CN110016460A - A kind of efficient chicken embryo tire primary fibroblast G2/M phase method for synchronizing - Google Patents
A kind of efficient chicken embryo tire primary fibroblast G2/M phase method for synchronizing Download PDFInfo
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Abstract
The invention discloses a kind of efficient chicken embryo tire primary fibroblast G2/M phase method for synchronizing.The present invention provides a kind of efficient chicken embryo tire primary fibroblast G2/M phase method for synchronizing, include the following steps: the nocodazole for converging addition final concentration 100ng/ml in the chicken embryo tire primary fibroblast that rate is 85%-90% to adhere-wall culture, suppress culture 4h, G2/M phase cell is collected by centrifugation after then patting culture dish and making M phase cell detachment.The experiment of the invention proves that the present invention is combined using nocodazole (100ng/ml) blocked method and mechanical concussion method, chicken embryo tire primary fibroblast can be made to synchronize to the ratio of G2/M phase and be up to 95.62%.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of efficient phase synchronization side chicken embryo tire primary fibroblast G2/M
Method more particularly to a kind of efficient chicken embryo tire primary fibroblast G2/M phase method for synchronizing suitable for chromosome sorting.
Background technique
The cell continuously divided terminates to complete entire mistake experienced to mitosis next time from last mitosis
Journey is a cell cycle (cell cyc1e).The entire cell cycle can be divided into interphase and m period, and the M phase is to have silk point
The phase is split, is that chromosome is really initially separated period.Interphase can be further subdivided into 3 G1 phase, S phase, G2 phase phases again.In cell
In incubation, cell many places are in different Cell Cycles, wherein only a few cell is carrying out mitotic event
(M phase), remaining cell are respectively at G1, S and G2 each phase.In order to obtain the biochemical material of moment, need to obtain the period one
The cell of cause property.Cell can be made largely to be in same cell stage using cell synchronization technology, and it is big to can get the period
The substance of amount.
Two class of chemical block method and physics back-and-forth method can be divided into from physical and chemical angle by obtaining M phase cellular processes.
Chemical block method is that the formation of micro-pipe is destroyed using certain specific chemical reagent, by cells blocks in the M phase.It is common
Blocking drugs have colchicine, demecolcine, nocodazole etc..The shortcomings that chemical block method is that these block reagent all can
There is certain toxicity, if activity or action time are improper, will cause cell and generate abnormal division, growth is lopsided or even dead
It dies.The different type cell of different plant species, the tolerance to these chemical block reagents are different, therefore to different plant species
Different types of cell obtains most preferably suppressing concentration and most preferably the time is suppressed to usually require largely to be tried for G2/M phase cell
It tests and gropes.
G2/M phase cell physics back-and-forth method is to be rounded protuberance using in M phase cell, low with the adhesion of culture dish
Characteristic is gently vibrated, and M phase cell is easily detached from wall, is suspended in culture solution, is collected culture solution and is obtained the required M phase
Cell.It is few that the shortcomings that physics back-and-forth method, is that adhere-wall culture cell is in the cell quantity of division stage, can not achieve short-term a large amount of
M phase cell is collected, and if oscillation amplitude is excessive may be collected into Interphase cells.
Summary of the invention
The object of the present invention is to provide a kind of efficient chicken embryo tire primary fibroblast G2/M phase method for synchronizing.
Method provided by the invention includes the following steps: to be added into the chicken embryo tire primary fibroblast of adhere-wall culture
The nocodazole of final concentration 100ng/ml carries out after suppressing culture 4h, pats culture dish, collect the cell to fall off and obtain the G2/M phase
Cell.
In the above method, the opportunity of the nocodazole that final concentration 100ng/ml is added is the chicken embryo tire to adhere-wall culture
When primary fibroblast converges 85 ﹪ -90 ﹪ of rate.
In the above method, the time for suppressing culture is 4h hours.
In the above method, culture medium used in the chicken embryo tire primary fibroblast culture is by volumn concentration 20%
80 ﹪ of FBS and volumn concentration DMEM/F12 culture medium composition.
In the above method, it is described suppress culture 4h after, concussion collect chicken embryo tire primary fibroblast G2/M phase cell；
The concussion, which is collected as first patting culture dish, makes G2/M phase cell detachment, then is collected by centrifugation.
In the above method, the chicken embryo tire primary fibroblast is any generation cell in 1-10 generation.
It is also the scope of protection of the invention by G2/M phase cell prepared by the above method.
The process of cell Proliferation is known as the cell cycle, and in the cell cycle, most important event is the carrier of hereditary information
DNA replication dna is copied at two parts, and by two parts of copies, to be assigned to two filial generations intracellular by mitotic mode.Proliferous type is thin
The cell cycle of born of the same parents was divided into for 4 phases, wherein the two crucial phases are that DNA synthesizes (S) phase and cell division (M) phase；After the M phase and
The S phase start before the gap phase be known as the G1 phase, and after the S phase and the M phase start before the gap phase be known as the G2 phase.G2 phase and M phase
Cell have gone through the duplication of S phase DNA, therefore the content of G2/M phase cell DNA is 2 times of G1 phase cell, and is in G1
S phase after phase before the G2/M phase is the synthesis period of cell DNA, and the content of cell DNA is generally higher than G1 phase cell and small
In G2/M phase cell.
Carrying out cell cycle analysis with flow cytometry (Flow Cytometry, FCM) is by amount of DNA in measurement cell
And complete.After measuring DNA content, cell cycle analysis software (such as the Becton Dickinson company that FCM is equipped with can be used
ModFit software and Flowjo software) it is automatically analyzed.The cell cycle of this measurement can be G1 according to the amount point of DNA
Phase, S phase and G2/M phase.
Present invention determine that nocodazole to the chicken embryo tire primary fibroblast G2/M phase synchronizes optimal activity, and
In conjunction with physical method, the method for foring a kind of efficient synchronization chicken embryo tire primary fibroblast to G2/M phase, the G2/M phase
It synchronizes efficiency and is up to 95.62%, lay a good foundation for the preparation and chromosome flow sorting of later period a large amount of metaphase chromosomes.This
It tests optimized nocodazole and opportunity is added, activity and action time are suitable for chicken embryo tire primary fibroblast.Greatly
The acquisition of amount M phase cell can prepare a large amount of chicken metaphase chromosome, be used for other subsequent experimentals, and such as chromosome karyotype analysis is special
Heterosomal sorting, the fluorescence in situ hybridization etc. of specific chromosomal.
Detailed description of the invention
Fig. 1 is the chicken embryo tire primary fibroblast that nocodazole is handled and handled without nocodazole.
(a) the normal chicken embryo tire primary fibroblast not add nocodazole processing；
It (b) is the test group chicken embryo tire primary fibroblast of nocodazole (final concentration 100ng/ml) processing.
Fig. 2 is that various concentration nocodazole handles the comparison of chicken embryo tire primary fibroblast G2/M phase cell proportion
Figure.
Fig. 3 is chicken embryo tire primary fibroblast each cell sample cell cycle distribution figure after different disposal.
It (A) is chicken embryo tire primary fibroblast each cell cycle point of the normal adherent growth handled without nocodazole
Butut；
(B) each thin for the cell suppressed through nocodazole (final concentration 100ng/ml) and directly collected without mechanical concussion
Born of the same parents' period profile figure；
It (C) is cell sample each cell cycle of the mechanical oscillation removing after nocodazole (final concentration 100ng/ml) is suppressed
Distribution map.
Fig. 4 is chicken embryo tire primary fibroblast each cell cycle ratio after different disposal.
Control group 1: the normal attached cell without nocodazole processing；
Control group 2: it is only directly collected thin after nocodazole (final concentration 100ng/ml) is suppressed without mechanical oscillation
Born of the same parents；
Test group: the cell that the test group of mechanical concussion is collected after nocodazole (final concentration 100ng/ml) is suppressed.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, unless otherwise specified, remaining reagent commercially obtain
It arrives.
Culture medium prescription used in following example is that volumn concentration is that 20 ﹪ FBS+ volumn concentrations are
80 ﹪ DMEM/F12 culture mediums.
The separation and culture of embodiment 1, chicken embryo tire primary fibroblast
Chicken embryo tire primary fibroblast, and passaging anchorage culture are separated from aseptic chicken embryo, the specific method is as follows:
(1) it after egg surface disinfection, is placed in sterile super-clean bench；
(2) eggshell for gently breaking gas chamber end into pieces with tweezers blunt end will wherein chicken embryo be taken out with aseptic nipper, and be placed in sterile training
It supports in ware；
(3) with the head and four limbs of sterile scissors removal embryo, trunk is shredded as far as possible；
(4) the trunk tissue shredded is transferred in sterile 15ml centrifuge tube, the pancreas that 10ml concentration is 0.25 ﹪ is added
Protease is placed in 37 DEG C of thermostat water baths and digests 15-20min, and 1000rpm is centrifuged 6min, discards pancreatin supernatant；
(5) 1ml complete medium is added, is sieved through filter through 40 μm of steril cells after precipitating is suspended, filtrate is placed in diameter
In 10cm sterile petri dish, i.e. containing the chicken embryo tire primary fibroblast largely isolated in filtrate；
(6) appropriate complete medium is added in the culture dish containing chicken embryo tire primary fibroblast in step (5), sets
In 37 DEG C, 5%CO2Incubator in cultivated；
(7) purifying of chicken embryo tire primary fibroblast: the chick-embryo cell just separated often contains various kinds of cell type, answers
Purify chicken embryo tire primary fibroblast with pancreatin differential digestion method, being generally passaged to 3-4 generation, to can get purer chicken embryo tire former
For fibroblast.
Shown in the chicken embryo tire primary fibroblast result such as Fig. 1 (a) isolated and purified.
Embodiment 2, nocodazole suppress chicken embryo tire primary fibroblast in the condition optimizing of G2/M phase
It will be passaged to the chicken embryo tire primary fibroblast of 2-10 generation, blocked with various concentration nocodazole, visited
Rope obtains the nocodazole activity of maximum ratio G2/M phase chicken embryo tire primary fibroblast, and the specific method is as follows:
One, various concentration nocodazole suppresses processing
1, the culture dish for being inoculated with the 4th generation chicken embryo tire primary fibroblast is divided into 7 groups, every group of 3 repetitions.
2, cell confluency rate, adherent area of the microscopically observation attached cell in culture dish bottom, adherent area are detected
Account for about the 85%-90% of entire culture dish area, i.e. cell confluency rate reaches 85 ﹪ -90 ﹪, is added not to each of 7 groups
With the nocodazole of final concentration:
Not plus the control group of nocodazole: the culture dish of inoculated into chick embryo tire primary fibroblast；
50ng/ml nocodazole processing group: Nuo Kaoda is added into the culture dish of inoculated into chick embryo tire primary fibroblast
Azoles makes its concentration 50ng/ml in the medium；
100ng/ml nocodazole processing group: Nuo Kaoda is added into the culture dish of inoculated into chick embryo tire primary fibroblast
Azoles makes its concentration 100ng/ml in the medium；
200ng/ml nocodazole processing group: Nuo Kaoda is added into the culture dish of inoculated into chick embryo tire primary fibroblast
Azoles makes its concentration 200ng/ml in the medium；
300ng/ml nocodazole processing group: Nuo Kaoda is added into the culture dish of inoculated into chick embryo tire primary fibroblast
Azoles makes its concentration 300ng/ml in the medium；
500ng/ml nocodazole processing group: Nuo Kaoda is added into the culture dish of inoculated into chick embryo tire primary fibroblast
Azoles makes its concentration 500ng/ml in the medium；
1000ng/ml nocodazole processing group: promise is added into the culture dish of inoculated into chick embryo tire primary fibroblast and examines
Up to azoles, make its concentration 1000ng/ml in the medium.
Culture medium is shaken up, incubator is put back to and continues to block culture 4h.Every group of effect duration is consistent.
The culture medium of above-mentioned use is 20%FBS+80%DMEM/F12 culture medium.
Two, Flow cytometry G2/M phase cell proportion
(1) culture medium is discarded, after cleaning 1 time with PBS buffer solution, pancreatin digestion is added, adds when most cells are rounded
Enter complete medium and terminates digestion.
(2) 1000rpm is centrifuged 6min, collects cell.
(3) sample of each collection is added in 1ml ice bath pre-cooling 70% ethanol water of volumn concentration, gently blows and beats
It mixes, 4 DEG C are fixed 2 hours or the longer time.
(4) 3500rpm is centrifuged 3-5 minutes, sedimentation cell.
(5) 0.5 milliliter of propidium iodide (Propidium Iodide, PI) dyeing liquor is added in every solencyte sample, slowly
And cell precipitation is sufficiently resuspended, 37 DEG C are protected from light warm bath 30 minutes.Then storage can be protected from light with 4 DEG C or ice bath.It is suitable after the completion of dyeing
Flow cytometer detection is completed in 24 hours, it is therefore desirable to complete flow cytometer detection on the same day.
(6) result that flow cytomery is formed with Flowjo software or Modfit fluidic cell cycle analysis software into
Row analysis, can obtain ratio shared by each cell cycle in each cell sample.Respectively using Count and PE-A as ordinate and abscissa
Make cell cycle distribution figure.The ratio shared by GraphPad Prism5 software statistics each group G2/M phase cell carries out data
It counts and makes cylindricality analysis chart.
As a result as Figure 1-Figure 2.
Cell state compares before and after Fig. 1 handles chicken embryo tire primary fibroblast for nocodazole, and Fig. 1 (a) is not add promise
The control group chicken embryo tire primary fibroblast up to azoles processing is examined, Fig. 1 (b) is the test group that nocodazole handles (100ng/ml)
Chicken embryo tire primary fibroblast.As seen from Figure 1, chicken embryo tire primary fibroblast form after nocodazole is handled obviously becomes
Circle, meets the form of medium cell.
Fig. 2 is that various concentration nocodazole handles the comparison of chicken embryo tire primary fibroblast G2/M phase cell proportion.
The proportional numerical value that each sample G2/M phase cell can be obtained after the analysis of Flowjo software, will use after each experimental group data statistics
GraphPad Prism5 software mapping analysis, as shown in Figure 2.
From Figure 2 it can be seen that final concentration of 100ng/ml nocodazole co-suppression effect is best, the ratio highest of G2/M phase cell
(58%).
It (illustrates: though the ratio of 1000ng/ml nocodazole processing group G2/M phase cell is high, a large amount of cells occurs
The phenomena of mortality will affect follow-up test, and cellular morphology variation is huge, and toxic effect is larger, is not considered as most preferably suppressing dense
Degree.)
Embodiment 3, chicken embryo tire the primary fibroblast efficient G2/M phase synchronize
After determining that optimal nocodazole suppresses concentration according to above-described embodiment 2, a large amount of G2/M phases are collected in conjunction with machinery concussion
Cell, the method is as follows:
One, collection mode optimizes after nocodazole processing chicken embryo tire primary fibroblast
After 4th generation chicken embryo tire primary fibroblast of embodiment 1 is inoculated with, it is divided into test group (more than 10 wares) and 2
Control group, control group 1 are the normal growth cell that any reagent processing is not added, and control group 2 is only to add nocodazole (final concentration
Drug 100ng/ml) suppresses group, 3 repetitions of every group of setting.
Individual mechanical oscillation group is not set up, because the ratio that the cell G2/M phase of the normal growth of drug-treated is not added is
18% or so, and existing G2 phase cell also has M phase cell, wherein only M phase cell can just fall off when vibration.According to text
Report is offered, it is few in M phase cell quantity, a large amount of cells cannot be collected in the short time, and may receive if oscillation amplitude is excessive
Collect Interphase cells, so not doing independent mechanical oscillation control group.
Control group 1: when cell confluency rate is 85%-90%, any drug-treated is not added, makes cell normal growth 4h again
Afterwards, remove culture medium, PBS is washed once, and the trypsin digestion of 0.1% concentration is rounded to most cells to fall off, and adds appropriate FBS
Digestion is terminated, 15ml centrifuge tube is collected in, room temperature 200g is centrifuged 8min, collects cell.
Control group 2: when cell confluency rate is 85%-90%, nocodazole is added, makes its concentration in the medium
100ng/ml removes culture medium after suppressing culture 4h, and PBS is washed once, and the trypsin digestion of 0.1% concentration is thin to major part
Born of the same parents, which are rounded, to fall off, and appropriate FBS is added to terminate digestion, is collected in 15ml centrifuge tube, and room temperature 200g is centrifuged 8min, collects cell.
Test group: when cell confluency rate is 85%-90%, nocodazole is added, makes its concentration in the medium
100ng/ml suppresses culture 4h；Pat culture dish, collect fall off G2/M phase cell (machinery concussion removing collect the G2/M phase it is thin
Born of the same parents), it finally converges in same centrifuge tube, room temperature 200g is centrifuged 8min, obtains the G2/M phase cell of test group collection.
Two, Flow cytometry G2/M phase cell proportion
(1) cell sample that above-mentioned each group is collected is added in 70% ethyl alcohol of 1ml ice bath pre-cooling, and gently piping and druming mixes, and 4 DEG C
Fix 2 hours or the longer time.
(2) 3500rpm is centrifuged 3-5 minutes, sedimentation cell.
(3) 0.5 milliliter of propidium iodide (Propidium Iodide, P I) dyeing liquor is added in every solencyte sample, slowly
And cell precipitation is sufficiently resuspended, 37 DEG C are protected from light warm bath 30 minutes.Then storage can be protected from light with 4 DEG C or ice bath.It is suitable after the completion of dyeing
Flow cytometer detection is completed in 24 hours, it is therefore desirable to complete flow cytometer detection on the same day.
(4) result that flow cytomery is formed is analyzed using modfit fluidic cell cycle analysis software, can
Obtain ratio shared by each cell cycle in each cell sample.Make cell week using Count and PE-A as ordinate and abscissa respectively
Phase distribution map.It is thin with GraphPad Prism5 software statistics test group and the G1 phase cell proportion of control group each sample, G2/M phase
Born of the same parents' ratio and S phase cell proportion carry out data statistics and make cylindricality analysis chart.
Flow cytometry analysis each group collects the cell cycle distribution result of cell as shown in figure 3, Fig. 3 (A) is without promise
Each cell cycle distribution figure of cell collected up to the control group 1 of azoles processing is examined, Fig. 3 (B) is only through nocodazole (final concentration
Each cell cycle distribution figure of cell sample that control group 2 after 100ng/ml) suppressing is collected；Fig. 3 (C) is (whole through nocodazole
Concentration 100ng/ml) suppress each cell cycle distribution figure of cell sample that the mechanical test group shaken is collected afterwards.
From fig. 4, it can be seen that the G2/ for the cell sample that mechanical oscillation are removed after nocodazole (final concentration 100ng/ml) is suppressed
The ratio of M phase cell is extremely significant to be higher than 1 cell G2/M phase cell proportion (P < 0.01) of control group and only examine through promise up to 95.62%
The G2/M phase cell proportion (P < 0.01) for the control group 2 suppressed up to azoles (final concentration 100ng/ml).
Flow cytometry analysis each group collect cell each cell cycle cell shared by ratiometric result as shown in figure 4,
Control group 1 indicates that the cell that the control group handled without nocodazole is collected, control group 2 are indicated through nocodazole (100ng/ml)
The cell for suppressing rear directly digestion to be collected, test group indicate that mechanical oscillation are removed thin after nocodazole (100ng/ml) is suppressed
Born of the same parents, from fig. 4, it can be seen that G2/M phase cell in the cell sample that mechanical oscillation removing is collected after nocodazole (100ng/ml) is suppressed
Ratio extremely significant be higher than 1 cell of control group (P < 0.01) and only nocodazole suppresses a group cell (control group 2) (P < 0.01).
Complex chart 3 and Fig. 4 explanation, mechanical concussion removing is collected after the nocodazole of final concentration of 100ng/ml is suppressed
G2/M phase cell is a kind of efficient efficient G2/M phase method for synchronizing of chicken embryo tire primary fibroblast.
Claims (7)
1. a kind of method for obtaining chicken embryo tire primary fibroblast G2/M phase cell, includes the following steps: to adhere-wall culture
The nocodazole of final concentration 100ng/ml is added in chicken embryo tire primary fibroblast, carries out suppressing culture, it is thin to obtain the G2/M phase
Born of the same parents.
2. according to the method described in claim 1, it is characterized by: it is described be added final concentration 100ng/ml nocodazole when
Between for when the chicken embryo tire primary fibroblast of adhere-wall culture converges 85 ﹪ -90 ﹪ of rate.
3. method according to claim 1 or 2, it is characterised in that: the time for suppressing culture is 4 hours.
4. method according to claim 1 to 3, it is characterised in that: the chicken embryo tire primary fibroblast culture
Culture medium used is made of the DMEM/F12 culture medium of 80 ﹪ of FBS and volumn concentration of volumn concentration 20%.
5. method according to any one of claims 1-4, it is characterised in that:
The method also includes following steps: it is described suppress culture after, concussion collect chicken embryo tire primary fibroblast G2/M
Phase cell；
The concussion, which is collected as first patting culture dish, makes G2/M phase cell detachment, then is collected by centrifugation.
6. any method in -5 according to claim 1, it is characterised in that: the chicken embryo tire primary fibroblast is the
Any generation cell in 1-10 generation.
7. the G2/M phase cell prepared by the method any in claim 1-6.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321724A (en) * | 2011-08-05 | 2012-01-18 | 厦门大学 | Method for screening spindle checkpoint inhibitor from cells |
| WO2017079029A1 (en) * | 2015-11-02 | 2017-05-11 | ORIG3N Inc. | Cell cycle block improves efficiency in generating induced pluripotent stem cells |
-
2018
- 2018-01-09 CN CN201810017822.9A patent/CN110016460A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321724A (en) * | 2011-08-05 | 2012-01-18 | 厦门大学 | Method for screening spindle checkpoint inhibitor from cells |
| WO2017079029A1 (en) * | 2015-11-02 | 2017-05-11 | ORIG3N Inc. | Cell cycle block improves efficiency in generating induced pluripotent stem cells |
Non-Patent Citations (2)
| Title |
|---|
| LYUBOMIR T VASSILEV: "Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1", 《CELL CYCLE》 * |
| 杨惠茹 等: "不同浓度秋水仙素和nocodazole 对绒山羊成纤维细胞周期同步化的影响", 《中国畜牧兽医》 * |
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Application publication date: 20190716 |