CN107132344A - A kind of method that DNA Damage is caused based on high intension technology quantitative analysis aldehyde material - Google Patents

Abstract

The invention discloses a method for quantitatively analyzing cell DNA damage caused by aldehydes based on high-content technology. The method includes the following steps: 1) cell culture, 2) cell poisoning, 3) immunofluorescent labeling of γH2AX, 4) High-content technical quantitative analysis. The advantages of the present invention are: the automatic imaging of the high-content imaging system is used to quantitatively analyze the DNA double-strand break marker γH2AX protein induced by aldehydes, and the direct and rapid detection of cells is realized, which makes sample processing more convenient; high resolution Imaging, not only makes the distribution of γH2AX in the nucleus can be directly observed but also makes the image data easy to store and reanalyze, and can realize the quantitative analysis of γH2AX induced by aldehydes in each nucleus, and the detection result is more sensitive and accurate .

Description

A method based on high-content technology for quantitative analysis of cellular DNA damage caused by aldehydes method

technical field

The invention belongs to the technical field of in vitro detection of DNA damage, and more specifically relates to an in vitro quantitative analysis method for cell DNA damage caused by aldehydes.

Background technique

Aldehydes are a class of active substances containing carbonyl functional groups that can produce various toxic effects without metabolism. Small molecule saturated and unsaturated aldehydes such as formaldehyde, acetaldehyde and acrolein are a kind of common air and environmental pollutants. At present, the International Organization for Research on Cancer (IARC) under the World Health Organization (WHO) has classified Acrolein is listed as Class 1, Class 2 and Class 3 carcinogens respectively, and the World Health Organization Framework Convention on Tobacco Control (FCTC) lists formaldehyde, acetaldehyde and acrolein as one of the priority smoke control pollutants. A large amount of evidence shows that aldehydes have a variety of genotoxic effects, such as gene mutation, DNA breakage, chromosome aberration, etc. Therefore, accurate detection of acetaldehyde-induced cellular DNA damage is of great significance for genotoxicity and hazard evaluation of acetaldehyde.

DNA double-strand break is the most serious form of DNA damage. If this damage cannot be repaired correctly or in time, it may lead to chromosomal aberration or cell apoptosis. As a biomarker of DNA double-strand breaks, phosphorylated histone γH2AX has been widely used in clinical medicine, radiology and toxicology research. Including many monomeric compounds and mixtures, their genotoxicity was determined and evaluated by γH2AX experiment. For example, Tanaka et al. used γH2AX experiment to detect and evaluate the genotoxicity of cigarette smoke condensate; The effect relationship was studied. Due to its high sensitivity, large-scale analysis and detection in combination with other instrument detection techniques, and many advantages that other genotoxicity detection techniques do not have, it has been widely used in the genotoxicity screening and screening of compounds and toxic substances. Evaluation.

At present, the main methods for analysis and detection of γH2AX include flow cytometry, ELISA (enzyme-linked immunosorbent assay), Western Blot (western blotting), microscopy and so on. Flow cytometry and Western Blot are cumbersome to operate. Adherent cells need to be enzymatically hydrolyzed into a single-cell suspension before detection, which destroys the structural and functional integrity of the cells, and the detection throughput is low. ELISA cannot provide the distribution of fluorescent focal points in cells and needs to add other detection proteins to correct the results, while the detection throughput of microscopy technology is low, and the error of human counting is large.

Contents of the invention

In view of the above problems, the purpose of the present invention is to provide a simple, effective and highly sensitive quantitative analysis method for DNA damage caused by aldehydes without destroying cells. The detection results of this method are accurate and visualized, so as to overcome the defects of the prior art .

The object of the present invention is achieved by combining high content technology with quantitative analysis of γH2AX. Specifically, the purpose of the present invention is achieved through the following technical solutions:

A method for quantitative analysis of cellular DNA damage caused by aldehydes based on high-content technology, the method comprising the following steps:

1) Cell culture: inoculate cells in cell culture medium for cell culture;

2) Cell poisoning: sucking and discarding the cell culture solution, adding cell poisoning solution to continue culturing, the cell poisoning solution contains aldehydes and cell culture solution;

3) Immunofluorescence labeling: aspirating and discarding the venom solution of the cells, performing immunofluorescence labeling of γH2AX in the cells and DAPI staining of the nuclei;

4) Quantitative analysis with high-content technology: Fluorescence measurement of the nuclei and γH2AX of the cells were carried out, and the content of γH2AX was characterized by the average fluorescence intensity detected in the effective nuclei of the identified effective cells.

Preferably, in the step 1), the cells are adherent growth cells; more preferably, when inoculated, the cells are in the logarithmic growth phase, and are inoculated in the cell culture medium as a single cell suspension;

More preferably, after cell inoculation, the cells are cultured in the cell culture solution for 24 hours at 37° C. and 5% CO ₂ .

Preferably, in the step 2), aldehydes refer to compounds containing saturated or unsaturated short-chain fatty aldehyde groups; preferably, the aldehydes are saturated or unsaturated C1-C3 fatty aldehydes, more preferably , the aldehyde substance is formaldehyde or acetaldehyde, or the aldehyde substance is acrolein;

More preferably, the concentration of aldehydes in the cell poisoning solution is 0-1000 μmoL/mL, preferably >0 and ≤1000 μmoL/mL, more preferably 7.81-1000 μmoL/mL; or, the aldehydes in the cell poisoning solution The concentration of the substance is 0-180 μmoL/mL, preferably >0 and ≤ 180 μmoL/mL, more preferably 20-180 μmoL/mL;

According to a specific embodiment of the present invention, the concentration of aldehydes in the cell poisoning solution may be 0, 7.81, 15.63, 31.25, 62.50, 125, 250, 500 or 1000 μmoL/L, or the concentration of aldehydes in the cell poisoning solution The concentration of the substance may be 0, 20, 40, 60, 80, 100, 120, 140, 160 or 180 μmol/L.

Preferably, the cells are cultured in the cell exposure solution for 1-24 hours, preferably 24 hours.

Preferably, said step 3) includes:

3-1) Absorb and discard the cell poisoning solution, wash the cells, and add paraformaldehyde to fix the cells;

3-2) Wash the cells, then add Triton-100X to permeabilize the cells;

3-3) Wash the cells, then add serum albumin to block, and then add the primary antibody, namely anti-γH2AX antibody, to incubate;

3-4) washing the cells, and then adding an immunofluorescence-labeled secondary antibody for incubation;

3-5) washing the cells, adding DAPI (4',6-diamidino-2-phenylindole) for staining;

3-6) Wash the cells and save them.

Preferably, in the step 4), the fluorescence measurement of the cell nucleus and γH2AX is carried out at two different excitation wavelengths and emission wavelengths;

Preferably, in the step 3-4), the immunofluorescence-labeled secondary antibody is an Alexa Fluor 488-labeled secondary antibody, and in the step 4), the cell nucleus is detected in channel one with an excitation wavelength of 358nm and an emission wavelength of 461nm. Fluorescence measurement, the fluorescence measurement of γH2AX is carried out in channel 2 with an excitation wavelength of 495nm and an emission wavelength of 519nm, so as to obtain the average fluorescence intensity detected by channel 2 in the effective nucleus of the effective cells identified by channel 1, thereby characterizing the content of γH2AX;

More preferably, in two channels, the measurement magnification is 200 times, and 9 fields of view are analyzed and determined per well.

According to a specific embodiment of the present invention, step 4) is performed using a high-content cell analysis system, such as a model ArrayScan VTI600 high-content cell analysis system purchased from Thermo Scientific.

Optionally, the method of the present invention also includes setting a blank control group in which no primary antibody is added in step 3-3), and only a secondary antibody is added in step 3-4), so that in step 4), the non-specific The blank signal due to adsorption is thereby subtracted from the detected fluorometric signal.

The methods of the invention may also include the preparation of dose-response curves. That is, by setting a variety of cell poisoning solutions containing different concentrations of aldehydes in step 2), the method is carried out, and finally a dose-effect curve is drawn according to the average fluorescence intensity of γH2AX and the concentration of aldehydes.

Alternatively, the method of the present invention may also include drawing a time-effect curve. That is, the method is carried out by culturing the cells in the cell poisoning solution containing the same concentration of aldehydes for different time in step 2), and finally the time-effect curve is drawn according to the average fluorescence intensity of γH2AX and the culture time.

According to a specific embodiment of the present invention, when the cell is human lung cancer cell line A549, the method of the present invention can be carried out as follows:

1) Cell culture: use RPMI-1640 culture solution containing 10% FBS and 2mmoL/L L-glutamine to cultivate human lung cancer cell line A549 in an incubator at 37°C and 5% CO ₂ , when the cell confluency When it reaches 70-80%, use 0.25% trypsin to digest and obtain a single cell suspension, and then inoculate a 96 ^- well cell culture plate with a concentration of 105 cells/mL in 100 μL per well. FBS and 2mmoL/L L-glutamine in RPMI-1640 culture medium were cultured at 37°C and 5% CO ₂ for 24h.

2) Cell poisoning: discard the cell culture solution after 24 hours of culture, add the cell poisoning solution to continue the culture, and the cell poisoning solution contains 0, 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 μmol/L respectively Formaldehyde or acetaldehyde or 0, 20, 40, 60, 80, 100, 120, 140, 160 and 180 μmol/L acrolein in the cell culture solution in step 1). At least two groups of blank controls were set in the concentration group. The blank control group was only added with RPMI-1640 culture solution containing 10% FBS and 2mmoL/L L-glutamine, and cultured at 37°C and 5% CO ₂ for 24h.

3) Immunofluorescence labeling:

3-1) Discard the cell poisoning solution, add 100 μL PBS (phosphate buffered saline, pH 7.2-7.4; the same for the text) to wash the cells twice, each time at least 5 min; then add 50 μL 4% paraformaldehyde to each well The solution was fixed at room temperature for 15 min;

3-2) The fixed cells were washed twice with PBS for at least 5 minutes each time; then 50 μL of 0.5% Triton-100X solution (in PBS) was added to permeabilize for 15 minutes at room temperature;

3-3) The permeabilized cells were washed twice with PBS for at least 5 minutes each time; then each well was blocked with 3% BSA blocking solution (in PBS) at 37°C for 1 hour, and then 50 μL of mouse anti-human γH2AX antibody solution (1:200, v/v), add only 50 μL 1% BSA to a group of blank wells as a negative control well, and add 50 μL mouse anti-human γH2AX antibody solution as a blank control well to another group of blank wells, and incubate The conditions are: incubate at 37°C for 2 hours or overnight at 4°C;

3-4) After the primary antibody incubation, the cells were washed three times with PBS for at least 5 minutes each time; then, 50 μL of Alexa Fluro 488-labeled goat anti-mouse antibody solution (1:200, v/v) was added to each well and kept at 37°C. Light incubation for 2h;

3-5) The cells incubated with the secondary antibody were washed three times with PBS for at least 5 minutes each time; then, 50 μL of 1 μg/mL DAPI (in PBS) was added to stain the nucleus for 10 minutes at room temperature;

3-6) After washing with PBS three times, add 100 μL of PBS to each well, store in the dark at 4°C, and wait for testing.

4) Quantitative analysis of high-content technology: set the excitation wavelength and emission wavelength to 358nm and 461nm in channel one (fluorescence measurement of cell nuclei), set the excitation wavelength and emission wavelength to 495nm and 519nm in channel two (fluorescence measurement of target γH2AX), and measure The multiplier was 200, and 9 fields of view were analyzed and measured in each well, and the content of γH2AX was characterized by the average fluorescence intensity detected in channel 2 of effective cells identified in channel 1 and in the effective nucleus of channel 2.

5) Data processing: set a blank control group in which no primary antibody was added in step 3-3), and only secondary antibody was added in step 3-4), the signal detected in step 4) was caused by non-specific adsorption Blank signal, subtract the blank signal from the detection signals of all sample test wells; finally draw the dose-effect curve according to the fluorescence intensity of the target γH2AX and the concentration of aldehydes.

Referring to Fig. 1, the present invention provides a method for quantitatively analyzing cellular DNA damage caused by aldehydes based on high-content technology, which overcomes the deficiencies of the existing methods for in vitro exposure of aldehydes and DNA damage detection. Specifically, the present invention provides a method for assessing DNA double-strand breaks caused by aldehydes, wherein phosphorylated histone γH2AX is used as a biomarker for aldehyde-induced DNA double-strand breaks , and the high content technology is used to detect γH2AX, which improves the detection efficiency and sensitivity. In the method of the present invention, after the cells are exposed to aldehydes, the direct and high-throughput detection of cell samples is realized through cell immunofluorescence staining γH2AX and high-content automatic imaging and analysis.

Compared with prior art, method of the present invention also has following excellent effect:

1) The method of the present invention can be carried out in a cell culture plate, such as a 96-well plate, so the required amount of cells and samples is small, and 96 samples can be detected at the same time, and the detection throughput is higher;

2) There is no need to destroy the cells before the test, and there is no need to prepare single-cell suspensions or extract proteins by enzymatic hydrolysis, so sample processing is simpler and faster;

3) High-content technology has the function of high-resolution imaging acquisition, so the distribution of γH2AX in the nucleus can be directly observed, and the image data is also convenient to store for re-analysis;

4) Through the identification of the nucleus, the fluorescently labeled γH2AX in each nucleus can be quantitatively analyzed, so the detection result is more sensitive and accurate.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Fig. 1 shows a flow chart of the method of the present invention.

Figure 2 shows the dose-effect curve of formaldehyde-induced production of γH2AX in A549 cells.

Figure 3 shows the dose-effect curve of acetaldehyde-induced γH2AX production in A549 cells.

Fig. 4 shows the dose-effect curve of acrolein-induced γH2AX production in A549 cells.

Figure 5 shows the time-effect curve of formaldehyde-induced production of γH2AX in A549 cells.

detailed description

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The pharmaceutical raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

Primary antibody: mouse anti-human γH2AX antibody, purchased from Biolegend, Cat. No. 613402;

Prepare a solution when used: take 100 μL of mouse anti-human γH2AX antibody and add it to 1% BSA solution, dilute it at 1:200 (v/v), and mix well.

Secondary antibody: goat anti-mouse antibody labeled with Alexa Fluro 488, purchased from Wuhan Jiayuan Quantum Dot Company, Cat. No. YM002;

Prepare a solution when used: take 100 μL of Alexa Fluro 488-labeled goat anti-mouse antibody and add it to PBS solution, dilute it at 1:200 (v/v), and mix well.

A high-content cell analysis system was purchased from Thermo Scientific, model ArrayScan VTI600.

Example 1

The induced γH2AX after 24h of formaldehyde exposure was measured.

A549 cells in logarithmic growth phase were collected, planted in 96-well plates at 10,000 cells per well, in RPMI-1640 medium containing 10% FBS and 2mmoL/L L-glutamine at 37°C, 5% CO ₂ incubator for 24h.

Aspirate and discard the cell culture fluid after culturing for 24 h, and use cell culture fluid containing 0, 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 μmol/L formaldehyde respectively (also containing 10% FBS and 2 mmol/L L- Glutamine RPMI-1640 culture medium) was used as the cell poisoning solution, and the cells were continued to be cultured for 24 hours.

After the poisoning, discard the cell poisoning solution, add 100 μL PBS to each well and wash twice for at least 5 minutes each time; then add 50 μL 4% paraformaldehyde solution to each well and fix at room temperature for 15 minutes; fix the cells with PBS buffer Wash twice for at least 5 min each time; then add 50 μL of 0.5% Triton-100X solution (in PBS) to each well for permeabilization at room temperature for 15 min; the permeabilized cells are washed twice with PBS for at least 5 min each time; then Wells were blocked by adding 3% BSA blocking solution (in PBS) at 37°C for 1 h, and then 50 μL of mouse anti-human γH2AX antibody solution (1:200, v/v) was added to each sample well, and only Add 50 μL of 1% BSA as a negative control well, and add 50 μL of mouse anti-human γH2AX antibody solution as a blank control well to another group of blank wells. The incubation conditions were: incubate at 37°C for 2 h or overnight at 4°C; the cells incubated with the primary antibody were washed three times with PBS for at least 5 min each time; then 50 μL of Alexa Fluro 488-labeled goat anti-mouse antibody solution (1: 200, v/v) and incubate at 37°C in the dark for 2 hours; the cells incubated with the secondary antibody were washed three times with PBS for at least 5 minutes each time; The nuclei were stained with light for 10 minutes; after washing with PBS three times, 100 μL of PBS was added to each well, and stored at 4°C in the dark.

After auto-focusing, set the excitation wavelength and emission wavelength of channel 1 (nucleus fluorescence measurement) of the high-content cell analysis system to 358nm and 461nm respectively, and set the excitation wavelength and emission wavelength of channel 2 (fluorescence measurement of target γH2AX) to 495nm respectively and 519nm, the measurement magnification is 200 times, 9 fields of view are analyzed and measured in each hole, and γH2AX is characterized by the average fluorescence intensity measured in channel 2 of the effective nucleus of the effective cells identified in channel 1.

A blank control group was set up in the test, that is, no primary antibody was added, only fluorescently labeled secondary antibody was added, and the detected signal was the blank signal caused by non-specific adsorption; the detection signal of all sample test wells should be deducted from the blank signal; finally according to The fluorescence intensity of the target γH2AX and the concentration of formaldehyde were used to draw the dose-effect curve.

Figure 2 shows the dose-effect relationship curve of γH2AX produced by A549 cells induced by different concentrations of formaldehyde 24 hours after exposure. It can be seen from the figure that with the increase of formaldehyde concentration, the γH2AX produced in the cells induced by it first increased and then decreased. When the concentration of formaldehyde was 250 μmoL/L, the content of γH2AX was the highest, which was 1.95 times that of the blank control group (that is, when the concentration of formaldehyde was 0).

Example 2

The induced γH2AX after 24h exposure to acetaldehyde was measured.

The experimental process was carried out as described in Example 1, the only difference being that the formaldehyde in the cell poisoning solution was replaced by acetaldehyde corresponding to the same concentration.

Figure 3 shows the dose-effect relationship curve of γH2AX produced by A549 cells induced by different concentrations of acetaldehyde 24 hours after exposure. It can be seen from the figure that as the concentration of acetaldehyde increases, the γH2AX produced in the cells induced by it gradually increases, showing a significant dose-effect relationship.

Example 3

The γH2AX induced by acrolein exposure for 24h was measured.

The experimental procedure was carried out as described in Example 1, the only difference being that the cell culture solution containing 0, 20, 40, 60, 80, 100, 120, 140, 160 and 180 μmol/L acrolein was used as the cell poisoning solution.

Figure 4 shows the dose-effect relationship curve of γH2AX produced by A549 cells induced by different concentrations of acrolein 24 hours after exposure. It can be seen from the figure that with the increase of acrolein concentration, the γH2AX produced by A549 cells first increased and then decreased. When the concentration of acrolein was 160 μmoL/L, the content of γH2AX produced in the cells was the highest, which was 10.3 times that of the normal group (ie when the concentration of acrolein was 0).

Example 4

The γH2AX induced by 500μmoL/mL formaldehyde after exposure for 1, 2, 4, 8, 12 and 24 hours were measured.

The experimental process was carried out as described in Example 1, the only difference being that the concentration of formaldehyde in the cell poisoning solution was fixed at 500 μmoL/mL, and the poisoning time was 1, 2, 4, 8, 12 and 24 hours, respectively.

Figure 5 shows the time-effect relationship curve of γH2AX produced by A549 cells induced by 500 μmoL/mL formaldehyde after 1, 2, 4, 8, 12 and 24 hours of exposure. It can be seen from the figure that as the exposure time increases, the γH2AX produced by A549 cells gradually increases, showing a significant time-effect relationship.

The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, all should belong to the scope of the appended claims of the present invention.

Claims (8)

1. a kind of method that DNA Damage is caused based on high intension technology quantitative analysis aldehyde material, methods described includes following Step：

1) cell culture：Cell is seeded in cell culture fluid and carries out cell culture；

2) cell is contaminated：The cell culture fluid is abandoned in suction, is added cell contamination liquid and is continued to cultivate, the cell contamination liquid includes aldehyde Class material and cell culture fluid；

3) immunofluorescence label：The cell contamination liquid is abandoned in suction, carries out the immunofluorescence label and nucleus of γ H2AX in cell DAPI dyeing；

4) high intension technology quantitative analysis：The fluoremetry of the nucleus and γ H2AX of the cell is carried out respectively, to be recognized Effective cell effective cell core in the average fluorescent strength that detects characterize γ H2AX content.

2. according to the method described in claim 1, it is characterised in that the step 1), the cell is adherent growth cell；

Preferably, during inoculation, the cell is in exponential phase, is inoculated in single cell suspension in cell culture fluid；

It is highly preferred that cell inoculation after by cell in cell culture fluid in 37 DEG C, 5%CO₂Under the conditions of cultivate 24h.

3. method according to claim 1 or 2, it is characterised in that the step 2) in, aldehydes in the cell contamination liquid Material is the compound containing saturation or unsaturated short-chain fat aldehyde radical；

Preferably, the aldehyde material is saturation or unsaturation C1-C3 fatty aldehydes；It is highly preferred that the aldehyde material is formaldehyde Or acetaldehyde, or the aldehyde material is methacrylaldehyde；

Preferably, the concentration of aldehyde material is 0-1000 μm of oL/mL in the cell contamination liquid, preferably>0 and≤1000 μm of oL/ ML, more preferably 7.81-1000 μm oL/mL；Or, the concentration of aldehyde material is 0-180 μm of oL/ in the cell contamination liquid ML, preferably>0 and≤180 μm of oL/mL, more preferably 20-180 μm oL/mL；

Preferably, the culture cell 1-24h, preferably 24h in cell contamination liquid.

4. include according to the method in any one of claims 1 to 3, it is characterised in that the step 3)：

3-1) inhale and abandon the cell contamination liquid, wash cell, add paraformaldehyde progress cell and fix；

Cell 3-2) is washed, Triton-100X is then added so that cell-permeant；

Cell 3-3) is washed, seralbumin closing is then added, it is that anti-γ H2AX antibody is incubated that primary antibody is added afterwards；

Cell 3-4) is washed, the secondary antibody for then adding immunofluorescence label is incubated；

Cell 3-5) is washed, DAPI is added and is dyed；

Cell 3-6) is washed, is preserved.

5. method according to any one of claim 1 to 4, it is characterised in that the step 4) in, the cell it is thin Karyon and γ H2AX fluoremetry are carried out under two groups of different excitation wavelengths and launch wavelength respectively；

Preferably, the step 3-4) in, the secondary antibody of immunofluorescence label is the secondary antibody that Alexa Fluor 488 are marked, and The step 4) in, the fluoremetry of nucleus is carried out with excitation wavelength 358nm and launch wavelength 461nm in passage one, γ H2AX fluoremetry is carried out in passage two with excitation wavelength 495nm and launch wavelength 519nm, is recognized with obtaining passage one Effective cell the average fluorescent strength that is detected of effective cell core internal channel two, thus characterize γ H2AX content；

Preferably, in two passages, measurement multiple is 200 times, and 9 visuals field are analyzed and determined per hole.

6. method according to any one of claim 1 to 5, it is characterised in that optionally, methods described also includes, if Put in step 3-3) in without add primary antibody, only in step 3-4) in add secondary antibody blank control group so that in step 4) in The blank signal as caused by non-specific adsorption is obtained, thus blank signal is deducted from the fluoremetry signal detected.

7. method according to any one of claim 1 to 6, it is characterised in that by step 2) the middle a variety of bags of setting Aldehyde material containing various concentrations cell contamination liquid, carry out methods described, finally according to step 4) obtain γ H2AX be averaged Fluorescence intensity and the concentration of aldehyde material draw dose-effect curve.

8. method according to any one of claim 1 to 6, it is characterised in that by step 2) in by cell in bag Different time is cultivated in the cell contamination liquid of aldehyde material containing same concentrations, methods described is carried out, finally according to step 4) obtain M- effect curve during γ H2AX average fluorescent strengths and the incubation time drafting obtained.