CN107219196A - A kind of cell cycle detection method based on light scattering measurement - Google Patents
A kind of cell cycle detection method based on light scattering measurement Download PDFInfo
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Abstract
The invention provides a kind of cell cycle detection method based on light scattering measurement, biological cell G1 phases in each stage in cell cycle process can truly be reflected by scattered light intensity distribution curve, the S phases, G2 phases and the change of M phases, and this species diversity is determined to the major parameter of cell cycle as scattered light intensity method, it easily can intuitively judge which cycle cell to be measured is in the light scattering characteristic difference of different angles according to different cycles cell, so as to which the growth behavior and cycle of biological cell are judged and described exactly, its operating process is simple, measuring speed is fast, dyeing processing is carried out also without to cell, cell is not damaged, cell sample after measurement can also be reused, be conducive to the popularization and application during biomedical research, it can also be used for surveying in physical examination, and it can be used in a variety of different types of biological cells, the cell cycle comparative analysis and correlative study of virocyte, with good technology application prospect.
Description
Technical field
The present invention relates to technical field of biological, more particularly to Cell Measurement Technique, and in particular to a kind of to be dissipated based on light
Penetrate the cell cycle detection method of measurement.
Background technology
Cell cycle refers to that cell terminates undergone overall process since being completed once division to division next time, is divided into
Between phase and two stages of division stage.Wherein, a phase is divided into three phases, i.e. DNA pre-synthesis phases again（The G1 phases）, DNA synthesis the phase（The S phases）With
DNA post-synthesis phases（The G2 phases）.Division stage（The M phases）It is divided into early stage, mid-term, later stage and latter stage.
Cancer is a kind of cell cycle disease, and the cell cycle is the Main Basiss of antineoplastic classification.Cell cycle manages
New target spot is provided by develop antineoplastic.Accurately antineoplastic can directly against different cycles cancer cell,
Directly kill, so as to reach the purpose for the treatment of.But can accurately kill cancer cell premise is that to correspondence cell week
Phase is detected.Therefore, treatment of the analysis measurement to cancer is carried out to the cancer cell of different cycles using correlation technique and diagnoses tool
It is significant.
The cell cycle detection method more commonly used at present is mainly flow cytometry, and its method is to cell dyeing
On the basis of, by being detected to the chromosome content in individual cells, so that judge the cycle residing for cell, but this method
Testing process it is complex, detection speed is slow, and need to rely on cell dyeing, has damage to cell, and can not accurately divide
The cell of G2 phases and M phases are distinguished, the cell cycle of colony's cell can not also be detected.
The content of the invention
For above shortcomings in the prior art, it is an object of the invention to provide a kind of based on light scattering measurement
Cell cycle detection method, the detection of cell cycle is simply and rapidly realized using light scattering characteristic, to solve existing cell
The problems such as dyeing damage cell, flow are complicated, detection speed is slow is needed in cycle detection technology.
In order to solve the above technical problems, present invention employs following technical scheme：
A kind of cell cycle detection method based on light scattering measurement, is respectively prepared using the cell sample of variant cell cycle
Sample cell suspension, light is scattered using wide-angle light scattering apparatus respectively
Ionization meter, obtains each self-corresponding scattered light intensity distribution curve of sample cell suspension of each cell cycle；Then it is directed to
Cell sample to be measured, is made the cell suspension to be measured with same prescribed concentration, is dissipated under identical measuring condition using wide-angle light
Penetrate instrument and be scattered luminous intensity measurement, obtain the scattered light intensity distribution curve of cell suspension to be measured, and it is different from each respectively
Each self-corresponding scattered light intensity distribution curve of sample cell suspension of cell cycle is contrasted, it is determined that with cell suspension to be measured
The sample cell suspension of a cell cycle that matches of scattered light intensity distribution curve, the cell cycle is determined as to be measured
The cell cycle of cell sample.
In the above-mentioned cell cycle detection method based on light scattering measurement, specifically, following step is specifically included：
A）Predefine the prescribed concentration of detection cell suspension used；
B）Using the cell sample of G1 phases, S phases, G2 phases and M phases, the corresponding G1 phases with prescribed concentration, S phases, G2 is respectively prepared
The sample cell suspension of phase and M phases simultaneously load in teat glass, after shaking up respectively, under 37 DEG C of temperature conditionss, utilize wide-angle light
The sample cell suspension that scatterometer measures each cell cycle respectively is interior at interval of the scattering on 5 ° in 0 ° ~ 180 ° range of scatter angles
Luminous intensity, according to measurement result, obtains each self-corresponding scattered light intensity of sample cell suspension of G1 phases, S phases, G2 phases and M phases
Distribution curve；
C）For cell sample to be measured, the cell suspension to be measured with same prescribed concentration is made, and loads in teat glass, point
After not shaking up, under 37 DEG C of temperature conditionss, cell suspension to be measured is measured in 0 ° ~ 180 ° range of scatter angles using wide-angle light scattering apparatus
The interior scattered light intensity at interval of on 5 °, according to measurement result, obtains the scattered light intensity distribution curve of cell suspension to be measured；
D）By the sample cell suspension of the scattered light intensity distribution curve of cell suspension to be measured cell cycles different from each respectively
Each self-corresponding scattered light intensity distribution curve is contrasted, it is determined that the scattered light intensity distribution curve phase with cell suspension to be measured
The sample cell suspension of one cell cycle of matching, is determined as the cell cycle of cell sample to be measured the cell cycle.
In the above-mentioned cell cycle detection method based on light scattering measurement, preferably, the step A）Specifically,
10 are respectively prepared using the cell sample of any cell cycle6Individual cell/mL, 2 × 105Individual cell/mL, 105Individual cell/mL and 5
×104The cell suspension of these four concentration of individual cell/mL simultaneously loads in teat glass, after shaking up respectively, utilizes wide-angle light scattering
Instrument measures scattered light intensity of the cell suspension of four kinds of concentration in 90 ° of angle of scatterings respectively, according to measurement result, selective scattering
A kind of concentration of the maximum cell suspension of luminous intensity is used as the prescribed concentration for detecting cell suspension used.
In the above-mentioned cell cycle detection method based on light scattering measurement, preferably, the step B）Specially：
b1）Using the cell sample of G1 phases, S phases, G2 phases and M phases, the corresponding G1 phases with prescribed concentration, S phases, G2 is respectively prepared
Phase and the sample cell suspension of M phases, and load in teat glass；
b2）After the sample cell suspension of the G1 phases, S phases, G2 phases and the M phases that are loaded into respectively in teat glass shake up, in 37 DEG C of temperature
Under the conditions of degree, the sample cell suspension of each cell cycle is measured respectively in 0 ° ~ 180 ° angle of scattering models using wide-angle light scattering apparatus
At interval of the scattered light intensity on 5 ° in enclosing, each self-corresponding scattered light intensity of sample cell suspension of variant cell cycle is obtained
Profile data is spent, and is recorded；
b3）Repeat step b2）5 ~ 10 times, for each cell cycle sample cell suspension on each scattering angle
Scattered light intensity angle value obtained by each measurement, the absolute value for rejecting wherein deviation from average rate is more than 10% scattered light intensity angle value, then will
Scattered light intensity angle value on the scattering angle obtained by other measurements is averaging, and obtains the sample cell suspension of corresponding cell cycle
Scattered light intensity average value on corresponding firing angle degree, so as to respectively obtain the sample cell suspension of corresponding cell cycle at 0 °
At interval of the scattered light intensity average value on 5 ° in ~ 180 ° of range of scatter angles, and curve data fit is carried out, then to fitting gained
Scattered light intensity profile data carry out the disposal of gentle filter, the sample cell suspension for obtaining the corresponding cell cycle is corresponding
Scattered light intensity distribution curve；Thus each self-corresponding scattering of sample cell suspension of G1 phases, S phases, G2 phases and M phases are respectively obtained
Light intensity distributions curve.
In the above-mentioned cell cycle detection method based on light scattering measurement, preferably, the step C）Specially：
c1）Using cell sample to be measured, the cell suspension to be measured with prescribed concentration is made, and load in teat glass；
c2）After the cell suspension to be measured being loaded into teat glass shakes up, under 37 DEG C of temperature conditionss, wide-angle light scattering apparatus is utilized
Cell suspension to be measured is measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °, cell suspension to be measured is obtained
Scattered light intensity profile data, and recorded；
c3）Repeat step c2）5 ~ 10 times, for cell suspension to be measured on each scattering angle each time measurement obtained by
Scattered light intensity angle value, reject wherein deviation from average rate absolute value be more than 10% scattered light intensity angle value, then by the scattering angle its
Scattered light intensity angle value obtained by its time measurement is averaging, and is obtained scattered light intensity of the cell suspension to be measured on corresponding firing angle degree and is put down
Average, is averaged so as to respectively obtain cell suspension to be measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °
Value, and curve data fit is carried out, then the disposal of gentle filter is carried out to the scattered light intensity profile data obtained by fitting, obtain
To the scattered light intensity distribution curve of cell suspension to be measured.
In the above-mentioned cell cycle detection method based on light scattering measurement, preferably, the step D）Specially：
d1）From 0 ° ~ 180 ° range of scatter angles, forward scattering angle range, lateral scattering angle range and back scattering angle are marked off
Interval, wherein, forward scattering angle range is 0 ° ~ 20 ° range of scatter angles, and lateral scattering angle range is 80 ° ~ 100 ° angle of scattering models
Enclose, back scattering angle range is 160 ° ~ 180 ° range of scatter angles；
d2）According to each self-corresponding scattered light intensity distribution curve of the sample cell suspension of G1 phases, S phases, G2 phases and M phases, difference
Each comfortable forward-scattering angle of sample cell suspension that statistics obtains G1 phases, S phases, G2 phases and M phases is interval, lateral scattering angle range and
The scattered light intensity distribution histogram of back scattering angle range；
d3）According to the scattered light intensity distribution curve of cell suspension to be measured, statistics obtains cell suspension to be measured in forward-scattering angle
The scattered light intensity distribution histogram of interval, lateral scattering angle range and back scattering angle range；
d4）By cell suspension to be measured forward scattering angle range, lateral scattering angle range and back scattering angle range scattered light
Sample cell suspension each comfortable forward-scattering angle of the strong distribution histogram respectively with G1 phases, S phases, G2 phases and M phases is interval, lateral scattered
Interval and back scattering angle range the scattered light intensity distribution histogram of firing angle is contrasted, it is determined that the scattering with cell suspension to be measured
The sample cell suspension for the cell cycle that light distribution histogram matches, cell sample to be measured is determined as by the cell cycle
This cell cycle.
In the above-mentioned cell cycle detection method based on light scattering measurement, specifically, the step d4）In, G1 phases, S
Each comfortable forward-scattering angle interval of sample cell suspension, lateral scattering angle range and the back scattering angle range of phase, G2 phases and M phases
Scattered light intensity distribution histogram matching according to being：
Scattered light of the sample cell suspension of M phases in forward scattering angle range, lateral scattering angle range and back scattering angle range
It is strong most strong；
The sample cell suspension of G1 phases is only second to the sample of M phases in the scattered light intensity of lateral scattering angle range and back scattering angle range
Product cell suspension, and only it is better than the sample cell suspensions of G2 phases in the scattered light intensity of forward scattering angle range；
The sample cell suspension of S phases is only second to the sample cell suspension of M phases in the scattered light intensity of forward scattering angle range, lateral
The scattered light intensity of scattering angle range is only better than the sample cell suspension of G2 phases, most weak in the scattered light intensity of back scattering angle range；
The sample cell suspension of G2 phases is only better than the sample cell suspension of S phases except the scattered light intensity of back scattering angle range,
The scattered light intensity of forward scattering angle range and back scattering angle range is most weak.
In the above-mentioned cell cycle detection method based on light scattering measurement, preferably, wide-angle light scattering apparatus is utilized
When being scattered luminous intensity measurement, light source is used as using laser.
Compared to prior art, the present invention has the advantages that：
1st, the cell cycle detection method of the invention based on light scattering measurement, its operating process is simple, and measuring speed is fast, is also not required to
Dyeing processing is carried out to cell, cell is not damaged, it is poor in the light scattering characteristic of different angles according to different cycles cell
It is different easily intuitively to judge which cell to be measured be in cycle, and cell sample after measuring can also repeat
Utilize, be conducive to the popularization and application during biomedical research, it can also be used to surveyed in physical examination.
2nd, the cell cycle detection method of the invention based on light scattering measurement, can be true by scattered light intensity distribution curve
The change of real reflection biological cell G1 phases in each stage, S phases, G2 phases and M phases in cell cycle process, the growth of cell, increase
It is all inseparable with the cell cycle to grow, because nucleus refractive index, size can occur in each phase of cell cycle
Change and the change of cytoplasm size and refractive index be the reason for causing cell light scattering profiles difference, therefore can be by this species diversity
The major parameter of cell cycle is determined as scattered light intensity method, so as to growth behavior exactly to biological cell and cycle
Judged and described.
3rd, because light scattering characteristic curve can be and different types of with the structure change and variations in refractive index of reacting cells
Biological cell, virocyte are not only variant on morphosis, and cellular refractive index is dissipated there is also difference, therefore the present invention based on light
The cell cycle detection method for penetrating measurement can also be used to a variety of different types of biological cells, the cell cycle pair of virocyte
Than analysis and correlative study, with good technology application prospect.
Brief description of the drawings
Fig. 1 is the flow chart of the cell cycle detection method of the invention based on light scattering measurement.
Fig. 2 is G1 phases, S phases, the cell suspension scattered light intensity point of four different cell cycles of G2 phases and M phases in embodiment
Cloth curve map.
Fig. 3 is scattered light intensity distribution curve and G1 phases, S phases, G2 phases and the M phases of Hela cell suspensions to be measured in embodiment
Each self-corresponding scattered light intensity distribution curve of Hela sample cell suspensions comparison diagram.
Fig. 4 be in embodiment G1 phases, S phases, the Hela sample cells suspension of G2 phases and M phases in forward scattering angle range, side
To scattered light intensity profiles versus's histogram of scattering angle range and back scattering angle range.
Fig. 5 be embodiment in Hela cell suspensions to be measured forward scattering angle range, lateral scattering angle range and after to dissipate
The interval scattered light intensity profiles versus's histogram with the Hela sample cell suspensions of G1 phases, S phases, G2 phases and M phases of firing angle.
Embodiment
For how simply and rapidly to realize the problem of cell cycle is detected, found by research, the different cell cycles
Biological cell, its light scattering characteristic is different.The light scattering of biological cell is mainly by light in cell and cell nuclear periphery
It is the reflecting of the different refractivity of diffraction, cytoplasm and nucleus and surrounding medium, the reflecting of different optical boundaries, intracellular
Absorption factor cause.These light scattering characteristic parameters all can be different in different Cell Cycles for cell, therefore
The scattered light signal difference caused using this difference just can judge which cell cycle cell is in.
Based on this, the invention provides a kind of cell cycle detection method based on light scattering measurement, embodiments thereof is,
The sample that the correspondence variant cell cycle with prescribed concentration is respectively prepared using the cell sample of variant cell cycle is thin
Born of the same parents' suspension, luminous intensity measurement is scattered using wide-angle light scattering apparatus respectively, obtains the sample cell suspension of each cell cycle
Each self-corresponding scattered light intensity distribution curve；Then cell sample to be measured is directed to, is made with the to be measured of same prescribed concentration
Cell suspension, luminous intensity measurement is scattered under identical measuring condition using wide-angle light scattering apparatus, obtains cell suspension to be measured
Scattered light intensity distribution curve, and each self-corresponding scattered light intensity of sample cell suspension of cell cycles different from each respectively
Degree distribution curve is contrasted, it is determined that the cell cycle matched with the scattered light intensity distribution curve of cell suspension to be measured
Sample cell suspension, the cell cycle is determined as to the cell cycle of cell sample to be measured.
Specifically, the cell cycle detection method flow of the invention based on light scattering measurement is as shown in figure 1, including following
Step：
A）Predefine the prescribed concentration of detection cell suspension used.
Predefine the prescribed concentration of detection cell suspension used, in order to be more beneficial for ensuring that the cell cycle is detected
Accuracy.During specific implementation operation, the step can be realized using such a way：Utilize the cell of any cell cycle
Sample is respectively prepared 106Individual cell/mL, 2 × 105Individual cell/mL, 105Individual cell/mL and 5 × 104These four are dense by individual cell/mL
The cell suspension of degree simultaneously loads in teat glass, after shaking up respectively, and four kinds of concentration are measured respectively using wide-angle light scattering apparatus
Scattered light intensity of the cell suspension in 90 ° of angle of scatterings, according to measurement result, a kind of maximum cell of selective scattering luminous intensity hangs
The concentration of liquid is used as the prescribed concentration for detecting cell suspension used.
When making cell suspension, each cell suspension can use PBS（Phosphate Buffer
Saline, phosphate buffered saline solution）10 mL are made as reagent, production method is to count 10 times of concentration with cell counter
Cell, is then diluted to 10mL with PBS again；For example, with 106Exemplified by the cell suspension of individual cell/mL concentration, one
10 are needed altogether7Individual cell, so calculating 10 with cell counter7Individual cell, is then diluted to 10mL with PBS again, produces
To 106The cell suspension of individual cell/mL concentration；The cell of other concentration is all first to obtain required total cell number, Ran Houzai
10mL is diluted to PBS.Selection 106Individual cell/mL, 2 × 105Individual cell/mL, 105Individual cell/mL and 5 ×
104The cell suspension of these four concentration of individual cell/mL, is the cell suspension because this four concentration ranges, and multiple scattering will not be right
Scattered light intensity is impacted, it is advantageously ensured that determining the repeatability and stability of cell cycle based on light scattering measurement.And through 90 °
A kind of concentration of cell suspension of scattered light intensity measurement selective scattering luminous intensity maximum in angle of scattering is used thin as detection
The prescribed concentration of born of the same parents' suspension, it is therefore an objective to preferably ensure that the follow-up prescribed concentration for measuring cell suspension used is resulted in brighter
Aobvious light scattering characteristic parameter, is more beneficial for ensuring the accuracy of cell cycle detection.
B）Using the cell sample of G1 phases, S phases, G2 phases and M phases, the corresponding G1 phases with prescribed concentration, S is respectively prepared
The sample cell suspension of phase, G2 phases and M phases simultaneously load in teat glass, after shaking up respectively, under 37 DEG C of temperature conditionss, using wide
The sample cell suspension that angle light scattering apparatus measures each cell cycle respectively is interior at interval of on 5 ° in 0 ° ~ 180 ° range of scatter angles
Scattered light intensity, according to measurement result, obtains each self-corresponding scattered light of sample cell suspension of G1 phases, S phases, G2 phases and M phases
Strength distribution curve.
In the step, when specifically performing scattered light intensity measurement using wide-angle light scattering apparatus, laser can be preferred to use
As light source, its advantage is good beam quality, with extraordinary monochromaticjty, directionality and stability.In measurement operation, G1
Phase, S phases, the measurement of each self-corresponding scattered light intensity distribution curve of the sample cell suspension of G2 phases and M phases, can pass through respectively
One-shot measurement and obtain.But in order to preferably ensure that the sample cell suspension of gained G1 phases, S phases, G2 phases and M phases are each self-corresponding
The referring to property of scattered light intensity distribution curve, can be using the step B optimized）Measurement scheme, be specially：
b1）Using the cell sample of G1 phases, S phases, G2 phases and M phases, the corresponding G1 phases with prescribed concentration, S phases, G2 is respectively prepared
Phase and the sample cell suspension of M phases, and load in teat glass；
b2）After the sample cell suspension of the G1 phases, S phases, G2 phases and the M phases that are loaded into respectively in teat glass shake up, in 37 DEG C of temperature
Under the conditions of degree, the sample cell suspension of each cell cycle is measured respectively in 0 ° ~ 180 ° angle of scattering models using wide-angle light scattering apparatus
At interval of the scattered light intensity on 5 ° in enclosing, each self-corresponding scattered light intensity of sample cell suspension of variant cell cycle is obtained
Profile data is spent, and is recorded；
b3）Repeat step b2）5 ~ 10 times, for each cell cycle sample cell suspension on each scattering angle
Scattered light intensity angle value obtained by each measurement, the absolute value for rejecting wherein deviation from average rate is more than 10% scattered light intensity angle value, then will
Scattered light intensity angle value on the scattering angle obtained by other measurements is averaging, and obtains the sample cell suspension of corresponding cell cycle
Scattered light intensity average value on corresponding firing angle degree, so as to respectively obtain the sample cell suspension of corresponding cell cycle at 0 °
At interval of the scattered light intensity average value on 5 ° in ~ 180 ° of range of scatter angles, and curve data fit is carried out, then to fitting gained
Scattered light intensity profile data carry out the disposal of gentle filter, the sample cell suspension for obtaining the corresponding cell cycle is corresponding
Scattered light intensity distribution curve；Thus each self-corresponding scattering of sample cell suspension of G1 phases, S phases, G2 phases and M phases are respectively obtained
Light intensity distributions curve.
In step B）In, in order to obtain accurate scattered light intensity information, first have to ensure that cell sample grows in nature
State, using light microscope observe the form of cell, obtains phases different in the cell cycle（G1 phases, S phases, G2 phases
With the M phases）Cell, as the cell sample of cell cycle standard.And for the sample cell suspension of each cell cycle,
The scattered light intensity profile data measurement of 5 ~ 10 times is all carried out respectively, and carries out average fit and the disposal of gentle filter,
Deviation from average rate is eliminated during average fit（Deviation from average rate=(individual values-entirety average)/entirety average）Absolute value be more than
10% scattered light intensity angle value is averaging again, in order to eliminate scattered light intensity distortion error caused by a variety of causes in measurement,
Ensuring the G1 phases, S phases, each self-corresponding scattered light intensity distribution curve of the sample cell suspension of G2 phases and M phases of gained has more
Good referring to property.
C）For cell sample to be measured, the cell suspension to be measured with same prescribed concentration is made, and load teat glass
It is interior, after shaking up respectively, under 37 DEG C of temperature conditionss, cell suspension to be measured is measured in 0 ° ~ 180 ° scatterings using wide-angle light scattering apparatus
At interval of the scattered light intensity on 5 ° in angular region, according to measurement result, the scattered light intensity distribution of cell suspension to be measured is obtained
Curve.
In the step in the step, when specifically performing scattered light intensity measurement using wide-angle light scattering apparatus, it is also preferred that using
Laser is used as light source.In measurement operation, the measurement of the scattered light intensity distribution curve of cell suspension to be measured can also only pass through
One-shot measurement and obtain.But in order to which the measurement for the scattered light intensity distribution curve for preferably ensuring gained cell suspension to be measured is accurate
Property, can be using the step C optimized）Measurement scheme, be specially：
c1）Using cell sample to be measured, the cell suspension to be measured with prescribed concentration is made, and load in teat glass；
c2）After the cell suspension to be measured being loaded into teat glass shakes up, under 37 DEG C of temperature conditionss, wide-angle light scattering apparatus is utilized
Cell suspension to be measured is measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °, cell suspension to be measured is obtained
Scattered light intensity profile data, and recorded；
c3）Repeat step c2）5 ~ 10 times, for cell suspension to be measured on each scattering angle each time measurement obtained by
Scattered light intensity angle value, reject wherein deviation from average rate absolute value be more than 10% scattered light intensity angle value, then by the scattering angle its
Scattered light intensity angle value obtained by its time measurement is averaging, and is obtained scattered light intensity of the cell suspension to be measured on corresponding firing angle degree and is put down
Average, is averaged so as to respectively obtain cell suspension to be measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °
Value, and curve data fit is carried out, then the disposal of gentle filter is carried out to the scattered light intensity profile data obtained by fitting, obtain
To the scattered light intensity distribution curve of cell suspension to be measured.
The scattered light intensity profile data measurement of 5 ~ 10 times is carried out for cell suspension to be measured, and carries out average fit
And the disposal of gentle filter, scattered light intensity angle value of the absolute value more than 10% of deviation from average rate is eliminated during average fit again
It is averaging, in order to eliminate scattered light intensity distortion error caused by a variety of causes in measurement, preferably ensures treating for gained
Survey the measurement accuracy of the scattered light intensity distribution curve of cell suspension.
D）By the sample cell of the scattered light intensity distribution curve of cell suspension to be measured cell cycles different from each respectively
Each self-corresponding scattered light intensity distribution curve of suspension is contrasted, it is determined that being distributed song with the scattered light intensity of cell suspension to be measured
The sample cell suspension for the cell cycle that line matches, is determined as the cell week of cell sample to be measured the cell cycle
Phase.
In step D）In, the sample of the scattered light intensity distribution curve of cell suspension to be measured cell cycles different from each respectively
Contrast matching way between each self-corresponding scattered light intensity distribution curve of product cell suspension, can be using contrast error analysis
Mode, the sample cell of find out the scattered light intensity distribution curve of cell suspension to be measured cell cycles different from each respectively hangs
To the sample cell suspension of a minimum cell cycle of ratio error between each self-corresponding scattered light intensity distribution curve of liquid, sentence
The scattered light intensity distribution curve being set to cell suspension to be measured matches.In order to further reduce the data fortune of contrast matching
Calculation amount, can use following preferred scheme：
d1）From 0 ° ~ 180 ° range of scatter angles, forward scattering angle range, lateral scattering angle range and back scattering angle are marked off
Interval, wherein, forward scattering angle range is 0 ° ~ 20 ° range of scatter angles, and lateral scattering angle range is 80 ° ~ 100 ° angle of scattering models
Enclose, back scattering angle range is 160 ° ~ 180 ° range of scatter angles；
d2）According to each self-corresponding scattered light intensity distribution curve of the sample cell suspension of G1 phases, S phases, G2 phases and M phases, difference
Each comfortable forward-scattering angle of sample cell suspension that statistics obtains G1 phases, S phases, G2 phases and M phases is interval, lateral scattering angle range and
The scattered light intensity distribution histogram of back scattering angle range；
d3）According to the scattered light intensity distribution curve of cell suspension to be measured, statistics obtains cell suspension to be measured in forward-scattering angle
The scattered light intensity distribution histogram of interval, lateral scattering angle range and back scattering angle range；
d4）By cell suspension to be measured forward scattering angle range, lateral scattering angle range and back scattering angle range scattered light
Sample cell suspension each comfortable forward-scattering angle of the strong distribution histogram respectively with G1 phases, S phases, G2 phases and M phases is interval, lateral scattered
Interval and back scattering angle range the scattered light intensity distribution histogram of firing angle is contrasted, it is determined that the scattering with cell suspension to be measured
The sample cell suspension for the cell cycle that light distribution histogram matches, cell sample to be measured is determined as by the cell cycle
This cell cycle.
By the scattered light intensity distribution curve of cell suspension in forward scattering angle range（0 ° ~ 20 ° range of scatter angles）, side
To scattering angle range（80 ° ~ 100 ° range of scatter angles）With back scattering angle range（160 ° ~ 180 ° range of scatter angles）Scattered light
Strong distribution situation, can be good at embodying its respective scattered light intensity distribution character, therefore, it is possible to effectively thin to carry out
The contrast matching and identification in born of the same parents' cycle；And such matching way of contrast, it is only necessary to by cell suspension to be measured in forward scattering
The scattered light intensity distribution histogram of angle range, lateral scattering angle range and back scattering angle range respectively with G1 phases, S phases, G2 phases
With the scattering of each comfortable forward-scattering angle interval of sample cell suspension of M phases, lateral scattering angle range and back scattering angle range
Light distribution histogram carries out contrast matching operation, helps to reduce contrast on the premise of cell cycle detection accuracy is ensured
The data operation quantity of matching.Specifically asking for dissipating for forward scattering angle range, lateral scattering angle range and back scattering angle range
When penetrating light distribution histogram, forward scattering angle range, lateral scattering angle range, back scattering can be calculated by counting respectively
Obtained in the respective range of scatter angles of angle range at interval of the total and/or average value of 5 scattered light intensity angle value of 5 ° of measurements.
It can see by above-mentioned flow, the cell cycle detection method of the invention based on light scattering measurement, its operation stream
Journey is simple, and measuring speed is fast, it is not required that carries out dyeing processing to cell, cell is not damaged, according to different cycles cell
It easily can intuitively judge which cycle cell to be measured is in the light scattering characteristic difference of different angles, and measure
Later cell sample can also be reused, and be conducive to the popularization and application during biomedical research, it can also be used in body
Detection.
Illustrate the inventive method with embodiment below.It should be understood that these embodiments are only used for into one
Step explanation embodiment of the present invention, rather than for limiting the present invention.
Embodiment：
Using the method for the present invention, cell cycle detection is carried out to Hela cells, following step is specifically included：
1）Predefine the prescribed concentration of detection cell suspension used.The Hela cell samples of variant cell cycle are obtained in advance
Product, 10 are respectively prepared using the Hela cell samples of any cell cycle6Individual cell/mL, 2 × 105Individual cell/mL, 105It is individual thin
Born of the same parents/mL and 5 × 104The cell suspension of these four concentration of individual cell/mL simultaneously loads in teat glass, after shaking up respectively, using wide
Angle light scattering apparatus measures scattered light intensity of the cell suspension of four kinds of concentration in 90 ° of angle of scatterings respectively, according to measurement result,
A kind of concentration of the maximum cell suspension of selective scattering luminous intensity is used as the prescribed concentration for detecting cell suspension used.
2）The measurement of each phase Hela cell sample scattering spectrums.Using the Hela cell samples of G1 phases, S phases, G2 phases and M phases
Product, are respectively prepared the sample cell suspension of the corresponding G1 phases with prescribed concentration, S phases, G2 phases and M phases and load teat glass
It is interior, after shaking up respectively, under 37 DEG C of temperature conditionss, measure the sample cell of each cell cycle respectively using wide-angle light scattering apparatus
Suspension, at interval of the scattered light intensity on 5 °, according to measurement result, obtains G1 phases, S phases, G2 in 0 ° ~ 180 ° range of scatter angles
Each self-corresponding scattered light intensity distribution curve of sample cell suspension of phase and M phases.The step is specially：
21）Using the Hela cell samples of G1 phases, S phases, G2 phases and M phases, the corresponding G1 phases with prescribed concentration, S is respectively prepared
The sample cell suspension of phase, G2 phases and M phases, and load in teat glass.
22）After the sample cell suspension of the G1 phases, S phases, G2 phases and the M phases that are loaded into respectively in teat glass shake up, 37
Under DEG C temperature conditionss, the sample cell suspension of each cell cycle is measured respectively in 0 ° ~ 180 ° scatterings using wide-angle light scattering apparatus
At interval of the scattered light intensity on 5 ° in angular region, each self-corresponding scattering of sample cell suspension of variant cell cycle is obtained
Light intensity distributions curve data, and recorded.
23）Repeat step 22）5 ~ 10 times, for each cell cycle sample cell suspension in each angle of scattering
The scattered light intensity angle value obtained by each measurement is spent, the absolute value for rejecting wherein deviation from average rate is more than 10% scattered light intensity angle value,
The scattered light intensity angle value obtained by other measurements on the scattering angle is averaging again, the sample cell of corresponding cell cycle is obtained
Scattered light intensity average value of the suspension on corresponding firing angle degree, so that the sample cell suspension for respectively obtaining the corresponding cell cycle exists
At interval of the scattered light intensity average value on 5 ° in 0 ° ~ 180 ° range of scatter angles, and curve data fit is carried out, then to fitting
The scattered light intensity profile data of gained carries out the disposal of gentle filter, obtains the sample cell suspension pair of corresponding cell cycle
The scattered light intensity distribution curve answered；Thus the sample cell suspension for respectively obtaining G1 phases, S phases, G2 phases and M phases is each self-corresponding
Scattered light intensity distribution curve.
Each self-corresponding scattered light of Hela sample cell suspensions of G1 phases, S phases, G2 phases and the M phases obtained in the present embodiment
Strength distribution curve is as shown in Figure 2.
3）The measurement of Hela cell samples scattering spectrum to be measured.Cell cycle unknown Hela cell samples to be measured are obtained,
The cell suspension to be measured with same prescribed concentration is made, and loads in teat glass, after shaking up respectively, in 37 DEG C of temperature conditionss
Under, cell suspension to be measured is measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 ° using wide-angle light scattering apparatus
Degree, according to measurement result, obtains the scattered light intensity distribution curve of cell suspension to be measured.The step is specially：
31）Using Hela cell samples to be measured, the cell suspension to be measured with prescribed concentration is made, and load in teat glass.
32）After the cell suspension to be measured being loaded into teat glass shakes up, under 37 DEG C of temperature conditionss, dissipated using wide-angle light
Penetrate instrument and measure cell suspension to be measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °, obtain cell to be measured
The scattered light intensity profile data of suspension, and recorded.
33）Repeat step 32）5 ~ 10 times, institute is measured each time on each scattering angle for cell suspension to be measured
Scattered light intensity angle value, the absolute value for rejecting wherein deviation from average rate is more than 10% scattered light intensity angle value, then by the scattering angle
Scattered light intensity angle value obtained by upper other measurements is averaging, and obtains scattered light intensity of the cell suspension to be measured on corresponding firing angle degree
Average value is spent, so as to respectively obtain cell suspension to be measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °
Average value, and curve data fit is carried out, then the scattered light intensity profile data obtained by fitting is carried out at smothing filtering
Reason, obtains the scattered light intensity distribution curve of cell suspension to be measured.
The Hela cell suspensions to be measured obtained in the present embodiment（Sample in Fig. 3）Scattered light intensity distribution curve with
G1 phases, S phases, comparison diagram such as Fig. 3 of each self-corresponding scattered light intensity distribution curve of the Hela sample cell suspensions of G2 phases and M phases
It is shown.
4）By the sample cell of the scattered light intensity distribution curve of cell suspension to be measured cell cycles different from each respectively
Each self-corresponding scattered light intensity distribution curve of suspension is contrasted, it is determined that being distributed song with the scattered light intensity of cell suspension to be measured
The sample cell suspension for the cell cycle that line matches, is determined as the cell week of cell sample to be measured the cell cycle
Phase.In the present embodiment, using the contrast matching way of optimization, it is specially：
41）From 0 ° ~ 180 ° range of scatter angles, forward scattering angle range, lateral scattering angle range and back scattering angle are marked off
Interval, wherein, forward scattering angle range is 0 ° ~ 20 ° range of scatter angles, and lateral scattering angle range is 80 ° ~ 100 ° angle of scattering models
Enclose, back scattering angle range is 160 ° ~ 180 ° range of scatter angles.
42）According to each self-corresponding scattered light intensity distribution curve of the sample cell suspension of G1 phases, S phases, G2 phases and M phases,
Statistics obtains each comfortable forward-scattering angle interval of sample cell suspension, the lateral scattering angular region of G1 phases, S phases, G2 phases and M phases respectively
Between and back scattering angle range scattered light intensity distribution histogram.The Hela of G1 phases, S phases, G2 phases and M phases that the present embodiment is obtained
Scattered light intensity profiles versus of the sample cell suspension in forward scattering angle range, lateral scattering angle range and back scattering angle range
Histogram is as shown in Figure 4.
43）According to the scattered light intensity distribution curve of cell suspension to be measured, statistics obtains cell suspension to be measured and dissipated in forward direction
The scattered light intensity distribution histogram in firing angle interval, lateral scattering angle range and back scattering angle range.What the present embodiment was obtained treats
Survey Hela cell suspensions（Sample in Fig. 5）In forward scattering angle range, lateral scattering angle range and back scattering angle range
Scattered light intensity profiles versus's histogram with the Hela sample cell suspensions of G1 phases, S phases, G2 phases and M phases is as shown in Figure 5.
44）By cell suspension to be measured dissipating in forward scattering angle range, lateral scattering angle range and back scattering angle range
Penetrate sample cell suspension each comfortable forward-scattering angle interval, side of the light distribution histogram respectively with G1 phases, S phases, G2 phases and M phases
Contrasted to scattering angle range and the scattered light intensity distribution histogram of back scattering angle range, it is determined that with cell suspension to be measured
The sample cell suspension for the cell cycle that scattered light intensity distribution histogram matches, the cell cycle is determined as to be measured thin
The cell cycle of born of the same parents' sample.
In this step, each comfortable forward-scattering angle of the sample cell suspension of G1 phases, S phases, G2 phases and M phases is interval, lateral
The matching of the scattered light intensity distribution histogram of scattering angle range and back scattering angle range is according to being：The sample cell suspension of M phases
It is most strong in the scattered light intensity of forward scattering angle range, lateral scattering angle range and back scattering angle range；The sample cell of G1 phases
Suspension is only second to the sample cell suspension of M phases in the scattered light intensity of lateral scattering angle range and back scattering angle range, and preceding
Only it is better than the sample cell suspension of G2 phases to the scattered light intensity of scattering angle range；The sample cell suspension of S phases is in forward-scattering angle
Interval scattered light intensity is only second to the sample cell suspension of M phases, is only better than the G2 phases in the scattered light intensity of lateral scattering angle range
Sample cell suspension, it is most weak in the scattered light intensity of back scattering angle range；The sample cell suspension of G2 phases is except back scattering angle
Interval scattered light intensity is only better than the sample cell suspension of S phases, in the scattering of forward scattering angle range and back scattering angle range
Light intensity is most weak.In the present embodiment, found by Fig. 5 contrast, Hela cell suspensions to be measured（Sample in Fig. 5）Lateral
Scatter angle range（80°~100°）With back scattering angle range（160°~180°）Scattered light intensity be only second to the Hela samples of M phases
Cell suspension, in forward scattering angle range（0°~20°）Scattered light intensity be only better than the Hela sample cell suspensions of G2 phases, with G1
The sample cell suspension scattered light intensity distribution characteristics of phase matches, therefore, it is determined that the Hela for the G1 phases of Hela cells to be measured is thin
Born of the same parents.
In above-mentioned Fig. 2 ~ Fig. 5, abscissa represents scatter angle values（angle）, ordinate represents scattered light intensity
Angle value（light scattering）.
In summary, it can be seen that the cell cycle detection method of the invention based on light scattering measurement, can be by scattering
The change of light intensity distributions curve truly reflects biological cell in cell cycle process G1 phases in each stage, S phases, G2 phases and M phases
Change, the growth of cell, propagation are all inseparable with the cell cycle, because nucleus is rolled in each phase of cell cycle
Penetrate that rate, size can change and the change of cytoplasm size and refractive index is the original for causing cell light scattering profiles difference
Cause, therefore this species diversity can be determined to the major parameter of cell cycle as scattered light intensity method, so as to thin to biology exactly
The growth behavior of born of the same parents and cycle are judged and described；Also, because light scattering characteristic curve can be become with the structure of reacting cells
Change and variations in refractive index, and different types of biological cell, virocyte not only variant, cellular refractive index on morphosis
There is also difference, thus the cell cycle detection method of the invention based on light scattering measurement can also be used to it is a variety of different types of
The cell cycle comparative analysis and correlative study of biological cell, virocyte, with good technology application prospect.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to skill of the invention
Art scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover at this
Among the right of invention.
Claims (8)
1. a kind of cell cycle detection method based on light scattering measurement, it is characterised in that use the thin of variant cell cycle
The sample cell suspension of the correspondence variant cell cycle with prescribed concentration is respectively prepared in born of the same parents' sample, utilizes wide-angle light scattering apparatus
Luminous intensity measurement is scattered respectively, obtains each self-corresponding scattered light intensity distribution of sample cell suspension of each cell cycle
Curve；Then cell sample to be measured is directed to, the cell suspension to be measured with same prescribed concentration is made, under identical measuring condition
Luminous intensity measurement is scattered using wide-angle light scattering apparatus, the scattered light intensity distribution curve of cell suspension to be measured is obtained, and divide
Each self-corresponding scattered light intensity distribution curve of sample cell suspension of cell cycles not different from each is contrasted, it is determined that with
The sample cell suspension for the cell cycle that the scattered light intensity distribution curve of cell suspension to be measured matches, by the cell week
Phase is determined as the cell cycle of cell sample to be measured.
2. the cell cycle detection method based on light scattering measurement according to claim 1, it is characterised in that specifically include down
State step：
A）Predefine the prescribed concentration of detection cell suspension used；
B）Using the cell sample of G1 phases, S phases, G2 phases and M phases, the corresponding G1 phases with prescribed concentration, S phases, G2 is respectively prepared
The sample cell suspension of phase and M phases simultaneously load in teat glass, after shaking up respectively, under 37 DEG C of temperature conditionss, utilize wide-angle light
The sample cell suspension that scatterometer measures each cell cycle respectively is interior at interval of the scattering on 5 ° in 0 ° ~ 180 ° range of scatter angles
Luminous intensity, according to measurement result, obtains each self-corresponding scattered light intensity of sample cell suspension of G1 phases, S phases, G2 phases and M phases
Distribution curve；
C）For cell sample to be measured, the cell suspension to be measured with same prescribed concentration is made, and loads in teat glass, point
After not shaking up, under 37 DEG C of temperature conditionss, cell suspension to be measured is measured in 0 ° ~ 180 ° range of scatter angles using wide-angle light scattering apparatus
The interior scattered light intensity at interval of on 5 °, according to measurement result, obtains the scattered light intensity distribution curve of cell suspension to be measured；
D）By the sample cell suspension of the scattered light intensity distribution curve of cell suspension to be measured cell cycles different from each respectively
Each self-corresponding scattered light intensity distribution curve is contrasted, it is determined that the scattered light intensity distribution curve phase with cell suspension to be measured
The sample cell suspension of one cell cycle of matching, is determined as the cell cycle of cell sample to be measured the cell cycle.
3. the cell cycle detection method based on light scattering measurement according to claim 2, it is characterised in that the step A）
Specifically, being respectively prepared 10 using the cell sample of any cell cycle6Individual cell/mL, 2 × 105Individual cell/mL, 105It is individual thin
Born of the same parents/mL and 5 × 104The cell suspension of these four concentration of individual cell/mL simultaneously loads in teat glass, after shaking up respectively, using wide
Angle light scattering apparatus measures scattered light intensity of the cell suspension of four kinds of concentration in 90 ° of angle of scatterings respectively, according to measurement result,
A kind of concentration of the maximum cell suspension of selective scattering luminous intensity is used as the prescribed concentration for detecting cell suspension used.
4. the cell cycle detection method based on light scattering measurement according to claim 2, it is characterised in that the step B）
Specially：
b1）Using the cell sample of G1 phases, S phases, G2 phases and M phases, the corresponding G1 phases with prescribed concentration, S phases, G2 is respectively prepared
Phase and the sample cell suspension of M phases, and load in teat glass；
b2）After the sample cell suspension of the G1 phases, S phases, G2 phases and the M phases that are loaded into respectively in teat glass shake up, in 37 DEG C of temperature
Under the conditions of degree, the sample cell suspension of each cell cycle is measured respectively in 0 ° ~ 180 ° angle of scattering models using wide-angle light scattering apparatus
At interval of the scattered light intensity on 5 ° in enclosing, each self-corresponding scattered light intensity of sample cell suspension of variant cell cycle is obtained
Profile data is spent, and is recorded；
b3）Repeat step b2）5 ~ 10 times, for each cell cycle sample cell suspension on each scattering angle
Scattered light intensity angle value obtained by each measurement, the absolute value for rejecting wherein deviation from average rate is more than 10% scattered light intensity angle value, then will
Scattered light intensity angle value on the scattering angle obtained by other measurements is averaging, and obtains the sample cell suspension of corresponding cell cycle
Scattered light intensity average value on corresponding firing angle degree, so as to respectively obtain the sample cell suspension of corresponding cell cycle at 0 °
At interval of the scattered light intensity average value on 5 ° in ~ 180 ° of range of scatter angles, and curve data fit is carried out, then to fitting gained
Scattered light intensity profile data carry out the disposal of gentle filter, the sample cell suspension for obtaining the corresponding cell cycle is corresponding
Scattered light intensity distribution curve；Thus each self-corresponding scattering of sample cell suspension of G1 phases, S phases, G2 phases and M phases are respectively obtained
Light intensity distributions curve.
5. the cell cycle detection method based on light scattering measurement according to claim 2, it is characterised in that the step C）
Specially：
c1）Using cell sample to be measured, the cell suspension to be measured with prescribed concentration is made, and load in teat glass；
c2）After the cell suspension to be measured being loaded into teat glass shakes up, under 37 DEG C of temperature conditionss, wide-angle light scattering apparatus is utilized
Cell suspension to be measured is measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °, cell suspension to be measured is obtained
Scattered light intensity profile data, and recorded；
c3）Repeat step c2）5 ~ 10 times, for cell suspension to be measured on each scattering angle each time measurement obtained by
Scattered light intensity angle value, reject wherein deviation from average rate absolute value be more than 10% scattered light intensity angle value, then by the scattering angle its
Scattered light intensity angle value obtained by its time measurement is averaging, and is obtained scattered light intensity of the cell suspension to be measured on corresponding firing angle degree and is put down
Average, is averaged so as to respectively obtain cell suspension to be measured in 0 ° ~ 180 ° range of scatter angles at interval of the scattered light intensity on 5 °
Value, and curve data fit is carried out, then the disposal of gentle filter is carried out to the scattered light intensity profile data obtained by fitting, obtain
To the scattered light intensity distribution curve of cell suspension to be measured.
6. the cell cycle detection method based on light scattering measurement according to claim 2, it is characterised in that the step D）
Specially：
d1）From 0 ° ~ 180 ° range of scatter angles, forward scattering angle range, lateral scattering angle range and back scattering angle are marked off
Interval, wherein, forward scattering angle range is 0 ° ~ 20 ° range of scatter angles, and lateral scattering angle range is 80 ° ~ 100 ° angle of scattering models
Enclose, back scattering angle range is 160 ° ~ 180 ° range of scatter angles；
d2）According to each self-corresponding scattered light intensity distribution curve of the sample cell suspension of G1 phases, S phases, G2 phases and M phases, difference
Each comfortable forward-scattering angle of sample cell suspension that statistics obtains G1 phases, S phases, G2 phases and M phases is interval, lateral scattering angle range and
The scattered light intensity distribution histogram of back scattering angle range；
d3）According to the scattered light intensity distribution curve of cell suspension to be measured, statistics obtains cell suspension to be measured in forward-scattering angle
The scattered light intensity distribution histogram of interval, lateral scattering angle range and back scattering angle range；
d4）By cell suspension to be measured forward scattering angle range, lateral scattering angle range and back scattering angle range scattered light
Sample cell suspension each comfortable forward-scattering angle of the strong distribution histogram respectively with G1 phases, S phases, G2 phases and M phases is interval, lateral scattered
Interval and back scattering angle range the scattered light intensity distribution histogram of firing angle is contrasted, it is determined that the scattering with cell suspension to be measured
The sample cell suspension for the cell cycle that light distribution histogram matches, cell sample to be measured is determined as by the cell cycle
This cell cycle.
7. the cell cycle detection method based on light scattering measurement according to claim 6, it is characterised in that the step
d4）In, the G1 phases, the S phases, G2 phases and M phases each comfortable forward-scattering angle of sample cell suspension is interval, lateral scattering angle range and after
It is to the matching foundation for the scattered light intensity distribution histogram for scattering angle range：
Scattered light of the sample cell suspension of M phases in forward scattering angle range, lateral scattering angle range and back scattering angle range
It is strong most strong；
The sample cell suspension of G1 phases is only second to the sample of M phases in the scattered light intensity of lateral scattering angle range and back scattering angle range
Product cell suspension, and only it is better than the sample cell suspensions of G2 phases in the scattered light intensity of forward scattering angle range；
The sample cell suspension of S phases is only second to the sample cell suspension of M phases in the scattered light intensity of forward scattering angle range, lateral
The scattered light intensity of scattering angle range is only better than the sample cell suspension of G2 phases, most weak in the scattered light intensity of back scattering angle range；
The sample cell suspension of G2 phases is only better than the sample cell suspension of S phases except the scattered light intensity of back scattering angle range,
The scattered light intensity of forward scattering angle range and back scattering angle range is most weak.
8. the cell cycle detection method based on light scattering measurement according to claim 1, it is characterised in that utilize wide-angle light
When scatterometer is scattered luminous intensity measurement, light source is used as using laser.
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