CN108226485A - Complex immunity magnetic bead that hepatic stellate cells isolates and purifies and preparation method thereof - Google Patents
Complex immunity magnetic bead that hepatic stellate cells isolates and purifies and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to cells to isolate and purify field of material technology, more particularly to complex immunity magnetic bead for isolating and purifying of hepatic stellate cells and preparation method thereof, the complex immunity magnetic bead is grafted antibodies in the magnetic bead particles of 50~1500nm of grain size, the antibody includes CD11b antibody and CD146 antibody, and the kernel of the magnetic bead particles is Fe3O4Microballoon, from the inside to the outside including carboxylated graphene layer, modification of chitosan layer, sodium alginate layer.The present invention passes through the grafted antibodies in magnetic bead particles, obtain complex immunity magnetic bead, contain more carboxyl functional group in magnetic bead particles, there is good dispersion performance in the solution, be less likely to occur to reunite, and grafting rate of the antibody in the magnetic bead particles is higher, with reference to stability it is stronger, available for isolating and purifying for hepatic stellate cells, to obtain, purity is higher, the stronger hepatic stellate cells of activity.
Description
Technical field
Isolate and purify field of material technology the present invention relates to cell more particularly to hepatic stellate cells isolate and purify it is compound
Immunomagnetic beads and preparation method thereof.
Background technology
Liver cell is divided into hepatic parenchymal cells and nonparenchymal cell, nonparenchymal cell include sinusoidal endothelial cell (LSECs),
Kupffer cells (KCs), hepatic stellate cells (HSCs) and pit cell.The form of hepatic stellate cells is irregular, and cell space is rounded
Or irregular shape, it often stretches out several starlike cytoplasmic processes and surrounds hepatic sinusoid.Have in hepatic stellate cells matter 1~14 diameter about 1.0~
2.0 μm of fat drips, fat drips the inside, can spontaneous blue-green fluorescents under ultraviolet light excitation rich in vitamin A.
Since nineteen fifty-three Anderson creates shearing method from liver isolating hepatocytes, each laboratory is to the separation side of liver cell
Method is continuously improved, and is broadly divided into the method for non-perfusion and the method for perfusion.The method of non-perfusion is simple and practicable with its
Feature has obtained certain application, but digests asking there are many cells agglomerate in incomplete, isolated cell due to existing
Topic, it is impossible to meet the requirement of many basic research or clinical practice；Perfusion can be such that digestive juice is more fully connect with hepatic tissue
It touches, the vigor and quantity for improving separative efficiency and gained liver cell greatly improve.At present, using hepatic stellate cells in liver
Frequently with two step perfusion method combination density fluid gradient centrifugations, it is starlike thin to isolate liver for the minimum principle of histocyte Midst density
Born of the same parents.But the averag density of hepatic stellate cells is 1.053g/ml, the averag density of KCs cells is 1.076g/ml, and LSECs
The density range of cell is 1.061~1.080g/ml, it is seen then that the density difference of hepatic stellate cells, KCs cells and LSECs cells
It is not very big, is difficult that sub-argument goes out the higher hepatic stellate cells of purity using two step perfusion methods and density fluid gradient centrifugation, separation
LSECs cells, KCs cells are contaminated in the hepatic stellate cells gone out.
Immunomagnetic beads method is the technical method occurred the 1980s.Immunomagnetic beads are used in nineteen eighty-three Ugelstad propositions
In cell sorting, nineteen ninety, Mihenyi establishes MACS.The core of this method is to be coated with tool immune response in magnetic bead surfaces
Property antibody carry out antigen-antibody reaction, form rosette in cell surface, these cells for combining magnetic bead are once placed in
Under powerful magnetic field, the cell that will not be combined with other divides group, disappears immediately behind the magnetic bead disengaging magnetic field for having superpower paramagnetic field
Loss of excitation thus can screen or remove marked cell, so as to achieve the purpose that positive or negative selects cell.This
Technology has been widely used in cell and molecular biology at present, and un-mixing bases are because of, target cell and candidate stem cell etc..It detaches effect
Fruit has obtained the confirmation of the methods of immunofluorescence, PCR, FISH and FACS.Immunomagnetic beads can effectively sort cell, so as to certainly
The technical feasibility that this method is applied in hepatic stellate cells isolates and purifies is determined.But current immunomagnetic beads presence point
Poor-performing is dissipated, is easily reunited, the antibody grafting rate on immunomagnetic beads is relatively low, and the stability combined with antibody is poor.
Invention content
In view of this, the complex immunity magnetic bead isolated and purified the object of the present invention is to provide hepatic stellate cells and its preparation
Method by the grafted antibodies in magnetic bead particles, obtains complex immunity magnetic bead, contains more carboxyl-functional in magnetic bead particles
Group, in the solution with good dispersion performance, it is not easy to reunite, and grafting rate of the antibody in the magnetic bead particles compared with
Height, with reference to stability it is stronger, available for isolating and purifying for hepatic stellate cells, to obtain, purity is higher, the stronger liver star of activity
Shape cell.
The present invention solves above-mentioned technical problem by following technological means：
The complex immunity magnetic bead that hepatic stellate cells isolates and purifies, the complex immunity magnetic bead are 50~1500nm's of grain size
Grafted antibodies in magnetic bead particles, the kernel of the magnetic bead particles is Fe3O4Microballoon, from the inside to the outside including carboxylated graphene layer, change
Property chitosan layer, sodium alginate layer.
The present invention is in Fe3O4Microballoon outer cladding carboxylated graphene layer can play magnetic protection to carboxylated graphene layer
Effect, with reference to modification of chitosan layer and sodium alginate layer, can provide more carboxyl functional groups, and the absorption grafting for antibody carries
For more site, while improve the stability that complex immunity magnetic bead is combined with antibody.The addition of sodium alginate both provided
For the site of antibody attachment, and the dispersion performance of complex immunity magnetic bead in the solution can be improved, so as to can preferably and antibody
With reference to.
Further, the antibody includes CD11b antibody and CD146 antibody.Using CD11b antibody and Kupffer cells into
The characteristics of row antigen-antibody combines, and CD146 antibody and sinus hepaticus inner cell progress antigen-antibody are combined, can be under magnetic fields
Other cells not being combined divide group, separate sinus hepaticus inner cell and Kupffer cells in hepatic stellate cells separation, from
And obtain hepatic stellate cells.
Further, the Fe that mass fraction is 10%~20% is contained in the magnetic bead particles3O4Microballoon, 25%~40%
Modification of chitosan, 20%~35% sodium alginate.
Further, the Fe3O4Microballoon is using egg white solution pore, plasma treated porous microsphere.
Egg white solution pore can make Fe3O4Microballoon has larger specific surface area, reduces bulk density；Plasma
Free electron, negative ions can be generated in processing procedure, intensify a variety of high energy active particles such as molecule or atom and its free radical,
Fe can be significantly improved3O4Absorption property, dispersion performance and the stability of microballoon.
Further, the modification of chitosan is that the dispersed nano silicon dioxide of carboxylated and chitosan pass through dehydration
It is made.
Further, the dispersed nano silicon dioxide is with γ-modified nanometer titanium dioxide of aminopropyl trimethoxy
Silicon.
Modification of chitosan energy made from dehydration is passed through using the dispersed nano silicon dioxide and chitosan of carboxylated
More carboxyl functional groups are enough provided, the combination for antibody and complex immunity magnetic bead provides more attachment point；Sodium alginate is
A kind of typical anion polysaccharide, the carboxyl that anion active contains in molecule is compound with modification of chitosan, increases
Adding carboxyl amount, further provided more attachment points for antibody so that complex immunity magnetic bead can combine closely with antibody,
And sodium alginate bioactivity is good, hydrophily is stronger, can improve the dispersion performance of complex immunity magnetic bead, make it easier to molten
It is uniformly dispersed in liquid.
In addition, the invention also discloses the preparation method of above-mentioned complex immunity magnetic bead, include the following steps：
Take Fe3O4Microballoon and carboxylated graphene are dispersed with stirring in the ethylene glycol solution for being 25% in mass concentration, in 200
10~13h is reacted in~240 DEG C of Muffle furnace, furnace cooling is taken out, and Magneto separate washs to obtain graphene/Fe3O4Microballoon；
Chitosan is taken to be dissolved in the acetic acid solution that mass concentration is 2%, then adds in graphene/Fe3O4Microballoon, stirring
It is uniformly dispersed, successively stirring adds in petroleum ether, formaldehyde, pentanediol, reacts 5~8h in 60~65 DEG C, filters, with ethyl alcohol and distillation
Water washing, vacuum drying obtain chitosan/graphene/Fe3O4Microballoon takes chitosan/graphene/Fe3O4Microballoon is dispersed in
In n-butanol, the dispersed nano silicon dioxide of carboxylated is added, is heated to 60~65 DEG C of isothermal reaction 8h, is cooled down, magnetic point
From washing, vacuum drying obtains modification of chitosan/graphene/Fe3O4Microballoon；
Sodium alginate is taken to be dissolved in distilled water, adds modification of chitosan/graphene/Fe3O4Microballoon stirs 2h, freezing
It is dried to obtain magnetic bead particles；
Using containing 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and n-hydroxysuccinimide
After PBS buffer solution activated magnetic beads particle, the magnetic bead particles of activation and antibody are added in, 15~20h is reacted in PBS buffer solution, magnetic point
From obtaining immunomagnetic beads, then with the bovine serum albumin(BSA) buffer blind immunomagnetic beads 30min containing 1wt%, it is stored in 2~5 DEG C
Environment in.
Further, the Fe3O4Preparing for microballoon is as follows：Take equimolar Fe2(SO4)3And FeSO4·7H2O is dissolved in steaming
It in distilled water, adds egg white solution and is stirred to react 30h in 60~65 DEG C, be dried under reduced pressure, grind, and be transferred into 450 DEG C of Muffle furnaces
6h is kept the temperature, then takes out and is cooled down with ice water mixed liquor, respectively washed 2 times with distilled water and absolute ethyl alcohol, the powder being dried to obtain is packed into
Corona treatment is carried out in reactor, obtains Fe3O4Microballoon.
Further, the Fe3O4Microballoon prepare in plasma process conditions be：80~120sccm of argon flow amount, pressure
3~8Pa of power, 10~15min of power 80~100W corona treatments.
Further, the carboxylated graphene prepare it is as follows：Graphene is taken to add in the hydroxide of a concentration of 0.3mol/L
Ultrasonic wave disperses 3h in sodium solution, then adds in monoxone ultrasonic response 3h and obtains reaction solution, by reaction solution deionized water
Washing is centrifuged repeatedly to neutrality, is dried to obtain carboxylated graphene.
The complex immunity magnetic bead of the present invention uses porous Fe3O4Microballoon is kernel, carboxylated graphene layer, modification of chitosan
Layer, sodium alginate layer are middle layer, and antibody is outer layer, wherein, porous Fe3O4Nanoparticle has larger specific surface area, can subtract
The weight of light complex immunity magnetic bead, in order to preferably suspended dispersed；Carboxylated graphene layer can keep Fe well3O4It is micro-
The magnetic stability of ball, and the graphene of carboxylated is easier to be combined with modification of chitosan, avoids coming off；In 1- (3- dimethylaminos
Propyl) under the action of -3- ethyl-carbodiimide hydrochlorides and n-hydroxysuccinimide, carboxyl can be combined with antibody,
And modification of chitosan contains a large amount of carboxyl, and more binding sites are provided for antibody, sodium alginate further increases carboxyl
The quantity of functional group can also adsorb more antibody, and can adsorb Multiple Antibodies simultaneously, improve antibody grafting rate, more
Carboxyl functional group can improve the stability of antibody and complex immunity magnetic bead grafting.The complex immunity magnetic bead of the present invention can be used for
Hepatic stellate cells isolates and purifies, and to obtain, purity is higher, the stronger hepatic stellate cells of activity.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in detail：
The complex immunity magnetic bead that isolates and purifies of hepatic stellate cells of the present invention, complex immunity magnetic bead for grain size 50~
Grafted antibodies in the magnetic bead particles of 1500nm, antibody include CD11b antibody and CD146 antibody, and the kernel of magnetic bead particles is Fe3O4
Microballoon, from the inside to the outside including carboxylated graphene layer, modification of chitosan layer, sodium alginate layer.Contain quality point in magnetic bead particles
Number is 10%~20% Fe3O4Microballoon, 25%~40% modification of chitosan, 20%~35% sodium alginate.Wherein,
Fe3O4Microballoon is using egg white solution pore, plasma treated porous microsphere, and modification of chitosan is the dispersion of carboxylated
Property nano silicon dioxide be made with chitosan by dehydration, dispersed nano silicon dioxide is with γ-aminopropyl trimethoxy
The modified nano silicon dioxide of base.
Preparing for the complex immunity magnetic bead that the hepatic stellate cells of the present invention isolates and purifies is as follows：
Embodiment one
Fe3O4The preparation of microballoon：Take 0.1moL Fe2(SO4)3With 0.1moL FeSO4·7H2O is uniformly mixed, and is added in
500mL distilled water is stirred to being completely dissolved, and obtains mixed liquor, after 100g egg white solutions is taken to stir into milky uniform state, stirring
It is added dropwise in mixed solution, 30h is stirred to react under 60~65 DEG C of temperature conditions, be dried under reduced pressure, grind, and be transferred to 450
6h is kept the temperature in DEG C Muffle furnace, then takes out and is cooled down with ice water mixed liquor, respectively washed 2 times, be dried to obtain with distilled water and absolute ethyl alcohol
Powder be fitted into reactor, under conditions of argon flow amount 80sccm, pressure 3Pa, power 80W, carry out corona treatment
10min obtains Fe3O4Microballoon.
Preparing for carboxylated graphene is as follows：Graphene is taken to add in ultrasound in the sodium hydroxide solution of a concentration of 0.3mol/L
Wavelength-division dissipates 3h, then adds in monoxone ultrasonic response 3h and obtains reaction solution, reaction solution is centrifuged repeatedly washing with deionized water
To neutrality, it is dried to obtain carboxylated graphene.
Graphene/Fe3O4The preparation of microballoon：Take 1gFe3O4Microballoon and 3g carboxylated graphenes are dispersed with stirring in 200mL matter
It measures in a concentration of 25% ethylene glycol solution, 10h is reacted in 200 DEG C of Muffle furnace, furnace cooling is taken out, what Magneto separate was washed
To graphene/Fe3O4Microballoon.
Modification of chitosan/graphene/Fe3O4The preparation of microballoon：It is 2% that 2g chitosans is taken, which to be dissolved in 200mL mass concentrations,
In acetic acid solution, 2g graphenes/Fe is then added in3O4Microballoon is dispersed with stirring uniformly, is warming up to 40 DEG C, is added in 40mL petroleum ethers and is stirred
20min is mixed, is warming up to 50 DEG C, 10mL formaldehyde is added in and stirs 20min, the pentanediol that addition 10mL mass concentrations are 30% is in 60 DEG C
5h is stirred to react, is filtered, with ethyl alcohol and distillation water washing, vacuum drying obtains chitosan/graphene/Fe3O4Microballoon；Take 1.6g
Dispersed nano silicon dioxide and 1g succinic anhydrides are added in per in 100mL diethyl ether solutions, and isothermal reaction 3h, filters at 20 DEG C
To solid be dried in vacuo to obtain the dispersed nano silicon dioxide of carboxylated；Take 2g chitosans/graphene/Fe3O4Microballoon is uniform
It is scattered in 200mL n-butanols, adds the dispersed nano silicon dioxide of 1g carboxylated, be heated to 65 DEG C of isothermal reaction 8h,
Cooling, Magneto separate washing, vacuum drying obtain modification of chitosan/graphene/Fe3O4Microballoon.
The preparation of magnetic bead particles：1g sodium alginates is taken to be dissolved in distilled water, add 4g modification of chitosan/graphene/
Fe3O4Microballoon stirs 2h, and freeze-drying obtains magnetic bead particles.
The preparation of complex immunity magnetic bead：5mg magnetic bead particles is taken to be washed more than three times with PBS buffer solution Magneto separate, take 2mg magnetic
Pearl add in 500 μ LPBS buffer solutions in, add 2mg1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and
2mgN- HOSu NHSs, the mixing on turbula shaker, room temperature activate the carboxyl 30min of complex immunity magnetic bead surfaces, with
It is washed afterwards with PBS buffer solution Magneto separate, the magnetic bead after washing is added in 500 μ LPBS buffer solutions, adds in the mixing of CD11b antibody
15h is reacted, CD11b antibodies Antibodies are coupled to magnetic bead surfaces, Magneto separate obtains immunomagnetic beads, then with 500 oxen of the μ L containing 1wt%
Immunomagnetic beads 30min is closed in seralbumin buffer solution, is stored in 2~5 DEG C of environment.
Embodiment two
Fe3O4The preparation of microballoon：Take 0.1moL Fe2(SO4)3With 0.1moL FeSO4·7H2O is uniformly mixed, and is added in
500mL distilled water is stirred to being completely dissolved, and obtains mixed liquor, after 100g egg white solutions is taken to stir into milky uniform state, stirring
It is added dropwise in mixed solution, 30h is stirred to react under 60~65 DEG C of temperature conditions, be dried under reduced pressure, grind, and be transferred to 450
6h is kept the temperature in DEG C Muffle furnace, then takes out and is cooled down with ice water mixed liquor, respectively washed 2 times, be dried to obtain with distilled water and absolute ethyl alcohol
Powder be fitted into reactor, under conditions of argon flow amount 120sccm, pressure 8Pa, power 100W, carry out plasma at
15min is managed, obtains Fe3O4Microballoon.
The preparation of carboxylated graphene is the same as embodiment one.
Graphene/Fe3O4The preparation of microballoon：Take 1.5gFe3O4Microballoon and 2.0g carboxylated graphenes be dispersed with stirring in
200mL mass concentrations are in 25% ethylene glycol solution, and 13h is reacted in 240 DEG C of Muffle furnace, and furnace cooling is taken out, magnetic point
Graphene/Fe is obtained from what is washed3O4Microballoon.
Modification of chitosan/graphene/Fe3O4The preparation of microballoon：It is 2% that 1.5g chitosans is taken, which to be dissolved in 200mL mass concentrations,
Acetic acid solution in, then add in 1.75g graphenes/Fe3O4Microballoon is dispersed with stirring uniformly, is warming up to 40 DEG C, adds in 40mL stones
Oily ether stirs 30min, is warming up to 50 DEG C, adds in 10mL formaldehyde stirring 30min, adds in the pentanediol that 10mL mass concentrations are 30%
8h is stirred to react in 65 DEG C, is filtered, with ethyl alcohol and distillation water washing, vacuum drying obtains chitosan/graphene/Fe3O4Microballoon；
1.6g dispersibilities nano silicon dioxide and 1g succinic anhydrides is taken to add in every 100mL diethyl ether solutions, the isothermal reaction 3h at 20 DEG C,
Obtained solid is filtered to be dried in vacuo to obtain the dispersed nano silicon dioxide of carboxylated；Take 1.8g chitosans/graphene/Fe3O4
Microballoon is dispersed in 200mL n-butanols, adds the dispersed nano silicon dioxide of 1g carboxylated, is heated to 65 DEG C of constant temperature
8h, cooling are reacted, Magneto separate washing is dried in vacuo, obtains modification of chitosan/graphene/Fe3O4Microballoon.
The preparation of magnetic bead particles：1.75g sodium alginates is taken to be dissolved in distilled water, add 3.25g modification of chitosan/stone
Black alkene/Fe3O4Microballoon stirs 2h, and freeze-drying obtains magnetic bead particles.
The preparation of complex immunity magnetic bead：5mg magnetic bead particles is taken to be washed more than three times with PBS buffer solution Magneto separate, take 3mg magnetic
Pearl add in 500 μ LPBS buffer solutions in, add 2mg1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and
2mgN- HOSu NHSs, the mixing on turbula shaker, room temperature activate the carboxyl 30min of complex immunity magnetic bead surfaces, with
Washed afterwards with PBS buffer solution Magneto separate, the magnetic bead after washing is added in 500 μ LPBS buffer solutions, add in CD11b antibody and
CD146 antibody hybrid reaction 18h, by antibody coupling in magnetic bead surfaces, Magneto separate obtains immunomagnetic beads, then contains 1wt% with 500 μ L
Bovine serum albumin(BSA) buffer solution in close immunomagnetic beads 30min, be stored in 2~5 DEG C of environment.
Embodiment three
Fe3O4The preparation of microballoon：Take 0.1moL Fe2(SO4)3With 0.1moL FeSO4·7H2O is uniformly mixed, and is added in
500mL distilled water is stirred to being completely dissolved, and obtains mixed liquor, after 100g egg white solutions is taken to stir into milky uniform state, stirring
It is added dropwise in mixed solution, 30h is stirred to react under 60~65 DEG C of temperature conditions, be dried under reduced pressure, grind, and be transferred to 450
6h is kept the temperature in DEG C Muffle furnace, then takes out and is cooled down with ice water mixed liquor, respectively washed 2 times, be dried to obtain with distilled water and absolute ethyl alcohol
Powder be fitted into reactor, under conditions of argon flow amount 100sccm, pressure 5Pa, power 90W, carry out corona treatment
13min obtains Fe3O4Microballoon.
The preparation of carboxylated graphene is the same as embodiment one.
Graphene/Fe3O4The preparation of microballoon：Take 2.0gFe3O4Microballoon and 3.5g carboxylated graphenes be dispersed with stirring in
200mL mass concentrations are in 25% ethylene glycol solution, and 12h is reacted in 220 DEG C of Muffle furnace, and furnace cooling is taken out, magnetic point
Graphene/Fe is obtained from what is washed3O4Microballoon.
Modification of chitosan/graphene/Fe3O4The preparation of microballoon：1.75g chitosans is taken to be dissolved in 200mL mass concentrations be
In 2% acetic acid solution, 2.25g graphenes/Fe is then added in3O4Microballoon is dispersed with stirring uniformly, is warming up to 40 DEG C, adds in 40mL
Petroleum ether and stirring 25min is warming up to 50 DEG C, adds in 10mL formaldehyde and stirs 25min, addition 10mL mass concentrations are the penta 2 of 30%
Alcohol is stirred to react 6h in 63 DEG C, filters, and with ethyl alcohol and distillation water washing, vacuum drying obtains chitosan/graphene/Fe3O4It is micro-
Ball；1.6g dispersibilities nano silicon dioxide and 1g succinic anhydrides is taken to add in every 100mL diethyl ether solutions, the isothermal reaction at 20 DEG C
3h, the solid filtered are dried in vacuo to obtain the dispersed nano silicon dioxide of carboxylated；Take 2g chitosans/graphene/
Fe3O4Microballoon is dispersed in 200mL n-butanols, is added the dispersed nano silicon dioxide of 1g carboxylated, is heated to 65 DEG C
Isothermal reaction 8h, cooling, Magneto separate washing, vacuum drying obtain modification of chitosan/graphene/Fe3O4Microballoon.
The preparation of magnetic bead particles is the same as embodiment one.
The preparation of complex immunity magnetic bead：5mg magnetic bead particles is taken to be washed more than three times with PBS buffer solution Magneto separate, take 1.5mg
Magnetic bead add in 500 μ LPBS buffer solutions in, add 2mg1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and
2mgN- HOSu NHSs, the mixing on turbula shaker, room temperature activate the carboxyl 30min of complex immunity magnetic bead surfaces, with
It is washed afterwards with PBS buffer solution Magneto separate, the magnetic bead after washing is added in 500 μ LPBS buffer solutions, adds in the mixing of CD146 antibody
20h is reacted, by CD146 antibody couplings in magnetic bead surfaces, Magneto separate obtains immunomagnetic beads, then with 500 cow's serums of the μ L containing 1wt%
Immunomagnetic beads 30min is closed in albumin buffer solution, is stored in 2~5 DEG C of environment.
Example IV
The magnetic bead particles that embodiment two is prepared are applied in the isolating and purifying of hepatic stellate cells, as follows：
The magnetic bead particles that 5mg embodiments two are prepared is taken to be washed more than three times with PBS buffer solution Magneto separate, add in 500 μ
In LPBS buffer solutions, 4mg1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 4mgN- hydroxysuccinimidyls are added
Acid imide, the mixing on turbula shaker, room temperature activate the carboxyl 30min of complex immunity magnetic bead surfaces, then use PBS buffer solution
Magneto separate washs, and the magnetic bead after washing is added in 1000 μ LPBS buffer solutions, adds in 1mgCD11b antibody and 1mg CD146 resist
Body mixes hybrid reaction 20h, CD11b antibody and CD146 antibody is coupled to magnetic bead surfaces simultaneously, Magneto separate is contained
The immunomagnetic beads of CD11b antibody and CD146 antibody, then it is immune with being closed in bovine serum albumin(BSA) buffer solutions of the 500 μ L containing 1wt%
Magnetic bead 30min is stored in 2~5 DEG C of environment.
It is as follows to prepare related solution：
Buffer solution I：Weigh NaCl 7g, KCl 0.40g, NaH2PO4H2O 0.065g, Na2HPO40.120g, 4- hydroxyl
Ethyl piperazidine ethanesulfonic acid 1.8g, NaHCO3 0.33g add in 1L ultra-pure waters, stir to being completely dissolved, obtain buffer solution I；
Buffer solution II：Weigh KCl 0.40g, MgSO47H2O 0.04g, MgCl26H2O 0.18g, Na2HPO4
0.08g, KH2PO4 0.04g, NaHCO3 0.30g, glucose 0.850g, CaCl22H2O0.20g add in 1L ultra-pure waters, stir
It mixes to being completely dissolved, obtains buffer solution II；
Perfusate I：Ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) 0.180g, glucose 0.850g are added in every liter of buffer solution I,
Stirring obtains perfusate I to being completely dissolved；
Perfusate II：CaCl22H2O 0.55g, pronase e 0.25g, stirring are added in every liter of buffer solution I
To being completely dissolved, perfusate II is obtained；
Perfusate III：CaCl22H2O 0.55g, clostridiopetidase A D 0.55g are added in every liter of buffer solution I, is stirred to complete
Fully dissolved obtains perfusate III；
Digestive juice：CaCl22H2O 0.20g, pronase e 0.46g, clostridiopetidase A D are added in every liter of buffer solution I
0.46g, deoxyribonuclease 0.018g stir to being completely dissolved, obtain digestive juice；
Cleaning solution：CaCl22H2O 0.20g, deoxyribonuclease 0.02g, stirring are added in every liter of buffer solution I
To being completely dissolved, cleaning solution is obtained；
Centrifugate I：160g Nycoden powder is added in every liter of buffer solution II, stirs to being completely dissolved, obtains centrifugate
Ⅰ；
Centrifugate II stirs to being completely dissolved to add in 80g Nycoden powder in every liter of buffer solution II, obtains centrifugate
Ⅱ。
The method that the hepatic stellate cells of the present embodiment isolates and purifies is as follows：
Sterile anesthesia：Disappear with 1% amobarbital intraperitoneal injection of anesthesia mouse, and with the alcohol immersion of 75% volumetric concentration
Poison, fixation postures open abdominal cavity, fully exposure portal vein and inferior caval vein, in nearly left kidney position and adipose tissue it is more under
At vena cave inject 0.2mL heparin, and place disinfection after cotton balls, away from hepatic portal about 2cm at insertion 24G venous detaining needles,
Needle core is extracted, fixed indwelling tube is ligatured at nearly hepatic portal, retention needle tube is connected to peristaltic pump.
Liver perfusion：It is swollen, and cut off down rapidly by perfusate I to the liver that peristaltic pump filling temperature is 34~37 DEG C
Vena cave bloodletting, it is seen that liver becomes khaki, perfusion rate 11mL/min, Hemoperfusion time 15min, is used in filling process
The expanded state of the cotton balls control liver of placement；It is further continued for the perfusate II that filling temperature is 34~37 DEG C, perfusion rate 11mL/
Min, Hemoperfusion time 15min, equally with the expanded state of cotton balls control liver placed in filling process；Finally filling temperature is
34~37 DEG C of perfusate III continues to be perfused, perfusion rate 11mL/min, Hemoperfusion time 15min, until liver softens, pressure
Recess be not easy to restore, in filling process with place cotton balls control liver expanded state.
Stripping digestion：Perfusion treated liver is cut, rejects liver coating and connective tissue, disinfection scissors fully shreds
Hepatic tissue adds in the digestive juice of 34 DEG C of preheatings, and constant temperature oscillation digests 25min in 34 DEG C of water-bath, and digestion hunting speed is
220rpm/min with after standing 5min in ice-water bath, after larger tissue block precipitates, is filtered, filtrate through 200 mesh screens
In 26 DEG C, 150g/min centrifugations 3min；Suction pipe carefully draws upper strata suspension, avoids being drawn onto the liquid for closing on precipitation, and by upper strata
Suspension is transferred in new sterile centrifugation tube, in 26 DEG C, 1500g/min centrifugations 5min；The upper strata suspension after last time centrifugation is drawn,
Isometric cleaning solution is added in, after mixing, in 26 DEG C, 1500g/min centrifugations 5min；It is upper after careful absorption is cleaned for the first time again
Layer suspension, adds in isometric cleaning solution, after mixing, in 26 DEG C, 1500g/min centrifugations 5min；Supernatant is abandoned, cell is obtained and hangs
Liquid.
Density gradient centrifugation：Obtained cell suspension priority addition I mixing of buffer solution II and centrifugate will be digested, delayed at this time
The volume that fliud flushing II adds in is 2 times of cell suspension, and the volume that centrifugate I adds in is 1.5 times of cell suspension, average after mixing
Divide into 5 centrifuge tubes, the centrifugate II isometric with liquid in centrifuge tube and 0.1 times of volume are added in each centrifuge tube
Buffer solution II, in 2~5 DEG C, 300g/min centrifugation 20min, recycle turbidity zone.
Immunological magnetic bead sorting：The buffer solution II of turbidity zone liquid bovine serum albumin(BSA) containing 0.1wt% of recycling is pressed into 90 μ l/
107A progress cell resuspension, adds in PE label rat anti-mouse F4/80 antibody, and 4 DEG C of incubation 30min are cleaned carefully with buffer solution II
Born of the same parents then add in the 15 μ l/10 of immunomagnetic beads of antibody containing CD11b and CD146 antibody7A cell mixing is incubated 30min in 4 DEG C,
Every 3min mixing cells, then with 500g/min centrifuge washing 8min, Magneto separate is carried out in magnetic field, separation Kupffer is thin
Born of the same parents and sinus hepaticus inner cell, isolated supernatant suspension and antigen-antibody magnetic bead, then with isometric II Magneto separate of buffer solution
It is primary to wash antigen-antibody magnetic bead, obtained cleaning solution is mixed with supernatant suspension to get to hepatic stellate cells solution, is centrifuged,
The stillness of night is abandoned to get to hepatic stellate cells.
The present embodiment is that the magnetic bead particles that embodiment two is prepared are combined simultaneously with CD11b antibody, CD146 antibody,
So as to which the immunomagnetic beads containing CD11b antibody, CD146 antibody be made.It isolates and purifies to obtain liver star using negative magnetic bead sorting method
By obtained hepatic stellate cells under inverted phase contrast microscope, hepatic stellate cells yield is counted with blood cell counting plate for shape cell,
And cell activity is measured with conventional trypan exclusion stain, as a result cell yield 92%, cell survival rate 94%, cell purity
96%.
Example IV first passes through three kinds of perfusates and liver is irrigated, and can reduce the damage of liver cell in separation process,
Improve the motility rate of liver cell；Density gradient separation is then carried out, using the smaller principle of hepatic stellate cells density, obtains enrichment liver
The turbidity zone liquid of sternzellen；Immunomagnetic beads feminine gender sorting is being carried out, by turbidity zone liquid and antibody containing CD11b and CD146 antibody
Immunomagnetic beads carry out mixing, using CD11b antibody and Kupffer cells progress antigen-antibody combined, CD146 antibody and sinus hepaticus
Inner cell carries out the characteristics of antigen-antibody combination, can divide group with other cells not being combined under magnetic fields, thus from
Sinus hepaticus inner cell and Kupffer cells are separated in turbidity zone liquid, the stillness of night left is that the higher liver of purity is starlike thin
Born of the same parents.It is sorted using immunomagnetic beads feminine gender, magnetic bead is non-toxic, not damaged to cell, does not interfere with the cell activity of hepatic stellate cells.
The above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to preferred embodiment to this hair
It is bright to be described in detail, it will be understood by those of ordinary skill in the art that, it can modify to technical scheme of the present invention
Or equivalent replacement, without departing from the objective and range of technical solution of the present invention, the claim in the present invention should all be covered
In range.The present invention be not described in detail technology, shape, construction part be known technology.
Claims (10)
1. the complex immunity magnetic bead that hepatic stellate cells isolates and purifies, which is characterized in that the complex immunity magnetic bead is grain size 50
Grafted antibodies in the magnetic bead particles of~1500nm, the kernel of the magnetic bead particles is Fe3O4Microballoon, from the inside to the outside including carboxylated
Graphene layer, modification of chitosan layer, sodium alginate layer.
2. the complex immunity magnetic bead that hepatic stellate cells according to claim 1 isolates and purifies, which is characterized in that described anti-
Body includes CD11b antibody and CD146 antibody.
3. the complex immunity magnetic bead that hepatic stellate cells according to claim 2 isolates and purifies, which is characterized in that the magnetic
Contain the Fe that mass fraction is 10%~20% in pearl particle3O4Microballoon, 25%~40% modification of chitosan, 20%~35%
Sodium alginate.
4. the complex immunity magnetic bead that hepatic stellate cells according to claim 3 isolates and purifies, which is characterized in that described
Fe3O4Microballoon is using egg white solution pore, plasma treated porous microsphere.
5. the complex immunity magnetic bead that hepatic stellate cells according to claim 4 isolates and purifies, which is characterized in that described to change
Property chitosan is that the dispersed nano silicon dioxide of carboxylated is made with chitosan by dehydration.
6. the complex immunity magnetic bead that hepatic stellate cells according to claim 5 isolates and purifies, which is characterized in that described point
It is with γ-modified nano silicon dioxide of aminopropyl trimethoxy to dissipate property nano silicon dioxide.
7. according to the preparation method of complex immunity magnetic bead that any hepatic stellate cells of claim 1~6 isolates and purifies,
It is characterized by comprising the following steps：
Take Fe3O4Microballoon and carboxylated graphene are dispersed with stirring in the ethylene glycol solution for being 25% in mass concentration, in 200~240
DEG C Muffle furnace in react 10~13h, furnace cooling is taken out, and Magneto separate washs to obtain graphene/Fe3O4Microballoon；
Chitosan is taken to be dissolved in the acetic acid solution that mass concentration is 2%, then adds in graphene/Fe3O4Microballoon is dispersed with stirring
Uniformly, successively stirring adds in petroleum ether, formaldehyde, pentanediol, reacts 5~8h in 60~65 DEG C, filters, is washed with ethyl alcohol and distillation
It washs, vacuum drying obtains chitosan/graphene/Fe3O4Microballoon takes chitosan/graphene/Fe3O4Microballoon is dispersed in positive fourth
In alcohol, the dispersed nano silicon dioxide of carboxylated is added, is heated to 60~65 DEG C of isothermal reaction 8h, is cooled down, Magneto separate is washed
It washs, is dried in vacuo, obtains modification of chitosan/graphene/Fe3O4Microballoon；
Sodium alginate is taken to be dissolved in distilled water, adds modification of chitosan/graphene/Fe3O4Microballoon stirs 2h, freeze-drying
Obtain magnetic bead particles；
Delayed using the PBS containing 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and n-hydroxysuccinimide
After fliud flushing activated magnetic beads particle, the magnetic bead particles of activation and antibody are added in, 15~20h is reacted in PBS buffer solution, Magneto separate obtains
To immunomagnetic beads, then with the bovine serum albumin(BSA) buffer blind immunomagnetic beads 30min containing 1wt%, it is stored in 2~5 DEG C of ring
In border.
8. the preparation method of complex immunity magnetic bead that hepatic stellate cells according to claim 7 isolates and purifies, feature
It is, the Fe3O4Preparing for microballoon is as follows：Take equimolar Fe2(SO4)3And FeSO4·7H2O is dissolved in distilled water, then
It adds in egg white solution and is stirred to react 30h in 60~65 DEG C, be dried under reduced pressure, grind, and be transferred to and keep the temperature 6h into 450 DEG C of Muffle furnaces, with
Afterwards take out with ice water mixed liquor cool down, respectively washed 2 times with distilled water and absolute ethyl alcohol, the powder being dried to obtain be fitted into reactor into
Row corona treatment, obtains Fe3O4Microballoon.
9. the preparation method of complex immunity magnetic bead that hepatic stellate cells according to claim 8 isolates and purifies, feature
It is, the Fe3O4Microballoon prepare in plasma-treating technology condition be：80~120sccm of argon flow amount, pressure 3~
8Pa, 10~15min of power 80~100W corona treatments.
10. the preparation method of complex immunity magnetic bead that hepatic stellate cells according to claim 9 isolates and purifies, feature
It is, preparing for the carboxylated graphene is as follows：Graphene is taken to add in the sodium hydroxide solution of a concentration of 0.3mol/L to surpass
Sound wave disperses 3h, then adds in monoxone ultrasonic response 3h and obtains reaction solution, reaction solution with deionized water is centrifuged repeatedly and is washed
It washs to neutrality, is dried to obtain carboxylated graphene.
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