CN107653221B - Tapetal cell separation and collection method suitable for protein science research - Google Patents
Tapetal cell separation and collection method suitable for protein science research Download PDFInfo
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Abstract
The separation of plant tapetal cell and collection method suitable for proteomics research.The technology includes histocyte technique for fixing and cell separation, extraction and collection method.Its histocyte fixed operation process are as follows: collect bud in ice pan vessel-being immersed in anther rapidly in the bottle for containing fixer from anther-is separated in bud-, which is covered lid and is evacuated, makes fixer infiltrate through inside anther tissue-fixed the certain time of room temperature rapidly.It is greater than 97% with the tapetal cell purity that investigative technique of the present invention separation obtains, and cellular morphology, structural integrity, quality is high, it is confirmed by subsequent protein electrophoresis detection and proteomics research, proteomics research requirement is met by the total protein quality of the tapetal cell separation and Extraction, total protein separation and Extraction rate is very high with the total protein quantity for separating acquisition.
Description
Technical field
The invention belongs to the targeting separation and collection method of plant organ in biological study field or organization internal cell
Scope, in particular to a kind of separation suitable for proteomics and the anther tapetum cell of Histological research and collection side
Method.
Background technique
The individual of higher plant is made of a variety of Different Organs, and every kind of organ is constituted by different tissues multiple in an orderly manner again
Miscellaneous line and staff control's body, and different tissues is by different types of cell composition.How to be separated from complicated line and staff control
The cell or tissue needed out is in current entire plant science area research as botany research experimental material (test material)
Perplex the great difficult problem of scientists.Various generations are constantly in since biological living (including plant) group is woven under animation
It thanks among activity, and the variation of these metabolic activities has Space-time speciality, i.e., various metabolic activities are in constantly dynamic change
Among.Therefore, biology just receives very big limitation for the acquisition of the targeting cell or tissue test material of research, especially for
The research of the various targetings of modern biotechnology, accurate molecule mechanism etc..It can not target and obtain cell or tissue test material obstruction in due course
The development of the accurate molecular biology research such as plant.
Biology mostly uses greatly paraffin section combination laser microprobe dating to separate suitable for targeting cell separation technology at present
Technology.The technology the last century 80's is invented by foreign countries, and is promoted the use of in medical pathologies molecular biology.But due to
The requirement of the technology itself limits it and is widely popularized.Specific defect shows that one side paraffin section technology belongs to profession
Microsection operating technology, operator need to grasp by the training and training of certain time, it is therefore desirable to which training has
The professional technician of element could complete；On the other hand, entire paraffin wax flaking technical operation process is complicated, more demanding, and week
Phase is longer, and general a cycle gets off to want one week or so, and then it is micro- to have to pass through special professional laser for target cell acquisition
Cutting device cutting operation can just be got.And expensive laser microprobe dating instrument equipment allows general laboratory to hang back.
Chinese patent " a method of isolate and purify plant tapetal tissue " (application number 201510797285.0) disclosure
A kind of method separating tapetal tissue, this method are by acquiring and cutting to anther, by a timing in cleaning solution
Between vortex clean and filter removal pollen, be vortexed by the enzymatic hydrolysis of certain time, filter out tapetal cell solution, then pass through
Final collect of centrifugation obtains tapetal cell.The technology is thin using enzymatic hydrolysis+scroll machine separation principle separation tapetum
Born of the same parents.Although the tapetal cell that the technology obtains is living cells, a series of by washing, enzymatic hydrolysis, the vortex of certain time etc.
The non-tapetal cell for sampling the moment of the tapetal cell obtained after operating procedure, it is thus impossible to which it is specific accurately to obtain some
The tapetal cell at moment, to define the accuracy of its result of study.In addition, the method is only applicable to point of tapetal cell
From without being widely popularized property.
Summary of the invention
The purpose of the present invention is being suitable for shortcoming existing for targeting cell separation technology for existing biology, mention
Method is separated and collected for a kind of tapetal cell suitable for proteomics and Histological research.
Realize the technical solution of the object of the invention are as follows: tapetal cell separation and collection suitable for proteomics research
Operating technology, including histocyte technique for fixing and cell separation, extraction and collection method, are distinguished by technical operation process below
It illustrates:
One, histocyte technique for fixing
The technology includes fixer constituent and histocyte fixing means.
1, fixed operation method:
Collect bud in ice pan vessel-separate anther-from bud anther be immersed in rapidly to splendid attire fixer
Bottle in-lid lid pumping makes fixer infiltrate through inside anther tissue-fixed the certain time of room temperature rapidly.
2, this histocyte technique for fixing principle:
The technique for fixing is using the biological immune group fixation principle usually used in biological immune group field, by solid
Surely make intracellular protein coagulating, terminate endogenous or exogenous enzymes reaction, in-stiu encapsulation antigen, antigen is avoided to inactivate or more
It dissipates.
3, histocyte fixed effect:
With the cell that investigative technique of the present invention is fixed, show the intact nothing of cellular morphology, structure by microscope detection
Damage, cell quality are high.And show the anther total protein and fresh flowers fixed using the present invention by anther total protein electrophoresis detection
Medicine total protein height is consistent.Therefore, tapetum that is fixed with investigative technique of the present invention progress anther and thus separating acquisition is thin
The entirely appropriate subsequent cell proteomics research of born of the same parents is drawn materials with sample and is required.
Preferably, the fixer constituent are as follows: paraformaldehyde, polyethylene glycol, glacial acetic acid, phosphate buffer；It is fixed
Liquid concentration of component mass percent are as follows: paraformaldehyde 2%-4%, preferably 2%；Polyethylene glycol 5%-20%, preferably 8%-15%, more
Preferably 10%；Phosphate buffer molar concentration is preferably 0.1M, and PH7-7.6(glacial acetic acid adjusts pH value, and glacial acetic acid is on the one hand
PH value is adjusted, cellular contraction on the other hand can be prevented).
Preferably, the sample and fixer volume ratio about 1:20.
Preferably, the set time is preferred are as follows:, most preferably 3-4 hours, can also be in 3-12 hours models in 1-24 hours
In enclosing.Specific fixed duration selection is then according to different plant tissue cell's types and Objective extraction substance (such as total protein, solvable
Property albumen etc.) determine the fixation duration for being suitable for target cell.
Cell tissue rigid condition is preferably room temperature condition operation.
Fixed sample pumping can be equal with injector for medical purpose (being preferably greater than or equal to 10ml syringe) or dedicated air extractor
It can.
Preferably, the sample air suction process step is necessary operation link for plant tissue, and other biological
Tissue, such as animal or medical tissue sample, this operating procedure does not belong to must content.
Two, cell separation, extraction and collection method
1, wash anther: by fixed anther sample with distilled water displacement washing 3 times, every time 10 minutes, centre is shaken
Mix 1-2 sample, washes clean anther；
2, cell is separated:
(1) anther is moved into small beaker, adds and lightly beats institute repeatedly with the top of flat spillikin after a small amount of distilled water
There is anther about 5 minutes, the tapetal cell in anther tissue is made to be detached from anther and spreads out.
(2) about more than ten milliliters of distilled waters are added, gently beaten, suction pipe pressure-vaccum, stirred repeatedly again with the top of flat spillikin
Dynamic anther tissue, about 5 minutes.
(3) with 80 mesh cell sieve filtration cell mixed liquors, filtrate is placed in compared in large beaker, and residue collection enters previous small burning
In cup.
(4) (2) and (3) operating procedure is repeated.
(5) it is filtered again with 80 mesh cell sieves, filtrate merges with filtrate before, discards filter residue.
Preferably, the spillikin preferred top used when the cell separates has the non-ferrous device of relatively large area platform
Tool, such as band is platelet, injecting propeller backside or other similar object.It is preferable to use injecting propellers for the technology of the present invention.
Preferably, beaing operation and gently to beat, the squeeze when cell separates, Bu Nengtai
Firmly, otherwise cause to extract because of clasmatosis and fail.Tapetal cell is by gently beaing, squeezing and will hold very much
Easily its hetero-organization separates with anther.
3, cell extraction and collection method:
(1) with the cell mixture in 200 mesh cell sieves filtering large beaker, filter residue is discarded, filtrate is retained.
(2) filtrate is filtered with 600 mesh cell sieves, discards the filtrate containing pollen, washed lower filter residue with distilled water, use again
600 mesh cell sieves filter, and the ingredient in cell sieve is tapetal cell.
(3) tapetal cell in cell sieve is carefully drawn with suction pipe, is collected in small centrifuge tube.
(4) 1000rpm/min, room temperature are centrifuged 5 minutes.
(5) supernatant in small centrifuge tube is discarded with suction pipe, the precipitating for being centrifuged bottom of the tube is that the tapetum that is collected into is thin
Born of the same parents.
Technical principle explanation:
The cellular morphology of plant different tissues is of different sizes, and it is also different to be completely embedded degree.According to the biology of tapetum
Characteristic is learned, the tapetal cell after fixation gently squeezes or taps by mechanicalness, it is easy to its hetero-organization of anther point
It separates out and.Then the big tissue block of anther is separated by filtration away with different meshes cell sieve by several times.Further according to tapetal cell and
Pollen size is different, and tapetal cell is filtered, separated by the cell sieve for choosing suitable size (diameter).Finally by centrifugation
It can be obtained the very high tapetal cell of purity.
The tapetal cell purity is high (being greater than 97%) obtained with investigative technique of the present invention separation, and cellular morphology, structure
Very complete, quality is high.By late protein group studies have shown that the total egg of tapetal cell obtained by this investigative technique
White quality meets proteomics research requirement, and its total protein separation and Extraction rate and total protein quantity are very high.
In short, using investigative technique of the present invention, (half a day) can easily to obtain a large amount of purposes thin within a very short time
Born of the same parents, agents useful for same are common laboratory common agents, and normal-temperature operation does not need specific apparatus, and cell sieve used is cheap,
It conveniently buys, and a whole set of tapetal cell fixes, separate and collects that operating technology is simple, is easy to grasp, operate.
The present invention has following remarkable result:
1, investigative technique of the present invention is compared with paraffin section combination laser microprobe dating isolation technics, and the used time is short, operation side
Just, simple and easy, it can be completed without specialized equipment or professional training personnel.
2, compared with existing tapetal cell isolation technics, the technology of the present invention has used biological immune fixation principle, will
The tapetal cell of particular moment is killed livestock rapidly fixation.Thus, it is possible to obtain the goals research cell of Perfect Time section, thus really
The accuracy for having protected target cell result of study, segment value more valuable for accurate biological study, especially in molecular biology
Advantage is had more in terms of mechanism study.
3, the present invention is compared with existing tapetal cell isolation technics, tapetal cell form, the structural integrity of acquisition
Property good, purity is high (be greater than 97%), quality is high.
4, the present invention is compared with existing tapetal tissue isolation technics, and environmental condition is easy, ambient operation, routine experiment
Room equipment, operating procedure are simple and easy.
5, the tapetal cell obtained with investigative technique of the present invention separation shows this by the detection of subsequent protein group
Tapetal cell total protein quality meets proteomics research requirement, and its total protein separation and Extraction rate with separate the total of acquisition
Protein quantity is very high.
6, application range of the present invention and field are extensive.On the one hand the technology of the present invention is solved and is targeted in botany research field
The test material sampling work of anther tapetum cell is separated and collected, is provided for the orientation of tapetal cell, accurate biological study
Good approach.On the other hand, the technology of the present invention details slightly adjusts and can also extend to higher plant other are histiocytic
It separates and collects and the specific cells targeting separation in the animal even complex tissues such as medicine sample, extracts.In addition to this, originally
The cell that inventive technique obtains can be used not only for the research in terms of proteomics and histology, can also slightly technology adjustment use
The test material of molecular biology research is carried out in extraction of DNA and RNA etc..Therefore, the technology of the present invention is plant or even bioscience
Carry out accurate molecular biology research and provides platform of very easily drawing materials.
Specific embodiment
Technical solution of the present invention is clearer, is readily appreciated that in order to make, and with reference to embodiments, carries out to the present invention
It is further described.Described specific embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
The separation of fructus lycii anther tapetal cell, extraction and collection method suitable for protein science research
1, anther is collected and fixed operation process:
Ice pan is got out on experiment table top, clean glass culture dish is placed on ice pan --- from the branch of lycium chinensis mill for having bud
The target bud for quickly removing certain amount on item with tweezers is placed in glass culture dish --- (it is placed in ice pan on a glass
On) be stripped out anther from bud with dissecting needle, and be immersed in rapidly in the small penicillin bottle for filling fixer --- institute
After having anther to collect, penicillin bottle cap is covered, infiltrates through fixer in anther tissue rapidly sample pumping with syringe
Portion --- room temperature is 3.5-4 hours fixed.
In the technology of the present invention, bud number about 10-100, anther number about 40-400, fixer about 5-15ml, Gu
It fixes time 3.5-4 hours.
Preferably, the fixer component and mass percent are as follows: paraformaldehyde 2%-4%, most preferably 2%；Polyethylene glycol
5%-20%, most preferably 10%；Phosphate buffer molar concentration is 0.1M, glacial acetic acid 0.01-0.1%.
The technology of the present invention fixative optimization formula: paraformaldehyde 2%, polyethylene glycol 10%, 0.1M phosphate buffer, glacial acetic acid
0.05%；PH7.2.
Sample rigid condition: room temperature, sample and fixer volume ratio are 1:20.
Preferably, the set time are as follows: 2-24 hours, most preferably 3.5-4 hours.The selection of specific set time according to
Different plant tissues, cell type and cell extract are suitable for the set time of target cell to determine.Set time is too
Short, then intracellular protein is degraded because not fixed effectively；If the set time is too long, intracellular albumen
Matter is not extracted not come out due to excessively will lead to following protein repairing failure because fixing.
Preferably, the sample air suction process step is necessary operation link for plant tissue, and other biological
Tissue, such as animal or medical tissue sample, this operating procedure does not belong to must content.
2, tapetal cell separation, extraction and collection operating technology
(1) fixer in bottle is sucked out with suction pipe, discarded, the distilled water of change about equivalent, intermediate jog for several times, 10
Distilled water in bottle is discarded with suction pipe after minute, then changes to the distilled water of about equivalent.Repetitive operation 3 times, clean anther.
(2) anther is transferred in small beaker (such as 20ml beaker), a small amount of distilled water (not having anther about) is added to use afterwards
The propeller backside of syringe gently beats anther about 5 minutes repeatedly, separates the tapetal cell in anther tissue,
About 10ml distilled water is added, after gently beaing for several times, then vibration of ultrasonic wave is for several times for several times or in short-term with the concussion of suction pipe pressure-vaccum.
(3) with 80 mesh cell sieve filtration cell mixed liquors, filtrate is placed in compared in large beaker, and residue collection enters previous small burning
In cup, add about 5ml distilled water, touched anther filter residue about 5 minutes with syringe backside again, adds about 10ml distilled water, light
Tapping is beaten for several times, then shakes for several times or in short-term with suction pipe pressure-vaccum that vibration of ultrasonic wave is for several times.
(4) it repeats (3) operating procedure 2 times, filtrate merges with filtrate before, discards filter residue.
(5) with filtrate in 200 mesh cell sieves filtering large beaker, filter residue is discarded.
(6) it is filtered with 600 mesh cell sieves, discarding filtrate, (this step is to filter pollen (microspore).It washes in lower cell sieve
Filter residue, then filtered 1 time with 600 mesh, discard filtrate.In cell sieve is tapetal cell.
(7) tapetal cell in cell sieve is carefully drawn with suction pipe, moves into and is collected in small centrifuge tube.
(8) 1000rpm/min, room temperature are centrifuged 5 minutes.
(9) liquid is discarded supernatant with suction pipe, small centrifuge tube bottom precipitation is the tapetal cell of separation and Extraction.Separation is received
The tapetal cell collected can be placed in refrigerator cold-storage room and store for future use.
As in tapetal cell lock out operation technology described in the technology of the present invention:
The method for separating tapetal cell from anther has: diluted acid, diluted alkaline+mechanical presses method, stripping and slicing+vibration of ultrasonic wave
Method, dissecting needle partition method under stereoscopic anatomical lens are gently beaten, extruding+suction pipe pressure-vaccum concussion method, are gently beaten, extruding+ultrasonic wave
Lash method etc..
It is preferred: gently to beat, extruding+suction pipe pressure-vaccum concussion method, gently beat, extruding+vibration of ultrasonic wave method, more preferably
Gently to beat, extruding+vibration of ultrasonic wave method.
As in tapetal cell extraction operation technology described in the technology of the present invention: being carried out using the combination of different meshes cell sieve
The extraction of tapetal cell.Wherein after repeated screening is tested, 2 operations are filtered preferably through 80 mesh and 200 mesh cell sieves
Step filters out big unwanted tissues agglomerate and small unwanted tissues agglomerate respectively.And it cannot directly carry out 200 mesh cell sieves
Filter operation.According to tapetal cell and Pollen size, tapetal cell is collected in preferably 600 mesh cell sieve separations, is filtered simultaneously
Fall pollen.Changed according to anther difference developmental condition, cell size, it is determined whether increase the cell sieves filtration steps such as 500 mesh.It is early
Stage of development phase only needs 600 mesh filtration steps, and stage of development in later period needs increase by 500 mesh filter operations behind 600 mesh.Separately
Outside, according to target cell and the difference of untargeted cells size, Lai Jinhang cell grit number selection and combination, to obtain mesh
Mark cell.
It is collected in operating technology as tapetal cell described in the technology of the present invention:
It is preferred that room temperature is centrifuged, 200-1000 prm/min is centrifuged 5-10 minutes；Most preferably 1000rpm/min is centrifuged 5 points
Clock；Centrifugal rotational speed cannot be excessively high, and otherwise, tapetal cell is crushed because strength squeezes.Room temperature, low-temperature centrifugation do not influence to tie
Fruit.
Claims (5)
1. be suitable for proteomics research tapetal cell separation and collection method, this method include cell fixation methods and
Cell separation, extraction and collection method, it is characterised in that: its cell fixation methods are as follows: it collects bud and is placed in ice pan vessel, from
Anther is separated in bud, anther is immersed in rapidly in the bottle for containing fixer, and lid lid takes out the bottle for containing fixer
Gas infiltrates through fixer inside anther tissue rapidly, and room temperature condition operation is fixed；The cell separation, extraction and collection method
Are as follows: (1) wash: by fixed anther sample with distilled water displacement washing 3 times, every time 10 minutes, centre, which is shaken, mixes 1-2
It is secondary, washes clean anther；(2) separate cell: A. moves into anther in small beaker, adds the top after a small amount of distilled water with flat spillikin
All anther are lightly beaten 5 minutes in end repeatedly, so that the tapetal cell in anther tissue is detached from anther and spread out；B. again
More than ten milliliters of distilled waters are added, are gently beaten again 5 minutes with the top of flat spillikin；C. mixed with 80 mesh cell sieve filtration cells
Liquid is closed, filtrate is placed in large beaker, and residue collection enters in previous small beaker；D. B and C operating procedure is repeated；E. thin with 80 mesh
Born of the same parents' sieve filters again, and filtrate merges with filtrate before, discards filter residue；F. with the cell in 200 mesh cell sieves filtering large beaker
Mixed liquor discards filter residue, retains filtrate；G. filtrate is filtered with 600 mesh cell sieves, discards the filtrate containing pollen grain, cell sieve
On ingredient be tapetal cell；H. the tapetal cell in cell sieve is carefully drawn with suction pipe, move into and collect into it is small from
In heart pipe；I.1000rpm/min, room temperature is centrifuged 5 minutes.
2. the tapetal cell separation and collection method according to claim 1 suitable for proteomics research, special
Sign is: fixer constituent are as follows: paraformaldehyde, polyethylene glycol, glacial acetic acid, phosphate buffer, fixer concentration of component matter
Measure percentage are as follows: paraformaldehyde 2%-4%, polyethylene glycol 5%-20%, phosphate buffer molar concentration are 0.1M, are adjusted with glacial acetic acid
PH value is 7-7.6.
3. the tapetal cell separation and collection method according to claim 1 suitable for proteomics research, special
Sign is: anther sample is 1:20 with fixer volume ratio.
4. the tapetal cell separation and collection method according to claim 1 suitable for proteomics research, special
Sign is: the set time is 3-4 hours, and cell tissue rigid condition is room temperature condition operation.
5. the tapetal cell separation and collection method according to claim 1 suitable for proteomics research, special
Sign is: fixed sample air extractor is injector for medical purpose.
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| CN105602885A (en) * | 2015-11-18 | 2016-05-25 | 扬州大学 | Method for separating and purifying plant tapetum tissue |
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| CN105602885A (en) * | 2015-11-18 | 2016-05-25 | 扬州大学 | Method for separating and purifying plant tapetum tissue |
Non-Patent Citations (1)
| Title |
|---|
| 宁夏枸杞异型绒毡层发育的超微结构特点;徐青等;《西北植物学报》;20091215;第29卷(第12期);第2452-2463页，参见全文 |
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