CN105085938B - The method that bletilla striata polyose water gelatin, culture matrix and its application are broken up with inducing umbilical cord mesenchymal stem to corneal epithelial cell - Google Patents
The method that bletilla striata polyose water gelatin, culture matrix and its application are broken up with inducing umbilical cord mesenchymal stem to corneal epithelial cell Download PDFInfo
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Abstract
The present invention relates to field of cell culture, more particularly to the method that bletilla striata polyose water gelatin, culture matrix and its application are broken up with inducing umbilical cord mesenchymal stem to corneal epithelial cell.Bletilla polysaccharide is crosslinked obtained polysaccharide hydrogel by the present invention after sulphation with carrying multi-functional ε polylysines.The bletilla striata polyose water gelatin be translucent it is membranaceous, wherein with good hole, the growth and differentiation of suitable cell.Experiment shows, using bletilla striata polyose water gelatin provided by the invention as support, after hUC MSCs and amniotic epithelial cells are co-cultured 7 days, hUC MSCs differentiation rate is up to 31.96%.The differentiation rate significantly (p<0.01) comparative example of gel stent is not used.Also, gel provided by the invention can also make cell growth vigorous, shorten induction time.
Description
Technical field
The present invention relates to field of cell culture, more particularly to bletilla striata polyose water gelatin, culture matrix and its application and induction
The method that umbilical cord mesenchymal stem cells break up to corneal epithelial cell.
Background technology
Sound corneal epithelial cell is the essential condition that cornea resists extraneous various adverse factor damages, structure and function
Sound limbal stem cell is the main source of corneal epithelial regeneration.A variety of pathogenic factors, such as：Eye traumas, operation wound
Wound, inflammation, drug toxicity can cause corneal limbus to damage, and cause limbal stem cell dysfunction, cause epithelial cell to lack,
So as to cause to increase corneal infection, perforation, the danger of neovascularization.Therefore, obtained by vitro culture and be largely used to cornea
This problem of the epithelial cell of reparation is urgently to be resolved hurrily.Clinical research shows, is used as by vitro cultured corneal limbal stem cells
The seed cell of corneal epithelium reparation achieves good efficacy.But required by corneal limbal stem cell autograft in-vitro cultivation
Nutrient solution complicated component, cell it is unstable easily differentiation, cell can not continue expand, cell phenotype changes, so every time
Transplanting will remove part corneal limbal tissue on the normal cornea of patient or its family members and carry out in vitro culture, cumbersome, and
Secondary pain is brought to patient, and has researcher to find, when taking for cornea edge tissue more than 2/3rds, to cause to be good for
The irreversible decline of eye surface diseases and eyesight of eye so that Most patients are difficult to receive.Therefore, it is highly desirable to find newly
The autologous and cell derived of heteroplastic transplantation.
The advantages that low stain that umbilical cord mesenchymal stem cells (hUC-MSCs) have, source are sufficient, molecular marker for increased proliferation, also,
Umbilical cord mesenchymal stem cells are not limited by any ethics and legal principle, thus have attracted numerous researchers.It is but external evoked
HUC-MSCs breaks up very difficult to corneal epithelial cell.
Dimensional culture technology grows microenvironment by analogue body inner cell, establish between cell and extracellular matrix it is extensive
System, by forming certain 3-D solid structure, promote cell be similar to the gene expression in body, extracellular matrix secretion and
Cell biological function activity, therefore it has very big hair in stem cell in regenerative medicine and clinical translational medicine application aspect
Exhibition prospect.Complete Three-dimensional cell culture system needs to be made up of three parts, and support, seed cell, culture systems are indispensable.
The research of timbering material shows in recent years, and hydrogel material turns into most popular research direction.
Hydrogel is a kind of material for having hydrophily but can not dissolving and can only be swelled in water, is in nature
A kind of physical form of generally existing.Water in hydrogel can make to be dissolved in low molecular weight substance therein diffusion therefrom,
Characteristic with film, this diffusion property of gel can realize oxygen, the transmission of nutriment and the discharge of metabolic waste, be cell
Existence provides the environment of a similar biological tissue.Hydrogel can be divided into hydrogel and the conjunction of natural material preparation by material source
Into material hydrogel and both compound hydrogels.Natural hydrogel material mainly includes collagen, chitosan, alginates, fine jade
Lipolysaccharide, hyaluronic acid etc..Its advantage is that cell signal identity is good, can promote sticking, breed and breaking up for cell, have good
Biocompatibility and biodegradability；Shortcoming is limited source, it is difficult to is processed, it is difficult to ensures the same of different batches material
Property, the problems such as immunogenicity, degradation speed, mechanical property are unmanageable be present.It is by hydrophilic polymer to synthesize hydrogel
Water-swellable body through being physically or chemically cross-linked to form.PNIPAM, polyvinyl alcohol, polyethylene glycol and its derivative are
Study more synthetic material.Artificial synthesized hydrogel material is not limited by source, is easily worked shaping, can be adjusted as needed
Whole physics, chemistry, biomethanics and degradation property.But many shortcomings are have also discovered in practice：Synthetic material is usual
It is poor to there is cellular affinity, hydrophily and cell adhesion, lack cell recognition signal site the defects of.
Therefore, one kind should be researched and developed and possesses extracellular matrix function, be adapted to the artificial bionic microenvironment of cell development
Hydrogel, and use it for cell culture and induction differentiation.
The content of the invention
In view of this, the technical problem to be solved in the present invention is to provide bletilla striata polyose water gelatin, culture matrix and its answered
With the method broken up with inducing umbilical cord mesenchymal stem to corneal epithelial cell.Bletilla striata polyose water gelatin provided by the invention is
Bletilla polysaccharide is crosslinked with epsilon-polylysine after sulphation modification and obtained, and the gel can provide for cell in vitro can press close to body
Interior microenvironment.It can be shown to corneal epithelial cell differentiation as stent-induced inducing umbilical cord mesenchymal stem using the gel
Write the differentiation rate for improving cell.
The preparation method of bletilla striata polyose water gelatin provided by the invention is：By bletilla polysaccharide after sulphation with ε-poly- bad ammonia
Bletilla striata polyose water gelatin is made in acid crosslinking.
The bletilla striata (Bletilla striata) is orchid family bletilla striata category herbaceos perennial, is used as medicine with dry stem tuber.In vain
The good hygroscopicity and hydrophily that splendid achnatherum polysaccharide has, can promote the penetrating of oxygen, nutriment and other water soluble metaboliteses
Effect, while the adhesiveness of epithelium and stroma cell can also be strengthened.The present invention by bletilla polysaccharide after sulphation with carrying more officials
Polysaccharide hydrogel is made in the epsilon-polylysine crosslinking that can be rolled into a ball.Bletilla striata polyose water gelatin provided by the invention and bletilla polysaccharide solution are certainly
The hydrogel that body is formed is compared, and has better hole, and higher viscosity.Also, the preparation method is simple to operate, into
The glue time is short.Bletilla striata polyose water gelatin provided by the invention can be provided as cell in vitro and provide and internal similar micro-loop
Border, so as to advantageously in the growth and differentiation of cell.
In an embodiment of the present invention, the preparation method of bletilla polysaccharide is：Sunk after the bletilla striata is crushed through water extract-alcohol precipitation, gained
Shallow lake is resuspended with water, after being extracted with the mixture of chloroform and butanol, is dialysed through 30000D~50000D, dries obtained bletilla polysaccharide.
In certain embodiments, water extract-alcohol precipitation is specially：By bletilla striata powder with flooding after, extract solution mixes with ethanol water
Close, stand overnight obtained precipitation.
In certain embodiments, the temperature of extraction is 80 DEG C, and the time of extraction is 4h.
In certain embodiments, the volume fraction of ethanol is 95% in ethanol water.
In certain embodiments, the volume ratio of ethanol water and extract solution is 1:3.
After the extracting of the mixed liquor of chloroform and butanol, the albumen in extract is removed.Fetched water after centrifugation phase.
In certain embodiments, in the mixture of chloroform and butanol, the volume ratio of chloroform and butanol is 5:1.
In certain embodiments, the volume that precipitation is dissolved in solution after water is 3 times of chloroform and butanol mixture.
In certain embodiments, the dialyzate used is dialysed as distilled water.
In certain embodiments, drying is lyophilized.
The molecular weight of the obtained bletilla polysaccharide of the present invention is 30000D~50000D.
In certain embodiments, dried bletilla polysaccharide is also by purifying.
In certain embodiments, purify for will it is lyophilized after bletilla polysaccharide dissolving after, cross cross-link dextran G-100 posts.
In an embodiment of the present invention, the method for sulphation is chlorosulfonic acid-pyridine method.
In certain embodiments, the reagent of sulphation is chlorosulfonic acid and pyridine.
In certain embodiments, the volume ratio of chlorosulfonic acid and pyridine is 1:6.
In an embodiment of the present invention, the sulphation of bletilla polysaccharide is specially：Bletilla polysaccharide is dissolved in anhydrous N, N- diformazans
Base formamide, after 30min is stirred at room temperature, adding sulfur acidizing reagent, (volume ratio of chlorosulfonic acid and pyridine is 1:6) 60 DEG C of water, are placed in
Bathe stirring reaction 3h.
In certain embodiments, after sulphation, adjustment pH value is 7.5, saturating by dialyzate of distilled water after being concentrated under reduced pressure
3d is analysed, sulfated polysaccharides is obtained through freeze-drying after dialyzate concentration.
In an embodiment of the present invention, the molecular weight of epsilon-polylysine solution is 600Da~4300Da, including 25
~30 lysine residues.
In an embodiment of the present invention, crosslinking is specifically, by the bletilla polysaccharide solution of sulphation and epsilon-polylysine solution
Stood after mixing.
In certain embodiments, the solvent of epsilon-polylysine solution is PBS, wherein the quality of epsilon-polylysine point
Number is 3%.
In an embodiment of the present invention, the solvent of the bletilla polysaccharide solution of sulphation is PBS, and wherein sulphation is white
The concentration of splendid achnatherum polysaccharide is 200 μ g/mL.
In an embodiment of the present invention, the temperature of crosslinking is 18 DEG C~35 DEG C, and the time is 20s~40s.
In certain embodiments, the time of crosslinking is 30s.
Bletilla striata polyose water gelatin prepared by the method provided by the present invention.
Bletilla striata polyose water gelatin provided by the invention be translucent it is membranaceous, wherein with good hole, suitable cell
Growth and differentiation.Experiment shows, using bletilla striata polyose water gelatin provided by the invention as support, hUC-MSCs is thin with amnioic epithelium
After born of the same parents co-culture 7 days, hUC-MSCs differentiation rate is up to 31.96%.It is circular in ball and cell growth is vigorous.Cell adherence in
On support, it is bundled together with support；It is amplified to 2000 times, it is seen that obvious cell, its " pseudopodium " clench support
Gap.
The application of bletilla striata polyose water gelatin prepared by the method provided by the present invention in cell culture or induction differentiation.
In an embodiment of the present invention, inducing umbilical cord mesenchymal stem is induced to differentiate into corneal epithelial cell to break up.
The invention provides the method that inducing umbilical cord mesenchymal stem breaks up to corneal epithelial cell, with bletilla polysaccharide water
Gel is support, and under nutrient solution existence condition, umbilical cord mesenchymal stem cells and amniotic epithelial cells are co-cultured；Nutrient solution bag
Include basal medium, EGF and insulin.
In an embodiment of the present invention, the mass ratio of EGF and insulin is (5~25)：(5000~
25000)。
Preferably, the mass ratio of EGF and insulin is 2:3000~3:2000.
Preferably, the mass ratio of EGF and insulin is 1:1000.
In an embodiment of the present invention, the content of EGF is the μ g/mL of 5ng/mL~25；The content of insulin is
The μ g/mL of 5ng/mL~25.
Preferably, the content of EGF is 5ng/mL；The content of insulin is 5 μ g/mL.
Preferably, the content of EGF is 10ng/mL；The content of insulin is 10 μ g/mL.
Preferably, the content of EGF is 25ng/mL；The content of insulin is 25 μ g/mL.
In an embodiment of the present invention, basic culture solution behaviour stem cell serum-free culture medium and the training of epithelial cells serum-free
The volume ratio of foster base, human stem cell serum free medium and epithelial cells serum free medium is (50~60):(44~50).
Preferably, the volume ratio of human stem cell serum free medium and epithelial cells serum free medium is 55:45.
Human stem cell serum free medium and epithelial cells serum free medium used in the present invention can be that self-control is alternatively
Purchase is obtained, and the present invention is not limited this, and it is implemented all within protection scope of the present invention.Wherein, human stem cell is without blood
Clear culture medium suitably cultivates stem cell, and the human stem cell serum free medium that the present invention uses is Lonza UltraCULTURETM。
Epithelial cells serum free medium suitably cultivates epithelial cell, and the epithelial cells serum free medium that the present invention uses is trained for KSFM
Nutrient solution.The basic culture solution that the present invention uses all does not contain allogeneic serum, reduces the risk that animal sources serum is brought to human body.
In an embodiment of the present invention, penicillin and streptomysin are also included in culture medium provided by the invention.
In certain embodiments, the content of penicillin is 100U/mL, and the content of streptomysin is 100U/mL.
In an embodiment of the present invention, co-culture as Indirect co-culture.
In an embodiment of the present invention, the number of umbilical cord mesenchymal stem cells and amniotic epithelial cells ratio is (1~1.5):
(4~6).
Cell Co culturing Techenique is the technology in same environment by two or more cell co-culture, due to
It has the advantages of preferably reflection vivo environment, therefore is applied in INVENTIONModern cell research.Indirect co-culture system is i.e. by 2
In or 2 in more than cell be inoculated in respectively on different carriers, then both carriers are placed in same culture environment, are
Different types of cell shares same cultivating system and is not directly contacted with.Indirect co-culture can use transwell cells.
In an embodiment of the present invention, umbilical cord mesenchymal stem cells were the 3rd~5 generation；The amniotic epithelial cells are the 2nd
Generation.
In an embodiment of the present invention, the primary separation method of umbilical cord mesenchymal stem cells is tissue mass cell culture；Amnion
The primary separation method of epithelial cell is enzyme digestion.
Specifically, hUC-MSCs primary separation method comprises the following steps：
Step 1：Umbilical cord is rinsed 2 times with containing the PBS of 100U/mL penicillin and 100U/mL streptomysins, with body
After the ethanol water that fraction is 75% soaks 1min~2min, umbilical cord outer membrane and blood vessel are removed；
Step 2：With human stem cell serum-free medium culture, condition of culture 5%CO2, 37 DEG C, humidity 95%；5th~
Half amount changes culture medium within 7 days, continues full dose after culture 12~14 days and change liquid to remove tissue block, collects passage culture.
Specifically, the primary separation method of amniotic epithelial cells comprises the following steps：
Step 1：By amnion with containing the PBS of 100U/mL penicillin and 100U/mL streptomysins rinse 2 times after, break
It is broken, using mass fraction as 0.25% Trypsin Induced；
Step 2：After clostridiopetidase A II and Dnas enzymic digestions, culture.
The concentration of clostridiopetidase A II is 2.5g/L；The concentration of Dnas enzymes is 0.05g/L；The condition of digestion is 37 DEG C, 200rpm
Concussion, the time is 15min~30min.
The nutrient solution of culture is the epithelial cells serum free medium comprising EGF；Its Concentrations of Epidermal Growth Factor
Concentration be 10ng/mL~15ng/mL.
The inoculum density of culture is 1 × 107Individual/L.
The condition of culture is 5%CO2, 37 DEG C, after humidity 95% cultivates 2h, continue culture 24 after removing not adherent cell
Hour.
In an embodiment of the present invention, the time of co-cultivation is 7 days.
The invention provides bletilla striata polyose water gelatin, culture matrix and its application with inducing umbilical cord mesenchymal stem to angle
The method of film epithelial cell differentiation.Bletilla polysaccharide is crosslinked by the present invention after sulphation with carrying multi-functional epsilon-polylysine
Polysaccharide hydrogel is made.The bletilla striata polyose water gelatin be translucent it is membranaceous, wherein with good hole, the growth of suitable cell
With differentiation.Experiment shows, using bletilla striata polyose water gelatin provided by the invention as support, hUC-MSCs and amniotic epithelial cells are total to
After culture 7 days, hUC-MSCs differentiation rate is up to 31.96%.The differentiation rate significantly (p<0.01) it is better than not using gel stent
Comparative example.Also, gel provided by the invention can also make cell growth vigorous, shorten induction time.
Brief description of the drawings
Fig. 1 shows the hUC-MSCs cellular morphologies (100 ×) through transmitting for 3~5 generations；
Fig. 2-a show original cuiture 12h amniotic epithelial cells form (40 ×)；
Fig. 2-b show Secondary Culture 12h amniotic epithelial cells form (40 ×)；
Fig. 2-c show Secondary Culture 24h amniotic epithelial cells HE coloration results (100 ×)；
Fig. 2-d show that Secondary Culture 24h amniotic epithelial cells identify (100 ×) by antibody mediated immunity groupization of CK7；
Fig. 2-e show that Secondary Culture 24h amniotic epithelial cells identify (100 ×) by antibody mediated immunity groupization of CK8；
Fig. 2-f show that Secondary Culture 24h amniotic epithelial cells identify (100 ×) by antibody mediated immunity groupization of CK18.
Embodiment
The invention provides bletilla striata polyose water gelatin, culture matrix and its application with inducing umbilical cord mesenchymal stem to angle
The method of film epithelial cell differentiation., those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.It is special
Not it is pointed out that all similar replacements and change it is apparent to those skilled in the art, they all by
It is considered as and is included in the present invention.The method of the present invention and application are described by preferred embodiment, and related personnel is obvious
Present invention can be not being departed from, methods herein and application is being modified or suitably changed with combining in spirit and scope,
To realize and using the technology of the present invention.
The instrument that the present invention uses is all common commercially available product, can all be bought in market.
It is AE1 and AE5 to be used for the antibody that corneal epithelial cell is identified in the embodiment of the present invention, and they are all cytokeratins
Antibody.AE1 can identify that relative molecular mass is respectively 56500,50000,48000 and 40000 acidic cell keratin, energy
The acid keratin that a kind of relative molecular mass with corneal epithelial cell is 48000 reacts, with vimentin, knot egg
In vain, the silk-like proteins no cross reaction such as GFAP and NF-M.AE5 can be with the angle of corneal epithelial cell
Albumen K3 (relative molecular mass 64000) is specifically bound, and has a reaction with the corneal epithelial cell of people, rabbit, ox, and and skin
Epithelial cell no cross reaction.Cytokeratin K3 is the marker protein of the higher corneal epithelial cell of differentiation state.Therefore, such as
When fruit cell AE1 and AE5 the immunohistochemical staining positive, then this cell is corneal epithelial cell.
With reference to embodiment, the present invention is expanded on further：
The preparation of the bletilla polysaccharide of embodiment 1
Bletilla striata medicine materical crude slice smashes to be filtered in 80 DEG C of distilled water after dispersing and dissolving 4h, filters off impurity.
Via 95% ethanol soaked overnight of 3 times of volumes, sediment is resuspended filtrate with distilled water.
Add the chloroform of 1/3 volume and the mixed liquor (volume ratio 5 of butanol:1.) be mixed centrifugation extract proteins composition, extraction
The liquid of aqueous layer.
Dialysed (dialyzate is distilled water) through 30000D-50000D dialysis cartridges, freeze-dried powder is made, it is thick to obtain bletilla polysaccharide
Product.Obtained bletilla polysaccharide will be further purified through cross-link dextran G-100 after bletilla polysaccharide dissolving crude product.
The sulphation of the bletilla polysaccharide of embodiment 2
Bletilla polysaccharide chlorosulfonic acid-pyridine method is made in embodiment 1 and carries out sulphation modification.Concretely comprise the following steps：
0.2g polysaccharide powders are dissolved in the anhydrous DMFs of 35ml, after 30min is stirred at room temperature, added
(volume ratio of chlorosulfonic acid and pyridine is 1 to 17.5ml sulfur acidizing reagents:6) 60 DEG C of stirring in water bath reaction 3h, are placed in.After reaction terminates
Room temperature is cooled to, reaction solution adds 100ml frozen water, is neutralized to pH7.5 with 1mol/L NaOH, is concentrated under reduced pressure into finite concentration, uses
Distilled water dialysis 3d, sulfated polysaccharides is obtained after dialyzate concentration through freeze-drying.
The preparation of the bletilla polysaccharide gel of embodiment 3
Sulphation bletilla polysaccharide made from embodiment 2 is dissolved in PBS, it is molten to be configured to the polysaccharide that concentration is 200 μ g/ml
Liquid.Epsilon-polylysine is dissolved in PBS, mass fraction 3%, crosslinking agent is made.By polysaccharide solution and crosslinking agent with volume
Than 1:1 mixing, stands 30s at room temperature, you can forms translucent membranaceous bletilla striata polyose water gelatin.
The umbilical cord mesenchymal stem cells of embodiment 4 are separately cultured
Umbilical cord (Serological testing such as hepatitis B, hepatitis, HIV, mycoplasma, syphilis is feminine gender) is placed in containing 100U/mL
Rinsed 2 times in the PBS of penicillin and 100U/mL streptomysins, add 75% alcohol of precooling, soaked 1-2min, during which do not stop to turn over
Dynamic umbilical cord；Addition PBS, which is rinsed 2 times, washes away alcohol.Umbilical cord is cut into 2mm or so segment with tissue shear, every section is indulged with eye scissors
Xiang Kai, three blood vessels (two arteries, a vein) in umbilical cord are removed with vessel forceps, while remove umbilical cord outer membrane；By separator well
Umbilical cord the tissue blocks of about 1mm3 sizes is cut into eye scissors, take and be put into right amount in a diameter of 10cm sterile petri dish, cover
The floor space of 70% culture dish.Add LONZA human stem cells serum-free medium (Lonza UltraCULTURETM), 5%CO2、
37 DEG C, the CO that humidity is 95%2Cultivated in incubator.Half amount changes culture medium within 5th day~7 days, continues to cultivate 12 days~14 days left sides
Right full dose changes liquid and removes tissue block while collect passage culture.
Through transmitting the hUC-MSCs cellular morphologies in 3~5 generations as shown in figure 1, hUC-MSCs growths are vigorous, border is smooth, is in
Existing nido growth, colony are in polygonal or ellipse mostly, and the arrangement of colony inner cell is close, obscure boundary；Cell boundaries are clear, born of the same parents
Slurry is relatively abundant, and nucleus is big, and kernel is big.
The 3rd generation exponential phase cell is taken, cell separating liquid digests, Flow cytometry surface antigen CD 105,
CD45, CD34, CD31, CD40, CD29, CD44, HLA-DR expression, Cell-Quest software analysis results.Each sample
Analyze 6000~8000 cells.As a result it is as shown in table 1：
The 3rd generation hUC-MSCs cell surface marker the positive expression rate of table 1
| Cell phenotype | CD29 | CD31 | CD34 | CD40 | CD44 | CD45 | CD105 | HLA-DR |
| hUC-MSCs | 94.94±1.06 | 3.27±0.39 | 2.13±0.35 | 1.24±0.06 | 91.8±1.27 | 1.22±0.05 | 99.84±0.17 | 2.93±0.21 |
Through Flow cytometry, as shown in table 1, the 3rd generation hUC-MSCs is high to express CD105, CD44 and CD29, low expression
Or the not label such as expression of HLA-DR, CD31, CD45, CD40 and CD34.
It is prepared by the amniotic epithelial cells of embodiment 5
People's amnion is derived from healthy puerpera (Serological testing such as hepatitis B, hepatitis, HIV, mycoplasma, syphilis is feminine gender), will
Amnion is placed in the PBS containing 100U/mL penicillin and 100U/mL streptomysins and rinsed 2 times, is cut into about 30-50cm2Size
Tissue block, add the tryptic digestive juice of sufficient amount 0.25%, in 37 DEG C, 200R constant temperature oscillators digest 15~30min,
Acutely rocked with hand per 10min taking-ups, 2000rpm centrifugation 5min, add enough+0.05g/L Dnas of 2.5g/L clostridiopetidase As II
Enzyme, 4h is digested at 37 DEG C, centrifuge, add (the Keratinocyte serum-free of serum free medium containing epithelial cells
Medium, KFSM), the nutrient solution of 10-15ng/ml EGFs (EGF), piping and druming is uniform, with 1 × 107Individual/L is inoculated in
In a diameter of 10cm culture dish, volume fraction is put as gently will be not adherent after culture 2h in 5%CO2,37 DEG C of cell culture incubators
Cell suction out, because amniotic epithelial cells are adherent relatively slow, the mesenchymal cell or fibroblast mixed is adherent very fast, at this moment inhales
Go out cell (i.e. difference sticks the cell that method obtains) 90% cultivated again above is amniotic epithelial cells.Continue culture to cell
After fusion reaches 80%~90%, passage.Original cuiture 12h cell Microscopic observation (40 times) as shown in Fig. 2-a.Secondary Culture
12h cell Microscopic observation (40 times) as shown in Fig. 2-b.(dyed being taken out after 2nd generation amniotic epithelial cells culture 24h through HE
Microscopic observation such as Fig. 2-c, amplify 100 times), SABC identification of cell keratin CK7 (Fig. 2-d), CK8 (Fig. 2-e) and CK18
(Fig. 2-f).
As a result show, Microscopic observation 2nd generation amniotic epithelial cells are rounded, bright, refractivity is relatively strong, rich in third dimension.
The primary cell more than 90% for sticking method culture through difference is human amnion membrane, the mesenchymal cell mixed or into fiber finer
Born of the same parents are less, and can sink adherent growth in inoculation 12h, and cell enters exponential phase of growth, cell quantity showed increased, in not after 2d
The cell fusion of polygonal, 4~5d or so 90% of rule is in blocks, forms individual layer (see Fig. 2-a).The human amnion membrane of passage
Can be very fast adherent, it is inoculated with after 4~6h and can be adherent and starts to grow, cell growth accelerates after being inoculated with 24h, and cell is flat to become big,
Nucleus is clear, positioned at kytoplasm center.3~d or so cells 90% merge, in typical paving stone sample outward appearance (see Fig. 2-c).Soviet Union
Lignin eosin stains, it is seen that cell size is more uniform, and in irregular polygonal, kytoplasm enriches, the blue dye of karyon, circular, nuclear membrane
Clearly, kernel is obvious, it is seen that 2~3 nuclear fissions (Fig. 2-c).SABC identifies that people's amnioic epithelium of the present embodiment culture is thin
CK7, CK8, CK18 monoclonal keratin antibody stained positive are seen in the endochylema of born of the same parents, endochylema is in brown color, and karyon is in bluish violet.Table
Bright be successfully separated has cultivated amniotic epithelial cells.
Embodiment 6
Bletilla polysaccharide gel is prepared in the lower room of transwell cells.Method such as embodiment 3.By 0.5mL polysaccharide solutions
Six orifice plates are added, isometric crosslinking agent is added, is stored at room temperature 30s.
The 3-5 of propagation logarithmic phase is taken to be inoculated in Transwell for hUC-MSCs and 2nd generation amniotic epithelial cells and train altogether
In the system of supporting, upper chamber implantation amniotic epithelial cells inoculum density is 4 × 105Individual/hole, lower room are implanted into hUC-MSCs, inoculum density
For 1 × 105Individual/hole.2.5ml penicillin containing 100U/mL and 100U/mL streptomysins, volume fraction 55%LONZA are added per hole
Human stem cell serum free medium (Lonza Ultra CULTURETM), 45% epithelial cells serum free medium
The nutrient solution of (Keratinocyte serum-free medium, KFSM), 10ng/ml EGFs (EGF), 10ug/
The complete culture solution of ml insulin.
The nutrient solution more renewed every 2-3 days.Culture takes out cell climbing sheet after 7 days.
Observed under inverted microscope：After cultivating 7d, cell growth is vigorous, balling-up round shape, and arrangement is close, it is seen that many collection
Fall；Continue culture to 14d, cell volume to significantly increase.
Scanning electron microscopic observation：Visible cell is adhered on support after cultivating 7 days, is bundled together with support；Amplified
To 2000 times, it is seen that obvious cell, its " pseudopodium " clench standoff gap.
Cytokeratin AE1 and AE5 and P63 immune group is carried out according to the specification of immunohistochemical staining SP method kits
Change dyeing, DAB colour developings.Immunohistochemical staining result shows, it is seen that a large amount of DAB pigmented cells.In be dispersed in distribution and also cell
Color is deeper.
Embodiment 7
Bletilla polysaccharide gel is prepared in the lower room of transwell cells.Method such as embodiment 3.By 0.5mL polysaccharide solutions
Six orifice plates are added, isometric crosslinking agent is added, is stored at room temperature 30s.
The 3-5 of propagation logarithmic phase is taken to be inoculated in Transwell for hUC-MSCs and 2nd generation amniotic epithelial cells and train altogether
In the system of supporting, upper chamber implantation amniotic epithelial cells inoculum density is 4 × 105Individual/hole, lower room are implanted into hUC-MSCs, inoculum density
For 1 × 105Individual/hole.2.5ml penicillin containing 100U/mL and 100U/mL streptomysins, volume fraction 55%LONZA are added per hole
Human stem cell serum free medium (Lonza Ultra CULTURETM), 45% epithelial cells serum free medium
The nutrient solution of (Keratinocyte serum-free medium, KFSM), 25ng/ml EGFs (EGF), 25ug/
The complete culture solution of ml insulin.
The nutrient solution more renewed every 2-3 days.Culture takes out cell climbing sheet after 7 days.
Observed under inverted microscope：After cultivating 7d, cell growth is slow, it is seen that a little colony；Cultivate to 14d, cell
Volume, which has, sees increase.
Scanning electron microscopic observation：Continue visible cell after cultivating 7 days to be adhered on support, be bundled together with support；By its
It is amplified to 2000 times, it is seen that obvious cell, its " pseudopodium " clench standoff gap.
The SABC that specification according to immunohistochemical staining SP method kits carries out cytokeratin AE1 and AE5 contaminates
Color, DAB colour developings.AE1 immunohistochemical staining results show, it is seen that a little AE1 antibody DAB pigmented cells, it are distributed in being dispersed in, and
Cell color is thin.The light coloring of AE5 antibody.
Embodiment 8
Bletilla polysaccharide gel is prepared in the lower room of transwell cells.Method such as embodiment 3.By 0.5mL polysaccharide solutions
Six orifice plates are added, isometric crosslinking agent is added, is stored at room temperature 30s.
The 3-5 of propagation logarithmic phase is taken to be inoculated in Transwell for hUC-MSCs and 2nd generation amniotic epithelial cells and train altogether
In the system of supporting, upper chamber implantation amniotic epithelial cells inoculum density is 4 × 105Individual/hole, lower room are implanted into hUC-MSCs, inoculum density
For 1.5 × 105Individual/hole.2.5ml penicillin containing 100U/mL and 100U/mL streptomysins, volume fraction 55% are added per hole
LONZA human stem cells serum free medium (Lonza Ultra CULTURETM), 45% epithelial cells serum free medium
The nutrient solution of (Keratinocyte serum-free medium, KFSM), 5ng/ml EGFs (EGF), 5ug/ml
The complete culture solution of insulin.
The nutrient solution more renewed every 2-3 days.Culture takes out cell climbing sheet after 7 days.
Observed under inverted microscope：After cultivating 7d, cell growth is slow, it is seen that a little colony；Cultivate to 14d, cell
Volume, which has, sees increase.
Scanning electron microscopic observation：Visible a small amount of cell adherence is bundled together on support with support after continuing culture 7 days；
It is amplified to 2000 times, it is seen that obvious cell, its " pseudopodium " clench standoff gap.
The SABC that specification according to immunohistochemical staining SP method kits carries out cytokeratin AE1 and AE5 contaminates
Color, DAB colour developings.Immunohistochemical staining result shows, it is seen that a little AE1 antibody DAB pigmented cells.It is distributed in being dispersed in, Er Qiexi
Born of the same parents' coloring is thin.The light coloring of AE5 antibody.
Comparative example 1
The 3-5 of propagation logarithmic phase is taken to be inoculated in Transwell for hUC-MSCs and 2nd generation amniotic epithelial cells and train altogether
In the system of supporting, upper chamber implantation amniotic epithelial cells inoculum density is 4 × 105Individual/hole, lower room are implanted into hUC-MSCs, inoculum density
For 1 × 105Individual/hole.
Any gel is not added on culture plate, 2.5ml penicillin containing 100U/mL and 100U/mL streptomysins, body are added per hole
Fraction is 55%LONZA human stem cells serum free medium (Lonza Ultra CULTURETM), 45% epithelial cells is without blood
The nutrient solution of clear culture medium (Keratinocyte serum-free medium, KFSM), 10ng/ml EGFs
(EGF), the complete culture solution of 10ug/ml insulin.
The nutrient solution more renewed every 2-3 days.Culture takes out cell climbing sheet after 7 days.
Observed under inverted microscope：After cultivating 7d, cell is in then fusiformis, and single layer of confluent grows.Continue culture to 14d, can
See that cell volume has and see increase.
Cytokeratin AE1 and AE5 and P63 immune group is carried out according to the specification of immunohistochemical staining SP method kits
Change dyeing, DAB colour developings.Immunohistochemical staining result shows, it is seen that a little DAB pigmented cells.In be dispersed in distribution and also cell
Color is shallower.
Embodiment 9
To embodiment 6~8 and the gained cell of comparative example 1 after SABC detects, the cell of AE1, AE5 dyeing is carried out
Count, calculate two groups of differentiation rate and be compared.Every group of sampling 3 times, statistics after detecting respectively.
Induce differentiation rate=staining positive cells number/TCS × 100%.
Statistical result such as table 2：
The each group cell differentiation rate of table 2
Note：The different letters of same column shoulder mark represent there is significant difference (p<0.01)
Experiment shows, umbilical cord mesenchymal stem cells and amniotic epithelial cells is co-cultured, umbilical cord mesenchymal stem cells after 7 days
Part is in differentiation due, and cell is in distribution is dispersed in, and after testing, the differentiation rate that hUC-MSCs is divided into corneal epithelial cell is reachable
31.96%.HUC-MSCs differentiation rate is 26.52% in comparative example without using gel stent；It can be seen that the present invention provides
Method can improve the percentage that hUC-MSCs breaks up to corneal epithelial cell, the effect has significant difference (p<
0.01)。
It the above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (8)
1. the preparation method of bletilla striata polyose water gelatin, it is characterised in that handed over by bletilla polysaccharide after sulphation with epsilon-polylysine
Bletilla striata polyose water gelatin is made in connection；
The molecular weight of the bletilla polysaccharide is 30000D~50000D；
The sulfuric acid turns to：Bletilla polysaccharide is dissolved in anhydrous DMF, after 30min is stirred at room temperature, adds sulfuric acid
Change reagent, be placed in 60 DEG C of stirring in water bath reaction 3h；The sulfur acidizing reagent is 1 by volume ratio:6 chlorosulfonic acid forms with pyridine；
The crosslinking is to be stood after the bletilla polysaccharide solution of sulphation is mixed with epsilon-polylysine solution；
The temperature of the crosslinking is 18 DEG C~35 DEG C, and the time is 20s~40s；
The solvent of the epsilon-polylysine solution is PBS, wherein, the mass fraction of epsilon-polylysine is 3%；
The solvent of the bletilla polysaccharide solution of the sulphation is PBS, wherein, the concentration of sulphation bletilla polysaccharide is 200 μ
g/mL。
2. preparation method according to claim 1, it is characterised in that the preparation method of the bletilla polysaccharide is：By the bletilla striata
Through water extract-alcohol precipitation after crushing, gained precipitation is resuspended with water, after being extracted with the mixture of chloroform and butanol, through 30000D~50000D
Dialysis, dry obtained bletilla polysaccharide.
3. bletilla striata polyose water gelatin made from any one of claim 1~2 preparation method.
4. bletilla striata polyose water gelatin made from any one of claim 1~2 preparation method is in cell culture or induction differentiation
In application.
5. application according to claim 4, it is characterised in that it is described be induced to differentiate into inducing umbilical cord mesenchymal stem to
Corneal epithelial cell breaks up.
6. the method that inducing umbilical cord mesenchymal stem breaks up to corneal epithelial cell, it is characterised in that with claim 1~2
Bletilla striata polyose water gelatin is support made from any one preparation method, under nutrient solution existence condition, by umbilical cord mesenchyma
Stem cell co-cultures with amniotic epithelial cells；The nutrient solution includes basal medium, EGF and insulin.
7. according to the method for claim 6, it is characterised in that the mass ratio of the EGF and insulin is (5
~25)：(5000~25000).
8. according to the method for claim 6, it is characterised in that the umbilical cord mesenchymal stem cells and amniotic epithelial cells
Number ratio is (1~1.5):(4~6).
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