CN103293321B - Kit for detecting DNA (Deoxyribose Nucleic Acid) damage induced early-stage nucleolus stress and application of kit - Google Patents
Kit for detecting DNA (Deoxyribose Nucleic Acid) damage induced early-stage nucleolus stress and application of kit Download PDFInfo
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- CN103293321B CN103293321B CN201310201625.XA CN201310201625A CN103293321B CN 103293321 B CN103293321 B CN 103293321B CN 201310201625 A CN201310201625 A CN 201310201625A CN 103293321 B CN103293321 B CN 103293321B
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Abstract
The invention relates to a kit for detecting DNA (Deoxyribose Nucleic Acid) damage induced early-stage nucleolus stress and application of the kit. The kit comprises a specific antibody to marker E2F1, a fluorescence secondary antibody corresponding to an anti-source, stationary liquid, permeabilizing liquid, confining liquid, cell nucleus dye, mounting medium and positive control. The operating method of the kit comprises the following steps of: marking E2F1 and DNA by an immumofluorescence method; measuring an immumofluorescence intensity ratio of E2F1 in nucleolus and nuclear matrix by a fluorescence microscope. The kit for detecting DNA damage induced early-stage nucleolus stress disclosed by the invention is economical, quick, low in device requirement and capable of digitizing observation results, so that an accurate and simple method is provided for researching the DNA damage induced early-stage nucleolus stress.
Description
Technical field
The present invention relates to a kind of detect DNA damage induction early stage kernel stress kit and application, be specifically related to by immunofluorescence quantitatively detect E2F1 the method for the ratio of entoblast and paralinin judge DNA damage induction early stage kernel stress, belong to detection kit field.
Background technology
Kernel is considered to the place of Ribosome biogenesis processing always.Kernel is an organelle just can observed under ordinary optical microscope.Under an electron microscope, kernel can be divided into three layers---fibrillar center, dense fibrillar component and grain fraction.2006,350 kinds of albumen in kernel were extended to 750 kinds by nucleolus protein group Epidemiological Analysis.And 2009, nucleolin is updated to more than 4500 kinds by this database.Along with increasingly mature, the development of high-throughput techniques, the development of epigenetics of Protocols in Molecular Biology, it is found that kernel is not only a region of carrying out ribosomal subunit assembling, it also participates in the multiple human disease processes such as cell cycle regulating, stress reaction, aging, tumour, virus infections.Various Cellular stress (comprising DNA damage) can cause nucleolar structure disorder and functional disturbance, and kernel occurs stress.The kernel that DNA damage causes to adjustment tumour cell cycle, Apoptosis and stress have great importance to the genomic stability of maintenance.Utilize Physiology and biochemistry, molecule and cell biology means researching DNA to damage and stress affect on kernel the concern just having caused association area scientist.
E2F1(or E2F-1, is below referred to as E2F1) belong to E2F transcription factor family member, cell cycle process control, autophagy and DNA damage reaction in promote that Apoptosis is all significant.E2F1, by a lot of gene expression relevant to Cycle Regulation of transcriptional activation, promotes cell cycle progression.In cell cycle progression, Retinoblastoma Protein RB and other pocket protein families member p107, p130 and E2F1 interact, and regulate the transcriptional activity of E2F1.Dissociating of RB-E2F1 compound can discharge, activate E2F1, and then activates the downstream target gene of E2F1 adjustment, thus promotes that cell cycle from the G1 phase is to S phase transition.E2F1, except playing an important role in cell cycle regulating, also plays an important role in DNA damage reaction.Research in recent years finds, E2F1 is also a kind of kernel transcription factor, participates in kernel internal ribosome DNA and transcribes.
In recent years, the development of immunofluorescence dyeing technology is very fast.In cell biology and molecular biology research, people usually adopt the nucleolins such as immunofluorescence label B23, Fibrillarin, p14ARF, carry out spike nucleolar structure.But, DNA damage induction kernel stress when, still do not have kernel stress mark prompting kernel stress.Although can adopt electron microscope observation nucleolar structure change prompting DNA damage induction kernel stress, this kind of method is extremely required great effort, and measurement data can not be quantized, very high to the requirement of material, equipment.
Summary of the invention
Inventor study find DNA damage induction early stage kernel stress process in, E2F1 is high aggregation in kernel; And DNA damage induction late period kernel stress process in, E2F1 assembles reduction in kernel.As can be seen here, E2F1 high aggregation in kernel can be used as DNA damage induction early stage kernel stress hallmark events.Thus, the invention provides a kind of detect DNA damage induction early stage kernel stress kit and application.
For achieving the above object, technical scheme of the present invention is as follows:
Detect DNA damage induction early stage kernel stress a kit, comprising: the fluorescence two in primary antibodie mark E2F1 specific antibody, corresponding primary antibodie source is anti-, immobile liquid, thoroughly change liquid, confining liquid, nucleus dyestuff, mountant, positive control.Preferably, described primary antibodie mark E2F1 specific antibody is primary antibodie mark rabbit source polyclone E2F1 antibody, the goat-anti rabbit Ig G that the anti-Alexa Fluor488 of described fluorescence two marks.
Preferably, described immobile liquid is 4% paraformaldehyde prepared by the PBS of pH7.2-7.4;
Described saturatingization liquid is the PBS(PBST of the pH7.2-7.4 containing 0.5%Triton-X100);
Described confining liquid is the 5%BSA(Albumin From Bovine Serum of the PBS preparation of pH7.2-7.4 containing 0.2%Triton-X100, bovine serum albumin(BSA));
Described nucleus dyestuff is Hoechst33342;
Described mountant is the volume ratio of the PBS of glycerine and pH7.2-7.4 is 9:1;
Described positive control is adriamycin (Adriamycin).
Above-mentioned kit detects the early stage kernel of DNA damage induction in preparation stress application in reagent;
Above-mentioned kit detect DNA damage induction early stage kernel stress in application, operation steps is as follows:
1 immunofluorescence technique mark E2F1 and DNA
The fluorescence two adding primary antibodie mark E2F1 specific antibody and corresponding primary antibodie source in the cell prepared is resisted, then discards two anti-Incubating Solutions, add Hoechst33342 and DNA is marked.
E2F1 immune fluorescence intensity ratio in kernel and paralinin measured by 2 fluorescent microscopes
When the ratio detecting sample is greater than the ratio of control sample, and one-way analysis of variance or T assay have the significant difference kernel that then DNA damage is induced to occur.
Preferably, described primary antibodie is labeled as rabbit source polyclone E2F1 antibody, and described fluorescence two resists the goat-anti rabbit Ig G for AlexaFluor488 mark.
The present invention by fluorescent microscope immunofluorescence quantitatively detect E2F1 the method for the ratio of entoblast and paralinin judge DNA damage induction early stage kernel stress generation.Providing the early stage kernel of cell that E2F1 induces as DNA damage stress the purposes of mark.For researching DNA wound inducement kernel stress mark provide easy, economic, method accurately and rapidly.
Accompanying drawing explanation
Fig. 1 kernel that to be E2F1 induce at adriamycin (ADR) stress in the immunofluorescence figure that assembles; Wherein A is control group, Ch1(DNA dyeing) immunofluorescence results figure; B is experimental group (ADR stimulates 6 hours), Ch1(DNA dyeing) immunofluorescence results figure; C is control group, Ch2(E2F1 dyeing) immunofluorescence results figure; D is experimental group (ADR stimulates 6 hours), Ch2(E2F1 dyeing) immunofluorescence results figure; In figure, numerical reference is linear No. ROI;
Fig. 2 is the fluorescence intensity value histogram to length of the corresponding linear ROI of special modality that Line Series analyzes; Wherein, A is control group, Ch1(DNA dyeing) the fluorescence intensity value histogram to length of linear ROI; B is adriamycin processed group, Ch1(DNA dyeing) the fluorescence intensity value histogram to length of linear ROI; C is control group, Ch2(E2F1 immunofluorescence dyeing) the fluorescence intensity value histogram to length of linear ROI; D is adriamycin processed group group, Ch2(E2F1 immunofluorescence dyeing) the fluorescence intensity value histogram to length of linear ROI;
Fig. 3 is the kernel of statistics 85 cells and the statistic analysis result figure of paralinin E2F1 fluorescence intensity ratio.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Test reagent: rabbit originates polyclone E2F1 antibody purchased from Santa cruz company, sc-193; Alexa Fluor488 mark goat-anti rabbit Ig G(purchased from Invitrogen company, A11008); Adriamycin available from Sigma.
Test apparatus: laser confocal scanning microscope is purchased from OLYMPUS, and model is FV1000S.
Embodiment 1 one kinds detect DNA damage induction early stage kernel stress kit, comprising:
(1) immobile liquid: 4% paraformaldehyde (the PBS preparation of pH7.2-7.4);
(2) thoroughly liquid is changed: the PBS(PBST of the pH7.2-7.4 containing 0.5%Triton-X100);
(3) confining liquid: the 5%BSA(Albumin From Bovine Serum of the PBS preparation of the pH7.2-7.4 containing 0.2%Triton-X100, bovine serum albumin(BSA));
(4) primary antibodie mark rabbit source polyclone E2F1 antibody;
(5) the goat-anti rabbit Ig G of Alexa Fluor488 mark;
(6) nucleus dyestuff: Hoechst33342;
(7) mountant: the ratio of the PBS of glycerine and pH7.2-7.4 is 9:1;
(8) positive control: adriamycin (Adriamycin).
Embodiment 2
1 sets up adriamycin induces early stage kernel Stress model
1.1 inoculating cells: at six orifice plate middle berths 2.5 × 10
5individual H1299 cell, is equipped with the square cover glass that the length of side is 1.8 centimetres in advance in each hole of 6 orifice plates.
1.2 process cells: when cell confluency degree reaches 60% on cover glass in 1.1, with 1 μM of adriamycin process cell, after not adding the physiological saline process cell of control group dissolving adriamycin of adriamycin, continue at 37 ° of C, 5%CO simultaneously
2and cultured cell 6 hours under saturated humidity condition.
2. immunofluorescence technique mark E2F1 and DNA
2.1 fixed cells: new 6 orifice plates cover glass in 1.2 being placed in the PBS containing ice-cold pH7.2-7.4, discard the PBS of pH7.2-7.4 and add (the PBS preparation of pH7.2-7.4) in 1ml4% paraformaldehyde, room temperature fixes 15 minutes.The PBS of pH7.2-7.4 washes 3 times, each 5 minutes.
2.2 change cell thoroughly: the PBS discarding pH7.2-7.4 in 2.1, add the PBS(PBST that 1ml contains the pH7.2-7.4 of 0.5%Triton-X100) room temperature changes cell 30 minutes thoroughly.
2.3 close: the PBST discarding pH7.2-7.4 in 2.2, add 1ml confining liquid (containing 5%BSA and the 0.2%Triton-X100 for preparing with the PBS of pH7.2-7.4) room temperature closed 1 hour.
2.4 discard confining liquid in 2.3, add 40 μ l be diluted in primary antibodie mark rabbit source polyclone E2F1 antibody in confining liquid (purchased from Santa cruz company, sc-193; Volume ratio 1:200), be 1.8 centimetres of sealing membrane cover faces on the cover slip by the length of side, drive bubble out of sealed membrane, 4 ° of C overnight incubation, or incubated at room 1 hour.
2.5 2 anti-marks: discard primary antibodie Incubating Solution in 2.4, wash three times with 600 μ l PBST, each 5 minutes.Add 40 μ l and be diluted in the goat-anti rabbit Ig G(of the Alexa Fluor488 mark in confining liquid purchased from Invitrogen company, A11008; Volume ratio 1:500), be 1.8 centimetres of sealing membrane cover faces on the cover slip by the length of side, drive bubble out of sealed membrane, lucifuge 4 ° of C overnight incubation, or incubated at room 1 hour.
2.6 marker DNAs: discard two anti-Incubating Solutions in 2.5, wash 3 times with 600 μ l PBST, each 5 minutes.Add the Hoechst33342 that the final concentration be diluted in the PBS of pH7.2-7.4 is 5 μ g/ μ l, incubated at room 10 minutes.
2.7 mountings: discard marker DNA Incubating Solution in 2.6, the PBS of 600 μ l pH7.2-7.4 washes 3 times, each 5 minutes.By mountant (glycerine: PBS volume is 9:1), mounting is on microslide, and cover glass faces down.
2.8 laser confocal scanning microscope shootings: by microslide face down in 2.7 in OLYMPUSFV1000S laser confocal scanning microscope 100 times of oily Microscopic observations.E2F1 excites with argon (ion) laser (488nm), and passage is Ch2; DNA excites with ultraviolet (364nm), and passage is Ch1.
3. measure E2F1 immune fluorescence intensity ratio in kernel and paralinin
3.1 for OLYMPUS FV1000S laser confocal scanning microscope, use the linear button (Line button) in the ROI instrument (ROI tool) in OLYMPUSFLUOVIEW Ver.1.6 software, Hoechst33342 is utilized not contaminate the characteristic of kernel, through the part setting-out (not signing in beyond paralinin) of single kernel and paralinin in the Ch1 that DNA dyestuff is corresponding in scan image, be designated as the (see figure 1)s such as 1,2,3 successively.
3.2 select Line Series in Analysis menu, will occur Line Series Analysis window.Click corresponding ROI No. and choose corresponding Ch1 or Ch2, click SAVE button.Bitmaps(*.bmp is selected in Save as type drop-down menu), the fluorescence intensity value histogram (see figure 2) to length of the corresponding linear ROI of special modality can be preserved.
3.3 select Line Series in Analysis menu, will occur Line Series Analysis window.Click corresponding ROI No. and choose corresponding Ch1 or Ch2, click SAVE button.Tabdelimited(*.xls is selected in Save as type), the Microsoft Excel of this channel fluorescence intensity value of the corresponding linear ROI specified point of special modality can be preserved.
3.4 according to the dyeing channel of Ch1(DNA described in 3.3) the corresponding fluorescence intensity level of neutral line ROI each point, preserves corresponding fluorescence intensity value histogram in conjunction with 3.2 institutes and judge that the corresponding point of unexpected decline are nucleolar zone.
3.5 find out Ch2(E2F1 dyeing channel according to the corresponding point of nucleolar zone described in 3.4 numbering) corresponding point numbering, this region fluorescence intensity level is done mean value; Again all the other points (i.e. paralinin district) fluorescence intensity level is done mean value.
3.6 by nucleolar zone fluorescence intensity mean value in 3.5 divided by paralinin district fluorescence intensity mean value, obtain kernel and paralinin fluorescence intensity ratio.
3.7 repeat 3.2 to 3.6 steps, measure kernel and the paralinin fluorescence intensity ratio of multiple cell, and carry out one-way analysis of variance.As shown in Figure 3, control group kernel and paralinin fluorescence intensity ratio are 1.4955965 to result, and adriamycin group kernel and paralinin fluorescence intensity ratio are 3.0780291.Relatively two groups of (n=85) difference P-value=6.25316 × 10
-18<0.01, has pole significant difference.
Claims (6)
1. kit detect DNA damage induction early stage kernel stress in application, described kit comprises: the fluorescence two in primary antibodie mark E2F1 specific antibody, corresponding primary antibodie source is anti-, immobile liquid, thoroughly change liquid, confining liquid, nucleus dyestuff, mountant, positive control, it is characterized in that
Operation steps is as follows:
(1) immunofluorescence technique mark E2F1 and DNA
The fluorescence two adding primary antibodie mark E2F1 specific antibody and corresponding primary antibodie source in the cell prepared is resisted, then discards two anti-Incubating Solutions, add Hoechst 33342 couples of DNA and mark;
(2) E2F1 immune fluorescence intensity ratio in kernel and paralinin measured by fluorescent microscope
When the ratio detecting sample is greater than the ratio of control sample, and one-way analysis of variance or T assay have the significant difference kernel that then DNA damage is induced to occur.
2. application according to claim 1, is characterized in that, described primary antibodie mark E2F1 specific antibody is primary antibodie mark rabbit source polyclone E2F1 antibody, the goat-anti rabbit Ig G that the anti-Alexa Fluor 488 of described fluorescence two marks.
3. application according to claim 1, is characterized in that, described immobile liquid is 4% paraformaldehyde prepared by the PBS of the pH7.2-7.4 of pH7.2-7.4.
4. application according to claim 1, is characterized in that, described saturatingization liquid is the PBS of the pH7.2-7.4 of pH7.2-7.4 containing 0.5%Triton-X100; Described confining liquid is the 5%BSA of the PBS preparation of pH7.2-7.4 containing 0.2%Triton-X100.
5. application according to claim 1, is characterized in that, described nucleus dyestuff is Hoechst33342; Described mountant is the volume ratio of the PBS of glycerine and pH7.2-7.4 is 9:1.
6. application according to claim 1, is characterized in that, described positive control is adriamycin.
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| CN102517282A (en) * | 2011-12-30 | 2012-06-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for enriching and separating endogenous transcription factors and compounds thereof and concatenated transcription factor response elements special for method |
| CN103119179A (en) * | 2010-07-23 | 2013-05-22 | 哈佛大学校长及研究员协会 | Methods for detecting signatures of disease or conditions in bodily fluids |
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| CN103119179A (en) * | 2010-07-23 | 2013-05-22 | 哈佛大学校长及研究员协会 | Methods for detecting signatures of disease or conditions in bodily fluids |
| CN102517282A (en) * | 2011-12-30 | 2012-06-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for enriching and separating endogenous transcription factors and compounds thereof and concatenated transcription factor response elements special for method |
Non-Patent Citations (1)
| Title |
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| E2F1 localizes to sites of UV-induced DNA damage to enhance nucleotide excision repair.;Ruifeng Guo et al.;《Journal of Biological Chemistry》;20100422;第285卷(第25期);19308-19315 * |
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