CN102787096A

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Info

Publication number: CN102787096A
Application number: CN2012102680975A
Authority: CN
Inventors: 王慧; 张进祥; 王国梁
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-07-30
Filing date: 2012-07-30
Publication date: 2012-11-21

Abstract

The invention discloses an extraction method of mouse liver sinusoidal endothelial cells. According to the method, the separation of the liver sinusoidal endothelial cells is realized through removing kupffer cells through gadolinium chloride injection, in combination with two-step perfusion, differential centrifugation, percoll centrifugation and differential attachment. The method has the advantages of simplicity, easiness, feasibility, high cellular purity and high pick-up rate, can keep complete structure and function activity in vitro culture, and can be used for biochemistry and immunology. In addition, the method, which can use general instruments in a laboratory, is low in cost and can be popularized to most laboratories.

Description

The process for extracting of mouse sinusoidal endothelial cell

Technical field

The present invention relates to a kind of process for extracting of mouse liver cell, refer to a kind of process for extracting of mouse sinusoidal endothelial cell particularly.

Background technology

Sinusoidal endothelial cell (Liver Sinusoidal Endothelial Cells) belongs to the unsubstantiality cell in the liver, in liver, plays barrier, substance metabolism, ultrafiltration endocytosis, effects such as antigen presentation.Chinese scholars has been carried out a large amount of research to the sinusoidal endothelial cell separation method in recent years, but will obtain the purity height, and cytoactive is good, and the sinusoidal endothelial cell of function-stable is still very difficult, especially the mouse sinusoidal endothelial cell.Because mouse species is abundant, can satisfy the needs of all kinds of researchs more fully, so it is significant to obtain the good mouse sinusoidal endothelial cell of cytoactive.

At present, the mouse sinusoidal endothelial cell adopts following three kinds of process for extracting: two step perfusion in situ combine density gradient centrifugation, the digestion of mechanically sepg desmoenzyme and Density Gradient Centrifugation, immunological magnetic bead sorting and airflow classification method.Two step perfusion in situ combine density gradient centrifugation mainly to concentrate on rat; Because the restriction of technical qualification; The less sinusoidal endothelial cell that is applied to mouse separates; And this method can't be separated sinusoidal endothelial cell with Kupffer Cell (kupffer cells), causes the final cell purity that obtains to be affected; Digestion of mechanically sepg desmoenzyme and the damage of Density Gradient Centrifugation pair cell are excessive, and can cause the digestion of tissue block surface excessively, and inner digestion is not enough; Though immunological magnetic bead sorting and the resulting mouse sinusoidal endothelial cell of airflow classification method purity are high; But because the surface marker of sinusoidal endothelial cell lack of specific; Make that cell acquisition amount is few; And must add before the cell sorting that some chemical labeling might cause the differentiation or the apoptosis of cell, simultaneously its expensive instrument and reagent costs constraints its in each breadboard application.

In sum, existing three kinds of mouse sinusoidal endothelial cell process for extracting all have defective separately, and prior art is not reported as yet has the suitable sinusoidal endothelial cell process for extracting to mouse.

Summary of the invention

The objective of the invention is to remedy and be prone to produce physical abuse when prior art is separated to the mouse sinusoidal endothelial cell; The cell viability that Chemical Pretreatment causes is poor; Defectives such as state decline and purity are not enough; Provide a kind of and can keep cell integrity and biochemical activity preferably, and the sinusoidal endothelial cell quantity of once extracting is more, the process for extracting of the high mouse sinusoidal endothelial cell of purity.

For realizing above-mentioned purpose, the process for extracting of the mouse sinusoidal endothelial cell that the present invention designed may further comprise the steps:

1) choose mouse tail vein injection Gadolinium trichloride solution, day injection volume of Gadolinium trichloride and the ratio of mouse body weight are 10 ~ 20mg/25g, inject continuously after two days and undergo surgery art fasting previous day;

2) disinfecting apparatus and animal worktable, 36 ~ 38 ℃ of preparatory temperature D-hanks liquid, RPMI-1640 and collagenase IV solution;

3) choose mouse peritoneal injection vetanarcol and heparin sodium, day injection volume of vetanarcol and the ratio of mouse body weight are 40 ~ 100mg/kg, and heparin sodium 40 ~ 60U/ only;

4) treat to be fixed in the animal surgery platform after the mouse holonarcosis, sterilization mouse web portion, sterile gauze drape prevent to pollute;

5) get median incision and advance abdomen, strut the abdominal cavity, intestinal tube is dialled to left lower quadrant, expose portal vein and postcava;

6) connect 50 milliliters of syringes, filling pump, prolongation pipe and venous detaining needles, open filling pump, let the D-hanks liquid of preparatory temperature be full of prolongation pipe and venous detaining needle, emptying air;

7) venous indwelling needle penetration portal vein is extracted nook closing member after the success, dual silk thread ligation, and D-hanks liquid is pumped to portal vein, when observing the liver color and becoming khaki color, cuts off postcava, and the adjustment perfusion rate is 30ml/h;

8) cut off liver ligament and diaphram on every side successively, with liver complete separating rapidly, liver injury is not placed in the petridish of having placed 36 ~ 38 ℃ of D-hanks liquid in advance, continues D-hanks perfusion 25 ~ 40min;

9) then, perfusion liquid is changed to the collagenase IV solution of preparatory temperature, the adjustment perfusion rate is 15 ~ 17ml/h, behind perfusion 20 ~ 30min, extracts venous detaining needle, places collagenase IV solution to place 10 ~ 20min in 37 ℃ of incubators in liver;

10) from incubator, take out liver; Liver is placed on 100 mesh filter screens, the diaphram, ligament, the hepatic hilar region blood vessel that deduct gall-bladder and link to each other with liver, the not damaged tweezer is peeled off Glisson's capsule; Choose wherein vascular system, wash repeatedly with warm in advance RPMI-1640 cell is filtered filter screen;

11) the centrifugal 3min of cell suspension low speed 50g after filtering makes the substantive cell precipitation of liver, get supernatant with the centrifugal 10min of 800g after, it is resuspended with 3 milliliters of RPMI-1640s to get cell precipitation, cell suspension is subsequent use;

12) in centrifuge tube, spread the 50%percoll solution of 3ml respectively, the 25%percoll solution of 3ml, and 3ml cell suspension, the centrifugal 1200g of normal temperature, 20 ~ 40min;

13) get cellular layer between the two-layer percoll liquid; Behind the resuspended mixing of RPMI-1640; The centrifugal 10min of 2000rmp removes supernatant, and gained deposition is resuspended and similarity condition is once centrifugal again with RPMI-1640; Get deposition and obtain treating culturing cell, in concentration of volume percent is 20% foetal calf serum, cultivate;

14) observe the adherent state treat culturing cell, treat cell attachment after 1 hour, get not the attached cell suspension and in new container, cultivate, change liquid after 12 hours, remove not attached cell and dead cell, be sinusoidal endothelial cell;

Wherein, the mass concentration of said collagenase IV solution is 0.05 ~ 0.1%, is preferably 0.05%.

The concentration of said Gadolinium trichloride solution is 90 ~ 120mg/ml, is preferably 100mg/ml.

The compound method of said Gadolinium trichloride solution is: the ratio of not having intracellular toxin 0.1M phosphate buffered saline buffer or every 1ml physiological saline solution 90 ~ 120mg Gadolinium trichloride according to every 1ml is prepared, and filtration sterilization.

In the said step 1), day injection volume of Gadolinium trichloride and the ratio of mouse body weight are preferably 10mg/25g.

The compound method of said 25%percoll solution and 50%percoll solution is: percoll stoste autoclave sterilization, and then, 1 part of mass concentration of 9 parts of percoll stoste mixing is 8.5% saline water, is 100%percoll solution behind the mixing; Get the saline water of the 100%percoll solution mixing 1.5ml of 1.5ml, be formulated as 50%percoll solution; Get the saline water of the 100%percoll solution mixing 2.25ml of 0.75ml, be formulated as 25%percoll solution.

Said mass concentration is that the compound method of 0.05 ~ 0.1% collagenase IV solution is: dissolve 100 ~ 200mg collagenase IV in the D-hanks liquid of every 200ml, and 4 ℃ of stirred overnight, after the filtration sterilization, packing is preserved.

The process for extracting of mouse sinusoidal endothelial cell provided by the invention is to utilize Gadolinium trichloride injection to remove Kupffer Cell, combines two to go on foot perfusions, differential centrifugation, percoll is centrifugal, differential the is adherent separation that realizes sinusoidal endothelial cell.Compare with prior art; The present invention adopted mouse preform injection Gadolinium trichloride solution two days, to promote the apoptosis of KC cell, protected sinusoidal endothelial cell complete sum of fenestration in sepn process active stable simultaneously; Thereby the cell purity of extraction is obviously raise, and activity is maintained; Select for use two steps perfusions assurance sinus hepaticus inner blood fully to remove, prevented enzymic digestion not exclusively to reach physical abuse.

Beneficial effect of the present invention: the process for extracting of the mouse sinusoidal endothelial cell that is provided, simple, cell purity is high, pick-up rate is high, and it is active to keep complete 26S Proteasome Structure and Function in vitro culture, can be used for biochemistry and immunology.In addition, the inventive method is used the conventional instrument in laboratory, and cost is low can be promoted in most of laboratory.

Description of drawings

Fig. 1 is with the isolating sinusoidal endothelial cell inverted microscope of the inventive method photo.

Fig. 2 is the ESEM picture of sinusoidal endothelial cell among Fig. 1.

Fig. 3 is the immunofluorescence picture of sinusoidal endothelial cell among Fig. 1.

Embodiment

Below in conjunction with accompanying drawing and specific embodiment the present invention is made further detailed description.

Embodiment

The step of extracting sinusoidal endothelial cell to mouse is:

1) choose normal male KM mouse, about 9 ~ 10 weeks in age in week, between body weight 20 ~ 25g, tail vein injection Gadolinium trichloride solution 0.1 ~ 0.2ml (about 10mg ~ 20mg/25g) undergo surgery after continuous two days, art fasting previous day is not restricted water supply;

2) disinfecting apparatus and animal worktable, ultraviolet pre irradiation experiment table 1 hour, 37 ℃ of preparatory temperature D-hanks, RPMI-1640, and mass concentration is 0.05% collagenase IV solution;

3) getting an abdominal injection 0.1ml of mouse mass concentration is the Sodital sodium solution of 1% (10mg/ml), about 40mg/kg, and reaching 0.2ml concentration is the heparin sodium aqua of 200U/ml, about 40U/ is only;

4) treat to be fixed in the animal surgery platform after the mouse holonarcosis, using mass concentration is 75% alcohol disinfecting mouse web portion three times, cuts the sterile gauze drape and prevents to pollute;

5) get median incision and advance abdomen, strut the abdominal cavity with eye speculum, mosquito clamp is mentioned xiphoid-process and is fixed, and fully exposes the abdominal cavity, drips a little saline water, prevents that the abdominal cavity is dry; Intestinal tube is dialled to left lower quadrant, exposed portal vein and postcava;

6) connect 50 milliliters of syringes, filling pump, prolongation pipe and No. 22 venous detaining needles, open filling pump, let 37 ℃ of warm in advance D-hanks liquid be full of and prolong pipe and venous detaining needle, the adjustment rate of flooding is 28 ~ 32ml/h, the emptying air;

7) 2 on silk thread is kept somewhere in portal vein below, and venous indwelling needle penetration portal vein is extracted nook closing member after the success, dual silk thread ligation, and D-hanks liquid is pumped to portal vein, when observing the liver color and becoming khaki color, cuts off postcava, and the adjustment perfusion rate is 30ml/h;

8) cut off liver ligament and diaphram on every side successively, with liver complete separating rapidly, liver injury is not placed in the petridish of having placed 37 ℃ of preparatory temperature D-hanks liquid in advance, continues D-hanks perfusion 30min;

9) then, perfusate is replaced with 37 ℃ of collagenase IV solution of temperature in advance, the adjustment perfusion rate is 15ml/h, behind the perfusion 25min, extracts venous detaining needle, places collagenase IV solution to place 10min in 37 ℃ of incubators in liver and guarantees fully digestion;

12) in 15 milliliters of centrifuge tubes, the first 50%percoll of shop 3ml is with the tube wall miter angle that tilts; The 25%percoll that carefully adds 3ml along tube wall; Cell suspension 3ml places the top, and the adding method is the same, the centrifugal 1200g of normal temperature; 20 ~ 40min, it is minimum making whizzer acceleration stresses and retarded velocity in the centrifugal process;

13) carefully discard last confluent monolayer cells in the centrifuge tube; Get the cellular layer between the two-layer percoll liquid, behind the resuspended mixing of RPMI-1640 10ml, the centrifugal 10min of 2000rmp; Remove supernatant; Gained deposition is resuspended and similarity condition is once centrifugal again with RPMI-1640, gets to precipitate to obtain treating culturing cell, in concentration of volume percent is 20% foetal calf serum, cultivates;

14) observe the adherent state treat culturing cell, treat cell attachment after 1 hour, get not the attached cell suspension and cultivate, change liquid after 12 hours, remove not attached cell and dead cell, be sinusoidal endothelial cell in target container.

Wherein, the compound method of above-mentioned Gadolinium trichloride solution is: the ratio of not having intracellular toxin 0.1M phosphate buffered saline buffer (PBS) dissolving 100mg Gadolinium trichloride according to every 1ml is prepared, and filtration sterilization.

The compound method of 25%percoll solution and 50%percoll solution is: percoll stoste autoclave sterilization.

9 parts of percoll stoste+1 part mass concentrations are 8.5% saline water, are 100%percoll solution behind the mixing;

1.5ml the saline water of 100%percoll solution+1.5ml, be formulated as 50%percoll solution;

0.75ml the saline water of 100%percoll solution+2.25ml, be formulated as 25%percoll solution.

Mass concentration is that the compound method of 0.05% collagenase IV solution is: 100mg collagenase IV (sigma) adds in the D-hanks liquid of 200ml, and 4 ℃ of stirred overnight are preserved every about 15ml of mouse consumption according to each consumption packing after the filtration sterilization.

Above-mentioned D-hanks liquid is the tri-distilled water preparation, and compound method is: KCl:0.4g, KH₂PO₄: 0.06g, NaCl:8.0g, NaHCO₃: 0.35g, Na₂HPO₄7H₂O:0.06g, be dissolved in be settled to 1L in the tri-distilled water after, autoclaving sealing is preserved.

The RPMI-1640 compound method is: 1640 dry powder (GIBICO), 1 bag+HEPES (AMERSCO) 3g+ sodium hydrogencarbonate 2g, after the dissolving, be settled to 1L, and filtration sterilization is packed as every bottle of refrigerator of 100ml and preserves.

Concentration of volume percent is that 20% foetal calf serum compound method is: foetal calf serum 20ml+1640 nutrient solution 80ml mixing.

Laboratory test results

Experimental example 1: microscopic examination

The mouse sinusoidal endothelial cell inverted microscope that obtains is observed, and it is circular, full that the down visible viable cell of low power lens is light, good transmittance, and karyon clear (Fig. 1) is estimated every mouse and can be obtained about 2 * 10⁶Individual sinusoidal endothelial cell.

Experimental example 2: ESEM (SEM) is observed

The sinusoidal endothelial cell that embodiment 1 is obtained is inoculated on the cell climbing sheet that has overlay mouse tail collagen I type, 4 ℃ of fixed overnight of LUTARALDEHYDE, the dehydration of 30% ~ 100% alcohol gradient; Gradient is 10%, the about 2h of each gradient, and dehydration finishes environmental scanning electronic microscope detection (Fig. 2) on the dry metal spraying of final vacuum; Visible cell is fusiformis; Nuclear is big, and is thin all around, visible fenestration not of uniform size.

Experimental example 3: immunofluorescence (IF) is identified

1) drip on the cell climbing sheet dry behind the I type mouse tail collagen subsequent use;

2) sinusoidal endothelial cell (LSEC) is inoculated on the cell climbing sheet;

3) cell washs 5min * 3 time with PBS;

4) 4% Paraformaldehyde 96 is fixed 1 hour for 4 ℃;

5) PBS washing 5min * 1 time;

6) 0.05% Triton-100 is in 4 ℃ of rupture of membranes 10min;

7) 4 ℃ of 5%BSA sealings are 1 hour;

8) cd31 antibody is 1: 50, and 4 ℃ are spent the night in the wet box;

9) PBS washing 5min * 3 time;

10) hatched 1 hour in FITC mark fluorescent two anti-4 ℃ of wet boxes;

11) DAPI 20ul drips and dyes nuclear 10min in the creep plate surface;

12) PBS washing 5min * 3 time;

13) glycerine mountant mounting, fluorescent microscope is taken a picture.

The result sees Fig. 3, and visible cell is fusiformis, and nuclear is big, and antibody staining concentrates on the after birth.

According to above embodiment and experimental example, a mouse is on average extracted sinusoidal endothelial cell about 2 * 10⁶, immunofluorescence and ESEM are accredited as sinusoidal endothelial cell and immunofluorescence is observed cell purity more than 95%.Explain that the isolating sinusoidal endothelial cell purity of the present invention is high, cell injury is little, and pick-up rate is high, and keeps the 26S Proteasome Structure and Function of sinusoidal endothelial cell within a certain period of time, is used for biochemistry and immunology research, helps promoting in common laboratory.

The above disclosed the preferred embodiments of the present invention that are merely can not limit the present invention's interest field certainly with this, so according to the equivalent variations that claim of the present invention is done, still belong to the scope that the present invention is contained.

Claims

1. the process for extracting of a mouse sinusoidal endothelial cell may further comprise the steps:

Wherein, the mass concentration of said collagenase IV solution is 0.05 ~ 0.1%.

2. the process for extracting of mouse sinusoidal endothelial cell according to claim 1 is characterized in that: the concentration of said Gadolinium trichloride solution is 90 ~ 120mg/ml.

3. the process for extracting of mouse sinusoidal endothelial cell according to claim 1 is characterized in that: the concentration of said Gadolinium trichloride solution is 100mg/ml.

4. according to the process for extracting of claim 2 or 3 described mouse sinusoidal endothelial cells; It is characterized in that: the compound method of said Gadolinium trichloride solution is: the ratio of not having intracellular toxin 0.1M phosphate buffered saline buffer or every 1ml physiological saline solution 90 ~ 120mg Gadolinium trichloride according to every 1ml is prepared, and filtration sterilization.

5. the process for extracting of mouse sinusoidal endothelial cell according to claim 1 is characterized in that: in the said step 1), day injection volume of Gadolinium trichloride and the ratio of mouse body weight are 10mg/25g.

6. the process for extracting of mouse sinusoidal endothelial cell according to claim 1; It is characterized in that: the compound method of said 25%percoll solution and 50%percoll solution is: percoll stoste autoclave sterilization; Then; 1 part of mass concentration of 9 parts of percoll stoste mixing is 8.5% saline water, is 100%percoll solution behind the mixing; Get the saline water of the 100%percoll solution mixing 1.5ml of 1.5ml, be formulated as 50%percoll solution; Get the saline water of the 100%percoll solution mixing 2.25ml of 0.75ml, be formulated as 25%percoll solution.

7. the process for extracting of mouse sinusoidal endothelial cell according to claim 1 is characterized in that: the mass concentration of said collagenase IV solution is 0.05%.

8. according to the process for extracting of claim 1 or 7 described mouse sinusoidal endothelial cells; It is characterized in that: said mass concentration is that the compound method of 0.05 ~ 0.1% collagenase IV solution is: dissolve 100 ~ 200mg collagenase IV in the D-hanks liquid of every 200ml; 4 ℃ of stirred overnight; After the filtration sterilization, packing is preserved.