CN104531611B - The specific surfaces molecular marker CD63 of liver stem cells and its application - Google Patents
The specific surfaces molecular marker CD63 of liver stem cells and its application Download PDFInfo
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Abstract
The present invention relates to medical bioengineering technical field, and the invention provides a kind of specific surfaces molecular marker CD63 of liver stem cells, the present invention still further provides the application of the specific surfaces molecular marker of the liver stem cells.Present invention method of quick separating and culture amplifying liver stem cells from liver using the molecular marker, specifically using the surface specific mark CD63 of liver stem cells, by flow cytometer, quick separating goes out liver stem cells from the Liver Cell Suspension of preparation.CD63+ liver stem cells have self-renewing and two-way differentiation characteristic in vitro, and have therapeutic action to the mouse liver of damage.The liver stem cells that present invention separation obtains can screen, texturize the seed cell of engineering liver and liver diseases cell therapy as liver drug, and the also research for liver development and the characteristics of cell biology of liver stem cells provides preferable cell model.
Description
Technical field
The present invention relates to medical bioengineering technical field, there is provided the specific surfaces molecular marker of liver stem cells
CD63 and its application, and pass through the method for surface marker quick separating and culture amplifying liver stem cells from liver.Specifically
Be related to the surface specific mark using liver stem cells, by the method for airflow classification from Liver Cell Suspension quick separating
Go out liver stem cells, and realize liver stem cells amplification in vitro and two-way differentiation, and in liver diseases animal model liver
Replant.
Background technology
Tissue stem cell refers to the neoblast being present in the tissue that individual has broken up, and this cell has self
Renewal and specialization form the ability of the cell of the tissue.Tissue stem cell is present in the various histoorgans of body, is tissue
The 26S Proteasome Structure and Function basis that organ stable state maintains.Tissue stem cell in adult tissue is under normal circumstances mostly in not
Dormancy state, it can be just activated only in pathological state or in the presence of extraneous factor and breed and divide to the cell type of the tissue
Change.Liver stem cells in liver are considered as being present in Hering ' s pipes and bile duct gland, have and produce liver cell and epithelial duct
The two-way differentiation potential of cell.In normal liver tissue, the quantity of liver stem cells is few, only when liver damage and liver cell
Propagation be suppressed or when liver is by lasting extensive chronic injury, the liver stem cells in liver can just be activated,
I.e. so-called " bile duct reaction ".Normal use in conjunction can suppress medicine (such as the climbing groundsel of hepatocyte growth in animal model
Alkali, paracetamol etc.) and 2/3rds livers cut to induce bile duct to react, and (such as chronic disease in the chronic liver disease of the mankind
Virus hepatitis, PBC etc.) often to the presence for observing bile duct reaction.
Liver stem cells are separately cultured and its furtherd investigate for liver development, regeneration after hepar damnification, liver neoplasm
The cell therapy of whole end-stage liver disease caused by the mechanism and a variety of causes of generation all has highly important Research Significance
And application value.Because liver stem cells quantity present in liver is few, the method for separation of liver stem cells is reported at present still
Imperfection.After Li etc. cuts induction bile duct reaction by senecionine 2/3 liver of joint, liver is prepared into cell suspension, through undue
After level centrifugation removes hepatic parenchymal cells, then by the method for differential velocity adherent purified liver stem cells (Li WL, Su J, Yao YC,
Tao XR,Yan YB,Yu HY,Wang XM,Li JX,Yang YJ,Lau JT,Hu YP,Isolation and
characterization of bipotent liver progenitor cells from adult mouse,Stem
Cells, 24 (2), 322-332), the method that this kind isolates and purifies liver stem cells is comparatively laborious, and is not easy from normal liver tissue
Isolate liver stem cells.In recent years, Foxl1, and the surface marker combination streaming of the liver stem cells such as Lgr5 or magnetic bead point are passed through
The method of choosing isolates liver stem cells (Shin S, Walton G, Aoki from normal liver or the liver of induction bile duct reaction
R,Brondell K,Schug J,Fox A,Smirnova O,Dorrell C,Erker L,Chu AS,Wells RG,
Grompe M,Greenbaum LE,Kaestner KH,Foxl1-Cre-marked adult hepatic progenitors
have clonogenic and bilineage differentiation potential,Genes Dev.25(11):
1185-1192;Dorrell C,Erker L,Schug J,Kopp JL,Canaday PS,Fox AJ,Smirnova O,
Duncan AW,Finegold MJ,Sander M,Kaestner KH,Grompe M,Prospective isolation of
a bipotential clonogenic liver progenitor cell in adult mice.Genes Dev.25
(11):1193-1203;Huch M,Dorrell C,Boj SF,van Es JH,Li VS,van de Wetering M,Sato
T,Hamer K,Sasaki N,Finegold MJ,Haft A,Vries RG,Grompe M,Clevers H,In vitro
expansion of single Lgr5+liver stem cells induced by Wnt-driven regeneration,
Nature.494(7436):247-250.), but these marks can not effectively distinguish liver stem cells and bile duct cell.Therefore,
The method operation of existing separation liver stem cells is complicated or the liver stem cells purity of separation is relatively low, at present still can not be quick
The method that liver stem cells are specifically isolated and purified from liver organization.
CD63 is the memebrane protein for including four hydrophobic domains (hydrophobic domains), is tetratransmembrane protein
One of Major Members of white family (tetraspanin family).CD63 mainly with integrin (integrins) family protein
Complex is formed, participates in the functions such as development, growth, activation and the migration of regulation cell.At present, CD63 is found mainly to be expressed in
T cell, macrophage and the blood platelet of activation.Present invention discover that CD63 is also the specific surfaces molecular marker of liver stem cells
Thing, and the method for quick, special separation and purifying CD63+ liver stem cells from Liver Cell Suspension is provided, meanwhile, realize
Liver stem cells amplification in vitro and two-way differentiation, and replanting in liver diseases animal model liver.CD63+ livers are dry thin
The acquisition of born of the same parents provides platform for the cell therapy of liver diseases and liver disease therapy drug screening, also to develop using CD63 as target
The research such as target diagnosis for liver disease kit and treatment method lays the foundation.
The content of the invention
It is an object of the invention to provide a kind of specific surfaces molecular marker CD63 of liver stem cells.
Another object of the present invention is to provide the specific surfaces molecular marker CD63 of liver stem cells in liver stem cells
Application in the method that quick separating expands and/or induction is broken up.
A further object of the present invention is that the specific surfaces molecular marker CD63 for providing liver stem cells is preparing liver
Application in the medicine of kit for diagnosing diseases and/or treatment liver diseases.
The first aspect of the present invention, there is provided a kind of specific surfaces molecular marker of liver stem cells, described liver do
The specific surfaces molecular marker of cell is CD63.
The specific surfaces mark of the specific surfaces molecular marker, also referred to as liver stem cells of the liver stem cells of the present invention
Surface molecular mark, the surface molecular mark etc. of liver stem cells of thing, liver stem cells.
The second aspect of the present invention, there is provided applications of the CD63 as the specific surfaces mark of liver stem cells, should answer
With applications of the specifically CD63 in liver stem cells quick separating expands and/or induces the method for differentiation.
Specific surfaces marks of the CD63 as liver stem cells, it can divide exactly, specifically from normal liver
Liver stem cells are separated out, can also be induced from DDC in the reacted liver of bile duct and isolate liver stem cells.
Specific surfaces molecular marker CD63 is expanded and/or induced in liver stem cells quick separating in the method for differentiation
Using.For the present invention by liver stem cells surface specific mark CD63, the method with reference to airflow classification is quick special from small
CD63 positive liver stem cells are isolated in mouse liver.Liver stem cells positive CD63 have powerful multiplication capacity and are divided into liver
The two-way differentiation capability of cell and bile duct cell.Wherein described liver refers to normal liver or the DDC induction reacted livers of bile duct
It is dirty.
The described DDC induction reacted livers of bile duct, abductive approach can be found in document Preisegger KH, Factor
VM,Fuchsbichler A,Stumptner C,Denk H,Thorgeirsson SS,Atypical ductular
proliferation and its inhibition by transforming growth factor beta1in the 3,
5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic
liver disease,Lab Invest.79(2):103-109。
Applications of the specific surfaces molecular marker CD63 in the method that liver stem cells quick separating expands, specific method
It is as follows:
A, the liver stem cells of immunofluorescence in situ and the immunohistochemical staining identification CD63 positives induce in normal liver or DDC
Positioning after bile duct reaction in liver.Immunofluorescence and immunohistochemical staining result show the positive liver stem cells of CD63 in liver
Inside it is distributed in Hering ' s pipes and bile duct gland.
B, by the method for selected by flow cytometry apoptosis, induce from normal liver or DDC and isolated after bile duct reaction in liver
The liver stem cells of the CD63 positives (CD63+), and realize the amplification of the positive liver stem cells of CD63 in vitro.First by normal hepatocytes
Dirty or DDC induction bile duct react 3 weeks after liver with two-step method original position collagenase perfusion digestion liver (Wang MJ1, Chen F,
Li JX,Liu CC,Zhang HB,Xia Y,Yu B,You P,Xiang D,Lu L,Yao H,Borjigin U,Yang GS,
Wangensteen KJ,He ZY,Wang X,Hu YP,Reversal of hepatocyte senescence after
continuous in vivo cell proliferation,Hepatology.60(1):349-361.), digestion mixture after
It is continuous to be incubated 30 minutes at 37 DEG C with 0.25% clostridiopetidase A, Hepatic nonparenchymal cell is then enriched with by gradient centrifugation.Pass through again
Selected by flow cytometry apoptosis goes out CD11b, CD31 and CD45 feminine gender, and liver stem cells positive CD63.Finally by liver positive CD63
Stem cell largely expands in SCM-A culture mediums, SCM-A culture mediums:DMEM/F12,10% hyclone (Fetal bovine
Serum, FBS), 1 × mycillin, 0.1mM2- mercaptoethanols (2-Mercaptoethanol, 2-Mer), 1 × insulin-turn
Ferritin-selenium solution (Insulin-Transferrin-Selenium Solution, ITS), 10ng/ml hepatocyte growth factors
Sub (Hepatocyte Growth Factor, HGF), 10ng/ml EGFs (Epidermal growth factor,
EGF), 10ng/ml niacinamides (Nicotinamide), 10-7M dexamethasone (dexamethasone, Dex) and 50ug/ml
Gentamicin (Gentamycin).
Described immunofluorescence in situ and immunohistochemical staining positioning, reference can be made to document Yu B, He ZY, You P, Han
QW,Xiang D,Chen F,Wang MJ,Liu CC,Lin XW,Borjigin U,Zi XY,Li JX,Zhu HY,Li WL,
Han CS,Wangensteen KJ,Shi Y,Hui LJ,Wang X,Hu YP,Reprogramming fibroblasts
into bipotential hepatic stem cells by defined factors,Cell Stem Cell.13(3):
328-340。
The method of described selected by flow cytometry apoptosis, reference can be made to document Wang MJ1, Chen F, Li JX, Liu CC,
Zhang HB,Xia Y,Yu B,You P,Xiang D,Lu L,Yao H,Borjigin U,Yang GS,Wangensteen
KJ,He ZY,Wang X,Hu YP,Reversal of hepatocyte senescence after continuous in
vivo cell proliferation,Hepatology.60(1):349-361.;Flow cytometer model Becton used
Dickinson FACs Calibur。
It is preferred that the wherein positive liver stem cells of step A situs immunofluorescence and immunohistochemical staining identification CD63 exist
Positioning after normal liver or DDC induction bile duct reactions in liver concretely comprises the following steps:
A1, normal liver and DDC induction bile duct react 3 weeks after liver with OCT embedding mediums embed after, frozen section 7um
It is thick.4% paraformaldehyde (PFA) room temperature fixes 10 minutes, removes fixer, and PBS is washed three times, and 4 DEG C save backup;
A2, immunofluorescence dyeing:By the liver section after step A1 processing with 1% bovine serum albumin(BSA) (bovine
Serum album, BSA) room temperature close 30 minutes after, dropwise addition confining liquid dilution the antibody of mouse anti-CD 63 and rabbit-anti CK19 resist
Body, 4 DEG C overnight.PBS-T is washed three times, 5 minutes every time, and goat anti-rabbit igg (the FITC-Goat anti of FITC marks are added dropwise
Rabbit IgG) and TRITC mark sheep anti-mouse igg (TRITC-Goat anti Mouse IgG), 37 DEG C react 30 minutes,
Washed 3 times, every time 5 minutes with PBS-T;DAPI redyes nucleus 1 minute, and PBS-T is washed 2 times.Anti- fluorescence decay mountant mounting,
Fluorescence microscopy Microscopic observation.
A3, immunohistochemical staining:By the liver section 0.3% dioxygen water seal 30 minutes after step A2 processing,
PBS-T is washed three times, and after 1%BSA room temperatures are closed 30 minutes, the antibody of mouse anti-CD 63 that dropwise addition confining liquid dilute, 4 DEG C overnight.
PBS-T is washed three times, 5 minutes every time.It is added dropwise in section and marks secondary antibody with the HRP of PBS-T dilutions, 37 DEG C is incubated 30 minutes, use
PBS-T is washed 3 times, every time 5 minutes.DAB dyeing liquor (the article No.s for the method Fresh that by specification provides are added dropwise in section:
Kit-0014, Fuzhou Maixin biotechnology Development Co., Ltd), Microscopic observation section color change, when color is obvious, immediately
Slice, thin piece is put into running water and rinsed, color development stopping reaction.After DAB colour developings, haematine (article No.:H3136, purchased from Sigma) it is multiple
Dye, mounting after dehydration.
It is preferred that wherein step B's concretely comprises the following steps:
B1, two-step method original position collagenase perfusion digestion liver (Wang MJ1, Chen F, Li JX, Liu CC, Zhang
HB,Xia Y,Yu B,You P,Xiang D,Lu L,Yao H,Borjigin U,Yang GS,Wangensteen KJ,He
ZY,Wang X,Hu YP,Reversal of hepatocyte senescence after continuous in vivo
cell proliferation,Hepatology.60(1):349-361.), digestion mixture continues to be existed with 0.25% clostridiopetidase A
37 DEG C are incubated 30 minutes.
B2, postdigestive cell suspension 50g centrifugal force low-speed centrifugal abandon precipitation (big liver cell) in 30 minutes twice, receive
After collecting supernatant, continue to use 300g centrifugal forces 5 minutes, cell is resuspended with serum free medium, counts, cell is diluted to
107/ ml, liquid (OptiPrep is layered using density gradient centrifugationTMDensity Gradient Medium) (article No.:D1556,
Sigma companies) enrichment nonparenchymal cell.
After the nonparenchymal cell that B3, collection are enriched to, PBS is washed twice, and serum free medium is resuspended.Use anti-mouse
CD16/32 antibody (every 106Cell 1ug antibody), 4 DEG C are closed 10 minutes.
B4,4 DEG C of lucifuges are incubated CD63-PE antibody (every 106Cell 0.5ug antibody), while CD11b, CD31 are incubated,
CD45-FITC antibody (every 106Cell 0.2ug antibody), 20 minutes.It is thin with the PBS containing 2%FBS (mass percent)
Born of the same parents three times.
B5, sorted using streaming instrument, to obtain the CD63-PE positives, CD11b, CD31, cell negative CD45-FITC.
96 holes of the liver stem cells extremely culture medium containing SCM-A for the CD63+ that 500 B6, inoculation selected by flow cytometry apoptosis go out
In plate, 37 DEG C, 5%CO2, 95% damp condition culture 2 weeks.
Further, after above-mentioned steps B, present invention also offers CD63 in the method for liver stem cells induction differentiation
Induced in vitro point using, liver stem cells positive CD63 after the liver stem cells positive CD63 that specifically isolates or amplification
Turn to mature hepatocytes or bile duct epithelial cell.
Above-mentioned steps B, the method by selected by flow cytometry apoptosis, from liver after normal liver or DDC induction bile duct reactions
In isolate the positive liver stem cells of CD63, and realize the amplification of the positive liver stem cells of CD63 in vitro.
The invention provides the method that the positive liver stem cells of CD63 are induced to differentiate into mature hepatocytes in vitro.This method
Specially step C.
C, liver stem cells positive CD63 liver under inductive condition (DMEM/F12,10%FBS, 10ng/ml HGF,
10ng/ml EGF, 10-7M Dex, 1% dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide, DMSO), 10ng/ml tumour inhibitors
(oncostatin M, OSM), 20% matrigel (Matrigel)) ripe hepatic lineage is induced to differentiate into, liver positive CD63 does
The hepatic lineage height that cell induction is differentiated to form is expressed liver cell specific function gene (CYP7a1, G6PD, AAT etc.) and had
Store the function of glycogen.
It is preferred that specific induction step is:
C1, by 5 × 104Cell/cm2By liver stem cells kind positive CD63 in 1 Collagen Type VI (article No.:354236, purchased from BD
Company) coated 35mm culture dishes, add SCM-A culture mediums, in 37 DEG C, 5%CO2Culture to cell covers with incubator, its
Between change liquid every other day.
After C2, cell cover with, liver is changed to inducing culture (DMEM/F12,10%FBS, 10ng/ml HGF, 10ng/ml
EGF, 10-7M Dex, 1%DMSO, 10ng/ml OSM, 20%Matrigel) continue culture 6 days, change liquid every other day therebetween.
Present invention also offers the positive liver stem cells of CD63 to be induced to differentiate into bile duct epithelial cell in vitro.This method has
Body is step D.
D, liver stem cells positive CD63 are induced to differentiate into the pipe sample knot of branch's sample under NTx three-dimensional cultivation condition
Structure, the mark Krt19 and EpCAM of the pipe spline structure expression bile duct cell of induced synthesis.
It is preferred that specific induction step is:
D1, solution used are placed in 4 DEG C of precoolings 30 minutes;
D2, prepare type i collagen gel solution:Type i collagen 800ul, 10 × PBS 100ul, 1N NaOH 20ul,
DdH2O80ul is mixed, and is placed on ice.
D3, by 1ml bile ducts induction broth (DMEM, 10%FBS, 1x mycillin, 1x ITS, 20ng/ml HGF,
50ug/ml Gentamycin) be resuspended 5 × 104Liver stem cells and the type i collagen gel of above-mentioned preparation positive individual CD63 are molten
Liquid mixes, and adds in 35mm culture dishes;
D4, culture dish is placed in 37 DEG C, 5%CO2Cultivated 2 hours in incubator, wait the gel sets containing cell;
D5,1.5ml bile duct induction broths are added into culture dish, and cell continued into culture (period changes every other day within 9 days
Liquid).
Feature daughter cell caused by CD63 positive liver stem cells that above-mentioned separation amplification method obtains and its differentiation.
Feature daughter cell caused by CD63 positive liver stem cells that above-mentioned separation amplification method obtains and its differentiation exists
Prepare the application in systematism engineering liver.
Feature daughter cell caused by CD63 positive liver stem cells that above-mentioned separation amplification method obtains and its differentiation exists
Prepare the application in the medicine of cell therapy acute hepatic failure and various whole end-stage liver diseases.
The third aspect of the present invention, there is provided another applications of the CD63 as the specific surfaces mark of liver stem cells,
The application specifically specific surfaces molecular marker CD63 is preparing diagnosis for liver disease kit and/or treatment liver diseases
Medicine in application.
Applications of the CD63 of the present invention in diagnosis for liver disease kit is prepared, inspection is included in described kit
Survey the reagent of CD63 expression quantity.
Marks of the CD63 as liver stem cells, with tracer and liver stem cells can be disclosed in liver neoplasm generation, liver fiber
The effect that liver stem cells are played during change and hepatic sclerosis, CD63 also are used as the early diagnosis mark of liver diseases
Thing.
Applications of the CD63 of the present invention in prevention or treatment liver diseases medicine is prepared, described medicine are foregoing
CD63 in the application during liver stem cells quick separating expands or induced the method for differentiation the obtained CD63 of separation amplification method
Feature daughter cell caused by positive liver stem cells and its differentiation.
CD63 positive liver stem cells can be as liver drug screening, systematism engineering liver and liver diseases cell therapy
Seed cell, the also research for liver development and the characteristics of cell biology of liver stem cells provide preferable cell model.
The courage that liver stem cells positive CD63 are induced hepatocellular injury model mice (Fah knock out mice) and DDC
Pipe damage model mouse has the ability that liver is grown again, and shows therapeutic action.
It can be replanted in damage in the model mice body that liver stem cells positive CD63 pass through Spleen transplantation to hepar damnification
Liver, and alleviate the damage of liver.After in liver stem cell transplantation to the mouse model body of liver cell chronic injury positive CD63,
Liver stem cells positive CD63 are colonized in liver and are divided into mature hepatocytes to alleviate the damage of liver, are embodied in damage
The recovery of wound model mouse liver function.And liver stem cell transplantation positive CD63 is to the model mice of the DDC bile duct injuries induced
Afterwards, then bile duct epithelial cell is divided into be colonized on bile duct.
Beneficial effects of the present invention:
The invention provides the positive liver stem cells of the CD63 obtained according to above-mentioned isolated culture method.Liver positive CD63
Stem cell possesses the most essential feature of liver stem cells, i.e., powerful multiplication capacity and can be divided into under specific inductive condition
Ripe liver cell and the two-way differentiation capability of bile duct epithelial cell.
The innovative point of the present invention is:1) CD63 is the surface specific mark of liver stem cells;2) CD63 is combined to indicate
Thing and flow cytometer can be isolated and purified out quickly with multiplication capacity from normal liver and the liver of DDC induction bile duct reactions
With the liver stem cells of two-way differentiation capability;3) liver stem cells positive CD63 have grows ability again to liver injury, and shows
Go out therapeutic action.
The specific surfaces mark CD63 tools of liver stem cells provided by the present invention have been widely used, and CD63 is as liver
The mark of stem cell, with tracer and process of the liver stem cells in liver neoplasm generation, liver fibrosis and hepatic sclerosis can be disclosed
The effect that middle liver stem cells are played, CD63 also are used as the early diagnosis marker of liver diseases.
CD63 positive liver stem cells provided by the present invention, which also have, to have been widely used.Liver drug screening, group can be used as
The seed cell of chemical industry journey liver and liver diseases cell therapy is knitted, is also the cell biology of liver development and liver stem cells
The research of characteristic provides preferable cell model.
Brief description of the drawings
Fig. 1 is the positioning figure of CD63 positive liver stem cells in wild-type mice liver.
Wherein A:CK19 immunofluorescence dyeings, the cell shown in white arrow on pipeline are CK19 positive cells, B:CD63
Immunofluorescence dyeing, the cell shown in white arrow on pipeline are CD63 positive cells, C:A and B stacking chart, white arrow institute
It is CK19 and CD63 double positive cells to show the cell on pipeline;D:CD63 immunohistochemical staining, 1 and 2 is in square frames in figure D
Partial enlarged drawing, cell shown in black arrow are CD63 positive cells.Scale:100 microns.
Fig. 2 is the positioning figure of CD63 positive liver stem cells in liver after DDC induction bile ducts react.
Wherein A:CK19 immunofluorescence dyeings, cell shown in white arrow are CK19 positive cells, B:CD63 immunofluorescences
Dyeing, cell shown in white arrow are CD63 positive cells, C:A and B stacking chart, cell shown in white arrow be CK19 and
CD63 double positive cells;D:CD63 immunohistochemical staining, 1 and 2 be the partial enlarged drawing in square frame, black arrow institute in figure D
It is CD63 positive cells to show cell.Scale:100 microns.
Fig. 3 is the scatter diagram by surface marker CD63 airflow classification liver stem cells.
Wherein A:In wild-type mice liver CD11b-CD31-CD45-CD63+ liver stem cells ratio be 0.2% (Q4 as
Limit);B:After DDC induction bile duct reactions in liver CD11b-CD31-CD45-CD63+ liver stem cells ratio be 0.5% (Q4 as
Limit).
Fig. 4 is the aspect graph of the positive liver stem cells of the CD63 of airflow classification.
Wherein A:It is selected from shape of the liver stem cells of the CD63 positives in wild-type mice liver under inverted phase contrast microscope
State;B:Liver stem cells positive CD63 in mouse liver after DDC induction bile ducts react are selected under inverted phase contrast microscope
Form.
Fig. 5 is the growth curve chart in CD63 positive the 10th generations of liver stem cells (P10) and the 30th generation (P30).Shown in figure
Liver stem cells positive p10 and P30 CD63 do not have difference in multiplication capacity.
Fig. 6 is the positive liver stem cells of CD63 related gene expression situation of change after liver breaks up to induction and courage to induction
Figure.Courage is to bile duct specific gene (CK19, Gja1, Ggt1) expression enhancing after induction, and liver is specific to liver cell after induction
Gene (Alb, Hnf4a, Cps1, Aat, Cyp7a1, G6p) expression enhancing.
Fig. 7 is staining for glycogen figure after the positive liver stem cells livers of CD63 break up to induction.
Wherein A:Cell after liver stem cells liver positive CD63 breaks up to induction after staining for glycogen display induction has storage
Deposit the function of glycogen;B:For the partial enlarged drawing in white box in figure A, it is that staining for glycogen is positive to scheme black dotted lines inframe in B
Cell.
Fig. 8 is the gallbladder tube structure figure that branch's sample is formed after the positive liver stem cells courages of CD63 break up to induction.
Wherein A:Immunofluorescence dyeing bile duct mark Krt19, cell shown in arrow are Krt19 positive cells;B:It is immune
Fluorescent staining bile duct mark EpCAM, cell shown in arrow are EpCAM positive cells;C:The branch's sample bile duct formed after induction
Form of the structure under inverted phase contrast microscope.
Fig. 9 is that the positive liver stem cells of CD63 indicate the form after green fluorescence and green fluorescence expression in vitro
Figure.
Forms of the wherein A for the liver stem cells positive CD63 after the upper green fluorescence of mark under inverted phase contrast microscope, B
To indicate the positive liver stem cells of the CD63 after upper green fluorescence all expression green fluorescences.
After Figure 10 grows Fah knock out mice livers again for the positive liver stem cells of the CD63 after the upper green fluorescence of mark,
The immunofluorescence dyeing figure of liver section.
Wherein A is that immunofluorescence dyeing is shown in Fah knock out mice liver from the liver stem cells that CD63 is positive
Cell expression Fah genes (in white dashed line);The serial section immunofluorescence dyeing that B is A shows Fah knock out mice livers
From the cell expression GFP of liver stem cells positive CD63 in dirty (in white dashed line);C is A serial section immunofluorescence
Dyeing is shown in Fah knock out mice liver from the cell expression liver cell specificity base of liver stem cells positive CD63
Because of Alb (in white dashed line).
Figure 11 is that the positive liver stem cells of CD63 grow the liver function schematic diagram after Fah knock out mice livers again.CD63
After positive liver stem cells grow Fah knock out mice livers again, Fah knock out mice liver functions are clearly better, liver
The index ALT (A) of damage, AST (B) and Tbil (D) are decreased obviously, and liver synthesis albumin A LB (C) ability substantially carries
Rise.
After Figure 12 grows the mouse liver of DDC damages again for the positive liver stem cells of the CD63 after the upper green fluorescence of mark, liver
The immunofluorescence dyeing figure of dirty section.
Wherein A is determining for the liver stem cells that DDC damages on the bile duct of mouse liver the CD63 positives for having green fluorescence mark
Plant (shown in arrow);B is that DDC damages in mouse liver the liver stem cells expression bile duct cell that the CD63 positives are come from bile duct
Specificity marker CK19;C is A and B fusion figure.
Specific implementation method
Elaborated in conjunction with the implementation of embodiments of the invention and accompanying drawing to the present invention, following examples are with this
Implemented under premised on the technical scheme of invention, and give detailed embodiment and specific operating process, but this hair
Bright protection domain is not limited to following embodiments.
Positioning of the embodiment 1.CD63 liver stem cells in liver
1st, normal liver and DDC induction bile duct react 3 weeks after liver (Preisegger KH, Factor VM,
Fuchsbichler A,Stumptner C,Denk H,Thorgeirsson SS,Atypical ductular
proliferation and its inhibition by transforming growth factor beta1in the 3,
5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic
liver disease,Lab Invest.79(2):103-109) use OCT embedding medium (article No.s:4583, purchased from SAKURA companies)
After embedding, frozen section 7um is thick.4% paraformaldehyde (PFA) (article No.:P-6148, purchased from Sigma companies) room temperature fixes 10 points
Clock, remove fixer, PBS (article No.s:BS7016, purchased from Sangon Biotech (Shanghai) Co., Ltd.) wash three times, 4 DEG C of guarantors
Deposit standby.
2nd, immunofluorescence dyeing:By the 1%BSA (article No.s of the liver section after step 1 is handled:A1933, purchased from sigma
Company) room temperature close 30 minutes after, be added dropwise with 1%BSA dilute the antibody (article No. of mouse anti-CD 63:Mab5417, it is public purchased from R&D
Department) and rabbit-anti CK19 antibody (article No.s:A3190, purchased from Abbomax companies), 4 DEG C are overnight.PBS-T (article No.s:Rbr01485, purchase
From Shanghai Rong Bai Bioisystech Co., Ltd) wash three times, 5 minutes every time, FITC-Goat anti Rabbit IgG are added dropwise
(goat anti-rabbit igg of FITC marks, article No.:111-095-003, purchased from Jackson companies) and TRITC-Goat anti Mouse
IgG (sheep anti-mouse igg of TRITC marks, article No.:115-025-003, purchased from Jackson companies), 37 DEG C are reacted 30 minutes, are used
PBS-T is washed 3 times, every time 5 minutes;DAPI (article No.s:D6584, purchased from Sangon Biotech (Shanghai) Co., Ltd.) it is multiple
Contaminate nucleus 1 minute, PBS-T is washed 2 times.Anti- fluorescence decay mountant (article No.:C1210, have purchased from Beijing Puli's lema gene technology
Limit company) mounting, fluorescence microscopy Microscopic observation.Immunofluorescence dyeing result is shown:In normal liver and DDC induction bile duct reactions
Liver in, cell coexpression CK19 (Figure 1A -1C and Fig. 2A -2C) positive CD63.
3rd, immunohistochemical staining:Liver after step 1 is handled is closed 30 minutes with 0.3% hydrogen peroxide (volume ratio),
PBS-T is washed three times, after 1%BSA room temperatures are closed 30 minutes, the antibody of mouse anti-CD 63 diluted with 1%BSA is added dropwise, 4 DEG C overnight.
PBS-T is washed three times, 5 minutes every time.It is added dropwise in section and marks secondary antibody (article No. with the HRP of PBS-T dilutions:Sc-2005, it is purchased from
Santa Cruz companies), 37 DEG C are incubated 30 minutes, are washed 3 times, every time 5 minutes with PBS-T.By specification is added dropwise in section to carry
DAB dyeing liquor (the article No.s of the method Fresh of confession:Kit-0014, Fuzhou Maixin biotechnology Development Co., Ltd), under mirror
Slice, thin piece, when color is obvious, is put into running water rinses immediately by observation section color change, color development stopping reaction DAB colour developings
Afterwards, haematine (article No.:H3136, purchased from Sigma) redye, mounting after dehydration.
As a result show the positive liver stem cells of CD63 in liver positioned at Hering ' s pipes (Fig. 1 D and Fig. 2 D-1) and bile duct
Gland (Fig. 2 D-2).
The quick separating and amplification cultivation of embodiment 2.CD63 positive liver stem cells
1st, two-step method original position collagenase perfusion digestion liver (Wang MJ1, Chen F, Li JX, Liu CC, Zhang HB,
Xia Y,Yu B,You P,Xiang D,Lu L,Yao H,Borjigin U,Yang GS,Wangensteen KJ,He ZY,
Wang X,Hu YP,Reversal of hepatocyte senescence after continuous in vivo cell
proliferation,Hepatology.60(1):349-361.), digestion mixture continues with 0.25% clostridiopetidase A (article No.:
11088858001, purchased from Roche) be incubated 30 minutes at 37 DEG C.
2nd, postdigestive cell suspension 50g centrifugal force low-speed centrifugal abandons precipitation (big liver cell) in 30 minutes twice, collects
Supernatant continues to use 300g centrifugal forces 5 minutes, with plasma-free DMEM medium (article No.:SH30022.01, it is purchased from
Hyclone companies) cell is resuspended, count, cell is diluted to 107/ ml, uses OptiPrepTM Density Gradient
Medium (article No.s:D1556, sigma company) layering liquid enrichment nonparenchymal cell.
3rd, after collecting the nonparenchymal cell being enriched to, PBS is washed twice, and serum free medium is resuspended.Use anti-mouse CD16/
32 antibody (article No.s:101309, purchased from Biolegend companies) (every 106Cell 1ug antibody), 4 DEG C are closed 10 minutes.
4th, 4 DEG C of lucifuges are incubated CD63-PE antibody (article No.s:143904, purchased from Biolegend companies) (every 106Cell
0.5ug antibody), while it is incubated CD11b-FITC (article No.s:11-0112, purchased from eBioscience companies), CD31 (article No.s:
CYT-31F2, purchased from Cytognos companies), CD45-FITC antibody (article No.s:553079, purchased from BD companies) (every 106Cell
0.2ug antibody), 20 minutes.With the PBS cell three times containing 2%FBS.
5th, sorted using streaming instrument (flow cytometer model Becton Dickinson FACs Calibur), to obtain
Obtain the PE positives, cell negative FITC.Liver stem cells positive CD63 are about 0.2%, DDC induction bile ducts in normal mouse liver
Liver stem cells positive CD63 are about 0.5% (Fig. 3) in the liver of reaction.
6th, the liver stem cells for the CD63+ that 500 selected by flow cytometry apoptosis go out are inoculated with to 96 orifice plates of the culture medium containing SCM-A
In, 37 DEG C, 5%CO2, after 95% damp condition culture 2 weeks, no matter normal liver or DDC induction bile duct reaction liver in
The positive liver stem cells of CD63 be respectively formed typical Epithelial clone (Fig. 4).
7th, liver stem cells positive CD63 show stronger multiplication capacity under the condition of culture of SCM-A culture medium,
The multiplication capacity no significant difference (Fig. 5) of the multiplication capacity of its 30th generation cell (P30) and the 10th generation cell (P10).
Embodiment 3.CD63 positive liver stem cells are divided into mature hepatocytes
1st, by 5 × 104Cell/cm2By liver stem cells kind positive CD63 in the coated 35mm culture dishes of 1 Collagen Type VI, addition
SCM-A culture mediums, in 37 DEG C, 5%CO2Culture is covered with to cell in incubator, changes liquid every other day therebetween.
2nd, after cell covers with, liver is changed to inducing culture (DMEM/F12,10%FBS, 10ng/ml HGF, 10ng/
MlEGF, 10-7M Dex, 1%DMSO, 10ng/ml OSM, 20%Matrigel) continue culture 6 days, change liquid every other day therebetween.
3rd, the detection for the mature hepatocytes that the induction of CD63 positive liver stem cells is differentiated to form:Collect the cell formed after induction
RNA is extracted, Real-Time detections are shown, the table of the mark (Abcg2, CK19, Gja1, Ggt1) of stem cell and bile duct cell
Significantly lowered before relatively being induced up to amount, and the expression quantity of the mark (Alb, Hnf4a, Cps1, Aat, Cyp7a1, G6P) of liver cell
Compared with significantly rising (Fig. 6) before induction.Cell after PAS dyeing display inductions has the function (Fig. 7) of glycogen deposit, result above
Show, under the induction system, cell positive CD63 can be induced to differentiate into mature hepatocytes.
Embodiment 4.CD63 positive liver stem cells are divided into bile duct epithelial cell
1st, solution used is placed in 4 DEG C of precoolings 30 minutes;
2nd, type i collagen gel solution is prepared:Type i collagen 800ul, 10 × PBS 100ul, 1N NaOH 20ul, ddH2O
80ul is mixed, and is placed on ice.
3rd, by 1ml bile ducts induction broth (DMEM, 10%FBS, 1x mycillin, 1x ITS, 20ng/ml HGF,
50ug/ml Gentamycin) be resuspended 5 × 104Liver stem cells and the type i collagen gel of above-mentioned preparation positive individual CD63 are molten
Liquid mixes, and adds in 35mm culture dishes;
4th, culture dish is placed in 37 DEG C, 5%CO2Cultivated 2 hours in incubator, wait the gel sets containing cell;
5th, 1.5ml bile duct induction broths are added into culture dish, and cell is continued into culture 9 days (period changes liquid every other day)
Cell forms typical branch's sample pipe spline structure (Fig. 8) afterwards.Branch's spline structure immunofluorescence dyeing shows, the branch of induced synthesis
Spline structure expresses the mark CK19 and EpCAM (Fig. 8 A and 8B) of bile duct cell.These results show in courage under inductive condition,
Liver stem cells positive CD63 can be induced to differentiate into bile duct epithelial cell.
Liver stem cells positive embodiment 5.CD63 treat chronic hepatocellular injury model mice
1st, with liver stem cells positive green fluorescence mark CD63, by 106Lentiviral particle (the goods of individual expression GFP genes
Number:12255, purchased from Addgene companies) add to 105In liver stem cells culture dish positive individual CD63, virus infection 72 hours
2ug/ml puromycin (PURO) (article No. is added afterwards:J593, purchased from Amresco companies) screen one week, after PURO screenings
Liver stem cells positive CD63 all express GFP (Fig. 9).
2nd, take 8-10 week old Normal-weights Fah knock out mice (Wang MJ1, Chen F, Li JX, Liu CC,
Zhang HB,Xia Y,Yu B,You P,Xiang D,Lu L,Yao H,Borjigin U,Yang GS,Wangensteen
KJ,He ZY,Wang X,Hu YP,Reversal of hepatocyte senescence after continuous in
vivo cell proliferation,Hepatology.60(1):349-361.), anaesthetized in super-clean bench, alcohol disinfecting;
3rd, abdominal cavity, exposure spleen are opened under rib on the left of abdominal cavity;
4th, will about 106Liver stem cells positive individual mark GFP CD63 are suspended from 100ul PBS, with 1ml micro syringes
Cell suspension is subjected to spleen injection;
5th, SPF level Animal Houses are put back to after mouse is sewed up a wound to continue to raise, and NTBC (2- nitros -4- are removed in its drinking water
Fluoroform stupid -1,3 cyclohexane iodine, after Fah knock out mice goes out in above-mentioned steps 2, NTBC is added in its drinking-water, to
The accumulation of caused metabolic poison after Fah gene delections processed, hepatic injury occurs from mouse).
6th, the 8th week after cell transplantation, mouse liver and venous blood are collected, and the liver of the analysis mark GFP CD63 positives does
Cell in liver grow again situation and to hepar damnification therapeutic action (specific experiment method is shown in Wang MJ1, Chen F,
Li JX,Liu CC,Zhang HB,Xia Y,Yu B,You P,Xiang D,Lu L,Yao H,Borjigin U,Yang GS,
Wangensteen KJ,He ZY,Wang X,Hu YP,Reversal of hepatocyte senescence after
continuous in vivo cell proliferation,Hepatology.60(1):349-361.).Liver section is exempted from
Epidemic disease fluorescent staining shows that the liver liver plate of Fah knock out mice has and grows (figure again from liver stem cells positive GFP
10B), and these cells grown again express the specific gene Fah (Figure 10 A) and Alb (Figure 10 C) of liver cells.Mouse is quiet simultaneously
The liver function test result of arteries and veins blood shows that index ALT, AST and Tbil of hepar damnification do not carry out the Fah genes of cell transplantation
Knock-out mice is decreased obviously, and liver synthesis Alb ability is obviously improved (Figure 11).Result above shows the positive livers of CD63
Stem cell can grow in being divided into mature hepatocytes in the liver of chronic hepatocellular injury again, and show therapeutic action.
Liver stem cells positive embodiment 6.CD63 replant DDC induction bile duct injury model mices
1st, the wild-type mice of DDC feedings 8-10 week old Normal-weights 3 days.
2nd, by 106Liver stem cells positive individual expression GFP CD63 be fed with 3 days by Spleen transplantation to DDC after mouse
In liver, implantation method is the same as embodiment 5.
3rd, the mouse after transplanting continues to be fed with DDC, and mouse liver is collected after 3 weeks.
4th, the CD63 positive liver stem cells that the expression GFP of transplanting is detected by immunofluorescence dyeing induce bile duct to damage in DDC
(specific experiment method is shown in Yu B, He ZY, You P, Han QW, Xiang D, Chen to situation of replanting in the mouse liver of wound
F,Wang MJ,Liu CC,Lin XW,Borjigin U,Zi XY,Li JX,Zhu HY,Li WL,Han CS,
Wangensteen KJ,Shi Y,Hui LJ,Wang X,Hu YP,Reprogramming fibroblasts into
bipotential hepatic stem cells by defined factors,Cell Stem Cell.13(3):328-
340)。
There are external source GFP positive cells on the bile duct that coloration result is shown in the mouse liver of DDC induction bile duct injuries
It is implanted into (Figure 12 A), and cell expression bile duct cell Specific marker CK19 (Figure 12 B and the figure of the GFP positives of these external sources
12C), result above shows that the positive liver stem cells of CD63 can replant the bile duct of the mouse liver into DDC induction bile duct injuries
Bile duct epithelial cell that is interior and being divided into maturation.
The preferred embodiment to the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (13)
- Applications of the 1.CD63 in liver stem cells quick separating expands or induces the method for differentiation, described application is to make CD63 For the specific surfaces molecular marker of liver stem cells, CD63 positive livers are isolated from liver do with reference to the method for airflow classification Cell.
- 2. applications of the CD63 according to claim 1 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, the liver refers to normal liver or the DDC induction reacted livers of bile duct.
- 3. applications of the CD63 according to claim 1 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, applications of the CD63 in the method that liver stem cells quick separating expands comprises the following steps that:A, the liver stem cells of immunofluorescence in situ and the immunohistochemical staining identification CD63 positives are in normal liver or DDC induction bile ducts Positioning after reaction in liver;B, by the method for selected by flow cytometry apoptosis, induced from normal liver or DDC and isolate CD63 in liver after bile duct reaction Positive liver stem cells, and realize the amplification of the positive liver stem cells of CD63 in vitro;Normal liver or DDC are induced first Bile duct react 3 weeks after liver two-step method original position collagenase perfusion digestion liver, digestion mixture continues with 0.25% collagen Enzyme is incubated 30 minutes at 37 DEG C, is then enriched with Hepatic nonparenchymal cell by gradient centrifugation;Pass through selected by flow cytometry apoptosis again Go out CD11b, CD31 and CD45 feminine gender, and liver stem cells positive CD63;Finally liver stem cells positive CD63 are trained in SCM-A Support and largely expanded in base;The SCM-A culture mediums are DMEM/F12,10% hyclone, 1 × mycillin, 0.1mM 2 mercapto ethanols, 1 × pancreas Island element-transferrins-selenium solution, 10ng/ml HGFs, 10ng/ml EGFs, 10ng/ml Buddhist nun gram acyl Amine, 10-7M dexamethasone and 50 μ g/ml Gentamicins.
- 4. applications of the CD63 according to claim 3 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, the wherein positive liver stem cells of step A situs immunofluorescence and immunohistochemical staining identification CD63 are normal Positioning after liver or DDC induction bile duct reactions in liver concretely comprises the following steps:A1, normal liver and DDC induction bile duct react 3 weeks after liver with OCT embedding mediums embed after, the μ m-thick of frozen section 7; 4% paraformaldehyde room temperature fixes 10 minutes, removes fixer, and PBS is washed three times, and 4 DEG C save backup;A2, immunofluorescence dyeing:Liver section after step A1 processing is closed 30 minutes with 1% bovine serum albumin(BSA) room temperature Afterwards, the antibody of mouse anti-CD 63 and rabbit-anti CK19 antibody that dropwise addition confining liquid dilutes, 4 DEG C overnight;PBS-T is washed three times, 5 points every time Clock, the goat anti-rabbit igg of FITC marks and the sheep anti-mouse igg of TRITC marks is added dropwise, 37 DEG C are reacted 30 minutes, and 3 are washed with PBS-T It is secondary, 5 minutes every time;DAPI redyes nucleus 1 minute, and PBS-T is washed 2 times;Anti- fluorescence decay mountant mounting, under fluorescence microscope Observation;A3, immunohistochemical staining:By the liver section 0.3% dioxygen water seal 30 minutes after step A2 processing, PBS-T is washed Three times, after 1%BSA room temperatures are closed 30 minutes, the antibody of mouse anti-CD 63 that dropwise addition confining liquid dilute, 4 DEG C overnight;PBS-T is washed Three times, every time 5 minutes;It is added dropwise in section and marks secondary antibody with the HRP of PBS-T dilutions, 37 DEG C is incubated 30 minutes, and 3 are washed with PBS-T It is secondary, 5 minutes every time;The DAB dyeing liquors of dropwise addition Fresh in section, Microscopic observation section color change, when color is obvious, Slice, thin piece is put into running water immediately and rinsed, color development stopping reaction;After DAB colour developings, haematoxylin redyeing, mounting after dehydration.
- 5. applications of the CD63 according to claim 3 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, wherein step B's concretely comprises the following steps:B1, two-step method original position collagenase perfusion digestion liver, digestion mixture continue to be incubated 30 at 37 DEG C with 0.25% clostridiopetidase A Minute;B2, postdigestive cell suspension 50g centrifugal force low-speed centrifugal abandon precipitation in 30 minutes twice, after collecting supernatant, continue to use 300g centrifugal forces 5 minutes, cell is resuspended with serum free medium, counts, cell is diluted to 107/ ml, uses density level bands Degree centrifugation layering liquid enrichment nonparenchymal cell;After the nonparenchymal cell that B3, collection are enriched to, PBS is washed twice, and serum free medium is resuspended;Use anti-mouse CD16/32 Antibody, every 106The μ g antibody of cell 1,4 DEG C are closed 10 minutes;B4,4 DEG C of lucifuges incubation CD63-PE antibody, every 106The μ g antibody of cell 0.5, while CD11b is incubated, CD31, CD45-FITC Antibody, every 106The μ g antibody of cell 0.2,20 minutes;With the PBS cell three times containing 2%FBS;B5, sorted using streaming instrument, to obtain the CD63-PE positives, CD11b, CD31, cell negative CD45-FITC;In liver stem cells extremely 96 orifice plates of the culture medium containing SCM-A for the CD63+ that 500 B6, inoculation selected by flow cytometry apoptosis go out, 37 DEG C, 5%CO2, 95% damp condition culture 2 weeks.
- 6. applications of the CD63 according to claim 3 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, after above-mentioned steps B, there is provided applications of the CD63 in the method for liver stem cells induction differentiation, be specifically Liver stem cells positive CD63 are induced to differentiate into mature hepatocytes or bile duct epithelial cell in vitro.
- 7. applications of the CD63 according to claim 6 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, liver stem cells positive wherein CD63 are the step of being induced to differentiate into mature hepatocytes in vitroStep C:Liver stem cells positive CD63 are in liver to ripe hepatic lineage is induced to differentiate under inductive condition, and wherein liver is to luring Sliver part is:DMEM/F12,10%FBS, 10ng/ml HGF, 10ng/ml EGF, 10-7M Dex, 1% dimethyl sulfoxide (DMSO), 10ng/ml tumour inhibitors, 20% matrigel.
- 8. applications of the CD63 according to claim 7 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, step C specific method is:C1, by 5 × 104Cell/cm2By liver stem cells kind positive CD63 in the coated 35mm culture dishes of 1 Collagen Type VI, addition SCM-A culture mediums, in 37 DEG C, 5%CO2Culture is covered with to cell in incubator, changes liquid every other day therebetween;After C2, cell cover with, change liver and continue culture 6 days to inducing culture, change liquid every other day therebetween.
- 9. applications of the CD63 according to claim 6 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, liver stem cells positive CD63 are the step of being induced to differentiate into bile duct epithelial cell in vitroStep D:Liver stem cells positive CD63 are induced to differentiate into the pipe sample knot of branch's sample under NTx three-dimensional cultivation condition Structure, the mark Krt19 and EpCAM of the pipe spline structure expression bile duct cell of induced synthesis.
- 10. applications of the CD63 according to claim 9 in liver stem cells quick separating expands or induces the method for differentiation, Characterized in that, step D specific method is:D1, solution used are placed in 4 DEG C of precoolings 30 minutes;D2, prepare type i collagen gel solution:μ l, the ddH2O80 μ l of 800 100 μ l, 1N NaOH of μ l, 10 × PBS of type i collagen 20 Mix, be placed on ice;D3,5 × 10 that 1ml bile ducts induction broth is resuspended4Liver stem cells positive individual CD63 and the type i collagen of above-mentioned preparation Gel solution mixes, and adds in 35mm culture dishes, wherein bile duct induction broth is:DMEM, 10%FBS, 1x mycillin, 1x ITS, 20ng/ml HGF, 50 μ g/ml Gentamicins;D4, culture dish is placed in 37 DEG C, 5%CO2Cultivated 2 hours in incubator, wait the gel sets containing cell;D5,1.5ml bile duct induction broths are added into culture dish, and cell is continued into culture 9 days, during which change liquid every other day.
- 11. the CD63 as described in any one of claim 3 to 5 is in liver stem cells quick separating expands or induced the method for differentiation Application in feature daughter cell caused by the obtained CD63 positive liver stem cells of separation amplification method and its differentiation.
- 12. feature daughter cell is in preparation group caused by CD63 positive liver stem cells as claimed in claim 11 and its differentiation Knit the application in chemical industry journey liver.
- 13. feature daughter cell is preparing carefully caused by CD63 positive liver stem cells as claimed in claim 11 and its differentiation Born of the same parents treat the application in acute hepatic failure medicine or various whole end-stage liver disease medicines.
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