an object to be detected, and replicating the recombinant virus;
step three, placing the infected second eukaryotic cell in the step one into a negative control solution, and replicating the recombinant virus; wherein the negative control solution does not contain a factor that induces a mutation in a second reporter gene;
step four, infecting the recombinant viruses obtained in the step two and the step three with first eukaryotic cells containing first reporter genes respectively to ensure that second reporter genes in the recombinant viruses have sufficient expression;
step five, activating the first reporter gene and observing the activity of the first reporter gene; activating the second reporter gene and observing its activity; obtaining the activity of the first reporter gene and the second reporter gene after the recombinant viruses of the object solution group to be detected (step two) and the negative control solution group (step three) infect the first eukaryotic cell containing the first reporter gene;
and taking the ratio of (the activity of the second reporter gene of the solution group to be detected/the activity of the first reporter gene of the solution group to be detected)/(the activity of the second reporter gene of the negative control solution group/the activity of the first reporter gene of the negative control solution group) as the eukaryotic cell genotoxicity index of the solution to be detected.
The detection method provided by the embodiment of the invention can comprehensively and accurately judge the eukaryotic cell genotoxicity of the object to be detected.
Example 5
The embodiment of the invention provides a system for detecting the genetic toxicity of eukaryotic cells, which comprises a first eukaryotic cell, a second eukaryotic cell and a recombinant virus, wherein the recombinant virus can replicate in the second eukaryotic cell; the first eukaryotic cell is reactive to infection by the recombinant virus.
The recombinant virus contains a second reporter gene (gfp gene) and a cre enzyme (cre gene) that causes gene recombination. The genome of the first eukaryotic cell contains a first reporter gene (tomato fluorescent protein).
The method for detecting the genetic toxicity of the eukaryotic cells comprises the following steps:
step one, infecting the second eukaryotic cell by the recombinant virus;
step two, placing the infected second eukaryotic cell in the step one into a solution of an object to be detected, and replicating the recombinant virus;
step three, placing the infected second eukaryotic cell in the step one into a negative control solution, and replicating the recombinant virus; wherein the negative control solution does not contain a mutagen that induces a mutation in a second reporter gene;
and step four, infecting the first eukaryotic cell with the recombinant viruses obtained in the step two and the step three respectively to ensure that the second reporter gene in the recombinant viruses has sufficient expression.
In this example, the cre gene can cleave the stop codon of the second reporter gene of the cell, which can make the cell display red under the excitation light; if the gfp gene of part of the recombinant virus is mutated under the stimulation of a mutagen, the virus will show green color. Red can be used to judge that the cell is infected by the virus, green can be used to judge cell mutation; silent mutations can be distinguished based on negative control tests. Fig. 1 and fig. 2 show the light emitting principle of the present embodiment.
That is, the present invention can determine whether the second reporter gene (e.g., gfp gene) is mutated by fluorescence development under excitation light, i.e., determine whether the analyte solution contains a mutagen.
Example 6
The main experimental materials: 1X 109pfu/mL recombinant adenovirus containing cre gene and gfp gene, tissue culture dish, DMEM medium containing 10% FBS, 5-bromouracil, cyclosporine, dimethyl sulfoxide, sterilized water, test tube rack, centrifuge, neural stem cell, pipettor, cell counter, cell culture box, 15mL centrifuge tube, 50mL calcined adenovirusCup, 1.75mL tube, centrifuge tube, microscope.
The experimental method and the steps are as follows:
1. preparation of cell suspensions
1.1) HEK293 cell count without reporter gene;
A. shaking the culture dish containing trypsin to disperse the cells;
B. adding 50mL of culture medium, and mixing by pipetting;
C. adding 10ul of cell suspension into 90ul of culture medium;
D. the diluted solution is removed to prepare a slide specimen, and cell counting is carried out under a microscope.
1.2) subpackaging the cell suspension with 1mL of each tube
1.3) placing the tube containing the cell suspension in a cell incubator for culture
2. Preparation of mutagenic solution
2.1) Cyclosporin
1mg/mL of cyclosporin was diluted to 1ug/mL with DMSO, and then 1ug/mL of cyclosporin was diluted to 100ng/mL and 400ng/mL, 5mL each, with cell culture medium.
2.2) 5-Bromopluracil
1mg/mL of 5-bromouracil was diluted to 1ug/mL with dimethyl sulfoxide, and then 1ug/mL of 5-bromouracil was diluted to 1ug/mL and 5ug/mL each with 5mL of cell culture medium.
3. Adenovirus infection, mutation induction
3.1) adding 1mL of cell suspension to each well of a 6-well cell culture plate;
3.2) after 24 hours, removing cell culture supernatant after the cells adhere to the wall;
3.3) adding 1mL of cell culture medium and a proper amount of solution to be detected (in the experiment, the final concentration is 1ug/mL or 5ug/mL of 15-bromouracil, and the final concentration is 100ng/mL and 400ng/mL of cyclosporine) or a drug solvent (in the experiment, dimethyl sulfoxide) into each hole of the cell culture plate;
3.4) adding adenovirus to the cells, the ratio of virus to cell number (MOI) being 10;
3.5) culturing the cells in a carbon dioxide incubator at 37 ℃ for 48 hours;
3.6) removing the cell culture medium, gently washing the cells with 2mL of the EDTA-containing medium, adding 0.2mL of Trypsin (Trypsin-EDTA), incubating at 37 ℃ for 3-5 minutes,
3.7) when the cells float, gently washing the cells by using a 1mL pipette to separate the cells from the bottom of the culture plate;
3.8) add 1.8mL DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin; cells were broken up with a 1mL pipette;
3.9) transferring the cells to a 15mL centrifuge tube, and centrifuging at 1000 g; removing the cell supernatant;
3.10) adding 1mL of sterile PBS, quickly freezing in a dry ice/alcohol bath, thawing in a constant-temperature water bath at 37 ℃, and repeating for three times;
3.11) centrifuging at 1500g for 2 minutes, taking the supernatant, and detecting the virus titer;
3.12) if desired, steps 3.1) -3.11) can be repeated a number of times;
4. cell fluorescence development
4.1) HEK293 cells were infected with the viruses 3.1) -3.12) at an MOI (ratio of virus to cell) of 10;
4.2) culturing the cells in a carbon dioxide incubator at 37 ℃ for 12-20h
4.3) shooting under the irradiation of bright field and blue exciting light respectively;
4.4) Total cell count and Green cells were counted to observe the proportion of Green cells.
5. And (4) analyzing results: as shown in fig. 3, the results of fluorescence experiments for infection of HEK293 cells with viruses replicated after treatment with different cell mutation-inducing agents are shown.
Two control experiments were also performed in this example, a blank control (no virus infection) and a negative control (adenovirus group with recombinant GFP gene but no mutagen).
Referring to fig. 3, it can be found that:
HEK293 cells in the blank control group (Untreated) did not develop fluorescence;
a considerable number of HEK293 cells in the negative control group (adonovirus only) appeared green under blue excitation light;
compared with the control group (a blank control group and a negative control group), the four groups of the 5ug/mL 5-bromouracil group, the 1ug/mL 5-bromouracil group, the 400ng/mL cyclosporine group and the 100ng/mL cyclosporine group can find that cells showing green under blue excitation light in the four groups of experiments are obviously reduced, which indicates that the GFP gene is subjected to more mutations after the four groups are treated by the mutagen.
Example 7
Referring to fig. 3, the results of the control experiment of 6 groups of neural stem cells in example 6 are shown, and 3 groups of experiments are performed in this example, which respectively include: the neural stem cells (the first eukaryotic cell, recombinant tdTomato is arranged in a genome, a terminator stop codon with floxed at two ends is arranged in front of the tdTomato), 5ug/mL 5-bromouracil-induced mutant adenovirus-infected neural stem cells containing recombinant GFP genes, and 400ng/mL cyclosporine-induced mutant adenovirus-infected neural stem cells containing recombinant GFP genes; referring to FIG. 4, the control group (without the mutagen), 5ug/mL 5-bromouracil group, and 400ng/mL cyclosporin group were designated in FIG. 4, respectively.
II, an experimental method:
5.1) adding 1mL of neural stem cell suspension to each well of a 6-well cell culture plate;
5.2) after 24 hours, removing cell culture supernatant after the cells adhere to the wall;
5.4) adding 3.1) -3.12) of the prepared adenovirus to the cells, the ratio of virus to cell number (MOI) being 1;
5.5) culturing the cells in a carbon dioxide incubator at 37 ℃ for 48 hours;
5.6) shooting under the irradiation of bright field, yellow-green exciting light (exciting tdTomato red fluorescence) and blue exciting light (exciting green fluorescence of GFP) respectively; cells with high brightness highly express the corresponding fluorescent protein in monochromatic photographs under each excitation light.
5.7) the number of red and green cells was counted and the ratio green/red cells was calculated.
Thirdly, analyzing results:
the results are shown in FIGS. 4 and 5.
As shown in fig. 4, there are 3 sets of experiments, which are:
a first group: neural stem cells without a mutagen;
second group: the mutant is 5ug/mL 5-fluorouracil neural stem cells;
third group: neural stem cells mutated to 400ng/mL cyclosporin.
As can be seen from FIG. 4, the virus-infected neural stem cells without the treatment of the mutagen showed more red and green, and the number and morphology of the cells of the two colors were substantially the same, thus it can be seen that the virus infecting the first group of cells had almost no mutation; ② the number of cells which show red fluorescence is obviously more than that of cells which show green by virus infected neural stem cells which are treated by 5ug/mL of 5-fluorouracil, thus showing that GFP of the second group of viruses has more mutation; ③ the virus-infected neural stem cells treated with cyclosporin in an amount of 400ng/mL showed a significantly higher number of red cells than green cells, indicating that the virus GFP in the third group of cells was also more mutated.
Based on the above results, the genetic toxicity of 5ug/mL 5-fluorouracil and 400ng/mL cyclosporin for eukaryotes could be interpreted. The mutation rate was 1- (sample group ratio of green red blood cells/treatment group ratio of green red blood cells).
Referring to fig. 5, there is shown the results of statistical analysis of 3 experiments (e.g., fig. 4) of this example, the ordinate represents the mean mutation rate at 95% confidence interval, and the abscissa represents the results of the first, third and second groups, respectively, wherein p <0.0001 for the third group and p <0.006 for the second group; all have statistical significance. According to the results shown in the figure, the experimental group has more mutations compared with the control group, so that the virus, the cell and the method provided by the invention can be used for detecting the genetic toxicity of the detected object to the eukaryotic cell.
The 5-bromouracil in the embodiment of the present invention, which is a base analog, is a known mutation inducer, and has a structure similar to that of dTMP except that the methyl group of T is substituted with bromine at the fifth carbon atom. 5-bromouracil produces two tautomers, one in the keto form and one in the enol form. The keto form can be complementarily paired with A, and the enol form can be complementarily paired with G, which results in base transition mutation when 5-bromouracil is incorporated into DNA for replication. Thus, 5-bromouracil can convert AT to GC, causing gene mutation.
Cyclosporin of the present embodiment is represented by formula C62H111N11O12In 2017, 10, 27, the national cancer research institute promulgates a list of carcinogens, cyclosporine is also known as a mutation inducer in a list of carcinogens.
In conclusion, the system for detecting the eukaryotic cell genotoxicity can detect the eukaryotic cell genotoxicity of the solution to be detected, and can comprehensively and accurately detect the genotoxicity. The invention has higher accuracy and practicability for the detection of the drug genotoxicity.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (12)
1. A system for detecting the genotoxicity of a eukaryotic cell, comprising a first eukaryotic cell, a second eukaryotic cell and a recombinant virus; said recombinant virus is capable of replication in said second eukaryotic cell;
the recombinant virus contains a second reporter gene and a nuclease that causes gene recombination.
2. The system for detecting the genetic toxicity of eukaryotic cells according to claim 1, wherein said first eukaryotic cell is reactive to infection by said recombinant virus; the genome of the first eukaryotic cell contains a first reporter gene; the first reporter gene is expressed only under conditions of infection by the recombinant virus.
3. The system for detecting the genotoxicity of eukaryotic cells according to claim 2, wherein the first reporter gene is at least one selected from the group consisting of a fluorescent protein, a chromogenic protein having enzymatic activity, and a protein that indirectly activates a detectable signal.
4. The system for detecting the genetic toxicity of the eukaryotic cells according to claim 3, wherein the color-developing protein having the enzymatic activity is at least one of peroxidase, alkaline phosphatase, and luciferase.
5. The system for detecting the genotoxicity of eukaryotic cells according to claim 1, wherein the nuclease is at least one selected from cre enzyme, zinc finger nuclease, transcription activator-like effector nuclease, CRISPR/Cas system.
6. The system for detecting the genotoxicity of eukaryotic cells according to claim 1, wherein the second reporter gene is mutated by a mutagenic factor during replication of the recombinant virus in the second eukaryotic cell.
7. The system for detecting the genotoxicity of eukaryotic cells according to claim 2 or 6, wherein the second reporter gene comprises at least one of a fluorescent protein different from the first reporter gene, a chromogenic protein having an enzymatic activity and a protein indirectly activating a detectable signal.
8. The system for detecting the genetic toxicity of the eukaryotic cell according to claim 1, wherein the recombinant virus is one of adenovirus, adeno-associated virus, lentivirus and retrovirus.
9. The system for detecting the genotoxicity of eukaryotic cells according to claim 1, wherein the first eukaryotic cell is one of a mammalian cell, an insect and plant cell, a fungal cell; and/or, the second eukaryotic cell is one of a mammalian cell, an insect, a plant cell, and a fungal cell.
10. Use of a system for the detection of the genotoxicity of eukaryotic cells according to any one of claims 1 to 9 for the detection of genotoxicity of eukaryotic cells.
11. A method for detecting the genetic toxicity of eukaryotic cells, which is characterized by adopting the system for detecting the genetic toxicity of eukaryotic cells as claimed in any one of claims 1 to 8, and comprises the following steps:
step one, infecting a second eukaryotic cell by adopting a recombinant virus;
step two, placing the infected second eukaryotic cell in the step one into a solution of an object to be detected, and replicating the recombinant virus;
step three, placing the infected second eukaryotic cell in the step one into a negative control solution, and replicating the recombinant virus; wherein the negative control solution does not contain a factor that induces a mutation in a second reporter gene;
step four, infecting the recombinant viruses obtained in the step two and the step three with first eukaryotic cells respectively to ensure that a second reporter gene in the recombinant viruses has sufficient expression;
step five, activating the second reporter gene, and observing the activity of the second reporter gene obtained in the step four; and respectively obtaining the activity of the second reporter genes after the recombinant viruses of the object solution group to be detected and the negative control solution group infect the first eukaryotic cell, wherein the activity ratio of the two groups of second reporter genes represents the genetic toxicity of the eukaryotic cells of the object solution to be detected.
12. The method for detecting the genetic toxicity of eukaryotic cells according to claim 11, wherein the method further comprises:
infecting the recombinant viruses obtained in the second step and the third step with first eukaryotic cells containing a first reporter gene respectively;
activating the first reporter gene and observing its activity; activating the second reporter gene and observing its activity; obtaining the activity of a first reporter gene and the activity of a second reporter gene after the recombinant viruses of the solution group of the substance to be detected and the negative control solution group infect a first eukaryotic cell containing the first reporter gene;
and taking the ratio of (the activity of the second reporter gene of the solution group to be detected/the activity of the first reporter gene of the solution group to be detected)/(the activity of the second reporter gene of the negative control solution group/the activity of the first reporter gene of the negative control solution group) as the eukaryotic cell genotoxicity index of the solution to be detected.
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