CN102382893A - Specificity quantitive detection for cell apoptosis - Google Patents
Specificity quantitive detection for cell apoptosis Download PDFInfo
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Abstract
The invention discloses a cell pre-incubation reagent box for detecting cell apoptosis and is characterized in that the cell pre-incubation reagent box comprises a terminal transferase (TdT) for marking a terminal 3' of a DNA, and a dideoxy nucleotide triphosphoric acid (ddNTP) or a triphosphoric acid deoxyribonucleoside (dNTP); the dideoxy nucleotide triphosphoric acid (ddNTP) or the triphosphoric acid deoxyribonucleoside (dNTP) is not marked and is marked in advance as a basic group in a dideoxy nucleotide triphosphoric acid (ddNTP) pair or a triphosphoric acid deoxyribonucleoside (dNTP) pair is added at the terminal3' of a fractured DNA segment after a cell membrane is penetrated through. The method provides a more specific way for detecting cell apoptosis.
Description
Technical field
The invention belongs to the cytobiology technology field, be specifically related to a kind of apoptotic specificity and quantize detection technique.
Background technology
Apoptosis is a path that receives tight regulation and control, and its purpose is when body is in a series of biological procedures that comprises tumour pressure, to remove the body unwanted cells.Therefore, when the normal cell apoptosis pathway of body got muddled, the abnormal apoptosis regulation and control just became a tumorigenic dominant mechanism.The more important thing is that apoptotic level has not only become the strong mark that detects the cancer therapy effect, also become the vital signs of prediction clinical treatment prognosis.Therefore, the exploitation of more accurate apoptosis detection technique just becomes the necessary link of a key in the discovery of knubble biological affinity tag.
Original position terminal enzyme (DNA) labeling technique (TUNEL) is the domestic method that is used for detecting the original position dna degradation of apoptotic cell; Its principle is that the fracture of nuclear chromatin is one of the sign in apoptosis late period; Can to cause the dna double chain to produce a large amount of 3 '-C-terminal, therefore can utilize this attributes to identify apoptotic cell.Specific practice is carried out mark through fluorescently-labeled dGMP (F-dUTP) to the dna break place and is identified.Deoxyribonucleotide terminal enzyme (DNA) (TdT) thereby can utilize expose on the DNA chain 3 '-deoxynucleotide that C-terminal catalysis does not rely on template to 3 of double-stranded DNA or single stranded DNA '-C-terminal generates double-stranded DNA.In addition, deoxythymidine analogue 5-bromo-2 '-pancreatic desoxyribonuclease 5 '-triphosphoric acid (BrdUTP) is applicable to that also TdT reacts the mark broken site.BrdU one is attached among the DNA, just can detect through immunohistochemistry technique through a kind of anti-BrdU antibody.Non-apoptotic cell does not just combine the ability of lot of F-dUTP, because this type cell lacks the DNA 3 '-C-terminal of exposure.Yet; In recent years along with the technological development of apoptosis; Increasing arguement has appearred in the particularity to the TUNEL technology; Major cause is that this technology can also be labeled as apoptotic cell with non-viable non-apoptotic cell by error, and for example original position terminal enzyme (DNA) labeling technique can not be distinguished apoptosis, necrocytosis and aqtocytolysis.Some susceptibility of improving original position terminal enzyme (DNA) labeling technique and nonspecific progress have been arranged in recent years.For example, cell follows hard on after Proteinase K carries out digestion process through the microwave exposure pre-treatment, and detected result shows susceptibility and the specificity that can improve detection greatly.Yet these improvement are only just being worked in cultured cells, and the non-specific dna break that detection apoptosis, necrocytosis and tissue slice etc. are caused cuts little ice.Utilize the morphological analysis method to go to recognize that apoptotic cell can help improve specificity really, but this make whole process time-consuming again the effort.And the evaluation of apoptosis form is the qualification process of a subjectivity.So, press for and a kind ofly can detect specifically and measure because the dna fragmentation of apoptosis-specific is cracked rather than the cracked technological method of dna fragmentation that caused by necrosis or non-specific fracture.
In tumor biopsy, by the cracked sign that has become a kind of important prediction clinical tumor reaction of oncotherapy inductive dna fragmentation.Therefore, can accurately have crucial effects with the method that detects the DNA fragment of apoptosis-specific repeatablely.Unfortunately; Commercial available original position terminal enzyme (DNA) labeling technique (TUNEL) not only can detect because the DNA fragment that apoptosis causes; The dna fragmentation that also can detect the non-specific dna break generation that is caused by tissue slice is cracked, and therefore researching and developing the apoptotic technology of special detection has crucial meaning.The present invention is just based on above thinking.
Summary of the invention
Based on the urgency of the problems referred to above, the invention provides a kind of new apoptosis detection technique, this technology can accurately also can repeatedly detect the DNA fragment fracture of apoptosis-specific.Utilize this technology in the tumour living tissue detects, can improve the accuracy that apoptosis detects, thus can predicted treatment efficient and treatment result, and it also will improve the apoptotic specificity of detection in clinical examination and Basic Experiment Study.
In order to solve these problems of the prior art, technical scheme provided by the invention is:
A kind ofly be used to detect apoptotic cell preincubate test kit, it is characterized in that said cell preincubate test kit comprises:
Be used for the end-labelled terminal enzyme (DNA) of 3 of DNA ' (Terminal transferase, TdT);
Unmarked dideoxy nucleotide triphosphoric acid (ddNTP) or triphosphoric acid dezyribonucleoside (dNTP), the dna fragmentation 3 ' dideoxy nucleotide triphosphoric acid of end adding that is used for the penetrating back fracture of cytolemma obtains mark in advance to (ddNTP) or triphosphoric acid dezyribonucleoside to the base of (dNTP).
Preferably, said cell preincubate test kit is counted with the reaction final concentration of its component:
KCl 10-100mM；
Tris-HCl 1-100mM；
MgCl
2 1-100mM；
DTT，pH?7.9 0.1-10mM；
TdT 1-500U；
Unlabelled ddCTP 10-1000 μ M;
Fluorescently-labeled dUTP 0.01-100nM.
Preferably, said cell preincubate test kit is counted with the reaction final concentration of its component:
KCl 10-100mM；
Tris-HCl 1-100mM；
MgCl
2 1-100mM；
DTT，pH?7.9 0.1-10mM；
TdT 1-500U；
Unlabelled dCTP 1-100nM;
Fluorescently-labeled dUTP 0.01-100nM.
Preferably, said cell preincubate test kit is counted with the reaction final concentration of its component:
KCl 50mM；
Tris-HCl 20mM；
MgCl
2 10mM；
DTT，pH?7.9 1mM；
TdT 50U；
Unlabelled ddCTP 10-1000 μ M;
Fluorescently-labeled dUTP 0.01-100nM.
Preferably, said dideoxy nucleotide triphosphoric acid (ddNTP) is selected from a kind of of ddATP, ddGTP, ddCTP, ddTTP, and the reaction final concentration of ddNTP is 500 μ M; Said triphosphoric acid dezyribonucleoside (dNTP) is selected from a kind of of dATP, dGTP, dCTP, dTTP; And the reaction final concentration of dNTP is 10nM.
Another object of the present invention is to provide a kind of and utilize described cell preincubate test kit to detect apoptotic method, it is characterized in that said method comprising the steps of:
(1) cell to be detected is carried out preincubate under the condition with described cell preincubate test kit in isolated culture liquid; The temperature of preincubate is 37 ℃; Incubation time is 1 hour, when hatching, cell to be detected is carried out the processing of penetratingization of cytolemma;
(2) add fluorescently-labeled dUTP in the cell isolated culture liquid to be detected after hatching and carry out terminal deoxynucleoside acyltransferase mediated dUTP nick end labeling otch end mark (TUNEL test), the dUTP final concentration is 10nM;
(3) fluorescent mark strength of signal in the cell to be detected after detection is hatched; The fluorescent mark strength of signal that obtains through detection with confirm apoptosis situation in the cell to be detected.
Preferably, cell to be detected is the HT-29 cell in the said method, and said fluorescent mark strength of signal is measured through laser scanning cell streaming appearance (LSC).
The reagent of penetratingization of the cytolemma processing of preferably, adopting in the said method steps (1) is DNaseI and Triton X-100; After first DNase I with 5～20 μ g/ml carries out hatching of cell to be detected, carry out dissolution process with 0.1～1%Triton X-100 cell membrane lipid, the treatment time is 10 minutes, and treatment temp is 37 ℃.
Preferably, fluorescently-labeled dUTP is Cy5-dUTP or FITC-dUTP in the said method steps (2).
Another purpose of the present invention is to provide the application of a kind of described cell preincubate test kit aspect the detection apoptosis.
Preferably, the reaction buffer of the preincubate of cell described in the present invention test kit part can be allocated through 1 * NEBuffer 4.Preferably, the reaction final concentration of said terminal enzyme (DNA) is 50U (unit).Preferably, the reaction final concentration of dideoxy nucleotide triphosphoric acid (ddNTP) is 500 μ M in the said test kit; The reaction final concentration of triphosphoric acid dezyribonucleoside (dNTP) is 10nM in the perhaps said test kit.
The preferred cell preincubate of the present invention test kit is following component and content:
KCl 10-100nM；
Tris-HCl 1-100mM；
MgCl
2 1-100mM；
DTT，pH?7.9 0.1-10mM；
TdT 1-500U；
ddCTP 10-1000μM；
Cy5-dUTP 0.01-100nM。
In the apoptotic cell, its cytolemma still is kept perfectly in early days, but cell is just engulfed rapidly by scavenger cell or peripheral cell before by cracking under lysis.Yet necrocytosis is another kind of process of cell death, is typical non-caspase dependency process, has the characteristic feature that permeability of cell membrane increase, cell and organoid increase.Apoptotic cell and non-viable non-apoptotic cell are different; Apoptotic cell has kept the integrity of cytolemma; Therefore unlabelled ddCTP carries out preincubate with TdT with cell and can let the DNA breach of non-viable non-apoptotic cell obtain mark in advance, and the fluorescently-labeled dUTP that carries out subsequently and TdT general only meeting detect dna break and can in non-viable non-apoptotic cell, not detect in apoptotic cell.
Apoptosis detection method specificity of the present invention is high, and it can detect the dna fragmentation cracked (rather than DNA fragment downright bad or that tissue slice causes) of apoptosis induction.This detection method will not only be improved the accurately apoptotic generation of detection under study for action greatly, and can more reliable governing principle be provided to the tumour personalized treatment.
The objective of the invention is to develop the film defective characteristics that a kind of more accurate apoptosis detection technique the present invention makes full use of non-viable non-apoptotic cell, reach the purpose that specificity is identified apoptotic cell.The unlabelled ddCTP (2 ' 3 '-dideoxycytidine) and the pre-treatment of terminal deoxynucleotidyl transferase (TdT) can be so that the dna break place in the non-viable non-apoptotic cell be labeled; Apoptotic cell and normal cell then there is not mark, because the cytolemma of these cells all is an impermeable to those two kinds of molecules.Like this, the pre-incubation step has guaranteed that the dna fragmentation of apoptosis-inducing is cracked by specific mark, and the DNA fragment that necrocytosis or non-specific tissue slice cause then is not labeled.
It is that necessary means the present invention of distinguishing the dna break of non-specific dna break and apoptosis-inducing carries out preincubate to unlabelled ddCTP and can block preformed dna break is detected that the present invention utilizes unlabelled ddCTP pair cell to carry out preincubate; Can carry out terminal deoxynucleoside acyltransferase mediated dUTP nick end labeling otch end mark experiment (TUNEL) earlier, and then carry out LSC and analyze; Measured the degree of dna break and compared with an external cell system.
The present invention is an example with the HT-29 cell, and unlabelled ddCTP of preincubate (500 μ M) and TdT have reduced the recall rate of preformed dna break widely.This quantitative observation is illustrated in the cell of unlabelled ddCTP of preincubate (500 μ M) and TdT, and the cell that the averaged magnitude relevant with FITC do not carried out preincubate has relatively descended 73%.Although FITC-dUTP is widely used in tissue culture and examination of living tissue, the high autofluorescence in the tissue, the especially wave spectrum of FIFC emission make judgment signal and background difficult unusually.In order to overcome this difficult problem, the present invention preferably uses the first-selection of Cy5-dUTP as the TdT substrate, because the near-infrared region of Cy5 emission only produces very shallow background in tumor biopsy.As shown in Figure 3, and unlabelled ddCTP is compared with the cell that TdT does not carry out preincubate, unlabelled ddCTP and TdT are being carried out in the cell of preincubate, the signal that Cy5 is associated has descended 55%.
The present invention has also used LSC (laser scanning cell streaming appearance) to measure that tumour cell is in the apoptosis per-cent in period in each center examination of living tissue.For the ease of analyzing, each slide glass all is placed on the robotization platform by computer control.Vision localization can be carried out through the microscope on the device in the purpose zone of being scanned, and the zone of tumour can be drawn through Wincyte software.Slide glass scanning and the contoured structure of nucleus are accomplished through argon laser and red detector (bromination third pyridine) scanning.The FITC/Cy5 positive cell is detected through argon laser and green detector.Similar with flow cytometer counting (fluorescent activation cell sorting system), fluorescence intensity level relatively is recorded in the four-quadrant scatter diagram.The slide glass of a negative control is used for measuring the analysis door of each sample.Visually confirm FITC or Cy5 positive cell with being positioned at again.In case door is set, thereby data file will be handled mensuration FITC or the percentage of Cy5 positive cell in the examination of living tissue of center automatically.Shown in Fig. 3 B,, caused 6648 and 6877 positive counting respectively through using the terminal deoxynucleoside acyltransferase mediation property dUTP otch end mark experiment of Cy5-dUTP and FITC-dUTP.Yet, the elimination that the preincubate of ddCTP is and then successful non-specific dna break.
In a word, these data show clearly, and unlabelled ddCTP of preincubate and TdT can block fluorescently-labeled ddCTP and the dna break fluorescent mark of TdT to being pre-existing in through subsequently.Therefore, preincubate is for reducing in the non-viable non-apoptotic cell because the signal that the DNA otch that film rupture causes forms is very important.
For the effect of specificity in the dna break of quantization cell apoptosis induction of distinguishing Cy5-dUTP and FITC-dUTP, the present invention also carries out Cy5-dUTP, FITC-dUTP to the comparison test of tumor biopsy dna break specificity.The present invention comes relatively to use Cy5-dUTP and FITC-dUTP to be determined at the intensity of dna break signal in the tumor biopsy through above-mentioned detection method.LSC (laser scanning cell instrument) can be used to detect the ratio of Cy5-(or FITC-) positive events and medium fluorescence intensity (MFI).The average of the total intensity of fluorescent probe in the medium fluorescence intensity representative tissue.Therefore, the clear SNR of measuring of the ratiometer between positive fluorescent event and the medium fluorescence intensity.As previously mentioned; The principle of this objective; General FITC-dUTP during promptly deoxynucleoside acyltransferase mediation property dUTP otch end mark is tested endways produces autofluorescence in tumor biopsy, thereby and owing to the susceptibility to photobleaching has relative instability.Simultaneously, Cy5 is a stable more and low background fluorescence probe, can strengthen the specificity that the present invention detects the relevant dna break of apoptosis.Therefore the SNR of Cy5-dUTP (Fig. 4) in detection method is significantly higher than FITC-dUTP apoptosis test kit, and this shows that this apoptosis detection method provides one to have more specific apoptosis detection approach.
The DNase I that the present invention uses, i.e. Deoxyribonuclease I, Chinese is a deoxyribonuclease I, is a kind ofly can digest the endonuclease that strand or double-stranded DNA produce monodeoxyribonucleotide or strand or double-stranded oligodeoxynucleotide.Product behind DNase I hydrolysis strand or the double-stranded DNA, 5 ' hold to be phosphate group, 3 '-hold to be hydroxyl.
DNase I activity depends on calcium ion, and can be activated by mg ion or divalent manganesetion.Under the mg ion existence condition, but any site of DNase I random shearing double-stranded DNA; Under the divalent manganesetion existence condition, DNase I can shear the dna double chain in same site, forms flat end, or 1-2 the sticking end that Nucleotide is outstanding.
DNase I does not contain RNase (RNase free), can be used for the processing of various RNA samples.Provide and be used for the required EDTA of DNase I inactivation.DNase I is used to prepare the RNA sample that does not contain DNA; Removing the equiprobable DNA of genomic dna before the RT-PCR reaction in the RNA sample pollutes; External T7, T3, RNA Polymerases catalyzed RNA such as SP6 are transcribed the back and are removed dna profiling; DNase I footprinting researching DNA-protein interaction; Nick translation (nick translatioin); Produce DNA segment library at random; Part was sheared genomic dna as positive control during apoptosis TUNEL detected.
DNase I derives from ox pancreas, through obtaining from the ox pancreas purifying.Like need enzyme is diluted, can use enzyme storage liquid (50mM Tris-acetate (pH 7.5), 10mM CaCl
2, 50% (v/v) glycerol) dilute.
Triton X-100 (polyoxyethylene octyl phenyl ether) is a kind of non-ionics (or claiming stain remover).Molecular weight is 646.86 (C
34H
62O
11).Its ability lipin dissolving is to increase the permeability of antibody cell membrane.Triton X-100 typical concentrations is 1% and 0.3% in the immunocytochemistry; Wherein 1% Triton X-100 is usually used in the rinsing tissue sample; 0.3% Triton X-100 then is usually used in dilute serum; 0.1% Triton X-100 is usually used in LAMP (ring mediated isothermal amplification), preparation BSA etc.But normally be mixed with 30% Triton X-100 storing solution earlier, face the time spent to be diluted to desired concn.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Fig. 1 is the flow process comparison of the method and the TUNEL method of the dna break of kit detection cell apoptosis of the present invention;
Fig. 2 be different concns ddCTP preincubate (going out to carry out block at dna break in advance handles) to the HT-29 cell in the influence of average fluorescent strength (MFI);
Fig. 3 is not for carrying out ddCTP preincubate and the influence of adopting the ddCTP preincubate to fluorescent mark dUTP in the HT-29 cell;
Fig. 4 utilizes Cy5-dUTP to detect the micrograph of the crack conditions of DNA in people's tumour living tissue through adopting method of the present invention and traditional TUNEL method; Green is a nucleus, and redness is a dna fragmentation;
Fig. 5 utilizes Cy5-dUTP to detect the crack conditions comparison of nucleus and DNA in people's tumour living tissue through adopting method of the present invention and traditional TUNEL method; Green is a nucleus, and redness is a dna fragmentation;
Fig. 6 detects the influence of the crack conditions of DNA in people's tumour living tissue for adopting different fluorescently-labeled dUTP to method of the present invention and traditional TUNEL method; One group is Cy5-dUTP wherein; Below one group be FITC-dUTP;
Fig. 7 detects the diverse microcosmic photo of the crack conditions of DNA in people's tumour living tissue for adopting different fluorescently-labeled dUTP to method of the present invention and traditional TUNEL method; Comprise Cy5-dUTP, FITC-dUTP;
Fig. 8 is the method for the dna break of kit detection cell apoptosis of the present invention and the idiographic flow comparison diagram of TUNEL method.
Embodiment
Below in conjunction with specific embodiment such scheme is further specified.Should be understood that these embodiment are used to the present invention is described and are not limited to limit scope of the present invention.The implementation condition that adopts among the embodiment can be done further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in the normal experiment.
The dna fragmentation selected marker test of embodiment 1 apoptosis cleaved
In the apoptotic cell, its cytolemma still is kept perfectly in early days, but cell is just engulfed rapidly by scavenger cell or peripheral cell before by cracking under lysis.Yet necrocytosis is another kind of process of cell death, is typical non-caspase dependency process, has the characteristic feature that permeability of cell membrane increase, cell and organoid increase.Therefore, present embodiment makes full use of the film defective characteristics of non-viable non-apoptotic cell, reaches the purpose of special evaluation apoptotic cell.
Specific practice is: with 5X10
3The HT-29 cell in/hole is used H under 37 ℃
2O
2Handled one hour, inducing cell is downright bad; Clean with PBS then, and in the substratum that contains 10%FCS (foetal calf serum), hatch.The 5X10 that lives
3The HT-29 cell in/hole joins H then
2O
2In the inductive non-viable non-apoptotic cell group, then come cell death inducing with gamma-radiation or the 100nM taxol treatment of 5Gy.The cell through any processing is not used as negative control group.The HT-29 cell is (specific practice is with being added on the cell among not fluorescently-labeled 500 μ M ddCTP and the 5U TdT) under the condition of cell preincubate test kit, places isolated culture liquid, 37 ℃ of preincubate 1h.The unlabelled ddCTP (2 ' 3 '-dideoxycytidine) and the pre-treatment of terminal deoxynucleotidyl transferase (TdT) can be so that the dna break place in the non-viable non-apoptotic cell be labeled; Apoptotic cell and normal cell then there is not mark, because the cytolemma of these cells is impermeable (Fig. 1) to the ddCTP molecule.Then, again 5 μ M Cy5-dUTP and TdT are added in the cell, hatched 30 minutes in 37 ℃.Like this, because non-viable non-apoptotic cell is by the ddCTP mark, Cy5-dUTP will only carry out mark to apoptotic cell.Therefore, the step of preincubate has guaranteed that the dna fragmentation of apoptosis-inducing is cracked by the specific fluorescence mark, and the DNA fragment that necrocytosis or non-specific tissue slice cause is not then by the specific fluorescence mark.
As shown in Figure 1, the inventive method has detected apoptotic dna break, but does not detect the dna break from non-viable non-apoptotic cell.Normal cell (N) does not have dna break in nucleus, and non-viable non-apoptotic cell (Nec), apoptotic cell (Apo) have dna break.Apoptotic cell and non-viable non-apoptotic cell all contain dna break in nucleus separately.Unlabelled dUTP and TdT preincubate marker DNA fracture in non-viable non-apoptotic cell.After cytolemma is changed processing thoroughly, dUTP and TdT mark dna break in nucleus of fluorescent probe coupling (or claiming mark).Use multiple detection system, show that all the inventive method only detects the dna break of apoptotic cell.And the TUNEL test detects the dna break of apoptotic cell and non-viable non-apoptotic cell.
Embodiment 2 detects apoptosis situation in the HT-29 cell
Carrying out preincubate with unlabelled ddCTP pair cell is the necessary means of distinguishing the dna break of non-specific dna break and apoptosis-inducing.
In order to verify hypothesis of the present invention: promptly unlabelled ddCTP is carried out preincubate and can block preformed dna break is detected, can carry out terminal deoxynucleoside acyltransferase mediated dUTP nick end labeling otch end mark experiment (TUNEL) earlier.That is to say,, can in cell, produce dna break, will detect the dna break that this acellular apoptosis causes with conventional TUNEL apoptosis detection technique with behind the DNase I processing cell (the present invention has adopted the HT-29 cell).But, through with the ddCTP preincubate after, this false positive rate significantly reduces.
Concrete operations are following: the HT-29 cell places 4 hole slide glasss (10000 cells/well); Earlier with the DNase I of 10 μ g/L in 37 ℃ hatch 30 minutes after; Handled 10 minutes under 37 ℃ of conditions with 0.2%Triton X-100 pair cell, thereby just can produce penetrating cytolemma and non-specific dna fragmentation.Next, the HT-29 cell (is added on the cell in not fluorescently-labeled 500 μ M ddCTP and 5U TdT) under the condition of cell preincubate test kit, places isolated culture liquid, 37 ℃ of preincubate 1h.The unlabelled ddCTP (2 ' 3 '-dideoxycytidine) and the pre-treatment of terminal deoxynucleotidyl transferase (TdT) can then not have mark to apoptotic cell and normal cell so that the dna break place in the non-viable non-apoptotic cell is labeled.Then, again 5 μ M Cy5-dUTP and TdT are added in the cell, hatched 30 minutes in 37 ℃.Like this, because non-viable non-apoptotic cell is by the ddCTP mark, Cy5-dUTP will only carry out mark to apoptotic cell.Data on Fig. 2 and Fig. 3 can be found out, detect the crack conditions of DNA in the HT-29 cell through Cy5-dUTP and TdT.Unlabelled ddCTP of preincubate (500 μ M) and TdT have reduced the recall rate of preformed dna break widely.This LSC quantitative observation is illustrated in the cell of unlabelled ddCTP of preincubate (500 μ M) and TdT, and the cell that the averaged magnitude relevant with FITC do not carried out preincubate has relatively descended 73%.
Embodiment 3 detects apoptosis situation in the tumour cell
In order to prove that in tumor biopsy this technology can be used for the detection specificity dna break equally, tumor tissue section is embedded in (this is comprising the cell of necrosis) on the slide glass, for tissue slice, handle as follows earlier:
1, dewaxing: YLENE dewaxing 2 times, each 5-10 minute; Hydrous ethanol (the good section that will dewax was put into 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol each 2 minutes successively)
2, immerse PBS rinsing 3 times, each 5 minutes to section.
3, traverse section adds the Proteinase K working fluid on tissue block, 21-37 ℃ acts on 15-30 minute.(the preparation of Proteinase K working fluid: 2 μ L, 50 * Proteinase K+98 μ L PBS)
4, the sample of handling Proteinase K well immerses PBS rinsing 3 times, each 5 minutes.
5, immerse section in the confining liquid room temperature (15-25 ℃) sealing 10 minutes.(the preparation of confining liquid: 3%H
2O
2Be dissolved in methyl alcohol)
6, immerse PBS rinsing 2 times, each 5 minutes to section.
And then with tissue slice 37 ℃ of preincubate 1h in unlabelled ddCTP and TdT, carry out incubation with Cy5-dUTP and TdT then.What immunofluorescence microscopy used is the epifluorescence microscope that is equipped with narrow passband excitation filter frame, and this fluorescent microscope can be selected red fluorescence and green fluorescence through roller.Image is to use the low-temperature CCD low camera to obtain.As shown in Figure 4, downright bad cell is by the dyeing of the terminal deoxynucleoside acyltransferase mediation property of the Cy5-dUTP dUTP otch end mark degree of depth, and ddCTP can block the signal that is produced by non-specific dna break simultaneously.When the neck tumour living tissue through the patient of gamma-ray therapy is used for the detection of dna break, as shown in Figure 4, Green=Nuclei nucleus wherein, Red=DNA fragmentation dna fragmentation.The dewaxing of embedding tumor tissues is also fixing.Carry out LSC through HeNe ray and infrared detector and analyze, then hatch 1h for 37 ℃ with Cy5-dUTP and TdT (Cy5-dUTP (+TdT)).Cy5-dUTP with independent is hatched as negative control.The LSC analytical results: last one group of Cy5-dUTP (Persongen Biomedicine (Suzhou) Co., Ltd.) that adopts, the method condition is: PMT=25, Thresh.=2500; The FITC-dUTP (Promega company) that next group adopts, the method condition is: PMT=13, Thresh.=1500.
The present invention has also used LSC (laser scanning cell streaming appearance) to measure that tumour cell is in the apoptosis per-cent in period in each center examination of living tissue.Each slide glass all is placed on the robotization platform by computer control.Vision localization can be carried out through the microscope on the device in the purpose zone of being scanned, and the zone of tumour can be drawn through Wincyte software.Slide glass scanning and the contoured structure of nucleus are accomplished through argon laser and red detector (bromination third pyridine) scanning.The FITC/Cy5 positive cell is detected through argon laser and green detector.Similar with flow cytometer counting (fluorescent activation cell sorting system), fluorescence intensity level relatively is recorded in the four-quadrant scatter diagram.The slide glass of a negative control is used for measuring the analysis door of each sample.Visually confirm FITC or Cy5 positive cell with being positioned at again.In case door is set, thereby data file will be handled mensuration FITC or the percentage of Cy5 positive cell in the examination of living tissue of center automatically.As shown in Figure 5, detect the crack conditions of DNA in people's tumour living tissue through Cy5-dUTP and TdT.Through using the terminal deoxynucleoside acyltransferase mediation property dUTP otch end mark experiment of Cy5-dUTP and FITC-dUTP, caused 6648 and 6877 positive counting respectively.Yet, the elimination that the preincubate of ddCTP is and then successful non-specific dna break.The Cy5 fluorescent probe has low background characteristics among Fig. 6, after the sick inspection of human hela sample preparations becomes paraffin section, through dewaxing and fixing the processing, respectively with Cy5-dUTP and Cy5-dUTP+TdT, hatches 1 hour at 37 ℃.Can see tumour cell on Cy5-dUTP mark under the TdT effect, and when not having the existing of TdT, Cy5 has shown extremely low background.
Fluorescently-labeled optimal conditions when embodiment 4 detects the apoptosis situation
Although FITC-dUTP is widely used in tissue culture and examination of living tissue, the high autofluorescence in the tissue, the especially wave spectrum of FIFC emission make judgment signal and background difficult unusually.In order to overcome this difficult problem, present embodiment provides the tumor biopsy dna break specific Cy5-dUTP higher than FITC-dUTP.Cy5-dUTP is because the near-infrared region of Cy5 emission only produces very shallow background in tumor biopsy as the meliority of TdT substrate.Come relatively to use Cy5-dUTP and FITC-dUTP to be determined at the intensity of dna break signal in the tumor biopsy through the method for present embodiment.LSC (laser scanning cell instrument) can be used to detect the ratio of Cy5-(or FITC-) positive events and medium fluorescence intensity (MFI).The average of the total intensity of fluorescent probe in the medium fluorescence intensity representative tissue.Therefore, the clear SNR of measuring of the ratiometer between positive fluorescent event and the medium fluorescence intensity.As previously mentioned; The principle of this objective; General FITC-dUTP during promptly deoxynucleoside acyltransferase mediation property dUTP otch end mark is tested endways produces autofluorescence in tumor biopsy, thereby and owing to the susceptibility to photobleaching has relative instability.Simultaneously, Cy5 is a stable more and low background fluorescence probe, can strengthen the specificity (Fig. 7) that method of the present invention detects the relevant dna break of apoptosis.In the specific implementation, with reference to embodiment 2 and embodiment 3, unique difference is that Cy5-dUTP is replaced with FTIC-dUTP.
Fig. 8 is the particular flow sheet of the inventive method; Wherein use method of the present invention detect apoptosis induction but not the dna break of necrosis induction detects the feasibility of this test design with this.Cell will be used the gamma-radiation (IR) of 5Gy radiation dose, the taxol of 100nM or H after the inventive method or TUNEL test processing
2O
2Handle.For bulk testing, the part cell with IR or taxol treatment after, the part cell is used H
2O
2Handle, hatch altogether with viable cell again.Normal cell does not have dna break (blueness) in nucleus; Apoptotic cell and non-viable non-apoptotic cell all contain dna break (grey) in nucleus.Unlabelled dUTP and TdT preincubate marker DNA fracture (green) in non-viable non-apoptotic cell.After cytolemma is changed processing thoroughly, dUTP and TdT mark dna break (redness) in nucleus of fluorescent probe coupling (or claiming mark).The apoptosis test that the inventive method is carried out will only detect the dna break of apoptotic cell.On the contrary, the TUNEL test detects the dna break of apoptotic cell and non-viable non-apoptotic cell.
In a word; Through different with non-viable non-apoptotic cell; Apoptotic cell has kept these characteristics of integrity of cytolemma; The present invention supposes that unlabelled ddCTP and TdT and cell carry out preincubate and can let the DNA breach of non-viable non-apoptotic cell obtain mark in advance, and fluorescently-labeled dUTP that carries out subsequently and TdT will only can detect dna break and can in non-viable non-apoptotic cell, not detect in apoptotic cell.On the basis of this assumption, the present invention has developed a kind of analytical technology of high specific, and it can detect the dna fragmentation of apoptosis induction cracked (rather than DNA fragment downright bad or that tissue slice causes) (Fig. 8).The inventive method is applied to the apoptosis detection technique, will not only improve greatly and accurately detect apoptotic generation under study for action, and can more reliable governing principle be provided to the tumour personalized treatment.
Above-mentioned instance only is explanation technical conceive of the present invention and characteristics, and its purpose is to let the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.
Claims (10)
1. one kind is used to detect apoptotic cell preincubate test kit, it is characterized in that said cell preincubate test kit comprises:
Be used for DNA the end-labelled terminal enzyme (DNA) of 3' (Terminal transferase, TdT);
Unmarked dideoxy nucleotide triphosphoric acid (ddNTP) or triphosphoric acid dezyribonucleoside (dNTP) are used for that the dna fragmentation 3' of the penetrating back fracture of cytolemma is terminal to add a dideoxy nucleotide triphosphoric acid (ddNTP) or triphosphoric acid dezyribonucleoside are obtained mark in advance to the base of (dNTP).
2. according to claim 1ly be used to detect apoptotic cell preincubate test kit, it is characterized in that said cell preincubate test kit counts with the reaction final concentration of its component:
KCl?10-100?mM；
Tris-HCl 1-100?mM；
MgCl
2 1-100?mM；
DTT，pH?7.9 0.1-10?mM；
TdT?1-500U；
Unlabelled ddCTP 10-1000 μ M;
Fluorescently-labeled dUTP 0.01-100nM.
3. according to claim 1ly be used to detect apoptotic cell preincubate test kit, it is characterized in that said cell preincubate test kit counts with the reaction final concentration of its component:
KCl?10-100?mM；
Tris-HCl 1-100?mM；
MgCl2 1-100?mM；
DTT，pH?7.9 0.1-10?mM；
TdT?1-500U；
Unlabelled dCTP 1-100nM;
Fluorescently-labeled dUTP 0.01-100nM.
4. according to claim 1ly be used to detect apoptotic cell preincubate test kit, it is characterized in that said cell preincubate test kit counts with the reaction final concentration of its component: KCl 50 mM; Tris-HCl 20 mM; MgCl
210 mM; DTT, pH 7.9 1mM; TdT 50U; Unlabelled ddCTP 10-1000 μ M; Fluorescently-labeled dUTP 0.01-100nM.
5. according to claim 1ly be used to detect apoptotic cell preincubate test kit; It is characterized in that said dideoxy nucleotide triphosphoric acid (ddNTP) is selected from a kind of of ddATP, ddGTP, ddCTP, ddTTP, and the reaction final concentration of ddNTP is 500 μ M; Said triphosphoric acid dezyribonucleoside (dNTP) is selected from a kind of of dATP, dGTP, dCTP, dTTP; And the reaction final concentration of dNTP is 10nM.
6. one kind is utilized any described cell preincubate test kit of claim 1～5 to detect apoptotic method, it is characterized in that said method comprising the steps of:
(1) cell to be detected is carried out preincubate under the condition with any described cell preincubate test kit of claim 1 ~ 5 in isolated culture liquid; The temperature of preincubate is 37 ℃; Incubation time is 1 hour, when hatching, cell to be detected is carried out the processing of penetratingization of cytolemma;
(2) add fluorescently-labeled dUTP in the cell isolated culture liquid to be detected after hatching and carry out terminal deoxynucleoside acyltransferase mediated dUTP nick end labeling otch end mark (TUNEL test), the dUTP final concentration is 10nM;
(3) fluorescent mark strength of signal in the cell to be detected after detection is hatched; The fluorescent mark strength of signal that obtains through detection with confirm apoptosis situation in the cell to be detected.
7. method according to claim 6 is characterized in that cell to be detected is the HT-29 cell in the said method, and said fluorescent mark strength of signal is measured through laser scanning cell streaming appearance (LSC).
8. method according to claim 7 is characterized in that the reagent of penetratingization of the cytolemma processing of employing in the said method steps (1) is DNase I and Triton X-100; After first DNase I with 5 ~ 20 μ g/ml carries out hatching of cell to be detected, carry out dissolution process with 0.1 ~ 1% Triton X-100 cell membrane lipid, the treatment time is 10 minutes, and treatment temp is 37 ℃.
9. method according to claim 7 is characterized in that fluorescently-labeled dUTP is Cy5-dUTP or FITC-dUTP in the said method steps (2).
10. the application of any described cell preincubate test kit of claim 1 ~ 5 aspect the detection apoptosis.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106337082A (en) * | 2016-04-28 | 2017-01-18 | 湖南大学 | Enzyme process generated random G-quadruplex-based cell apoptosis label-free detection method |
| CN107367455A (en) * | 2017-07-10 | 2017-11-21 | 中国科学院合肥物质科学研究院 | A kind of measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation |
| CN107367455B (en) * | 2017-07-10 | 2020-10-16 | 中国科学院合肥物质科学研究院 | Method for measuring absolute quantification of number of tumor cell tolerance pro-apoptosis protein molecules |
| CN111579538A (en) * | 2020-04-22 | 2020-08-25 | 山东第一医科大学(山东省医学科学院) | Apoptosis kit applied to circulating tumor cell detection and detection method thereof |
| WO2021213259A1 (en) * | 2020-04-22 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Apoptosis kit for detecting circulating tumor cells and detection method therefor |
| CN111579538B (en) * | 2020-04-22 | 2022-12-30 | 山东第一医科大学(山东省医学科学院) | Method for detecting circulating tumor cells by using apoptosis kit |
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