CN107367455A - A kind of measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation - Google Patents
A kind of measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation Download PDFInfo
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Abstract
The invention provides a kind of measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation.This method marks pro apoptotic protein matter by EGFP, and EGFP numerical value when being resistant to pro apoptotic protein matter molecule by flow cytometry measure tumour cell, by the conversion for exciting fluorescence intensity of the fluorescent microsphere and FITC and EGFP of FITC marks, the measurement of the absolute quantitation of tumour cell tolerance pro apoptotic protein matter molecule amount is realized.The inventive method not only solves the problem of protein content in tradition research increases how many times and causes Apoptosis and lack absolute figure, the difference problem between the activity problems and BH3 small peptides and BH3 protein using the BH3 small peptides run into BH3 analysis methods is it also avoid, realizes the absolute quantitation test of tumor living cell tolerance pro apoptotic protein matter molecule.
Description
Technical field
The invention belongs to analyze detection field, and in particular to tumour cell tolerance pro apoptotic protein matter molecule amount is definitely fixed
The measuring method of amount.
Background technology
Tumour seriously endangers human health.Numerous studies show the generation of tumour in recent years, development and treatment all with cell
Apoptosis has close contact.Apoptosis pathway mainly includes death receptor4 and the mitochondrial pathways.Death receptor
Work as death receptor in path（Such as DR4, DR5 etc.）Combined with its aglucon（Such as TRAIL）Afterwards, it is possible to promote Apoptosis,
So these death receptors are all pro apoptotic protein matter.Mitochondrial apoptosis approach is mainly by BCL2 family listed business.
BCL2 protein families members are divided into anti-apoptotic proteins matter and pro apoptotic protein matter, and pro apoptotic protein matter includes only tying containing BH3 again
The pro apoptotic protein in structure domain（BIM, PUMA, NOXA, BMF, BAD, HRK etc.）With Multidomain pro apoptotic protein BAX and BAK.
It is a series of that BCL2 families regulate and control the permeability of mitochondrial membrane, control line grain cylinder cell pigment c etc. by protein interaction
The release of pro apoptotic protein matter, caspase is activated, so as to cause a series of Apoptosis to react.Other intracellular many eggs
White matter is also pro apoptotic protein matter（Such as P53, ERK）, they by promote the pro apoptotic protein matter in downstream transcriptional expression or
Person improves the modes such as the stability of downstream pro apoptotic protein matter in the cell to promote Apoptosis.
Apoptosis is most important for tumor research.On the one hand, anti-apoptotic BCL2 family proteins are in kinds of tumors
Height expression.On the other hand, many antineoplastics, including DNA damage class medicine, kinase inhibitor and micropipe aggregation inhibitor
Deng, and by inducing mitochondrial apoptotic pathways kill tumour cell.Moreover, many pro apoptotic protein matter
Analog has become antineoplastic or carries out clinical test.For example, pro apoptotic protein matter BAD BH3 analog
Navitoclax, can specificity suppression BCL2, BCLxLAnd BCLw, it is currently a variety of swollen so as to cause apoptosis of tumor cells
Knurl carries out clinical test.And to be approved for recurrent chronic lymphatic thin by more specific BCL2 inhibitor venetoclax
The treatment of born of the same parents' property leukaemia.So the absolute molecular number of measurement apoptosis of tumor cells tolerance pro apoptotic protein matter not only helps
Treatment curative effect of the antineoplastic to certain specific tumors is predicted in us, it helps we judge which protein molecule is
Effective drug target molecule.
Which pro apoptotic protein matter is the research method for comparing early stage find thin typically by the method for Western blotting
Intracellular expression increase, then verifies that these protein have apoptosis-promoting effect really by the method for overexpression, can not be real
Existing quantitative analysis（Puthalakath H, wait Cell death and differential, 2002,9 (5):505-512；
Li R, wait Cell death and differential, 2005,12 (3): 292-303）.Develop successively in recent years
The method of BH3 analyses（Deng J, wait Cancer Cell, and 2007,12: 171-185；Vo TT, Cell are waited,
2012, 151: 344-355）, however, this method has its limitation：(1) the corresponding BH3 small peptides of BH3 protein can not be complete
Full response BH3 protein, research show that the activity of BH3 small peptides and the activity only between protein containing BH3 are distinguishing（Dai
H, waits .The Journal of Biological Chemistry, and 2014,289:89-99）；(2) because this method is to use
What the mitochondria of separation was completed, so the method detected using BH3 small peptides is only applicable to mitochondrial apoptosis approach, and can not
Used in other pro apoptotic protein matter.
The content of the invention
The present invention provides a kind of measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation.Pass through
EGFP marks pro apoptotic protein matter, and when being resistant to pro apoptotic protein matter molecule by flow cytometry measure tumour cell
EGFP numerical value, by the conversion for exciting fluorescence intensity of the fluorescent microsphere and FITC and EGFP of FITC marks, realize that tumour is thin
Born of the same parents are resistant to the measurement of the absolute quantitation of pro apoptotic protein matter molecule amount.
The technical solution adopted by the present invention is as follows：
（1）Promote apoptosis molecular dna first with EGFP end marks, be transferred to by the method for transduction in living cells；
（2）After cell growth certain time（24 hours or 48 hours）, these is promoted apoptosis molecule overexpression；
（3）Then the Annexin V of fluorescent dye APC mark are added to mark apoptotic cell, pass through flow cytomery point
Analyse the EGFP fluorescence intensities of the rush apoptosis molecule of tumour cell tolerance EGFP couplings；
（4）While flow cytomery is analyzed, the reference pipe that a pipe carries standard FITC fluorescent microspheres, this fluorescence are done
Microballoon can be obtained on the associated straight lines of FITC quantity and fluorescence intensity on each fluorescent microsphere；
（5）Again by XRF determine between a certain amount of FITC and EGFP in measured by flow cytometry wave-length coverage
The conversion relation of emitted luminescence intensity, the absolute figure that tumour cell tolerance promotees the EGFP of apoptosis molecule can be obtained；Because one
EGFP promotees apoptosis molecule with one and is connected, and the numerical value is the absolute figure that tumour cell tolerance promotees apoptosis point.
The specific work process of the detection method of the present invention is as follows：（1）By the method for molecular cloning by required measurement
The cDNA of cDNA and the EGFP molecule of target rush apoptosis molecule, which is connected, to be cloned in high-expression vector, according to demand can be by it
It is placed on EGFP N-terminal or C-terminal；（2）By electricity turn, virus transfection the methods of by plasmid transfection to be tested tumour cell
In；（3）After 24 hours or 48 hours, cell is collected, is dyed with the Annexin V of APC- marks, and pass through flow cytometer
The fluorescence intensity information of EGFP/APC in tumour cell is gathered, so as to cause tumour cell tolerance to promote apoptosis molecule critical point
EGFP fluorescence intensity information；（4）While sample is gathered, need to utilize flow cytometer under same condition of work simultaneously
The fluorescence intensity information of standard fluorescence microballoon of the collection containing a number of gradient FITC, makes FITC fluorescence intensities and quantity
Linear relation figure；（5）The EGFP protein of purification standard, FITC is bought, detected and flowed by sepectrophotofluorometer
The conversion relation of the EGFP protein and FITC excitating light strengths of the consistent identical molecular number of formula cell instrument Detection wavelength scope；
（6）The fluorescence intensity information that tumour cell is resistant to the EGFP of rush apoptosis molecule critical point is converted into EGFP molecule amount, from
And obtain the absolute figure of required rush apoptosis molecule.
Difference and advantage compared with prior art of the invention is：
（1）The number for intracellular pro apoptotic protein matter molecule of this method measurement, more can be anti-different from extracellular experiment
Answer the rush apoptosis molecule amount of the tolerance of living cells；（2）This method measurement is an absolute number, is not protein content phase
To how many times of growth can cause Apoptosis, therefore can be according to the absolute number to the rush apoptosis required for tumour cell
Molecule similar medicine concentration is estimated；（3）What this method measured is the molecule that active protein causes Apoptosis
Number, can solve the problems, such as that the activity problems of small peptide method Small Peptides and small peptide are distinguished with protein active；（4）This method
The absolute magnitude that apoptosis molecule not only can be promoted to BH3 analogs is calculated, and can also be resistant to other oroteins to tumour cell
Molecule（Such as DR4, ERK, P53 etc.）Absolute magnitude calculated.
（2）The innovation of the present invention is：By the way that pro apoptotic protein matter is connected with EGFP molecules, EGFP marks
Thick apoptin is combined monitoring Apoptosis with APC-Annexin V, the application of the fluorescent microsphere of quantitative FITC marks, with
And the ingenious conversion between FITC and EGFP, successfully realize the exhausted of measurement tumor living cell tolerance pro apoptotic protein matter molecule
To quantitative.This method, which not only solves protein content in tradition research and increases how many times, to be caused Apoptosis and lacks absolute number
The problem of value, it also avoid activity problems and BH3 small peptides and BH3 albumen using the BH3 small peptides run into BH3 analysis methods
Difference problem between matter, realize the absolute quantitation test of tumor living cell tolerance pro apoptotic protein matter molecule.
Brief description of the drawings
Fig. 1 is the flow chart that the inventive method is implemented；
Fig. 2 is to be resistant to promote apoptosis molecule BIM by flow cytometry analysis Jurkat cellEL, PUMA, NOXA, BMF threshold value shows
It is intended to；
Fig. 3 is the schematic diagram by FITC standard fluorescences microballoon conversion fluorescence intensity and FITC molecule amounts；
Fig. 4 is that the EGFP and FITC of conversion purifying launch the schematic diagram of photocathode；
Fig. 5 is the threshold value absolute number of the Jurkat cell and SKW6.4 cell tolerances rush apoptosis molecule obtained by this method
Measurement result figure.
Embodiment
The inventive method implementing procedure is as shown in Figure 1.Concretely comprise the following steps（1）By the method for molecular cloning by required measurement
The cDNA of the target cDNA and the EGFP molecule that promote apoptosis molecule be connected and be cloned in high-expression vector, according to demand can general
It is placed on EGFP N-terminal or C-terminal；（2）By electricity turn, virus transfection the methods of by plasmid transfection to be tested tumour cell
In；（3）After 24 hours or 48 hours, cell is collected, is dyed with the Annexin V of APC- marks, and pass through flow cytometer
The fluorescence intensity information of EGFP/APC in tumour cell is gathered, so as to gather the critical point of tumour cell tolerance rush apoptosis molecule
EGFP fluorescence intensity information；（4）While sample is gathered, need to utilize flow cytometer under same condition of work simultaneously
The fluorescence intensity information of standard fluorescence microballoon of the collection containing a number of gradient FITC, makes FITC fluorescence intensities and quantity
Linear relation figure；（5）The EGFP protein of purification standard, FITC is bought, detected and flowed by sepectrophotofluorometer
The conversion relation of the EGFP protein and FITC excitating light strengths of the consistent identical molecular number of formula cell instrument Detection wavelength scope；
（6）The fluorescence intensity information that tumour cell is resistant to the EGFP for the critical point for promoting apoptosis molecule is converted into EGFP molecule amount,
So as to obtain the absolute figure that tolerance promotees apoptosis molecule.
In implementation process, we are by BIMEL, after PUMA, NOXA, BMF these pro apoptotic protein matter are connected to EGFP
Face, it is building up in fibrocyte expression vector.We have selected two kinds of tumor cell lines Jurkat and SKW6.4, be converted respectively by electricity
Method by these plasmid transductions enter tumour cell in.Cell culture 24 hours, corresponding pro apoptotic protein matter is set to cross scale
Reach.Use to add or be added without apoptosis inhibitor QVD groups in order to observe Apoptosis, in experiment and analyze respectively.Then, receive
Collect cell, and the Annexin V of cell and APC marks are reacted, mark apoptotic cell.Led to by flow cytometry analysis GFP
Road and APC passages, select the negative cells of Annexin V.
With EGFP-BIM in Jurkat tumour cellsELTolerance measurement exemplified by, choose Annexin V negative cells not wither
Cell is died, the border EGFP fluorescence intensities for being not added with QVD groups are made by analysis software（Such as Fig. 2 a）.Answered with same method
On EGFP-PUMA, these pro apoptotic protein matter molecules of EGFP-NOXA, EGFP-BMF（Fig. 2 b, Fig. 2 c, Fig. 2 d）.In streaming
While Cytometric Analysis sample cell, while the fluorescent microsphere containing a number of FITC is tested with reference to pipe（Fig. 3 a）.Pass through
The molecule amount of FITC on the fluorescent microsphere of standard, the standard curve of FITC molecular numbers and FITC fluorescence intensities can be obtained
（Fig. 3 b）, so as to which the quantity of FITC corresponding to the EGFP on above-mentioned border fluorescence intensity is calculated.Utilize purifying
EGFP protein（Fig. 4 a）With the standard items FITC of purchase flow cytometer it is corresponding detect it is glimmering in the range of wavelength of transmitted light
The relation of luminous intensity（Fig. 4 b）, you can to obtain the conversion of the fluorescence intensity obtained by each molecule FITC and each molecule EGFP
Relation（Fig. 4 c）, so as to which the molecular amounts of EGFP corresponding to the EGFP on above-mentioned border fluorescence intensity be calculated.Because one
EGFP molecules are connected with a pro apoptotic protein matter, it is hereby achieved that tumour cell tolerance promotees the absolute number of apoptosis molecule threshold value
Value.According to the above method, the Jurkat cell that we measure, and SKW6.4 cell tolerances promote the absolute number of apoptosis molecule threshold value
Value such as Fig. 5.
Description of the invention does not elaborate and partly belongs to techniques well known.
It is described above, part embodiment only of the present invention, but protection scope of the present invention is not limited thereto, and is appointed
What those skilled in the art the invention discloses technical scope in, the change or replacement that can readily occur in should all be covered
Within protection scope of the present invention.
Claims (3)
- A kind of 1. measuring method of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation, it is characterised in that including Following steps：（1）Promote apoptosis molecular dna to realize the mark to promoting apoptosis molecule using EGFP end marks, be the default measurement of living cells Amount is prepared；（2）The Annexin V that are marked by fluorescent dye APC mark apoptotic cell, realize the detection of apoptotic cell；（3）The corresponding EGFP of tumour cell tolerance rush apoptosis molecule fluorescence intensity is analyzed by flow cytomery；（4）Using the standard FITC fluorescent microspheres with varying number absolute quantitation is realized with reference to pipe；（5）A certain amount of FITC and EGFP molecular emissions luminous intensity is determined by XRF to realize changing for FITC and EGFP Calculate, obtain obtaining the absolute figure for the EGFP that tumour cell tolerance promotees apoptosis molecule.
- A kind of 2. measurement side of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation according to claim 1 Method, it is characterised in that（1）Promote apoptosis molecular dna first with EGFP end marks, be transferred to by the method for transduction in living cells；（2）These are made to promote apoptosis molecule overexpression after cell growth certain time；（3）Then the Annexin V of fluorescent dye APC mark are added to mark apoptotic cell, pass through flow cytomery point Analyse the EGFP fluorescence intensities of the rush apoptosis molecule of tumour cell tolerance EGFP couplings；（4）While flow cytomery is analyzed, the reference pipe that a pipe carries standard FITC fluorescent microspheres, this fluorescence are done Microballoon can be obtained on the associated straight lines of FITC quantity and fluorescence intensity on each fluorescent microsphere；（5）Again by XRF determine between a certain amount of FITC and EGFP in measured by flow cytometry wave-length coverage The conversion relation of emitted luminescence intensity, the absolute figure that tumour cell tolerance promotees the EGFP of apoptosis molecule can be obtained.
- A kind of 3. measurement side of tumour cell tolerance pro apoptotic protein matter molecule amount absolute quantitation according to claim 1 Method, it is characterised in that concrete operation method is as follows：（1）The cDNA that the target of required measurement is promoted to cDNA and the EGFP molecule of apoptosis molecule by the method for molecular cloning is connected It is cloned in high-expression vector, EGFP N-terminal or C-terminal can be placed it according to demand；（2）By electricity turn, virus transfection method by plasmid transfection into tested tumour cell；（3）After 24 hours or 48 hours, cell is collected, is dyed with the Annexin V of APC- marks, and pass through flow cytometer The fluorescence intensity information of EGFP/APC in tumour cell is gathered, so as to gather the critical point of tumour cell tolerance rush apoptosis molecule EGFP fluorescence intensity information；（4）While sample is gathered, need to utilizing flow cytometer, collection contains a fixed number simultaneously under same condition of work The fluorescence intensity information of the gradient FITC of amount standard fluorescence microballoon, make the linear relation figure of FITC fluorescence intensities and quantity；（5）The EGFP protein of purification standard, FITC is bought, examined by sepectrophotofluorometer detection with flow cytometer Survey the conversion relation of the EGFP protein and FITC excitating light strengths of the consistent identical molecular number of wave-length coverage；（6）The fluorescence intensity information that tumour cell is resistant to the EGFP for the critical point for promoting apoptosis molecule is converted into EGFP molecular number Mesh, so as to obtain the absolute figure that tolerance promotees apoptosis molecule.
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Citations (5)
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| WO2009085216A2 (en) * | 2007-12-20 | 2009-07-09 | Squicor | Compositions and methods for detecting or elimninating senescent cells to diagnose or treat disease |
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