CN106520672A - Extraction method of mouse hepatic sinus endothelial cells - Google Patents
Extraction method of mouse hepatic sinus endothelial cells Download PDFInfo
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Abstract
The invention discloses an extraction method of mouse hepatic sinus endothelial cells. According to the method, Kupffer cells are removed through gadolinium chloride injection, and separation of the hepatic sinus endothelial cells is achieved by combining two-step perfusion, differential centrifugation, percoll centrifugation and differential attachment. The extraction method of the mouse hepatic sinus endothelial cells is simple, easy to operate and high in cell purity and harvest rate, complete structural and functional activity is kept in in-vitro culture, and therefore the method can be applied in biochemistry and immunology. In addition, laboratorial conventional instruments are used in the method, the cost is low, and therefore the method can be popularized in most laboratories.
Description
Technical field
The present invention relates to a kind of extracting method of cell, in particular to a kind of extracting method of mouse sinusoidal endothelial cell.
Background technology
Sinusoidal endothelial cell (Liver Sinusoidal Endothelial Cells）Belong to the unsubstantiality in liver
Cell, plays the effect such as barrier, metabolism, ultrafiltration endocytosis, antigen presentation in liver.Recent domestic scholar is to sinus hepaticus
Endothelial cell separation method has carried out substantial amounts of research, but will obtain purity height, and cytoactive is good, the hepatic sinusoidal endothelium of function-stable
Cell is still highly difficult, especially mouse sinusoidal endothelial cell.As mouse species are abundant, all kinds of researchs can be more fully met
Needs, so it is significant to obtain the good mouse sinusoidal endothelial cell of cytoactive.
At present, mouse sinusoidal endothelial cell adopts following three kinds of extracting methods：Two step perfusion in situ combine density gradient from
Heart method, mechanically decoupled combination enzymic digestion and Density Gradient Centrifugation, immunological magnetic bead sorting and airflow classification method.Two step perfusion in situ are combined
Density-gradient centrifugation method focuses primarily upon rat, due to the restriction of technical conditions, the less sinusoidal endothelial cell for being applied to mouse
Separate, and sinusoidal endothelial cell and Kupffer Cell (kupffer cells) cannot be separated by the method, cause the thin of final acquisition
Born of the same parents' purity is affected；Mechanically decoupled combination enzymic digestion and Density Gradient Centrifugation are excessive to cellular damage, and can cause tissue block
Surface digestion is excessive, and internal digestion is not enough；Though immunological magnetic bead sorting and the mouse sinusoidal endothelial cell obtained by airflow classification method
So purity is high, but as sinusoidal endothelial cell lacks the surface marker of specificity so that cell acquisition amount is few, and cell point
Differentiation or the apoptosis that some chemical labelings are likely to cause cell must be added before choosing, while its expensive instrument and reagent expense
Limit its application in each laboratory.
In sum, existing three kinds of mouse sinusoidal endothelial cells extracting method has respective defect, and prior art is still
The suitable sinusoidal endothelial cell extracting method for mouse is not reported.
The content of the invention
It is an object of the invention to make up when prior art is separated for mouse sinusoidal endothelial cell that mechanical damage is also easy to produce,
Caused by Chemical Pretreatment, cell viability is poor, and state declines and the purity defect such as not enough, there is provided one kind can preferably keep thin
Born of the same parents' integrality and biochemical activity, and the sinusoidal endothelial cell quantity once extracted is more, the high mouse sinusoidal endothelial cell of purity
Extracting method.
For achieving the above object, the extracting method of the mouse sinusoidal endothelial cell designed by the present invention, comprises the following steps：
1 choose mouse tail vein injection gadolinium chloride solution, gadolinium chloride day injection volume and Mouse Weight ratio be 10 ~ 20mg/
25g, continuous injection were performed the operation after two days, art fasted for one day prior.
2 disinfecting apparatuses and animal workbench, 36 ~ 38 DEG C of pre-temperature D-hanks liquid, RPMI-1640 and clostridiopetidase A IV solution.
3 choose mouse peritoneal injection yellow Jackets and liquaemin, yellow Jackets day injection volume and the ratio of Mouse Weight be
40 ~ 100mg/kg, 40 ~ 60U/ of liquaemin are only.
4 are fixed on animal operating table after mouse holonarcosis, and mouse web portion of sterilizing, sterile gauze drape prevent pollution.
5 take median incision enters abdomen, struts abdominal cavity, and intestinal tube is allocated to left lower quadrant, exposure portal vein and inferior caval vein.
6 connection 50 milliliters of syringes, charge pump, extension tube and venous detaining needles, open charge pump, allow the D- of pre-temperature
Hanks liquid is full of extension tube and venous detaining needle, emptying air.
7 venous indwelling pin puncture portal veins, extract nook closing member after success, dual silk thread is ligatured, and it is quiet that D-hanks liquid is pumped to door
Arteries and veins, it was observed that when liver color becomes khaki, cutting off inferior caval vein, adjustment perfusion rate is 30ml/h.
8 cut off liver surrounding ligaments and diaphram successively, separate liver complete rapidly, not liver injury, by which
It is placed in and has been pre-placed in the culture dish of 36 ~ 38 DEG C of D-hanks liquid, continues D-hanks 25 ~ 40min of perfusion.
9 then, and perfusion liquid is changed to the clostridiopetidase A IV solution of pre-temperature, and adjustment perfusion rate is 15 ~ 17ml/h, perfusion 20
After ~ 30min, venous detaining needle is extracted, liver is placed in clostridiopetidase A IV solution and 10 ~ 20min is placed in 37 DEG C of incubators.
10 take out liver from incubator, and liver is placed on 100 mesh filter screens, the diaphram that deducts gall-bladder and be connected with liver, ligament,
Hepatic hilar region blood vessel, not damaged tweezer peel off Glisson's capsule, choose wherein vascular system, and being rinsed with the RPMI-1640 of pre-temperature repeatedly will be thin
Born of the same parents filter filter screen.
Cell suspension low speed 50g centrifugation 3min after 11 filtrations, are made liver parenchyma cell precipitation, take supernatant and be centrifuged with 800g
After 10min, it is resuspended with 3 milliliters of RPMI-1640s to take cell precipitation, obtains cell suspension standby.
12 in centrifuge tube, spreads the 50%percoll solution of 3ml, the 25%percoll solution of 3ml, and 3ml cells respectively
Suspension, normal temperature centrifugation 1200g, 20 ~ 40min.
13 take the cellular layer between two-layer percoll liquid, after the resuspended mixing of RPMI-1640,2000rmp centrifugation 10min,
Remove supernatant, gained precipitation RPMI-1640 is resuspended and similarity condition is centrifuged once again, take precipitation and obtain treating cultured cells,
Concentration of volume percent be 20% hyclone in cultivate.
The adhered state of cultured cells is treated in 14 observations, after cell attachment 1 hour, takes non-attached cell suspension in new container
Middle culture, changes liquid after 12 hours, remove non-attached cell and dead cell, as sinusoidal endothelial cell.
Wherein, the mass concentration of the clostridiopetidase A IV solution is 0.05 ~ 0.1%, preferably 0.05%.
The concentration of the gadolinium chloride solution is 90 ~ 120mg/ml, preferably 100mg/ml.
The compound method of the gadolinium chloride solution is：Give birth to according to every 1ml endotoxin-frees 0.1M phosphate buffer or per 1ml
The proportions of reason salt water dissolves 90 ~ 120mg gadolinium chlorides, and filtration sterilization.
In the step 1, gadolinium chloride day injection volume and the ratio of Mouse Weight be preferably 10mg/25g.
The compound method of the 25%percoll solution and 50%percoll solution is：Percoll stoste autoclave sterilizations, so
Afterwards, 9 parts of percoll stostes mix the physiological saline that 1 part of mass concentration is 8.5%, are 100%percoll solution after mixing；Take
The physiological saline of the 100%percoll solution mixing 1.5ml of 1.5ml, is formulated as 50%percoll solution；Take the 100% of 0.75ml
The physiological saline of percoll solution mixing 2.25ml, is formulated as 25%percoll solution.
The mass concentration is that the compound method of 0.05 ~ 0.1% clostridiopetidase A IV solution is：D-hanks liquid per 200ml
Middle dissolving 100 ~ 200mg clostridiopetidase A IV, 4 DEG C are stirred overnight, and after filtration sterilization, packing is preserved.
The extracting method of the mouse sinusoidal endothelial cell that the present invention is provided is to remove Kupffer Cell, knot using gadolinium chloride injection
Close two step perfusions, differential centrifugation, percoll centrifugations, the separation to realize sinusoidal endothelial cell of differential velocity adherent.And prior art
Compare, the present invention adopts mouse preform injection gadolinium chloride solution two days, to promote the apoptosis of KC cells, while protecting hepatic sinusoidal endothelium thin
Born of the same parents' the complete and active of fenestration in separation process is stablized, so that the cell purity for extracting is significantly raised, active
To holding；Ensure that blood is fully removed in sinus hepaticus from two step perfusions, prevent enzymic digestion not exclusively and mechanical damage.
Beneficial effects of the present invention：The extracting method of the mouse sinusoidal endothelial cell for being provided, simple, cell purity
High, pick-up rate is high, and culture retains complete 26S Proteasome Structure and Function activity in vitro, can be used for biochemistry and immunology.Additionally, this
Bright method uses laboratory conventional instrument, low cost promote in most of laboratory.
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment
For mouse extract sinusoidal endothelial cell the step of be：
1 chooses normal male KM mouse, week old about 9 ~ 10 weeks, between 20 ~ 25g of body weight, tail vein injection gadolinium chloride solution 0.1 ~
0.2ml（About 10mg ~ 20mg/25g）Performed the operation after continuous two days, art fasted for one day prior is not restricted water supply.
2 disinfecting apparatuses and animal workbench, ultraviolet pre-irradiation experimental bench 1 hour, 37 DEG C of pre-temperatures D-hanks, 1640 trainings
Nutrient solution, and mass concentration be 0.05% clostridiopetidase A IV solution.
3 take one lumbar injection 0.1ml mass concentration of mouse for 1%（10mg/ml）Nembutal sodium solution, about 40mg/
Kg, and the heparin sodium aqua that 0.2ml concentration is 200U/ml, about 40U/ is only.
4 are fixed on animal operating table after mouse holonarcosis, are 75% alcohol disinfecting mouse web portion three with mass concentration
Time, cutting sterile gauze drape prevents pollution.
5 take median incision enters abdomen, struts abdominal cavity with eye speculum, and mosquito clamp lifts xiphoid-process to be fixed, and fully exposes abdominal cavity,
A little physiological saline is added dropwise, prevents abdominal cavity to be dried；Intestinal tube is allocated to into left lower quadrant, exposure portal vein and inferior caval vein.
6 connection 50 milliliters of syringes, charge pump, extension tube and No. 22 venous detaining needles, open charge pump, allow 37 DEG C in advance
The D-hanks liquid of temperature is full of extension tube and venous detaining needle, and adjustment rate of flooding is 28 ~ 32ml/h, emptying air.
2, indwelling silk thread below 7 portal veins, venous indwelling pin puncture portal vein extract nook closing member, dual silk thread knot after success
Prick, D-hanks liquid is pumped to portal vein, it was observed that when liver color becomes khaki, cutting off inferior caval vein, adjust perfusion rate
For 30ml/h.
8 cut off liver surrounding ligaments and diaphram successively, separate liver complete rapidly, not liver injury, by which
It is placed in and has been pre-placed in the culture dish of 37 DEG C of pre-temperature D-hanks liquid, continues D-hanks perfusion 30min.
9 then, and perfusate replaced with the clostridiopetidase A IV solution of 37 DEG C of pre-temperatures, and adjustment perfusion rate is 15ml/h, perfusion
After 25min, venous detaining needle is extracted, liver is placed in clostridiopetidase A IV solution and ensures fully to disappear in 37 DEG C of incubators placement 10min
Change.
10 take out liver from incubator, and liver is placed on 100 mesh filter screens, the diaphram that deducts gall-bladder and be connected with liver,
Ligament, hepatic hilar region blood vessel, not damaged tweezer peel off Glisson's capsule, choose wherein vascular system, are rushed with the RPMI-1640 of pre-temperature repeatedly
Wash and cell is filtered into filter screen.
Cell suspension low speed 50g centrifugation 3min after 11 filtrations, make liver parenchyma cell precipitation, take supernatant with 800g
After centrifugation 10min, it is resuspended with 3 milliliters of RPMI-1640s to take cell precipitation, obtains cell suspension standby.
12 in 15 milliliters of centrifuge tubes, first spread the 50%percoll of 3ml, tube wall is inclined 45 degree of angles, is carefully added along tube wall
Enter the 25%percoll of 3ml, cell suspension 3ml is placed in the top, and Adding Way is the same, normal temperature centrifugation 1200g, 20 ~ 40min,
Centrifuge acceleration and deceleration is made to be minimum in centrifugal process.
13 carefully discard the upper cell in centrifuge tube, take the cellular layer between two-layer percoll liquid, with 1640 cultures
After the resuspended mixings of liquid 10ml, 2000rmp centrifugation 10min remove supernatant, and gained precipitation uses RPMI-1640 resuspended and similarity condition
It is centrifuged again once, takes precipitation and obtain treating cultured cells, cultivates in the hyclone that concentration of volume percent is 20%.
The adhered state of cultured cells is treated in 14 observations, after cell attachment 1 hour, is taken non-attached cell suspension and is held in target
Device culture, changes liquid after 12 hours, remove non-attached cell and dead cell, as sinusoidal endothelial cell.
Wherein, the compound method of above-mentioned gadolinium chloride solution is：According to every 1ml endotoxin-frees 0.1M phosphate buffer
（PBS）The proportions of dissolving 100mg gadolinium chlorides, and filtration sterilization.
The compound method of 25%percoll solution and 50%percoll solution is：Percoll stoste autoclave sterilizations.
+ 1 part of mass concentration of 9 parts of percoll stostes is 8.5% physiological saline, is 100%percoll solution after mixing；
The physiological saline of the 100%percoll solution+1.5ml of 1.5ml, is formulated as 50%percoll solution；
The physiological saline of the 100%percoll solution+2.25ml of 0.75ml, is formulated as 25%percoll solution.
Mass concentration is that the compound method of 0.05% clostridiopetidase A IV solution is：100mg clostridiopetidase A IV（sigma）Add
In the D-hanks liquid of 200ml, 4 DEG C are stirred overnight, and preserve according to the packing of each consumption after filtration sterilization, and every mouse consumption is about
15ml。
Above-mentioned D-hanks liquid is prepared for tri-distilled water, and compound method is：KCl：0.4g, KH2PO4：0.06g, NaCl：8.0g,
NaHCO3：0.35g, Na2HPO4 7H2O：0.06g, after being settled to 1L in being dissolved in tri-distilled water, autoclaving sealing preserve.
RPMI-1640 compound method is：1640 dry powder（GIBICO）1 bag+HEPES（AMERSCO）3g+ sodium acid carbonates
2g, after dissolving, is settled to 1L, and filtration sterilization is packed as every bottle of Refrigerator store of 100ml.
Concentration of volume percent is that 20% hyclone compound method is：Hyclone 20ml+1640 nutrient solutions 80ml is mixed
It is even.
Laboratory test results
Experimental example 1：Micro- sem observation
By the mouse sinusoidal endothelial cell inverted microscope observation for obtaining, under low power lens, visible living cells is circular in light, full,
Good transmittance, karyon are clear, evaluate every mouse and can obtain about 2 × 106 sinusoidal endothelial cells.
Experimental example 2：ESEM（SEM）Observation
The sinusoidal endothelial cell that embodiment 1 is obtained is inoculated in and has been overlay on the cell climbing sheet of Collagen type-I I types, 4 DEG C of glutaraldehyde
Fix overnight, 30% ~ 100% alcohol serial dehydration, gradient is 10%, each gradient about 2h, dehydration is vacuum dried on metal spraying after terminating
Environmental scanning electronic microscope is detected, it is seen that cell is in fusiformis, and greatly, surrounding is thin, it is seen that fenestration not of uniform size for core.
Experimental example 3：Immunofluorescence（IF）Identification
1）Dry after I type Collagen type-Is are added dropwise on cell climbing sheet standby；
2）By sinusoidal endothelial cell（LSEC）It is inoculated on cell climbing sheet；
3）Cells rinsed with PBS 5min × 3 time；
4）4% 4 DEG C of paraformaldehyde fixes 1 hour；
5）PBS washs 5min × 1 time；
6）0.05% Triton-100 is in 4 DEG C of rupture of membranes 10min；
7）5%BSA closes 4 DEG C 1 hour；
8）Cd31 antibody 1: 50, in wet box, 4 DEG C overnight；
9）PBS washs 5min × 3 time；
10）FITC mark fluorescents two are incubated 1 hour in resisting 4 DEG C of wet box；
11）DAPI 20ul drops contaminate core 10min in creep plate surface；
12）PBS washs 5min × 3 time；
13）Glycerine mountant mounting, fluorescence microscope photograph.
Cell is in fusiformis, and greatly, antibody staining is concentrated on after birth core.
According to above example and experimental example, a mouse averagely extracts sinusoidal endothelial cell about 2 × 106, immunofluorescence
And ESEM is accredited as sinusoidal endothelial cell and immunofluorescence observes cell purity more than 95%.Illustrate that the present invention is detached
Sinusoidal endothelial cell purity is high, and cellular damage is little, and pick-up rate is high, and keep within a certain period of time sinusoidal endothelial cell structure and
Function, for biochemical and immunological investigation, is conducive to promoting in common laboratory.
Above disclosed is only the preferred embodiments of the present invention, and the right model of the present invention can not be limited certainly with this
Enclose, therefore the equivalent variations made according to scope of the present invention patent, still belong to the scope that the present invention covers.
Claims (8)
1. a kind of extracting method of mouse sinusoidal endothelial cell, comprises the following steps：
1）Choose mouse tail vein injection gadolinium chloride solution, gadolinium chloride day injection volume and Mouse Weight ratio be 10 ~ 20mg/
25g, continuous injection were performed the operation after two days, art fasted for one day prior；
2）Disinfecting apparatus and animal workbench, 36 ~ 38 DEG C of pre-temperature D-hanks liquid, RPMI-1640 and clostridiopetidase A IV solution；
3）Choose mouse peritoneal injection yellow Jackets and liquaemin, yellow Jackets day injection volume and the ratio of Mouse Weight be
40 ~ 100mg/kg, 40 ~ 60U/ of liquaemin are only；
4）Animal operating table is fixed on after mouse holonarcosis, mouse web portion of sterilizing, sterile gauze drape prevent pollution；
5）Take median incision and enter abdomen, strut abdominal cavity, intestinal tube is allocated to into left lower quadrant, exposure portal vein and inferior caval vein；
6）Connect 50 milliliters of syringes, charge pump, extension tube and venous detaining needles, open charge pump, allow the D-hanks of pre-temperature
Liquid is full of extension tube and venous detaining needle, emptying air；
7）Venous indwelling pin puncture portal vein, extracts nook closing member after success, dual silk thread ligation, D-hanks liquid are pumped to portal vein,
It was observed that when liver color becomes khaki, cutting off inferior caval vein, adjustment perfusion rate is 30ml/h；
8）Liver surrounding ligaments and diaphram being cut off successively, being separated liver complete rapidly, liver injury, is not placed on
It has been pre-placed in the culture dish of 36 ~ 38 DEG C of D-hanks liquid, has continued D-hanks 25 ~ 40min of perfusion；
9）Then, perfusion liquid is changed to the clostridiopetidase A IV solution of pre-temperature, adjustment perfusion rate is 15 ~ 17ml/h, perfusion 20 ~
After 30min, venous detaining needle is extracted, liver is placed in clostridiopetidase A IV solution and 10 ~ 20min is placed in 37 DEG C of incubators；
10）Liver is taken out from incubator, liver is placed on 100 mesh filter screens, it is the diaphram that deducts gall-bladder and be connected with liver, tough
Band, hepatic hilar region blood vessel, not damaged tweezer peel off Glisson's capsule, choose wherein vascular system, are rinsed with the RPMI-1640 of pre-temperature repeatedly
Cell is filtered into filter screen；
11）After filtration cell suspension low speed 50g centrifugation 3min, make liver parenchyma cell precipitation, take supernatant with 800g from
After heart 10min, it is resuspended with 3 milliliters of RPMI-1640s to take cell precipitation, obtains cell suspension standby；
12）In centrifuge tube, it is outstanding that the 50%percoll solution of 3ml, the 25%percoll solution of 3ml, and 3ml cell are spread respectively
Liquid, normal temperature centrifugation 1200g, 20 ~ 40min；
13）The cellular layer between two-layer percoll liquid is taken, after the resuspended mixing of RPMI-1640,2000rmp centrifugation 10min,
Remove supernatant, gained precipitation RPMI-1640 is resuspended and similarity condition is centrifuged once again, take precipitation and obtain treating cultured cells,
Concentration of volume percent be 20% hyclone in cultivate；
14）The adhered state of cultured cells is treated in observation, after cell attachment 1 hour, takes non-attached cell suspension in new container
Culture, changes liquid after 12 hours, remove non-attached cell and dead cell, as sinusoidal endothelial cell；Wherein, the clostridiopetidase A IV
The mass concentration of solution is 0.05 ~ 0.1%.
2. the extracting method of mouse sinusoidal endothelial cell according to claim 1, it is characterised in that：The gadolinium chloride solution
Concentration be 90 ~ 120mg/ml.
3. the extracting method of mouse sinusoidal endothelial cell according to claim 1, it is characterised in that：The gadolinium chloride solution
Concentration be 100mg/ml.
4. the extracting method of the mouse sinusoidal endothelial cell according to Claims 2 or 3, it is characterised in that：The gadolinium chloride
The compound method of solution is：According to every 1ml endotoxin-frees 0.1M phosphate buffer or per 1ml 90 ~ 120mg of physiological saline solution
The proportions of gadolinium chloride, and filtration sterilization.
5. the extracting method of mouse sinusoidal endothelial cell according to claim 1, it is characterised in that：The step 1）In,
Gadolinium chloride day injection volume and Mouse Weight ratio be 10mg/25g.
6. the extracting method of mouse sinusoidal endothelial cell according to claim 1, it is characterised in that：The 25%percoll
The compound method of solution and 50%percoll solution is：Percoll stoste autoclave sterilizations, then, 9 parts of percoll stostes mixing 1
Part mass concentration is 8.5% physiological saline, is 100%percoll solution after mixing；The 100%percoll solution for taking 1.5ml is mixed
The physiological saline of 1.5ml is closed, 50%percoll solution is formulated as；Take the 100%percoll solution mixing 2.25ml's of 0.75ml
Physiological saline, is formulated as 25%percoll solution.
7. the extracting method of mouse sinusoidal endothelial cell according to claim 1, it is characterised in that：The clostridiopetidase A IV is molten
The mass concentration of liquid is 0.05%.
8. the extracting method of the mouse sinusoidal endothelial cell according to claim 1 or 7, it is characterised in that：The quality is dense
The compound method for spending the clostridiopetidase A IV solution for 0.05 ~ 0.1% is：100 ~ 200mg collagens are dissolved in D-hanks liquid per 200ml
Enzyme IV, 4 DEG C are stirred overnight, and after filtration sterilization, packing is preserved.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055305A (en) * | 2018-09-17 | 2018-12-21 | 河南科技大学 | A kind of separating and extracting process of milk cow liver stem cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055305A (en) * | 2018-09-17 | 2018-12-21 | 河南科技大学 | A kind of separating and extracting process of milk cow liver stem cells |
| CN109055305B (en) * | 2018-09-17 | 2021-03-26 | 河南科技大学 | Method for separating and extracting milk cow liver stem cells |
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Application publication date: 20170322 |