CN102507580A - Method for quantitatively measuring cell spreading rate based on idealized cell radius - Google Patents
Method for quantitatively measuring cell spreading rate based on idealized cell radius Download PDFInfo
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- CN102507580A CN102507580A CN2011103186297A CN201110318629A CN102507580A CN 102507580 A CN102507580 A CN 102507580A CN 2011103186297 A CN2011103186297 A CN 2011103186297A CN 201110318629 A CN201110318629 A CN 201110318629A CN 102507580 A CN102507580 A CN 102507580A
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Abstract
The invention relates to a method for quantitatively measuring cell spreading rate based on idealized cell radius. The method is characterized by comprising the steps of: planting adherent cells in a suspended state on culture holes or plates, capturing the morphology of the cells at different time points by using imaging equipment during the cell adhering and spreading process, measuring the adhering area of each cell, idealizing the cells before and after spreading to be round, and calculating the cell spreading rate (growth rate of the cell radius within unit time). According to the invention, the idealized cell radius is used as a standard, the spreading rate is calculated according to the cell spreading time and the change of the cell radius, and the defects of the traditional method are solved; and compared with the traditional cell spreading measuring method, the method has the advantages that the method is more accurate, the required measuring equipment is simple, and the calculation method is simple and feasible, and can be integrated to measuring equipment.
Description
Technical field
The invention belongs to biological technical field, be used to measure attached cell sprawl speed and various medicine pair cell is sprawled the method for the influence of speed.
Technical background
It is one of important physiological activity or function of attached cell that cell is sprawled, and many human physiological activities or disease are relevant with sprawling of cell.For example; Vascular endothelial cell makes up blood vessel or repairs damaged blood vessel endothelium layer through sprawling of cell; Therefore; The change that endothelial cell is sprawled is often relevant with generation, development and the metastases etc. of angiocardiopathy, and the influence that the medicine pair cell is sprawled also must be to assess one of important indicator in the test of the cell in vitro of curative effect of medication.
Regrettably, at present pair cell is sprawled mechanism, and particularly the research of the medicine pair cell influence of sprawling also is not very clear, wherein cell sprawl speed accurately, the shortage of method for quantitative measuring is exactly one of the main reasons.
At present; The method that the quantitative measurment cell of bibliographical information is sprawled has only one: the state that cell the is sprawled form of cell (just according to) is divided into not to be sprawled, partly sprawls and sprawl three kinds fully, calculates the quantity or the quantity number percent of the cell that is in these three kinds of states again.Yet; The method that this quantitative measurement cell is sprawled is very coarse: at first, confirm that not sprawling, partly sprawl and sprawl fully three kinds of states has very large subjectivity, but the neither one quantitative standards; Every kind of cell even cell that each is independent after all; Form when they are sprawled fully or adherent area all maybe be different, judge that whether a cell is sprawled fully is very difficult, promptly can produce certain deviation thus.Secondly, the state of partly sprawling has comprised the state in a lot of interstages in fact, thereby, only cell is sprawled and be divided into three kinds of states and can't sprawl along with change of time by the quantitative comparison cell.In addition, sprawling through calculating cell quantity or the recently quantitative cell of quantity percentage is round-about way, comes directly and there is not quantitative measurment to sprawl speed.
Summary of the invention
The purpose of this invention is to provide and a kind ofly sprawl the method for speed, to replace existing traditional method for quantitative measuring based on idealized cell radius quantitative measurment cell.
The present invention realizes through following technical scheme.
The attached cell that will be in suspended state is planted on culture hole or the plate, cell attachment with sprawl in the process, use imaging device, catch a large amount of cells at different time points (T
n) shape appearance figure, measure the adherent area (A) of each cell, the form of cell before and after sprawling is idealized as circle, and calculates the change in radius speed of all cells in the unit interval, this promptly is defined as cell and sprawls speed.It is following that cell is sprawled the rate calculations formula:
Wherein, Rs representes the speed of sprawling of cell; T
N+1The n+1 that sprawls behind the expression cell attachment (measures the moment of n after the moment in setting) constantly, T
nThe n that sprawls behind the expression cell attachment constantly; A
N+1The expression cell is sprawled the adherent area (or mean value of the adherent area of all cells) of the n+1 moment individual cells in the process, A
nThe expression cell is sprawled the adherent area (or mean value of the adherent area of all cells) of the n moment individual cells in the process.
Imaging device of the present invention can be the microscope that can differentiate the individual cells form and have the image recording function, comprises ordinary optical microscope or laser confocal microscope and atomic force microscope etc.
Principal feature of the present invention is: do not divide and do not sprawl, partly sprawl and sprawl fully three kinds of cells and sprawl state, but be standard with the idealized radius of cell, the degree that the big more explanation cell of radius is sprawled is big more; Sprawl the speed of sprawling that the variation of used time and radius calculates according to cell, can explain that the different time inner cell sprawls the speed of variation.Thereby, the invention solves the shortcoming of classic method recited above, can replace.
In addition, it is more accurate that the present invention sprawls measuring method than traditional cell, and classic method can only be measured the state that cell is sprawled, and the speed of can't the quantitative measurment cell sprawling more can't compare the cell of different time sections and sprawl speed.Measuring equipment required for the present invention is also very simple, is equipment commonly used in the biological study; Computing method are simple equally, feasible, as designing in the future a cover algorithm or a software, also are readily integrated in the measuring equipment and go.
Specific embodiments
The present invention will be described further through following examples.
The employed imaging device of embodiment of the present invention is Carl Zeiss 710 laser confocal microscopes that Germany produces; The wherein measurement of cell attachment area is to use this microscopical area measurement function (the LSM 710 Release Version 5.5 sp2 softwares that this microscope carries).
Embodiment 1: the quantitative measurment that cell is sprawled.
The Human umbilical vein endothelial cells that suspends is planted on 24 orifice plates that contain nutrient solution, is positioned over CO
2Incubator (37
0C, 5% CO
2) the middle cultivation.After cultivating 6 hours, 24 hours and 48 hours, observe and obtain the cell feature image respectively with Laser Scanning Confocal Microscope; And measure the adherent area of all individual cells in the image and the average adherent area of all cells, on average sprawl speed through what the top formula that provides calculated the different time sections cell.Calculate at last: the average average ideal radius when cell is sprawled in the process 6 hours, 24 hours and 48 hours is respectively 12.95,18.19 and 19.01 microns; In the time of 0-6 hour, 6-24 hour and 24-48 hour, cell on average to sprawl speed (change in radius in the unit interval) be respectively 2.16,0.29 and 0.03 microns/hour.Data presentation, cell was sprawled the fastest in 0-6 hour, and prolonged spreading rate in time and descend gradually, and cell is still being sprawled after 48 hours, though it is very low to sprawl speed.
Embodiment 2: the quantitative measurment of the influence that the medicine pair cell is sprawled.
The Human umbilical vein endothelial cells that suspends is planted on 24 orifice plates that contain nutrient solution, is positioned over CO
2Incubator (37
0C, 5% CO
2) the middle cultivation.Cultivate and obtain the cell feature image with Laser Scanning Confocal Microscope after 24 hours; In cell solution, add 20 ug/ml Ox-LDL (oxidisability low-density lipoprotein) subsequently; Continue to cultivate again 48 hours, and respectively 6 hours, 12 hours, 24 hours and the feature image of using Laser Scanning Confocal Microscope to obtain cell in 48 hours.And measure the adherent area of all individual cells in the image and the average adherent area of all cells, calculate the idealized radius of different time sections cell and on average sprawl speed through the top formula that provides.Calculate the situation that influences that the medicine pair cell is sprawled at last: before adding medicine; The idealized radius of cell is about 20.04 microns, and the idealized radius of cell is respectively 19.87,21.20,21.35 and 21.70 microns when adding behind the medicine 6 hours, 12 hours, 24 hours and 48 hours; Add behind the medicine in the time of 0-6 hour, 6-12 hour, 12-24 hour and 24-48 hour on average the sprawling speed and be respectively of cell-O.O3,0.22,0.01 and 0.02 micron/hour.Data presentation; 0-6 hour inner cell retraction (sprawled and suppressed after adding medicine; Sprawling speed is negative value); The cell of retraction just begins to sprawl rapidly come (sprawling speed is 0.22) again in 6-12 hour, in 12-24 hour with sprawl all very slowly (sprawl speed and have only 0.01 and 0.02 respectively) of 24-48 hour inner cell.
Claims (1)
1. method of sprawling speed based on idealized cell radius quantitative measurment cell; It is characterized in that the attached cell that will be in suspended state is planted on culture hole or the plate; Cell attachment with sprawl in the process, use imaging device, catch the shape appearance figure of a large amount of cells in different time points; Measure the adherent area of each cell, and be calculated as follows out cell and sprawl speed:
Wherein, Rs representes the speed of sprawling of cell; T
N+1The n+1 moment of sprawling behind the expression cell attachment, T
nThe n that sprawls behind the expression cell attachment constantly; A
N+1The expression cell is sprawled the n+1 mean value of the adherent area of adherent area or all cells of individual cells constantly in the process, A
nThe expression cell is sprawled the n mean value of the adherent area of adherent area or all cells of individual cells constantly in the process.
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|---|---|---|---|
| CN2011103186297A CN102507580A (en) | 2011-10-19 | 2011-10-19 | Method for quantitatively measuring cell spreading rate based on idealized cell radius |
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| CN2011103186297A CN102507580A (en) | 2011-10-19 | 2011-10-19 | Method for quantitatively measuring cell spreading rate based on idealized cell radius |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104700394A (en) * | 2014-11-04 | 2015-06-10 | 中国石油天然气股份有限公司 | High-throughput screening method of duckweed |
-
2011
- 2011-10-19 CN CN2011103186297A patent/CN102507580A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| DAMIEN CUVELIER ET. AL: "The Universal Dynamics of Cell Spreading", 《CURRENT BIOLOGY》 * |
| 陈勇 等: "基于AFM形态学观察的细胞铺展机理的假想", 《中国生物医学工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104700394A (en) * | 2014-11-04 | 2015-06-10 | 中国石油天然气股份有限公司 | High-throughput screening method of duckweed |
| CN104700394B (en) * | 2014-11-04 | 2018-05-04 | 中国石油天然气股份有限公司 | A kind of duckweed high-throughput screening method |
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Application publication date: 20120620 |