CN105586307A - Separating method and culturing method for hepatic stellate cells of Mongolian gerbil - Google Patents
Separating method and culturing method for hepatic stellate cells of Mongolian gerbil Download PDFInfo
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Abstract
The invention discloses a separating method and culturing method for hepatic stellate cells of a Mongolian gerbil. The separating method includes the steps that 1, the livers of the Mongolian gerbil are moved into a combined enzyme solution to be subjected to perfused digestion, then liver capsules and Glisson pedicles are removed, the livers are smashed to be pasty, the paste is moved into a combined enzyme solution, the mixture is stirred for digestion, and a stirred digestion product is obtained; 2, a first Hank's solution is added, the mixture is filtered, liver tissues are obtained, then a second Hank's solution is added, primary centrifuging is carried out, sediment is obtained, the sediment is suspended again through a third Hank's solution, then a Nycodenz solution is added, the mixture is mixed to be even, centrifuging is carried out again, and a turbid band, namely, the separated hepatic stellate cells is obtained. The separating and culturing method includes the steps of separating and culturing. By means of the methods, the yield and the survival rate of the hepatic stellate cells can be increased, the purity of the HSCs can be guaranteed, and a foundation is laid for further researching of deposition of cell matrixes and forming of fibrosis of the hepatic stellate cells after the livers are injured.
Description
Technical field
Field is cultivated in the separation that the present invention relates to HSCs, is specifically related to the liver star of a kind of meriones unguiculatusThe separation of shape cell and cultural method.
Background technology
HSCs (hepaticstellatecell, HSC), claim again Ito Cells (Fat-storecell,FSC), interstitial cell, be mainly distributed in the perisinusoidal space of Disse between liver cell and sinusoidal endothelial cell(Disse gap). HSC has central role in the developing of liver fibrosis, and is to promote liver fiberChange the principal element of development. The metabolism that the HSC of quiescent condition mainly participates in vitamin A regulates, and works as liverDirtyly be subject to that chemistry, physics and biological factor stimulate and after damaging, HSC generation is bred and is activated,Synthetic taking collagen as main extracellular matrix (extracellularmatrix, ECM) and various types of cellsThe factor, as MMPs, TNF-α etc., participates in the reparation of impaired hepatic tissue. Stimulate while continuing to occur HSCA large amount of activation, cause the synthetic unbalance with degraded of ECM, and then cause the interior connective tissue of liver extremely heavyLong-pending, there is liver fibrosis.
HSCs is the main effects cell of liver fibrosis, in sinus hepaticus, also have sinusoidal endothelial cell,Kupffer cell etc. HSCs accounts for 8.00～13.00% left and right of whole liver cell, is considered toMain storage and the cell of metabolism vitamin A in liver. Hepatic stellate cell activator is also converted into flesh fibroblastDimension cell like cell, various fibrosis factors all using HSC (HSCs) as final target cell,HSCs remains static under normal circumstances. In the time that liver is subject to inflammation or mechanical stimulus equivalent damage,HSCs is activated, and its phenotype changes activated form into by silent oscillation. HSCs one side who activatesFace participates in the formation of liver fibrosis and the reconstruction of liver inner structure by hyperplasia and extracellular matrix secretion, anotherAspect makes in sinus hepaticus voltage rise high by cellular contraction.
More domestic authors adopt perfusion in situ method to separate HSC, and because of liver In situ perfusion method, perfusion liquid is difficult forReclaim, the large usage quantity of enzyme, and temperature while being difficult to grasp enzyme perfusion, win difficulty after liver digestion. OrderThe front method for large and small mouse separation cultivation HSC is comparatively ripe, but domestic there is no about lengthThe research of pawl gerbil jird HSC, and the external also cultivating system of Erecting and improving not yet, and meriones unguiculatus HSCResearch be different from large and small mouse, can not mutually use for reference each other.
Application publication number is China's invention of CN103805553A (application number is 201210455728.4)Patent Application Publication a kind of separation method of Mouse Liver sternzellen, by intubate fix, perfusion digestion,The steps such as cell dispersion, the separation of Mouse Liver sternzellen complete the separation to Mouse Liver sternzellen. UseThe method of stating has obtained the Mouse Liver sternzellen needing, and quality is fine, better than the disintegrate-quality of traditional ratA lot, and more convenient in whole process, just can complete in common lab, very stable, owing to adoptingWith mouse, without some special equipment, reduced the cost of this separation method, obtain high-qualityThe Mouse Liver sternzellen of amount is the further biological behaviour creation of further investigation Mouse Liver sternzellenCondition. This technical scheme adopts perfusion in situ method to separate HSC, and because of liver In situ perfusion method, perfusion liquid is notEasily reclaim, the large usage quantity of enzyme, and temperature while being difficult to grasp enzyme perfusion, win difficulty after liver digestion.This technical scheme is not suitable for meriones unguiculatus HSC simultaneously.
Summary of the invention
For defect of the prior art, the liver that the object of this invention is to provide a kind of meriones unguiculatus is starlikeThe separation method of cell, can improve output and the motility rate of HSCs, can ensure HSCs purity.
The present invention is achieved by the following technical solutions:
A separation method for the HSCs of meriones unguiculatus, comprising:
1) by the liver of meriones unguiculatus, first move into perfusion digestion in associating enzyme liquid, then remove Glisson's capsuleWith Glisson sheath, pulverize liver in the pasty state, then move into mixed enzyme solution, stir digestion, obtain stirring and disappearProduct after change;
2) add a Hank ' s liquid to stirring in postdigestive product, filter, obtain hepatic tissue; ThenIn hepatic tissue, add the 2nd Hank ' s liquid, tentatively centrifugal, obtain precipitation; Resuspended heavy with the 3rd Hank ' s liquidForm sediment, then add Nycodenz liquid to mix to it, again centrifugal, obtain turbidity zone, turbidity zone is separationThe HSCs obtaining.
Separation method of the present invention, has successfully separated the HSCs of meriones unguiculatus, simple and feasible,And be that the relation of further studying HSC and liver fibrosis is laid a good foundation. Meanwhile, the inventive method energyThe output and the motility rate that enough improve HSCs, can ensure HSCs purity, for further studying liver starShape cell cytostromatic deposition and Fibrotic formation after hepatic injury are laid a good foundation.
Following as the preferred technical solution of the present invention:
Step 1) in, the acquisition of the liver of described meriones unguiculatus comprises: meriones unguiculatus is opened to abdomen,Free abdominal aorta pours into pre-perfusion liquid in free abdominal aorta, takes off liver, obtains long pawl sandThe liver of mouse.
Described meriones unguiculatus make a living before about body weight 400g (400g ± 50g), be in a good state of nutritionMeriones unguiculatus; Body weight, compared with the lighter, can be used vitamin A processing in advance, by vitamin A gavage,Guarantee that HSCs has certain buoyancy.
Described pre-perfusion liquid is 4 DEG C, containing Hank ' the s liquid of heparin 25U/ml.
The consumption of described pre-perfusion liquid is 50ml/.
Described associating enzyme liquid, is made up of the component of following percentage by volume: 0.02%～0.08% chain enzymeProtease and 0.05%～0.2% clostridiopetidase A and Hank ' the s liquid of surplus, further preferred, by belowThe component composition of percentage by volume: 0.05% pronase, 0.1% clostridiopetidase A and surplusHank ' s liquid.
The condition of described perfusion digestion is: 35 DEG C～39 DEG C perfusion digestion 5～20min; Further preferably,The condition of described perfusion digestion is: 37 DEG C of perfusion digestion 10min.
Described mixed enzyme solution, is made up of the component of following percentage by volume: 0.02%～0.08% chain enzymeProtease, 0.05%～0.2% clostridiopetidase A, 0.0005%～0.003% DNaseI and surplusHank ' s liquid. More preferably, formed by the component of following percentage by volume: 0.05% chain enzyme eggWhite enzyme, 0.1% clostridiopetidase A, 0.001% DNaseI and Hank ' the s liquid of surplus.
The condition of described stirring digestion is: 35 DEG C～39 DEG C are stirred digestion 10～20min; Further excellentChoosing, the condition of described stirring digestion is: 37 DEG C are stirred digestion 10～15min.
Step 2) in, a Hank ' s liquid, the 2nd Hank ' s liquid and the 3rd Hank ' s liquid are Hank ' sLiquid, can adopt prior art.
Described filtration adopts 80 order～120 object piano-wire screens, further preferred, and described filtration adopts100 order piano-wire screens.
Described preliminary centrifugal condition is: 300g～400g, 5min～20min, and further preferred, instituteThe preliminary centrifugal condition of stating is: 350g, 10min.
Described centrifugal condition is again: 2 DEG C～6 DEG C, 1200g～1600g, 13min～23min. EnterOne step is preferred, and described centrifugal condition is again: 4 DEG C, 1400g, 18min.
Described Nycodenz liquid can be selected 20%Nycodenz liquid, by percent by volume 20%Hank ' the s liquid of Nycodenz parting liquid and percent by volume 80%, 20%Nycodenz liquid is by bodyAmass and make than the Nycodenz parting liquid of 20%:80% and Hank ' s liquid.
The present invention also provides the isolated culture method of the HSCs of a kind of meriones unguiculatus.
An isolated culture method for the HSCs of meriones unguiculatus, comprising: described meriones unguiculatusThe separation method of HSCs and cultivation;
Described cultivation comprises: get and separate the HSCs obtaining, with 1 × 105～1×106Individual/mlInoculum density carries out former culture; In the time that Fusion of Cells degree is 80%～90%, the cultivation of going down to posterity.
Preferably, the culture medium of described former culture by the hyclone of percentage by volume 5%～15% and85%～95% DMEM nutrient solution composition; Described former culture and the condition of cultivating that goes down to posterity are:35 DEG C～39 DEG C, by percentage by volume 2%～8%CO2With in the atmosphere of 92%～98% air composition, cultivate.Further preferably, the culture medium of described former culture is by the hyclone and 90% of percentage by volume 10%DMEM nutrient solution composition; Described former culture and go down to posterity cultivate condition be: 37 DEG C, byPercentage by volume 5%CO2With in the atmosphere of 95% air composition, cultivate. Air can be preferably humid air,Humidity 50%～90%.
This law adopts in vitro clostridiopetidase A, pronase perfusion, and every male meriones unguiculatus liver approximately obtainsObtain sternzellen 1.5 × 107Individual.
Mainly improve from the following aspect for improving cell yield:
1, the male meriones unguiculatus that generally select body weight 400g left and right, is in a good state of nutrition is comparatively suitable.Body weight, compared with the lighter, can be processed male meriones unguiculatus with vitamin A in advance, guarantees that HSCs has oneFixed buoyancy, in the time of density gradient centrifugation preferably with other cell separation.
2, with the pre-perfusion liquid abdominal aorta perfusion of 4 DEG C, perfusion liquid can be entered by portal vein and arteria hepaticaEnter liver, haemocyte rushes Xian and obtains comparatively totally in liver, and hypothermia perfusion can reduce warm ischemia of liver to liverDirty damage, takes off the perfusion of the fixing row again of row portal catheterization and liver surface clean after liver, as much as possibleReduce the harmful effect that haemocyte produces the activity of digestive ferment.
3, whether liver digestion is appropriate, is the key factor that affects cell yield and vigor. This law adoptsIn vitro collagenase digesting, liver pours in 37 DEG C of water-baths, and liver and liver endoenzyme digestive juice are keptAt 37 DEG C, can ensure the activity of enzyme, can reduce digestion time, ensureing that portal catheterization is well sameTime, suitable blocking-up infrahepatic vena cava, allows enzymic digestion liquid become high pressure liver expert, and digestive ferment is by doorVein system system acts on hepatic tissue equably, can improve the utilization rate of enzyme.
4, when Nycodenz density gradient centrifugation separates HSCs, thin according to precipitation when resuspended sedimentation cellBorn of the same parents' amount adds Hank ' s liquid, in order to avoid the layering of cell viscosity influence cell is drawn the rear cell of separation the least possibleDraw cellular layer liquid under interface, otherwise can be mingled with Kupffer cell, affect the purity of sternzellen.
5, separate about large and small mouse HSC the method report of cultivating both at home and abroad a lot, but have customs director's pawl sandThe foundation of the separation cultivating system of mouse HSC still belongs to the first time, the present invention's process method improvement, and repetition test,Final form a set of be applicable to the economy of meriones unguiculatus, rationally, HSC extracting method easily.
Brief description of the drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, the present inventionOther features, objects and advantages will become more obvious:
Fig. 1 is that the former culture 2h of meriones unguiculatus HSCs is adherent, contains the state diagram of the HSC of refractive particle(×100)；
Fig. 2 is the former culture 24h of meriones unguiculatus HSCs upgrowth situation figure (× 200);
Fig. 3 is that the former culture 3d of meriones unguiculatus HSCs is focal growth figure (× 200);
Fig. 4 is that the former culture 7d of meriones unguiculatus HSCs is fibrocyte sample aspect graph (× 200);
Fig. 5 is HSC autofluorescence figure (× 200), wherein, left (a), in (b), right (c) figureBe respectively HSC autofluorescence × 200, DAPI × 200, the two merging × 200;
Fig. 6 is that meriones unguiculatus HSCs is cultured to the 3rd day row α-SMA immunocytochemical stain figure,Wherein, left (d) figure is × 200; Right (e) figure is × 400;
Fig. 7 is Desmin immunofluorescence dyeing (24h after going down to posterity) figure, wherein, left (f), in (g),Right (h) figure is respectively Desmin immunofluorescence dyeing × 200, DAPI × 200, the two merging × 200;
Fig. 8 is α-SMA immunofluorescence dyeing (24h after going down to posterity) figure, wherein, left (j), in (k),Right (l) be respectively α-SMA immunofluorescence dyeing (24h after going down to posterity) × 200, DAPI × 200,Merge × 200;
Fig. 9 is that meriones unguiculatus HSCs is cultured to the 3rd day, oil red colored graph (× 200).
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail. Following examples will contribute to abilityThe technical staff in territory further understands the present invention, but does not limit in any form the present invention. Should be understood thatBe, to those skilled in the art, without departing from the inventive concept of the premise, all rightMake some distortion and improvement. These all belong to protection scope of the present invention.
1 materials and methods
1.1.1 animal is prepared
The male meriones unguiculatus of male SD, Zhejiang Province's Experimental Animal Center provides, production licenceNumber: SCXK (Zhejiang) 2014-0001, body weight 400g left and right, gives conventional feed and feeds, normally drinking-water. ?Separate front 2W (the last fortnight) the VitaminA200U/100g every day body weight gavage of HSCs.
1.1.2 main agents
Pronase e (PronaseE), IV Collagenase Type (TypeIVcollagenase), DNaseI,Nycodenz parting liquid is purchased from Sigma company; DMEM nutrient solution is purchased from Invitrogen company; TireCow's serum is Hangzhou Chinese holly company product.
1.1.3 key instrument
Fluorescence microscope, ultracentrifuge, inverted microscope, CO2Incubator, clean bench.
1.2 experimental technique
1.2.1 the separation of HSCs preparation
The separation of HSCs: the preoperative fasting 8h of male meriones unguiculatus, freely drinks water. With hydration chlorine ChuoIntraperitoneal injection is anaesthetized male meriones unguiculatus or is selected just dead male meriones unguiculatus. Chest belly is removedBy hair, skin degerming. The fixing grand cross of dorsal position is opened abdomen, upper to xiphoid-process, down to pubic symphysis, and left and rightTo both sides midaxillary line. Flap turns up to fix and expands wound, appears all internal organs in abdominal cavity. Small intestine is turned over to a mouse body left sideSide, pre-free liver, free infrahepatic vena cava above vena renalis dextra. Pressing down liver, to cut off falciform toughBring to liver superior and inferior vena cava.
Free abdominal aorta, venipuncture needle puncture, injects 4 DEG C of Hank ' s liquid (including heparin 25U/ml)50ml, folder closes aorta pectoralis and cuts infrahepatic vena cava and the broken heart simultaneously, as far as possible red by what deposit in liverCell is gone out. In the time that liver color shoals, ligation liver superior and inferior vena cava, row portal catheterization is fixed, and getsLower liver, rinses liver surface, 4 DEG C of capable portal vein perfusion livers of Hank ' s liquid 50ml.
Liver moves into the 250ml beaker in 37 DEG C of water-baths, continues the pre-temperature of circumfusion to the associating of 37 DEG C(associating enzyme liquid is by percentage by volume 0.05% pronase e, 0.1%IV Collagen Type VI for enzyme liquid 50mlHank ' the s liquid composition of enzyme and surplus), digestion liver 10 minutes. In culture dish, remove Glisson's capsuleWith Glisson sheath. Liver is ground into pasty state, and (mixed enzyme solution is by percent by volume to put into mixed enzyme solutionHank ' the s of 0.05% pronase e, 0.1%IV Collagenase Type, 0.001%DNaseI and surplusLiquid) in, 37 DEG C of water-baths are stirred 10～15min with tweezers, and hepatic tissue is fully digested, and are disappearedThe hepatic tissue of having changed.
The hepatic tissue having digested is added to 4 DEG C of Hank ' s liquid 50ml, with 100 order piano-wire screens filtrations. FilterThe hepatic tissue of crossing is distributed in two 50ml centrifuge tubes, adds Hank ' s liquid to 50ml, 350g centrifugal 10Min. Get precipitation and be resuspended in 50mlHank ' s liquid, the centrifugal 10min of 350g again. Merge precipitationAdd 50mlHank ' s liquid 10ml resuspended, draw 2ml and be placed in 10ml glass centrifuge tube, add 3ml20%Nycodenz liquid (by Nycodenz parting liquid and Hank ' the s liquid of volume ratio 20%:80%Make) mix, in the complete whole precipitations of this ratio packing, every pipe liquid level covers 2mlHank ' s liquid, 1400g,4 DEG C of centrifugal 18min, take out gently, do not shake liquid level, have as seen a muddy band under Hank.s liquid.Insert and under liquid levels, aspirate muddiness herein and be with the centrifuge tube in 10ml with No. 16 syringe needles, turbidity zone is and separatesThe HSCs arriving. Add DMEM nutrient solution, the centrifugal 10min of 900g, precipitation is resuspended in 10mlIn the DMEM nutrient solution of left and right, then add 1.11ml hyclone. Counting cells output and row platformExpect that indigo plant refuses to dye test, judgement vigor.
1.2.2 cell is cultivated
With 1 × 105～1×106The density of individual/ml is inoculated in 50ml plastic culture bottle, 37 DEG C,Volume fraction is 5%CO2, 95% humid air CO2In constant incubator, cultivate. Training 1d after firstChange liquid, optionally every 2～3d changes liquid once later. Do the analyses such as SABC as need, cell can be connectPlant in interior 6 orifice plates that have slide. When reaching 80%～90% left and right, Fusion of Cells can go down to posterity. PassFor time use routinely 0.125% pancreatin.
1.2.3 cell qualification
325nm length ultraviolet optical excitation cell autofluorescence. Om observation cellular morphology. Immuning tissueMethod is identified primary HSC and Desmin (desmin), the A-SMA (α-smooth muscle of the HSC that goes down to posterityActin) expression.
2 results
The survival rate of 2.1HSC and yield
Adopt Nycodenz density－gradient centrifuga－tion method to separate meriones unguiculatus HSC, meriones unguiculatus average weightBe 60 grams, the cell yield of every mouse liver is about (0.5～1) × 107Individual. According to trypan blue exclusion test,Cell viability is (98.6) %.
The morphologic observation of 2.2HSC
Under ordinary optical inverted microscope, observe, the HSC of fresh separated drips owing to being rich in fat, hasVery strong refractivity, presents the little spherical shape bright, third dimension is stronger, sees Fig. 1, and Fig. 1 is long pawl sandThe former culture 2h of mouse HSCs is adherent, containing the figure of the HSC of refractive particle, × 100. Fig. 2 is long pawl sandThe former culture 24h of mouse HSCs upgrowth situation figure, × 200, pasting appears in cell 30～60min after inoculationWall, 85% cell attachment after cultivation 24h. Fig. 3 is that the former culture 3d of meriones unguiculatus HSCs is focalThe figure of property growth, × 200,72h attached cell presents fusiformis, polygon. Fig. 9 is meriones unguiculatusHSCs is cultured to the 3rd day, the figure of oil red dyeing, and × 200, normal optical Microscopic observation oil red coloration resultShow, red fat drips and is mainly distributed in endochylema. Fig. 5 is the figure of HSC autofluorescence, × 200, and thinBorn of the same parents are spontaneous green fluorescence under the exciting of 325nm length ultraviolet light. Fig. 4 is that meriones unguiculatus HSCs is formerCulture 7d is fibrocyte sample aspect graph, and × 200, after 5～7d, cell continues to stretch, most absolutelyNumber cell is star, and part cell occurs that sheet merges; The fusion of 10～11d left and right reaches 80%～90%,At the bottom of being paved with bottle, cell can go down to posterity.
2.3HSC qualification
Immuncytochemical detection demonstration, it is cloudy that α-smooth muscle filamentous actin (α-SMA) of adherent 24h isProperty, after former culture 3d, α-SMA positive rate is 75% (Fig. 6). After HSCs had digestive transfer culture,Cell shows as the state of activation, immunofluorescence detection display Desmin (Fig. 7) and α-SMA (Fig. 8)Positive staining reaches 100%, and endochylema is graininess or fills the air painted. After going down to posterity, 1d cell shows as activationPhenotype.
3. conclusion
HSC is a kind of nonparenchymal cell in liver, and the HSC of activation is the master who synthesizes, secretes ECMOriginate, in the course advancement of liver fibrosis in critical role. As previously mentioned, meriones unguiculatus lipidMetabolic patterns and the mankind are similar, to high fat diet sensitivity, with respect to large and small mouse, easily form liver fiberChange, be considered to the ideal animals of Liver Fibrosis Model research. Therefore the separation of meriones unguiculatus HSC trainingSupport and will lay the foundation for study of incident mechanism and the reversal therapies thereof of liver fibrosis.
Separate about large and small mouse HSC the method report of cultivating both at home and abroad a lot, but relevant meriones unguiculatusThe foundation of the separation cultivating system of HSC still belongs to the first time. Our repetition test, finally forms a set of being applicable toThe economy of meriones unguiculatus, rationally, HSC extracting method easily:
1, the selection of meriones unguiculatus and pretreatment
The closed colony that this seminar preserves has part gerbil jird to have spontaneous obesity symptom, i.e. after 3 monthly ages,Figure is obviously partially fat, and body weight mouse more of the same age is obviously higher, therefore select the long pawl sand of 60g left and right in this experimentMouse, male or female, and use in advance the processing of vitamin A gavage, guarantee that HSCs has certain floatingPower, so that in the time of density gradient centrifugation, can be preferably and other cell separation.
2. the improvement of method for filling
We on the basis of method for filling, improve in position, adopt liver extracorporal circulatory system perfusionMethod, specific as follows: with the pre-perfusion liquid abdominal aorta perfusion of 4 DEG C, perfusion liquid can be passed through portal veinEnter liver with arteria hepatica, haemocyte rushes Xian and obtains comparatively totally in liver, and hypothermia perfusion can reduce liverThe damage of hot ischemic to liver. Take off after liver, the perfusion of the fixing row again of row portal catheterization is clear with liver surfaceWash, reduce as much as possible haemocyte digestive enzyme activity is produced to harmful effect. In whole filling process alsoShould avoid bubble to enter the perfusion that blood vessel causes in liver insufficient.
3. the points for attention in enzymic digestion
Whether liver digestion is appropriate, is the key factor that affects cell yield and vigor. This law adopts in vitroEnzyme digestion. The method mainly contains following 3 advantages, and temperature control is easily realized in (1) liver part,Liver pours in 37 DEG C of water-baths, makes liver and liver endoenzyme digestive juice remain on 37 DEG C, Ke YibaoThe activity of card enzyme, can reduce digestion time. (2) can avoid in original position enzyme digestion due to liver side shootThe abundant enzyme dosage causing of circulation increases, cost up. (3) ensureing the good while of portal catheterization,Suitable blocking-up infrahepatic vena cava, allows enzymic digestion liquid in liver, form high pressure, and digestive ferment passes through portalSystem acts on hepatic tissue equably, can improve the utilization rate of enzyme. Mixed enzyme solution used in the methodProportioning is: clostridiopetidase A: DNA enzyme: chain protease=0.1%:0.001%:0.05%, clostridiopetidase A is mainly usedCollagen in digestion liver, the DNA discharging after DNA enzyme degradable hepatic parenchymal cells breaks, reduces thinThe adhesion of intercellular, and chain protease is mainly used in removing liver cell, improves the concentration of HSC. Digestive fermentMust configure before use, and concentration is accurate.
4. the selection of separating medium and interpolation
Drip because HSC is rich in fat, cell density is minimum in all liver cells, therefore can adopt density level bandsSpend centrifugal method and separate HSC, therefore, in the separation and purification that is chosen in HSC of separating medium, be alsoVery important. Conventional separating medium has Metrizamidee, Percoll, Nycodenz. We pass throughPreliminary experiment is found Metrizamidee, the obvious band of the more difficult formation of Percoll, and need high speed centrifugation,And Nycodenz layering is stable, configuration is simple, and therefore we adopt Nycodenz as separating medium,Its typical concentrations is 18%, but the centrifugal rear HSC cellular layer of this concentration do not concentrate, and by 20% concentrationComparatively desirable, reproducible, therefore adopt in this experiment 20%Nycodenz as separating medium.
The adding method of Nycodenz has 2 kinds, and the first is suspension method, is placed in pipe by NycodenzThe end,, cover the above cell suspension of respective volume gently, two liquid layers that formation can not mix, and the second isMode of averaging, mixes by cell suspension and Nycodenz, then covers a small amount of cell culture fluid in the above,Or Hank ' s, inventor through alternative mode of averaging, after this method density gradient centrifugation, cell quantity is many,Band is comparatively concentrated.
Other
The hepatic tissue viscosity having digested is very high, is difficult for filtering, and affects yield and the vigor of HSC, because ofWhen this filters, we add 4 DEG C of Hank ' s liquid, not only reduce the viscosity of cell suspension, also simultaneouslyCan reduce chain protease activity, reduce the toxic action to HSC, impel yield, the purity of HSCGreatly improve with vigor.
In a word, this method does not need expensive reagent, special instrument and equipment, density of single layer gradient centrifugationOne-step method gets final product separation and purification HSC, and cell yield is also higher, is suitable for applying, for further grindingStudying carefully HSCs cytostromatic deposition and Fibrotic formation mechanism after hepatic injury lays a good foundation.
Above specific embodiments of the invention are described. It will be appreciated that, the present invention alsoBe not limited to above-mentioned specific implementations, those skilled in the art can be within the scope of the claimsMake various distortion or amendment, this does not affect flesh and blood of the present invention.
Claims (10)
1. a separation method for the HSCs of meriones unguiculatus, is characterized in that, comprising:
1) by the liver of meriones unguiculatus, first move into perfusion digestion in associating enzyme liquid, then remove Glisson's capsuleWith Glisson sheath, pulverize liver in the pasty state, then move into mixed enzyme solution, stir digestion, obtain stirring and disappearProduct after change;
2) add a Hank ' s liquid to stirring in postdigestive product, filter, obtain hepatic tissue; ThenIn hepatic tissue, add the 2nd Hank ' s liquid, tentatively centrifugal, obtain precipitation; Resuspended heavy with the 3rd Hank ' s liquidForm sediment, then add Nycodenz liquid to mix to it, again centrifugal, obtain turbidity zone, turbidity zone is separationThe HSCs obtaining.
2. the separation method of the HSCs of meriones unguiculatus according to claim 1, its featureBe step 1) in, the acquisition of the liver of described meriones unguiculatus comprises: to the long pawl sand of firm deathMouse opens abdomen, and free abdominal aorta pours into pre-perfusion liquid in free abdominal aorta, takes off liver,Obtain the liver of meriones unguiculatus.
3. the separation method of the HSCs of meriones unguiculatus according to claim 2, its featureBe step 1) in, described pre-perfusion liquid is 4 DEG C, containing Hank ' the s liquid of heparin 25U/ml.
4. the separation method of the HSCs of meriones unguiculatus according to claim 1, its featureBe step 1) in, described associating enzyme liquid, is made up of the component of following percentage by volume:Hank ' the s liquid of 0.02%～0.08% pronase and 0.05%～0.2% clostridiopetidase A and surplus;
The condition of described perfusion digestion is: 35 DEG C～39 DEG C perfusion digestion 5～20min.
5. the separation method of the HSCs of meriones unguiculatus according to claim 1, its featureBe step 1) in, described mixed enzyme solution, is made up of the component of following percentage by volume:0.02%～0.08% pronase, 0.05%～0.2% clostridiopetidase A, 0.0005%～0.003%Hank ' the s liquid of DNaseI and surplus;
The condition of described stirring digestion is: 35 DEG C～39 DEG C are stirred digestion 10～20min.
6. the separation method of the HSCs of meriones unguiculatus according to claim 1, its featureBe step 2) in, described filtration adopts 80 order～120 object piano-wire screens.
7. the separation method of the HSCs of meriones unguiculatus according to claim 1, its featureBe step 2) in, described preliminary centrifugal condition is: 300g～400g, 5min～20min;
Step 2) in, described centrifugal condition is again: 2 DEG C～6 DEG C, 1200g～1600g,13min～23min。
8. an isolated culture method for the HSCs of meriones unguiculatus, is characterized in that, comprising:The separation method of the HSCs of the meriones unguiculatus described in claim 1～7 any one and cultivation;
Described cultivation comprises: get and separate the HSCs obtaining, with 1 × 105～1×106Individual/mlInoculum density carries out former culture; In the time that Fusion of Cells degree is 80%～90%, the cultivation of going down to posterity.
9. the isolated culture method of the HSCs of meriones unguiculatus according to claim 8, itsBe characterised in that, the culture medium of described former culture by the hyclone of percentage by volume 5%～15% and85%～95% DMEM nutrient solution composition.
10. the isolated culture method of the HSCs of meriones unguiculatus according to claim 8,It is characterized in that, described former culture and go down to posterity cultivate condition be: 35 DEG C～39 DEG C, by volume hundredMark 2%～8%CO2With in the atmosphere of 92%～98% air composition, cultivate.
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