CN101363836A - Method for measuring cell cycle by cell autofluorescence spectrum - Google Patents
Method for measuring cell cycle by cell autofluorescence spectrum Download PDFInfo
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- CN101363836A CN101363836A CNA2008100703035A CN200810070303A CN101363836A CN 101363836 A CN101363836 A CN 101363836A CN A2008100703035 A CNA2008100703035 A CN A2008100703035A CN 200810070303 A CN200810070303 A CN 200810070303A CN 101363836 A CN101363836 A CN 101363836A
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Abstract
The invention discloses a method for measuring the cell cycle by adopting the cell autofluorecence spectrum. By utilizing the influence of the continuous variation of an intracellular fluorescent substance (such as amino acid, nucleic acid, protein and the like) during the variation process of the cell cycle (G1 cycle, S cycle, G2 cycle and M cycle) on the peak position, the spectrum peak and the pear intensity of the fluorecence spectrum thereof, the method finds the corresponding relationship between the parameters and the cycle the cell locates in, establishes a cell cycle fluorecence spectrum model and finally implements the accurate measurement of a single cell cycle by utilizing the model. The method has the advantages of simple sampling process, convenient operation, common instrument and equipment, high accuracy and high measurement speed, and can conveniently judge the cell cycle of a cell by applying the relationship that the cell absorption peak intensity varies along with the cell cycle. No fluorescent dye is needed to dye a cell sample, thereby eliminating the adverse effect due to the application of the fluorescent dye, so that the cell can be used for the subsequent cultivation and research after being measured.
Description
Technical field
The present invention relates to a kind of method of measuring the cell cycle, particularly a kind of cell autofluorescence spectrum that utilizes accurately reflects the method that the cell cycle changes.
Background technology
Cell is as the basis of life science, and the key issue of all life all will be sought from cell.The research of great biological phenomenas such as biological reproductive development, heredity, nerve (brain) activity all will be based on cell.Plants and Animals grows and relies on cell proliferation, cell differentiation and Apoptosis to realize that the pathogenesis of all diseases also will be based on cytopathy research.Cell division is the substantially the most most important process of origin of life.Cell growth and cell division are the citation forms of all biological propagation, and cell growth and fissional cycle are is called for short the cell cycle (cellcycle) cell generation cycle.For eukaryotic, its cell cycle was made up of G1 phase, S phase, G2 phase and M phase, and it is the comprehensive embodiment of the whole physiology courses of cell, is prevalent in the higher organism.At present, cell cycle and regulatory mechanism thereof demonstrate huge directive significance in fields such as tumour, stem cell transplantation, aging, reproduction and science of heredity.
At present, the research method of judging for the cell cycle mainly contains following several: optical microscopy, flow cytometry analysis method (dna content analysis), 3H-TdR labelling method etc.Optical microscopy mainly is to observe to judge the cell cell cycle of living in by the form of microscope pair cell, and this method is by eye-observation, and judgement speed is slow; The flow cytometry analysis method is widely used in cell cycle research, but costs an arm and a leg.Start with from dna content, G1 phase and G2/M phase are contained fixing dna content, are respectively 1 times and 2 times, and the dna content of S phase cell is used the length that flow cytometer just can be determined the cell cycle by the variation of monitoring cell DNA content between 1 times and 2 times.But because the dna content of G2 phase and M phase cell all is 2 times, so flow cytometer can't be distinguished G2 phase and M phase cell.The 3H-TdR labelling method is a kind of classical way of measuring cell.But the radioactive isotope that uses during it is measured requires Special Equipment, is easy to the operator is damaged and environment is polluted, and the cell cycle of radioactive isotope own also has interference and inevitable simultaneously.
Cell is in the change procedure of cell cycle, and materials such as the amino acid of cell interior, protein, nucleic acid all will huge variation take place along with the growth change of cell.Histiocytic fluorescence has two kinds: a kind of is autofluorescence, and a kind of is secondary fluorescence.Autofluorescence is meant that cell or tissue dyes without fluorchrome, the fluorescence that is sent under the irradiation of ultraviolet light or short-wavelength light.Histocyte is after fluorchrome dyeing, and the fluorescence that some composition and fluorchrome present in conjunction with the back in the cell is called secondary fluorescence.The variation of above-mentioned two kinds of fluorescence spectrums can the reacting cells cycle variation, wherein the variation of autofluorescence is mainly caused by aromatic amino acid in the protein and nucleic acid, secondary fluorescence is fluorchrome and intracellular some special component present different colours in conjunction with the back a selectivity fluorescence, though this fluorescence is convenient to more by force observe, but be used to produce the fluorchrome of secondary fluorescence or the spinoff that stain may bring the unknown to cell, growth impacts to cell.Therefore utilize the fluorophore in the direct pair cell of autofluorescence spectrum method of cell to study.Nucleic acid and protein are the very important two class materials that produce fluorescence in the cell.The fluorescence quantum yield of nucleic acid is very low, brings bigger difficulty to detection.Protein constitutes the major part of cell, accounts for the over half of dry cell weight, demonstrates hyperfluorescence in 300～350nm scope, and it and vital movement are closely bound up, have diversified function in vivo.The intrinsic photoluminescent property of protein makes that utilizing fluorescent spectrometry that it is studied has special advantages, and it has natural fluoresence mainly is because aromatic amino acid---tyrosine, tryptophane and phenylalanine.Their relative intensity of fluorescence is than being 100:9:0.5, and the maximum emission wavelength of phenylalanine is 282nm, tyrosine 304nm, tryptophane 340nm.
At present, both at home and abroad judge with the cell cycle of pair cell and yet there are no report by setting up cell autofluorescence spectrum model.
Summary of the invention
At above shortcomings in the prior art, the purpose of this invention is to provide a kind of short-cut method that can judge eukaryotic cell cycle in each stage (G1 phase, S phase, G2 phase and M phase), the influence of biochemical reaction pair cell internal protein in the fluorescence spectrum information of same cell growth behavior of cell and the cell proliferation process is connected cell cycle judge.
The object of the present invention is achieved like this: the method for utilizing the fluorescence spectrum method for measuring cell cycle, it is characterized in that comprising the steps: 1) with the individual cells of certain cell as a variation system, make standard model with being in the individual cells of cell cycle G1 phase, S phase, G2 phase and M phase under the normal growth state.Utilize fluorospectrophotometer or fluorescence spectrophotometer to measure the maximum excitation wavelength of tested cell standard model, during promptly with this wavelength illumination sample, can obtain the fluorescence Spectra of relative intensity maximum;
Be that excitation wavelength scans in 250～700nm scope at first with 250nm, obtain cell after the emission spectrum under the 250nm situation, measure the excitation spectrum between 250nm first fluorescence peak wavelength in the emission spectrum, find the strongest peak of fluorescence intensity in the excitation spectrum, the pairing wavelength in this peak is the maximum excitation wavelength of tested cell sample, at last do exciting light and measure this wavelength to the fluorescence spectrum between 700nm, sweep spacing 0.1nm with maximum excitation wavelength;
For the situation of in the fluorescence spectrum scope, having only a fluorescence peak, then directly utilize this fluorescence peak intensity and the relation of cell cycle to set up model and differentiate by fluorescence peak intensity cell cycle; For the situation that a plurality of absorption peaks are arranged in the fluorescence spectrum scope, then set up apart from nearest fluorescence peak intensity of excitation wavelength and the relation curve of cell cycle, utilize this to concern that cell cycle differentiates again;
2) utilize fluorospectrophotometer or fluorescence spectrophotometer in maximum excitation wavelength arrives the 700nm scope, sample is scanned, after the gained curve of spectrum carried out pre-service, with the absorption peak strength contrast of the absorption peak strength of the sample curve of spectrum and the fluorescence spectrum model of described certain cell cycle, can judge which that tested cell is in the cell cycle in stage.
This method is utilized the cell influence that the continuous variation of fluorescent material (as amino acid, nucleic acid, protein etc.) causes by force the peak position of its fluorescence spectrum, spectrum peak and peak in the born of the same parents in cell cycle (G1 phase, S phase, G2 phase and M phase) change procedure, find out these parameters and the corresponding relation in cell cycle of living in, set up the cell cycle fluorescence spectrum model of specific cells, finally utilize the accurate mensuration of this model realization the individual cells cycle.This method is used for a kind of conventional method the measurement of cell cycle, solve and relied on the comparatively loaded down with trivial details method of discrimination that the flow cytometer cell cycle is measured in the past, and be in the cell of G2 phase and M phase in can cell cycle and distinguish, this is that flow cytometer can't be realized.
The inventive method sample preparation is simple, easy to operate, instrument and equipment is general, accuracy is high, measuring speed is fast, the relation that application cell fluorescence peak intensity changed with the cell cycle, the cell cycle of pair cell is judged very easily, cell sample need not to adopt fluorescein stain to dyeing, thereby the spinoff that the use of having eliminated fluorescein stain brings, make cell after measurement, also can be used for follow-up cultivation and research, help in the biomedical research process, applying.
Description of drawings
Fig. 1 is each stage autofluorescence spectrum curve map of Hela cell cycle;
Fig. 2 is the graph of a relation of fluorescence peak intensity and cell cycle.
Embodiment
The invention will be further described below by specific embodiment.
Embodiment: utilize fluorescent spectrometry the Hela cell to be carried out the method for cell cycle measurement:
1, at first, set up the fluorescence spectrum model of Hela cell cycle, each the time phase cell standard spectrum curve obtain according to the following steps:
1. the acquisition of sample.For obtaining spectroscopic data accurately, to guarantee that at first cell sample is under the self-sow state, adopt observation by light microscope according to metamorphosis, obtain and be in the cell cycle individual cells of phase (G1 phase, S phase, G2 phase and M phase) simultaneously not, with it as standard model.
2. each the time the obtaining of phase cell maximum excitation wavelength.Be that excitation wavelength scans in 250～700nm scope at first with 250nm, obtain cell after the emission spectrum under the 250nm situation, measure the excitation spectrum between first fluorescence peak wavelength 360nm of 250nm in the emission spectrum, find the strongest peak of fluorescence intensity in the excitation spectrum, the pairing wavelength 290nm in this peak is the maximum excitation wavelength of tested cell sample.
3. each the time phase cell fluorescence spectrum measurement.Utilize fluorospectrophotometer or fluorescence spectrophotometer under physiological temp (36.5 ± 0.5 ℃), the 300nm of measurement standard sample is to the fluorescence spectrum between 700nm, sweep spacing 0.1nm, scanning times 5 times.(the measurement result curve as shown in Figure 1).
4. the pre-service of cell fluorescence spectrum.5 groups of spectroscopic datas of each cell cycle are averaged standard spectrum curve as this sample sets cell, the average gained curve of spectrum are carried out The disposal of gentle filter, with reduce eliminate measure in the spectrum distortion that causes of a variety of causes.
5. the foundation of cell fluorescence spectral model.Obtain two characteristic fluorescence peaks after finishing the measurement of the curve of spectrum of each stage sample of cell cycle, utilize the fluorescence peak intensity at the 360nm wavelength place nearest to set up corresponding model (as shown in Figure 2) with the relation of cell cell cycle respective stage of living in apart from excitation wavelength 290nm.
2, after the spectral model of finishing cell cycle in each stage is set up, but judge with regard to the pair cell cycle of living in.At first, utilize fluorospectrophotometer or fluorescence spectrophotometer in 290～700nm scope, tested Hela cell sample is scanned, the gained curve of spectrum is carried out after smothing filtering etc. handles, with absorption peak strength and the model contrast that the curve of spectrum is set up the used wavelength correspondence of model, can judge the G1 phase (as shown in Figure 2) that tested cell is in the cell cycle.
The inventive method with the individual cells of certain cell as a variation system, make standard model with the individual cells that is in cell cycle G1 phase, S phase, G2 phase and M phase under the normal growth state, utilize fluorospectrophotometer or fluorescence spectrophotometer to measure each cell sample greater than the fluorescence spectrum of its maximum excitation wavelength 10nm in the 700nm scope.Main fluorescence peak place in this scope, the intensity of fluorescence peak can along with the cell cycle change in intracellular organic matter (as amino acid, nucleic acid, protein etc.) continuous variation obvious variation arranged.For the situation of in this scope, having only a fluorescence peak, then directly utilize this fluorescence peak intensity and the relation of cell cycle to set up model and differentiate by fluorescence peak intensity cell cycle.For the situation that a plurality of fluorescence peaks are arranged, then, set up the relational model of its intensity and cell cycle to differentiate from that nearest fluorescence peak cell cycle of excitation wavelength in this scope.After finishing the foundation of model, in the time will measuring, only need to measure its fluorescence spectrum, gained fluorescence peak intensity and correlation model contrast can be judged its cell cycle of living at maximum excitation wavelength～700nm to the residing cell cycle of this kind cell.
Cell fluorescence spectroscopic methodology provided by the present invention is the variation tendency of can reflect eukaryotic in cell cycle process G1 phase in each stage, S phase, G2 phase and M phase.The growth of cell, breeding all with the cell cycle interwoveness, when each of cell cycle mutually in, since in the cell materials such as amino acid, protein change with the cell cycle and each the time uneven distribution and concentration change in mutually, caused cell each the time mutually in the peak value of fluorescence spectrum and the difference of intensity thereof.So this species diversity can be measured the major parameter of cell cycle as the fluorescence spectrum method, thereby the growth behavior of pair cell is described and judges exactly.
Utilize the variation of the protein of fluorescence spectrum in can the cell cycle process to follow the tracks of accurately and then draw carefully Born of the same parents' cycle is the characteristic spectrum of phase simultaneously not, and with this as the foundation of setting up cell cycle spectral information model. Simultaneously, sample Need not to adopt fluorescein stain to dyeing, thereby eliminated the side effect that the use of fluorescein stain brings, so that cell is after measurement Also can be used for follow-up cultivation and research. It also has the advantages such as equipment is simple, cheap, easy to operate, sensitivity height.
Claims (5)
1, utilizes the cell autofluorescence spectrum to measure the method for cell cycle, it is characterized in that, comprise the steps:
1) with the individual cells of certain cell as a variation system, with being in the individual cells of cell cycle G1 phase, S phase, G2 phase and M phase under the normal growth state as standard model;
2) utilize fluorospectrophotometer or fluorescence spectrophotometer to measure the maximum excitation wavelength of tested cell standard model, do exciting light with maximum excitation wavelength and measure this wavelength to the fluorescence spectrum between 700nm;
3) utilize fluorospectrophotometer or fluorescence spectrophotometer in maximum excitation wavelength arrives the 700nm scope, to scanning with the congener sample of described standard model, after the gained curve of spectrum carried out pre-service, with the absorption peak strength contrast of the absorption peak strength of the sample curve of spectrum and the fluorescence spectrum model of described certain cell cycle, can judge which that tested cell is in the cell cycle in stage;
For the situation of in the fluorescence spectrum scope, having only a fluorescence peak, then directly utilize this fluorescence peak intensity and the relation of cell cycle to set up model and differentiate by fluorescence peak intensity cell cycle; For the situation that a plurality of peaks are arranged in the fluorescence spectrum scope, then adopt the fluorescence peak nearest apart from excitation wavelength, utilize this to concern that cell cycle differentiates again.
2, according to the described method of utilizing the cell autofluorescence spectrum to measure the cell cycle of claim 1, it is characterized in that, the acquisition of described standard model, guarantee that cell sample is under the self-sow state, adopt observation by light microscope according to metamorphosis, obtain and be in the cell cycle cell of phase simultaneously not, be made into standard model.
3, according to the described method of utilizing the cell autofluorescence spectrum to measure the cell cycle of claim 1, it is characterized in that, the measuring method of described maximum excitation wavelength is: be that excitation wavelength scans tested cell standard model in 250～700nm scope with 250nm at first, obtain this standard model cell after the emission spectrum under the 250nm situation, measure the excitation spectrum between 250nm first fluorescence peak wavelength in the emission spectrum, find the strongest peak of fluorescence intensity in the excitation spectrum, the pairing wavelength in this peak is the maximum excitation wavelength of tested standard model cell, at last do exciting light and measure this wavelength to the fluorescence spectrum between 700nm, sweep spacing 0.1nm with maximum excitation wavelength.
4, according to the described method of utilizing the fluorescence spectrum method for measuring cell cycle of claim 1, it is characterized in that, described step 2) measurement of fluorescence spectrum is to utilize fluorospectrophotometer or fluorescence spectrophotometer under 36.5 ± 0.5 ℃ of physiological temps in, carry out the fluorescence spectral measuring of standard model, setting spectrophotometric sweep limit is that maximum excitation wavelength is to 700nm, scanning step 0.1nm, scanning times 5 times.
5, according to the described method of utilizing the fluorescence spectrum method for measuring cell cycle of claim 1, it is characterized in that, the pre-service of described cell spectrum is that 5 groups of spectroscopic datas with each cell cycle are averaged the standard spectrum curve as this sample sets cell, and the average gained curve of spectrum is carried out The disposal of gentle filter.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107202773A (en) * | 2017-06-01 | 2017-09-26 | 重庆大学 | A kind of cell cycle scattered light intensity method for establishing model of utilization polystyrene spheres |
| CN109762868A (en) * | 2019-01-15 | 2019-05-17 | 大连大学 | A method of based on the micro-fluidic chip Accurate Analysis cell cycle |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107202773A (en) * | 2017-06-01 | 2017-09-26 | 重庆大学 | A kind of cell cycle scattered light intensity method for establishing model of utilization polystyrene spheres |
| CN107202773B (en) * | 2017-06-01 | 2019-07-05 | 重庆大学 | A kind of cell cycle scattered light intensity method for establishing model using polystyrene spheres |
| CN109762868A (en) * | 2019-01-15 | 2019-05-17 | 大连大学 | A method of based on the micro-fluidic chip Accurate Analysis cell cycle |
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