CN102115741A - Method and system for classifying and extracting biomacromolecules in situ - Google Patents
Method and system for classifying and extracting biomacromolecules in situ Download PDFInfo
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Abstract
The invention relates to a method and a system for classifying, separating and purifying different proteins in a biological tissue in situ. The system comprises a mixer for preparing an extraction medium, an extraction chamber for extracting a biomacromolecule-containing sample, a detector for performing spectrophotometric detection on the extracted biomacromolecules, and a collector for collecting the extracted biomacromolecules, wherein the mixer is communicated with the extraction chamber through a high-pressure constant-current pump and an extract conveying pipe; the extraction chamber is communicated with the fraction collector through a reversing valve and an extract collecting pipe; the detector is communicated with the extraction chamber through a high-pressure constant-current pump and a reversing valve; and the sample is obtained by cutting the biomacromolecule-containing biological tissue into slices and fixing the slices by using a clamping device. By the method and the system for classifying and extracting the proteins in situ, protein components in all tissues of cytoplasm, cytomembrane and extracellular matrix protein and the like can be quickly and efficiently separated.
Description
Technical field
The invention belongs to the Medical Molecular Biology field, relate to a kind of proteic extracting method and system thereof, relate in particular to a kind of method and system thereof that from biological tissue, different proteins is carried out original position classification separation purification.
Background technology
Basic comprising unit of biological tissue is a cell, and different cells and extracellular composition constitute specific weave construction.The basic material of these structures is organic macromolecules such as nucleic acid, protein, lipid and carbohydrate, and studying these biomacromolecules all has very important meaning to biological phenomena and human health.The direct carrier of various physiological functions and biological phenomena is a protein, will directly illustrate the change mechanism of life under physiology or pathological conditions to the research of protein structure and function; Proteinic existence form and mechanics as problems such as posttranslational modification, protein-protein interaction and protein conformations, must depend on proteinic direct research; Simultaneously, to the complicated active full appreciation of biological phenomena, be unable to do without the research on protein integral body, the dynamic level.
Along with the arriving of genome times afterwards comprehensively, people diversion on protein groups studies.Up-to-date studies show that, than genome research, the difficulty of protein groups research is people's the imagination head and shoulders above.This is determined by the protein self character: 1, proteinic kind is far more than the kind of gene, and same gene can be expressed as multiple proteins, and widely different each other; 2, proteinic abundance alters a great deal, many protein contents that critical function arranged seldom, but the pair cell activity plays an important role; 3, proteinic degraded or renewal speed difference are very big, have the protein degradation of important regulating and controlling function very fast, not easily separated; 4, protein properties difference is very big, with other various biomacromolecules very complex interactions is arranged, comprise water, ion, carbohydrate, lipid, nucleic acid etc., and interactional intensity difference, same protein can be with a plurality of other different types of interactions of molecules; 5, protein can also be modified by variety of way, as phosphorylation, acetylize, methylate, glycosylation etc.; 6, under the different states, the albumen kind and the number change of cell expressing are very big, and for example: only lack certain nutritive substance, cell changes a series of protein expressions possibly.
People studies show that, in-house proteinic kind, quantity, distribution and metabolism have significant difference.Tissue is to be made of cell and extracellular matrix, wherein: 1, in the extracellular, be extracellular matrix, be mainly structural protein, as collagen protein, protein-polysaccharide etc., its kind is limited, and content is big, and metabolism is slow, and is water-soluble low; 2, cytolemma is mainly the various membranins that contain fat-soluble TMD, as acceptor, passage, translocator etc., also comprise the various albumen of extracellular near close cytolemma part in cytolemma and the cell, its function is very important, kind is abundant, the content rareness, two property (transmembrane protein) or water-soluble (near membranin) height, metabolism difference is big; 3, comprise multiple composition in the cell, remove nucleus, outside the membranin in the organoid, be hydrophilic protein, comprise cytoskeleton, various metabolism proteins, signaling pathway protein etc., its kind is many, and content is big; 4, be mainly nucleic acid binding protein in the nucleus.
Protein groups research at be the protein of all kinds in the biological tissue, object is very extensive, comprises simple prokaryotic organism, as intestinal bacteria, to complicated many cells higher organism, as Mammals and people.Although the object of research is very extensive, the basic fundamental means of research are very similar with flow process, are specially:
1, determine to study biological or organize is as cultured cells or the tissue that collects, organ;
2, at the difference of research object, by various means, comprise methods such as physics (as extruding, ultrasonic etc.), the chemistry scale remover of broken cell membrane (various can), broken histocyte, discharge molecule in whole tissues, avoid protein degradation (as adding proteinase inhibitor) simultaneously;
3, from cell pyrolysis liquid, extract albumen, it is a most complicated step, must be according to the difference of albumen kind, select diverse ways, for example: extract according to proteic water-soluble, fat-soluble, the different iso-electric point of different sorts, ionic strength etc., simultaneously avoid proteic loss as far as possible, but the complicated operations process must cause albumen to reclaim the increasing of loss;
4, proteic analysis comprises structure, kind and content, and the progress of this respect is the most obvious, and its technology comprises various electrophoresis, chromatogram, mass spectrometric improvement and combined utilization, the foundation of polypeptide database, storage, retrieval is analyzed etc.
At present, progress along with sample technology of preparing and detection means, protein groups research can be determined height, middle abundance protein kind and the content in culturing cell easily, as various structural protein and metabolism protein, but when the research cellular function plays the low-abundance protein (as epicyte protein) of important regulating and controlling effect and more complicated in-vivo tissue, but lack effective means, this point has the animal tissues of complex construction in research, when comprising tissue, more obvious.
In fact, how to obtain the low-abundance protein of sufficient amount, become a bottleneck of protein groups research.Address this problem two kinds of approach at present: 1, improve and be used to the sample size extracted at first, though like this in the leaching process low-abundance protein have loss, finally can obtain the target protein of sufficient amount, can be for the analysis use.This method is feasible to the tissue that can obtain in a large number, but because many tissues are difficult to a large amount of acquisitions, or itself is just very rare, very precious, makes that this mode is difficult to realize.2, improve the sensitivity that target protein detects, along with the further raising of detection means, the detection of low-abundance protein is also relatively easy; But a problem that faces in the present detection is that the existence of a large amount of high-abundance proteins is very obvious to the background interference that the detection of low-abundance protein forms, and has also increased detection difficulty and burden, has reduced detection efficiency.Improve the extraction efficiency of low-abundance protein selectively, this is a kind of optimal state, can reduce sample size, alleviates the detection burden.But this also is to make progress a slowest step, because intracellular protein ingredient is very complicated, and the proteic method of select target specifically, as antibodies, methods such as marker mark, these methods or of a high price, efficient is low; Can't be applicable to complicated tissue (method as the cytolemma mark can only be applicable to culturing cell).
By the analysis to prior art, the contriver thinks, in present protein extraction process, owing to adopted first broken the mixing, make all protein ingredients mix, separate the method for purifying again, this makes the zone of the relative enrichment of albumen that script exists in tissue disappear.This method has increased follow-up extraction difficulty, has reduced detection efficiency, and the whole extraction efficiency of albumen, particularly low-abundance protein does not only improve, and seriously descends on the contrary.
Summary of the invention
In order to solve the above-mentioned technical problem that exists in the background technology, the invention provides a kind ofly can reach fast, the high efficiency separation endochylema, after birth and extracellular matrix protein etc. all in the tissue original position classification of protein ingredient extract proteic method and systems thereof.
Technical solution of the present invention is: the invention provides a kind of original position classification and extract the method for biomacromolecule, its special character is: said method comprising the steps of:
1) biological tissue that will contain biomacromolecule cuts into slices; The described biological tissue of containing biomacromolecule is epithelium, nervous tissue, muscle tissue, reticular tissue and by the organ of these organizational compositions; Described biomacromolecule is nucleic acid, protein, lipid or polyose; Described protein is plasmosin matter, membrane-associated protein matter, cytoskeletal protein and/or extracellular matrix proteins; The thickness of described section is less than or equal to the diameter that the biological tissue of containing biomacromolecule constitutes cell or target cell;
2) tissue slice is fixed;
3) with extracting medium tissue slice is carried out the biomacromolecule extraction; Described extraction medium comprises basic extracting solution and extracting solution additive; Described basic extracting solution is that nucleic acid extraction damping fluid, protein extraction damping fluid or carbohydrate extract damping fluid; Described extracting solution additive is the composite nucleic acid enzyme, proteinase inhibitor; Described extraction conditions is: 4～200 ℃, and 0～400 normal atmosphere, the flow that extracts medium is 10～1500ml/min.
Above-mentioned steps 3) specific implementation is:
3.1) tissue slice is carried out precooling;
3.2) make and extract medium back and forth by the fixed tissue slice after the precooling;
3.3) judge to adopt spectrophotometric detection method by the extraction medium behind the tissue slice whether extraction biomacromolecule process reaches balance.
Above-mentioned steps 3) be in the environment of fully-closed, to carry out.
Above-mentioned steps 1) also comprise biological tissue's fixing step that section is preceding:
When the biological tissue of containing biomacromolecule is hollow tissue, in the biological tissue specimens of hollow, cut into slices behind the injection molding medium; Described shaping medium is an agarose;
Perhaps, when the biological tissue of containing biomacromolecule is blood tissues, the isolated cells composition is mixed with shaping medium, shaping medium is cut into slices after being shaped, and described blood cell density is 100～10
5/ ul.
Above-mentioned steps 3) extraction step or extraction step are respectively successively adopted in extraction:
Described extraction step respectively is: when biomacromolecule comprises plasmosin matter, membrane-associated protein matter, cytoskeletal protein and extracellular matrix proteins, extract plasmosin matter, membrane-associated protein matter, cytoskeletal protein and/or extracellular matrix proteins respectively;
Described extraction step successively is: when biomacromolecule comprises plasmosin matter, membrane-associated protein matter, cytoskeletal protein and extracellular matrix proteins, extract successively according to the order of plasmosin matter, membrane-associated protein matter, cytoskeletal protein and extracellular matrix proteins.
Above-mentioned extraction conditions is: 4-200 ℃, and the 0-400 normal atmosphere, the flow that extracts medium is 10-1500ml/min, the thickness of described section is less than the target cell diameter.
The system of biomacromolecule is extracted in the classification of a kind of original position, and its special character is: the system that biomacromolecule is extracted in described original position classification comprises the collector that carries out the detector of Spectrophotometric Assays, the biomacromolecule that is extracted is collected to the extraction chamber that extracts mixing tank that medium prepares, the sample that contains biomacromolecule is extracted, to the biomacromolecule that is extracted; Be communicated with by high pressure constant flow pump and extracting solution transfer lime between described mixing tank and the extraction chamber; Be communicated with by reversing valve and extracting solution collection tube between described extraction chamber and the categorised collection device; Described detector and extraction chamber are communicated with by high pressure constant flow pump and reversing valve; Described sample is to carry out the fixed sample after the biological tissue of containing biomacromolecule is cut into slices and with clamping device.
Above-mentioned clamping device comprises overcoat and interior cover, and described overcoat comprises overcoat top and sleeve side wall, cover bottom and sidewall of the inside race in described interior cover comprises; Described sidewall of the inside race is embedded in the sleeve side wall; Described overcoat top and interior cover bottom are that filter membrane is for being not less than 2000-3000 purpose filter screen.
The above mixing tank also comprises the filtration defoaming device that is driven by vacuum pump, and described filtration defoaming device is communicated with mixing tank.
Above-mentioned mixing tank, extraction chamber and collector are in the closed environment.
Advantage of the present invention is: method and device thereof that plasmosin, membrane-associated protein and extracellular matrix protein are extracted in original position classification provided by the present invention are to utilize different albumen to have the characteristic of relative rich region and the physical strength that tissue itself has in tissue, the structural performance that is had according to tissue itself, different protein ingredients are carried out original position to be separated, can reach quick, high efficiency separation endochylema, after birth and extracellular matrix protein.The present invention can be easy to combine with existing various technology, is adapted to comprise the extensive studies object of various animals and plants, and leaching process carries out in the system of fully-closed, can reduce proteic loss to greatest extent.Be suitable for organizing the analysis of whole protein spectrum or specific protein spectrum, its process can realize microminiaturization, automatization, digitizing easily simultaneously.
Description of drawings
Fig. 1 is the preferred embodiment synoptic diagram of system provided by the present invention;
Fig. 2 is the structure cut-away diagram of clamping device provided by the present invention;
Fig. 3 is the structural representation of clamping device provided by the invention.
Embodiment
The invention provides the classification of a kind of original position and extract the method for biomacromolecule, this method can be applied to the separation and Extraction of biomacromolecule, nucleic acid especially, protein and polyose etc.The biological tissue of containing biomacromolecule is epithelium, nervous tissue, muscle tissue, reticular tissue and by the organ (neural system (brain, spinal cord and nerve) of these organizational compositions; The recycle system (heart and blood vessel), respiratory system (lungs, tracheae); Digestive tract (stomach, intestines, liver, pancreas); Urinary system (kidney and urinary tract, bladder); Endocrine system; Blood system; Protein is plasmosin matter, membrane-associated protein matter, cytoskeletal protein and/or extracellular matrix proteins.
What the present invention is directed to is the shortcoming of existing protein extraction, and topmost is exactly by extracting medium by tissue slice, can dividing kind to extract stage by stage like this.Tissue slice, fixing, extraction is exactly the core of present method.In theory, any tissue that can be cut into section can be used present method.And cut into slices, be the fundamental method of modern biomedical research.
In present biomedical research, tissue slice is research organization and cyto-architectural the most basic, indispensable technology.Because fine structures such as cell can not be finding of naked eye, must be by microscope, therefore, these tissue slicies purposes at ordinary times is the structure of tissues observed and cell, with location and the mutual relationship of the various biomacromolecules of painted methods analyst.Existing histology, the technical foundation of subjects such as cytology are exactly tissue slice and tissue staining.Existing section is not applied to the research of analysis of protein extraction aspect, and protein extraction all is with tissue block or culturing cell homogenate, is with all biomacromolecules, comprises that various albumen all mix earlier, progressively separate by different methods again.
The purpose of cutting into slices in present method makes in-house each cell damaged face all occur, thereby makes that extracting medium can pass section, extracts albumen, reduces the extraction medium flow simultaneously and crosses the resistance of section.
Slice thickness in present method depends on will extract proteic target cell size, and on-fixed.The purpose of section is to make all cells all be switched to, and damaged face occurs.When cell varies in size, also can adopt other thickness, when big (can reach the 20-30 micron sometimes), slice thickness also can be increased to the 20-30 micron as cell dia.Usually selecting 4 microns, is all can switch to because most eukaryotic diameters all greater than 4 microns, can guarantee to be close to whole cells like this.
Section can utilize existing any present method purpose slicing machine that meets, for example the most frequently used come card slicing machine.The requirement of slicing machine only is the section that can cut out desired thickness, can guarantee simultaneously to organize relative complete (cyrogenic equipment etc. for example being arranged, because can reduce the Decomposition of the interior oneself protease of tissue like this) with cell.
For convenience of explanation, the biological tissue of containing biomacromolecule that adopts during the invention process is respectively cerebral tissue, muscle tissue, blood tissues, lungs tissue, liver organization or renal tissue; The present invention extracts protein to these different biological tissues, has formed different embodiment, and specific embodiment is as described below.
Embodiment 1, and biological tissue is a cerebral tissue:
1, people's clinical operation cerebral tissue sample, or laboratory animal cerebral tissue sample can be flesh tissue or tissue block after treatment;
But 2, flesh tissue direct slicing is if fixing organization blocks then needs through dewaxing steps such as degraded fixing agent after section.For preventing tissue degradation, sectioning is carried out at low temperatures usually.Conventional brain tissue slice is cut into the thick section of 4um with freezing microtome, most brain cells can be switched to like this, finishes the shattering process of all cells;
3, the structure rigidity and the intensity that have of brain tissue slice itself mainly is made of extracellular matrix and intracellular cytoskeleton.Certain thickness tissue slice can support for rigid material (as filter membrane, filter screen etc.); These support and constitute the sample folder.The purpose of sample folder is: tissue samples, Bao tissue samples particularly, be very soft, fragile and be difficult to carry out (imagine and shakeout the very thin sliced meat that only have 1mm thick) of operation, and tissue slice can be very big (for example section of full brain), at this moment to go to wash away repeatedly the section that fetters in other words that is not fixed be unpractical with extracting medium (as extracting solution etc.) fluid.The purpose of sample folder allows tissue slice open and flat exactly, allows to extract medium simultaneously and passes through, and interacts with section.Certainly, also can fix, might not fix with sample folder provided by the present invention with other modes, its purpose is exactly that tissue slice is open and flat, allow to extract medium simultaneously and pass through, interact, to the extraction that is well on of the biomacromolecule that will extract with section.
4, protein extract accounts for the useful space 60% in the extraction chamber after input terminus imports, under 2 normal atmosphere air-operated drive, flow through the tissue slice that is positioned at the sample folder, flow direction can come and go control, make extracting solution flow through section repeatedly, the sample extraction degree is by detecting a mouthful evaluation, after reaching requirement, extracting solution is exported from delivery port, is collected by the categorised collection device.
5, the protein extraction process is plasmosin, membrane-associated protein, cytoskeletal protein and extracellular matrix successively usually.The extracting parameter of various protein ingredients can be according to the adjustment of experiment needs.The leaching process of range protein composition is as follows in cerebral tissue: 1) plasmosin; 2) membrane-associated protein; 3) cytoskeletal protein; 4) extracellular matrix protein.The sequential process here can guarantee to realize the purpose of the classification original position isolated protein that present method proposes.And present separation method is earlier with all weave constructions and cytoclasis, allows the stripping as much as possible of all macromole, mixes, and the various compositions that will mix by the whole bag of tricks are separated respectively again.Because when beginning to extract, the complexity of sample does not only reduce, raise on the contrary, this with regard to artificial increase difficulty.
Cerebral tissue albumen classification leaching process parameter:
Tissue is prepared: 1) fresh mouse brain tissue, immerse freezing sclerosis in the liquid nitrogen; 2) will freeze good cerebral tissue piece and put into cryostat microtome, be cut into the thick section of 4um; 3) in cryostat, sample is put in section and pressed from both sides (constituting) by 3000 order stainless (steel) wires; 4) in cryostat, sample is folded up into sample extraction chamber (stainless steel tube is made) the sealing extraction chamber.The step of tissue extraction is: 1) insert precooling suppressor proteins extracting solution (endochylema extracting solution) through the extracting solution input aperture, extracting solution enter sample extraction chamber flushing sample (4 degrees centigrade, normal pressure, 10ml/min, parameter can also be temperature: 0-200 degree centigrade; Pressure: 0-400 normal atmosphere; Flow: 10-1500ml/min.); 2) high pressure constant flow pump drives extracting solution back and forth by the extraction chamber, determines that through the pattern detection chamber leaching process has arrived equilibrium state (employing uv detection method); 3) from extracting solution delivery port output saturation extract, through categorised collection device income suppressor proteins collector; 4) to the indoor input endochylema of sample extraction extracting solution, the same terms flushing sample is collected sample albumen.Repeat to be extracted into promising result (ultraviolet detection shows that protein concentration no longer raises).
The purpose of precooling: one of albumen research and analysis important aspect be exactly to extract abundant complete protein molecule.But contain a large amount of various proteolytic enzyme in histocyte, after disorganization, proteolytic enzyme meeting protein degradation molecule so just can't obtain complete protein molecular.So in the protein extraction process, must suppress the activity of these enzymes, precooling is exactly by cooling, reduces a kind of means of these protease activities.
Adopt uv detection method to judge that it is that protein concentration no longer raises that leaching process reaches the equilibrated standard.Because when the extraction medium flows through the sample section repeatedly, constantly have protein dissolution to come out, when reaching capacity, the protein concentration in the extracting solution no longer raises, and shows that this extraction has reached balance, and it is nonsensical to prolong extraction time again.
The multiple meaning: step 4) is the repetition to step 1), and generally through 1,2,3 steps just can be extracted plasmosin, and step 4) is in order to extract more albumen, to make extraction effect better.If extraction effect is satisfied, can save repeating step.
5, repeat to use same step, change the extracting solution composition, extract membrane-associated protein respectively, cytoskeletal protein and extracellular matrix protein.
6, remaining component, after the said extracted process, the section of sample folder inner tissue still has remaining component, can according to circumstances preserve and wait until with post analysis.
7, in whole leaching process, proteic outflow efficient and discharge in the tissue are with the section extracting liquid volume and the number of times positive correlation of flowing through of flowing through.When selecting different extracting solutions, can separate respectively and extract endochylema, after birth, cytoskeleton and extracellular matrix protein.Because adopt the mode of cut mechanically, this method can be applicable to various physiological and pathological processes, the research and analysis of simple and complex organization's cell all can be made into section because these are organized.
The basis extracting solution is a protein extraction damping fluid reagent (as Tris-HCl, phosphoric acid, carbonic acid etc.) commonly used in the Biochemical Research, and concrete parameter can be determined by experimenter oneself according to the experiment demand, also can be disposed voluntarily by it.Container is stored by glass or other chemical container.Owing to there are not unified all proteic extracting solutions that are applicable to, the implementer must select suitable extracting solution according to self needs and tissue characteristics.The extracting solution additive is the conjugated protein enzyme inhibitors, and this extracting solution additive can be prepared voluntarily, also can buy commercial prod from each big biochemical reagents company), before the extraction, it is directly added basic extracting solution.
Embodiment 2, and the biological tissue that chooses is the lungs tissues:
Because lungs are hollow tissues, in order to guarantee the effect of cutting into slices, in lung tissue sample, injects the agarose of thawing by tracheae, after agarose solidifies, promptly can carry out by the slicing mode identical with embodiment 1.
Because blood cell is a suspension cell, does not form the conventional organization structure.In order to extract the albumen of blood cell, with blood cell with the plasma component centrifugation after, mix cell density 10 with the agarose that melts
5/ ul after agarose solidifies, promptly can carry out by the slicing mode identical with embodiment 1.
Embodiment 4, and the biological tissue that chooses is other organs and tissue:
Liver, kidney, the mode of muscle etc. and above-mentioned similar is seen embodiment 1.
Carry out albumen for different tissues, when especially plasmosin, membrane-associated protein, cytoskeletal protein and extracellular matrix protein extract, can extract respectively, also can extract successively.
When extracting successively, what at first extract is plasmosin, and just process one:
The plasmosin extracting solution is: Tris-HCl (pH7.5) 50mM, and NaCl 50mM, proteolytic enzyme composite inhibitor (EDM company) proteolytic enzyme composite inhibitor is commercial preparation, uses to get final product according to product description, also can be according to the needs increase and decrease of oneself.
Process two: membrane-associated protein separates:
Tissue is prepared: with the endochylema albumen sepn.
Tissue extraction: tissue samples is the samples remaining of extracting behind the plasmosin 1) insert precooling epicyte protein extracting solution (after birth extracting solution) through the extracting solution input aperture, extracting solution enter sample extraction chamber flushing sample (4 degrees centigrade, normal pressure, 10ml/min); 2) high pressure constant flow pump drives extracting solution back and forth by the extraction chamber, determines that through the pattern detection chamber leaching process has arrived equilibrium state (employing uv detection method); 3) from extracting solution delivery port output saturation extract, through categorised collection device income epicyte protein collector; 4) to the indoor input after birth of sample extraction extracting solution, the same terms flushing sample is collected sample albumen.Repeat to extract until promising result (ultraviolet detection shows that protein concentration no longer raises).Membrane-associated protein extracting solution: the subcellular fractionation kit fraction of after birth extracting solution Calbiochem company 2, proteolytic enzyme composite inhibitor (EDM company).
Process three: cytoskeletal protein separates:
Tissue is prepared: with the endochylema albumen sepn.
Tissue extraction: tissue samples was for extracting plasmosin, residue sample 1 behind the membrane-associated protein) inserts precooling cytoskeletal protein extracting solution (cytoskeletal protein extracting solution) through the extracting solution input aperture, extracting solution enter sample extraction chamber flushing sample (4 degrees centigrade, normal pressure, 10ml/min); 2) high pressure constant flow pump drives extracting solution back and forth by the extraction chamber, determines that through the pattern detection chamber leaching process has arrived equilibrium state (employing uv detection method); 3) from extracting solution delivery port output saturation extract, through categorised collection device income cytoskeletal protein collector; 4) to the indoor input cytoskeletal protein of sample extraction extracting solution, the same terms flushing sample is collected sample albumen.Repeat to extract until promising result (ultraviolet detection shows that protein concentration no longer raises).Cytoskeletal protein extracting solution: the subcellular fractionation kit fraction of Calbiochem company 4, proteolytic enzyme composite inhibitor (EDM company)
Process four: extracellular matrix protein separates:
Tissue is prepared: with the endochylema albumen sepn.
Tissue extraction: tissue samples was for extracting plasmosin, membrane-associated protein, residue sample behind the cytoskeletal protein: 1) insert precooling extracellular matrix protein extracting solution (extracellular matrix protein extracting solution) through the extracting solution input aperture, extracting solution enters (4 degrees centigrade in sample extraction chamber flushing sample, normal pressure, 10ml/min); 2) high pressure constant flow pump drives extracting solution back and forth by the extraction chamber, determines that through the pattern detection chamber leaching process has arrived equilibrium state (employing uv detection method); 3) from extracting solution delivery port output saturation extract, through categorised collection device income extracellular matrix protein collector; 4) to the indoor input extracellular matrix protein of sample extraction extracting solution, the same terms flushing sample is collected sample albumen.Repeat to extract until promising result (ultraviolet detection shows that protein concentration no longer raises).5) open the extraction chamber, take out the sample folder, the sample folder is gone up remaining albumen collect, put into remaining composition collector.Extracellular matrix protein extracting solution: extracellular matrix extracting solution Invitrogen company's T RIzol reagent, chloroform (Sigma company).
Referring to Fig. 1, the present invention also provides a kind of original position classification to extract the system of biomacromolecule, and this system comprises: tissue samples preparation and slicing device; Section stationary installation (filter membrane and filter screen); The sample extraction chamber; Extract medium; Detection control apparatus; Extract medium transport, sample collection device; Device for analyzing samples; Take-off equipment as a result.Wherein, the tissue slice unit, tissue samples can be cut into the section of different thickness, the thickness of section depends on the purpose of experiment, be generally less than or equal to organize the size of internal object cell, guarantee that most cells are all switched to, have multiple mode to keep the albumen in the sample not to be degraded in the slicing device simultaneously, comprise quick-frozen, low temperature etc.
Referring to Fig. 2 and Fig. 3, the invention provides a kind of clamping device that contains the biomacromolecule section that is used for fixing, this clamping device comprises overcoat 1 and the interior cover 2 that is connected with overcoat 1; Overcoat 1 is with after interior cover 2 is connected, and overcoat 1 and interior cover 2 constitute one and be used to deposit the lock chamber that contains the biomacromolecule section.Overcoat 1 is connected with interior cover 2 with regard to realizing fully and carries out clamping to containing biomacromolecule section.In use, will contain the lock chamber that the section of biomacromolecule places overcoat 1 and interior cover 2 to be constituted, convenient fixing or clamping, if follow-up needs are arranged, can also shift to an earlier date or add afterwards some subsequent analysis liquid in this lock chamber, for example protein extract etc. all is good selection.
As preferably, the overcoat 1 of this clamping device provided by the present invention also comprises overcoat top 3 and the outward extending sleeve side wall 6 from the edge at overcoat top 3; Sleeve side wall 6 and interior cover 2 link together, and overcoat top 3, sleeve side wall 6 and interior cover 2 form one and be used to deposit the lock chamber that contains the biomacromolecule section.In use, be the same with foregoing use-pattern.
Above embodiment in use, for more convenient in follow-up use, at overcoat top 3 and/or interior cover 2 also be provided with filter element, this filter element can be that filter membrane or filter screen or the functional materials that plays a role in filtering constitute, when the filtration parts were filter screen, filter screen can be to be not less than 2000-3000 purpose filter screen.
No matter be the implementation of which kind of embodiment, overcoat 1 and interior cover 2 are clampings or are threaded.
When overcoat 1 and interior cover 2 are clamping, sleeve side wall 6 inside are provided with the projection 5 that is used for cover 2 in the clamping, and this projection 5 can be one or more, are using a plurality of protruding 5 o'clock, be undoubtedly preferably, to form a built-in bulge loop also be good selection if be cascaded a plurality of protruding 5.
When overcoat 1 and interior cover 2 were clamping, interior cover 2 edges were provided with the projection that is used for clamping sleeve side wall 6.Projection on interior cover 2 edges is one or more.
As a preferred embodiment, the clamping device that is used for fixing biomacromolecule section provided by the present invention is made of two threaded stainless steel tubes, and the bottom is with the fixing stainless steel filtering net (3000 order) of strong adhesive.Stainless steel tube one inwall has screw thread, is equivalent to overcoat 1; Stainless steel tube two outer walls have screw thread, cover 2 in being equivalent to.After by screw thread steel pipe two being screwed in steel pipe one, distance is 0.1mm between the both sides stainless (steel) wire of steel pipe bottom, constitutes the section put area.The big I of sample folder is divided into the model of different diameter according to the difference of sample section.
The sample extraction chamber is that stainless steel tube constitutes, and it is draw-in groove that steel pipe 1 dress sample presss from both sides an end, and the sample folder can snap in the draw-in groove internal fixing, has the screw thread screwing hermetic to constitute the sample extraction chamber between two steel pipes, lock chamber just, and the two sections sealings in extraction chamber have interface to insert extracting solution.The length overall 1cm of extraction chamber, sample folder each 0.5cm of both sides.The purpose of extraction chamber has provided extracts medium with cut into slices interactional place of the tissue samples in the sample folder.Other material preparation sample extraction chamber also is fine, as long as can satisfy the demand, and does not influence subsequent analysis.Sample folder and extraction chamber adopt stainless steel to make because preliminary experiment is to use them to finish, and the processing of phase commute.Use other material from now on, as glass, pottery also can when plastics etc. are made sample extraction.
This clamping device is mainly the upholder that rigidity has filtering function, and with in-house composition with extract medium and do not react, the macromole of not degrading can tolerate the various conditions of leaching process.Be divided into upper and lower surface, respectively the both sides of fixed tissue slices.The preparation material must meet above-mentioned various condition.The present invention screws in overcoats 1 by screw thread with interior cover 2 in use, after screwing, forms the lock chamber of sealing between overcoat spacer screen and interior cover top net, and tissue slice is put into wherein, and extracting solution flows through tissue slice in the sample room by spacer screen top net, finishes leaching process.
The sample extraction chamber, just the sample extraction module mainly is to interact for extracting medium and tissue slice, makes the target molecule in the tissue slice dissolve in the place of extracting medium, condition is airtight, can tolerate multiple extraction conditions, comprises acid, alkali, temperature, variations such as pressure.Indeformable, do not reveal.
Extracting medium, is to make the target molecule in the tissue slice dissolve in fluid wherein and promote this various materials that dissolve in process, extracts medium and tissue slice and interacts in that sample extraction is indoor, finishes the extraction of target molecule, sepn process.Extract medium and comprise gas, liquid etc.Require not destroy extraction element, meet the extraction requirement.
Detector can be monitored the device of leaching process terms and conditions and parameter, comprises whether the indoor a certain leaching process of sample extraction is finished, if finish, will extract medium output, import next round and extract medium and parameter, and the carrying out of control flow, make leaching process carry out smoothly.The abundance of target molecule detects in the output medium, further purifies etc.
Extract the conveying of medium, applicator mainly is that a kind of power set drives extraction medium flowing in sample room and whole extraction element, adjusts flow direction.Sample collection device is according to the different media in the sample extraction process, collects the device that flows out medium respectively at heterogeneity, and the sample of collection is through having the cellular constituent of special properties after the enrichment.
Mixing tank comprises the filtering net of removing impurity and the vacuum environment of removing bubble, all can be considered reagent and purifies.When agents useful for same is to be pre-mixed, this part can omit.Can directly insert input sample extraction device with extracting medium.This part is to consider that full-automatic regulation and control are required in the application in future, is about to different sample extraction media and allocates automatically by control device and parameter.
Collector can be to artificially collect or the automatically collecting mode.
The present invention in use, the function of mixing tank is before extraction, with basic extracting solution and extracting solution added ingredients thorough mixing.The conveying of basis extracting solution both can also can be sent to mixing tank by transferpump by action of gravity, simply can be with basic extracting solution itself as mixing tank.Bubble is removed in the effect of vacuum pump from extracting solution, prevent that bubble from blocking the extraction efficiency in transport pipe and the reduction extraction chamber.De-bubble is to utilize at a certain temperature, and the solubleness of gas in liquid with the principle that air pressure reduces, is made a kind of vacuum environment, but makes the gas solubility in the liquid descend, thereby removes gas in the liquid.Filtration is with the possible particulate leaching in the extracting solution by filter membrane.Since protein extraction mostly in the physiological temp scope, mild condition, keeping the simple method of extraction chamber's temperature is that ice bath or water-heater are put in the sample extraction chamber, complicated method is to add a heat-insulating airtight sleeve pipe in the sample extraction chamber.The junction is airtight with screw thread or silicone tube folder.Detector is commercial ultraviolet protein nucleic acid detector, can detect albumen and nucleic acid component.The categorised collection device both can be full automatic commercialization instrument, also can be manual fully, after having a kind of new protein ingredient to be extracted, place a new receiving flask to the extracting solution delivery port, and collect new composition and get final product.
Claims (10)
1. the method for biomacromolecule is extracted in an original position classification, it is characterized in that: said method comprising the steps of:
1) biological tissue that will contain biomacromolecule cuts into slices; The described biological tissue of containing biomacromolecule is epithelium, nervous tissue, muscle tissue, reticular tissue and by the organ of these organizational compositions; Described biomacromolecule is nucleic acid, protein, lipid or polyose; Described protein is plasmosin matter, membrane-associated protein matter, cytoskeletal protein and/or extracellular matrix proteins; The thickness of described section is less than or equal to the diameter that the biological tissue of containing biomacromolecule constitutes cell or target cell;
2) tissue slice is fixed;
3) with extracting medium tissue slice is carried out the biomacromolecule extraction; Described extraction medium comprises basic extracting solution and extracting solution additive; Described basic extracting solution is that nucleic acid extraction damping fluid, protein extraction damping fluid or carbohydrate extract damping fluid; Described extracting solution additive is the composite nucleic acid enzyme, proteinase inhibitor; Described extraction conditions is: 4～200 ℃, and 0～400 normal atmosphere, the flow that extracts medium is 10～1500ml/min.
2. the method for biomacromolecule is extracted in original position classification according to claim 1, and it is characterized in that: the specific implementation of described step 3) is:
3.1) tissue slice is carried out precooling;
3.2) make and extract medium back and forth by the fixed tissue slice after the precooling;
3.3) judge to adopt spectrophotometric detection method by the extraction medium behind the tissue slice whether extraction biomacromolecule process reaches balance.
3. the method for biomacromolecule is extracted in original position classification according to claim 2, and it is characterized in that: described step 3) is to carry out in the environment of fully-closed.
4. extract the method for biomacromolecule according to claim 1 or 2 or 3 described original position classification, it is characterized in that: described step 1) also comprises the biological tissue's fixing step before the section:
When the biological tissue of containing biomacromolecule is hollow tissue, in the biological tissue specimens of hollow, cut into slices behind the injection molding medium; Described shaping medium is an agarose;
Perhaps, when the biological tissue of containing biomacromolecule is blood tissues, the isolated cells composition is mixed with shaping medium, shaping medium is cut into slices after being shaped, and described blood cell density is 100～10
5/ ul.
5. the method for biomacromolecule is extracted in original position classification according to claim 4, and it is characterized in that: extraction step or extraction step are respectively successively adopted in the extraction of described step 3):
Described extraction step respectively is: when biomacromolecule comprises plasmosin matter, membrane-associated protein matter, cytoskeletal protein and extracellular matrix proteins, extract plasmosin matter, membrane-associated protein matter, cytoskeletal protein and/or extracellular matrix proteins respectively;
Described extraction step successively is: when biomacromolecule comprises plasmosin matter, membrane-associated protein matter, cytoskeletal protein and extracellular matrix proteins, extract successively according to the order of plasmosin matter, membrane-associated protein matter, cytoskeletal protein and extracellular matrix proteins.
6. the method for biomacromolecule is extracted in original position classification according to claim 4, it is characterized in that: described extraction conditions is: 4-200 ℃, the 0-400 normal atmosphere, the flow that extracts medium is 10-1500ml/min, the thickness of described section is less than the target cell diameter.
7. the system of biomacromolecule is extracted in original position classification, it is characterized in that: the system that biomacromolecule is extracted in described original position classification comprises the collector that carries out the detector of Spectrophotometric Assays, the biomacromolecule that is extracted is collected to the extraction chamber that extracts mixing tank that medium prepares, the sample that contains biomacromolecule is extracted, to the biomacromolecule that is extracted; Be communicated with by high pressure constant flow pump and extracting solution transfer lime between described mixing tank and the extraction chamber; Be communicated with by reversing valve and extracting solution collection tube between described extraction chamber and the categorised collection device; Described detector and extraction chamber are communicated with by high pressure constant flow pump and reversing valve; Described sample is to carry out the fixed sample after the biological tissue of containing biomacromolecule is cut into slices and with clamping device.
8. the system of biomacromolecule is extracted in original position classification according to claim 7, and it is characterized in that: described clamping device comprises overcoat and interior cover, and described overcoat comprises overcoat top and sleeve side wall, cover bottom and sidewall of the inside race in described interior cover comprises; Described sidewall of the inside race is embedded in the sleeve side wall; Described overcoat top and interior cover bottom are that filter membrane is for being not less than 2000-3000 purpose filter screen.
9. extract the system of biomacromolecule according to claim 7 or 8 described original position classification, it is characterized in that: described mixing tank also comprises the filtration defoaming device that is driven by vacuum pump, and described filtration defoaming device is communicated with mixing tank.
According to Claim 8 or the classification of 9 described original positions extract the system of biomacromolecules, it is characterized in that: described mixing tank, extraction chamber and collector are in the closed environment.
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| CN113834869A (en) * | 2016-09-02 | 2021-12-24 | 得克萨斯大学体系董事会 | Collection probes and methods of use thereof |
| CN114431197A (en) * | 2022-01-20 | 2022-05-06 | 南方海洋科学与工程广东省实验室(广州) | Marine benthos in-situ fixing system and method |
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| CN1414106A (en) * | 2001-10-24 | 2003-04-30 | 北京大学 | Method of clone gene and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107567637A (en) * | 2015-04-30 | 2018-01-09 | 皇家飞利浦有限公司 | Brain tissue is classified |
| CN107567637B (en) * | 2015-04-30 | 2022-01-25 | 皇家飞利浦有限公司 | Brain tissue classification |
| CN113834869A (en) * | 2016-09-02 | 2021-12-24 | 得克萨斯大学体系董事会 | Collection probes and methods of use thereof |
| CN114431197A (en) * | 2022-01-20 | 2022-05-06 | 南方海洋科学与工程广东省实验室(广州) | Marine benthos in-situ fixing system and method |
| CN114431197B (en) * | 2022-01-20 | 2022-11-04 | 南方海洋科学与工程广东省实验室(广州) | Marine benthos in-situ fixing system and method |
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