CN104862272B - Application of the Y-27632 inhibitor in the sorting of CD44 positive intestinal stem cells - Google Patents
Application of the Y-27632 inhibitor in the sorting of CD44 positive intestinal stem cells Download PDFInfo
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Abstract
It is to sort intestinal stem cell using CD44 as label the present invention relates to application of 27632 inhibitor of Y in the sorting of CD44 positive intestinal stem cells.By using 27632 inhibitor of Y and magnetic bead sorting CD44 positive cells are used in combination, to keep the method for separation intestinal stem cell more convenient, operation is simpler.Present invention application membranous antigen sorts the intestinal stem cell in crypts, does not injure eucaryotic cell structure, moreover, separation method using the present invention once can get sufficient amount of intestinal stem cell, material base has been established for the development of subsequent applications.
Description
Technical field
The present invention relates to stem cells to be separately cultured technical field more particularly to a kind of Y-27632 inhibitor in the CD44 positives
Application in intestinal stem cell sorting.
Background technology
In recent years, high expression of Lgr5 (GPCR49) albumen in intestinal crypts is widely accepted to identify the mark of intestinal stem cell
Note.2009, the scholars such as Japanese Sato were from parental generation C57/B6JLgr5-IRES-CRE-eGEFPHigh expression is isolated in mouse intestinal
The cell of Lgr5 albumen, and establish a set of perfect Differentiation Induction in vitro system.They confirm that mouse intestinal crypts base portion is high
The cell of expression Lgr5 albumen can independently be proliferated and be divided into a complete small enteric epithelium structure in vitro.
Currently, the major technique for being applied to separation intestinal stem cell in the world is fluidic cell sorting technology.But it is domestic at present
Research of the scholar in this field is more rare.But, because colorectal cancer originates from the gene mutation of intestinal stem cell, domestic scholars pair
The sorting of large intestine cancer stem cell has carried out corresponding research.For example, the primary cancerous tissue to 10 colorectal cancer fresh specimens such as Jia Ru
It is detected and sorts with the content of tumor stem cell in metastatic carcinoma tissue (cancer stem cell, CSC).They flow in application
The primary cancerous tissue of formula antibody CD133-PE label colorectal cancers and metastatic carcinoma tissue cell suspension, go out using flow cytomery
The CSC cell contents of the CD133 positives.But, they think so to use fluidic cell sorting technology bigger to the damage of cell, point
Select the period long, it is of high cost, complicated for operation (referring to Jia Ru etc., the detection of tumor stem cell in large intestine primary carcinoma and metastatic carcinoma tissue
With sorting, clinical and experimental pathology magazine, the 9th phase, on September 16th, 2014).
In existing research, also there are some investigators to find that using magnetic bead sorting technology that can also realize does colorectal cancer
The sorting of cell.For example, after roc etc. is incubated using monoclonal T cell differentiation antigen primary antibody with tumor cell suspension, then with immunomagnetic beads mark
The secondary antibody of note combines.The characteristics of being specifically bound using primary antibody, secondary antibody, when positive cell suspension flows through sorting magnetic field,
It can be adsorbed in magnetic-type sorting column.Magnetic-type sorting column magnetic field is removed out of column again to elute, collect large intestine cancer stem cell (referring to
Roc etc., the progress of large intestine cancer stem cell sorting and identification, world Chinese digestion magazine, the 36th phase of volume 16,2008 12
The moon 28).However, the magnetic bead sorting technology depends on the identification to colorectal cancer stem cell surface marker, do not find yet so far
The specific surfaces marker of large intestine cancer stem cell, thus there are still certain difficulties when sorting aim cell.
At present studies have found that characteristic of the intestinal stem cell with " empty nest apoptosis ", needs that Y- is added in cultivating system
27632 inhibitor inhibit the generation of this phenomenon.The chemical molecular formula of Y-27632 inhibitor is C14H21N3O.2HCl, is
The inhibitor of ROCK apoptotic signal accesses, this inhibitor can inhibit " the empty nest apoptosis " of intestinal stem cell.
However, during sorting, it is impossible to enough Y-27632 inhibitor be added in sheath fluid to inhibit intestines dry thin
The empty nest apoptosis of born of the same parents, this is the major technique limitation of Lgr5 positive intestinal stem cells sorting.In addition, due to C57/B6JLgr5 -IRES-CRE-eGEFPThe expression of mouse CRE genes needs tamoxifen to induce, and after tamoxifen induces 10% Lgr5
Intestinal stem cell i.e. apoptosis immediately, this can cause sorting stem cell population to be reduced；Secondly, relative to inbred strais wild-type mice
For, C57/B6JLgr5-IRES-CRE-eGEFPTransgenic mice is expensive and rearing conditions are harsh；Third, market is on sale at present
The binding site of Lgr5 monoclonal antibodies only in the cell, certainly will need to carry out rupture of membranes to cell by the methods of antibody label,
The activity of cell will certainly be influenced after rupture of membranes.Therefore, intestinal stem cell is sorted by above a variety of using this target protein of Lgr5
The limitation of technical conditions.
One kind intestinal stem cell method for separating of low cost, easy to operation, simple, quick how is found to have become at present urgently
Problem to be solved.
Invention content
The present invention provides a kind of methods of sorting CD44 positive intestinal stem cells, and especially Y-27632 inhibitor is in CD44
Application in positive intestinal stem cell sorting, has the characteristics that of low cost, easy to operation, simple and quick.
To reach the invention purpose, the present invention uses following technical scheme：
In a first aspect, the application the present invention provides Y-27632 inhibitor in the sorting of CD44 positive intestinal stem cells, described
Using include use the culture medium containing Y-27632 inhibitor.
Used culture medium mainly has basal medium, dissociation culture medium etc. in application in the present invention, typically
Contain above-mentioned Y-27632 inhibitor in dissociating culture medium.
Y-27632 inhibitor used in the present invention is not particularly limited, such as can come from the U.S.
The Y-27632 inhibitor of Sigma-Aldrich companies.
When the present invention is added to CD44 positive intestinal stem cells sorting culture medium used by using Y-27632 inhibitor,
But it may be implemented that intestinal stem cell is inhibited to occur " empty nest apoptosis " in assorting room, to greatly improve surviving for aim cell
Rate.
Application of the present invention includes that the separation of CD44 positive intestinal stem cells is carried out using magnetic bead.
Preferably, the CD44 positives intestinal stem cell is detached from intestinal crypts obtains.
For airflow classification, magnetic bead sorting method is time saving, easy to operate；Cell contamination probability is low, of low cost,
Product purity is high.
The expression of Lgr5 is regulated and controled by Wnt/ β-catenin signal paths, and the access causes intestinal stem cell to be proliferated
Major impetus.Recent study shows that another target for modulation of Wnt/ β-catenin signal paths is CD44 genes.Due to intestines
Stem cell has the activity of high proliferation, then the expression product CD44 albumen of CD44 genes can be present in the surface of intestinal stem cell.
Using this feature, the present invention has carried out point wild inbred mouse intestinal crypts CD44 positive cells using magnetic bead sorting technology
Choosing.Later, it is cultivated using relevant intestinal stem cell cultivating system.It was found that there are a group intestines in CD44 positive cells
Stem cell.Therefore, CD44 antigen is one of surface markers of intestinal stem cell.Using this labelling technique, it, which is avoided, sorted
Rupture of membranes is carried out to cell in journey, does not injure cell activity；It is done in addition, this labelling technique is conveniently used for wild inbred mouse intestines
The sorting of cell.Importantly, due to small (the cell suspension saturation of the system of magnetic bead sorting：1~2ml), it is allowed in point
Enough Y-27632 inhibitor are added to inhibit " the empty nest apoptosis " of intestinal stem cell during choosing.Therefore, the present invention passes through application
Magnetic bead technology and Y-27632 inhibitor sort CD44 positive intestinal stem cells, overcome all in the presence of the prior art
More problems.
The present invention can individually use in addition Y-27632 inhibitor to culture medium or combine the side using magnetic bead sorting
Formula carries out, and when using two ways combination, separating effect will protrude more.For example, not adding Y-27632 inhibitor
To the group of culture medium, the colony-forming efficiency of intestinal stem cell is significantly lower than the group for adding Y-27632 inhibitor.
The present invention is that intestinal stem cell is sorted from intestinal tissue using CD44 as label.Due to CD44 genes in intestinal stem cell
Expression adjusted by Wnt/ β-catenin signal paths, meanwhile, the expression of CD44 genes power is in each position of crypts
It is different in cell, it is expressed strongly in the cell close to crypt base, and the location presentation far from substrate is weaker.By
There is higher expression in intestinal stem cell as a kind of membranous antigen in CD44, thus the present invention passes through this molecule of CD44
The target spot as sorting intestinal stem cell is marked, to carry out the separation of mouse intestinal stem cell.
In the present invention, the method includes CD44+ cells are sorted from the enteron aisle of inbred mouse.Why the present invention adopts
It is since relative to transgenic mice, inbred mouse is cheap, is easy to get, and genetic background is steady with inbred mouse
It is fixed.Preferably, the inbred mouse is C57BJ/6 mouse.
Heretofore described application mainly includes the following steps that：
(1) crypts is detached from intestinal tissue；
(2) crypts single cell suspension is prepared；
(3) crypts CD44 positive intestinal stem cells are sorted.
Specifically, the step (1) in application of the present invention includes the following steps：
A) under aseptic condition, intestinal tissue is splitted, removes intestinal contents, and remove the villus ingredient in epithelium；
B) the obtained intestinal tissues of step a) are placed in centrifuge tube, rinse supreme limpid clear, addition separating liquid, incubation repeatedly
20-40min；
C) it waits for that intestinal tissue is settled down to centrifuge tube bottom, separating liquid is sucked out, supernatant is abandoned in centrifugation；
D) the D-hanks liquid of precooling is added, cell is resuspended and being aggressively shaken makes crypts be released from intestinal tissue, it is micro-
Under the microscope until being full of abundant crypts in supernatant；
E) supernatant is sucked out, is filtered, filtrate is collected into the coated centrifuge tubes of BSA and centrifuged, and supernatant is discarded；
F) cell is resuspended with basal medium, cell suspension is transferred in centrifuge tube, centrifuge, discard supernatant, repeat 2-3
It is secondary, to remove the individual cells in cell suspension to purify crypts.
Preferably, the step b) separating liquids are the D-hanks solution of the EDTA containing 2mM, pH value 8.0.
Preferably, the step f) basal mediums be containing 1 × Glutamax (such as Invitrogen companies of the U.S.),
1 × HEPES (such as Invitrogen companies of the U.S.) and l × mycillin (such as Invitrogen companies of the U.S.)
Advanced DMEM/F12 serum free mediums (such as Invitrogen companies of the U.S.).
Heretofore described step (1) specifically includes following steps as a preferred technical solution,：
A) under aseptic condition, whole section of small intestine is detached from intestinal tissue, it is splitted along the longitudinal axis, using the D- of precooling
Hanks liquid washes away intestinal contents；
B) the light postcibal diarrhea Cavity surface of coverslip to sterilize, to remove the villus ingredient in epithelium；
C) intestinal tissue that step b) is obtained is cut with sterile scissors to the big fractionlets of 3mm and is placed in 50mL centrifuge tubes, used
Precooling D-hanks liquid rinses supreme limpid clear repeatedly；
D) separating liquid is added, 4 DEG C of shaking table concussions are incubated 30min；
E) it after being incubated, waits for that tissue is settled down to centrifuge tube bottom, separating liquid is carefully sucked out, 1000rpm centrifuges 5min, discards
Supernatant；
F) the D-hanks liquid of 30mL precoolings is added, cell is resuspended and being aggressively shaken makes crypts be released from intestinal tissue,
Microscopically observation is until be full of abundant crypts in supernatant；
G) supernatant is sucked out, with 70 μm of strainer filterings, filtrate is collected into the coated centrifuge tubes of BSA, 1000rpm from
Heart 5min, discards supernatant；
H) it uses l0mL basal mediums that cell is resuspended, cell suspension is transferred in 15mL centrifuge tubes, 500rpm centrifugations
2min discards supernatant, repeats 2-3 times, to remove the individual cells in cell suspension to purify crypts.
Specifically, the step (2) in application of the present invention includes the following steps：
A) crypts that step (1) detaches is placed in dissociation culture medium, is incubated 30-45min, blown and beaten repeatedly carefully per 10min
Born of the same parents' suspension 5-10 times, to promote individual cells to be released from crypts；
B) after being incubated, suspension is filtered, collect filtrate and is centrifuged, supernatant is discarded, obtains crypts single cell suspension；
Preferably, the dissociation culture medium be containing 1 × Glutamax (such as Invitrogen companies of the U.S.), 1 ×
HEPES (such as Invitrogen companies of the U.S.), 1 × mycillin (such as Invitrogen companies of the U.S.), 1 ×
N2supplement (such as Invitrogen companies of the U.S.), 1 × B27supplement (such as Invitrogen companies of the U.S.)
With the Advanced DMEM/F12 free serum cultures of 10 μM of Y-27632 inhibitor (such as Sigma-Aldrich)
Base (such as Invitrogen companies of the U.S.).
As optimal technical scheme, heretofore described step (2) specifically includes following steps：
A) crypts that step (1) detaches being placed in the dissociation culture medium of 5mL precoolings, 4 DEG C of shaking tables are incubated 45 minutes, and every 10
Minute piping and druming cell suspension 5-10 times repeatedly, to promote individual cells to be released from crypts；
B) it after being incubated, successively with 40 μm of strainers, 20 μm of strainer filtering suspensions, collects filtrate and 1000rpm centrifuges 5 points
Clock discards supernatant.
Specifically, the step (3) in separation method of the present invention includes the following steps：
A) CD44 monoclonal antibodies are added into the dissociation culture medium of step (2), are incubated 25-30min, centrifugation washes away residual
Antibody is stayed, it is primary to repeat this step；
B) magnetic bead coupled antibody is added into dissociation culture medium again, is incubated 15-20min；
C) cell suspension that oneself incubation finishes is loaded to Magnetic Isolation column, the cell being adsorbed in column is collected in elution, i.e.,
For CD44 positive intestinal stem cells；
Preferably, the dissociation culture medium be containing 1 × Glutamax (such as Invitrogen companies of the U.S.), 1 ×
HEPES (such as Invitrogen companies of the U.S.), 1 × mycillin (such as Invitrogen companies of the U.S.), 1 ×
N2supplement (such as Invitrogen companies of the U.S.), 1 × B27supplement (such as Invitrogen companies of the U.S.)
With the Advanced DMEM/F12 free serum cultures of 10 μM of Y-27632 inhibitor (such as Sigma-Aldrich)
Base (such as Invitrogen companies of the U.S.).
As optimal technical scheme, heretofore described step (3) specifically includes following steps：
A) the dissociation culture medium for drawing 1-2mL precoolings, with 1:It is mono- that rabbit anti-mouse CD44 is added in the ratio of 50 (w/v) thereto
Clonal antibody is incubated 30 minutes on ice, and 1000rpm is centrifuged 2 minutes, washes away remaining antibody, and it is primary to repeat this step；
B) the dissociation culture medium for drawing 1-2mL precoolings again, with 1:Goat anti-rabbit igg is added in the ratio of 4 (v/v) thereto
Magnetic bead coupled antibody is protected from light incubation 15 minutes on ice；
C) cell suspension that oneself incubation finishes is loaded to Magnetic Isolation column, the cell being adsorbed in column is collected in elution, i.e.,
For CD44 positive intestinal stem cells.
Isolated intestinal stem cell height expresses Lgr5 albumen in the present invention, and can be divided into and constitute the four of enteric epithelium
Type cell：Paneth's cell absorbs cell, endocrine cell and goblet cell.
Second aspect, the present invention also provides a kind of methods preparing small enteric epithelium structure comprising following steps：
(1) pass through the isolated CD44 positives intestinal stem cell of application described in first aspect；
(2) CD44 positives intestinal stem cell described in in-vitro multiplication and it is divided into complete small enteric epithelium structure.
As currently preferred technical solution, in the method for preparing small enteric epithelium structure, step (2) includes：
A) matrigel melted on ice will be previously placed in and the ratio of 10 μ L matrigels is added in ice with every 100 intestinal stem cells
Upper resuspension cell；
B) after cell premix, matrigel and cell suspension are added by 37 DEG C of preheatings with the ratio in 10 μ L matrigels/hole
96 well culture plates；When inoculation, matrigel is located at the center of board bottom, and glue is avoided to touch wall；
C) cell of inoculation is placed in 37 DEG C of incubators and is incubated 5min, matrigel is made to shape；
D) after being incubated, 100 μ L complete mediums, 37 DEG C, 5%CO are added into every hole22 are incubated in wet environment
Collect small enteric epithelium structure in week.
Preferably, the matrigel is addition 1mM Jagged-1 (such as Ana Spec companies of the U.S.) without phenol red no life
The semisolid culturemedium of the long factor (Growth factor reduced)；The complete medium is to contain 1 × Glutamax
(such as Invitrogen companies of the U.S.), 1 × HEPES (such as Invitrogen companies of the U.S.), l × mycillin are (such as beautiful
Invitrogen companies of state), 1 × N2supplement (such as Invitrogen companies of the U.S.), 1 × B27supplement (examples
Such as Invitrogen companies of the U.S.), 10 μM of Y-27632 inhibitor (such as Sigma-Aldrich), l μ g/mL
Human R-spondin (such as PeproTech companies of the U.S.), (such as the U.S. 100ng/mL murine Noggin
PeproTech companies), 100ng/mLmurine EGF (such as PeproTech companies of the U.S.), 100ng/ml Murine Wnt-
3a's (such as PeproTech companies of the U.S.) and 1mM N-Acetylcysteine (such as Sigma-Aldrich)
Advanced DMEM/F12 serum free mediums (such as Invitrogen companies of the U.S.).
Compared with prior art, the present invention at least has the advantages that：
(1) present invention inhibits intestinal stem cell in assorting room using Y-27632 inhibitor is added into culture medium
" empty nest apoptosis " improves the colony-forming efficiency about 50% of aim cell.
(2) present invention is more convenient using the relatively traditional intestinal stem cell method for separating of separation method operation of magnetic bead.
(3) present invention sorts the intestinal stem cell in crypts using membranous antigen, does not injure eucaryotic cell structure；Moreover, adopting
Sufficient amount of intestinal stem cell is once can get with the separation method of the present invention, substance base has been established for the development of subsequent applications
Plinth.
Description of the drawings
Fig. 1 is the CD44 positive cell phenotypic maps using flow cytometry identification magnetic bead sorting；
Fig. 2 is the ectogenesis figure of CD44 positive intestinal stem cells；Wherein, A is the high equimultiple image developed the 0th to 9 day
(400 times)；B is the medium-power image (200 times) developed the 10th to 14 day；C is that CD44 negative cells develop the 14th day low
Equimultiple image (40 times)；D is the high equimultiple image (40 times) that CD44 positive cells are developed the 14th day；
Fig. 3 is the differentiation figure of CD44 positive intestinal stem cells；Wherein, DAPI is label nucleus；Villin is that label absorbs
Cell；Muc2 is label goblet cell；Chr-A is label enteroendocrine cell；LysozymeC is label paneth's cell；Merge
It is blending image；
It is positive thin to CD44 that Fig. 4 is that fluidic cell sorting technology combines Y-27632 inhibitor sorting technology with magnetic bead respectively
The influence of cytoactive；Wherein, left figure is magnetic bead combined Y-27632 inhibitor sorting technology；Right figure is fluidic cell sorting technology；
Fig. 5 is the colony-forming efficiency of CD44 positive cells when having no added Y-27632 inhibitor during magnetic bead sorting；Its
In, left figure is not add Y-27632 inhibitor groups；Right figure is addition Y-27632 inhibitor groups.
Specific implementation mode
The technical solution further illustrated the present invention below by specific implementation mode.
Those skilled in the art understand the present invention it will be clearly understood that the embodiment is only to aid in, and are not construed as to this hair
Bright concrete restriction.
Preparation of reagents
1. separating liquid：The D-hanks solution (pH 8.0) of the EDTA containing 2mM.
2. basal medium：Advanced DMEM/F12 serum free mediums (Invitrogen companies of the U.S.), wherein containing
There are 1 × Glutamax (Invitrogen companies of the U.S.), 1 × HEPES (Invitrogen companies of the U.S.), 1 × mycillin (beautiful
Invitrogen companies of state).
3. dissociating culture medium：Advanced DMEM/F12 serum free mediums (Invitrogen companies of the U.S.), wherein containing
There are 1 × Glutamax (Invitrogen companies of the U.S.), 1 × HEPES (Invitrogen companies of the U.S.), 1 × mycillin (beautiful
Invitrogen companies of state), 1 × N2supplement (Invitrogen companies of the U.S.), (U.S. 1 × B27 supplement
Invitrogen companies), 10 μM of Y-27632 (Sigma-Aldrich).
4. matrigel：U.S. company BD production without growth factor (Growth factor reduced) and without phenol red
Semisolid culturemedium need to individually add 1mM Jagged-1 (Ana Spec companies of the U.S.).
5. complete medium：Advanced DMEM/F12 serum free mediums (Invitrogen companies of the U.S.), wherein containing
There are 1 × Glutamax (Invitrogen companies of the U.S.), 1 × HEPES (Invitrogen companies of the U.S.), 1 × mycillin (beautiful
Invitrogen companies of state), 1 × N2supplement (Invitrogen companies of the U.S.), (U.S. 1 × B27 supplement
Invitrogen companies), 10 μM of Y-27632 (Sigma-Aldrich), 1 μ g/mL human R-spondin it is (beautiful
PeproTech companies of state), 100ng/ml murine Noggin (PeproTech companies of the U.S.), 100ng/mL murine
EGF (PeproTech companies of the U.S.), 100ng/mL Murine Wnt-3a (PeproTech companies of the U.S.) and 1mM N-
Acetylcysteine (Sigma-Aldrich).
6. primary antibody：Rabbit anti-mouse CD44 monoclonal antibodies (Santa Cruz companies of the U.S.).
7. secondary antibody：Goat anti-rabbit igg magnetic bead coupled antibody (German Miltenyi companies).
The separation of embodiment 1C57BL/6 mouse CD44 positive cells and phenotypic evaluation
1. under aseptic condition, obtaining other people and testing the C57BL/6 mouse (Chinese people that healthy control group used has just been put to death
Liberation army Military Medical Science Institute Experimental Animal Center) whole section of small intestine, it is splitted along the longitudinal axis, the D-hanks liquid of precooling washes away
Intestinal contents.
2. the light postcibal diarrhea Cavity surface of coverslip of sterilizing, to remove the villus ingredient in epithelium.
It is placed in 50mL centrifuge tubes 3. being cut intestinal tissue to the big fractionlets of 3mm with sterile scissors, with precooling D-hanks liquid
It rinses repeatedly supreme limpid clear.
4. separating liquid is added, 4 DEG C of shaking table concussions are incubated 30 minutes.
5. after being incubated, waits for that tissue is settled down to centrifuge tube bottom, separating liquid is carefully sucked out；1000 revs/min, centrifuge 5 points
Clock discards supernatant.
6. the D-hanks liquid of 30mL precoolings is added, cell is resuspended and being aggressively shaken makes crypts be released from intestinal tissue,
Microscopically observation is until be full of abundant crypts in supernatant.
7. supernatant is sucked out, with 70 μm of strainer filterings, filtrate is collected into the coated centrifuge tubes of BSA, 1000 revs/min
Clock centrifuges 5 minutes, discards supernatant.
8. cell is resuspended in 10mL basal mediums, cell suspension is transferred in 15mL centrifuge tubes, 500 revs/min, from
The heart 2 minutes, discards supernatant；Repeat this step 2-3 times, it is therefore intended that the individual cells in removal cell suspension are to purify crypts.
9. the crypts of separation is placed in the dissociation culture medium of 5mL precoolings, 4 DEG C of shaking tables are incubated 45 minutes, and every 10 minutes anti-
Piping and druming cell suspension 5-10 times again, to promote individual cells to be released from crypts.
10. after being incubated, use 40 μm of strainers, 20 μm of strainers successively filtering suspensions successively, collection filtrate and with 1000 turns/
Minute, it centrifuges 5 minutes, discards supernatant.
11. drawing the dissociation culture medium of 1-2mL precoolings, rabbit anti-mouse CD44 monoclonal antibodies are added thereto, ratio is
1:50(w/v)；After being incubated 30 minutes on ice, 1000 revs/min, centrifuges 2 minutes, wash away remaining antibody, it is primary to repeat this step.
12. drawing the dissociation culture medium of 1-2mL precoolings again, the magnetic bead secondary antibody of goat antirabbit is added thereto, ratio is:
1:4(v/v)；It is protected from light incubation 15 minutes on ice.
13. by step 12 oneself be incubated the direct Magnetic Isolation column (U.S. day Ni, LD types) of crossing of the cell suspension that finishes and collect point
From column inner cell (positive sorting), dissociation culture medium elutes column inner cell, counts.
14. with the phenotype for the CD44+ cells that flow cytometry sorts, the results are shown in Figure 1.Magnetic bead sorting is obtained
CD44 positive cells respectively with monoclonal anti-mouse CD24-FITC, CD31-PE, CD34-PE, CD44-APC and CD45-APC
Flow cytometer showed is carried out after antibody label；Mouse IgG 2c-FITC, IgG2a-PE and IgG2b-APC are Isotype control.As a result it shows：
CD44 positive cells do not express CD31, CD34 and CD45, show CD44 positive cell endothelium systems and hematopoiesis system source；Positive expression
CD24 and CD44 shows that CD44 positive cells are regulated and controled by COX-2 related pathways and Wnt signal paths, and the access is that intestines are dry thin
The major impetus of born of the same parents' proliferation.
The Fiber differentiation of embodiment 2CD44 positive cells
1. with step 1-13 in embodiment 1, magnetic bead sorting C57BL/6 mouse CD44 positive cells (positive selection), with
CD44 negative cells (Solid phase) are as a contrast.
2. being resuspended the matrigel melted on ice is previously placed in the ratio that 10 μ L matrigels are added in every 100 sorting cells
Cell, this step should operate on ice, to prevent temperature raising from matrigel being caused to solidify.
3. after cell premixes, matrigel -37 DEG C of cell suspension addition is preheated with the ratio in 10 μ L matrigels/hole
96 well culture plates (100 sorting cells/10 μ l matrigels/holes)；When inoculation, matrigel should be located at the center of board bottom, avoid glue
Touch wall.
It is incubated 5 minutes 4. the cell of inoculation is placed in 37 DEG C of incubators, matrigel is made to shape；Prepare complete medium.
5. after being incubated, the complete medium of 100 μ L, 37 DEG C, 5%CO are added into every hole2It is carried out in wet environment
Culture.
6. after culture in 14 days, the development of dynamic observation CD44 positive intestinal stem cells under inverted microscope.As a result, it has been found that
After Fiber differentiation in 14 days, part cell can be in constantly expanding and form ' cryptomere ' structure in vitro in CD44 positive cell groups.
In contrast, CD44 negative cells are then formed without ' cryptomere ' structure.(as shown in Figure 2)
Embodiment 3CD44 positive cells are divided into 4 type cells of enteric epithelium
1. after in vitro culture 2 weeks, complete medium is discarded；And the neutral formalin solution (200 of precooling is added into 96 orifice plates
The holes μ L/) " mini intestines " that CD44 positive cells differentiate are fixed, 4 DEG C, 30min.
2. washing away neutral formalin fixer (2-3 times) with the DPBS of precooling；And it is added into 96 orifice plates preconfigured
0.025% Triton X-100 solution (200 holes μ L/) is incubated 10min at 4 DEG C.
3. blowing and beating Matrigel with the DPBS solution of precooling, making it dissolve and releasing internal " mini intestines "；
1000rpm centrifuges 2min.
After 4.DPBS washings " mini intestines " precipitate 2-3 times, it is transferred in new EP pipes.
5. being incubated " mini intestines " with the PBS solution 1mL containing 5%BSA, under the conditions of 4 DEG C precipitates 30min.
6. pre-configured primary antibody working fluid is added, including：Anti-mouse Villin, Muc-2, Chr-A and
Lysozyme C monoclonal antibody (amount of antibody：Antibody diluent amount=1:250)；It 4 DEG C, is incubated overnight.
7. next day is precipitated 2-3 times with PBS solution washing " mini intestines ".
8. " mini intestines " are transferred on glass slide, 50 μ L contain the mounting glue mounting of DAPI, take pictures under mirror.As a result as schemed
Shown in 3.Wherein, CD44 positive cells of the invention are divided into the four type cells for constituting enteric epithelium：Absorb cell (Villin
It is positive), goblet cell (Muc2 is positive), endocrine cell (Chr-A is positive) and paneth's cell (the Lysozyme C positives).
Embodiment 4Y-27632 inhibitor can inhibit the apoptosis of CD44 positive cells
1. the step of with embodiment 1, anti-mouse CD44 monoclonal antibody magnetic bead sorting methods obtain CD44 positive cells.
2. CD44 positive cell (items in application anti-mouse CD44-APC labeling of monoclonal antibody C57BL/6 mouse enteric epitheliums
Part：Antibody ratios：1 microgram/106 cell is incubated 15 minutes on ice), carry out the airflow classification of CD44 positive cells.
3. application Annexin V/PI to the CD44 positive cells of the CD44 positive cells of magnetic bead sorting and airflow classification into
Line flag, markers step is with reference to Invitrogen companies of U.S. Dead Cell Apoptosis Kit with Annexin V
Alexa Fluor 488＆PI-for Flow Cytometry (Replaces PHN1008, PHN1010) kit (article No.：
V13241)。
4. flow cytometer analyzes Apoptosis.As a result such as Fig. 4.As a result, it has been found that being sorted using flow cytometry
CD44 positive cell cells late apoptic (Annexin V/PI bis- positive) quantity compared with being sorted through magnetic bead combined Y-27632
CD44 positive cell late apoptic quantity showed increaseds (amplitude of increasing is about 20%).
Embodiment 5Y-27632 inhibitor can improve the Clone formation power of CD44 positive cells
1. the step of with embodiment 1, anti-mouse CD44 monoclonal antibody magnetic bead sorting methods obtain CD44 positive cells.With
The sorting group of Y-27632 inhibitor is not added as a contrast.
2. the step of with embodiment 2, carries out two groups of CD44 positive cells external evoked culture in 14 days.
3. the development of dynamic observation CD44 positive cells under inverted microscope is compared after Fiber differentiation the 14th, each group
The colony-forming efficiency of CD44 positive cells.As a result, it has been found that 10 μ Μ Y-27632 inhibitor of use in conjunction during magnetic bead sorting
CD44 positive cell colony-forming efficiencies are about 12%；This numerical value less adds the CD44 positive cell shapes of Y-27632 inhibitor
50% is improved at rate (about 6%).
It can be seen that in CD44 positive cells that there are intestinal stem cells by embodiment 1-5；And magnetic bead sorting method is combined
Y-27632 inhibitor can improve the survival rate and colony-forming efficiency of aim cell, have important application value.
Applicant states that the present invention illustrates the detailed features and method detailed of the present invention by above-described embodiment, but
The invention is not limited in above-mentioned detailed features and method detaileds, that is, do not mean that the present invention has to rely on above-mentioned detailed features
And method detailed could be implemented.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to this hair
The addition of the bright equivalence replacement for selecting component and auxiliary element, the selection etc. of concrete mode, all fall within protection scope of the present invention
Within the open scope.
Claims (8)
- Application of the 1.Y-27632 inhibitor in the sorting of CD44 positive intestinal stem cells, which is characterized in that the application includes using Culture medium containing Y-27632 inhibitor further includes the separation that CD44 positive intestinal stem cells are carried out using magnetic bead；Specifically include with Lower step：(1) crypts is detached from intestinal tissue；(2) crypts single cell suspension is prepared, is specifically included：A) crypts that step (1) detaches is placed in dissociation culture medium, is incubated 30-45min, it is outstanding to blow and beat cell repeatedly per 10min Liquid 5-10 times, to promote individual cells to be released from crypts；The dissociation culture medium be containing 1 × Glutamax, 1 × HEPES, 1 × mycillin, 1 × N2 supplement, 1 × B27 supplement and 10 μM of Y-27632 inhibitor Advanced DMEM/F12 serum free mediums；B) after being incubated, suspension is filtered, collect filtrate and is centrifuged, supernatant is discarded, obtains crypts single cell suspension；(3) crypts CD44 positive intestinal stem cells are sorted, are specifically included：A) CD44 monoclonal antibodies are added into the dissociation culture medium of step (2), are incubated 25-30min, it is anti-to wash away residual for centrifugation It is primary to repeat this step for body；B) magnetic bead coupled antibody is added into dissociation culture medium again, is incubated 15-20min；C) cell suspension that oneself incubation finishes is loaded to Magnetic Isolation column, the cell being adsorbed in column is collected in elution, as CD44 positive intestinal stem cells.
- 2. application according to claim 1, which is characterized in that the step (1) includes：A) under aseptic condition, intestinal tissue is splitted, removes intestinal contents, and remove the villus ingredient in epithelium；B) the obtained intestinal tissues of step a) are placed in centrifuge tube, rinse supreme limpid clear, addition separating liquid, incubation 20- repeatedly 40min；C) it waits for that intestinal tissue is settled down to centrifuge tube bottom, separating liquid is sucked out, supernatant is abandoned in centrifugation；D) the D-hanks liquid of precooling is added, cell is resuspended and being aggressively shaken makes crypts be released from intestinal tissue, under microscope Observation is until be full of abundant crypts in supernatant；E) supernatant is sucked out, is filtered, filtrate is collected into the coated centrifuge tubes of BSA and centrifuged, and supernatant is discarded；F) cell is resuspended with basal medium, cell suspension is transferred in centrifuge tube, centrifuge, discard supernatant, repeat 2-3 times, To remove the individual cells in cell suspension to purify crypts.
- 3. application according to claim 2, which is characterized in that the step b) separating liquids are the D- of the EDTA containing 2mM Hanks solution, pH value 8.0.
- 4. application according to claim 2, which is characterized in that the step f) basal mediums be containing 1 × The Advanced DMEM/F12 serum free mediums of Glutamax, 1 × HEPES and l × mycillin.
- 5. application according to claim 1, which is characterized in that step b) dissociation culture mediums in step (3) be containing There are 1 × Glutamax, 1 × HEPES, 1 × mycillin, 1 × N2 supplement, 1 × B27 supplement and 10 μM The Advanced DMEM/F12 serum free mediums of Y-27632 inhibitor.
- 6. a kind of method preparing small enteric epithelium structure, which is characterized in that it includes the following steps：(1) isolated CD44 positives intestinal stem cell is applied according to claim 1-5 any one of them；(2) CD44 positives intestinal stem cell described in in-vitro multiplication and it is divided into complete small enteric epithelium structure.
- 7. according to the method described in claim 6, it is characterized in that, the step (2) includes：A) matrigel melted on ice will be previously placed in weigh on ice with the ratio of every 100 intestinal stem cells 10 μ L matrigels of addition Outstanding cell；B) after cell premix, matrigel and cell suspension are added the 96 of 37 DEG C of preheatings with the ratio in 10 μ L matrigels/hole Well culture plate；When inoculation, matrigel is located at the center of board bottom, and glue is avoided to touch wall；C) cell of inoculation is placed in 37 DEG C of incubators and is incubated 5min, matrigel is made to shape；D) after being incubated, 100 μ L complete mediums, 37 DEG C, 5%CO are added into every hole2It is incubated 2 weeks, collects in wet environment Small enteric epithelium structure.
- 8. the method according to the description of claim 7 is characterized in that the matrigel be add 1mM Jagged-1 without phenol red Semisolid culturemedium without growth factor；The complete medium is to contain 1 × Glutamax, 1 × HEPES, l × blueness strepto- Element, 1 × N2 supplement, 1 × B27 supplement, 10 μM of Y-27632 inhibitor, l μ g/mL human R- Spondin, 100ng/mL murine Noggin, 100ng/mL murine EGF, 100ng/ml Murine Wnt-3a and The Advanced DMEM/F12 serum free mediums of 1mM N-Acetylcysteine.
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