WO2016029783A1 - 保持细胞应力平衡条件下检测SSBs结合状态的装置及方法 - Google Patents

保持细胞应力平衡条件下检测SSBs结合状态的装置及方法 Download PDF

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WO2016029783A1
WO2016029783A1 PCT/CN2015/086252 CN2015086252W WO2016029783A1 WO 2016029783 A1 WO2016029783 A1 WO 2016029783A1 CN 2015086252 W CN2015086252 W CN 2015086252W WO 2016029783 A1 WO2016029783 A1 WO 2016029783A1
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cise
cell
electrophoresis
cells
buffer
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国前
于金明
廖湘鲁
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山东省肿瘤医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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    • G01N27/447—Systems using electrophoresis

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  • the invention relates to a device and a method for detecting the binding state of SSBs under the condition of maintaining stress balance of cells.
  • Single strand DNA-binding proteins include replication protein A (RPA) and telomerase protection protein telomeres protein 1 (POT1).
  • RPA replication protein A
  • POT1 telomerase protection protein
  • SSBs Single strand DNA-binding proteins
  • SSBs that produce conformal changes detach from single-stranded DNA.
  • the SSBs that are out of bound state are separated from the cells under conditions that maintain cell integrity, and the isolated SSBs are kept from re-entering the cells, and there is no such device to date.
  • DNA is the carrier of the organism's genetic code-gene, and the stability of the genome is crucial to the biological behavior of the organism. Genomic instability or genetic variation is closely related to many human diseases, such as cancer, aging and so on. While SSBs are present in all eukaryotic cells, the binding state of SSBs plays a key role in ensuring proper DNA replication, DNA damage repair, and genome stability. However, there are no reports on the technical means and experimental methods to detect the binding state of SSBs inside the nucleus with intact structure and maintaining its stress balance.
  • a device for detecting the binding state of SSBs under the condition of maintaining stress balance of cells comprising: a cell electrophoresis tank: the groove has an inner diameter of 0.76 to 0.84 cm (preferably 0.8 cm), an outer diameter of 0.90 to 1.00 cm (preferably 0.95 cm), and a length of 18.9 to ⁇ 17.1cm (preferably 18cm) polystyrene round tube, in the middle of the tube wall, a rectangular notch of 8.55 ⁇ 0.57cm ⁇ 9.45 ⁇ 0.63cm (preferably 9 ⁇ 0.6cm) along the longitudinal axis thereof; electrophoresis buffer pool: There is one anode and one cathode, the same size, 9.5 ⁇ 3.33 ⁇ 4.75cm ⁇ 10.5 ⁇ 3.68 ⁇ 5.25cm (preferably 10 ⁇ 3.5 ⁇ 5cm); material: polystyrene; side connecting channel: one on the anode and one on the cathode side, a silicone tube having the same size, an inner diameter of 0.76 to 0.84 cm (
  • Cell in situ electrophoresis is a method in which a cell to be tested is prepared as a single cell suspension, and a single free cell is dispersed in an isotonic or stress-balanced medium, and the cell is placed under an electric field.
  • An electrophoretic method in which a substance is separated from a cell.
  • the above device for detecting the binding state of SSBs under the condition of maintaining cell stress balance is a horizontal electrophoresis device.
  • the method for detecting the binding state of the SSBs by using the above device specifically includes the following steps:
  • Cell pre-fixation The cells in the growth phase were separated and treated, and a cell suspension was prepared by adding phosphate buffer (PBS). The above cell suspension was applied to the bottom of the culture dish, and the culture dish was placed at 0 ° C and irradiated with a UV lamp. (Preferably 254 nm UV lamp is irradiated with 1.84 W/cm 2 intensity, the total dose is 165.6 J/cm 2 ), then the cells in the culture dish are collected with phosphate buffer (PBS), centrifuged (260 g for 5 min), and the supernatant is discarded. Adding 4% paraformaldehyde pre-fixed 1-6 (preferably 3) sec;
  • pre-fixation is to undergo the in situ electrophoresis process without stress in the state of stress structure and non-denatured proteins in the cells, so as to maintain cell integrity to facilitate subsequent punching.
  • Cell punching The above pre-fixed cells are added to an emptying agent, which can perforate the cell membrane and the nucleus, preferably polyethylene glycol octyl phenyl ether (Triton X-100) at a concentration of 0.2. %, cell perforation time 5-15 (preferably 10) min;
  • an emptying agent preferably polyethylene glycol octyl phenyl ether (Triton X-100) at a concentration of 0.2. %, cell perforation time 5-15 (preferably 10) min;
  • the purpose of perforation is to dissolve the lipid on the cell biofilm, so that the single-stranded DNA-binding protein can be removed from the cell, so that the shed single-stranded binding protein can be removed from the cell without destroying the overall structure of the cell.
  • CISE Cell in situ electrophoresis
  • the single-stranded binding protein detached from the single strand of DNA flows out through the pores in the cell, thereby separating the detached single-stranded binding protein from the unshed single-stranded binding protein;
  • the cell sample point is 1.5 cm away from the cathode end in the middle lane (the purpose is to prevent cells from moving to the filter membrane to aggregate when the electrophoresis is performed), to ensure that the cells do not rupture during electrophoresis, and the single-stranded DNA is reduced.
  • the time for electrophoresis is preferably 3 min.
  • Detection Detection by flow cytometry analysis.
  • the PBS used in the present invention has a pH of 7.4.
  • the above-mentioned culture dish is irradiated with UV lamp under the condition of 0 ° C, and the original state of the SSBs is strengthened to ensure the accuracy of the subsequent detection results.
  • the device of the present invention enables cells to separate bound SSBs from unbound SSBs while maintaining a stress equilibrium condition.
  • the electrophoresis buffer prepared by the invention can maintain cell isotonicity and ensure cell integrity. Glucose is selected because it has no polarity, and the molecular structure does not affect subsequent centrifugation, which is convenient for cell recovery.
  • the invention ingeniously combines cell in situ electrophoresis with cell flow analysis, and the detection result is objective and accurate.
  • the device of the invention has the advantages of simple structure, convenient operation and objective analysis results.
  • the detection of the binding state of SSBs by the method of the present invention exhibits two results of "keep binding” and “dissociation” which are easy to judge.
  • the dissociation of SSBs caused by the nuclear shrinkage caused by the change of stress in the device of the present invention has important biological significance.
  • the cascade process and biological effects can be summarized as: genomic instability caused by cell contraction-nuclear compression-chromatin conformal changes.
  • the invention adopts the first pre-fixing with 4% paraformaldehyde 200 ⁇ l seconds, and the second fixation with 70% ethanol 15ml before the flow analysis, thereby ensuring the cell integrity in the later flow analysis. At the same time, the accuracy of the flow analysis results is made.
  • the molecular mechanism leading to endogenous genomic instability discovered by the originality of the present invention belongs to the natural law at the basic level of life sciences. It reveals the pathogenesis of many major diseases (such as cancer, aging degenerative diseases - Alzheimer's disease, etc.), clarifies life phenomena (such as cell dryness, protoplast variation, etc.) and breaks through the bottleneck that limits the treatment of stem cells (such as tumors). Occurs), with important theoretical and practical significance.
  • the present invention may also provide an experimental means of detecting other DNA binding molecules, as well as molecular binding assays suitable for the methods of the present invention.
  • the present invention can explain that cell detachment from the substrate on which it is grown under the intervention of non-physiological chemical, physical and inflammatory factors, cell contraction, nuclear compression and conformal changes of chromatin can lead to changes in the binding state of SSBs, ie, unbound Separation of SSBs from combined SSBs.
  • This separation demonstrates the disassembly of DNA replication and repair complexes assembled by SSBs, resulting in instability of the cellular genome.
  • telomere shortening is still the mainstream academic viewpoint to explain the limit mechanism of Heifer. That is, the cell will produce a certain amount of shortening after each round of DNA replication telomere, and when shortened to a certain extent, the cell will have proliferative aging death. However, the length to which the telomere is shortened triggers the cell proliferative aging death, and so far only the length is inferred, and the length is not strictly determined.
  • the detection result of the technology of the present invention can expandly explain the mechanism of action of inflammatory carcinogenesis as: in the case of inflammation, especially in chronic inflammation, typical cell and extracellular matrix damage and repair occur, and these pathological changes have contributed to Another characteristic of inflammation is the detachment, rounding, and re-adhesion and spreading.
  • These repeated pathological processes lead to changes in the binding state of the SSBs disclosed in the present invention, resulting in DNA damage and gene mutations, which have an evolutionary effect.
  • the mechanism disclosed by the present technology suggests that genomic instability can lead to tumorigenesis, and the result of natural selection and tumor evolution can stabilize the tumor genome (Hela cells are exemplified).
  • many intervention factors may lead to instability of the tumor genome, such as chemotherapeutic drugs; in terms of the stemness of the cells, the detection results of the present invention show that mouse embryonic stem cells and Hela cells having the characteristics of tumor stem cells pass through.
  • Classical trypsin digestion in the case of a single cell suspension, the DNA-bound SSBs in the cells remain bound. Under the same treatment conditions, DNA-bound SSBs in human embryonic lung fibroblasts and mouse embryonic fibroblasts dissociated.
  • Retroviral replication requires reverse transcription of the RNA into DNA, integration of the DNA into the genome of the host cell, transcription of the mRNA to produce a viral structural protein, and polymerization to generate viral RNA, which is then assembled into a progeny virus.
  • DNA damage occurs during host cell replication, it will also lead to DNA damage integrated into the host cell genome, and the result of natural selection is either lethal or viral; in plant protoplasts Asexual breeding.
  • Protoplast asexual breeding is an important method of plant breeding. A feature of this method is the randomness and uncontrollability of mutations. The mechanism is unknown.
  • Figure 1 is a front view of a cell in situ electrophoresis apparatus, wherein: 1. electrophoresis buffer pool, 2. filter membrane, 3. side connection channel, 4. cell electrophoresis tank;
  • Figure 2 is a graph showing the effect of normal human embryonic lung fibroblast nuclear size on the binding state of SSB.
  • 2a shows the results of RPA32, RPA70 and POT1;
  • 2b and 2c are the nuclear staining of HFLF in the adherent and trypsin-digested suspension state, respectively. ;
  • Figure 3 is a diagram showing the relationship between the nuclear size of mouse embryonic stem cells and the SSB binding state, wherein 3a shows the results of RPA32, RPA70 and POT1; 3b and 3c are the nuclear fluorescence staining of mES in the adherent and trypsin-digested suspension state, respectively;
  • Figure 4 is a graph showing the effect of tumor cell size on the binding state of SSB.
  • 4a shows the results of RPA32, RPA70 and POT1;
  • 4b and 4c are the nuclear staining patterns of Hela in the adherent and trypsin-digested suspension state, respectively;
  • Figure 5 is a nuclear fluorescence staining and flow cytometry analysis of HFLF treated with drug VCR and DTX.
  • Figure 5a left and 5a right are HFLF cell adherent and trypsin-digested suspension after drug VCR treatment.
  • Nuclear fluorescence staining, 5b left and 5b right are HFLF cell adherence and trypsin after drug DTX treatment Enzyme digestion and suspension of nuclear fluorescence staining, Figure 5c from left to right for the RPA32, RPA70 and POT1 flow cytometry results after drug VCR treatment, Figure 5d from left to right respectively after the drug VCR treatment of RPA32 , RPA70 and POT1 flow cytometry test results;
  • Figure 6 shows the nuclear fluorescence staining and flow cytometry analysis of Hela after drug VCR and DTX treatment.
  • Figure 6a left and 6a right are Hela cell nuclear cell adherence and trypsin digestion suspension after drug VCR treatment. Nuclear staining of the cells, 6b left and 6b right are the nuclear staining patterns of Hela cell nuclear cell adherent and trypsin-digested suspension after drug DTX treatment.
  • Figure 6c shows the RPA32 after drug VCR treatment from left to right.
  • Figure 6d from left to right are the RPA32, RPA70 and POT1 flow cytometry results after drug VCR treatment;
  • Figure 7a shows the Western blot of mES, MEF (mouse embryo fibroblasts), HFLF and Hela
  • Figure 7b shows the nuclear staining of MEF without drug treatment and after treatment with VCR and DTX
  • Figure 7c is a flow cytometric analysis of MEF without drug treatment and after treatment with drugs VCR and DTX.
  • the apparatus used includes a cell electrophoresis tank 4: the tank is a polystyrene round tube having an inner diameter of 0.8 cm, an outer diameter of 0.95 cm, and a length of 18 cm. In the middle of the pipe wall, there is a rectangular slot of 9 ⁇ 0.6cm along the longitudinal axis thereof; electrode buffer tank 1: one anode and one cathode; the same size, 10 ⁇ 3.5 ⁇ 5cm; material: polystyrene; side connection Channel 3: one on each side of the anode and the cathode; a silicone tube of the same size, inner diameter 0.8 cm, outer diameter 0.95 cm, length 10 cm; filter membrane 2: one on the anode and cathode sides; the same size, 0.45 ⁇ m hydrophilic polyphenylene Hydrophilic polyethersulfone filter; electrode: one anode and one cathode; 6 cm long, 0.5 mm diameter electrophoresis instrument with platinum wire.
  • electrode buffer tank 1 one anode and one cath
  • HFLF normal human embryonic lung fibroblasts
  • the cells were rinsed once by centrifugation in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the discarded cell pellet was added to 200 ⁇ l of PBS and divided into two equal portions, which were separately added to the bottom of two 3.5 cm culture dishes, and pipetted with a pipette.
  • the cell suspensions at the bottom of the two culture dishes were evenly distributed throughout the bottom of the culture dish.
  • Cell pellets 936 ⁇ l of CISE buffer (Tris: 25 mM; glycine Glycine: 192 mM; glucose Glucose: 43.2 mM; pH 8.3) was added, and the cells were gently suspended by suction.
  • CISE sample tube was added with 300 ⁇ l of PBS, the cells were suspended, divided into three equal portions, and transferred into three 1.5 ml Eppendorf tubes, respectively, and 1 ml of PBS was added to each Eppendorf tube, centrifuged at 560 g for 5 minutes, and the supernatant was discarded.
  • Add 400 ⁇ l PBS to the CISE control tube suspend the cells, divide into four equal portions, and transfer into four 1.5 ml Eppendorf tubes, respectively, in each Eppendorf. 1 ml of PBS was added to the tube, centrifuged at 560 g for 5 minutes, and the supernatant was discarded.
  • a 4th Eppendorf tube containing CISE control was added to 3 ⁇ PBS instead of primary antibody for antibody isotype control (Note: cells treated with CISE and CISE control cells were used as antibody isotype control, and the results were not statistically significant. The difference was the CISE control cells used as antibody isotype controls).
  • a conventional two-parameter FCM test (the flow cytometer used in this example is FACSCalibur).
  • the DDM mode is used to exclude cell debris and cell aggregation. 80,000 cells were obtained per test sample.
  • FIG. 2a shows the RPA32, RPA70, and POT1 detection results from left to right.
  • the trailing edge of the CISE sample is shifted forward compared with the trailing edge of the CISE control peak, indicating that the binding state of the SSBs changes due to the shrinkage of the nucleus, that is, the dissociation of the SSBs occurs.
  • Figure 2b shows the nuclear fluorescence staining of normal-grown HFLF cells.
  • Figure 2c shows the fluorescein staining of HFLF cells after trypsinization. 2b and 2c show that HFLF cells shrink after HFLF digestion by trypsin.
  • FIG. 3a shows the RPA32, RPA70, and POT1 detection results from left to right.
  • the trailing edge of the CISE sample coincides with the trailing edge of the CISE control peak, indicating that the binding state of the SSBs has not changed due to the absence of shrinkage in the nucleus. That is, there is no dissociation of SSBs.
  • Figure 3b shows the nuclear fluorescence staining of normal growing mES cells.
  • Figure 3c shows the fluorescence staining of mES cells after trypsin digestion. 3b and 3c can be seen that mES is trypsinized after excision, mES The nucleus did not contract.
  • FIG. 4a shows the RPA32, RPA70, and POT1 detection results from left to right.
  • the trailing edge of the CISE sample coincides with the trailing edge of the CISE control peak, indicating that the binding state of the SSBs did not change due to the absence of shrinkage in the nucleus, ie, the SSBs did not dissociate
  • Figure 4b shows the nuclear fluorescence of the normal-grown Hela cells.
  • the staining map, Figure 4c is the nuclear fluorescence staining of Hela cells after trypsin digestion, 4b, 4c can be seen that Hela cells have not contracted after trypsin digestion.
  • HRP-coupled goat anti-mouse IgG sc-2031, Santa Cruz Biotechnology, Inc.
  • HRP-coupled goat anti-rabbit IgG sc-2004, Santa Cruz Biotechnology, Inc.
  • the ECL test was performed using an Amersham ECL plus kit (GE Healthcare).
  • the experimental cells adhering to the 3 cm culture dish in the logarithmic growth phase were washed twice with PBS.
  • 1 ml of Hoechst 33342 stain (5 ⁇ g/ml in PBS) was added to the dish and incubated for 20 minutes at room temperature in the dark.
  • the cells were subjected to UV excitation, fluorescence observation, and photographing under an inverted microscope of Nikon TE300.
  • the suspension cells which were routinely digested with 0.25% trypsin the cells were collected by conventional centrifugation, rinsed twice with PBS, and the precipitated cells were suspended by adding 1 ml of Hoechst 33342 solution, and incubated at room temperature for 20 minutes in the dark.
  • the cells were suspended in 200-300 ⁇ l PBS, and the cells were dropped on a slide, and subjected to UV excitation, fluorescence observation, and photographing under a Nikon TE300 inverted microscope.
  • Microtubles have the effect of compressing stress on the nucleus, thereby changing the size of the nucleus.
  • the experiment of the present invention uses docetaxel (microtubule stabilizer, DTX) and vincristine (microtubule depolymerization agent, VCR) as a tool to regulate experimental cells HFLF, MEF, Hela and mESC.
  • the size of the nucleus The final concentrations of the drugs used in the experiments were: 50 nM (Docetaxel) and 100 ng/ml (Vincristine). Both drugs were treated for 1.5 hours.
  • the drug VCR can destroy the microtubule tissue of the cell and keep the cell in a non-shrinking state.
  • the cell can strengthen the microtubule tissue of the cell to cause the cell to shrink.
  • the cell does not occur after the HFLF treated by the drug VCR.
  • the trailing edge of CISE sample coincides with the trailing edge of CISE control peak.
  • the cells contracted significantly.
  • the trailing edge of CISE sample moved forward compared with the trailing edge of CISE control peak, which indicates that the cell is in Under the condition of contraction, the corresponding nucleus shrinks and the binding state of SSBs changes.

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Abstract

一种保持细胞应力平衡条件下检测SSBs结合状态的装置及方法，该装置包括：圆管细胞电泳槽，管壁中部，沿其纵轴方向开有一矩形槽口，电泳缓冲液池，侧连接通道，滤膜，孔径为0.2μm或0.45μm亲水聚苯醚砜滤膜，所述电泳缓冲液池与侧连接通道铸成一体，侧连接通道与电泳槽通过可拆卸的方式密封连接，滤膜插在侧连接通道与电泳槽的连接处。该装置能够使细胞在保持原细胞结构完整性的条件下分离结合态SSBs与非结合SSBs。

Description

保持细胞应力平衡条件下检测SSBs结合状态的装置及方法 技术领域
本发明涉及一种保持细胞应力平衡条件下检测SSBs结合状态的装置及方法。
背景技术
单链DNA结合蛋白(single strand DNA-binding proteins,SSBs)包括replication protein A(RPA)和端粒保护蛋白protection of telomeres protein 1(POT1)。当DNA复制的时候，双链DNA需要解旋成单链DNA并且与单链DNA结合蛋白结合来防止形成错误结构，维护端粒及基因组的稳定性。然而，已分化了的正常体细胞在非生理的化学、物理以及炎症因素干预下，细胞会从其生长的基质上脱离，其细胞核会产生剧烈的缩小，与此同时，与SSBs结合的染色质/DNA会受到这种缩小和拥挤的细胞核的压迫，从而产生适形变化,产生适形变化的SSBs会从单链DNA上脱离。将脱离结合状态的SSBs在保持细胞完整性的条件下，使其从细胞中分离出来，并且保持分离出的SSBs不会重新进入细胞，至今没有这样的装置。
众所周知，DNA是生物体遗传密码-基因的载体，基因组的稳定性对生物体的生物学行为是至关重要的。基因组的不稳定性或基因的变异与人类众多疾病密切相关，如癌症、衰老等等。而SSBs存在于一切真核生物细胞，SSBs的结合状态对保证DNA的正确复制、DNA的损伤修复以及基因组的稳定性具有关键性的作用。然而目前未发现有关技术手段和实验方法能够实现对结构完整且保持其应力平衡的细胞核内部的SSBs结合状态进行检测的文献报道。
发明内容
本发明的目的就是为了提供一种保持细胞应力平衡条件下检测SSBs结合状态的装置及方法。
为了实现上述目的，本发明采用如下技术方案：
一种保持细胞应力平衡条件下检测SSBs结合状态的装置，包括：细胞电泳槽：该槽为内径0.76～0.84cm(优选0.8cm)、外径0.90～1.00cm(优选0.95cm)、长度18.9～17.1cm(优选18cm)的聚苯乙烯圆管，管壁中部，沿其纵轴方向开有一8.55×0.57cm～9.45×0.63cm(优选9×0.6cm)的矩形槽口；电泳缓冲液池：阳极、阴极各有一个，尺寸相同，9.5×3.33×4.75cm～10.5×3.68×5.25cm(优选10×3.5×5cm)；材质：聚苯乙烯；侧连接通道：阳极与阴极侧各有一个，尺寸相同，内径0.76～0.84cm(优选0.8cm)，外径0.90～1.00cm(优选0.95cm)，长9.5～10.5cm(优选10cm)的硅胶管；滤膜：阳极与阴极侧各有一个， 尺寸相同，0.45μm或0.2μm亲水聚苯醚砜(Hydrophilic polyethersulfone)滤膜；电极：阳极与阴极各有一根，5.7～6.3cm(优选6cm)长，直径0.475～0.525cm(优选0.5mm)电泳仪用铂丝。
细胞原位电泳(cell in situ electrophoresis,CISE)，是将待检细胞制备成单细胞悬液，使其单个游离的细胞分散于等渗即应力平衡的介质中，在电场作用下，将细胞中的物质从细胞中分离出来的一种电泳方式。
上述保持细胞应力平衡条件下检测SSBs结合状态的装置，为水平式电泳装置。
利用上述装置检测SSBs结合状态的方法，具体包括以下步骤：
细胞预固定：分离处理生长期细胞，加入磷酸盐缓冲液(PBS)制成细胞悬液，将上述细胞悬液涂布于培养皿底部，将上述培养皿置于0℃条件下用UV灯照射(优选254nm UV灯以1.84W/cm2强度照射，总剂量165.6J/cm2)后，用磷酸盐缓冲液(PBS)收集培养皿中的细胞，离心(260g离心5min)弃上清液后加入4％多聚甲醛预固定1-6(优选3)sec；
预固定的目的为：既有应力结构以及细胞内待检蛋白非变性的状态下，经受细胞原位电泳过程，而不发生破碎，以保持细胞完整性，以方便接下来的打孔。
细胞打孔：将上述预固定的细胞，加入打空剂，所述打空剂能够使细胞膜和细胞核都打孔，优选聚乙二醇辛基苯基醚(Triton X-100)，浓度为0.2％，细胞打孔时间5-15(优选10)min；
打孔的目的为：溶解细胞生物膜上的脂质，使单链DNA结合蛋白能够从细胞中移出，以方便脱落的单链结合蛋白从细胞中移出，不破坏细胞的整体结构。
细胞原位电泳(cell in situ electrophoresis，CISE)：将上述打孔后的细胞团加入CISE缓冲液(三羟甲基氨基甲烷Tris 25mM；甘氨酸Glycine:192mM；葡萄糖Glucose：43.2mM；pH8.3)制成细胞悬液，恒压模式下，在电泳槽阴极侧滴入细胞悬液，电泳完成后用移液器吸净电泳槽中的液体，并将吸出液汇集于一离心管中，用电泳缓冲液冲洗电泳槽，重复电泳过程，直至细胞悬液全部完成电泳；
在电泳条件下，从DNA单链上脱落的单链结合蛋白通过细胞上的孔道流出，从而实现脱落的单链结合蛋白与未脱落的单链结合蛋白的分离；
优选点样时，细胞样品点在中间泳道距离阴极端1.5cm处(目的是：电泳时，防止细胞移动至滤膜处从而聚集成团)，保证电泳时细胞不破裂，且减少了单链DNA结合蛋白从一个细胞移出后再通过细胞上的孔道进入另一个细胞的概率。电泳的时间优选为3min。
检测：使用流式细胞术分析检测。
本发明中所用到的PBS，pH值都为7.4。
本发明的有益效果：
在预固定时，将上述培养皿置于0℃条件下用UV灯照射，强化了SSBs原有的状态，保证后续检测结果的准确。
本发明的装置能够使细胞在保持应力平衡状态的条件下分离结合态SSBs与非结合SSBs。
本发明制备的电泳缓冲液可以保持细胞等渗，保证细胞完整，选用Glucose是因为其无极性，且分子结构不影响后续的离心，便于细胞的回收。
本发明巧妙的将细胞原位电泳与细胞流式分析术结合，检测结果客观准确。
本发明的装置结构简单、操作方便和分析结果客观的特点。应用本发明方法对SSBs结合状态的检测，呈现出易于判断的“保持结合”与“出现解离”两种结果。
本发明装置的使用应力改变所致细胞核缩小造成的SSBs解离具有重要的生物学意义。其级联过程以及生物学效应可概括为：细胞收缩-细胞核压缩-染色质适形变化所导致的基因组不稳定性。
本发明采用了首先用4％的多聚甲醛200μl秒级预固定，在进行流式分析前又用70％的乙醇15ml进行二次固定，保证了细胞在后期的流式分析中细胞的完整，同时使得流式分析结果的准确性。
用本发明技术原创性发现的导致内源性基因组不稳定的分子机制属于生命科学基础层面上的自然规律。它对于揭示许多重大疾病(如癌症、衰老退化性疾病-阿尔茨海默症等)的发病机制，阐明生命现象(如细胞干性、原生质变异等)，突破限制干细胞治疗治疗的瓶颈(如肿瘤发生)，具有重要的理论与实际意义。
从技术层面或方法学上，本发明还可为研究其它DNA结合分子，以及适合于本发明技术方法的分子结合作用分析提供一种实验检测手段。
本发明能够说明细胞在非生理的化学、物理以及炎症因素干预下，从其生长的基质上脱离所产生的细胞收缩、细胞核压缩以及染色质适形变化会导致SSBs结合状态的改变，即未结合的SSBs与结合的SSBs的分离。这种分离说明由SSBs所参与组装的DNA复制与修复复合体的去组装，结果是导致细胞基因组的不稳定。
应用本发明所揭示的这种SSBs结合状态改变的自然规律，可解释一系列困扰生命科学领域的众多重要与疾病以及应用研究相关的科学谜题。在经典假说与理论方面，这一发现使得一项半个多世纪的科学谜题的背后机理从科学逻辑上得到更完善的解释。1961年由美国科 学家海弗里克(Leonard Hayflick)提出的著名假说----海弗里克极限(Hayflick limit)被誉为了生物学的顶梁柱(pillar of biology)。该假说认为细胞只能群体倍增(population doublings)约50代，其后细胞便进入增殖性衰老死亡。关于这一假说的机理，诺贝尔奖得主卡洛尔.格雷德(Carol W.Greider)等人于1990年在国际著名杂志Nature上发表文章提示这种增殖性衰老是由端粒的缩短造成的。这一端粒缩短理论至今仍成为解释海弗里克极限机理的主流学术观点。即细胞每经一轮DNA复制端粒会出现一定量的缩短，当缩短到一定程度时，细胞将出现增殖性衰老死亡。但是端粒缩短到何等长度即触发细胞增殖性衰老死亡，迄今只有推断长度，没有严格的确定长度。然而，根据由本发明方法所得实验结果进行的深入实验研究证实，当细胞群体倍增只有7次(远未达到海弗里克极限所述的50次左右)，而细胞传代达50次左右(与达到海弗里克极限时，细胞所经历的传代数一致)的时候，细胞即出现增殖性衰老死亡。说明海弗里克极限并非由群体倍增产生。实质上，由于细胞传代所引起的SSBs结合状态改变可造成细胞DNA的损伤，这种损伤的累积是造成海弗里克极限的决定性因素；这一发现亦为解释另一项重要生物学谜题---小鼠胚胎成纤维细胞的体外增殖性衰老死亡提供了实验与理论依据。1998年卡曼等人发现叙利亚仓鼠胚胎成纤维细胞在经历20-30次群体倍增后，虽保留相当长(23kb)的端粒长度及端粒酶活性，却仍然呈现增殖性衰老死亡。科学家因无法用端粒缩短理论来解释这一现象，故推测老鼠的细胞增殖性衰老死亡机理可能与人的不一样，但确切机理不详。然而，用本发明技术所得到的实验结果阐明了这一科学谜题的机理；在干细胞方面，其干细胞治疗已成为全世界科学家与民众关心的再生医学热点问题。人们寄希望于用干细胞治疗来解决目前许多疑难绝症。但是，该项技术的临床应用目前仍存在许多问题，其中，生物安全问题是重要一项，而干细胞治疗可导致癌症是安全问题的重中之重。实验证实，在动物治疗模型中，部分治疗的细胞在动物体内会形成肿瘤，即使在未长成肿瘤的治疗细胞中，其基因变异也是一种潜在的致病威胁。目前因缺乏理论认识，干细胞的致瘤问题已成为制约干细胞临床应用的瓶颈。通过本专利技术所揭示的SSBs结合状态改变造成细胞DNA损伤与基因组不稳定性的发现，为解决干细胞治疗的障碍问题提供了一项重要的理论突破。
当前，人们普遍采用将未分化的多能干细胞先诱导为用于治疗的分化细胞，再植入体内。并认为这些分化的细胞中混入了未分化的干细胞是致瘤的问题所在。然而，本发明技术的检测结果从机理和实验上提示，在为满足治疗数量要求所进行的细胞扩增过程中，即使采用现在的经典手段进行一次扩增，其增殖的分化细胞基因发生突变的概率也为100％，即存在致癌风险。这一发现既为干细胞治疗带来了挑战，也为干细胞治疗怎样突破障碍指明了解决问题 的方向；在癌症发生与基因突变方面，细胞的基因突变是大家公认的致癌机理。传统理论认为，癌症的发生是细胞经历多次突变事件，与癌症发生有关的基因突变逐渐积累的结果。但是，近来科学家通过大规模高通量基因组测序分析发现，许多癌症的发生是大量与癌症相关的基因经历一次事件即发生突变所致。这一发现被命名为“染色体碎裂”(chromothripsis)、“染色体风暴”(kataegis)。然而，造成这一现象的背后机理对于科学家却是一个谜题。本发明技术检测的结果恰好为其发生机理提供科学解释。一个细胞的DNA复制起点有40，000到50，000个之多。因为人的细胞要在一个细胞周期内完成约30亿碱基对的DNA巨大复制工作，所以需要数万个复制点同时工作才能完成。在这一复制过程中，一次造成处于该复制阶段的体细胞脱离其生长基质的事件，均可导致众多的DNA损伤与基因突变，这可解释为造成“染色体风暴”、“染色体碎裂”的原因；在炎症致癌方面，大量的研究证实炎症是导致癌症的重要原因。但对于炎症致癌的机理各种解释不一。其中最主要的解释是炎症细胞所释放的炎症因子经过繁杂的途径与步骤间接性地损伤细胞的DNA。
而本发明技术的检测结果可将炎症致癌的作用机制拓展性地解释为：在炎症发生时，尤其在慢性炎症时，会出现典型的细胞与细胞外基质的损伤与修复，这些病理变化促成了炎症典型的另一特征---细胞脱落、变圆以及再粘着与铺展，这些反复的病理过程会导致本发明所揭示的细胞SSBs结合状态改变，从而造成DNA损伤与基因突变，出现具有进化作用的“染色体风暴”、“染色体碎裂”并进行自然选择与致癌进化。这种解释从逻辑角度来分析也更为直接；在肿瘤异质性方面，研究证实肿瘤异质性是肿瘤治疗的难点之一，已成为肿瘤研究的热点问题。本发明技术所揭示的机理，为肿瘤异质性的产生提供了科学的解释。细胞周期的自然分布从理论上提示肿瘤内部的细胞各自处于不同的周期时相位置，即非同步状态。若出现“染色体风暴”事件，每个细胞将产生不同的DNA损伤，因此细胞自然会出现异质性；在肿瘤进化与基因组不稳定性方面，科学家对基因组不稳定性是肿瘤发生的原因还是结果存在争议。本发明技术所揭示的机理提示基因组不稳定性可导致肿瘤的发生，而自然选择与肿瘤进化的结果可以使肿瘤的基因组趋于稳定(Hela细胞即是例证)。而许多干预因素又可导致肿瘤基因组出现不稳定性，如化疗药物等；在细胞的干性(stemness)方面，本发明技术检测结果显示，小鼠胚胎干细胞和具有肿瘤干细胞特性的Hela细胞在经过经典的胰蛋白酶消化，制成单细胞悬液的情况下，其细胞中与DNA结合的SSBs仍保持结合状态。同样的处理条件下，人胚胎肺成纤维细胞和小鼠胚胎成纤维细胞中与DNA结合的SSBs则出现解离。提示避免细胞核的应力收缩是维持细胞干性的一个重要机制；在单细胞测序方面，鉴于研究证实肿瘤细胞具有高度的异质性，所以近年来单细胞测序迅速发展，以期反映细胞亚群的异质性，为后续研究 提供数据依据。然而，本发明技术检测结果提示，如果选择用于单细胞测序的肿瘤细胞尚未处于进化的基因组稳定阶段，其单细胞测序结果只能反映该细胞测序时的DNA序列关系，若用作序列数据指示后续的研究，则须谨慎对待。因为该序列随时面临着进化的改变；在阿尔茨海默症(老年痴呆)致病机理方面，研究证实β-淀粉样蛋白的沉积是阿尔茨海默症的致病因素，其致病过程可造成神经细胞的DNA损伤，致细胞衰老死亡。有研究显示神经细胞在接触β-淀粉样蛋白仅数分钟内即会出现收缩。根据本发明技术的检测结果可提示，除了其余的致病机理之外，理论上讲，仅这种收缩就能够造成其神经细胞的DNA损害；在逆转录病毒变异方面。逆转录病毒的复制，须将其RNA逆转录成DNA，再将该DNA整合到宿主细胞的基因组，转录mRNA生成病毒结构蛋白，并通过聚合生成病毒RNA，然后装配成子代病毒。理论上，在宿主细胞复制过程中若出现上述的DNA损伤，同样会导致病毒整合到宿主细胞基因组的DNA损伤，而自然选择的结果要么是致死性的，要么是病毒的变异；在植物原生质体无性育种方面。原生质体无性育种是植物育种的一种重要方法。该方法的一个特点是突变的随机性和不可控性。其机理不详。本发明技术检测结果提示，植物原生质体在制备过程中会出现强烈的收缩与变圆，这种处于增殖状态细胞内部应力的剧烈变化，理论上很可能造成细胞基因组DNA的损伤与变异。这种理论上的解释将为植物原生质体无性育种机理的深入研究提供线索与帮助。
附图说明
图1为细胞原位电泳装置主视图，其中1.电泳缓冲液池，2.滤膜，3.侧连接通道，4.细胞电泳槽；
图2为正常人胚肺成纤维细胞核大小影响SSB结合状态的检测图，其中2a显示RPA32、RPA70和POT1检测结果；2b和2c分别为HFLF在贴壁与胰蛋白酶消化悬浮状态的细胞核荧光染色图；
图3为小鼠胚胎干细胞核大小影响SSB结合状态的检测图，其中3a显示RPA32、RPA70和POT1检测结果；3b和3c分别为mES在贴壁与胰蛋白酶消化悬浮状态的细胞核荧光染色图；
图4为肿瘤细胞核大小影响SSB结合状态的检测图，其中4a显示RPA32、RPA70和POT1检测结果；4b和4c分别为Hela在贴壁与胰蛋白酶消化悬浮状态的细胞核荧光染色图；
图5为HFLF经过药物VCR和DTX处理后的细胞核荧光染色图和流式细胞术分析检测图，其中图5a左、5a右分别为经过药物VCR处理后的HFLF细胞贴壁与胰蛋白酶消化悬浮的细胞核荧光染色图，5b左、5b右分别为经过药物DTX处理后的HFLF细胞贴壁与胰蛋白 酶消化悬浮的细胞核荧光染色图，图5c从左向右分别为经过药物VCR处理后的RPA32、RPA70和POT1流式细胞术检测结果，图5d从左向右分别为经过药物VCR处理后的RPA32、RPA70和POT1流式细胞术检测结果；
图6为Hela经过药物VCR和DTX处理后的细胞核荧光染色图和流式细胞术分析检测图，其中图6a左、6a右分别为经过药物VCR处理后的Hela细胞核细胞贴壁与胰蛋白酶消化悬浮的细胞核荧光染色图，6b左、6b右分别为经过药物DTX处理后的Hela细胞核细胞贴壁与胰蛋白酶消化悬浮的细胞核荧光染色图，图6c从左向右分别为经过药物VCR处理后的RPA32、RPA70和POT1流式细胞术检测结果，图6d从左向右分别为经过药物VCR处理后的RPA32、RPA70和POT1流式细胞术检测结果；
图7a为mES、MEF(小鼠胚胎成纤维细胞)，HFLF和Hela的蛋白质印迹(Western Blot图)，图7b为MEF不经过药物处理，以及经过药物VCR和DTX处理后的细胞核荧光染色图，图7c为MEF不经过药物处理，以及经过药物VCR和DTX处理后的流式细胞术分析检测图。
具体实施方式
下面结合附图与实施例对本发明作进一步说明。
实施例1
如图1所示，所用的装置：包括细胞电泳槽4：该槽为一内径0.8cm、外径0.95cm、长度18cm的聚苯乙烯圆管。管壁中部，沿其纵轴方向开有一9×0.6cm的矩形槽口；电极缓冲液槽1：阳极、阴极各有一个；尺寸相同，10×3.5×5cm；材质：聚苯乙烯；侧连接通道3：阳极与阴极侧各有一个；尺寸相同，内径0.8cm，外径0.95cm,长10cm的硅胶管；滤膜2：阳极与阴极侧各有一个；尺寸相同，0.45μm亲水聚苯醚砜(Hydrophilic polyethersulfone)滤膜；电极：阳极与阴极各有一根；6cm长，直径0.5mm电泳仪用铂丝。
胰蛋白酶消化对培养的正常人胚肺成纤维细胞(HFLF)SSBs(检测3种，分别为：复制蛋白A32：RPA32、复制蛋白A70：RPA70和端粒保护蛋白1：POT1)结合状态影响的检测：
(1)用常规0.25％胰蛋白酶消化法获取指数生长期的2×107HFLF。
(2)细胞经磷酸盐缓冲液(PBS)离心漂洗一次，将弃上清的细胞团加入200μl PBS后分成两等分，分别滴加于两个3.5cm培养皿底部，用移液器吸头分别将两个培养皿底部的细胞悬液均匀地摊布于整个培养皿底部。
(3)将培养皿置于冰上，用254nm UV灯以1.84W/cm2强度照射，总剂量165.6J/cm2。
(4)分别用20ml PBS将两个培养皿中被照射的细胞冲洗收集于一个50ml的离心管中。
(5)收集的细胞经260g离心5分钟，弃上清后，加入4％的多聚甲醛200μl预固定3秒钟，加入46ml PBS终止固定。
(6)经260g离心5分钟弃上清后，加入0.2％Triton X-100 200μl进行细胞打孔，时间10分钟，之后加入46ml PBS，470g离心5分钟，弃上清。
(7)细胞团加入936μl CISE缓冲液(三羟甲基氨基甲烷Tris:25mM；甘氨酸Glycine:192mM；葡萄糖Glucose:43.2mM；pH8.3)，并通过吹吸轻柔地将细胞分散悬浮。
(8)取500μl细胞悬液加入另一支50ml离心管作为CISE的对照，待后续的固定处理。其余细胞悬液被用作CISE样本。
(9)装配CISE装置，如见图1，并连接电泳电源。
(10)按图1所示加入CISE缓冲液。
(11)开启电源，设置为恒压模式，调整电压设置到500V并开始电泳运行。
(12)戴橡胶或塑料手套，穿绝缘底鞋具，保证人体与地面绝缘。用塑料把手移液器吸取126μl CISE样品，轻柔小心地贴近液面，在距阴极侧电泳槽开口1.5cm处滴入CISE缓冲液。
(13)样品滴入后立即开始计时，电泳时间3分钟。计时结束，中止电源。
(14)用移液器尽量吸净电泳槽中的液体，并将吸出液汇集于一50ml离心管中。
(15)用CISE缓冲液彻底冲洗电泳槽，吸净残留液，按步骤10标准重新将电泳槽注入CISE电泳缓冲液。
(16)重复步骤10)-15)，直至全部CISE样品被CISE处理完毕。若最后一轮CISE样品不足126μl，仍按前步骤操作。
(17)分别用CISE缓冲液补加CISE样品汇集管和CISE对照管的液量至50ml。
(18)将CISE样品和CISE对照以1275g转速离心5分钟，弃上清。
(19)分别将CISE样品和CISE对照加入0.3ml PBS，并轻柔地将细胞分散悬浮。
(20)分别将CISE样品和CISE对照用70％的乙醇15ml固定。
(21)固定24小时后，将CISE样品和CISE对照按常规方式行流式细胞术分析检测。流式细胞术分析CISE结果：
(1)将固定的CISE样品和CISE对照以1275g离心5分钟，弃上清。
(2)CISE样品管加入300μl PBS，混悬细胞，分成三等分，分别移入三个1.5ml Eppendorf管，分别在每个Eppendorf管中加入1ml PBS，560g离心5分钟，弃上清。CISE对照管中加入400μl PBS，混悬细胞，分成四等分，分别移入四个1.5ml Eppendorf管，分别在每个Eppendorf 管中加入1ml PBS，560g离心5分钟，弃上清。
(3)将所有Eppendorf管分别加入20μl PBS配制的3％牛血清白蛋白。
(4)将三个装有CISE样品的Eppendorf管分别标记并加入兔抗RPA32，SANTA CRUZ，终浓度按说明书推荐量实验确定)、兔抗RPA70(BETHYL，终浓度按说明书推荐量实验确定)和兔抗POT(abcam，终浓度按说明书推荐量实验确定)抗体。同样，将三个装有CISE对照的Eppendorf管分别标记并加入兔抗RPA32(SANTA CRUZ，浓度剂量与CISE样品管相同)、兔抗RPA70(BETHYL，浓度剂量与CISE样品管相同)和兔抗POT(abcam，浓度剂量与CISE样品管相同)抗体。另外，将第4个装有CISE对照的Eppendorf管加入3μPBS替代一抗用作抗体同型对照(注：经实验证实，用CISE处理的细胞和CISE对照的细胞用作抗体同型对照，结果无统计学差异，故采用CISE对照的细胞作抗体同型对照)。
(5)上述Eppendorf管室温孵育2小时。
(6)上述Eppendorf管分别加入1ml PBS，500g离心漂洗2次，弃上清。
(7)将所有Eppendorf管分别加入20μl PBS配制的3％牛血清白蛋白。
(8)将所有Eppendorf管分别加入2μl FITC标记的羊抗兔IgG二抗。室温、避光孵育30分钟。
(9)上述Eppendorf管分别加入1ml PBS，500g离心漂洗2次，弃上清。
(10)将所有Eppendorf管分别加入PI(碘化丙啶)染液(浓度50μg/ml，按常规DNA染液方法配制)0.5ml，置4℃避光1小时。
(11)行常规双参数FCM检测(本实施例所用流式细胞仪为FACSCalibur)。DDM模式用于排除细胞碎片和细胞聚集。每个检测样品获取80000个细胞。
(12)结果采用Flowjo软件分析，如图2所示，虚线为抗体同型对照，深色实线为CISE对照，浅色实线为CISE样品。图2a从左向右分别为RPA32、RPA70和POT1检测结果。如图2所示，CISE样品峰后沿较CISE对照峰后沿前移，说明由于细胞核的缩小，SSBs的结合状态发生改变，即SSBs出现解离。图2b为正常生长的HFLF细胞核荧光染色图，图2c为经过胰蛋白酶消化后的HFLF细胞核荧光染色图，2b、2c可以看出HFLF经过胰蛋白酶消化后，HFLF细胞核发生收缩。
实施例2
除所检测分析细胞为小鼠胚胎干细胞(mES)，所用装置、实验与分析过程与实施例1相同。图3a从左向右分别为RPA32、RPA70和POT1检测结果。如图3a所示，CISE样品峰后沿与CISE对照峰后沿重合，说明由于细胞核没有出现缩小，SSBs的结合状态未发生改变， 即SSBs未出现解离，图3b为正常生长的mES细胞核荧光染色图，图3c为经过胰蛋白酶消化后的mES细胞核荧光染色图，3b、3c可以看出mES离体经过胰蛋白酶消化后，mES细胞核未发生收缩。
实施例3
除所检测分析细胞为肿瘤细胞(Hela)外，所用装置、实验与分析过程与实施例1相同。图4a从左向右分别为RPA32、RPA70和POT1检测结果。如图4所示，CISE样品峰后沿与CISE对照峰后沿重合，说明由于细胞核没有出现缩小，SSBs的结合状态未发生改变，即SSBs未出现解离，图4b为正常生长的Hela细胞核荧光染色图，图4c为经过胰蛋白酶消化后的Hela细胞核荧光染色图，4b、4c可以看出Hela离体经过胰蛋白酶消化后，Hela细胞核未发生收缩。
Western blot analysis
参照文献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):1057-60.中所描述的Western blot方法对ESC,MEF,HELF and Hela细胞进行分析。实验所用一抗为：兔抗β-tubulin单克隆抗体(#2128,Cell Signaling Technology,Inc.)和小鼠抗β-actin单克隆抗体(sc-4778,Santa Cruz Biotechnology,Inc.)，二抗为：HRP偶连的羊抗小鼠IgG(sc-2031,Santa Cruz Biotechnology,Inc.)和HRP偶连的羊抗兔IgG(sc-2004,Santa Cruz Biotechnology,Inc.)。ECL检测采用Amersham ECL plus kit(GE Healthcare)。
细胞核荧光染色与显微图像获取
用PBS冲洗两遍处于对数生长期的、贴壁生长于3cm培养皿的实验细胞。在培养皿中加入1ml Hoechst 33342染液(5μg/ml in PBS)，室温、避光孵育20分钟。用PBS冲洗两遍后，在Nikon TE300倒置显微镜下，经UV激发，荧光观测、拍照。对于经0.25％胰蛋白酶常规消化处理的悬浮细胞，采用常规离心收集，PBS离心漂洗两遍，加入1ml Hoechst 33342染液将沉淀细胞悬浮，室温、避光孵育20分钟。经PBS离心漂洗，用200-300μl PBS悬浮细胞，将细胞滴加于载波片上，在Nikon TE300倒置显微镜下，经UV激发，荧光观测、拍照。
细胞的药物处理
微管(microtubles)具有对细胞核产生压缩应力，从而改变细胞核大小的作用。据此原理，本发明所行实验采用多西紫杉醇Docetaxel(微管稳定剂，DTX)和长春新碱Vincristine(微管解聚剂,VCR)作为工具药物来调节实验细胞HFLF、MEF、Hela和mESC的细胞核大小。 实验采用的药物在培养液中的终浓度分别为：50nM(Docetaxel)和100ng/ml(Vincristine)。两种药物处理时间均为1.5小时。
药物VCR能够破坏细胞的微管组织，使细胞保持不收缩的状态，相反DTX处理后细胞能够加强细胞的微管组织使细胞收缩，由图5可知，经过药物VCR处理后的HFLF，细胞未发生收缩，CISE样品峰后沿与CISE对照峰后沿重合，经过药物DTX处理后的HFLF，细胞发生明显的收缩，CISE样品峰后沿较CISE对照峰后沿前移，由此可以说明，细胞处于收缩状态条件下，相应的细胞核出现缩小，SSBs的结合状态发生改变。
由图6可知，经过药物VCR处理后的Hela，细胞未发生收缩，CISE样品峰后沿与CISE对照峰后沿重合，经过药物DTX处理后的Hela，细胞发生明显的收缩，CISE样品峰后沿较CISE对照峰后沿前移，由此可以说明，细胞处于收缩状态条件下，相应的细胞核出现缩小，SSBs的结合状态发生改变。
由图7b和图7c可知，离体的MEF以及经过药物DTX处理后的MEF，细胞收缩，CISE样品峰后沿较CISE对照峰后沿前移，而经过药物VCR处理后的MEF，细胞未发生收缩，CISE样品峰后沿较CISE对照峰后沿前移。由图7a可以看出，mES的微管蛋白表达极少，这是解释实施例mES经过离体后细胞未收缩的原因。
上述虽然结合附图对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，所属领域技术人员应该明白，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。

Claims (14)

  1. 一种保持细胞应力平衡条件下检测SSBs结合状态的装置，其特征在于，包括：细胞电泳槽：该槽为一内径0.76～0.84 cm、外径0.90～1.00 cm、长度18.9～17.1cm的圆管，管壁中部，沿其纵轴方向开有一8.55×0.57cm～9.45×0.63cm的槽口；电泳缓冲液池：阳极、阴极各有一个，尺寸相同，9.5×3.33×4.75cm～10.5×3.68×5.25cm；侧连接通道：阳极与阴极侧各有一个，尺寸相同，内径0.76～0.84 cm，外径0.90～1.00 cm，长9.5～10.5cm；滤膜：阳极与阴极侧各有一个，尺寸相同，0.2μm或0.45μm亲水聚苯醚砜滤膜。
  2. 如权利要求1所述的装置，其特征在于，所述细胞电泳槽内径0.8 cm、外经0.95 cm、长度18 cm的圆管，管壁中部，沿其纵轴方向开有一9×0.6 cm槽口；电泳缓冲液池：阳极、阴极各有一个，尺寸相同，10×3.5×5 cm；所述侧连接通道，阳极与阴极侧各有一个，尺寸相同，内经0.8cm，外经0.95cm,长10cm；滤膜，阳极与阴极侧各有一个，尺寸相同，孔径为0.45μm亲水聚苯醚砜滤膜。
  3. 如权利要求1或2所述的装置，其特征在于，所述细胞电泳槽和电泳缓冲液池的材质为聚苯乙烯，所述侧连接通道为硅胶管。
  4. 如权利要求1所述的装置，其特征在于，所述装置为水平式电泳装置。
  5. 一种保持细胞应力平衡条件下检测SSBs结合状态的方法，具体包括以下步骤：
    (1)细胞预固定：分离细胞，加入磷酸盐缓冲液制成细胞悬液，将上述细胞悬液涂布于培养皿底部，将上述培养皿置于≤0℃条件下用UV灯照射后，用磷酸盐缓冲液收集培养皿中的细胞，离心弃上清液后加入4％的多聚甲醛预固定1-6sec，预固定完成后加入打空剂，所述打空剂能使细胞膜和细胞和都打孔，打孔5-15min；
    (2)细胞原位电泳：将上述打孔后的细胞团加入电泳缓冲液制成细胞悬浮，恒压模式下，在电泳槽阴极侧滴入细胞悬液，电泳完成后用移液器吸净电泳槽中的液体，并将吸出液汇集于一离心管中，用电泳缓冲液冲洗电泳槽，重复电泳过程，直至细胞悬液全部完成电泳；
    (3)检测：使用流式细胞术分析检测。
  6. 如权利要求5所述的方法，其特征在于，所述步骤1)中用254 nm UV灯以1.84W/cm2强度照射，总剂量165.6J/cm2。
  7. 如权利要求5所述的方法，其特征在于，所述步骤1)中离心为在260g离心5min。
  8. 如权利要求5所述的方法，其特征在于，所述步骤1)中所述预固定3sec。
  9. 如权利要求5所述的方法，其特征在于，所述步骤1)中所述打空为用0.2％Triton X-100打孔10min。
  10. 如权利要求5所述的方法，其特征在于，所述步骤2)中CISE缓冲液为浓度为Tris： 25 mM；Glycine：192 mM；Glucose：43.2 mM，pH8.3。
  11. 如权利要求5所述的方法，其特征在于，所述细胞原位电泳的具体步骤为：
    (1)细胞团加入CISE缓冲液制成分散悬浮液；
    (2)取上述部分细胞悬液加入离心管作为CISE的对照，剩余细胞悬液用作CISE样本；
    (3)装配CISE装置，并连接电泳电源；
    (4)加入CISE缓冲液；
    (5)开启电源，设置为恒压模式，调整电压设置到500V并开始电泳运行；
    (6)根据CISE装置的大小吸取CISE样品滴入阴极侧电泳槽；
    (7)样品滴入后立即开始计时，计时结束，中止电源；
    (8)用移液器吸净电泳槽中的液体，并将吸出液汇集于离心管中；
    (9)用CISE缓冲液冲洗电泳槽，吸净残留液，按步骤4标准重新将电泳槽注入CISE电泳缓冲液；
    (10)重复步骤6-9，直至全部CISE样品被CISE处理完毕；
    (11)分别用CISE缓冲液补加CISE样品汇集管和CISE对照管的液量至相同；
    (12)将CISE样品和CISE对照以1275g转速离心5分钟，弃上清；
    (13)分别将CISE样品和CISE对照加入PBS制成细胞分散悬浮；
    (14)分别将CISE样品和CISE对照用70％的乙醇或4％的多聚甲醛固定。
  12. 如权利要求11所述的方法，其特征在于，所述步骤6)阴极侧电泳槽开口1.5cm处滴入CISE缓冲液。
  13. 如权利要求11所述的方法，其特征在于，所述步骤14)固定24h。
  14. 如权利要求5-13任一所述的方法，在细胞增值性衰老死亡中的应用；在检验干细胞治疗中致癌风险程度中应用；在检验炎症发生后中致癌风险程度中应用；在验证肿瘤异质性中的应用；在维持细胞干性中的应用；在分析单细胞测序中的应用；在分析阿尔茨海默症致病机理方面的应用；在研究逆转录病毒变异方面的应用；在植物无性育种中的应用。
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