CN103293321A - 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 PDF

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CN103293321A
CN103293321A CN201310201625XA CN201310201625A CN103293321A CN 103293321 A CN103293321 A CN 103293321A CN 201310201625X A CN201310201625X A CN 201310201625XA CN 201310201625 A CN201310201625 A CN 201310201625A CN 103293321 A CN103293321 A CN 103293321A
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kit
stress
kernel
mark
dna
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CN103293321B (en
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金雅琼
李淑艳
贾弘禔
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Peking University
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Peking University
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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

A kind of detect early stage kernel that dna damage induces stress kit and application thereof
Technical field
The present invention relates to a kind of detect early stage kernel that dna damage induces stress kit and application thereof, be specifically related to by immunofluorescence quantitatively detect E2F1 the method for the ratio of entoblast and paralinin judge early stage kernel that dna damage induces stress, belong to the detection kit field.
Background technology
Kernel is considered to the place of the synthetic processing of ribosomes always.Kernel be one just can observed organelle under ordinary optical microscope.Under electron microscope, kernel can be divided into three layers---fibrillar center, dense fibrillar component and grain fraction.2006, the credit of nucleolus protein group was analysed 350 kinds of albumen in the kernel is extended to 750 kinds.And 2009, this database is updated to nucleolin more than 4500 kinds.Along with increasingly mature, the development of high-throughput techniques of Protocols in Molecular Biology, the development of epigenetics, it is found that kernel is not only a zone of carrying out the ribosomal subunit assembling, it also participates in multiple human disease processes such as cell cycle regulating, stress reaction, aging, tumour, virus infections.Various cells are coerced (comprising dna damage) and can be caused nucleolar structure disorder and functional disturbance, and kernel takes place stress.The kernel that dna damage causes stress be to regulating tumour cell cycle, Apoptosis and having great importance to keeping genomic stability.Utilize Physiology and biochemistry, molecule and the damage of cell biology means researching DNA stress influence the concern that has just caused the association area scientist to kernel.
E2F1(or E2F-1 below are referred to as E2F1) belong to E2F transcription factor family member, the regulation and control of cell cycle process, autophagy and in the dna damage reaction, promote that Apoptosis is all significant.E2F1 can promote cell cycle progression by a lot of gene expressions relevant with Cycle Regulation of transcriptional activation.In cell cycle progression, retinoblastoma albumen 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 that E2F1 regulates, thereby promotes cell cycle from the G1 phase to S phase transition.E2F1 also plays an important role in the dna damage reaction except playing an important role in cell cycle regulating.In recent years discover that E2F1 also is a kind of kernel transcription factor, participate in kernel internal ribosome DNA and transcribe.
In recent years, the development of immunofluorescence dyeing technology is very fast.In cell biology and molecular biology research, people usually adopt nucleolins such as immunofluorescence label B23, Fibrillarin, p14ARF, come the spike nucleolar structure.But, the kernel that dna damage is induced stress situation under, still do not have kernel stress mark the prompting kernel stress.Though can adopt the variation prompting dna damage of electron microscope observation nucleolar structure induce kernel stress, this kind method is extremely required great effort, and measurement data can not be quantized, and is very high to the requirement of material, equipment.
Summary of the invention
The inventor discover early stage kernel that dna damage is induced stress process in, E2F1 highly assembles in kernel; And dna damage is induced late period kernel stress process in, E2F1 assembles reduction in kernel.This shows, E2F1 in kernel, highly assemble can be used as early stage kernel that dna damage induces stress significant event.Thus, the invention provides a kind of detect early stage kernel that dna damage induces stress kit and application thereof.
For achieving the above object, technical scheme of the present invention is as follows:
A kind of detect early stage kernel that dna damage induces stress kit, comprising: the fluorescence in primary antibodie mark E2F1 specific antibody, corresponding primary antibodie source is two anti-, immobile liquid, change liquid, confining liquid, nucleus dyestuff, mountant, positive control thoroughly.Preferably, described primary antibodie mark E2F1 specific antibody is primary antibodie mark rabbit source polyclone E2F1 antibody, the goat-anti rabbit Ig G of described fluorescence two anti-Alexa Fluor488 marks.
Preferably, described immobile liquid is 4% paraformaldehyde by the PBS preparation of pH7.2-7.4;
Describedization liquid is the PBS(PBST that contains the pH7.2-7.4 of 0.5%Triton-X100);
Described confining liquid is the 5%BSA(Albumin From Bovine Serum of PBS preparation that contains the pH7.2-7.4 of 0.2%Triton-X100, bovine serum albumin(BSA));
Described nucleus dyestuff is Hoechst33342;
Described mountant is that 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 application in stress reagent of early stage kernel that dna damage induces in preparation;
Above-mentioned kit detect early stage kernel that dna damage induces stress in application, operation steps is as follows:
1 immunofluorescence technique mark E2F1 and DNA
The fluorescence two that adds primary antibodie mark E2F1 specific antibody and corresponding primary antibodie source in the cell for preparing is anti-, discard two anti-Incubating Solutions then, add the DNA of Hoechst33342 and carry out mark.
2 fluorescent microscopes are measured E2F1 immune fluorescence intensity ratio in kernel and paralinin
When the ratio of test sample during greater than the ratio of control sample, and one-way analysis of variance or T assay have significant difference then the dna damage kernel of inducing stress take place.
Preferably, described primary antibodie is labeled as rabbit source polyclone E2F1 antibody, and described fluorescence two is anti-to be the goat-anti rabbit Ig G of AlexaFluor488 mark.
The present invention by the fluorescent microscope immunofluorescence quantitatively detect E2F1 the method for the ratio of entoblast and paralinin judge early stage kernel that dna damage induces stress generation.The purposes that cell early stage kernel that E2F1 induces as dna damage stress mark is provided.The kernel of inducing for researching DNA damage stress mark easy, economic, method accurately and rapidly is provided.
Description of drawings
Fig. 1 for E2F1 kernel that adriamycin (ADR) is induced stress in the immunofluorescence figure of gathering; Wherein A is control group, Ch1(DNA dyeing) immunofluorescence figure as a result; B is experimental group (ADR stimulate 6 hours), Ch1(DNA dyeing) immunofluorescence figure as a result; C is control group, Ch2(E2F1 dyeing) immunofluorescence figure as a result; D is experimental group (ADR stimulate 6 hours), Ch2(E2F1 dyeing) immunofluorescence figure as a result; Numerical reference is linear ROI number among the figure;
Fig. 2 is the fluorescence intensity value histogram to length of the corresponding linear ROI of special modality of Line Series analysis; 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 85 cells of statistics 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 of the present invention and characteristics will be more clear along with description.But embodiment only is exemplary, scope of the present invention is not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace the details of technical solution of the present invention and form without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention.
Test reagent: rabbit source polyclone E2F1 antibody is available from Santa cruz company, sc-193; The goat-anti rabbit Ig G(of Alexa Fluor488 mark is available from Invitrogen company, A11008); Adriamycin is available from Sigma company.
Test apparatus: laser confocal scanning microscope is available from OLYMPUS, and model is FV1000S.
1 one kinds of embodiment detect early stage kernel that dna damage induces stress kit, comprising:
(1) immobile liquid: 4% paraformaldehyde (the PBS preparation of pH7.2-7.4);
(2) change liquid thoroughly: the PBS(PBST that contains the pH7.2-7.4 of 0.5%Triton-X100);
(3) confining liquid: contain the 5%BSA(Albumin From Bovine Serum of PBS preparation of the pH7.2-7.4 of 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 cell: at six orifice plate middle berths 2.5 * 10 5Individual H1299 cell is equipped with the length of side and is 1.8 centimetres square cover glass in advance in each hole of 6 orifice plates.
1.2 processing cell: treat in 1.1 that cell degree of converging reaches at 60% o'clock on the cover glass, handle cell with 1 μ M adriamycin, after the control group that does not add adriamycin is simultaneously handled cell with the physiological saline of dissolving adriamycin, continue at 37 ° of C, 5%CO 2And cultured cell 6 hours under the saturated humidity condition.
2. immunofluorescence technique mark E2F1 and DNA
2.1 fixed cell: the cover glass in 1.2 is placed new 6 orifice plates of the PBS that contains 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 the 1ml4% paraformaldehyde, room temperature is fixed 15 minutes.The PBS of pH7.2-7.4 washes 3 times, each 5 minutes.
2.2 change cell thoroughly: discard the PBS of 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 changed cell 30 minutes thoroughly.
2.3 sealing: discard the PBST of pH7.2-7.4 in 2.2, add 1ml confining liquid (contain 5%BSA and with the 0.2%Triton-X100 of the PBS preparation of pH7.2-7.4) room temperature and sealed 1 hour.
2.4 discard confining liquid in 2.3, add 40 μ l and be diluted in primary antibodie mark rabbit source polyclone E2F1 antibody in the confining liquid (available from Santa cruz company, sc-193; Volume ratio 1:200), the length of side is 1.8 centimetres seals membrane cover on cover glass, bubble is driven out of sealed film, 4 ° of C overnight incubation, or incubated at room 1 hour.
2.5 two anti-marks: discard primary antibodie Incubating Solution in 2.4, it is inferior to give a baby a bath on the third day after its birth 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 the confining liquid available from Invitrogen company, A11008; Volume ratio 1:500), the length of side is 1.8 centimetres seals membrane cover on cover glass, bubble is driven out of sealed film, 4 ° of C overnight incubation of lucifuge, or incubated at room 1 hour.
2.6 marker DNA: discard two anti-Incubating Solutions in 2.5, wash 3 times each 5 minutes with 600 μ l PBST.The final concentration that adds among the PBS that is diluted in pH7.2-7.4 is the Hoechst33342 of 5 μ g/ μ l, incubated at room 10 minutes.
2.7 mounting: discard marker DNA Incubating Solution in 2.6, the PBS of 600 μ l pH7.2-7.4 washes 3 times, each 5 minutes.Mounting is to microslide with mountant (glycerine: the PBS volume is 9:1), and cover glass faces down.
2.8 laser confocal scanning microscope shooting: microslide face down in 2.7 is observed under 100 times of oily mirrors of OLYMPUSFV1000S laser confocal scanning microscope.E2F1 excites with argon (ion) laser (488nm), and passage is Ch2; DNA excites with ultraviolet ray (364nm), and passage is Ch1.
3. measure E2F1 immune fluorescence intensity ratio in kernel and paralinin
3.1 be example with OLYMPUS FV1000S laser confocal scanning microscope, use the linear button (Line button) in the ROI instrument (ROI tool) in the OLYMPUS FLUOVIEW Ver.1.6 software, utilize Hoechst33342 not dye the characteristic of kernel, in scan image, pass the part setting-out (not signing in beyond the paralinin) of single kernel and paralinin among the Ch1 of DNA dyestuff correspondence, be designated as (see figure 1)s such as 1,2,3 successively.
3.2 in the Analysis menu, select Line Series, Line Series Analysis window will occur.Click corresponding ROI No. and choose corresponding Ch1 or Ch2, click the SAVE button.In Save as type drop-down menu, select Bitmaps(*.bmp), the fluorescence intensity value histogram (see figure 2) to length that can preserve the corresponding linear ROI of special modality.
3.3 in the Analysis menu, select Line Series, Line Series Analysis window will occur.Click corresponding ROI No. and choose corresponding Ch1 or Ch2, click the SAVE button.Selection Tab delimited(*.xls in Save as type), can preserve the Microsoft Excel of this passage fluorescence intensity level of the corresponding linear ROI specified point of special modality.
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, judge that in conjunction with the 3.2 corresponding fluorescence intensity value histograms of preserving the corresponding point that descend suddenly are nucleolar zone.
3.5 find out the Ch2(E2F1 dyeing channel according to the corresponding point of nucleolar zone described in 3.4 numbering) the corresponding point numbering, should do mean value by the zone fluorescence intensity level; Again all the other points (being the paralinin district) fluorescence intensity level is done mean value.
3.6 nucleolar zone fluorescence intensity mean value in 3.5 divided by paralinin district fluorescence intensity mean value, is obtained kernel and paralinin fluorescence intensity ratio.
3.7 repeat 3.2 to 3.6 steps, measure kernel and the paralinin fluorescence intensity ratio of a plurality of cells, and carry out one-way analysis of variance.The result as shown in Figure 3, control group kernel and paralinin fluorescence intensity ratio are 1.4955965, adriamycin group kernel and paralinin fluorescence intensity ratio are 3.0780291.Compare two groups of (n=85) difference P-value=6.25316 * 10 -18＜0.01, have utmost point significant difference.

Claims (10)

  1. One kind detect early stage kernel that dna damage induces stress kit, comprising: the fluorescence in primary antibodie mark E2F1 specific antibody, corresponding primary antibodie source is two anti-, immobile liquid, change liquid, confining liquid, nucleus dyestuff, mountant, positive control thoroughly.
  2. 2. kit 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 of described fluorescence two anti-Alexa Fluor488 marks.
  3. 3. kit according to claim 1 is characterized in that, described immobile liquid is 4% paraformaldehyde by the PBS preparation of the pH7.2-7.4 of pH7.2-7.4.
  4. 4. kit according to claim 1 is characterized in that, describedization liquid is the PBS of pH7.2-7.4 that contains the pH7.2-7.4 of 0.5%Triton-X100; Described confining liquid is the 5%BSA of PBS preparation that contains the pH7.2-7.4 of 0.2%Triton-X100.
  5. 5. kit according to claim 1 is characterized in that, described nucleus dyestuff is Hoechst33342; Described mountant is that the volume ratio of the PBS of glycerine and pH7.2-7.4 is 9: 1.
  6. 6. kit according to claim 1 is characterized in that, described positive control is adriamycin.
  7. 7. the described kit of claim 1-7 detects the application in stress reagent of early stage kernel that dna damage induces in preparation.
  8. The described kit of claim 1-7 detect early stage kernel that dna damage induces stress in application.
  9. 9. application according to claim 8 is characterized in that, operation steps is as follows:
    (1) immunofluorescence technique mark E2F1 and DNA
    The fluorescence two that adds primary antibodie mark E2F1 specific antibody and corresponding primary antibodie source in the cell for preparing is anti-, discard two anti-Incubating Solutions then, add the DNA of Hoechst33342 and carry out mark;
    (2) fluorescent microscope is measured E2F1 immune fluorescence intensity ratio in kernel and paralinin
    When the ratio of test sample during greater than the ratio of control sample, and one-way analysis of variance or T assay have significant difference then the dna damage kernel of inducing stress take place.
  10. 10. application according to claim 9 is characterized in that, described primary antibodie is labeled as rabbit source polyclone E2F1 antibody, and described fluorescence two is anti-to be the goat-anti rabbit Ig G of Alexa Fluor488 mark.
CN201310201625.XA 2013-05-27 2013-05-27 Kit for detecting DNA (Deoxyribose Nucleic Acid) damage induced early-stage nucleolus stress and application of kit Expired - Fee Related CN103293321B (en)

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CN109187146A (en) * 2018-08-27 2019-01-11 青岛北大新世纪言鼎生物医学科技有限公司 Human body cell holotype state immunofluorescence dyeing method and kit

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Publication number Priority date Publication date Assignee Title
CN109187146A (en) * 2018-08-27 2019-01-11 青岛北大新世纪言鼎生物医学科技有限公司 Human body cell holotype state immunofluorescence dyeing method and kit
CN109187146B (en) * 2018-08-27 2021-03-30 青岛言鼎生物医疗科技有限公司 Human body cell full-form immunofluorescence staining method and kit

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