CN109988745A - A kind of extracting method of nucleus - Google Patents

A kind of extracting method of nucleus Download PDF

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Publication number
CN109988745A
CN109988745A CN201810004997.6A CN201810004997A CN109988745A CN 109988745 A CN109988745 A CN 109988745A CN 201810004997 A CN201810004997 A CN 201810004997A CN 109988745 A CN109988745 A CN 109988745A
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Prior art keywords
nucleus
core
cell
cardiac
vitro
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Chinese (zh)
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张雪梅
王文伟
祁晓燕
张静
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Fudan University
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Fudan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Abstract

The invention belongs to field of biomedicine technology, it is related to a kind of method of nucleus extraction, the method that cytosol, film and nuclear leve are divided is separated especially from vitro dog ventricular muscles and isolated dog ventricular cardiac myocytes, the method of the present invention includes steps: Step 1: in vitro dog cardiac muscle cell is obtained, Step 2: subcellular is fractionated.This method helps to get largely the people's functional cell core that can be used for basic research and clinical application；It can safely and effectively realize the screening and enrichment to specific type human cell, obtained cell is made to have better biological safety；The biological characteristics that the present invention comprehensively has detected separated nucleus are sought peace function, provide reliable data for the research and application of nucleus separation, for other novel cells nucleus extraction assess provide it is a set of can method system for reference.

Description

A kind of extracting method of nucleus
Technical field
The invention belongs to field of biomedicine technology, are related to the method for nucleus extraction, and in particular to a kind of from vitro reality The method for separating and extracting nucleus is tested in animal hearts cardiac muscle and ventricular muscle cell sample.
Background technique
Prior art discloses the duplication in nuclear membrane including genome and responsible DNA and transcription and nascent RNA are mature Molecular mechanism.Some researches show that be attributed to the fact that nuclear membrane for more and more functions, including pass through receptor and its agent, ion Channel and ionic pump and transporter separation DNA in nuclear membrane itself.This field knows, although nucleus is in structure and function It is critically important on energy, but due to challenge relevant to internal accessibility, complete separating nucleus is simultaneously completely pure from heart cell Change to allow the relevant knowledge of in vitro study etc. still seldom.
Studies have shown that and most of mammalian cells are conversely, because core tuberculosis during fetal growth, cardiac muscle cell In there are non-pathology multinucleation, core is closed in dual double-layer of lipoid, is provided and is selectively entered carefully by nuclear Pore Complex The nucleus nuclear membrane of cytoplasm, the platform that can also provide additional endocrine signal process control gene expression dose. Nuclear membrane itself includes outer nuclear membrane and interior nuclear membrane, is separated by the core week space of 40-50nm, core and space between cells are by nuclear pore complex It is covered, the inner surface of interior nuclear membrane is lined with the core intermediate inheritance structural network formed by lamin, in outer nuclear membrane and periphery Matter net is connected and contains there are many protein common with periphery endoplasmic reticulum, and in contrast, interior nuclear membrane contains special with nucleus Many different integrated membrane protein, with chromatin have direct interaction.Nearest research experiment evidence shows to include Ah Piece sample substance, adrenergic, Endothelin and angiotensin receptor many g protein coupled receptors be positioned at nuclear membrane and adjust Gene expression.However, being confined to the molecular pharmacology of heart nuclear membrane receptor and physiologic function is still seldom exploited, this is very big It is attributed to that accessibility is relevant in vivo challenges and whether survive after separating with the receptor in degree, with high purity is complete Core is from heart cell to external research.
Status based on the prior art, present inventor is quasi- to provide a kind of method of nucleus extraction, and in particular to A method of nucleus is separated and extracted from vitro experimental animal cardiac myocytes and ventricular muscle cell sample.
Summary of the invention
It is an object of the invention to the statuses based on the prior art, provide a kind of method of nucleus extraction, and in particular to A method of nucleus is separated and extracted from vitro experimental animal cardiac myocytes and ventricular muscle cell sample.
The invention proposes separate from from vitro experimental animal cardiac muscular tissue and isolated adult ventricular cardiac myocytes The technical solution of complete core, the method for the present invention is based on plasma membrane for the part permeability and difference of lutern and smudge cells With discontinuous Sucrose density centrifugation, the method for the quick separating of the cytosols such as non-nuclear membrane and nucleus is provided.
In this method, discloses and separated carefully from vitro experimental animal dog ventricular muscles and isolated dog ventricular cardiac myocytes The method (as shown in table 1) that cell lysis matter, film and nuclear leve are divided；Since nuclear membrane is cholesterol-free, integrality is not by Radix Astragali extract for treating Influence, in order to separate core complete and with enzymatic activity, this method is selectively mutual with sterol using infiltration plasma membrane separation The cardiac glycoside and cardigin of effect after permeabilization, using Dounce homogenizer, then pass through differential sucrose density gradient centrifugation (as shown in Figure 1) discharges nucleus by machinery smashing from remaining cell body, and resulting core enriching section is suitable for having Close functional study.
Table 1: the size and density of heart subcellular organelle
Specifically, a kind of extracting method of nucleus of the invention comprising step:
Step 1: in vitro dog cardiac muscle cell is obtained,
Step 2: subcellular is fractionated.
Include step by step in step one of the invention:
1. preparing oxygenatedchemicals buffer and in relation to instrument and device for casting；
2. conventional treatment experimental animal obtains isolated experiment animal hearts；
3. in vitro experimental animal heart perfusion perfusion and digestion, obtain cardiac muscle cell.
It is all in step two of the invention to carry out on ice or under 4 degree step by step, including step by step:
1. in vitro experimental animal cardiac muscular tissue and cardiac muscle cell's homogenization；
2. tissue and cell homogenates differential centrifugation obtain the thick core and film and cytoplasm of separation；
3. discontinuous sucrose density gradient centrifugation is centrifuged again after obtaining the thick core beds of precipitation, obtains the core of separation and show thin Long almond-shaped or circle, and to DNA stained positive；
It is all in the step 2 to carry out on ice or under 4 degree step by step.
In the present invention, isolated core is further characterized by immunofluorescence or immunoblotting.
In the present invention, the material and instrument used includes:
When cardiac muscle cell separates:
1. all programs must use ultrapure (1 type) water；
2. at room temperature, 2mM calcium ion+Tyrode solution: 136mM NaCl, 5.4mM KCl, 1mM MgCl2·6H2O, 2mM CaCl2, 0.33mM NaH2PO4·H2O, 5mM HEPES, 10mM glucose, pH7.4；
3. room temperature not calcium ions+Tyrode solution: 136mM NaCl, 5.4mM KCl, 1mM MgCl2·6H2O, 0.33mM NaH2PO4·H2O, 5mM HEPES, 10mM glucose, pH7.4；
4. collagenase solution: not calcium ions+Tyrode solution in 100U/mL clostridiopetidase A II type, 0.1% cow's serum Albumin (BSA)；
5. at room temperature, phosphate buffered saline (PBS) (PBS): 137mM NaCl, 2.7mM KCl, 4.2mM NaH2PO4· H2O, 1.8mM KH2PO4, pH7.4.
For isolated film, when cytosol and nuclear fraction:
1. Solubilization buffer: 300mM sucrose, 60mM KCl, 0.15mM spermine, 0.5mM EGTA, 2mM EDTA, 1mM Dithiothreitol (DTT) (DTT), 10mg/mL BSA, 1mM MgCl2·6H2O, 50mM HEPES, pH 7.4 (at room temperature)；
2. 100 × phosphatase inhibitor cocktail: 20mM NaF, 0.2M Na3VO4, 20mM beta-glycerophosphate, 0.5mM AEBSF, 25 μ g/mL leupeptins, 10 μ g/mL Aprotinins, 1 μ g/mL pepstatin, 1 μM of Microcystin；
3. 100 × inhibitors of phosphatases and half permeabilization mixture: 20mM NaF, 0.2M Na3VO4, 20mM β-phosphoglycerol Salt, 0.5mM AEBSF, 25 μ g/mL leupeptins, 10 μ g/mL Aprotinins, 1 μ g/mL pepstatin, 1 micro- Microcystin, 45 μ G/mL digitonin；
4. room temperature storage buffer: 300mM sucrose, 60mM KCl, 0.5mM EGTA, 2mM EDTA, 1mM DTT, 1mM MgCl2·6H2O, 50mM HEPES, pH 7.4 and inhibitors of phosphatases；
5. store buffer liquid: 250mM sucrose, 25mM KCl, 5mM MgCl2·6H2O, 1mM DTT, 50mM HEPES, pH 7.4 (room temperatures) and phosphatase inhibitor cocktail；
6. low Sucrose buffer: 1.85M sucrose, 25mM KCl, 5mM MgCl2·6H2O, 1mM DTT, 50mM HEPES, PH 7.4 and inhibitors of phosphatases；
7. high-sucrose buffer: 2.15M sucrose, 25mM KCl, 5mM MgCl2·6H2O, 1mM DTT, 50mM HEPES, PH 7.4 and inhibitors of phosphatases.
Equipment includes: Lang Dengduofu perfusion equipment；Doubles homogenizer, close and loose pestle；Polytron PT3100 is equal Change device, model PT-DA 3012/2 disperses aggregate；Refrigeration desk centrifuge with angle and swing bucket type rotor； Beckman Avanti ultracentrifuge band swing-rotor；Beckman Coulter Optima MAX ultracentrifuge；Poly- third Alkene ultracentrifugation pipe (1.5,15,50mL)；Copolymerization coke or inverted light microscope；Needed for Western Blot；Other experiments When, including nylon wire (20,60,500 μm), liquid nitrogen, ice bucket, scoop, laboratory pipettor, funnel, digital balance, culture dish, Microscopic slide, ultrasonic sound appratus, incubator, 100%O2Deng.
The present invention provides a kind of extracting methods of nucleus, especially the dog ventricle heart from dog ventricular muscles and fresh separated The method that cytosol, film and nuclear leve are divided is separated in myocyte, method of the invention, which helps to get, largely can be used for basis People's functional cell core of research and clinical application；It can safely and effectively realize the screening and richness to specific type human cell Collection, makes obtained cell have better biological safety；The present invention comprehensively has detected the biological property of separated nucleus And function, reliable data are provided for the research and application of nucleus separation, are mentioned for the nucleus of other novel cells Take assessment provide it is a set of can method system for reference.
Detailed description of the invention
Fig. 1: subcellular is fractionated schematic diagram.
Fig. 2: the structural characterization of isolated heart cell and nucleus, wherein
Left hand view: (DIC) microscope is compared by differential interference and observes isolated rodlike cardiac muscle cell, right side two is opened Figure: the complete purifying cardiac cell nucleus marked with fluorescent DNA dyes DRAQ5 shown with DIC or fluorescence microscope, Shang Tubao By the enlarged drawing of the field of white edge instruction in respective image containing the following figure.
Fig. 3: the characterization of dog cardiac cell nucleus is separated by immunocytochemical stain, wherein heart core Nkx2.5, The antibody of GATA4, HDAC5, AT1R and AT2R are decorated, and are marked with the antibody for Lamin A/C and TO-PRO-3 Core shows interior nuclear membrane and DNA respectively.
Fig. 4: the characterization that the film from dog cardiac muscle cell, cytoplasm and nuclear leve are divided, wherein
Left figure: it uses cytoplasm marker (HSP70), plasma membrane (caveolin 3), interior nuclear membrane (lamin B and A/ C), nucleopore (nucleoporin 62), and reside in transcription factor in cardiac muscle cell western blot analysis core (GATA4, Nkx2.5)；Right figure: thin by SDS-PAGE (12 hole precast gel of 4-20% gradient, every swimming lane load 20 μ g total proteins) analysis Karyon separates the score during (film, cytoplasm and core), and is made dirty using Coomassie brilliant blue；Wherein, T, homogenate；C, cytosol； M, membrane fraction；N, core.
Specific embodiment
Embodiment 1
Nucleus is extracted in the steps below:
Step 1: obtain in vitro dog cardiac muscle cell, including: prepare oxygenatedchemicals buffer and in relation to instrument and Device for casting；Conventional treatment experimental animal obtains isolated experiment animal hearts；In vitro experimental animal heart perfusion perfusion and Digestion obtains cardiac muscle cell；
Step 2: subcellular is fractionated, and including: in vitro experimental animal cardiac muscular tissue and cardiac muscle cell homogenize； Tissue and cell homogenates differential centrifugation obtain the thick core and film and cytoplasm of separation；Discontinuous sucrose density gradient centrifugation obtains thick It is centrifuged again after the core beds of precipitation, obtains the core of separation and shows elongated almond-shaped or circle, and to DNA stained positive；In step 2 It is all to carry out on ice or under 4 degree step by step.
More specifically, comprising:
(1) water-bath is opened, is preheated and oxygenatedchemicals buffer (100%O2): preparing suture and surgical instrument；
(2) it rinses Perfused isolated heart method device for casting with 75% ethyl alcohol to recycle 15 minutes, then with diluted pure water After continuous flushing, rinsed with 2mM calcium ion+Tyrode primer solution；In all programs, removes all bubbles and keep constant Flow
Obtain experimental animal heart: morphine 2mg/kg subcutaneous injection, α-chloral 120mg/kg be intravenous, heparin 10000U and Mechanical ventilation, anaesthetizes the mongrel dogs of any gender 20-30kg, and the experimental animal carries out test tube of hepari, energy before surgery The clot reduced in vascular system forms and prevents from being formed thrombus, and solidifies in catheterization procedure or introduce bubble and fill prevention The tissue that fluid injection enters obstruction downstream enters the generation of cardiac muscle uniformly digested；
Routine disinfection row cardiectomy, by the above-mentioned in vitro heart of acquisition be put into the ice-cold calcium ion of 500mL+ It is rinsed in Tyrode solution, removes the tissue of remaining fat, tracheae and connection；A part of atrium sinistrum and left ventricle is perfused； The tissue being removed is preserved for subsequent subcellular fractionation；
The perfusion of Perfused isolated heart method is with digestion: the in vitro heart aortic sinus of acquisition is connected inserting for perfusion system Pipe, it is fixed, with calcium ion+Tyrode infusion 15 minutes, rinse remaining blood in heart；Catheter position should be able to avoid mouth Chamber occludes and limits irrigation flow, while keeping the constant infusion temperature of 36-38 degree；Sufficient perfusion hydraulic coupling is kept, is used in combination Tyrode infusion without calcium ion 10 minutes, by continuous circulation solution, with 200mL collagenase solution by heart perfusion about After sixty minutes, small pieces are obtained and knit heart tissue grinding separation cell mass, suitable clostridiopetidase A and corresponding is used in digestion process Ground modify digestion condition such as, liquor capacity, coronary artery fusion rate, best enzyme infusion time etc., with obtain it is an appropriate number of can Row cardiac muscle cell, static rodlike heart cell show apparent tubular cross striped under an optical microscope；When necessary, it takes Precautionary measures (1) carefully clean Lang Dengduofu equipment and replacement Tygon pipeline；(2) storage life of inventory solutions is limited； (3) pH value of distillation water source and oxidation buffer liquid is checked at room temperature, and uses ultrapure (1 type) water, to ensure these solution Quality etc., prevent rodlike Ca2+Tolerance cardiac muscle cell stops vigor surprisingly and reduces or calcium contamination or endotoxin are discharged into perfusion liquid Generation；
Cell suspending liquid is filled into 50mL pipe by 500 μM of nylon cell strainers, and is centrifuged 5 points with 500rpm Clock slowly slows down with pellet viable cells cardiac muscle cell；Cell is resuspended in the ice-cold PBS of 15mL, 5min is centrifuged with 500rpm, It repeats once, to remove all remaining fibroblasts；
Subcellular fractionation:
1. homogenization
Cardiac muscular tissue: dissecting left ventricular tissues from the in vitro dog heart (about 1g) obtained, thoroughly clear with ice-cold PBS It washes, and fritter is madeAfterwards, it is transferred in the 50mL Falcon pipe containing 15mL homogenate buffer, uses Polytron PT-3100 homogenizes tissue；The homogenizer disperses skeleton equipped with 3012/2 type of PT-DA, with maximum speed (20000rpm) sets 20s, makes pestle to the bottom of pipe at least ten times；
Cardiac muscle cell: cleaning myocyte with ice-cold PBS after digestion, at room temperature with 500rpm centrifugation 5 minutes, and will be thin Born of the same parents' precipitating is resuspended in 15mL homogenate buffer:
2. tissue and cell homogenates differential centrifugation:
It is being slowly stirred cell or 4 DEG C in the homogenization buffer and semi-permeable mixed liquor for being supplemented with inhibitors of phosphatases Rotation is permeated in 45 minutes, and the isotonic buffer solution chelated mineral containing 300mM sucrose Yu EDTA and EGTA is used in the present embodiment Ion, to prevent, organelle from being damaged and protein degradation, the homogenization buffer contain spermine to stablize chromatin；
It is transferred to Dounce homogenizer, has the homogenization buffer of inhibitors of phosphatases (without red mould with isometric supplement Element) dilution homogenate, further destroy tissue and cell and discharge nucleus, with the detergent of permeable membrane (for example, Triton-X100, Tween 20, deoxycholic acid, digitonin), it is complete to retain nuclear membrane while permeating heart cell plasma membrane；
With 300rpm centrifugation 3 minutes (centering rotor, refrigerate desk centrifuge) at 4 DEG C, precipitates and remove uncracked Cell, connective tissue and biggish concentration element；It is primary by 60 μm of nylon net filter supernatants, then pass through 20 μm of nylon wires Twice, further filtering is as needed to remove most waste material and tissue residues for filtering；Centrifuge tube is transferred to centrifugation Guan Zhong, and by being centrifuged 2000rpm centrifuge separation in swing-rotor (Sorvall 75-006-434 rotor) with 4,000 DEG C Thick core；Supernatant pours into clean centrifuge tube, removes any remaining substance inside pipe with dust-free paper, leaves soft particle alone (core)；Keep supernatant, the thick core for the separation that suspends again in 5mL core store buffer liquid, it is ensured that collect the entire core of bottom of the tube Particle isolates the quality of core using microscopic evaluation；At 4 DEG C (Beckman, TLA-100.3 rotors) with 38500rpm (80000 Translate) supernatant 60 minutes of centrifugation step 7, obtain film (precipitating) and cytoplasm (supernatant)；The sediment of above-mentioned steps is resuspended In film store buffer liquid, and keep film and cytoplasm fraction (supernatant) for further analyzing；
Discontinuous sucrose density gradient centrifugation:
It is drawn in the ultra-fine pipe of the ultra-fine pipe of 30mL (25 μm of 889mm), is made by the high-sucrose buffer for preparing 10ml Standby discontinuous saccharose gradient, high-sucrose pad is Chong Die with 10 milliliters of low Sucrose buffers, the thick core always to suspend is precipitated and is laminated Onto discontinuous saccharose gradient, when needing, at 4 DEG C with 10000rpm (12000 translate) centrifugation initial supernatant liquid (Sorvall, SS-34 rotor) 15 minutes, make mitochondria, lysosome, peroxisome and other Cell organelle pellets, then using continuous Saccharose gradient such as 15-35%, separates above-mentioned organelle；Optimize saccharose gradient and centrifugal condition in the present embodiment as needed (such as time, RCF), or, the substitutes such as Percoll, Nycodens or Metrizamide substitute sugarcane using such as Ficoll Sugar is as the culture medium for generating density gradient；
In swing type rotating cylinder (Beckman SW 28) (Beckman SW 28), ultracentrifugation pipe and 28000rpm are centrifuged Machine is placed 60 minutes at 4 DEG C, after the completion of centrifugation, takes pipe, and is abandoned upper containing microsomal membrane and other cytoplasmic contaminants Clear liquid wipes the remaining clast of pipe internal surface (see note 12) with dust-free paper, is needed according to experiment in nucleus store buffer liquid In suspend again precipitating, contain pure core, be transferred to microcentrifugal tube, and fast freezing is to carry out subsequent biochemical analysis；
Isolated core does not have pollutant, and shows elongated almond or circle, and to DNA stained positive (as shown in Figure 2), Isolated core carries out further characterization (as shown in Figure 4) by immunofluorescence (as shown in Figure 3) or immunoblotting.

Claims (7)

1. a kind of method of nucleus extraction, which is characterized in that this method is, from vitro experimental animal cardiac myocytes and ventricle Nucleus is separated and extracted in myocyte's sample comprising is selectively interacted using infiltration plasma membrane separation with sterol strong Heart glycosides and cardigin after permeabilization, using Dounce homogenizer, then by differential sucrose density gradient centrifugation, pass through machinery Smashing discharges nucleus from remaining cell body, obtains core enriching section.
2. the method for nucleus extraction according to claim 1, which is characterized in that itself comprising steps of
Step 1: in vitro dog cardiac muscle cell is obtained, Step 2: subcellular is fractionated.
3. the method for nucleus extraction as described in claim 1 or 2, which is characterized in that include substep in the step one It is rapid:
1) prepare oxygenatedchemicals buffer and in relation to instrument and device for casting；
2) conventional treatment experimental animal obtains isolated experiment animal hearts；
3) perfusion of in vitro experimental animal heart perfusion and digestion, obtain cardiac muscle cell.
4. the method for nucleus extraction as described in claim 1 or 2, which is characterized in that include substep in the step two It is rapid:
1) in vitro experimental animal cardiac muscular tissue and cardiac muscle cell's homogenization；
2) tissue and cell homogenates differential centrifugation obtain the thick core and film and cytoplasm of separation；
3) discontinuous sucrose density gradient centrifugation is centrifuged again after obtaining the thick core beds of precipitation, is obtained the core of separation and is shown elongated Almond-shaped or circle, and to DNA stained positive.
5. the method for nucleus extraction as described in claim 1 or 2, which is characterized in that in the step 2 it is all step by step Carry out on ice or under 4 degree.
6. the method for nucleus extraction as described in claim 1 or 2, which is characterized in that the separation of the method cardiac myocyte When, all programs use ultrapure 1 type water.
7. the method for nucleus extraction as described in claim 1 or 2, which is characterized in that the core separated in the method passes through Immunofluorescence or immunoblotting are further characterized.
CN201810004997.6A 2018-01-03 2018-01-03 A kind of extracting method of nucleus Pending CN109988745A (en)

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Publication number Priority date Publication date Assignee Title
CN111763765A (en) * 2020-02-18 2020-10-13 张缨 Novel method for preventing RNA degradation of novel coronavirus sample
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