CN100395330C - In vitro induction of embryo stem cell source elliptocyte and separation purification culturing method - Google Patents

Abstract

This invention provides a efficiently new induction model of hepatic oval cell. In this model significantly inducing the production of embryonic stem cell endogenous oval cell by using HGF and EGF, the whole induction method is fairly convenient, induction cycle is also very short. Utilizing very little embryonic stem cell provenance source can induce to generate sufficient hepatic oval cell which is contented to clinical requirement by using the invented induction model of hepatic oval cell, it also can be used for clinical category such as hepatic oval cell transplant, hepatic tissue engineering and so on, it can play a part in assistant supportive treatment or curing disease for Liver function failure, hepatic metabolic disease caused by various kinds of reason.

Description

External evoked and the separation purification culturing method of embryo stem cell source elliptocyte

One, technical field

The present invention relates to that cells in vitro is induced and separation purification culturing method, relate in particular to the external evoked and separation purification culturing method of embryo stem cell source elliptocyte.

Two, background technology

(embryonic stem cell is that an isolated class is not broken up diploid cell in the inner cell mass by the early stage blastocyst of Mammals ESC) to embryonic stem cell, has the potential of self and all-round differentiation ^[1]Embryonic stem cell can keep undifferentiated state also can infinitely increase under the specified conditions, have be divided into constitute body in a organized way with the potential of cell, be a kind of unlimited cell source, its potentiality on clinical cell, tissue, organ reparation and transplantation treatment is very huge.Generate various required purpose cells and be used for cell therapy and make up required organ even can control the directed differentiation of embryonic stem cell, these numerous just investigators dream of.Building of human ES cell is to make stem-cell research be chosen as the world's ten big science and technology by the Science magazine in continuous 2 years to make progress that the research boom of embryonic stem cell also starts at this point ^[2]Existing multinomial result of study shows that embryonic stem cell can break up the generation mature liver cells. ^[3]--[5]We have also confirmed this point at previous research ^[6]As a kind of liver stem cells, elliptocyte (Hepatic oval cells) is the adult stem cell that has the self ability, can break up hepatic parenchymal cellses such as generating liver cell and bile duct epithelial cell.Leduc in 1958 and Wilson have found elliptocyte when local form changes after with the drug-induced mouse liver injury of electron microscopy study.It is that a class volume is less, oval type nucleus, less basophilia, higher, the underdeveloped cell of organoid of karyoplasmic ratio of being of kytoplasm.Cause liver cell most of downright bad if liver is badly damaged, or when (effect of Hepatoxic substance, carcinogenic substance) suppressed residual hepatocyte growth for some reason, elliptocyte was activated in the liver.Just be based on this theory, people are that object has been set up more sophisticated elliptocyte and induced experimental model with the rodent: (2-Acetylaminofluorene 2-AAF) adds 2/3rds hepatectomies to continue to add the low dosage 2-acetamidofluorene in the food ^[7], perhaps add 3 in the food, 5-two B carbonyls-1,4-dihydro-trimethylpyridine (3,5-diethoxycarbonyl-1,4-dihydro-collidine, DDC) ^[8]Deng.By these animal models, people are greatly improved to the understanding of rodent elliptocyte.Induce the separation method of back to elliptocyte, now two step collagenase perfusion methods of improvement that adopt are separated hepatic parenchymal cells and nonparenchymal cell more, use isopycnic gradient centrifugation centrifugal (percoll liquid being made into the gradient centrifugation liquid of discontinuous concentration) then, obtain elliptocyte in 50% to 70%percoll interlayer at last. ^[9]-[10]A series of body is interior, experiment in vitro has confirmed that elliptocyte has two-way differentiation potential, can generate liver cell by proliferation and differentiation, can generate bile duct cell by proliferation and differentiation again ^[11]-[14]Also there is research to show that it is cell that elliptocyte also can change the non-liver of differentiation generation, as pancreatic islet endocrine ^[15]Deng.Existing known liver injury stimulates and can cause in the liver as stellate cells nonparenchymal cells such as (hepatic stellate cells) the secretion various kinds of cell factor, comprise pHGF (Hepatocyte Growth factor, HGF), the long factor-alpha of reincarnation (Transforming growth factor α, TGF-α), the long factor-beta of reincarnation (Transforming growth factor β, TGF-β), Urogastron (epidermal growth facter, EGF), acid fibroblast growth factor (acid fibroblast growth factof, α-FGF) etc., they activate elliptocyte, hyperplasia, atomization has the important regulating and controlling effect.Elliptocyte is expressed HGF part c-met, and stellate cell excretory HGF promotes the propagation (make its DNA synthetic water dawn show increase) of elliptocyte after in conjunction with c-met consumingly.The recombinant adenovirus transfection elliptocyte of HGF cDNA is carried in use, and the elliptocyte amount of proliferating cell nuclear antigen PCNA (+) can obviously increase.When list was used HGF, elliptocyte was obviously induced the hyperplasia of epithelial duct, can obviously induce the formation of liver plate or liver rope spline structure during couplings such as HGF and EGF, SCF, and EGF has key effect to elliptocyte to hepatocellular directional induction. ^[16]-[18]

Up to the present, in the process of embryonic stem cell directional induction in vitro generation hepatic parenchymal cells, whether exist this middle differential period of embryo stem cell source elliptocyte of two-way differentiation capability, and whether HGF and EGF have effect to the generation of embryo stem cell source elliptocyte, never has relevant report both at home and abroad.

Three, summary of the invention

The object of the invention provides a new guidance model of elliptocyte efficiently.

The technical problem to be solved in the present invention is: solve in the prior art rodent elliptocyte guidance model and induce loaded down with trivial details consuming time, the inductor of process to be the chemical toxicity material, have disadvantageous effect, thereby limit the defective of its range of application; Confirmation exists two-way differentiation capability in the embryonic stem cell directional induction in vitro generates the process of hepatic parenchymal cells embryo stem cell source elliptocyte differential period in the middle of this, and HGF and EGF have the significance inducing action to the generation of embryo stem cell source elliptocyte; And induce and sub-elect suitable elliptocyte quickly and easily.

The present invention is achieved through the following technical solutions:

Select mouse embryo stem cell for use, change into embryoid body differentiation grouping after 3 days, induce group interpolation pHGF (HGF), Urogastron (EGF) to do the directional induction differentiation; Detect the expression of elliptocyte mark A6 etc. during this time with immunocytochemistry, with flow cytometry analysis and use Sca-1 and the two index screening A6+ elliptocyte of CD34, the capable RT-PCR of the elliptocyte that is screened, transmission electron microscope detect, and calculate the elliptocyte differentiation rate; The A6+/Sca-1+/CD34+ elliptocyte of screening continues vitro culture and checks it whether to have two-way differentiation capability; With the positive elliptocyte film of atomic force microscope research A6 surface ultrastructure.

Technique effect of the present invention:

The present invention confirms first that the embryonic stem cell directional induction in vitro generates in the process of hepatic parenchymal cells and exists A6 male embryo stem cell source elliptocyte (RT-PCR and transmission electron microscope detect and also confirm its existence); The A6 of FACS screening is positive, and elliptocyte can break up generation mature liver cells and bile duct cell.

HGF and EGF can significance inducing embryo stem cell source gamogenetic egg circle cell generation, induce group elliptocyte differentiation rate all to be higher than control group significantly, Gao Shikeda about 4.46%; Use two index flow cytometries can filter out higher proportion A6+ elliptocyte (about 92.48%), the A6+ elliptocyte that filters out shared ratio in total cell is about 1.12%, and whole induction method is easier, and induction duration is also very short.

Utilize elliptocyte method for inducing and cultivating set forth in the present invention, utilize embryonic stem cell provenance seldom just can induce generation elliptocyte abundant, that can meet clinical needs, and then can be used for clinical categories such as elliptocyte transplanting, liver tissue engineering, liver failure, hepatic metabolism disease etc. due to a variety of causes are played the function of auxiliary supportive treatment or cure diseases.

Four, description of drawings

Fig. 1 .ESC induces cell growth state in the atomization to hepatic parenchymal cells.(A) ESC is the growth of colony sample, closely arranges boundary unclear (200 *); (B) ESC is transparent sphere (400 *); (C) induce group-Day8, form is paving stone sample (100 *) than homogeneous; (D) control group-Day8, form is complicated, and trilateral, circle, spindle cell mix growth (100 *) mutually

Fig. 2. embryonic stem cell is induced daughter cell A6 antigen and the antigenic expression of ALB behind the cell and airflow classification cell cultures in the atomization to hepatic parenchymal cells.(A)-(E) show the 9th day and induce component cell mass A6 (+) cell (arrow shows).(B)-(D) amplify the part that is (A).Single A6 (+) cell is oval, and the positive elliptocyte of A6 mainly is distributed in (Fig. 2 B, C) among the formed cell colony in the adherent back of embryoid body, and the periphery differentiation is than (Fig. 2 E) in the microcolony between the mature cell group.The positive epithelial duct like cell (Fig. 2 D) of the A6 of visible two spindle shapes in the lower left of cell colony.(F) show the 15th day and induce ALB (+) liver cell in the component cell mass, wherein visible a plurality of double-core liver cells (arrow shows).(G) show ALB (+) liver cell of CD34+/Sca-1+ cell cultures after the 15th day of selected by flow cytometry apoptosis, wherein visible a plurality of double-core liver cells (arrow shows).(H) negative control.Positive staining is brown (DAB colour developing) among the figure, is blue behind the phenodin transfect cell nuclear.A、E、H×100，F、G×200，B、C、D×400。

Fig. 3. three look facs analysis of embryo stem cell source elliptocyte.The R1 frame is selected the cell mass of facs analysis, and the R2 frame is elected the choosing of Sca-1 door as, and the two marks of the further capable CD34/CD45 of Sca-1+ cell are analyzed.UL is CD34+/CD45-, and UR is CD34+/CD45+, and LL is CD34-/CD45-, and LR is CD34-/CD45+.Induce that Sca-1+/CD34+/CD45+ accounts for 2.38% of total cell count (R1) in the differentiation daughter cell of group, and three positive cells of control group have only 0.10%.The almost equal coexpression CD45 (cell count among UL and the UR is 0) of the cell of Sca-1+.

Fig. 4. the double-colored facs analysis of embryo stem cell source elliptocyte.Get the 4th day to the 10th day the differentiation daughter cell of inducing group and control group of differentiation, analyze the expression (is a choosing with Sca-1) of antigen Sca-1 and CD34.Induce that Sca-1+/CD34+ cell positive rate began to rise from breaking up on the 4th day in the differentiation daughter cell of group, (1.20%) back of peaking to the 8th day descends; And the maintaining basically of control group one low-level (0.03%-0.21%).Cell in the R1 district is based on minicell, and the most cells structure is simpler, and control group breaks up the 8th day cell and is divided into two groups, induces group then not have this phenomenon.The cellular control unit point diagram disperses always in the R2 district (Sca-1 Gated), and induces the passing cell point diagram along with between differentiation phase of group to trend towards concentrating and moving right.Cellular control unit peak faciation changed little to the position always and is in low-levelly, and induces the passing cell peak group along with between differentiation phase of group constantly to move right (two positive cell proportions increase), reached maximum value to the 8th day.

Fig. 5. break up and induced the antigenic expression of group flow cytometer screening cell A6 on the 8th day.(A) Sca-1+/CD34+ cell (92.48% ± 2.01%), the overwhelming majority is the A6 positive, and part is strong positive.(B) non-two positive cell only minority be the A6 positive (1.66% ± 1.57%).(C) negative control.Positive staining is brown (DAB colour developing) among the figure.A、B、C×100

Fig. 6. the transmission electron microscope of the two positive cells of flow cytometer screening detects.Cell volume less (about 13 μ m), the karyoplasmic ratio height; Nuclear is oval, visible spissated chromatin; The caryoplasm inner cell organ is less, visible a small amount of plastosome and rough surfaced endoplasmic reticulum; The visible a small amount of microvillus of cell edges.Scale: 1 μ m

Fig. 7 .RT-PCR analyzes.A is the RT-PCR result of the two positive cells of flow cytometer screening, liver cell mark AFP, ALB, bile duct cell mark ck19, liver is that cell sign thing ck8, ck18 are all positive, and ripe liver cell mark G6Pase and ripe bile duct cell mark BG are negative; B is the RT-PCR result of two 20 days the cell masses of positive cell cultures of screening, and different with A is that AFP does not have expression, and G6Pase and BG have expression.BG refers to biliary glycoprotein, and G6Pase refers to glucose-6-phosphatase.Marker is 100-2000.Confidential reference items are β-actin.

The positive elliptocyte film Ultrastructural atomic force microscope analysis in surface of Fig. 8 .A6.(A), (B) induce the 9th day single elliptocyte of group by locating (arrow shows), (A * 100 with the dyeing of A6 monoclonal antibody; B * 400).(C)-(F) be the Ultrastructural atomic power scintigram of this surface of cell membrane, the latter amplifies for the part of the former frame favored area.(G)-(I) be the Ultrastructural atomic power scintigram in single embryonic stem cell film surface in contrast, the latter amplifies for the part of the former frame favored area.Embryonic stem cell is rounded, and diameter is about 9 μ m, and surface of cell membrane has most ellipsoidal particles, and granular size is 40-80nm.The elliptocyte ovalize, volume bigger (diameter is about 11 μ m), surface of cell membrane amounts of particles more and bigger (size is 200-400nm) is thick prolate ellipsoid shape.Sweep limit: (C) 20 * 20 μ m ²(D), (H) 5 * 5 μ m ²(E), (I) 2 * 2 μ m ²(F) 1 * 1 μ m ²(G) 15 * 15 μ m ²

Five, embodiment

Following specific embodiment just is further described technical scheme, does not limit the scope of the invention.

(1) experiment material and equipment:

1. the source of embryonic stem cell:

Choosing the Balb/c that commerce can get is mouse embryo stem cell.

2. cell culture reagent:

2.1 cell culture medium:

(1) embryonic stem cell medium component:

(Dubecco ' s modifided Eagle ' is (U.S. Hyclone company) smedium) to contain the DMEM of high sugar and glutamine;

20% foetal calf serum (Fetal bovine serum, FBS) (clear company of the Hangzhou four seasons);

1000U/ml recombined small-mouse leukaemia inhibitory factor (recombinant murineleukemia inhibitory factor, LIF) (U.S. ESGRO

-Chemicon company);

0.1mmol/L beta-mercaptoethanol (β-Mercaptoethanol, β-ME) (American I nvitrogen company);

25mmol/L HEPES (American I nvitrogen company);

100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin) (American I nvitrogen company).

(2) differential period minimum medium:

Compares with the embryonic stem cell substratum, do not contain outside the LIF, and the concentration of foetal calf serum becomes 15%, and added 0.1mmol/L non-essential amino acid (nonessentialamino acids) (American I nvitrogen company), all the other compositions with.

2.2 cell growth factor:

PHGF (Hepatocyte Growth factor, HGF) (U.S. Peprotech company),

Urogastron (epidermal growth factor, EGF) (U.S. Peprotech company).

2.3 other reagent, equipment

Disposable plastic culturing bottle, disposable plastic culture plate (U.S. Corning company),

Digestive system 0.25%Trypsin-1mM EDTA4Na (American I nvitrogen company).

3. immunocytochemistry and flow cytometry reagent:

Rat A6 monoclonal antibody is that commerce can get;

Anti-mouse/human albumin (albumin, ALB) monoclonal antibody (U.S. R﹠amp; D company);

SABC immunohistochemical methods test kit (Wuhan doctor's moral company);

The anti-mouse Sca-1 of PE-Cy5 conjugated monoclonal antibody (U.S. eBioscience company);

The anti-mouse CD34 of R-PE conjugated monoclonal antibody (U.S. BD Pharmingen company);

The anti-mouse CD45 of FITC conjugated monoclonal antibody (U.S. Biolegend company);

The inverted phase contrast microscope (Japanese Nikon Eclipse TS100) of band camera system (Japanese NIKON E4500);

Flow cytometer is U.S. BECTON DICKINSON FACScalibur FlowCytometers.

4.RT-PCR reagent:

TROZOL (TRI REAGENT

-U.S. MRC company);

CDNA synthetic agent box (U.S. Fermentas company);

PCR test kit (U.S. Fermentas company);

Primer is synthetic synthetic by the living worker in Shanghai bio-engineering corporation.Primer sequence and primer length, annealing temperature are as follows:

a-fetoprotein，5’-ACT CAC CCC AAC CTT CCT GTC-3’forward，

5’-CAG CAG TGG CTG ATA CCA GAG-3’reverse，

423bp, annealing temperature is 56 ℃;

albumin，5’-CAT GAC ACC ATG CCT GCT GAT-3’forward，

5’-CTC TGA TCT TCA GGA AGT GTA C-3’reverse，

452bp, annealing temperature is 53 ℃;

cytokeratin 19，5’-GTC CTA CAG ATT GAC AAT GC-3’forward，

5’-CAC GCT CTG GAT CTG TGA CAG-3’reverse，

569bp, annealing temperature is 53 ℃;

cytokeratin 18，5’-GGA CCT CAG CAA GAT CAT GGC-3’forward，

5’-CCA CGA TCT TAC GGG TAG TTG-3’reverse，

515bp, annealing temperature is 55 ℃;

cytokerat in 8，5’-AGT CTC AGA TCT CAG ACA CG-3’forward，

5’-CCA TAG GAT GAA CTC AGT CC-3’reverse，

561bp, annealing temperature is 55 ℃;

glucose-6-phosphatase，

5’-AAC CCA TTG TGA GGC CAG AGG-3’forward，

5’-TAC TCA TTA CAC TAG TTG GTC-3’reverse，

438bp, annealing temperature is 55 ℃;

biliary glycoprotein，

5’-GAA CTA GAC TCT GTC ACC CTG-3’forward，

5’-GCC AGA CTT CCT GGA ATA GA-3’reverse，

407bp, annealing temperature is 53 ℃;

β-actin，5’-TTC CTT CTT GGG TAT GGA AT-3’forward，

5’-GAG CAA TGA TCT TGA TCT TC-3’reverse，

203bp。

5. transmission electron microscope is PHILIPS TECNAL 10;

Atomic force microscope is Auto-probe CP Research (Si ₃N ₄Cantilevers with integral tips; Thermomicroscopes, the U.S.).

(2) the embryo stem cell source elliptocyte external evoked with separate the purification culturing process

1. the cultivation of embryonic stem cell:

Balb/c embryonic stem cell frozen in the liquid nitrogen is taken out fast, place 37 ℃ of warm water quick-thawing rewarmings (in 1-2 minute) immediately, stop buffer cleans, the centrifugal 5min of 1000rpm, abandon supernatant, add the embryonic stem cell substratum, the optimum density transferred species places 37 ℃, 5%CO in the disposable plastic culturing bottle ₂Cultivate in the incubator, change liquid every day 1 time.Go down to posterity when the cell stand density reaches about 70%-80%, average per 2～3d goes down to posterity 1 time.

2. generation embryoid body:

Embryonic stem cell is gone in the glass culturing bottle with optimum density, with differential period minimum medium resuspended, shake culture is cultivated (the wave and culture bottle was 1 time in average per 1 hour, and keeping cell to be in the suspended state growth is embryoid body with the growth).Change liquid every day 1 time, cultivated 3 days.The same day that begins to cultivate embryoid body is as inducing the 1st day of differentiation.

3. break up to the hepatic parenchymal cells directional induction:

Embryoid body was cultivated after 3 days, and the embryoid body size is appropriateness evenly.Its optimum density is gone to plastic culture plate or culturing bottle and grouping, and the substratum of inducing group is for containing the differential period minimum medium of HGF (30ng/ml) and EGF (100ng/ml); The substratum of control group only is the differential period minimum medium, and embryoid body will break up naturally at random.

Immunocytochemistry (Immunocytochemistry, ICC)

4.1. dyeing procedure is as follows:

(1) culturing cell (adherent growth) is with the fixing 20min of 4% cold Paraformaldehyde 96 room temperature, distillation washing;

The 0.3%Triton X100 room temperature rupture of membranes 20min that uses when (2) detecting ALB antigen, the distillation washing;

(3) 30%H ₂O ₂Pure methyl alcohol (1: 50) deactivating endogenous peroxydase, room temperature 20min, distillation washing 3 times;

(4) 5%BSA room temperature sealing 20min gets rid of unnecessary liquid (not washing);

(5) drip one of dilution and resist (A6-McAb 1: 20; ALB-McAb 1: 100) 4 ℃ of overnight incubation, PBS (PH7.2-7.6) washes 3 times, each 2min;

(6) drip the biotinylation goat anti-mouse igg, hatch 20min for 37 ℃, PBS (PH7.2-7.6) washes 3 times, each 2min;

(7) drip SABC (Streptavidin-superoxide enzyme complex), hatch for 37 ℃, 20min, PBS (PH7.2-7.6) washes 4 times, each 5min;

(8) freshly prepared DAB color development at room temperature (controlling reaction time under the mirror), Hematorylin is slightly redyed, conventional gradient alcohol dehydration mounting microscopy.

Negative control then substitutes anti-hatching with PBS, and all the other steps are constant.

4.2.A6 positive elliptocyte percentage:

Induce the capable A6 monoclonal antibody dyeing of group and the postdigestive single cell suspension of control group and selected by flow cytometry apoptosis cell, its dyeing procedure is substantially with above-mentioned ICC, and DAB drops in microscopy and cell counting on the slide glass after developing the color.The cell of different incubation times (inducing differentiation the 5th, 7,9,11 day) is respectively got 3 samples, and each sample standard deviation is got 5 visual field counting A6 positive cell numbers and total cell count at random, and both ratios are the positive elliptocyte percentage of A6.Induce the difference row statistical analysis (t check) between group and control group result, P＜0.05 o'clock is the significance of difference.

5. RT-polymerase chain reaction (RT-PCR):

The CD34+/Sca-1+ cell of convection type cell instrument sorting and the cell of cultivating 20 days thereof carry out RT-PCR and analyze (pressing the operation of test kit specification sheets).General steps is as follows:

With Trizol extracting cell total rna;

Reverse transcription reaction;

The PCR reaction is specially:

Add (50ul system) 2 * PCR Mix 25ul on ice,

forward primer 1ul，

reverse primer 1ul，

Template DNA 5ul，

H ₂O 18ul.

Of short duration centrifugal behind the mixing, place the PCR instrument to increase.94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, annealing 1min, 72 ℃ prolong 1min, totally 30 circulations; Last 72 ℃ prolong 10min.

The PCR product is in 1% agarose gel electrophoresis imaging, and ultraviolet observations is down also taken pictures.

6. flow cytometry (FACS):

Induce the at first suitably digestion (0.25%Trypsin-1mM EDTA4Na) of culturing cell group of group and control group, and fully blow and beat into single cell suspension.0.1mM PBS (PH7.2-7.4) clean cell after, counting cells gets about 1 * 10 ⁶Individual, add anti-mouse Sca-1-PE-Cy5, anti-mouse CD34-PE, anti-mouse CD45-FITC and an amount of PBS (reaction system is 20 μ l) successively, the lucifuge incubated at room is 20 minutes behind the abundant mixing.PBS fully cleans and machine testing is gone up in resuspended back.The capable FACS of the cell of different incubation times (breaking up the 4th day to the 10th day) detects, to analyze Sca-1, CD34 and antigenic expression degree of CD45 and trend; The cell that broke up the 4th, 6,8,10 day is respectively got 3 samples, and the numerical value of acquisition is got average as the CD34+/Sca-1+ cell positive rate of this day, induces the difference row statistical analysis (t check) between group and control group result, and P＜0.05 o'clock is the significance of difference.

7. transmission electron microscope detects (TEM):

The CD34+/Sca-1+ cell of selected by flow cytometry apoptosis is down fixed about 4 hours with 2.5% glutaraldehyde in 4 ℃, fully cleans with the PBS of 0.01M.Use 1%OsO then ₄Pre-fixed 30 minutes, conventional gradient alcohol dehydration is used the Resins, epoxy embedding afterwards.Prepare ultrathin section(ing), and dye transmission electron microscope observing with two hydrogen uranium of acetic acid and lead citrate.

8. atomic force microscope detects (AFM):

Get and induce the 9th day noble cells of componentization to carry out A6 antigen dyeing (the same ICC of step).Undifferentiated embryonic stem cell in contrast.Embryonic stem cell is digested to single cell suspension with 0.25% pancreas enzyme-EDTA, and (concentration is 1 * 10 ⁶/ ml), the glutaraldehyde room temperature with 2.5% is fixedly behind the 30min, and it is standby to drop on the cover glass dry air 20min.Cell to be checked places on the sheet mica, inserts then in the scanning area of sample table.On watch-dog, determine the approximate location of purpose cell, with careful the moving to of scanning probe tip in purpose cell region.Be adjusted under the Autoprobe CP Tapping pattern, and cantilever (ParkScientific Instruments, Microlever) the needle point radius-of-curvature is 10nm, and force constant is about 2.8N/m, and scanning speed is 0.5-1Hz, observes under normal atmosphere (An), the room temperature.Afm image utilizes the line analysis and the surface analysis function of software only through smoothing processing (using Thermomicroscopes ProscanImage Processing Software Version 2.1 softwares), and image is analyzed.

(3) result

1. embryonic stem cell and induce cell growth state in the atomization to hepatic parenchymal cells

Embryonic stem cell clonal expansion, growth rapidly, cell is closely arranged, the unclear (Fig. 1-A) of boundary.Digestion back with differential period minimum medium resuspended, the row suspension system is cultivated, embryonic stem cell is assembled mutually and is formed transparent spherical embryoid body (Fig. 1-B).After 3 days, treat that embryoid body grows into modest size, with its grouping and change in plastic culture plate or the culturing bottle and cultivate.After about 3-4 hour embryoid body is adherent, change the substratum of inducing group into contain HGF and EGF substratum, begin to induce differentiation.It is radiation growth around the middle mind-set that differentiated cell population the wall place with embryoid body, and the differentiated cell population of inducing group is the epithelial cell of form than homogeneous, is paving stone sample (Fig. 1-C).And the differentiated cell population of control group is complicated, and form is various, and the mixed mutually growth of visible trilateral, circle, spindle cell (Fig. 1-D).

2. embryonic stem cell is induced cell A6 antigen and the antigenic expression of ALB in the atomization to hepatic parenchymal cells

ESC directional induction differone A6 antigen and the antigenic expression of ALB had been detected for cell mass with immunocytochemistry in the 6th, 9,12,15 day of differentiation.Differentiation can measure the antigenic expression of A6 on the 6th day, Fig. 2 (A)-(E) shows that differentiation induced the antigenic expression of component cell mass A6 on the 9th day.The positive elliptocyte of A6 mainly is distributed in (Fig. 2 B, C) among the formed compact arranged cell colony in the adherent back of embryoid body, and the differentiation of colony periphery position is than (Fig. 2 E) in the microcolony between the mature cell group.The top of visible cell colony is being mingled with the positive elliptocyte of an A6, the visible double-core liver cell in its next door among Fig. 2 B.The positive epithelial duct like cell (Fig. 2 D) of the A6 of visible two spindle shapes in the lower left of cell colony.Fig. 2 (F) shows the 15th day and induces the antigenic expression of ALB in the component cell mass, the positive double-core liver cell of visible a plurality of ALB.

3. the liver of embryonic stem cell is the flow cytometry in the directional induction atomization

Get the 4th day to the 10th day the differentiation daughter cell of inducing group and control group of differentiation, the expression with facs analysis antigen Sca-1, CD34 and CD45 the results are shown in Figure 3,4.Sca-1 selects index as door.The nearly all Sca-1+ cell of Fig. 3 results suggest is all expressed CD45 again, that is to say that CD45 does not have obvious effect to the sorting of elliptocyte.So only analyze the 4th day to the 10th day differentiation daughter cell of two groups of differentiation with Sca-1 and the two indexs of CD34.(Fig. 4) substantially induces that Sca-1+/CD34+ cell positive rate began to rise from breaking up on the 4th day in the differentiation daughter cell of group, and (1.20%) back of peaking to the 8th day descends; And the maintaining basically of control group one low-level (0.03%-0.21%).Data point distribution from the R1 district, the cell of inducing group are based on minicell, and the most cells structure is simpler; The cell of control group was divided into two groups on the 8th day in differentiation, induced group then not have this phenomenon always.The cellular control unit point diagram disperses always in the R2 district (Sca-1 Gated), and induces the passing cell point diagram along with between differentiation phase of group to trend towards concentrating and moving right.Induce the overall fluorescent intensity of group cell constantly to strengthen and peaked, and always being in of control group is low-level in the 8th day.The cell that broke up the 4th, 6,8,10 day is respectively got 3 samples, gets average as the CD34+/Sca-1+ cell positive rate of this day, induces the difference row statistical analysis between group and control group result, the results are shown in Table 1.

Table 1. embryonic stem cell is induced the statistics of Sca-1+/CD34+ cell positive rate in the atomization to hepatic parenchymal cells

	Induced the 4th day	Induced the 6th day	Induced the 8th day	Induced the 10th day
					Induce group	0.15％±0.01％	0.76％±0.05％	1.13％±0.04％	0.74％±0.20％
Control group	0.05％±0.01％	0.12％±0.03％	0.27％±0.03％	0.14％±0.07％
					The P value	＜0.05	＜0.01	＜0.01	＜0.01

P＜0.05 is shown with significant difference; P＜0.01 is shown with utmost point significant difference.

4. the detection of two capable A6 antigens of positive cell of flow cytometer screening and transmission electron microscope

We have detected to break up to induce in the 8th day in the group flow cytometer screening Sca-1+/CD34+ cell and have expressed the shared ratio (see figure 5) of A6 cell antigen.The A6+ cell accounts for 92.48% ± 2.01% of total cell count in the Sca-1+/CD34+ cell, and the part cell is strong positive; But not two positive cells account for 1.66% ± 1.57%.Transmission electron microscope results is seen Fig. 6, and the elliptocyte volume is less, and about general 7-15 μ m, karyon is big and endochylema is less, the karyoplasmic ratio height; Nuclear is oval, visible spissated chromatin; The caryoplasm inner cell organ is less, visible a small amount of plastosome and rough surfaced endoplasmic reticulum; The visible a small amount of microvillus of cell edges.

5. the RT-PCR of two positive cells of flow cytometer screening and cultivation daughter cell thereof detects

RT-PCR the results are shown in Figure 7.Fig. 7 A is the RT-PCR result of the fresh screening of flow cytometer Sca-1+/CD34+ cell, as seen liver cell mark AFP, ALB, bile duct cell mark ck19, liver is that cell sign thing ck8, ck18 are all positive, and ripe liver cell mark glucose-6-phosphatase (G6Pase) and ripe bile duct cell mark biliary glycoprotein (BG) are negative; Fig. 7 B for screening Sca-1+/CD34+ cell cultures 20 days the RT-PCR result of cell mass, different with A is, AFP does not express, glucose-6-phosphatase and biliary glycoprotein then have positive band.

6. the embryo stem cell source gamogenetic egg is justified the differentiation rate of cell

To induce group and each sampling of control group to be digested to the capable A6-McAb dyeing of single cell suspension, drop in microscopy counting on the slide glass after the DAB colour developing in the 5th, 7,9,11 day of differentiation.The differentiation rate of two groups of A6 (+) elliptocyte of gained sees Table 2.Inducing the differentiation rate variation range of group A6 (+) elliptocyte is 1.25%-4.46%, induces about the 7th day and peaks; And always the remaining on of control group low-level (0.14%-0.25%).The t assay is shown the differentiation rate of inducing group A6 (+) elliptocyte the same period height than control group, and difference has significance.

The differentiation rate of table 2. embryo stem cell source gamogenetic egg circle cell

	Induced the 5th day	Induced the 7th day	Induced the 9th day	Induced the 11st day
					Induce group	1.79％±0.18％	4.46％±0.94％	3.64％±0.49％	1.25％±0.36％
Control group	0.16％±0.07％	0.25％±0.07％	0.20％±0.10％	0.14％±0.06％
					The P value	＜0.01	＜0.01	＜0.01	＜0.05

P＜0.05 is shown with significant difference; P＜0.01 is shown with utmost point significant difference.

7.A6 the positive elliptocyte film Ultrastructural atomic force microscope analysis in surface

Induce the 9th day elliptocyte of group by locating (Fig. 8 .A, B) with the dyeing of A6 monoclonal antibody, Fig. 8 .C-F is the Ultrastructural atomic power scintigram in elliptocyte film surface.Embryonic stem cell is (Fig. 8 .G-I) in contrast, and is rounded, and diameter is about 9 μ m, and surface of cell membrane has most ellipsoidal particles, and granular size is 40-80nm.The elliptocyte ovalize, volume bigger (diameter is about 11 μ m), surface of cell membrane amounts of particles more and bigger (size is 200-400nm) is thick prolate ellipsoid shape.For further analysis of cells film surface particles distribution situation, elliptocyte and embryonic stem cell are respectively got the localized membrane surface (Fig. 8 .E and I) of equal area (2 * 2 μ m) and are carried out quantitative analysis.Fig. 8. (1) and (2) is respectively the histogram of whole scanning area surface of cell membrane particle diameters among Fig. 8 .E and the I, and statistics sees Table 3.The film surface particles of elliptocyte (Fig. 8. (1)) camber is that the data point (data points) of 335nm is maximum, and embryonic stem cell (Fig. 8. (2)) highly be that the data point of 602nm is maximum.Surface of cell membrane size distribution situation in the line analysis of use IP2.1 software and surface analysis functional measurement Fig. 8 .E and the I zone, and by four parameters---mean roughness (average roughness, R _a), maximum peak valley spacing (maximumpeak-to-valley distance, R _P-v), r.m.s. roughness (root-mean-squared roughness, R _Rms), (mean height z) comes further quantitative analysis elliptocyte film and the intermembranous feature difference (table 3) of embryonic stem cell to center line average.Four parameters of embryonic stem cell film (R especially _aAnd R _Rms) all greater than the elliptocyte film, that is to say the more coarse of embryonic stem cell localized membrane surface ratio elliptocyte.

The quantitative analysis of table 3. elliptocyte film and the intermembranous feature difference of embryonic stem cell

	The elliptocyte film	The embryonic stem cell film
			Maximum peak valley spacing, R p-v(nm)	418.4	751.9
R.m.s. roughness 1, R rms(nm)	91.26	157.6
			Mean roughness, R a(nm)	78.74	127.0
Center line average, z (nm)	219.3	399.6

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Claims (7)

1. the external evoked method of a stem cell source elliptocyte is characterized in that: with pHGF HGF, Urogastron EGF embryonic stem cell is carried out the directional induction differentiation; The cell of inducing differentiation is carried out the screening of A6+ elliptocyte.

2. external evoked method as claimed in claim 1, wherein embryonic stem cell is to change into the embryonic stem cell of embryoid body differentiation after 3 days.

3. external evoked method as claimed in claim 1 or 2, wherein embryonic stem cell is a mammalian embryonic stem cell.

4. external evoked method as claimed in claim 1 or 2, wherein embryonic stem cell is a mouse embryo stem cell.

5. external evoked method as claimed in claim 1, screening wherein utilize Sca-1 and CD34 to be two indexs.

6. external evoked method as claimed in claim 5, screening wherein utilize flow cytometer to carry out.

7. external evoked method as claimed in claim 1, wherein HGF is that 30ng/ml and EGF are 100ng/ml.

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