CN105445354B - Keep detecting the device and method of SSBs bonding states under cell stress equilibrium condition - Google Patents

Abstract

The invention discloses a kind of device and method for keeping detecting SSBs bonding states under cell stress equilibrium condition, the device includes：Pipe cell electrophoresis groove, in the middle part of tube wall, direction is provided with a rectangular notch along its longitudinal axis, running buffer liquid pool, side interface channel, filter membrane, aperture is 0.2 μm or 0.45 μm hydrophilic Poly-s 179 filter membrane, the running buffer liquid pool and side interface channel cast solid, side interface channel are tightly connected with electrophoresis tank by dismountable mode, and filter membrane is inserted in the junction of side interface channel and electrophoresis tank.The device of the present invention can make cell separate reference state SSBs and non-binding SSBs under conditions of archaeocyte structural intergrity is kept.The present invention is cleverly combined cell in-situ electrophoresis with cell flow cytometer showed art, and testing result is accurate.

Description

Keep detecting the device and method of SSBs bonding states under cell stress equilibrium condition

Technical field

The present invention relates to a kind of device and method for keeping detecting SSBs bonding states under cell stress equilibrium condition.

Background technology

Single-stranded DNA binding protein (single strand DNA-binding proteins, SSBs) includes Replication protein A (RPA) and telomere protected protein protection of telomeres protein 1 (POT1).When DNA replication dna, double-stranded DNA needs to untwist into single stranded DNA and combined to prevent with single-stranded DNA binding protein Wrong structure is only formed, safeguards the stability of telomere and genome.However, differentiated normal somatic cell is in unphysiologicization Learn, under physics and inflammatory factor intervention, cell can depart from from the matrix of its growth, and its nucleus can produce violent contracting Small, at the same time, chromatin/DNA for being combined with SSBs can be oppressed by this diminution and crowded nucleus, so as to produce Conformal change, producing the SSBs of conformal change can depart from from single stranded DNA.The SSBs that will be disengaged from bonding state is keeping cell complete Under conditions of whole property, it is separated from cell, and keep the SSBs isolated not reenter cell, do not have so far There is such device.

It is well known that DNA is the carrier of organism genetic code-gene, the biology of the stability of genome to organism It is vital that scholarship and moral conduct, which is,.The unstability of genome or the variation of gene and the numerous diseases of the mankind are closely related, as cancer, Aging etc..And SSBs is present in all eukaryotic cells, SSBs bonding state is to ensureing DNA correct duplication, DNA Injury repair and the stability of genome have critical effect.But find no close technological means and experiment side at present Method can be realized to structural integrity and the document that keeps the SSBs bonding states inside the nucleus of its stress equilibrium to be detected Report.

The content of the invention

The purpose of the present invention is just to provide for detecting SSBs bonding states under a kind of holding cell stress equilibrium condition Device and method.

To achieve these goals, the present invention adopts the following technical scheme that：

A kind of device for keeping detecting SSBs bonding states under cell stress equilibrium condition, including：Cell electrophoresis groove：The groove For 0.76~0.84cm of internal diameter (preferably 0.8cm), 0.90~1.00cm of external diameter (preferably 0.95cm), 18.9~17.1cm of length The polystyrene pipe of (preferably 18cm), in the middle part of tube wall, along its longitudinal axis direction be provided with one 8.55 × 0.57cm~9.45 × 0.63cm (preferably 9 × 0.6cm) rectangular notch；Running buffer liquid pool：Anode, negative electrode respectively have one, and size is identical, 9.5 × 3.33 × 4.75cm~10.5 × 3.68 × 5.25cm (preferably 10 × 3.5 × 5cm)；Material：Polystyrene；Side interface channel： Anode respectively has one with cathode side, and size is identical, and 0.76~0.84cm of internal diameter (preferably 0.8cm), 0.90~1.00cm of external diameter are (excellent Select 0.95cm), the silicone tube of long 9.5~10.5cm (preferably 10cm)；Filter membrane：Anode respectively has one with cathode side, and size is identical, 0.45 μm or 0.2 μm hydrophilic Poly-s 179 (Hydrophilic polyethersulfone) filter membrane；Electrode：Anode is each with negative electrode There is one, 5.7~6.3cm (preferably 6cm) is long, 0.475~0.525cm of diameter (preferably 0.5mm) electrophoresis apparatus platinum filament.

Cell in-situ electrophoresis (cell in situ electrophoresis, CISE), is that cell to be checked is prepared into list Cell suspension, its single free cell is set to be scattered in the medium of isotonic i.e. stress equilibrium, under electric field action, by cell A kind of electrophoretic for being separated from cell of material.

The device of SSBs bonding states is detected under above-mentioned holding cell stress equilibrium condition, is horizontal electrophoretic apparatus.

Using the method for said apparatus detection SSBs bonding states, following steps are specifically included：

Cell pre-fixes：Separating treatment growth period cell, add phosphate buffer (PBS) and cell suspension is made, will be upper State cell suspension and be coated on culture dish bottom, irradiate (preferably 254nm UV lamps with UV lamp under the conditions of above-mentioned culture dish is placed in into 0 DEG C With 1.84W/cm²Intensity illumination, accumulated dose 165.6J/cm²) after, the cell in culture dish is collected with phosphate buffer (PBS), Centrifugation (260g centrifuge 5min), which is abandoned after supernatant, to be added 4% paraformaldehyde and pre-fixes 1-6 (preferably 3) sec；

The purpose pre-fixed is：Existing stress structure and it is intracellular treat that Reichl's test is non denatured in the state of, be subjected to cell In-situ electrophoresis process, it is broken without occurring, to keep cell integrity, to facilitate ensuing punching.

Cell-transmission model：By the above-mentioned cell pre-fixed, the agent that fans the air is added, the agent that fans the air can make cell membrane and nucleus All punch, preferably Triton X-100 (Triton X-100), concentration 0.2%, Cell-transmission model time 5-15 is (preferably 10)min；

The purpose of punching is：The lipid on cell biological film is dissolved, single-stranded DNA binding protein is moved from cell Go out, to facilitate the single strand binding protein to come off to be removed from cell, do not destroy the overall structure of cell.

Cell in-situ electrophoresis (cell in situ electrophoresis, CISE)：By the cell mass after above-mentioned punching Add CISE buffer solutions (trishydroxymethylaminomethane Tris 25mM；Glycine Glycine:192mM；Glucose Glucose： 43.2mM；PH8.3) it is made cell suspension, under constant voltage mode, instills cell suspension in electrophoresis tank cathode side, used after the completion of electrophoresis Liquid in pipettor exhaustion electrophoresis tank, and come together in liquid is suctioned out in a centrifuge tube, with electrophoresis wash buffer electrophoresis tank, weight Multiple electrophoresis process, until cell suspension is fully completed electrophoresis；

Under deposition condition, from DNA it is single-stranded on the single strand binding protein that comes off flowed out by the duct on cell, so as to real The separation of the single strand binding protein now to come off and the single strand binding protein not fallen off；

It is preferred that during point sample, in middle swimming lane, (purpose is cell sample point at cathode terminal 1.5cm：During electrophoresis, prevent thin Born of the same parents are moved at filter membrane agglomerating so as to assemble), ensure that cell does not rupture during electrophoresis, and reduce single-stranded DNA binding protein from one Individual cell enters the probability of another cell by the duct on cell again after removing.The time of electrophoresis is preferably 3min.

Detection：Detected using flow cytometry.

Used PBS in the present invention, pH value are all 7.4.

Beneficial effects of the present invention：

When pre-fixing, irradiated under the conditions of above-mentioned culture dish is placed in into 0 DEG C with UV lamp, enhance the original states of SSBs, Ensure the accurate of subsequent detection result.

The device of the present invention can make cell separate reference state SSBs and non-knot under conditions of original stress equilibrium state is kept Close SSBs.

Electrophoretic buffer prepared by the present invention can keep cell isotonic, ensure that cell is complete, be from Glucose because Its is nonpolarity, and molecular structure does not influence follow-up centrifugation, is easy to the recovery of cell.

The present invention is cleverly combined cell in-situ electrophoresis with cell flow cytometer showed art, and testing result is objective and accurate.

The present invention apparatus structure be simple and convenient to operate with analysis result it is objective the characteristics of.Using the inventive method pair The detection of SSBs bonding states, show " keeping combining " for being easy to judge and " dissociation " two kinds of results.

Apparatus of the present invention use caused by stress changes nucleus reduce caused by SSBs dissociation there is important biology Meaning.Its cascade process and biological effect can be summarized as：The conformal change of cellular contraction-nucleus compression-chromatin is led The genomic instability of cause.

Present invention employs being pre-fixed first with the 4% μ l second levels of paraformaldehyde 200, used again before flow cytometer showed is carried out 70% ethanol 15ml carries out secondary fixation, ensure that cell in the flow cytometer showed in later stage cell it is complete, while cause stream Formula precision of analysis.

Belong to life science with the original molecular mechanism for causing endogenous gene group unstable found of the technology of the present invention The natural law on fundamental aspect.It is for disclosing many major disease (such as cancer, aging degenerative disorders-Alzheimers Disease etc.) pathogenesis, illustrate biological phenomena (such as cell dryness, plasm make a variation), break through limitation stem-cell therapy treatment Bottleneck (such as tumour generation), there is important theory and practical significance.

From technological layer or methodology, the present invention can also be to study other DNA binding molecules, and be suitable for the present invention The molecular binding events analysis of technical method provides a kind of experiment detection means.

The present invention can illustrate cell in the case where unphysiologic chemistry, physics and inflammatory factor are intervened, from the base of its growth Cellular contraction, nucleus compression and the conformal change of chromatin can cause changing for SSBs bonding states caused by departing from matter Become, i.e. uncombined SSBs and the SSBs combined separation.This separation explanation participates in the DNA replication dna of assembling with repairing by SSBs Multiple complex goes to assemble, and result is cause cellular genome unstable.

The natural law changed using disclosed this SSBs bonding states, a series of puzzlement life can be explained Numerous important science puzzles related to disease and application study of scientific domain.In classical hypothesis and theoretical side, this It was found that the behind mechanism of the science puzzle of an over half a century is caused to obtain more perfect explanation from logic of science.1961 Famous the hypothesis ----Hai Fulike limit (Hayflick proposed by American scientist Hai Fulike (Leonard Hayflick) Limit) it has been described as the pillar (pillar of biology) of biology.The hypothesis thinks that cell can only population doublings (population doublings) about 50 generation, thereafter cell just enter proliferative aging death.On the mechanism of this hypothesis, Your Grays moral (Carol W.Greider) of Nobel Laureate Carlow et al. is in nineteen ninety on internationally famous magazine Nature This proliferative aging of prompting of publishing an article is as caused by the shortening of telomere.This telomere, which shortens theoretical still turn into so far, to be explained The main flow academic viewpoint of Hai Fulike limit mechanism.I.e. often a certain amount of shortening occurs through a wheel DNA replication dna telomere in cell, when When shortening to a certain extent, cell will appear from proliferative aging death.But telomere shortens to how length and triggers cell increasing Growing property aging death, length is only inferred so far, not strict measured length really.However, according to real obtained by the inventive method That tests result progress gos deep into experimental study confirmation, (reaches far away described in the Hai Fulike limit when cell population doublings only have 7 times 50 times or so), and passage is up to 50 times or so (during with reaching the Hai Fulike limit, the passage number that cell is undergone is consistent) When, there is proliferative aging death in cell.Illustrate that the Hai Fulike limit are not produced by population doublings.Substantially, due to SSBs bonding states caused by passage change the damage that can cause cell DNA, and the accumulation of this damage is to cause sea not In gram limit deciding factor；Also for explanation, another important biomolecule puzzle --- mouse embryo fibroblast is thin for this discovery The in-vitro multiplication aging death of born of the same parents provides experiment and theoretical foundation.Kaman in 1998 et al. find syrian hamster embryo into Fibrocyte is after 20-30 population doublings are undergone, though retain the telomere length and telomerase activation of considerably long (23kb), still Proliferative aging death is so presented.Scientist speculates the thin of mouse because that can not shorten theory with telomere to explain this phenomenon Born of the same parents' proliferative aging death mechanism may be different with people, but exact mechanism is unknown.However, with obtained by the technology of the present invention Experimental result illustrates the mechanism of this science puzzle；In terms of stem cell, its stem-cell therapy turns into whole world scientist The regenerative medicine hot issue being concerned about with the common people.People are placed hope on stem-cell therapy to solve current many difficult incurable diseases. But the clinical practice of this technology still suffers from many problems at present, wherein, bio-safety problem is important one, and dry thin Born of the same parents' treatment can cause the most important thing that cancer is safety problem.It is experimentally confirmed that in treatment of animals model, the cell of part treatment Tumour can be formed in animal body, and even in not growing up in the treatment cell of tumour, its genetic mutation is also a kind of potential cause Disease threatens.At present due to a lack of theoretic knowledge, the tumorigenesis problem of stem cell turns into the bottleneck for restricting stem cell clinical practice.Pass through SSBs bonding states disclosed in the art of this patent change the discovery for causing DNA Damage and genomic instability, for solution Certainly the problem on obstacle of stem-cell therapy provides an important theoretical breakthrough.

Currently, people, which generally use, first induces undifferentiated multipotential stem cell as the noble cells for treatment, replants Enter in vivo.And think to be mixed into where the problem of undifferentiated stem cell is tumorigenesis in the cell of these differentiation.It is however, of the invention The testing result of technology is prompted from mechanism and experimentally, in meet to treat the cell expansion process that quantitative requirement is carried out, Even if once being expanded using present classical means, the probability that its noble cells gene bred is undergone mutation also is 100%, that is, carcinogenic risk be present.This discovery both brought challenge for stem-cell therapy, also how to be broken through for stem-cell therapy Obstacle specifies the direction solved the problems, such as；In terms of cancer occurs with gene mutation, the gene mutation of cell is acknowledged Mechanism of carcinogenesis.Traditional theory thinks that cancer is that cell undergoes multiple catastrophic event, and the gene relevant with cancer generation is dashed forward Become the result gradually accumulated.But recent scientist is had found by extensive high throughput genome sequencing analysis, many cancers It is caused by a gene event of experience largely related to cancer is undergone mutation.This discovery is named as " chromosome Fragmentation " (chromothripsis), " chromosome storm " (kataegis).However, cause the behind mechanism of this phenomenon for Scientist is a puzzle.The result of the technology of the present invention detection is exactly its genesis mechanism and provides scientific explarnation.One cell DNA replication dna starting point have as many as 40,000 to 50,000.Because the cell of people will complete about 3,000,000,000 within a cell cycle The huge replication works of DNA of base-pair, so needing tens thousand of individual copy-points to work and could complete simultaneously.In this reproduction process In, once cause the body cell in the duplicate stage to depart from the event of its growth substrate, can cause numerous DNA damages with Gene mutation, the reason for this may be interpreted as causing " chromosome storm ", " chromosome fragmentation "；At the carcinogenic aspect of inflammation, largely Research confirms that inflammation is to cause the major reason of cancer.But differ for the various explanations of the carcinogenic mechanism of inflammation.It is wherein main Explanation be the inflammatory factor that is discharged of inflammatory cell by numerous and diverse approach and step indirectly damaging cells DNA.

And the carcinogenic mechanism of action expansion of inflammation can be construed to by the testing result of the technology of the present invention：Occur in inflammation When, especially in chronic inflammation, it may appear that the damage and reparation of typical cell and extracellular matrix, these pathological changes are facilitated The typical another feature of inflammation --- cell detachment, it is rounded and adheres and sprawl again, these pathologic processes repeatedly can causes Disclosed cell SSBs bonding states change, and so as to cause DNA damage and gene mutation, occur having evolution effect " chromosome storm ", " chromosome fragmentation " and carry out natural selection and carcinogenic evolution.This explanation is analyzed from logical perspective Also it is more direct；In terms of Tumor Heterogeneity, research confirms that Tumor Heterogeneity is one of difficult point of oncotherapy, it has also become tumour The hot issue of research.Mechanism disclosed in the technology of the present invention, the explanation of science is provided for the generation of Tumor Heterogeneity.Cell The phase position when NATURAL DISTRIBUTION in cycle theoretically prompts the cell of inside tumor each in the different cycles, i.e., asynchronous shape State.If there is " chromosome storm " event, each cell will produce different DNA damages, thus cell occur naturally it is heterogeneous Property；Tumour evolve with genomic instability in terms of, scientist be to genomic instability tumorigenic reason or As a result dispute be present.Mechanism prompting genomic instability disclosed in the technology of the present invention can lead oncogenic generation, and natural The result that selection is evolved with tumour can make the genome of tumour tend towards stability (Hela cells are illustration).And many interventions because Element can cause Oncogenome unstability, such as chemotherapeutics occur again；In terms of the dryness (stemness) of cell, this hair Bright technology for detection result shows that mouse embryo stem cell and the Hela cells with tumor stem cell characteristic are passing through classical pancreas Protease digestion, in the case of single cell suspension is made, the SSBs combined in its cell with DNA still keeps bonding state.Equally Treatment conditions under, the SSBs combined in human embryonic lung fibroblast and MEC with DNA is then solved From.It is an important mechanisms for maintaining cell dryness that prompting, which avoids the stress of nucleus from shrinking,；In terms of unicellular sequencing, in view of Research confirms that tumour cell has the heterogeneity of height, so unicellular sequencing develops rapidly in recent years, to reflect cell Asia The heterogeneity of group, data foundation is provided for follow-up study.However, the technology of the present invention testing result is prompted, if selecting to be used for list The tumour cell of cell sequencing is not yet in the genome stabilization sub stage evolved, and its unicellular sequencing result can only reflect the cell DNA sequence dna relation during sequencing, must careful attention if indicating follow-up research as sequence data.Because the sequence is at any time It is faced with the change of evolution；In terms of Alzheimer's disease (senile dementia) pathogenesis, research confirms amyloid-beta Deposition is the pathogenic factor of Alzheimer's disease, and its pathogenic course can cause the DNA damage of nerve cell, causes cell ageing dead Die.There is research to show that contraction occurs in only several minutes of amyloid-beta of contact in nerve cell.According to the technology of the present invention Testing result can prompt, in addition to remaining pathogenesis, theoretically, it is thin that only this contraction just can result in its nerve The DNA damage of born of the same parents；In terms of retrovirus variation.The duplication of retrovirus, must be by its RNA reverse transcription into DNA, then should DNA is incorporated into the genome of host cell, transcript mRNA generation virus structural protein, and generates viral RNA by polymerizeing, then It is assembled into progeny virus.In theory, if occurring above-mentioned DNA damage in host cell reproduction process, virus is also resulted in Be incorporated into the DNA damage of host cell gene group, or and the result of natural selection is lethal, or be virus change It is different；In terms of the asexual breeding of plant protoplast.The asexual breeding of protoplast is a kind of important method of plant breeding.This method A feature be mutation randomness and uncontrollability.Its mechanism is unknown.The technology of the present invention testing result is prompted, plant proto Strong contraction occurs with being rounded in plastid in preparation process, this violent change in vegetative state cell interior stress Change, be likely to cause the damage and variation of cell genomic dna in theory.This theoretic explanation will be plant protoplast The further investigation of asexual breeding mechanism is given a clue and helped.

Brief description of the drawings

Fig. 1 is cell in-situ electrophoretic apparatus front view, wherein 1. running buffer liquid pools, 2. filter membranes, 3. side interface channels, 4. Cell electrophoresis groove；

Fig. 2 is the detection figure that normal human embryonic lung fibroblasts core size influences SSB bonding states, and wherein 2a is shown RPA32, RPA70 and POT1 testing result；2b and 2c is respectively HFLF in adherent and Trypsin Induced suspended state cell Fluorecence dye figure；

Fig. 3 be mouse embryo stem cell core size influence SSB bonding states detection figure, wherein 3a show RPA32, RPA70 and POT1 testing results；3b and 3c is respectively that mES contaminates adherent with the cell nuclear fluorescence of Trypsin Induced suspended state Chromatic graph；

Fig. 4 be neoplastic cell nuclei size influence SSB bonding states detection figure, wherein 4a show RPA32, RPA70 and POT1 testing results；4b and 4c is respectively Hela in adherent and Trypsin Induced suspended state cell fluorecence dye figure；

Fig. 5 is cell fluorecence dye figures and flow cytometry detection of the HFLF after medicine VCR and DTX processing Figure, it is thin that wherein Fig. 5 a are left, the right HFLF cell attachments respectively after medicine VCR processing of 5a suspend with Trypsin Induced Karyon fluorescent staining figure, 5b is left, the right HFLF cell attachments and Trypsin Induced respectively after medicine DTX processing of 5b hangs Floating cell fluorecence dye figure, Fig. 5 c are respectively RPA32, RPA70 and POT1 stream after medicine VCR processing from left to right Formula cell art testing result, Fig. 5 d are respectively that RPA32, RPA70 and POT1 streaming after medicine VCR processing are thin from left to right Born of the same parents' art testing result；

Fig. 6 is cell fluorecence dye figures and flow cytometry detection of the Hela after medicine VCR and DTX processing Figure, wherein Fig. 6 a are left, the right Hela nucleus cell attachments and Trypsin Induced respectively after medicine VCR processing of 6a hangs Floating cell fluorecence dye figure, 6b is left, the right Hela nucleus cell attachments and pancreas respectively after medicine DTX processing of 6b Protease digestion suspend cell fluorecence dye figure, Fig. 6 c from left to right be respectively by medicine VCR processing after RPA32, RPA70 and POT1 Flow cytometry results, Fig. 6 d from left to right be respectively by medicine VCR processing after RPA32, RPA70 and POT1 Flow cytometry results；

Fig. 7 a are mES, MEF (MEC), HFLF and Hela Western blotting (Western Blot Figure), Fig. 7 b be MEF without drug-treated, and by medicine VCR and DTX processing after cell fluorecence dye figure, Fig. 7 c It is MEF without drug-treated, and the flow cytometry after medicine VCR and DTX processing detects figure.

Embodiment

The invention will be further described with embodiment below in conjunction with the accompanying drawings.

Embodiment 1

As shown in figure 1, device used：Including cell electrophoresis groove 4：The groove is an internal diameter 0.8cm, external diameter 0.95cm, length Spend 18cm polystyrene pipe.In the middle part of tube wall, direction is provided with one 9 × 0.6cm rectangular notch along its longitudinal axis；Electrode buffer Groove 1：Anode, negative electrode respectively have one；Size is identical, 10 × 3.5 × 5cm；Material：Polystyrene；Side interface channel 3：Anode with Cathode side respectively has one；Size is identical, internal diameter 0.8cm, external diameter 0.95cm, long 10cm silicone tube；Filter membrane 2：Anode and negative electrode Side respectively has one；Size is identical, 0.45 μm of hydrophilic Poly-s 179 (Hydrophilic polyethersulfone) filter membrane；Electricity Pole：Anode respectively has one with negative electrode；6cm grows, diameter 0.5mm electrophoresis apparatus platinum filaments.

Trypsin Induced (detects 3 kinds, is respectively to normal human embryonic lung fibroblasts (HFLF) SSBs of culture：It is multiple Albumin A 32 processed：RPA32, replication protein A 70：RPA70 and telomere protected protein 1：POT1) the detection that bonding state influences：

(1) the 2 × 10 of exponential phase of growth is obtained with conventional 0.25% trypsinization⁷HFLF。

(2) cell through phosphate buffer (PBS) centrifugal rinsing once, will abandon supernatant cell mass add 200 μ l PBS After be divided into two deciles, be added dropwise respectively in two 3.5cm culture dishes bottoms, with suction pipette head respectively by two culture dish bottoms The uniform stall with goods spread out on the ground for sale of cell suspension is distributed in whole culture dish bottom.

(3) culture dish is placed on ice, with 254nm UV lamps with 1.84W/cm²Intensity illumination, accumulated dose 165.6J/cm²。

(4) cell illuminated in two culture dishes is rinsed to the centrifuge tube for being collected in a 50ml with 20ml PBS respectively In.

(5) cell collected centrifuges 5 minutes through 260g, after abandoning supernatant, adds 4% μ l of paraformaldehyde 200 and pre-fixes 3 seconds Clock, add 46ml PBS and terminate fixation.

(6) after 260g is centrifuged 5 minutes and abandoned supernatant, 0.2%Triton X-100200 μ l is added and carry out Cell-transmission model, when Between 10 minutes, add 46ml PBS afterwards, 470g is centrifuged 5 minutes, abandons supernatant.

(7) cell mass adds 936 μ l CISE buffer solutions (trishydroxymethylaminomethane Tris:25mM；Glycine Glycine:192mM；Glucose Glucose:43.2mM；PH8.3), and by pressure-vaccum gently by the scattered suspension of cell.

(8) take 500 μ l cell suspensions to add control of another 50ml centrifuge tube as CISE, treat follow-up bearing Reason.Remaining cell suspension is used as CISE samples.

(9) CISE devices are assembled, such as see Fig. 1, and connect electrophoresis power.

(10) CISE buffer solutions are added as shown in Figure 1.

(11) power-on, is arranged to constant voltage mode, and adjustment voltage sets to 500V and starts electrophoresis operation.

(12) rubber or plastic glove are worn, wears insulation bottom footwear, ensures that human body insulate with ground.With plastic handles pipettor 126 μ l CISE samples are drawn, softly carefully press close to liquid level, away from instillation CISE bufferings at cathode side electrophoresis channel opening 1.5cm Liquid.

(13) sample immediately begins to timing, electrophoresis time 3 minutes after instilling.Timing terminates, and stops power supply.

(14) with pipettor try one's best exhaust electrophoresis tank in liquid, and by suction out liquid come together in a 50ml centrifuge tubes.

(15) CISE buffer solution cleaning down electrophoresis tanks are used, and exhaust Liquid Residue, again electrophoresis tank is noted by step 10 standard Enter CISE electrophoretic buffers.

(16) repeat step 10) -15), until whole CISE samples are disposed by CISE.If last wheel CISE sample Less than 126 μ l, still by preceding step operation.

(17) liquid measure of CISE samples manifold trunk and CISE control tubes is added to 50ml with CISE buffer solutions respectively.

(18) CISE samples and CISE controls are centrifuged 5 minutes with 1275g rotating speeds, abandons supernatant.

(19) CISE samples and CISE controls are added into 0.3ml PBS respectively, and gently by the scattered suspension of cell.

(20) CISE samples and CISE controls are fixed with 70% ethanol 15ml respectively.

(21) after fixing 24 hours, by CISE samples and CISE controls row flow cytometry detection in the usual way. Flow cytometry CISE results：

(1) fixed CISE samples and CISE controls are centrifuged 5 minutes with 1275g, abandons supernatant.

(2) CISE sample cells add 300 μ l PBS, suspension cell, are divided into trisection, move into three 1.5ml respectively Eppendorf is managed, and adds 1ml PBS in each Eppendorf pipes respectively, and 560g is centrifuged 5 minutes, abandons supernatant.CISE is compareed 400 μ l PBS are added in pipe, suspension cell, are divided into the quartering, four 1.5ml Eppendorf pipes are moved into respectively, respectively every 1ml PBS are added in individual Eppendorf pipes, 560g is centrifuged 5 minutes, abandons supernatant.

(3) all Eppendorf pipes are separately added into 3% bovine serum albumin(BSA) of 20 μ l PBS preparations.

(4) three Eppendorf pipes equipped with CISE samples are marked respectively and add rabbit-anti RPA32, SANTA CRUZ, The experiment of final concentration by specification recommended amounts determines), rabbit-anti RPA70 (BETHYL, final concentration by specification recommended amounts experiment determine) With rabbit-anti POT (abcam, the experiment of final concentration by specification recommended amounts determine) antibody.Equally, by three equipped with CISE controls Eppendorf pipes mark and add rabbit-anti RPA32 (SANTA CRUZ, concentration dose are identical with CISE sample cells), rabbit-anti respectively RPA70 (BETHYL, concentration dose are identical with CISE sample cells) and rabbit-anti POT (abcam, concentration dose and CISE sample cell phases Antibody together).It is used as antibody morphism pair in addition, the 4th Eppendorf pipe equipped with CISE controls is added into 3 μ PBS and substitutes primary antibody According to (note：It is verified by experiments, is compareed with the cell of the CISE cells handled and CISE controls as antibody morphism, as a result without statistics Difference is learned, therefore uses the cell of CISE controls to make antibody morphism control).

(5) above-mentioned Eppendorf pipes are incubated at room temperature 2 hours.

(6) above-mentioned Eppendorf pipes are separately added into 1ml PBS, 500g centrifugal rinsings 2 times, abandon supernatant.

(7) all Eppendorf pipes are separately added into 3% bovine serum albumin(BSA) of 20 μ l PBS preparations.

(8) all Eppendorf pipes are separately added into the goat anti-rabbit igg secondary antibody of 2 μ l FITC marks.Room temperature, lucifuge are incubated 30 minutes.

(9) above-mentioned Eppendorf pipes are separately added into 1ml PBS, 500g centrifugal rinsings 2 times, abandon supernatant.

(10) by all Eppendorf pipes be separately added into PI (propidium iodide) dye liquor (the μ g/ml of concentration 50, routinely DNA contaminate Liquid method is prepared) 0.5ml, puts 4 DEG C of lucifuges 1 hour.

(11) the conventional two-parameter FCM detections of row (flow cytometer used in the present embodiment is FACSCalibur).DDM patterns For excluding cell fragment and cell aggregation.Each detection sample obtains 80000 cells.

(12) result uses Flowjo software analysis, as shown in Fig. 2 dotted line compares for antibody morphism, dark solid is CISE is compareed, and light solid line is CISE samples.Fig. 2 a are respectively RPA32, RPA70 and POT1 testing result from left to right.Such as Fig. 2 Shown, edge moves forward along after compareing peak compared with CISE after CISE sample peaks, illustrates the diminution due to nucleus, SSBs bonding state hair Raw to change, i.e., SSBs is dissociated.Fig. 2 b are the HFLF cell fluorecence dye figures of normal growth, and Fig. 2 c are by trypsase Postdigestive HFLF cells fluorecence dye figure, 2b, 2c can be seen that HFLF after Trypsin Induced, HFLF nucleus hair It is raw to shrink.

Embodiment 2

Except tested and analyzed cell is mouse embryo stem cell (mES), equipment therefor, experiment are with analyzing process and embodiment 1 is identical.Fig. 3 a are respectively RPA32, RPA70 and POT1 testing result from left to right.As shown in Figure 3 a, after CISE sample peaks along with Along overlapping behind CISE controls peak, illustrate that SSBs bonding state does not change, i.e. SSBs because nucleus does not reduce Do not dissociate, Fig. 3 b are the mES cell fluorecence dye figures of normal growth, and Fig. 3 c are the mES after Trypsin Induced Cell fluorecence dye figure, 3b, 3c can be seen that mES in vitro after Trypsin Induced, and mES nucleus is not shunk.

Embodiment 3

In addition to tested and analyzed cell is tumour cell (Hela), equipment therefor, experiment and analysis process and the phase of embodiment 1 Together.Fig. 4 a are respectively RPA32, RPA70 and POT1 testing result from left to right.As shown in figure 4, edge and CISE after CISE sample peaks Edge overlaps after compareing peak, illustrates that SSBs bonding state does not change, i.e., SSBs does not go out because nucleus does not reduce Now dissociate, Fig. 4 b are the Hela cell fluorecence dye figures of normal growth, and Fig. 4 c are that the Hela after Trypsin Induced is thin Karyon fluorescent staining figure, 4b, 4c can be seen that Hela in vitro after Trypsin Induced, and Hela nucleus is not shunk.

Western blot analysis

Reference literature Guo Q, Tang W, Inagaki Y, Kokudo N, Sugawara Y, Karako H, Nakata M, Makuuchi M.Subcellular localization of KL-6mucin in colorectal carcinoma cell lines:association with metastatic potential and cell morphology.Oncol Rep.2007May；17(5):Western blot methods described in 1057-60. are thin to ESC, MEF, HELF and Hela Born of the same parents are analyzed.Primary antibody is used in experiment：Rabbit-anti β-tubulin monoclonal antibodies (#2128, Cell Signaling Technology, Inc.) and the anti-β-actin monoclonal antibodies of mouse (sc-4778, Santa Cruz Biotechnology, Inc.), secondary antibody is：Goat anti-mouse igg (sc-2031, Santa Cruz Biotechnology, Inc.) coupled HRP and HRP Coupled goat anti-rabbit igg (sc-2004, Santa Cruz Biotechnology, Inc.).ECL detections use Amersham ECL plus kit(GE Healthcare)。

Cell fluorecence dye obtains with micro-image

Twice of in exponential phase, adherent growth is rinsed in the experimental cell of 3cm culture dishes with PBS.In culture dish The middle dye liquors of addition 1ml Hoechst 33342 (5 μ g/ml in PBS), room temperature, lucifuge are incubated 20 minutes.Rinsed twice with PBS Afterwards, under Nikon TE300 inverted microscopes, excite, fluorescence observation, take pictures through UV.For conventional through 0.25% trypsase The suspension cell of digestion process, is collected using conventional centrifugal, twice of PBS centrifugal rinsings, adds the dye liquors of 1ml Hoechst 33342 Sedimentation cell is suspended, room temperature, lucifuge are incubated 20 minutes., will be thin with 200-300 μ l PBS suspension cells through PBS centrifugal rinsings Born of the same parents are added dropwise on wave carrier piece, under Nikon TE300 inverted microscopes, are excited, fluorescence observation, are taken pictures through UV.

The drug-treated of cell

Micro-pipe (microtubles) has produces compression stress to nucleus, so as to change the effect of nucleus size.According to This principle, the present invention, which goes to test, uses Docetaxel Docetaxel (microtubule stabilizer, DTX) and vincristine Vincristine (microtubule depolymerization agent, VCR) adjusts the thin of experimental cell HFLF, MEF, Hela and mESC as tool drug Karyon size.Testing final concentration of the medicine used in nutrient solution is respectively：50nM (Docetaxel) and 100ng/ml (Vincristine).Two kinds of drug exposure times are 1.5 hours.

Medicine VCR can destroy the micro-pipe tissue of cell, cell is kept non-shrinking state, thin after opposite DTX processing The micro-pipe tissue that born of the same parents can strengthen cell makes cellular contraction, and as shown in Figure 5, the HFLF after medicine VCR processing, cell is not sent out Raw to shrink, along after compareing peak with CISE along overlapping after CISE sample peaks, the HFLF after medicine DTX processing, cell occurs bright Aobvious contraction, compareed along compared with CISE behind peak along reach after CISE sample peaks, it is possible thereby to illustrate, cell is in contraction state condition Under, corresponding nucleus reduces, and SSBs bonding state changes.

It will be appreciated from fig. 6 that the Hela after medicine VCR processing, cell are not shunk, edge and CISE after CISE sample peaks Compare behind peak along overlapping, the Hela after medicine DTX processing, cell occur it is obvious shrink, after CISE sample peaks along compared with Along reach behind CISE controls peak, it is possible thereby to illustrate, cell is under the conditions of contraction state, and corresponding nucleus reduces, SSBs bonding state changes.

From Fig. 7 b and Fig. 7 c, in vitro MEF and the MEF after medicine DTX processing, cellular contraction, CISE samples Edge is moved forward along after compareing peak compared with CISE behind product peak, and the MEF after medicine VCR processing, cell are not shunk, CISE samples Edge moves forward along after compareing peak compared with CISE behind peak.MES tubulin expression is few it can be seen from Fig. 7 a, and this is to explain to implement Example mES the reason for cell is not contracted after in vitro.

Although above-mentioned the embodiment of the present invention is described with reference to accompanying drawing, model not is protected to the present invention The limitation enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not Need to pay various modifications or deformation that creative work can make still within protection scope of the present invention.

Claims (14)

1. a kind of method for keeping detecting SSBs bonding states under cell stress equilibrium condition, specifically includes following steps：

（1）Cell pre-fixes：Cell is separated, phosphate buffer is added and cell suspension is made, above-mentioned cell suspension is coated on Culture dish bottom, after using UV light irradiations under the conditions of above-mentioned culture dish is placed in into≤0 DEG C, culture dish is collected with phosphate buffer In cell, centrifugation, which is abandoned after supernatant, to be added 4% paraformaldehyde and pre-fixes 1-6sec, and punching agent, institute are added after the completion of pre-fixing Cell membrane and cell can be made and all punch by stating punching agent, punch 5-15min；

（2）Cell in-situ electrophoresis：Cell mass after above-mentioned punching is added into electrophoretic buffer cell suspension, constant voltage mode is made Under, cell suspension is instilled in electrophoresis tank cathode side, the liquid in the electrophoresis tank that exhausted after the completion of electrophoresis with pipettor, and liquid will be suctioned out Come together in a centrifuge tube, with electrophoresis wash buffer electrophoresis tank, repeat electrophoresis process, until cell suspension is fully completed electricity Swimming；

（3）Detection：Detected using flow cytometry.

2. the method as described in claim 1, it is characterised in that the step 1）It is middle with 254 nm UV lamps with 1.84W/cm² Intensity illumination, accumulated dose 165.6J/cm²。

3. the method as described in claim 1, it is characterised in that the step 1）Middle centrifugation is to centrifuge 5min in 260g.

4. the method as described in claim 1, it is characterised in that the step 1）Described in pre-fix 3sec.

5. the method as described in claim 1, it is characterised in that the step 1）Described in punching be with 0.2% Triton X- 100 punching 10min.

6. the method as described in claim 1, it is characterised in that the step 2）Middle CISE buffer solutions are that concentration is Tris：

25 mM；Glycine：192 mM；Glucose：43.2 mM, pH8.3.

7. the method as described in claim 1, it is characterised in that the cell in-situ electrophoresis concretely comprises the following steps：

（1）Cell mass adds CISE buffer solutions and scattered suspension is made；

（2）Above-mentioned part cell suspension is taken to add control of the centrifuge tube as CISE, remaining cell suspension is used as CISE samples；

（3）CISE devices are assembled, and connect electrophoresis power；

（4）Add CISE buffer solutions；

（5）Power-on, is arranged to constant voltage mode, and adjustment voltage sets to 500V and starts electrophoresis operation；

（6）CISE samples are drawn according to the size of CISE devices and instill cathode side electrophoresis tank；

（7）Sample immediately begins to timing after instilling, and timing terminates, and stops power supply；

（8）With pipettor exhaust electrophoresis tank in liquid, and by suction out liquid come together in centrifuge tube；

（9）With CISE wash buffer electrophoresis tanks, exhausted Liquid Residue, and electrophoresis tank is injected into CISE electricity again by step 4 standard Swimming buffer solution；

（10）Repeat step 6-9, until whole CISE samples are disposed by CISE；

（11）The liquid measure of CISE samples manifold trunk and CISE control tubes is added to identical with CISE buffer solutions respectively；

（12）CISE samples and CISE controls are centrifuged 5 minutes with 1275g rotating speeds, abandon supernatant；

（13）CISE samples and CISE controls are added into PBS respectively the scattered suspension of cell is made；

（14）CISE samples and CISE controls are fixed with 70% ethanol or 4% paraformaldehyde respectively.

8. method as claimed in claim 7, it is characterised in that the step 6）Instilled at cathode side electrophoresis channel opening 1.5cm CISE buffer solutions.

9. the method as described in claim 7, it is characterised in that the step 14）Fixed 24h.

10. the method as described in claim 1-9 is any, it is characterised in that the device that this method is based on, including：Cell electricity Swimming groove：The groove is the cm of an internal diameter 0.76~0.84, the cm of external diameter 0.90~1.00,18.9~17.1cm of length pipe, tube wall Middle part, along its longitudinal axis direction be provided with one 8.55 × 0.57cm~9.45 × 0.63cm notch；Running buffer liquid pool：Anode, the moon Extremely respectively there is one, size is identical, 9.5 × 3.33 × 4.75cm~10.5 × 3.68 × 5.25cm；Side interface channel：Anode and the moon Pole side respectively has one, and size is identical, the cm of internal diameter 0.76~0.84, the cm of external diameter 0.90~1.00, long 9.5~10.5cm；Filter membrane： Anode respectively has one with cathode side, and size is identical, 0.2 μm or 0.45 μm hydrophilic Poly-s 179 filter membrane.

11. the method as described in claim 10, it is characterised in that the cm of cell electrophoresis groove internal diameter 0.8, outer through 0.95 Cm, the cm of length 18 pipe, tube wall middle part, direction is provided with one 9 × 0.6 cm notches along its longitudinal axis；Running buffer liquid pool：Sun Pole, negative electrode respectively have one, and size is identical, 10 × 3.5 × 5 cm；The side interface channel, anode respectively have one with cathode side, chi It is very little identical, it is interior through 0.8cm, it is outer through 0.95cm, long 10cm；Filter membrane, anode respectively have one with cathode side, and size is identical, and aperture is 0.45 μm of hydrophilic Poly-s 179 filter membrane.

12. method as claimed in claim 11, it is characterised in that the material of the cell electrophoresis groove and running buffer liquid pool is Polystyrene, the side interface channel are silicone tube.

13. the method as described in claim 10, it is characterised in that described device is horizontal electrophoretic apparatus.

14. the method as described in claim 1-13 is any, application in cell proliferative aging death, cell is being maintained Application in dryness, the application in unicellular sequencing is analyzed, the application in terms of Alzheimer's disease pathogenesis is analyzed, Application in terms of research retrovirus variation or the application in the asexual breeding of plant.