CN105586307B - A kind of separation of the hepatic stellate cells of meriones unguiculatus and cultural method - Google Patents
A kind of separation of the hepatic stellate cells of meriones unguiculatus and cultural method Download PDFInfo
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Abstract
The invention discloses a kind of separation of the hepatic stellate cells of meriones unguiculatus and cultural methods, separation method includes: the 1) liver by meriones unguiculatus, first move into perfusion digestion in joint enzyme solution, then Glisson's capsule and Glisson sheath are removed, crush liver in the pasty state, mixed enzyme solution is moved into again, and stirring digestion obtains stirring postdigestive product；2) the first Hank ' s liquid is added, filtering obtains hepatic tissue；Then plus the 2nd Hank ' s liquid, initial centrifugation must precipitate；It is resuspended and is precipitated with the 3rd Hank ' s liquid, then Nycodenz liquid is added to it and mixes, be centrifuged again, obtain turbidity zone, turbidity zone is isolated hepatic stellate cells.Isolated culture method includes: separation and culture.The method of the present invention can be improved the yield and motility rate of hepatic stellate cells, it is ensured that HSCs purity is laid a good foundation for further research hepatic stellate cells formation of the deposition and fibrosis of cellular matrix after hepatic injury.
Description
Technical field
Field is separately cultured the present invention relates to hepatic stellate cells, and in particular to a kind of hepatic stellate cells of meriones unguiculatus
Separation and cultural method.
Background technique
Hepatic stellate cells (hepatic stellate cell, HSC), also known as Ito Cells (Fat-store cell,
FSC), interstitial cell is distributed mainly in the perisinusoidal space of Disse between liver cell and sinusoidal endothelial cell (gap Disse).HSC is in liver
There is central role in the occurrence and development of fibrosis, be the principal element for promoting hepatic fibrosis progression.The HSC of quiescent condition is main
The Metabolism regulation for participating in vitamin A, after liver is damaged by chemistry, physics and biological factor stimulation, HSC increases
It grows and is activated, synthesize the extracellular matrix (extracellular matrix, ECM) and various types of cells based on collagen
Factor such as MMPs, TNF-α etc. participates in the reparation of impaired hepatic tissue.When stimulation persistently occurs, HSC is largely activated, and causes ECM's
Synthesis and degradation are unbalance, and then lead to connective tissue abnormal deposition in liver, and liver fibrosis occurs.
Hepatic stellate cells is the main effects cell of liver fibrosis, and in sinus hepaticus, there are also sinusoidal endothelial cells, kupffer
Cell etc..Hepatic stellate cells accounts for 8.00~13.00% or so of entire liver cell, it is considered to be in liver it is main storage and
It is metabolized the cell of vitamin A.Hepatic stellate cell activator is simultaneously converted into myofibroblast like cell, and various fibrosis factors are equal
Using HSC (hepatic stellate cells) as final target cell, hepatic stellate cells remains static under normal circumstances.When liver is by inflammation
When disease or mechanical stimulus equivalent damage, hepatic stellate cells is activated, and phenotype is changed into activated form by silent oscillation.The liver of activation is starlike
On the one hand cell passes through hyperplasia and extracellular matrix secretion participates in the reconstruction of structure in the formation and liver of liver fibrosis, on the other hand
Sinus hepaticus internal pressure is increased by cellular contraction.
Domestic some authors separate HSC using perfusion in situ method, and because of liver In situ perfusion method, perfusion liquid is not easily recycled, enzyme
Dosage is larger, and temperature when being difficult to grasp enzyme perfusion, wins difficulty after liver digestion.HSC is separately cultured for large and small mouse at present
Method have been relatively mature, but the domestic research that there is no about meriones unguiculatus HSC, and external also not yet establish perfect training
The system of supporting, and the research of meriones unguiculatus HSC is different from large and small mouse, cannot mutually use for reference each other.
Application publication number is that the Chinese invention patent application of CN103805553A (application No. is 201210455728.4) is public
The separation method for having opened a kind of mouse hepatic stellate cells is starlike thin by intubation fixation, perfusion digestion, cell dispersion, Mouse Liver
Born of the same parents' separation and etc. complete separation to mouse hepatic stellate cells.The mouse hepatic stellate cells needed in aforementioned manners,
Quality is fine, better than the disintegrate-quality of traditional rat many and relatively convenient in whole process, complete in common lab energy
At, it is very stable, the cost of this separation method is reduced without some special equipment due to using mouse, is obtaining height
The mouse hepatic stellate cells of quality, the biological behaviour for further further investigation mouse hepatic stellate cells create condition.It should
Technical solution separates HSC using perfusion in situ method, and because of liver In situ perfusion method, perfusion liquid is not easily recycled, and the dosage of enzyme is larger, and difficult
To grasp temperature when enzyme perfusion, difficulty is won after liver digestion.The technical solution is not suitable for meriones unguiculatus HSC simultaneously.
Summary of the invention
For the defects in the prior art, the object of the present invention is to provide a kind of separation of the hepatic stellate cells of meriones unguiculatus
Method can be improved the yield and motility rate of hepatic stellate cells, it is ensured that HSCs purity.
The present invention is achieved by the following technical solutions:
A kind of separation method of the hepatic stellate cells of meriones unguiculatus, comprising:
1) by the liver of meriones unguiculatus, perfusion digestion in joint enzyme solution is first moved into, then removes Glisson's capsule and Glisson
Sheath crushes liver in the pasty state, then moves into mixed enzyme solution, and stirring digestion obtains stirring postdigestive product；
2) to the first Hank ' s liquid of addition in postdigestive product is stirred, filtering obtains hepatic tissue；Then add into hepatic tissue
2nd Hank ' s liquid, initial centrifugation must precipitate；It is resuspended and is precipitated with the 3rd Hank ' s liquid, then Nycodenz liquid is added to it and mixes,
It is centrifuged again, obtains turbidity zone, turbidity zone is isolated hepatic stellate cells.
Separation method of the invention has had successfully been isolated the hepatic stellate cells of meriones unguiculatus, simple and feasible, and is further
Research HSC and the relationship of liver fibrosis are laid a good foundation.Meanwhile the method for the present invention can be improved the yield and work of hepatic stellate cells
Rate, it is ensured that HSCs purity, for further research hepatic stellate cells deposition and fibrosis of cellular matrix after hepatic injury
Formation is laid a good foundation.
It is used as the preferred technical solution of the present invention below:
In step 1), the acquisition of the liver of the meriones unguiculatus includes: to carry out out abdomen to meriones unguiculatus, dissociates abdomen actively
Inoculated fluid injection is perfused into free abdominal aorta for arteries and veins, removes liver to get the liver of meriones unguiculatus is arrived.
It weight 400g or so (400g ± 50g) before the meriones unguiculatus is made a living, the meriones unguiculatus that is in a good state of nutrition；Body
Weight can be handled with vitamin A in advance compared with less serious case, that is, pass through vitamin A stomach-filling, it is ensured that hepatic stellate cells has certain buoyancy.
The inoculated fluid injection is Hank ' the s liquid of 4 DEG C, the 25U/ml containing heparin.
The dosage of the inoculated fluid injection is 50ml/.
The joint enzyme solution, is grouped as by the group of following volumes percentage: 0.02%~0.08% pronase
It is further preferably grouped by the group of following volumes percentage with 0.05%~0.2% clostridiopetidase A and Hank ' the s liquid of surplus
At: Hank ' the s liquid of 0.05% pronase, 0.1% clostridiopetidase A and surplus.
The condition of the perfusion digestion are as follows: 35 DEG C~39 DEG C 5~20min of perfusion digestion；Further preferably, the filling
Infuse the condition of digestion are as follows: 37 DEG C of perfusion digestion 10min.
The mixed enzyme solution is grouped as by the group of following volumes percentage: 0.02%~0.08% pronase,
Hank ' the s liquid of 0.05%~0.2% clostridiopetidase A, 0.0005%~0.003% DNaseI and surplus.Further preferably
To be grouped as by the group of following volumes percentage: 0.05% pronase, 0.1% clostridiopetidase A, 0.001%
Hank ' the s liquid of DNaseI and surplus.
The condition of the stirring digestion are as follows: 35 DEG C~39 DEG C stirrings digest 10~20min；Further preferably, described
Stir the condition of digestion are as follows: 37 DEG C of stirrings digest 10~15min.
In step 2), the first Hank ' s liquid, the 2nd Hank ' s liquid and the 3rd Hank ' s liquid are Hank ' s liquid, be can be used existing
There is technology.
The filtering uses 80 mesh~120 mesh piano-wire screen, and further preferably, the filtering uses 100 mesh steel wires
Sieve.
The condition of the initial centrifugation are as follows: 300g~400g, 5min~20min, further preferably, it is described it is preliminary from
The condition of the heart are as follows: 350g, 10min.
The condition being centrifuged again are as follows: 2 DEG C~6 DEG C, 1200g~1600g, 13min~23min.Further preferably,
The condition being centrifuged again are as follows: 4 DEG C, 1400g, 18min.
20%Nycodenz liquid can be selected in the Nycodenz liquid, by the Nycodenz separating liquid of percent by volume 20%
With Hank ' the s liquid of percent by volume 80%, i.e., 20%Nycodenz liquid by volume ratio 20%:80% Nycodenz separating liquid
And Hank ' s liquid is made.
The present invention also provides a kind of isolated culture methods of the hepatic stellate cells of meriones unguiculatus.
A kind of isolated culture method of the hepatic stellate cells of meriones unguiculatus, comprising: the liver of the meriones unguiculatus is starlike thin
The separation method of born of the same parents and culture；
The culture includes: to take isolated hepatic stellate cells, with 1 × 105~1 × 106A/ml inoculum density into
Row originally culture；When cell fusion degree is 80%~90%, secondary culture is carried out.
Preferably, the culture medium of the originally culture by the fetal calf serum of percentage by volume 5%~15% and 85%~
95% DMEM culture solution composition；The condition of the originally culture and secondary culture are as follows: at 35 DEG C~39 DEG C, by volume basis
2%~8%CO of number2It is cultivated in the atmosphere of 92%~98% air composition.Further preferably, the culture of the originally culture
Base is made of the fetal calf serum of percentage by volume 10% and 90% DMEM culture solution；The originally culture and secondary culture
Condition are as follows: at 37 DEG C, by percentage by volume 5%CO2It is cultivated in the atmosphere of 95% air composition.Air is preferably moist empty
Gas, humidity 50%~90%.
This law uses in vitro clostridiopetidase A, pronase perfusion, and every male meriones unguiculatus liver about obtains starlike thin
Born of the same parents 1.5 × 107It is a.
It is mainly improved from the following aspect to improve cell yield:
1, generally selection weight 400g or so, the male meriones unguiculatus being in a good state of nutrition are more appropriate.Weight compared with less serious case,
Male meriones unguiculatus can be handled with vitamin A in advance, it is ensured that hepatic stellate cells has certain buoyancy, in density gradient centrifugation
Preferably separated with other cells.
2, it is perfused with 4 DEG C of inoculated fluid injection abdominal aorta, perfusion liquid can enter liver, liver by portal vein and arteria hepatica
Interior haemocyte rushes Xian and obtains comparatively clean, and hypothermia perfusion can reduce damage of the warm ischemia of liver to liver, removes row after liver
The fixed perfusion of row again of portal catheterization and liver surface clean, as far as possible reduction haemocyte generate the activity of digestive ferment bad
It influences.
3, whether liver digestion is appropriate, is the key factor for influencing cell yield and vigor.This law uses in vitro clostridiopetidase A
Digestion, liver are irrigated in 37 DEG C of water-baths, so that enzymic digestion liquid in liver and liver is maintained at 37 DEG C, it is ensured that the work of enzyme
Property, it is possible to reduce digestion time is guaranteeing portal catheterization well simultaneously, blocking infrahepatic vena cava appropriate, allows enzymic digestion
For liquid in liver expert at high pressure, digestive ferment is uniformly applied to hepatic tissue by portal system, and the utilization rate of enzyme can be improved.
4, when Nycodenz density gradient centrifugation separation HSCs, Hank ' s is added according to sedimentation cell amount when sedimentation cell is resuspended
Liquid, in order to avoid cell viscosity influence cell is layered, cell draws cellular layer liquid under interface less as far as possible after drawing separation, otherwise can
It is mingled with Kupffer cell, influences the purity of sternzellen.
5, it is reported much about the large and small mouse HSC method being separately cultured both at home and abroad, but the separation in relation to meriones unguiculatus HSC
The foundation of cultivating system still belongs to the first time, and the present invention passes through method improvement, and repetition test ultimately forms a set of suitable for meriones unguiculatus
Economic, reasonable, convenient HSC extracting method.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is that meriones unguiculatus HSCs originally culture 2h is adherent, the state diagram (× 100) of the HSC containing refractive particle；
Fig. 2 is meriones unguiculatus HSCs originally culture upgrowth situation figure (× 200) for 24 hours；
Fig. 3 is meriones unguiculatus HSCs originally culture 3d in focal growth figure (× 200)；
Fig. 4 is that meriones unguiculatus HSCs originally culture 7d is in fibrocyte sample aspect graph (× 200)；
Fig. 5 is HSC autofluorescence figure (× 200), wherein left (a), in (b), right (c) figure be respectively HSC autofluorescence ×
200, DAPI × 200, the two merge × 200；
Fig. 6 is that meriones unguiculatus HSCs is cultivated 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 (after passage for 24 hours) figure, wherein left (f), in (g), right (h) figure be respectively
Immunofluorescence dyeing × 200 Desmin, DAPI × 200, the two merge × 200；
Fig. 8 is α-SMA immunofluorescence dyeing (after passage for 24 hours) figure, wherein left (j), in (k), right (l) be respectively α-SMA
Immunofluorescence dyeing (after passage for 24 hours) × 200, merges × 200 at DAPI × 200；
Fig. 9 is that meriones unguiculatus HSCs is cultivated to the 3rd day, oil red colored graph (× 200).
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
1 materials and methods
1.1.1 animal prepares
Male SD male meriones unguiculatus, Zhejiang Province's Experimental Animal Center provide, production licence number: SCXK (Zhejiang) 2014-
0001, weight 400g or so give conventional feed nursing, normal water.The daily V itamin of 2W (the last fortnight) before separating HSCs
A 200U/100g weight stomach-filling.
1.1.2 main agents
Pronase e (Pronase E), IV Collagenase Type (TypeIV collagenase), DNase I,
Nycodenz separating liquid is purchased from Sigma company；DMEM culture solution is purchased from Invitrogen company；Fetal calf serum is Hangzhou Chinese holly
Products.
1.1.3 key instrument
Fluorescence microscope, ultracentrifuge, inverted microscope, CO2Incubator, clean bench.
1.2 experimental method
1.2.1 the separation preparation of hepatic stellate cells
The separation of hepatic stellate cells: male meriones unguiculatus pre-operative anxiety 8h, free water.With Shui He Lv Chuo intraperitoneal injection
Anaesthetised male meriones unguiculatus selects just dead male meriones unguiculatus.Thorax abdomen removes coat, skin degerming.Dorsal position is solid
Determine grand cross and open abdomen, up to xiphoid-process, down toward pubic symphysis, left and right to two sides midaxillary line.Flap, which turns up to fix, expands wound, appears abdominal cavity
All internal organs.Small intestine is turned on the left of mouse body, pre- free liver, dissociate infrahepatic vena cava above vena renalis dextra.Push liver
Dirty cutting falciform ligament is to liver superior and inferior vena cava.
Free abdominal aorta, venipuncture pin puncture inject 4 DEG C of Hank ' s liquid (including heparin 25U/ml) 50ml, and folder closes
Aorta pectoralis cuts infrahepatic vena cava and the broken heart simultaneously, as far as possible goes out the red blood cell to deposit in liver.Become to liver color
When shallow, liver superior and inferior vena cava is ligatured, row portal catheterization is fixed, and liver is removed, and rinses liver surface, 4 DEG C of Hank ' s liquid 50ml
Row portal vein perfusion liver.
Liver moves into the 250ml beaker in 37 DEG C of water-baths, continues cycling through the joint enzyme solution 50ml (connection of perfusion pre-temperature to 37 DEG C
Synthase liquid is made of Hank ' the s liquid of 0.05% pronase e of percentage by volume, 0.1%IV Collagenase Type and surplus), disappear
Change liver 10 minutes.Glisson's capsule and Glisson sheath are removed in culture dish.Liver is ground into paste, it is (mixed to be put into mixed enzyme solution
Synthase liquid is by 0.05% pronase e of percent by volume, 0.1%IV Collagenase Type, 0.001%DNaseI and surplus
Hank ' s liquid) in, 37 DEG C of water-baths stir 10~15min with tweezers, digest hepatic tissue sufficiently, obtain the liver group digested
It knits.
4 DEG C of Hank ' s liquid 50ml are added in the hepatic tissue digested, are filtered with 100 mesh piano-wire screens.The hepatic tissue of filtration point
It is fitted into two 50ml centrifuge tubes, adds Hank ' s liquid to 50ml, 350g centrifugation 10min.Precipitating is taken to be resuspended in 50ml Hank ' s liquid
In, 350g is centrifuged 10min again.Merge precipitating plus 50ml Hank ' s liquid 10ml is resuspended, draws 2ml and be placed in the centrifugation of 10ml glass
The 20%Nycodenz liquid (being made of the Nycodenz separating liquid and Hank ' s liquid of volume ratio 20%:80%) of 3ml is added in Guan Zhong
It mixes, has dispensed in this ratio and all precipitated, every pipe liquid level covering 2ml Hank ' s liquid, 1400g, 4 DEG C of centrifugation 18min, gently
It takes out, not shake liquid level, it is seen that have a muddy band under Hank.s liquid.Suction muddiness herein is inserted under liquid level with No. 16 syringe needles
For band in 10ml centrifuge tube, turbidity zone is isolated hepatic stellate cells.Add DMEM culture solution, 900g is centrifuged 10min, precipitating
It is resuspended in 10ml or so DMEM culture solution, 1.11ml fetal calf serum is then added.It counts cell yield and row trypan blue is refused
Dye test, judges vigor.
1.2.2 cell culture
With 1 × 105~1 × 106The density of a/ml is inoculated into 50ml plastic culture bottle, in 37 DEG C, volume fraction 5%
CO2, 95% humid air CO2It is cultivated in constant incubator.Liquid is changed for the first time after training 1d, and optionally every 2~3d changes liquid one later
It is secondary.It, can be by cell inoculation in the interior 6 orifice plates for having slide if you need to make the analysis such as immunohistochemistry.When cell fusion reaches 80%~
90% or so can be passed on.0.125% pancreatin is routinely used when passage.
1.2.3 cellular identification
325nm length ultraviolet light activated cell autofluorescence.Om observation cellular morphology.Immunohistochemical method identification
The expression of the Desmin (desmin) of primary HSC and passage HSC, A-SMA (α-smooth muscle actin).
2 results
The survival rate and yield of 2.1HSC
Meriones unguiculatus HSC is separated using Nycodenz density-gradient centrifugation method, meriones unguiculatus average weight is 60 grams, every
The cell yield of mouse liver is about (0.5~1) × 107It is a.It is tested according to trypan blue exclusion, cell viability is (98.6) %.
The morphologic observation of 2.2HSC
It is observed under ordinary optical inverted microscope, the HSC of fresh separated has very strong refractive power due to being rich in fat drips
Property, bright, the stronger small spherical shape of three-dimensional sense is presented, sees that Fig. 1, Fig. 1 are that meriones unguiculatus HSCs originally culture 2h is adherent, contain refractive power
The figure of the HSC of particle, × 100.Fig. 2 is meriones unguiculatus HSCs originally culture upgrowth situation figure, × 200 for 24 hours, and cell is after inoculation
30~60min appearance is adherent, and 85% cell is adherent after culture for 24 hours.Fig. 3 is that meriones unguiculatus HSCs originally culture 3d is in focal life
Long figure, × 200,72h attached cell show shuttle shape, polygon.Fig. 9 is that meriones unguiculatus HSCs is cultivated to the 3rd day, oil red dye
The figure of color, × 200, normal light microscopic observation oil red coloration result shows that red fat drips are distributed mainly in endochylema.Fig. 5 is HSC
The figure of autofluorescence, × 200, cell is under the excitation of 325nm length ultraviolet light from fluoresced green.Fig. 4 is meriones unguiculatus
HSCs originally culture 7d is in fibrocyte sample aspect graph, and × 200, after 5~7d, cell continues to stretch, and most cells are in star
There is sheet fusion in shape, part cell；10~11d or so fusion reaches 80%~90%, and cell is paved with bottom of bottle and can pass on.
2.3HSC identification
Immuncytochemical detection shows that the smooth actin (α-SMA) of adherent α-for 24 hours is negative, originally culture 3d
Afterwards, α-SMA positive rate is 75% (Fig. 6).After hepatic stellate cells had digestive transfer culture, cells show is the state of activation, immunofluorescence inspection
It surveys display Desmin (Fig. 7) and α-SMA (Fig. 8) positive staining and reaches 100%, endochylema is in granular form or diffuses coloring.After passage
1d cells show is activated phenotype.
3. conclusion
HSC is a kind of nonparenchymal cell in liver, and the HSC of activation is the main source of synthesis, secretion ECM, in liver fiber
Critical role is in the disease progression of change.As previously mentioned, meriones unguiculatus lipid-metabolism mode and the mankind are similar, to high fat diet
Sensitivity, relative to large and small mouse, liver fibrosis easy to form, it is considered to be the ideal animals of Liver Fibrosis Model research.Therefore long
Being separately cultured for pawl gerbil jird HSC will lay the foundation for the study of incident mechanism of liver fibrosis and its reversal therapies.
It reports much about the large and small mouse HSC method being separately cultured, but is trained in relation to the separation of meriones unguiculatus HSC both at home and abroad
The foundation for the system of supporting still belongs to the first time.Our repetition tests ultimately form a set of suitable for the economic, reasonable, conveniently of meriones unguiculatus
HSC extracting method:
1, the selection and pretreatment of meriones unguiculatus
The closed colony that this seminar is saved has part gerbil jird to have spontaneous obesity symptom, i.e. after 3 monthly ages, posture is obvious
Partially fat, weight mouse more of the same age is obviously higher, therefore the meriones unguiculatus of 60g or so is selected in this experiment, male or female, and in advance with dimension
Raw element A stomach-filling processing, it is ensured that hepatic stellate cells is with certain buoyancy, so as in density gradient centrifugation, can preferably with its
He separates cell.
2. the improvement of method for filling
We on the basis of method for filling, are improved, using the method for liver Cardiopulmonary bypass, specifically in situ
It is as follows: to be perfused with 4 DEG C of inoculated fluid injection abdominal aortas, perfusion liquid can enter liver, blood in liver by portal vein and arteria hepatica
Cell rushes Xian and obtains comparatively clean, and hypothermia perfusion can reduce damage of the warm ischemia of liver to liver.After removing liver, row Men Jing
The fixed perfusion of row again of arteries and veins intubation and liver surface clean, as far as possible reduction haemocyte generate adverse effect to digestive enzyme activity.?
Also bubble should be avoided in entire filling process, and to enter perfusion caused by blood vessel in liver insufficient.
3. the points for attention in enzymic digestion
Whether liver digestion is appropriate, is the key factor for influencing cell yield and vigor.This law uses in vitro enzyme digestion.
This method mainly has following 3 advantages, and locally temperature control easy to accomplish, liver are irrigated (1) liver in 37 DEG C of water-baths,
Enzymic digestion liquid in liver and liver is set to be maintained at 37 DEG C, it is ensured that the activity of enzyme, it is possible to reduce digestion time.(2) it can avoid original
Since the abundant caused enzyme dosage of liver offshoot circulation increases in the enzyme digestion of position, cost increases.(3) guaranteeing portal catheterization
Well simultaneously, blocking infrahepatic vena cava appropriate, allows enzymic digestion liquid to form high pressure in liver, and digestive ferment is equal by portal system
Hepatic tissue is acted on evenly, and the utilization rate of enzyme can be improved.The proportion of mixed enzyme solution used in the method are as follows: clostridiopetidase A: DNA
Enzyme: chain protease=0.1%:0.001%:0.05%, clostridiopetidase A are mainly used for digesting collagen in liver, the degradable liver of DNA enzymatic
The DNA discharged after parenchyma rupture reduces intercellular adhesion, and chain protease is mainly used for removing liver cell, improves HSC
Concentration.Digestive ferment must configure before use, and concentration is accurate.
4. the selection and addition of separating medium
Since HSC is rich in fat drips, cell density is minimum in all liver cells, therefore the method that density gradient centrifugation can be used
HSC is separated, therefore, the selection of separating medium is also particularly significant in the isolating and purifying of HSC.Common separating medium has
Metrizamidee, Percoll, Nycodenz.We have found Metrizamidee, the more difficult formation of Percoll by preliminary experiment
Apparent band, and high speed centrifugation is needed, and Nycodenz layering is stablized, configuration is simple, therefore we are made using Nycodenz
For separating medium, HSC cellular layer is not concentrated after typical concentrations 18%, but the concentration are centrifuged, and more with 20% concentration
Ideal, it is reproducible, therefore in this experiment using 20%Nycodenz as separating medium.
The adding method of Nycodenz has 2 kinds, the first is suspension method, i.e., Nycodenz is placed in tube bottom, gently covered
The cell suspension of upper respective volume forms two liquid layers cannot mixing, and second is mode of averaging, i.e., by cell suspension with
Nycodenz is mixed, then is covered a small amount of cell culture fluid or Hank ' s, inventor above and selected mode of averaging through comparing,
After the method density gradient centrifugation, cell quantity is more, and band is more concentrated.
5. other
The hepatic tissue viscosity digested is very high, is not easy to filter, when influencing the yield and vigor of HSC, therefore filtering, we
4 DEG C of Hank ' s liquid are added, not only reduce the viscosity of cell suspension, while chain protease activity can also be reduced, reduce to HSC
Toxic effect, promote the yield, purity and vigor of HSC to greatly improve.
In short, this method does not need expensive reagent, special instrument and equipment, density of single layer gradient centrifugation one-step method
HSC is isolated and purified, and cell yield is also higher, is suitable for popularization and application, it is thin after hepatic injury for further research hepatic stellate cells
The deposition of cytoplasmic matrix and the Forming Mechanism of fibrosis are laid a good foundation.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (6)
1. a kind of separation method of the hepatic stellate cells of meriones unguiculatus characterized by comprising
1) by the liver of meriones unguiculatus, perfusion digestion in joint enzyme solution is first moved into, then removes Glisson's capsule and Glisson sheath, powder
Broken liver in the pasty state, then moves into mixed enzyme solution, and stirring digestion obtains stirring postdigestive product；
Weight 60g before the meriones unguiculatus is made a living；
The acquisition of the liver of the meriones unguiculatus includes: to carry out out abdomen to the meriones unguiculatus of rigid death, and dissociate abdominal aorta, to
Inoculated fluid injection is perfused in free abdominal aorta, removes liver to get the liver of meriones unguiculatus is arrived；
The inoculated fluid injection is Hank ' the s liquid of 4 DEG C, 25 U/ml containing heparin；
The joint enzyme solution, is grouped as by the group of following volumes percentage: 0.02% ~ 0.08% pronase and 0.05% ~
0.2% clostridiopetidase A and Hank ' the s liquid of surplus；
The condition of the perfusion digestion are as follows: 37 DEG C of perfusion digestion 10min；
The mixed enzyme solution is grouped as by the group of following volumes percentage: 0.02% ~ 0.08% pronase, 0.05% ~
Hank ' the s liquid of 0.2% clostridiopetidase A, 0.0005% ~ 0.003% DNaseI and surplus；
The condition of the stirring digestion are as follows: 37 DEG C of stirrings digest 10~15min；
2) to the first Hank ' s liquid of addition in postdigestive product is stirred, filtering obtains hepatic tissue；Then add second into hepatic tissue
Hank ' s liquid, initial centrifugation must precipitate；It is resuspended and is precipitated with the 3rd Hank ' s liquid, then 20%Nycodenz liquid is added to it and mixes,
It is centrifuged again, obtains turbidity zone, turbidity zone is isolated hepatic stellate cells；
First Hank ' the s liquid, the 2nd Hank ' s liquid and the 3rd Hank ' s liquid are 4 DEG C of Hank ' s liquid.
2. the separation method of the hepatic stellate cells of meriones unguiculatus according to claim 1, which is characterized in that in step 2,
The filtering uses 80 mesh ~ 120 mesh piano-wire screen.
3. the separation method of the hepatic stellate cells of meriones unguiculatus according to claim 1, which is characterized in that in step 2,
The condition of the initial centrifugation are as follows: 300g ~ 400g, 5min ~ 20min；
In step 2, the condition being centrifuged again are as follows: 2 DEG C ~ 6 DEG C, 1200g ~ 1600g, 13min ~ 23min.
4. a kind of isolated culture method of the hepatic stellate cells of meriones unguiculatus characterized by comprising claim 1 ~ 3 is any
The separation method of the hepatic stellate cells of meriones unguiculatus described in and culture；
The culture includes: to take isolated hepatic stellate cells, with 1 × 105~1 × 106A/ml inoculum density carries out former
It is commissioned to train feeding；When cell fusion degree is 80% ~ 90%, secondary culture is carried out.
5. the isolated culture method of the hepatic stellate cells of meriones unguiculatus according to claim 4, which is characterized in that described
The culture medium of originally culture is made of the fetal calf serum of percentage by volume 5% ~ 15% and 85% ~ 95% DMEM culture solution.
6. the isolated culture method of the hepatic stellate cells of meriones unguiculatus according to claim 4, which is characterized in that the original
Be commissioned to train support and secondary culture condition are as follows: at 35 DEG C ~ 39 DEG C, by 2% ~ 8%CO of percentage by volume2With 92% ~ 98% air composition
It is cultivated in atmosphere.
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