CN105807063A - Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases - Google Patents
Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases Download PDFInfo
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Abstract
The invention relates to the technical field of medical bioengineering, provides a liver stem cell specific surface molecular marker CD63 and a use thereof and especially relates to a use of CD63 in preparation of a kit for liver disease diagnosis or a drug for preventing or treating liver diseases. A method for fast separating, culturing and amplifying liver stem cells from liver by the molecular marker comprises fast separating liver stem cells from a liver cell suspension through the liver stem cell specific surface molecular marker CD63 through a flow cytometer. The liver stem cell with a CD63 positive result has the characteristics of self-renewal and bidirectional differentiation in vitro and has the effects of treating a damaged mouse liver. The separated liver stem cells can be used as seed cells for liver drug screening, tissular engineering liver and liver disease cell therapy and provide an ideal cell model for liver development and liver stem cell biological characteristic research.
Description
Technical field
The present invention relates to medical bioengineering technical field, provide specific surfaces molecular marker CD63 and the application thereof of liver stem cells, particularly CD63 at preparation diagnosis for liver disease test kit or prepares the application in prevention or treatment hepatic disease medicine, and by this surface marker method of sharp separation and cultivation amplifying liver stem cells from liver.It is specifically related to the surface specific mark of application liver stem cells, liver stem cells is gone out by the method for airflow classification sharp separation from Liver Cell Suspension, and realize liver stem cells amplification in vitro and two-way differentiation and replanting in hepatic disease animal model liver.
Background technology
Tissue stem cell refers to the undifferentiated cell being present in the individual tissue broken up, and this cell has a self renewal and specialization forms the ability of cell of this tissue.Tissue stem cell is present in the various histoorgans of body, is that the 26S Proteasome Structure and Function that histoorgan stable state maintains is basic.Tissue stem cell in adult tissue under normal circumstances mostly in a dormant state, only in pathological state or the propagation to the cell type differentiation of this tissue of being just activated under the effect of extraneous factor.Liver stem cells in liver is considered to be present in Hering ' s pipe and bile duct gland, has the two-way differentiation potential producing hepatocyte and bile duct epithelial cell.In normal liver tissue, the quantity of liver stem cells is few, and only when liver damage and hepatocellular propagation are suppressed or liver is subject to lasting extensive chronic injury, the liver stem cells in liver just can be activated, namely so-called " bile duct reaction ".In animal model, normal use in conjunction can suppress the medicine (such as retrorsine, acetaminophen etc.) of hepatocyte growth and 2/3rds livers to cut and induce bile duct to react, and (such as chronic viral hepatitis, primary biliary cirrhosis etc.) often to the existence observing that bile duct reacts in the chronic hepatopathy of the mankind.
The separation and Culture of liver stem cells and further investigation thereof for liver development, regenerate after hepar damnification, the cell therapy of whole end-stage liver disease that the mechanism of the generation of liver neoplasm and a variety of causes cause all has highly important Research Significance and using value.The quantity existed in liver due to liver stem cells is few, reports that the method for the separation of liver stem cells is not perfect at present.nullLi etc. by senecionine combine 2/3 liver cut induction bile duct reaction after，Liver is prepared into cell suspension，Remove after hepatic parenchymal cells through fractional centrifugation，Again through the method for differential velocity adherent by liver stem cells purification (LiWL,SuJ,YaoYC,TaoXR,YanYB,YuHY,WangXM,LiJX,YangYJ,LauJT,HuYP,Isolationandcharacterizationofbipotentliverprogenitorcellsfromadultmouse,StemCells,24(2),322-332)，The method that this kind separates purification liver stem cells is comparatively laborious，And from normal liver tissue, not easily isolate liver stem cells.nullIn recent years，Pass through Foxl1，The surface marker of the liver stem cells such as CD133 and Lgr5 isolates liver stem cells (ShinS in conjunction with the method for streaming or magnetic bead sorting from the liver of normal liver or induction bile duct reaction,WaltonG,AokiR,BrondellK,SchugJ,FoxA,SmirnovaO,DorrellC,ErkerL,ChuAS,WellsRG,GrompeM,GreenbaumLE,KaestnerKH,Foxl1-Cre-markedadulthepaticprogenitorshaveclonogenicandbilineagedifferentiationpotential,GenesDev.25(11):1185-1192；DorrellC,ErkerL,SchugJ,KoppJL,CanadayPS,FoxAJ,SmirnovaO,DuncanAW,FinegoldMJ,SanderM,KaestnerKH,GrompeM,Prospectiveisolationofabipotentialclonogenicliverprogenitorcellinadultmice.GenesDev.25(11):1193-1203；HuchM, DorrellC, BojSF, vanEsJH, LiVS, vandeWeteringM, SatoT, HamerK, SasakiN, FinegoldMJ, HaftA, VriesRG, GrompeM, CleversH, InvitroexpansionofsingleLgr5+liverstemcellsinducedbyWnt-drivenregeneration,, Nature.494 (7436): 247-250.) but these marks can not effectively distinguish liver stem cells and bile duct cell.Therefore, the method for existing separation liver stem cells operation liver stem cells purity that is complicated or that separate is relatively low, and still not having at present can the quick method of special separation purification liver stem cells from liver organization.
CD63 is the memebrane protein including four hydrophobic domains (hydrophobicdomains), is one of the Major Members of tetratransmembrane albumen family (tetraspaninfamily).CD63 is main forms complex with integrin (integrins) family protein, participates in the functions such as the adjustment growth of cell, growth, activation and migration.At present, CD63 is found mainly to be expressed in the platelet of T cell, macrophage and activation.Present invention discover that CD63 is also the specific surfaces molecular marker of liver stem cells, and provide separation quick from Liver Cell Suspension, special and the method for purification CD63+ liver stem cells, simultaneously, achieve liver stem cells amplification in vitro and two-way differentiation and replanting in hepatic disease animal model liver.Cell therapy and the screening of liver disease therapeuticing medicine for hepatic disease that obtain of CD63+ liver stem cells provides platform, also lays the foundation with the research such as the CD63 diagnosis for liver disease test kit being target and Therapeutic Method for exploitation.
Summary of the invention
It is an object of the invention to provide the specific surfaces molecular marker CD63 of a kind of liver stem cells.
Another object of the present invention is to the specific surfaces molecular marker CD63 that liver stem cells is provided application in the method for the amplification of liver stem cells sharp separation and/or induction differentiation.
A further object of the present invention is in that the specific surfaces molecular marker CD63 providing liver stem cells application in the medicine of preparation diagnosis for liver disease test kit and/or treatment hepatic disease.
A first aspect of the present invention, it is provided that the specific surfaces molecular marker of a kind of liver stem cells, the specific surfaces molecular marker of described liver stem cells is CD63.
The specific surfaces molecular marker of the liver stem cells of the present invention, also referred to as the specific surfaces mark of liver stem cells, the surface molecular labelling of liver stem cells, liver stem cells surface molecular mark etc..
A second aspect of the present invention, it is provided that CD63 is as the application of the specific surfaces mark of liver stem cells, this application specifically CD63 application in the method that the amplification of liver stem cells sharp separation and/or induction are broken up.
CD63 is as the specific surfaces mark of liver stem cells, it is possible to isolate liver stem cells exactly, specifically from normal liver, it is possible to induces the reacted liver of bile duct from DDC and isolates liver stem cells.
Specific surfaces molecular marker CD63 application in the method for the amplification of liver stem cells sharp separation and/or induction differentiation.The present invention passes through liver stem cells surface specific mark CD63, isolates CD63 positive liver stem cells in conjunction with the method for airflow classification is quickly special from mouse liver.Liver stem cells positive for CD63 has powerful multiplication capacity and is divided into the two-way differentiation capability of hepatocyte and bile duct cell.Wherein said liver refers to that normal liver or DDC induce the reacted liver of bile duct.
nullDescribed DDC induces the reacted liver of bile duct，Abductive approach can referring to document PreiseggerKH,FactorVM,FuchsbichlerA,StumptnerC,DenkH,ThorgeirssonSS,Atypicalductularproliferationanditsinhibitionbytransforminggrowthfactorbeta1inthe3,5-diethoxycarbonyl-1,4-dihydrocollidinemousemodelforchronicalcoholicliverdisease,LabInvest.79(2):103-109.
Specific surfaces molecular marker CD63 application in the method that liver stem cells sharp separation expands, concrete grammar is as follows:
A, original position immunofluorescence and immunohistochemical staining identify liver stem cells positive for CD63 location in liver after normal liver or DDC induce bile duct reaction.The liver stem cells of immunofluorescence and the immunohistochemical staining result display CD63 positive is distributed in Hering ' s pipe and bile duct gland in liver.
B, by the method for selected by flow cytometry apoptosis, induce, from normal liver or DDC, the liver stem cells isolating the CD63 positive (CD63+) after bile duct reaction liver, and achieve liver stem cells positive for CD63 amplification in vitro.nullFirst the liver two-step method original position collagenase perfusion digestion liver (WangMJ1 after inducing bile duct to react 3 weeks normal liver or DDC,ChenF,LiJX,LiuCC,ZhangHB,XiaY,YuB,YouP,XiangD,LuL,YaoH,BorjiginU,YangGS,WangensteenKJ,HeZY,WangX,HuYP,Reversalofhepatocytesenescenceaftercontinuousinvivocellproliferation,Hepatology.60(1):349-361.)，Digestion mixture continues to hatch 30 minutes with 0.25% collagenase at 37 DEG C，Then pass through gradient centrifugation enrichment Hepatic nonparenchymal cell.CD11b, CD31 and CD45 is gone out negative again through selected by flow cytometry apoptosis, and the liver stem cells that CD63 is positive.nullFinally the liver stem cells that CD63 is positive is expanded in a large number in SCM-A culture medium，SCM-A culture medium: DMEM/F12、10% hyclone (Fetalbovineserum,FBS)、1 × mycillin、0.1mM2-mercaptoethanol (2-Mercaptoethanol，2-Mer)、1 × Insulin-Transferrin-selenium solution (Insulin-Transferrin-SeleniumSolution,ITS)、10ng/ml hepatocyte growth factor (HepatocyteGrowthFactor,HGF)、10ng/ml epidermal growth factor (Epidermalgrowthfactor,EGF)、10ng/ml niacin amide (Nicotinamide)、10-7M dexamethasone (dexamethasone,And 50ug/ml Gentamicin (Gentamycin) Dex).
Described original position immunofluorescence and immunohistochemical staining location, can referring to document YuB, HeZY, YouP, HanQW, XiangD, ChenF, WangMJ, LiuCC, LinXW, BorjiginU, ZiXY, LiJX, ZhuHY, LiWL, HanCS, WangensteenKJ, ShiY, HuiLJ, WangX, HuYP, Reprogrammingfibroblastsintobipotentialhepaticstemcellsb ydefinedfactors, CellStemCell.13 (3): 328-340.
The method of described selected by flow cytometry apoptosis, can referring to document WangMJ1, ChenF, LiJX, LiuCC, ZhangHB, XiaY, YuB, YouP, XiangD, LuL, YaoH, BorjiginU, YangGS, WangensteenKJ, HeZY, WangX, HuYP, Reversalofhepatocytesenescenceaftercontinuousinvivocellp roliferation, Hepatology.60 (1): 349-361.；Flow cytometer model used is BectonDickinsonFACsCalibur.
It is preferred that wherein step A situ immunofluorescence and immunohistochemical staining identify liver stem cells positive for CD63 the concretely comprising the following steps of the location in liver after normal liver or DDC induce bile duct reaction:
A1, normal liver and DDC induce bile duct to react 3 weeks after the embedding of liver OCT embedding medium after, frozen section 7um is thick.4% paraformaldehyde (PFA) room temperature fixes 10 minutes, removes fixative, and PBS washes three times, and 4 DEG C save backup；
A2, immunofluorescence dyeing: by the liver section after step A1 processes with 1% bovine serum albumin (bovineserumalbum, BSA) after room temperature is closed 30 minutes, the mouse-anti CD63 antibody of dropping confining liquid dilution and the anti-CK19 antibody of rabbit, 4 DEG C overnight.PBS-T washs three times, each 5 minutes, the sheep anti-mouse igg (TRITC-GoatantiMouseIgG) that the goat anti-rabbit igg (FITC-GoatantiRabbitIgG) of dropping FITC labelling and TRITC indicate, 37 DEG C are reacted 30 minutes, wash 3 times with PBS-T, each 5 minutes；DAPI redyes nucleus 1 minute, and PBS-T washes 2 times.Anti-fluorescence decay mountant mounting, fluorescence microscopy Microscopic observation.
A3, immunohistochemical staining: being closed 30 minutes with 0.3% hydrogen peroxide by the liver section after step A2 processes, PBS-T washes three times, after 1%BSA room temperature is closed 30 minutes, the mouse-anti CD63 antibody that dropping is diluted with confining liquid, 4 DEG C overnight.PBS-T washs three times, each 5 minutes.In section, the dropping HRP labelling two of PBS-T dilution resists, and hatches 30 minutes for 37 DEG C, washs 3 times with PBS-T, each 5 minutes.Section drips freshly prepared DAB dyeing liquor (article No.: the Kit-0014 of method that by specification provides, Fuzhou Maixin biotechnology Development Co., Ltd), Microscopic observation section color change, when color is obvious, being put into by slice, thin piece immediately in tap water and rinse, color development stopping is reacted.After DAB colour developing, hematoxylin (article No.: H3136, purchased from Sigma) is redyed, mounting after dehydration.
It is preferred that wherein the concretely comprising the following steps of step B:
B1, two-step method original position collagenase perfusion digestion liver (WangMJ1, ChenF, LiJX, LiuCC, ZhangHB, XiaY, YuB, YouP, XiangD, LuL, YaoH, BorjiginU, YangGS, WangensteenKJ, HeZY, WangX, HuYP, Reversalofhepatocytesenescenceaftercontinuousinvivocellp roliferation, Hepatology.60 (1): 349-361.), digestion mixture continues to hatch 30 minutes with 0.25% collagenase at 37 DEG C.
B2, postdigestive cell suspension 50g centrifugal force low-speed centrifugal 30 minutes twice, abandon precipitation (big hepatocyte), after collecting supernatant, continues to use 300g centrifugal force 5 minutes, uses serum-free medium re-suspended cell, and counting, by cell dilution to 107/ ml, uses density gradient centrifugation layering liquid (OptiPrepTMDensityGradientMedium) (article No.: D1556, sigma company) is enriched with nonparenchymal cell.
After the nonparenchymal cell that B3, collection are enriched to, PBS washes twice, and serum-free medium is resuspended.Use anti-mouse CD16/32 antibody (every 106Cell 1ug antibody), close 10 minutes for 4 DEG C.
B4,4 DEG C of lucifuges hatch CD63-PE antibody (every 106Cell 0.5ug antibody), hatch CD11b, CD31, CD45-FITC antibody (every 10 simultaneously6Cell 0.2ug antibody), 20 minutes.With the PBS cell containing 2%FBS (mass percent) three times.
B5, use streaming instrument sorting, to obtain the CD63-PE positive, CD11b, CD31, the cell that CD45-FITC is negative.
The liver stem cells of the CD63+ that B6,500 selected by flow cytometry apoptosis of inoculation go out to 96 orifice plates of the culture medium containing SCM-A, 37 DEG C, 5%CO2, 95% damp condition is cultivated 2 weeks.
Further, after above-mentioned steps B, the liver stem cells that present invention also offers CD63 application in the method for liver stem cells induction differentiation, specifically positive for isolated CD63 liver stem cells or the CD63 after amplification positive is induced to differentiate into mature hepatocytes or bile duct epithelial cell in vitro.
Above-mentioned steps B, by the method for selected by flow cytometry apoptosis, induce from normal liver or DDC and liver isolated after bile duct reaction the positive liver stem cells of CD63, and achieve liver stem cells positive for CD63 amplification in vitro.
The invention provides the method that liver stem cells positive for CD63 is induced to differentiate into mature hepatocytes in vitro.The method is specially step C.
The positive liver stem cells of C, CD63 liver to (DMEM/F12,10%FBS, 10ng/mlHGF, 10ng/mlEGF, 10-7MDex, 1% dimethyl sulfoxide (Dimethylsulfoxide under inductive condition, DMSO), 10ng/ml tumour inhibitor (oncostatinM, OSM), 20% matrigel (Matrigel)) be induced to differentiate into ripe hepatic lineage, hepatic lineage high expressed hepatocyte specific function gene (CYP7a1, G6PD, AAT etc.) that liver stem cells induction positive for CD63 is differentiated to form and there is the function storing glycogen.
It is preferred that concrete induction step is:
C1, by 5 × 104Cell/cm2By liver stem cells kind positive for CD63 in 1 Collagen Type VI (article No.: 354236, purchased from BD company) coated 35mm culture dish, add SCM-A culture medium, in 37 DEG C, 5%CO2Incubator is cultured to cell cover with, changes liquid therebetween every other day.
After C2, cell cover with, change liver and continue to cultivate 6 days to inducing culture (DMEM/F12,10%FBS, 10ng/mlHGF, 10ng/mlEGF, 10-7MDex, 1%DMSO, 10ng/mlOSM, 20%Matrigel), change liquid therebetween every other day.
Present invention also offers liver stem cells positive for CD63 and be induced to differentiate into bile duct epithelial cell in vitro.The method is specially step D.
The liver stem cells that D, CD63 are positive is induced to differentiate into the pipe spline structure of branch's sample under NTx three-dimensional cultivation condition, and the pipe spline structure of induced synthesis expresses mark Krt19 and the EpCAM of bile duct cell.
It is preferred that concrete induction step is:
D1, solution used are placed in 4 DEG C of pre-coolings 30 minutes；
D2, prepare type i collagen gel solution: type i collagen 800ul, 10 × PBS100ul, 1NNaOH20ul, ddH2O80ul mix, and are placed on ice.
D3, by resuspended for 1ml bile duct induction broth (DMEM, 10%FBS, 1x mycillin, 1xITS, 20ng/mlHGF, 50ug/mlGentamycin) 5 × 104The liver stem cells of the individual CD63 positive mixes with the type i collagen gel solution of above-mentioned preparation, adds in 35mm culture dish；
D4, culture dish is placed in 37 DEG C, 5%CO2Incubator is cultivated 2 hours, waits the gel sets containing cell；
D5, in culture dish, add 1.5ml bile duct induction broth, and continue cell to cultivate 9 days (period changes liquid every other day).
CD63 positive liver stem cells that above-mentioned separation amplification method obtains and the functional daughter cell that differentiation produces thereof.
CD63 positive liver stem cells that above-mentioned separation amplification method obtains and the functional daughter cell that differentiation produces application in preparing systematism engineering liver thereof.
CD63 positive liver stem cells that above-mentioned separation amplification method obtains and the functional daughter cell that differentiation produces application in the medicine preparing cell therapy acute hepatic failure and various whole end-stage liver disease thereof.
A third aspect of the present invention, it is provided that CD63 as the Another Application of the specific surfaces mark of liver stem cells, this application specifically specific surfaces molecular marker CD63 preparation diagnosis for liver disease test kit and/or treatment hepatic disease medicine in application.
The CD63 of the present invention application in preparation diagnosis for liver disease test kit, comprises the reagent of detection CD63 expression in described test kit.
CD63 is as the mark of liver stem cells, it is possible to spike also discloses the liver stem cells effect that liver stem cells plays in the process of liver neoplasm generation, hepatic fibrosis and liver cirrhosis, and CD63 also is used as the early diagnosis marker of hepatic disease.
CD63 of the present invention prevents in preparation or treats the application in hepatic disease medicine, and described medicine is that aforesaid CD63 expands at liver stem cells sharp separation or induce the CD63 positive liver stem cells that in the application in the method for differentiation, separation amplification method obtains and the functional daughter cell breaking up generation thereof.
CD63 positive liver stem cells can screen as liver drug, the seed cell of systematism engineering liver and the treatment of liver disease cells, and also the research for the characteristics of cell biology of liver development and liver stem cells provides desirable cell model.
The bile duct injury model mice that hepatocyte injury model mice (Fah knock out mice) and DDC are induced by liver stem cells positive for CD63 has the ability that liver is grown again, and demonstrates therapeutical effect.
Liver stem cells positive for CD63 can be replanted in the liver of damage by Spleen transplantation to the model mice body of hepar damnification, and alleviates the damage of liver.Liver stem cell transplantation positive for CD63 to the mouse model body of hepatocyte chronic injury after, liver stem cells positive for CD63 is colonizated in liver and is divided into mature hepatocytes to alleviate the damage of liver, is embodied in the recovery of damage model Mouse Liver function.And the liver stem cell transplantation of the CD63 positive is to the model mice of the DDC bile duct injury induced, then it is divided into bile duct epithelial cell and is colonizated on bile duct.
The expression of CD63 and the detection of the dependency of hepatic disease, be embodied in:
Collect different developing period (early, middle and late phase) specimen of dissimilar hepatic disease (such as liver neoplasm, liver cirrhosis, hepatic fibrosis etc.) and (include blood, urine and other secretions, liver and focus thereof etc.), the expression of detection CD63, concrete detection method includes SABC and immunofluorescence, western blotting (WesternBlot), elisa (ELISA) and real-time quantitative fluorescence PCR (Real-TimePCR).
The detection method of SABC and immunofluorescence can referring to document YuB, HeZY, YouP, HanQW, XiangD, ChenF, WangMJ, LiuCC, LinXW, BorjiginU, ZiXY, LiJX, ZhuHY, LiWL, HanCS, WangensteenKJ, ShiY, HuiLJ, WangX, HuYP, Reprogrammingfibroblastsintobipotentialhepaticstemcellsb ydefinedfactors, CellStemCell.13 (3): 328-340.
Western blotting (WesternBlot) concretely comprises the following steps:
1, the entity specimen of liver and focus thereof presses the method cracking specimen that protein lysate (article No. P0013BRIPA, green skies biotechnology research institute) description provides, and collects protein lysate.
2, the concentration of total protein in detection protein lysate, the detection method provided by miniature BCA protein detection kit (MicroBCAProteinAssayKit, article No.: 23235, Thermo companies) description measures the total protein concentration of specimen.
null3、Western blotting (WesternBlot) detects the expression of CD63，Concrete grammar is with reference to WangMJ1,ChenF,LiJX,LiuCC,ZhangHB,XiaY,YuB,YouP,XiangD,LuL,YaoH,BorjiginU,YangGS,WangensteenKJ,HeZY,WangX,HuYP,Reversalofhepatocytesenescenceaftercontinuousinvivocellproliferation,Hepatology.60(1):349-361.)，The product that CD63 antibody is SantaCruz company used in western blotting (WesternBlot)，Article No. is sc-15363.
The concrete detection method of elisa (ELISA) is with reference to people's CD63 enzyme-linked immunosorbent assay (ELISA) reagent box (Humanclusterofdifferentiation63 (CD63) ELISAKit, article No.: MBS702053, purchased from mybiosource company) description that provides carries out.
The concrete operation method of real-time quantitative fluorescence PCR (Real-TimePCR) is with reference to SchmittgenTD, LivakKJ.Analyzingreal-timePCRdatabythecomparativeC (T) method.NatProtoc.3 (6): 1101-1108.
CD63 can as the early diagnosis marker of multiple hepatic disease (including liver liver neoplasm, liver cirrhosis, hepatic fibrosis etc.).CD63 also can as one of evaluation index of prognosis of hepatic disease simultaneously.Therefore, CD63 can as the target of the detection kit such as SABC and immunofluorescence, western blotting (WesternBlot), elisa (ELISA) and real-time quantitative fluorescence PCR (Real-TimePCR), for the judging prognosis of the early diagnosis sum of hepatic disease.The liver stem cells of the CD63 positive and the daughter cell of differentiation thereof are the desirable cell platforms of the screening of hepatic disease medicine, pharmacology, toxicity and clinical trial.Liver stem cells and the daughter cell of differentiation thereof that CD63 is positive can be used for treating the screening of the medicine of hepatic disease and the evaluation of the pharmacology of medicine, toxicity and curative effect.
Beneficial effects of the present invention:
The invention provides the liver stem cells positive for CD63 obtained according to above-mentioned isolated culture method.Liver stem cells positive for CD63 possesses the feature that liver stem cells is the most essential, namely powerful multiplication capacity and can be divided into the hepatocyte of maturation and the two-way differentiation capability of bile duct epithelial cell under specific inductive condition.
The innovative point of the present invention is in that: 1) CD63 is the surface specific mark of liver stem cells；2) quickly can induce from normal liver and DDC in conjunction with CD63 mark and flow cytometer the liver of bile duct reaction separates and be purified into the liver stem cells with multiplication capacity and two-way differentiation capability；3) liver stem cells positive for CD63 has the ability of growing again to liver injury, and demonstrates therapeutical effect.
The specific surfaces mark CD63 tool of liver stem cells provided by the present invention has been widely used, CD63 is as the mark of liver stem cells, can spike disclose the effect that liver stem cells occurs, liver stem cells plays in the process of hepatic fibrosis and liver cirrhosis in liver neoplasm, CD63 also is used as the early diagnosis of hepatic disease and the mark of Index for diagnosis.
Also having for cell that CD63 positive liver stem cells provided by the present invention and differentiation thereof produce has been widely used.Can screen as liver drug, the seed cell of systematism engineering liver and the treatment of liver disease cells, also the research for the characteristics of cell biology of liver development and liver stem cells provides desirable cell model.
Accompanying drawing explanation
Fig. 1 is the network for location of CD63 positive liver stem cells in wild-type mice liver.
Wherein A:CK19 immunofluorescence dyeing, cell on pipeline shown in white arrow is CK19 positive cell, B:CD63 immunofluorescence dyeing, cell on pipeline shown in white arrow is CD63 positive cell, the stacking chart of C:A and B, the cell on pipeline shown in white arrow is the double; two positive cell of CK19 and CD63；The immunohistochemical staining of D:CD63,1 and 2 is the partial enlarged drawing in figure D in square frame, and cell shown in black arrow is CD63 positive cell.Scale: 100 microns.Fig. 2 is that DDC induces after bile duct reaction the network for location of CD63 positive liver stem cells in liver.
Wherein A:CK19 immunofluorescence dyeing, cell shown in white arrow is CK19 positive cell, B:CD63 immunofluorescence dyeing, and cell shown in white arrow is CD63 positive cell, the stacking chart of C:A and B, cell shown in white arrow is the double; two positive cell of CK19 and CD63；The immunohistochemical staining of D:CD63,1 and 2 is the partial enlarged drawing in figure D in square frame, and cell shown in black arrow is CD63 positive cell.Scale: 100 microns.
Fig. 3 is the scatterplot by surface marker CD63 airflow classification liver stem cells.
Wherein A: in wild-type mice liver, CD11b-CD31-CD45-CD63+ liver stem cells ratio is 0.2% (Q4 quadrant)；After B:DDC induction bile duct reaction, in liver, CD11b-CD31-CD45-CD63+ liver stem cells ratio is 0.5% (Q4 quadrant).
Fig. 4 is the aspect graph of the liver stem cells positive for CD63 of airflow classification.
Wherein A: the liver stem cells that to be selected from wild-type mice liver CD63 positive form under inverted phase contrast microscope；B: be selected from the liver stem cells positive for CD63 in mouse liver after DDC induces bile duct to react form under inverted phase contrast microscope.
Fig. 5 is liver stem cells the 10th generation (P10) positive for CD63 and the growth curve chart in the 30th generation (P30).Figure showing, the liver stem cells positive for CD63 of p10 and P30 does not have difference at multiplication capacity.
Fig. 6 is liver stem cells related gene expression situation of change figure after liver breaks up to induction and gallbladder to induction positive for CD63.Gallbladder to induction after bile duct specific gene (CK19, Gja1, Ggt1) express strengthen, and liver to induction after hepatocyte-specific gene (Alb, Hnf4a, Cps1, Aat, Cyp7a1, G6p) express strengthen.
Fig. 7 is positive for CD63 liver stem cells liver to staining for glycogen figure after induction differentiation.
After wherein positive for A:CD63 liver stem cells liver breaks up to induction, staining for glycogen shows that the cell after inducing has the function storing glycogen；B: for the partial enlarged drawing in white box in figure A, be the cell of the staining for glycogen positive in black dotted lines frame in figure B.
The liver stem cells gallbladder that Fig. 8 is positive for CD63 forms the gallbladder tube structure figure of branch's sample after induction differentiation.
Wherein A: immunofluorescence dyeing bile duct mark Krt19, cell shown in arrow is Krt19 positive cell；B: immunofluorescence dyeing bile duct mark EpCAM, cell shown in arrow is EpCAM positive cell；C: the branch's sample gallbladder tube structure formed after induction form under inverted phase contrast microscope.
The liver stem cells that Fig. 9 is positive for CD63 indicates the form after green fluorescence and green fluorescence expression figure in vitro.
Wherein A is the form under inverted phase contrast microscope of the liver stem cells positive for CD63 after the upper green fluorescence of mark, and the liver stem cells positive for CD63 that B is after the upper green fluorescence of mark all expresses green fluorescence.
Figure 10 is after the liver stem cells positive for CD63 after the upper green fluorescence of mark grows Fah knock out mice liver again, the immunofluorescence dyeing figure of liver section.
Wherein A is that the cell deriving from liver stem cells positive for CD63 in immunofluorescence dyeing display Fah knock out mice liver expresses Fah gene (in white dashed line)；The cell that B is the liver stem cells deriving from the CD63 positive in the serial section immunofluorescence dyeing display Fah knock out mice liver of A expresses GFP (in white dashed line)；The cell that C is the liver stem cells deriving from the CD63 positive in the serial section immunofluorescence dyeing display Fah knock out mice liver of A expresses hepatocyte-specific gene Alb (in white dashed line)
The liver stem cells that Figure 11 is positive for CD63 grows the liver function schematic diagram after Fah knock out mice liver again.After liver stem cells positive for CD63 grows Fah knock out mice liver again, Fah knock out mice liver function is clearly better, the index ALT (A) of hepar damnification, AST (B) and Tbil (D) is decreased obviously, and the ability of liver synthesis albumin A LB (C) is obviously improved..
Figure 12 is after the mouse liver that the liver stem cells positive for CD63 after the upper green fluorescence of mark grows DDC damage again, the immunofluorescence dyeing figure of liver section.
Wherein A is the DDC field planting (shown in arrow) damaging the liver stem cells positive for CD63 having green fluorescence mark on the bile duct of mouse liver；B is that DDC damages the specificity marker CK19 coming from liver stem cells expression bile duct cell positive for CD63 in mouse liver on bile duct；C is the fusion figure of A and B.
Specific implementation method
In conjunction with embodiments of the invention and accompanying drawing, the enforcement of the present invention is elaborated; following example are to be carried out under premise the technical scheme is that; and give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1.CD63 liver stem cells location in liver
null1、Normal liver and DDC induce bile duct to react 3 weeks after liver (PreiseggerKH,FactorVM,FuchsbichlerA,StumptnerC,DenkH,ThorgeirssonSS,Atypicalductularproliferationanditsinhibitionbytransforminggrowthfactorbeta1inthe3,5-diethoxycarbonyl-1,4-dihydrocollidinemousemodelforchronicalcoholicliverdisease,With OCT embedding medium (article No.: 4583 LabInvest.79 (2): 103-109)，Purchased from SAKURA company) embedding after，Frozen section 7um is thick.4% paraformaldehyde (PFA) (article No.: P-6148, available from Sigma) room temperature fixes 10 minutes, removes fixative, PBS (article No.: BS7016, purchased from Sangon Biotech (Shanghai) Co., Ltd.) wash three times, 4 DEG C save backup.
2, immunofluorescence dyeing: by liver section 1%BSA (article No.: the A1933 after step 1 processes, purchased from sigma company) room temperature closes after 30 minutes, drip mouse-anti CD63 antibody (article No.: the mab5417 with 1%BSA dilution, purchased from R&D company) and anti-CK19 antibody (article No.: the A3190 of rabbit, purchased from Abbomax company), 4 DEG C are overnight.PBS-T (article No.: rbr01485, purchased from Shanghai Rong Bai Bioisystech Co., Ltd) wash three times, each 5 minutes, dropping FITC-GoatantiRabbitIgG (goat anti-rabbit igg of FITC mark, article No.: 111-095-003, purchased from Jackson company) and the TRITC-GoatantiMouseIgG (sheep anti-mouse igg of TRITC mark, article No.: 115-025-003, purchased from Jackson company), 37 DEG C are reacted 30 minutes, wash 3 times with PBS-T, each 5 minutes；DAPI (article No.: D6584, purchased from Sangon Biotech (Shanghai) Co., Ltd.) redyes nucleus 1 minute, and PBS-T washes 2 times.Anti-fluorescence decay mountant (article No.: C1210, purchased from Beijing Puli's lema gene Technology Co., Ltd.) mounting, fluorescence microscopy Microscopic observation.Immunofluorescence dyeing result shows: induce in the liver of bile duct reaction at normal liver and DDC, the cell coexpression CK19 (Figure 1A-1C and Fig. 2 A-2C) that CD63 is positive.
3, immunohistochemical staining: 0.3% hydrogen peroxide (volume ratio) of the liver after step 1 processes is closed 30 minutes, and PBS-T washes three times, after 1%BSA room temperature is closed 30 minutes, drips the mouse-anti CD63 antibody with 1%BSA dilution, and 4 DEG C overnight.PBS-T washs three times, each 5 minutes.In section, the dropping HRP labelling two anti-(article No.: sc-2005, purchased from SantaCruz company) of PBS-T dilution, hatches 30 minutes for 37 DEG C, washs 3 times with PBS-T, each 5 minutes.Section drips freshly prepared DAB dyeing liquor (article No.: the Kit-0014 of method that by specification provides, Fuzhou Maixin biotechnology Development Co., Ltd), Microscopic observation section color change, when color is obvious, immediately slice, thin piece is put in tap water and rinse, after color development stopping reaction DAB colour developing, hematoxylin (article No.: H3136, purchased from Sigma) redye, mounting after dehydration.
Liver stem cells positive for result display CD63 is positioned at Hering ' s in liver and manages (Fig. 1 D and Fig. 2 D-1) and bile duct gland (Fig. 2 D-2).
The sharp separation of embodiment 2.CD63 positive liver stem cells and amplification cultivation
null1、Two-step method original position collagenase perfusion digestion liver (WangMJ1,ChenF,LiJX,LiuCC,ZhangHB,XiaY,YuB,YouP,XiangD,LuL,YaoH,BorjiginU,YangGS,WangensteenKJ,HeZY,WangX,HuYP,Reversalofhepatocytesenescenceaftercontinuousinvivocellproliferation,Hepatology.60(1):349-361.)，Digestion mixture continues with 0.25% collagenase (article No.: 11088858001，Purchased from Roche) hatch 30 minutes at 37 DEG C.
2, postdigestive cell suspension 50g centrifugal force low-speed centrifugal 30 minutes twice, abandon precipitation (big hepatocyte), collect supernatant to continue to use 300g centrifugal force 5 minutes, with plasma-free DMEM medium (article No.: SH30022.01, purchased from Hyclone company) re-suspended cell, counting, by cell dilution to 107/ ml, uses OptiPrepTMDensityGradientMedium (article No.: D1556, sigma company) is layered liquid enrichment nonparenchymal cell.
3, after collecting the nonparenchymal cell being enriched to, PBS washes twice, and serum-free medium is resuspended.Use anti-mouse CD16/32 antibody (article No.: 101309, purchased from Biolegend company) (every 106Cell 1ug antibody), close 10 minutes for 4 DEG C.
4,4 DEG C of lucifuges hatch CD63-PE antibody (article No.: 143904, purchased from Biolegend company) (every 106Cell 0.5ug antibody), hatch CD11b-FITC (article No.: 11-0112 simultaneously, purchased from eBioscience company), CD31 (article No.: CYT-31F2, purchased from Cytognos company), CD45-FITC antibody (article No.: 553079, purchased from BD company) (every 106Cell 0.2ug antibody), 20 minutes.With the PBS cell containing 2%FBS three times.
5, streaming instrument (flow cytometer model is BectonDickinsonFACsCalibur) sorting is used, to obtain the PE positive, the cell that FITC is negative.Liver stem cells positive for CD63 in normal mouse liver is about 0.2%, and DDC induces liver stem cells positive for CD63 in the liver that bile duct reacts to be about 0.5% (Fig. 3).
6, the liver stem cells of the CD63+ that 500 selected by flow cytometry apoptosis of inoculation go out is to 96 orifice plates of the culture medium containing SCM-A, 37 DEG C, 5%CO2, after 95% damp condition cultivates 2 weeks, no matter normal liver or DDC induce the liver stem cells positive for CD63 in the liver of bile duct reaction to be respectively formed typical Epithelial clone (Fig. 4).
7, liver stem cells positive for CD63 demonstrates stronger multiplication capacity under the condition of culture of the culture medium of SCM-A, the multiplication capacity of its 30th generation cell (P30) and the multiplication capacity no significant difference (Fig. 5) of the 10th generation cell (P10).
Embodiment 3.CD63 positive liver stem cells is divided into mature hepatocytes
1, by 5 × 104Cell/cm2By liver stem cells kind positive for CD63 in the 1 coated 35mm culture dish of Collagen Type VI, add SCM-A culture medium, in 37 DEG C, 5%CO2Incubator is cultured to cell cover with, changes liquid therebetween every other day.
2, after cell covers with, change liver and continue to cultivate 6 days to inducing culture (DMEM/F12,10%FBS, 10ng/mlHGF, 10ng/mlEGF, 10-7MDex, 1%DMSO, 10ng/mlOSM, 20%Matrigel), change liquid therebetween every other day.
3, the detection of the mature hepatocytes that the induction of CD63 positive liver stem cells is differentiated to form: the cell extraction RNA formed after collecting induction, Real-Time detects display, the expression of the mark (Abcg2, CK19, Gja1, Ggt1) of stem cell and bile duct cell is notable before relatively inducing to be lowered, and the expression of hepatocellular mark (Alb, Hnf4a, Cps1, Aat, Cyp7a1, G6P) significantly rises (Fig. 6) before relatively inducing.Cell after PAS dyeing display induction has the function (Fig. 7) of glycogen deposit, and result above shows, under this induction system, cell positive for CD63 can be induced to differentiate into mature hepatocytes.
Embodiment 4.CD63 positive liver stem cells is divided into bile duct epithelial cell
1, solution used is placed in 4 DEG C of pre-coolings 30 minutes；
2, type i collagen gel solution is prepared: type i collagen 800ul, 10 × PBS100ul, 1NNaOH20ul, ddH2O80ul mix, and are placed on ice.
3, by resuspended for 1ml bile duct induction broth (DMEM, 10%FBS, 1x mycillin, 1xITS, 20ng/mlHGF, 50ug/mlGentamycin) 5 × 104The liver stem cells of the individual CD63 positive mixes with the type i collagen gel solution of above-mentioned preparation, adds in 35mm culture dish；
4, culture dish is placed in 37 DEG C, 5%CO2Incubator is cultivated 2 hours, waits the gel sets containing cell；
5, in culture dish, add 1.5ml bile duct induction broth, and cell is continued cultivates 9 days (period changes liquid every other day) cells afterwards form typical branch's sample pipe spline structure (Fig. 8).Branch's spline structure immunofluorescence dyeing shows, branch's spline structure of induced synthesis expresses mark CK19 and the EpCAM (Fig. 8 A and 8B) of bile duct cell.These results show at gallbladder under inductive condition, and liver stem cells positive for CD63 can be induced to differentiate into bile duct epithelial cell.
Liver stem cells treatment Chronic Liver cell injury model mice positive for embodiment 5.CD63
1, with the liver stem cells that green fluorescence mark CD63 is positive, by 106The individual lentiviral particle (article No.: 12255, purchased from Addgene company) expressing GFP gene adds to 105In the liver stem cells culture dish that individual CD63 is positive, viral infection adds puromycin (PURO) (article No.: the J593 of 2ug/ml after 72 hours, purchased from Amresco company) screen one week, the liver stem cells positive for CD63 after PURO screening all expresses GFP (Fig. 9).
2, Fah knock out mice (WangMJ1, ChenF, LiJX, the LiuCC of 8-10 week old Normal-weight are taken, ZhangHB, XiaY, YuB, YouP, XiangD, LuL, YaoH, BorjiginU, YangGS, WangensteenKJ, HeZY, WangX, HuYP, Reversalofhepatocytesenescenceaftercontinuousinvivocellp roliferation,, super-clean bench anaesthetize, alcohol disinfecting Hepatology.60 (1): 349-361.)；
3, on the left of abdominal cavity, under rib, open abdominal cavity, expose spleen；
4, by about 106The liver stem cells positive for CD63 of individual mark GFP is suspended from 100ulPBS, with 1ml microsyringe, cell suspension is carried out spleen injection；
5, mice puts back to the continuation raising of SPF level Animal House after sewing up a wound, its drinking water removes NTBC (2-nitro-4-fluoroform stupid-1,3 cyclohexane iodine, after in above-mentioned steps 2, Fah knock out mice goes out, its drinking-water adds NTBC, the accumulation of the metabolic poison in order to cause after resisting Fah gene delection, avoids mice generation hepatic injury).
null6、After cell transplantation the 8th week，Collect mouse liver and venous blood，And the liver stem cells positive for CD63 of analysis mark GFP situation of growing again in liver and (specific experiment method is shown in WangMJ1 to the therapeutical effect of hepar damnification,ChenF,LiJX,LiuCC,ZhangHB,XiaY,YuB,YouP,XiangD,LuL,YaoH,BorjiginU,YangGS,WangensteenKJ,HeZY,WangX,HuYP,Reversalofhepatocytesenescenceaftercontinuousinvivocellproliferation,Hepatology.60(1):349-361.).The liver liver plate of the immunofluorescence dyeing display Fah knock out mice of liver section has growing again (Figure 10 B) of the liver stem cells deriving from the GFP positive, and these cells grown again express hepatocellular specific gene Fah (Figure 10 A) and Alb (Figure 10 C).Simultaneously index ALT, AST and the Tbil of the liver function test result display hepar damnification of mouse vein blood does not carry out the Fah knock out mice of cell transplantation and is decreased obviously, and the ability of liver synthesis Alb is obviously improved (Figure 11).Result above shows that liver stem cells positive for CD63 can be grown again and is divided into mature hepatocytes in the liver of Chronic Liver cell injury, and demonstrates therapeutical effect.
Liver stem cells positive for embodiment 6.CD63 is replanted DDC and is induced bile duct injury model mice
1, the wild-type mice of DDC feed 8-10 week old Normal-weight 3 days.
2, by 106The individual liver stem cells positive for CD63 expressing GFP is by the mouse liver after Spleen transplantation to DDC feed 3 days, and implantation method is with embodiment 5.
3, the mice after transplanting continues feed DDC, collects mouse liver after 3 weeks.
null4、The CD63 positive liver stem cells expressing GFP transplanted by immunofluorescence dyeing detection induces the situation of replanting in the mouse liver of bile duct injury at DDC, and (specific experiment method is shown in YuB,HeZY,YouP,HanQW,XiangD,ChenF,WangMJ,LiuCC,LinXW,BorjiginU,ZiXY,LiJX,ZhuHY,LiWL,HanCS,WangensteenKJ,ShiY,HuiLJ,WangX,HuYP,Reprogrammingfibroblastsintobipotentialhepaticstemcellsbydefinedfactors,CellStemCell.13(3):328-340).
Coloration result is shown in the implantation (Figure 12 A) having external source GFP positive cell on the bile duct in the mouse liver of DDC induction bile duct injury, and the cell positive for GFP of these external sources expresses bile duct cell Specific marker CK19 (Figure 12 B and Figure 12 C), result above shows that liver stem cells positive for CD63 can be replanted in the bile duct of the mouse liver inducing bile duct injury into DDC and be divided into the bile duct epithelial cell of maturation.
Below the preferred embodiment of the invention has been illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art it may also be made that under the premise without prejudice to the invention spirit all equivalent modification or replacement, these equivalent modification or replacement are all contained in the application claim limited range.
Claims (6)
1.CD63 application in preparation diagnosis for liver disease test kit.
2. the CD63 according to claim 1 application in preparation diagnosis for liver disease test kit, it is characterised in that comprise the reagent of detection CD63 expression in described test kit.
The liver stem cells positive for 3.CD63 application in preparation prevention or treatment hepatic disease medicine.
4. the liver stem cells positive for CD63 according to claim 3 application in preparation prevention or treatment hepatic disease medicine, it is characterized in that, the application of the liver stem cells that described CD63 is positive is based on the screening, pharmacology, toxicity and the clinical trial that carry out on the platform of liver stem cells positive for CD63.
5. the liver stem cells positive for CD63 according to claim 3 application in preparation prevention or treatment hepatic disease medicine, it is characterized in that, the primary cell that liver stem cells is CD63 positive liver stem cells that described CD63 is positive and the functional daughter cell that differentiation produces thereof.
6. the liver stem cells positive for CD63 according to claim 5 application in preparation prevention or treatment hepatic disease medicine, it is characterized in that, the functional daughter cell following methods that the primary cell of described CD63 positive liver stem cells and differentiation thereof produce obtains:
By liver stem cells surface specific mark CD63, from liver, isolate the primary cell of CD63 positive liver stem cells in conjunction with the method for airflow classification；
The primary cell of CD63 positive liver stem cells breaks up the functional daughter cell of generation further.
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| CN106520678A (en) * | 2016-11-30 | 2017-03-22 | 张晓南 | Kit used for culturing liver-derived stem cells |
| CN112080560A (en) * | 2020-05-27 | 2020-12-15 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | Application of CD177 in preparation of product for diagnosing biliary atresia |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520678A (en) * | 2016-11-30 | 2017-03-22 | 张晓南 | Kit used for culturing liver-derived stem cells |
| CN106520678B (en) * | 2016-11-30 | 2019-12-24 | 张晓南 | Kit for culturing hepatic stem cells |
| CN112080560A (en) * | 2020-05-27 | 2020-12-15 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | Application of CD177 in preparation of product for diagnosing biliary atresia |
| CN112080560B (en) * | 2020-05-27 | 2024-01-26 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | Use of CD177 for the preparation of a product for diagnosing biliary tract occlusion |
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