CN103710302B - Prepare method and the special culture media combination thereof of pancreatic stem cells - Google Patents

Prepare method and the special culture media combination thereof of pancreatic stem cells Download PDF

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CN103710302B
CN103710302B CN201210371241.8A CN201210371241A CN103710302B CN 103710302 B CN103710302 B CN 103710302B CN 201210371241 A CN201210371241 A CN 201210371241A CN 103710302 B CN103710302 B CN 103710302B
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CN103710302A (en
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赵春华
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中国医学科学院基础医学研究所
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Abstract

The present invention relates to prepare the method for pancreatic stem cells and special culture media combination thereof.Specifically, the invention discloses a kind of method inducing human mesenchymal stem cell to obtain pancreatic stem cells and the combination of supporting culture medium thereof.Method of the present invention can the most once obtain limited endoderm cell and pancreatic stem cells through two one-step inducing methods, each phase cell can express corresponding specific gene, and the pancreatic stem cells finally obtained can break up pancreatic endocrine cell and the Exocrine Pancreas In Rats becoming meritorious energy.The pancreatic stem cells that the present invention obtains may be used for treating the injury of pancreas that the diseases such as diabetes cause, and the cell therapy for injury of pancreas disease provides experimental basis and Clinical Evidence, provides new approach for cellular transplantation therapy.

Description

Prepare method and the special culture media combination thereof of pancreatic stem cells

Technical field

The present invention relates to a kind of method preparing pancreatic stem cells and special culture media combination thereof.More specifically, The present invention relates to a kind of utilize special culture media combination by mescenchymal stem cell induction be pancreatic stem cells method and This special culture media combines.

Background technology

Diabetes have become the multiple and metabolic disease of refractory in the whole world, are the easy heaps of the glucose in a kind of blood Long-pending too much disease.Abroad give its another name " reticent killer " (Silent Killer).Particularly " adult type Diabetes ", it is the highest that the middle age of more than 40 years old catches rate.In Japan, in the population of more than 40 years old 10% Containing this disease, in the middle of i.e. ten people, just there is " diabetes " patient.Once suffer from " diabetes ", the life-span ten will be reduced More than Nian, thereby increases and it is possible to the complication of generation is throughout whole body.

For many years, medical circle is devoted to the research to diabetes always.This is because once suffer from " diabetes ", people The trouble of body will come one after another, owing to immunologic function weakens, human body easily infect by cold virus, streptococcus pneumoniae, The various infectious disease that pulmonary bacillus etc. are caused, and be difficult to cure.These diseases and optionally Destroy cell, phagocyte.The defensive enginery of inhibiting tumor cell can weaken significantly, causes cancerous cell to enliven, assembles. No wonder having such saying, once obtained " diabetes ", the life-span deducts more than ten years.Therefore diabetes are talked by many people And complexion changed, carrying out by every possible means is treated.

Diabetes (Diabetes) point type 1 diabetes, type 2 diabetes mellitus, gestational diabetes and the sugar of other specific types Urine disease.Its morbidity relates to the many factors such as inherited genetic factors, environmental factors, and with hypoinsulinism and pancreas Insulin resistance is main performance.

Insulin is unique blood sugar lowering hormone in the health that human pancreas's B cell is secreted.Insulin is by islets of langerhans β Cell is by endogenous or the thorn of exogenous material such as glucose, lactose, ribose, arginine, glucagon etc. A kind of proteohormone swashed and secrete.Insulin is the hypoglycemic hormone of unique fall in body, simultaneously facilitates sugar Former, fatty, protein synthesis.Exogenous insulin is mainly used in treating diabetes, and diabetics makes in early days It is expected to that the long period occurs with insulin and superpower antioxidant (such as injection thioctic acid, oral astaxanthin etc.) Honeymooners, injection of insulin does not have addiction and dependency.

Pancreatic stem cells is tissue specifc stem cells, and it can be divided into all types of pancreatic cell, Thus can be as the potential donor's cells of pancreatic diseases damaging cells replacement therapy.But, pancreatic stem cells It is difficult to obtain from pancreatic tissue and expand in vitro.Pancreas is obtained as seed cell using mescenchymal stem cell Gland stem cell will not limited by cell derived.

Stem cell is a kind of germinal cell with self-renewal capacity and polyphyly differentiation potential, is cell transplantation Preferable cell.In ontogenetic process, there is various forms of stem cell, such as embryonic stem cell, how competent Committed stem cell in cell and various tissue, such as hematopoietic stem cell, neural stem cell etc., but should in reality Use the challenge but facing ethics or the restriction drawn materials.Mescenchymal stem cell (MSC) is present in tissue In there is a kind of Subaerial blue green algae group of polyphyly differentiation potential, can be induced to differentiate into many in certain circumstances because of it Kind of histiocyte, and have that immunogenicity is low, transplant after can form the features such as the most chimeric, therefore can be as one Preferably for the cell of allotransplantation.Because MSC Subaerial blue green algae can obtain the differentiation of inside embryonic tissue Potential ability, easily draw materials, the more horn of plenty and do not limited by ethics and do not become tumor dangerous of originating, therefore use Derived mesenchymal stem cells in vitro induction obtains pancreatic stem cells and has higher using value clinically.

The present invention intends developing a kind of culture medium combination and methodology the most directionally to be lured by mescenchymal stem cell Lead as pancreatic stem cells, thus the treatment for diabetes and complication thereof provides a kind of new selection.

Summary of the invention

Therefore, the technical purpose of the present invention is that seeking one can be by mescenchymal stem cell directional induction in vitro The method of activated pancreatic stem cells and the combination of corresponding culture medium.

Therefore, a first aspect of the present invention relates to a kind of culture medium C, and it is by zooblast basal medium and solute Composition;Described solute and the concentration in zooblast basal medium thereof are as follows: 0.50 × 10-5mol/L-2.0× 10-5Mol/L retinoic acid, 10ng/ml-30ng/ml EGF, 10ng/ml-100ng/ml bFGF, 20ng/ml-200ng/ml Dkk1,0.2mmol/L-10mmol/L glutamine, volume ratio be 0.2%-5% non essential amino acid and can The B27 that volume ratio is 0.5%-5% of choosing;Preferably, 0.95 × 10-5mol/L-1.05×10-5Mol/L retinoic acid, 19ng/ml-21ng/ml EGF、32ng/ml-64ng/ml bFGF、80ng/ml-120ng/ml Dkk1、 1.9mmol/L-2.1mmol/L the non essential amino acid that glutamine, volume ratio are 0.95%-1.05% and optional The B27 that volume ratio is 1.9%-2.1%;And wherein said zooblast basal medium is DMEM in high glucose Culture medium or DMEM/F12 culture medium.

Preferably, described culture medium C is made up of DMEM/F12 culture medium and solute;Described solute and Concentration in culture medium B is as follows: 10-5Mol/L retinoic acid, 20ng/ml EGF, 60ng/ml bFGF, 100ng/ml Dkk1,2mmol/L glutamine, volume ratio be 1% non essential amino acid and optional volume ratio be 2% B27.

A second aspect of the present invention relates to a kind of by the test kit that mescenchymal stem cell induction is pancreatic stem cells, its It is made up of culture medium A, culture medium B and culture medium C, wherein

Described culture medium A is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 1.00ml/L-20.00ml/L hyclone, 1.00ng/mL-15.00ng/mL Activin A and 10-200ng/mL wnt3a;Preferably, 4.75ml/L-5.25ml/L hyclone, 4.75ng/mL-5.25ng/mL activin A and 40-60ng/mL wnt3a;

Described culture medium B is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 1.00ml/L-20.00ml/L hyclone and 1.00ng/mL-15.00ng/mL activin A;Preferably, 4.75ml/L-5.25ml/L hyclone and 4.75ng/mL-5.25ng/mL activin A

Described culture medium C is as described in above-mentioned first aspect;

And wherein said zooblast basal medium is DMEM in high glucose culture medium or DMEM/F12 cultivation Base.

Preferably, described culture medium A is made up of DMEM in high glucose culture medium and solute;Described solute and training Support the concentration in base A as follows: 5ml/L hyclone, 5ng/mL activinA and 5ng/mL wnt3a;

Described culture medium B is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium A In concentration as follows: 5ml/L hyclone and 5ng/mL activin A;

Described culture medium C is as described in above-mentioned first aspect.

Preferably, described the test kit that mescenchymal stem cell induction is pancreatic stem cells is also included culture medium D Or E, wherein:

Culture medium D is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium D Concentration is as follows: 5ml/L hyclone, 20ng/ml exendin-4,10ng/ml activin A, 10mmol/L Buddhist nun Gram amide, volume ratio be 2% B27 and volume ratio be the N2 of 1%;

Culture medium E is made up of DMEM in high glucose culture medium and solute;Described solute and dense in culture medium E thereof Spend as follows: 5ml/L hyclone, 20ng/ml exendin-4,15ng/ml FGF7,10mmol/L niacin amide, Volume ratio be 2% B27 and volume ratio be the N2 of 1%.

Preferably, described mescenchymal stem cell is fat mesenchymal stem cell, it is preferable that described mesenchyme is dry thin Born of the same parents are the 3rd generation mescenchymal stem cell.

A third aspect of the present invention relates to a kind of by the method that mescenchymal stem cell induction is pancreatic stem cells, its bag Include step: by mescenchymal stem cell successively with the culture medium A in the test kit described in claim 3 or 4, training Support the culture medium C described in base B and claim 1 or 2 to cultivate, obtain pancreatic stem cells, Qi Zhongsuo Stating mescenchymal stem cell uses conventional method to obtain.

Preferably, described method comprises the steps:

1) mescenchymal stem cell that conventional method obtains is inoculated in described culture medium A, cultivates 1d;

2) cell completing step 1) is transferred in described culture medium B, cultivate 4-5d;

3) step 2 will be completed) cell transfer in described culture medium C, cultivate 4-6d, obtain pancreatic stem thin Born of the same parents.

Preferably, in described step (1), described mescenchymal stem cell initial concentration in described culture medium A It is 2 × 105Individual/ml-1 × 106Individual/ml;In described method, the condition of culture of whole incubation is: 37 DEG C, 50ml/L CO2, relative air humidity be 95% incubator in cultivate.

Preferably, described method also comprises the steps:

The pancreatic stem cells obtained is separately added into culture medium D or culture medium E, every hole 2000 μ L, 37 DEG C, 50ml/L CO2, relative air humidity be 95% incubator in cultivate 8d, every 2d inhale abandon supernatant and add 2000 culture medium D new for μ L or E.

It is pancreatic stem cells according to above-mentioned being induced by mescenchymal stem cell that a fourth aspect of the present invention relates to a kind of The pancreatic stem cells that method obtains.

In other words, it is an object of the invention to provide a kind of side utilizing mescenchymal stem cell to prepare pancreatic stem cells Method and special culture media combination thereof.

The method for inducing and cultivating of the present invention is applicable to the mescenchymal stem cell of various tissue-derived each generation.This lures Lead the virus-free importing of cultural method, easily operation, efficiency height.The method for inducing and cultivating of the present invention is first with routine Method obtains the mescenchymal stem cell of certain tissue-derived (such as fatty tissue or myeloid tissue), then utilizes training Support base A and B and cultivate the mescenchymal stem cell of above-mentioned acquisition, obtain limited endoderm cell, in this is limited Endoderm cell expresses limited entoderm mark Foxa2, Sox17, it is thus achieved that efficiency can reach more than 95%;With After utilize culture medium C to cultivate to obtain pancreatic stem cells, described pancreatic stem cells expresses pancreatic stem cells mark Pdx1, Obtain efficiency and can reach more than 90%, pancreatic stem cells positive for Pdx1 can not only vitro differentiation for there being function Pancreatic endocrine cell, and can vitro differentiation be to have the Exocrine Pancreas In Rats of function, this mark is also Judge that the cell that the present invention is obtained is the quality standard of pancreatic stem cells.Wherein culture medium A consists of high sugar DMEM culture medium, 4.75ml/L-5.25ml/L hyclone, 4.75ng/mL-5.25ng/mL activin A and 40-60ng/mL wnt3a;Culture medium B consists of DMEM in high glucose culture medium, 4.75ml/L-5.25ml/L tire cattle Serum, 4.75ng/mL-5.25ng/mL activin A;Culture medium C consist of DMEM/F12 culture medium, 0.95 ×10-5mol/L-1.05×10-5Mol/L retinoic acid, 19ng/ml-21ng/ml EGF, 32ng/ml-64ng/ml FGF2(bFGF), 80ng/ml-120ng/mL Dkk1,1.9mmol/L-2.1mmol/L glutamine, volume It is the B27 of 1.9%-2.1% than non essential amino acid and the optional volume ratio for 0.95%-1.05%.This area skill Art personnel understand, the most specifically illustrate the culture medium composition of above-mentioned two concentration of height all in the embodiment of the present application In the case of realizing the present invention well, the concentration range of described culture medium can be to lower concentration and Geng Gao Concentration carry out suitable extension after remain to realize technical purpose of the present invention.The lowest or higher concentration The technique effect of the culture medium of composition may be better than, is equal to or is inferior to the experimental result indicated by the embodiment of the present application, But as long as can realize the technical purpose that the induction of described mescenchymal stem cell is pancreatic stem cells, the most such proportioning Culture medium still fall within the scope of protection of present invention.

Can be sketched by the step of mescenchymal stem cell acquisition pancreatic stem cells and be: the fatty tissue that fat absorption method is obtained Clean and obtain after conventional digestion the cell of separation, the cell of above-mentioned acquisition is seeded in equipped with expansion with suitable density Increase in the Tissue Culture Flask of culture fluid, obtain the 1st, 2,3 generation mescenchymal stem cells, the 3rd will obtained successively The most sequentially it is placed in culture medium A for mescenchymal stem cell and cultivates 1d, culture medium B cultivation 4-6d, culture medium C Cultivate 4-6d, finally obtain pancreatic stem cells.Those skilled in the art know, the above-mentioned training listed by the present invention The method step supported and induce is only the purpose of illustration, may not realize the preferred plan of the method for the invention, Said method step can be made suitable change and remain to realize the purpose of the present invention by those skilled in the art, Such change also falls into the scope of protection of present invention.Certainly, fill between the method for the invention is used Matter stem cell is not limited to the 3rd generation mescenchymal stem cell, and the mescenchymal stem cell of other generations can also use. So-called 3rd generation mescenchymal stem cell refers to the mescenchymal stem cell in the 3rd fat subsitutes source, and described mesenchyme is dry thin Born of the same parents can be adipose-derived mescenchymal stem cell can also be other source mescenchymal stem cells.Described amplification Culture fluid is not limited to the amplification cultivation liquid that the present invention is specifically used, the amplification training that other mescenchymal stem cells are suitable for Nutrient solution all can use.

Therefore, mescenchymal stem cell through two one-step inducing methods can the most once obtain limited endoderm cell, Pancreatic stem cells, each phase cell can express corresponding specific gene, and the pancreatic stem cells obtained has Differentiation becomes pancreatic endocrine cell and the function of Exocrine Pancreas In Rats of meritorious energy.

The pancreatic stem cells that the inventive method obtains can be applicable to treat the injury of pancreas that the diseases such as diabetes cause, Cell therapy for injury of pancreas disease provides experimental basis and Clinical Evidence, provides for cellular transplantation therapy New approach.

Accompanying drawing explanation

Fig. 1: for the form of human mesenchymal stem cell (seed cell of pancreatic stem cells).

Fig. 2: for the immunophenotype testing result of human mesenchymal stem cell (seed cell of pancreatic stem cells).Respectively The top of accompanying drawing indicates the immunophenotype type of correspondence.

Fig. 3: for the form of the limited endoderm cell that the differentiation of people's derived mesenchymal stem cells in vitro obtains.

Fig. 4: for the immunocyte fluorescence dye of the limited endoderm cell that the differentiation of people's derived mesenchymal stem cells in vitro obtains Color testing result.A is the cell after using culture medium A inducing culture, and B is the 3rd generation mescenchymal stem cell.

Fig. 5: for the real-time quantitative PCR of the limited endoderm cell that the differentiation of people's derived mesenchymal stem cells in vitro obtains Testing result.

Fig. 6: for the form of the pancreatic stem cells that limited endoderm cell's vitro differentiation obtains.

Fig. 7: for the immunocyte fluoroscopic examination knot of the pancreatic stem cells that limited endoderm cell's vitro differentiation obtains Really.A is the limited endoderm cell that the 3rd generation mescenchymal stem cell induction obtains, and B is for using culture medium C Cell after inducing culture.

Fig. 8: the pancreatic stem cells cell real-time quantitative PCR inspection obtained for limited endoderm cell's vitro differentiation Survey result.

Fig. 9: for the immunity of the pancreatic endocrine secretory cell that pancreatic stem cells/pancreatic progenitor cell vitro differentiation obtains Cell fluorescence dyeing testing result.A is the Pdx1 positive cell that the 3rd generation mescenchymal stem cell induction obtains, B For the cell after employing culture medium D inducing culture.

Figure 10: for the real-time quantitative PCR detection knot of the pancreatic endocrine cell that pancreatic stem cells vitro differentiation obtains Really.

Figure 11: for the insulin secretion total amount detection of the pancreatic endocrine cell that pancreatic stem cells vitro differentiation obtains Result.

Figure 12: for the insulin releasing situation detection of the pancreatic endocrine cell that pancreatic stem cells vitro differentiation obtains Result.

Figure 13: for the immunocyte fluorescence dye of the exocrine pancreas secretory cell that pancreatic stem cells vitro differentiation obtains Color testing result.A is the Pdx1 positive cell that the 3rd generation mescenchymal stem cell induction obtains, and B cultivates for using Cell after base E inducing culture.

Figure 14: for the real-time quantitative PCR detection knot of the Exocrine Pancreas In Rats that pancreatic stem cells vitro differentiation obtains Really.

Figure 15: for the amylase secretion total amount detection of the Exocrine Pancreas In Rats that pancreatic stem cells vitro differentiation obtains Result.

Detailed description of the invention

Below example facilitates a better understanding of the present invention, but is not meant to for limiting the present invention.Following Experimental technique in embodiment, if no special instructions, is conventional method.Test used in following embodiment Material, if no special instructions, is and is commercially available from routine biochemistry reagent shop.Determining in following example Amount test, is respectively provided with three times and repeats experiment, results averaged.

Embodiment

Embodiment 1 experiment material

Penicillin, streptomycin and trypsin-EDTA are purchased from GIBCO company.Trizol is purchased from American I nvitrogen Company, Oligo dT, M-MLV reverse transcriptase, Taq archaeal dna polymerase, dNTP and RNase inhibitor are purchased From Takara company of Japan.

Mouse anti human Foxa2 monoclonal antibody, rabbit anti-human Insulin monoclonal antibody, rabbit anti-human C-peptide polyclone Antibody, rabbit anti-human AMY monoclonal antibody are purchased from Abcam company of the U.S..Goat anti human's Sox17 monoclonal anti Body, Goat anti human's pdx1 monoclonal antibody are purchased from R&D company of the U.S..Chicken anti-mouse IgG of isothiocyanic acid labelling, Chicken anti-rabbit IgG of isothiocyanic acid labelling, rhodamine labelling donkey anti goat igg purchased from Santa cruz company, sieve The red goat anti-mouse IgG of bright labelling, the goat anti-rabbit igg of rhodamine labelling, the little mouse-anti of isothiocyanic acid labelling Goat IgG is purchased from Beijing company of Zhong Shan Golden Bridge.

Embodiment 2, the preparation of culture medium

DMEM/F12 culture medium (also known as DF12 culture medium) and DMEM in high glucose culture medium are purchased from GIBCO Company.Hyclone (FCS) is purchased from GIBCO company.Niacin amide is purchased from Sigma company.Glutamine Purchased from GIBCO company, catalog number is 25030-081.Retinoic acid is purchased from Sigma company, catalogue Number it is R2625.

Human activin A(ActivinA) purchased from Pepro Tech company of Britain, catalog number is 120-14.

EGF (epidermal growth factor, epithelical cell growth factor) is purchased from Pepro Tech company, product Catalog number (Cat.No.) is 100-15.

BFGF (basic fibroblast growth factor, basic fibroblast growth factor) is public purchased from Sigma Department, catalog number is F0291.

Exendin-4 is purchased from Sigma company, and catalog number is E7144.

FGF7 is purchased from Pepro Tech company, and catalog number is 100-19.

DKK1 is purchased from R&D company, and catalog number is 5439-DK-010.

B27 is purchased from GIBCO company, and catalog number is 17504-044.

N2 is purchased from GIBCO company, and catalog number is 17502-048.

Non essential amino acid is purchased from GIBCO company, and catalog number is 11140.

Following culture medium A-C is all configured by low content and high-load.

Culture medium A is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium A Concentration is as follows: 4.75ml/L(is low) or 5.25ml/L(high) hyclone, 4.75ng/mL(be low) or 5.25ng/mL (high) activinA and 40ng/mL(is low) or 60ng/mL(height) wnt3a..

Culture medium B is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium B Concentration is as follows: 4.75ml/L(is low) or 5.25ml/L(high) hyclone, 4.75ng/mL(be low) or 5.25ng/mL (high) activinA.

Culture medium C is made up of DMEM/F12 culture medium and solute;Described solute and dense in culture medium C thereof Spend as follows: 0.95 × 10-5Mol/L(is low) or 1.05 × 10-5Mol/L(is high) retinoic acid, 19ng/ml(be low) Or 21ng/ml(is high) EGF, 32ng/ml(be low) or 64ng/ml(high) FGF2(bFGF), 80ng/ml (low) or 120ng/mL(are high) Dkk1,1.9mmol/L(be low) or 2.1mmol/L(high) glutamine, Volume ratio is that 0.95%(is low) or 1.05%(high) non essential amino acid and volume ratio be that 1.9%(is low) or 2.1%(is high) B27.Wherein, retinoic acid, FGF2, EGF, Dkk1, glutamine and non-essential amino Acid is for obtaining composition necessary to pancreatic stem cells.

Culture medium D is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium D Concentration is as follows: 5ml/L hyclone, 20ng/ml exendin-4,10ng/ml activin A, 10mmol/L Buddhist nun Gram amide, volume ratio be 2% B27 and volume ratio be the N2 of 1%.

Culture medium E is made up of DMEM in high glucose culture medium and solute;Described solute and dense in culture medium E thereof Spend as follows: 5ml/L hyclone, 20ng/ml exendin-4,15ng/ml FGF7,10mmol/L niacin amide, Volume ratio be 2% B27 and volume ratio be the N2 of 1%.

Embodiment 3, the sequential inducing culture of insulin human secretory cell

Step one, preparation the 3rd generation mescenchymal stem cell

1, fat absorption method fatty tissue D-Hanks that gathers out will be used ' (can also use PBS) wash away blood Cell and anesthetics, 0.2%II Collagenase Type 37 DEG C digestion 30min, 100 eye mesh screens filter and collect filtrate.

2, the cell obtained by D-Hank ' the resuspended step 1 of s liquid repeated washing 2 times are to remove collagenase, room temperature 1200rpm is centrifuged 10min, collects cell.

3, by the cell of step 2 with 2 × 106The density of individual/ml is inoculated in equipped with the amplification cultivation liquid (example of formula One of be containing 58%DF12,40%MCDB, 2% hyclone, 1 × 10-9MITS、1×10-9M ground plug rice Pine, 1 × 10-4M2-phosphoric acid ascorbic acid, 20ng/mL interleukin-6,10ng/mL EGF, 10ng/mL PDGF-BB, 100U/mL penicillin and 100 μ g/mL streptomycins, other have the expansion of similar effect certainly Increase culture fluid the most all can use) T75 Tissue Culture Flask in, at 37 DEG C, 50ml/L CO2, air relative Humidity be 95% incubator cultivate (24-48hr) after 36hr and discard culture fluid (cell containing the most adherent), And supplementing new amplification cultivation liquid, the most every 3d partly measures the amplification cultivation liquid more renewed, when cell reaches 70%-80% When converging, with 1g/L trypsin Gibco company) conventional digestion, cell passes on according to 1:3, To 1st generation mesenchymal cell.

4,1st generation mesenchymal cell is cultivated in amplification cultivation liquid 2-3d(at 37 DEG C, 50ml/L CO2, empty Gas relative humidity is the incubator cultivation of 95%), reach 70%-80% and converge, wash 2 times with D-Hank ' s liquid, use 1g/L trypsin Gibco company) conventional digestion, cell passes on according to 1:3, obtains between 2nd generation Mesenchymal cells.

5,2nd generation mesenchymal cell is cultivated in amplification cultivation liquid 2-3d(at 37 DEG C, 50ml/L CO2, empty Gas relative humidity be 95% incubator cultivate), reach 70%-80% and converge, wash 2 times with D-Hank ' s liquid, so Afterwards with the trypsin Gibco company of 1g/L) conventional digestion, room temperature 1200rpm is centrifuged 6min, collects thin Born of the same parents.

6, by the cell of step 5 with 1 × 106The density in individual/hole is inoculated in 6 orifice plates equipped with amplification cultivation liquid and (purchases From Nunclon company) in, at 37 DEG C, 50ml/L CO2, relative air humidity be 95% incubator cultivate 4-6hr, reject culture medium after cell attachment, wash 2 times with D-Hank ' s, be denoted as the 3rd generation mescenchymal stem cell.

Certainly, the present invention can also use the isolated culture method of other mescenchymal stem cells well known in the art Obtain the 3rd generation mescenchymal stem cell or directly use commercially available 3rd generation mescenchymal stem cell and without from Row separation and Culture obtains.

Step 2, employing culture medium combination carry out sequential inducing culture

1, the cultivation of first stage

(1) in 6 orifice plates, add culture medium A (to note that and employ high concentration and two proportionings of low concentration Culture medium A, culture medium B and C below are also such), every hole 2000 μ L.

(2) in 6 orifice plates of step (1), the 3rd generation mescenchymal stem cell is inoculated so that it is in the medium Concentration is 6 × 105Individual/ml, at 37 DEG C, 50ml/L CO2, relative air humidity be 95% incubator in train Support 1d.

(3) inhale the culture medium A in 6 orifice plates abandoning step (2), add culture medium B(every 2d suction and abandon supernatant And add 2000 culture medium B new for μ L).

2, the cultivation of second stage

The supernatant in 6 orifice plates of first stage step (3) is abandoned in suction, adds culture medium C, every hole 2000 μ L, 37℃、50ml/L CO2, relative air humidity be 95% incubator in cultivate the every 2d of 4-6d(inhale abandon supernatant also Add 2000 culture medium C new for μ L).

3, the cultivation of phase III

Inhale the supernatant abandoned in 6 orifice plates of second stage, be separately added into culture medium D or culture medium E, every hole 2000 μ L, At 37 DEG C, 50ml/L CO2, relative air humidity be 95% incubator in cultivate the every 2d of 8d(inhale abandon supernatant And add 2000 culture medium D new for μ L or E), room temperature 1200rpm is centrifuged 6min, collects cell (precipitation). (being combined as: A → B → C → D or E) (uses D and E culture medium respectively merely to prove the pancreas obtained Stem cell has the function being divided into ripe Exocrine cells.After the sequential use being induced to culture medium C the completeest Cheng Liao.)

Step 3, (following experimental result is all that the culture medium A of low concentration, B, C are carried out in the qualification of inducing effect The result of induction)

1, the values of immunophenotyping of the 3rd generation mescenchymal stem cell

The cellular morphology of the 3rd generation mescenchymal stem cell that step one obtains is shown in Fig. 1.

By the phenotype of indirect immunofluorescence detection the 3rd generation mescenchymal stem cell, with Fluorescein isothiocyanate (FITC) Mouse anti human CD29 of labelling, CD34, CD44, CD73, CD90, CD105, CD106, CD117, Flow cytometer detection is carried out, stream after Flk-1, HLA-ABC and HLA-DR antibody (antibody is purchased from BD company) labelling Formula cell instrument is ACCURI C6 (Becton Dickinson).

Result is shown in that Fig. 2, abscissa represent that cell fluorescence intensity, vertical coordinate represent cell number;P2 represents selected thin Born of the same parents colony.Phenotypic results shows, the CD29 of the 3rd generation mescenchymal stem cell, CD44, CD73, CD90, CD105, Flk-1 and HLA-ABC are the positive, and CD34, CD106, CD117 and HLA-DR molecule is equal For feminine gender.Compared with prior art, the peculiar phenotype of the mescenchymal stem cell that the present invention is obtained is that Flk-1 is positive.

2, human mesenchymal stem cell is induced to differentiate into the qualification of limited endoderm cell

(1) identification of morphology

In culture medium A and B, cultivate cell after 6d reach 80%-90% and converge, the form of cell in incubation Change as follows: cultivating 1-2d, cell is essentially fusiformis, and minority is cobblestone sample epithelial cell form, and fusiformis is thin The percentage ratio of born of the same parents is more than 90%;After cultivating 4-6d, cell volume significantly increases, and arrangement is tight, spindle cell ratio Example reduces, and most cells present cobblestone sample epithelial cell form, and the percentage ratio of cobblestone like cell is more than 90%, The cell cultivating 5d in culture medium A and B is shown in Fig. 3.

(2) immunofluorescence dyeing is identified

The cell (using the 3rd generation mescenchymal stem cell as comparison) cultivating 5d in the 1 of step 2 is carried out immunity Fluorescence staining, detects the expression of limited entoderm mark Foxa2 and Sox17.

The concrete grammar of detection Foxa2 is as follows: cell is fixed with 80% ice ethanol, with containing 1%BSA after PBS PBST buffer blind;Then with mouse anti human Foxa2 monoclonal antibody (working concentration is 1:50 dilution) As anti-incubated at room 1hr;After cleaning with PBS, then with isothiocyanic acid (green fluorescence) labelling Goat anti-mouse IgG is as two anti-incubated at room 30min;Hoechst33342 is used after cleaning with PBS Dyeing liquor (Sigma) redyes nucleus (blue-fluorescence), at fluorescence microscopy Microscopic observation (Olympus).3rd Green fluorescence is not the most shown, for negative findings for mescenchymal stem cell.Use after culture medium A inducing culture is thin Born of the same parents more than 90% show green fluorescence.

The concrete grammar of detection Sox17 is essentially identical with the concrete grammar of detection Foxa2, and difference is only that employing Goat anti human's Sox17 monoclonal antibody (working concentration is 1:100 dilution) resists as one, uses rhodamine (red Color fluorescence) labelling mice anti goat igg as two resist.3rd generation mescenchymal stem cell does not the most show red glimmering Light, for negative findings.The cell more than 90% after culture medium A inducing culture is used to show red fluorescence.

Result is shown in Fig. 4 (A is the cell after using culture medium A inducing culture, and B is the 3rd generation mescenchymal stem cell). Result shows, after employing culture medium A inducing culture, most cells is Foxa2 and Sox17 is positive.

(3) Real-time PCR Analysis

Take the cell (using the 3rd generation mescenchymal stem cell as comparison) cultivating 5d in the 1 of step 2, extract total RNA reverse transcription are cDNA, identify Foxa2 gene and the expression of Sox17 gene by real-time quantitative PCR Situation.Using people GAPDH as the comparison of each gene, primer used is as follows:

For expanding the primer of Foxa2 gene it is:

Forward primer: 5 '-CTGAGCGAGATCTACCAGTGGA-3 ' (SEQ ID NO:1);

Downstream primer: 5 '-CAGTCGTTGAAGGAGAGCGAGT-3 ' (SEQ ID NO:2).

For expanding the primer of Sox17 gene it is:

Forward primer: 5 '-GCATGACTCCGGTGTGAATCT-3 ' (SEQ ID NO:3);

Downstream primer: 5 '-TCACACGTCAGGATAGTTGCAGT-3 ' (SEQ ID NO:4).

For expanding the primer of GAPDH it is:

Forward primer: 5 '-GGTCACCAGGGCTGCTTTTA-3 ' (SEQ ID NO:5);

Downstream primer: 5 '-GAGGGATCTCGCTCCTGGA-3 ' (SEQ ID NO:6).

Use△△CT method calculates the relative expression quantity of each gene.Foxa2 gene with the 3rd generation mescenchymal stem cell The expression of (or sox17 gene) is 1, calculates in the cell after using culture medium A and B inducing culture each The relative expression quantity of individual gene.Use in the cell after culture medium A and the sequential inducing culture of B, foxa2 gene Relative expression quantity is 15.43, and the relative expression quantity of sox17 gene is 4.70.Result is shown in Fig. 5.

Above qualification result shows, uses the cell after culture medium A and B inducing culture mainly to behave limited interior Endoderm cell (as it has been described above, have expressed the specificity marker gene of limited endoderm cell, and shows limit The form of qualitative endoderm cell).

3, limited endoderm cell is divided into the qualification of pancreatic stem cells

(1) identification of morphology

In culture medium C cultivate 6d, in incubation, the metamorphosis of cell is as follows: cell taper into and in Bulk coherent condition.The cell cultivating 6d in culture medium C is shown in Fig. 6.

(2) immunofluorescence dyeing is identified

The cell (using the 3rd generation mescenchymal stem cell as comparison) cultivating 6d in the 2 of step 2 is carried out immunity Fluorescence staining, detection pancreatic stem cells mark Pdx1 and limited endoderm cell indicate the expression of Foxa2 respectively Situation.

The concrete grammar of detection Pdx1 is essentially identical with the concrete grammar of detection Foxa2, and difference is only that employing mountain Goat-anti people's Pdx1 monoclonal antibody (working concentration is 1:100 dilution) resists as one.3rd generation mesenchyme is dry thin Born of the same parents the most do not show red fluorescence, for negative findings.The cell after culture medium C inducing culture more than 90% is used to show Show red fluorescence.

Result is shown in that (A is the limited endoderm cell that the 3rd generation mescenchymal stem cell induction obtains to Fig. 7, and B is for adopting Cell with after culture medium C inducing culture).Result shows, uses most cells after culture medium C inducing culture Positive for Pdx1 immunocyte fluorescence staining.

(3) Real-time PCR Analysis

Take the cell (using the 3rd generation mescenchymal stem cell as comparison) cultivating 6d in the 2 of step 2, extract total RNA reverse transcription are cDNA, identified respectively by real-time quantitative PCR Pdx1, P48, Sox9, Hnf6, The expression of Hnf1b, Nkx6.1 and Nkx2.2 gene.Use people GAPDH as the comparison of each gene, Primer used is as follows:

For expanding the primer of Pdx1 it is:

Forward primer: 5 '-TACTGGATTGGCGTTGTTTGTGGC-3 ' (SEQ ID NO:7);

Downstream primer: 5 '-AGGGAGCCTTCCAATGTGTATGGT-3 ' (SEQ ID NO:8).

For expanding the primer of P48 it is:

Forward primer: 5 '-TTCACCGACCAGTCTTCACG-3 ' (SEQ ID NO:9);

Downstream primer: 5 '-GTGGCTAAGGAACTCCACCT-3 ' (SEQ ID NO:10).

For expanding the primer of Sox9 it is:

Forward primer: 5 '-GGCGGAGGAAGTCGGTGAAG-3 ' (SEQ ID NO:11);

Downstream primer: 5 '-GGGTGCGGTGCTGCTGAT-3 ' (SEQ ID NO:12).

For expanding the primer of Hnf6 it is:

Forward primer: 5 '-TGTGGAAGTGGCTGCAGGA-3 ' (SEQ IDNO:13);

Downstream primer: 5 '-TGTGAAGACCAACCTGGGCT-3 ' (SEQ ID NO:14).

For expanding the primer of Hnf1b it is:

Forward primer: 5 '-GCACCTCTCCCAGCATCTCA-3 ' (SEQ IDNO:15);

Downstream primer: 5 '-GTCGGAGGATCTCTCGTTGC-3 ' (SEQ ID NO:16).

For expanding the primer of Nkx6.1 it is:

Forward primer: 5 '-AGAGAGTCAGGTCAAGGTCTGGTT-3 ' (SEQ ID NO:17);

Downstream primer: 5 '-TGTCTCCGAGTCCTGCTTCTTCTT-3 ' (SEQ IDNO:18).

For expanding the primer of Nkx2.2 it is:

Forward primer: 5 '-GACAACTGGTGGCAGATTTCGCTT-3 ' (SEQ ID NO:19);

Downstream primer: 5 '-AGCCACAAAGAAAGGAGTTGGACC-3 ' (SEQ IDNO:20).

For expanding the primer of GAPDH it is:

Forward primer: 5 '-GGTCACCAGGGCTGCTTTTA-3 ' (SEQ ID NO:5);

Downstream primer: 5 '-GAGGGATCTCGCTCCTGGA-3 ' (SEQ ID NO:6).

Use△△CT method calculates the relative expression quantity of each gene.Pdx1 gene with the 3rd generation mescenchymal stem cell (or p48 gene or sox9 gene or hnf6 gene or hnf1b gene or nkx6.1 gene or nkx2.2 gene) Expression be 1, calculate and use the relative expression quantity of each gene in the cell after culture medium C inducing culture.Adopt With in the cell after culture medium C inducing culture, the relative expression quantity of pdx1 gene is 49.53, the phase of p48 gene Being 58.04 to expression, the relative expression quantity of sox9 gene is 2.24, and the relative expression quantity of hnf6 gene is 4.30, The relative expression quantity of hnf1b gene is 24.41, and the relative expression quantity of nkx6.1 gene is 6.50, nkx2.2 gene Relative expression quantity be 17.71.Result is shown in Fig. 8.Result shows, after using culture medium C to cultivate, and human mesenchyme Pancreatic stem cells gene Pdx1, endoderm and the pancreatic precursor of the limited endoderm cell of source of human stem cell The expression of cytogene P48, Sox9, Hnf6, Hnf1b, Nkx6.1 and Nkx2.2 gene is all raised.Above Qualification result shows, after using culture medium C inducing culture, the cell in bulk coherent condition is pancreatic stem cells (this cell expresses pancreatic stem cells specificity marker gene, and demonstrates bulk coherent condition, and this cell enters One one-step inducing can obtain pancreatic endocrine cell and Exocrine Pancreas In Rats).

4, pancreatic stem cells vitro differentiation is the qualification of pancreatic endocrine cell

(1) immunofluorescence dyeing is identified

The cell (using the 3rd generation mescenchymal stem cell as comparison) cultivating 8d in the 3 of step 2 is carried out immunity Fluorescence staining, detection endocrine cell mark insulin (Insulin) and the expression of C peptide (C-peptide).

The concrete grammar of detection insulin is essentially identical with the concrete grammar of detection Foxa2, and difference is only that employing Rabbit anti-Human Insulin's monoclonal antibody (working concentration is 1:50 dilution) resists as one, uses isothiocyanic acid (green Color fluorescence) labelling goat anti-rabbit igg as two resist.3rd generation mescenchymal stem cell does not the most show green fluorescence, For negative findings.The cell more than 95% after culture medium D inducing culture is used to show green fluorescence.

The concrete grammar of detection C peptide is essentially identical with the concrete grammar of detection Foxa2, and difference is only that employing rabbit Anti-C-P polyclonal antibody (working concentration is 1:100 dilution) resists as one, uses rhodamine (red glimmering Light) labelling goat anti-rabbit igg as two resist.3rd generation mescenchymal stem cell does not the most show red fluorescence, for Negative findings.The cell more than 95% after culture medium D inducing culture is used to show red fluorescence.

Result is shown in that (A is the Pdx1 positive cell that the 3rd generation mescenchymal stem cell induction obtains to Fig. 9, and B is for using Cell after culture medium D inducing culture).Result shows, uses most cells after culture medium D inducing culture Positive for insulin and C peptide.

(3) Real-time PCR Analysis

Take the cell in the 3 of step 2, the 3rd generation mesenchyme (is done by the cell cultivating 8d in culture medium D Cell is as comparison) to extract total serum IgE reverse transcription be cDNA, identifies insulin base by real-time quantitative PCR Cause, glucagon gene, somatostatin gene, pancreatic polypeptide (Pan-pol) gene, ghrelin gene, MafA Gene and the expression of Glut-2 gene.Use people GAPDH as the comparison of each gene, primer used As follows

For expanding the primer of insulin gene it is:

Forward primer: 5 '-AGAGGCCATCAAGCAGATCACTGT-3 ' (SEQ ID NO:21);

Downstream primer: 5 '-CACAGGTGTTGGTTCACAAAGGCT-3 ' (SEQ ID NO:22).

For expanding the primer of glucagon gene it is:

Forward primer: 5 '-TCTTGATAATCTTGCCGCCAGGGA-3 ' (SEQ ID NO:23);

Downstream primer: 5 '-CATGCAAAGCAATGTGGCCTCAGA-3 ' (SEQ ID NO:24).

For expanding the primer of somatostatin gene it is:

Forward primer: 5 '-TGAACCCAACCAGACGGAGAATGA-3 ' (SEQ ID NO:25);

Downstream primer: 5 '-GAAATTCTTGCAGCCAGCTTTGCG-3 ' (SEQ ID NO:26).

For expanding the primer of Pan-pol gene it is:

Forward primer: 5 '-AAAGACACAAAGAGGACACGCTGG-3 ' (SEQ ID NO:27);

Downstream primer: 5 '-TCGTAGGAGACAGAAGGTGGCATT-3 ' (SEQ ID NO:28).

For expanding the primer of ghrelin gene it is:

Forward primer: 5 '-AAGTGATCGCCCACAAGCCTTACT-3 ' (SEQ ID NO:29);

Downstream primer: 5 '-TGTACAACAGTCGTGGGAGTTGCT-3 ' (SEQ ID NO:30).

For expanding the primer of MafA gene it is:

Forward primer: 5 '-CTTCAGCAAGGAGGAGGTCATC-3 ' (SEQ ID NO:31);

Downstream primer: 5 '-CTCGTATTTCTCCTTGTACAGGTCC-3 ' (SEQ ID NO:32).

For expanding the primer of Glut-2 gene it is:

Forward primer: 5 '-AGCTGCATTCAGCAATTGGACCTG-3 ' (SEQ ID NO:33);

Downstream primer: 5 '-ATGTGAACAGGGTAAAGGCCAGGA-3 ' (SEQ ID NO:34).

For expanding the primer of GAPDH it is:

Forward primer: 5 '-GGTCACCAGGGCTGCTTTTA-3 ' (SEQ ID NO:5);

Downstream primer: 5 '-GAGGGATCTCGCTCCTGGA-3 ' (SEQ ID NO:6).

Use△△CT method calculates the relative expression quantity of each gene.Insulin gene with the 3rd generation mescenchymal stem cell (or glucagon gene or somatostatin gene or Pan-pol gene or ghrelin gene or MafA gene or Glut-2 gene) expression be 1, calculate and use the phase of each gene in the cell after culture medium D inducing culture To expression.Using in the cell after culture medium D inducing culture, the relative expression quantity of insulin gene is 6.39, The relative expression quantity of glucagon gene is 2.17, and the relative expression quantity of somatostatin gene is 39.60, Pan-pol The relative expression quantity of gene is 1.42, and the relative expression quantity of ghrelin gene is 3.22, the relative table of MafA gene The amount of reaching is 7.66, and the relative expression quantity of Glut-2 gene is 8.22.Result is shown in Figure 10.Result shows, uses training After supporting base D cultivation, the pancreatic stem cells in fat mesenchymal stem cell source expresses pancreatic endocrine function cell phase Correlation gene insulin gene, glucagon gene, somatostatin gene, pancreatic polypeptide gene (Pan-pol), ghrelin, MafA and Glut-2.

(4) insulin secretion function detection

1. insulin assay

The cell of 8d will be cultivated (using the 3rd generation mescenchymal stem cell as right through culture medium D in the 3 of step 2 According to) test as follows:

For each cell hole, take all culture supernatant, and the cell in this hole is counted.By in cultivation Clear 2000rpm is centrifuged 10min, take supernatant and with insulin ELISA detection kit (EZHIASF-14K, MILLIPORE) detection insulin content (concrete operations are carried out according to test kit description).

Every 6 × 105The insulin total amount that emiocytosis after the induction of individual employing culture medium D obtains is (373.37 ± 8.47) (20.14 ± 4.81) μ U/ml, the insulin total amount that the 3rd generation mescenchymal stem cell secretion obtains is (20.14 ± 4.81) μ U/ml(P < 0.01).Result is shown in Figure 11.

2. cell C-peptide secretory function measures

By in the 3 of step 2 through culture medium D cultivate 8d cell test as follows:

By 5 × 105After individual cell PBS washs 3 times, in 1mL KRBH buffer, hatch 6hr (3-6hr), cell is placed in the 1mL KRBH buffer containing 5.5mM glucose and hatches 1hr, collects supernatant Liquid (solution first);Again cell is placed in the 1mL KRBH buffer containing 22mM glucose and hatches 1hr, collect (collecting supernatant here to need not be centrifuged, simply cell counting needs centrifugal counting to supernatant (solution second), centrifugal Condition is 1200rpm, 6min).

C peptide ELISA kit (EZHCP-20K, MILLIPORE) is used to detect solution first and solution respectively The content (concrete assay method reference reagent box description) of C peptide in second.

In solution first, C peptide emission levels is (203.98 ± 15.68) pmol/L/h (P < 0.05), this level representation pancreas Island element de novo synthesis amount.In solution second, C peptide emission levels reaches (325.23 ± 14.37) pmol/L/h(P < 0.05). Result is shown in Figure 12.Result shows, uses the cell after the induction of culture medium D to have in vitro glucose response Insulin secretion function.Result shows, using the cell after culture medium D inducing culture is insulin secretory cell (this cell expresses insulin secretory cell specificity marker gene, and demonstrate the ability of secreted in vitro insulin And the reactivity to glucose).

4, pancreatic stem cells vitro differentiation is the qualification of Exocrine Pancreas In Rats

(1) immunofluorescence dyeing is identified

The cell (using the 3rd generation mescenchymal stem cell as comparison) cultivating 8d in the 3 of step 2 is carried out immunity Fluorescence staining, detects the diastatic expression of Exocrine Pancreas In Rats mark.

Detecting diastatic concrete grammar essentially identical with the concrete grammar of detection Foxa2, difference is only that employing Rabbit anti-human amylase monoclonal antibody (working concentration is 1:50 dilution) resists as one, uses rhodamine (red Fluorescence) labelling goat anti-rabbit igg as two resist.3rd generation mescenchymal stem cell does not the most show red fluorescence, For negative findings.The cell more than 95% after culture medium C inducing culture is used to show red fluorescence.

Result is shown in that (A is the Pdx1 positive cell that the 3rd generation mescenchymal stem cell induction obtains to Figure 13, and B is for using Cell after culture medium E inducing culture).Result shows, after using culture medium E inducing culture, most cells is Amylase positive.

(3) Real-time PCR Analysis

Take the cell in the 3 of step 2, culture medium E is cultivated the cell of 8d (by dry for the 3rd generation mesenchyme thin Born of the same parents are as comparison), extract total serum IgE and reverse transcription is cDNA, identify gene starch by real-time quantitative PCR Enzyme (AMY), elastoser 1, Carboxypeptidase A (CPA) and the expression of chymotrypsinogen B (CTRB). Using people GAPDH as the comparison of each gene, primer used is as follows:

For expanding the primer of amylase gene it is:

Forward primer: 5 '-CTTTGTGGATAACCATGACAATCAA-3 ' (SEQ ID NO:35);

Downstream primer: 5 '-CAACTGCCATTTTGTACAGCCTAG-3 ' (SEQ ID NO:36).

For expanding the primer of elastoser 1 gene it is:

Forward primer: 5 '-AGCACAACCTGAGCCAGAAT-3 ' (SEQ ID NO:37);

Downstream primer: 5 '-TTGTTAGCCAGGATGGTTCC-3 ' (SEQ ID NO:38).

For expanding the primer of Carboxypeptidase A gene it is:

Forward primer: 5 '-ACTACGCCACCTACCACACC-3 ' (SEQ ID NO:39);

Downstream primer: 5 '-GGTGTTGCCAATCTGGATCT-3 ' (SEQ ID NO:40).

For expanding the primer of chymotrypsinogen 1 B gene it is:

Forward primer: 5 '-GAACTGGCTTCCATTTCTGC-3 ' (SEQ ID NO:41);

Downstream primer: 5 '-ATGTCATTACGCACGGTGAA-3 ' (SEQ IDNO:42).

For expanding the primer of GAPDH it is:

Forward primer: 5 '-GGTCACCAGGGCTGCTTTTA-3 ' (SEQ ID NO:5);

Downstream primer: 5 '-GAGGGATCTCGCTCCTGGA-3 ' (SEQ ID NO:6).

UseΔΔCT method calculates the relative expression quantity of each gene.Amylase gene with the 3rd generation mescenchymal stem cell The table of (or elastoser 1 gene or Carboxypeptidase A gene or chymotrypsinogen 1 B gene or ACTB gene) The amount of reaching is 1, calculates the relative expression quantity of each gene in the cell after using culture medium E inducing culture.Use training Supporting in the cell after base E inducing culture, the relative expression quantity of amylase gene is 6.45, elastoser 1 base The relative expression quantity of cause is 9.28, and the relative expression quantity of Carboxypeptidase A gene is 4.89, chymotrypsinogen 1 B gene Relative expression quantity be 4.79.Result is shown in Figure 14.Result shows, after using culture medium E to cultivate, fills between fat Pancreatic stem cells exocrine pancreatic function cell relating gene-1 amylase (AMY) of matter source of human stem cell, elastic egg White enzyme 1, Carboxypeptidase A (CPA) and chymotrypsinogen B (CTRB) express and raise.

(4) amylase secretion Function detection

The cell (using the 3rd generation mescenchymal stem cell as comparison) of 8d will be cultivated through culture medium E in the 3 of step 2 Test as follows:

For each cell hole, take all culture supernatant, and the cell in this hole is counted.By in cultivation Clear 2000rpm is centrifuged 10min, takes supernatant and (has with amylase ELISA detection kit detection amylase content Gymnastics is made to carry out according to test kit description).

Every 6 × 105The amylase total amount that emiocytosis after the induction of individual employing culture medium E obtains is (82.55 ± 1.01) μM, the insulin total amount that the 3rd generation mescenchymal stem cell secretion obtains is (27.23 ± 0.93) μM (P < 0.05). Result is shown in Figure 15.

Third generation mesenchyme is done by above the results show employing culture medium A of the present invention, B and C Cell the most disposably obtains limited endoderm cell after carrying out sequential induction and pancreatic stem is thin Born of the same parents, each phase cell can express corresponding specific gene, and the pancreatic stem cells finally obtained can break up Become pancreatic endocrine cell and the Exocrine Pancreas In Rats having function.Simultaneously with the culture medium A of high concentration, B and The result that C carries out sequential induction is quite similar with the result carrying out inducing with the culture medium A of low concentration, B and C, Do not repeat them here.

As well known to those skilled in the art, can be to culture medium A of the present invention, the composition of B, C, D and E Composition (the especially constituent of culture medium A, B and C) and concentration are made various change and are remained to realize this Invent technique effect required for protection, such as in order to make mescenchymal stem cell grow more quickly or more permanently Maintain its phenotype, can add corresponding somatomedin such as HGF in the medium, or other nutrients such as β- Mercaptoethanols etc., the concentration of the various solutes in described culture medium can also be lower or higher.Although carrying out so Adjustment after, the using effect of described culture medium may be deteriorated, but as long as it can realize the purpose of the present invention The most within the scope of the present invention.

Claims (10)

1., by the test kit that mescenchymal stem cell induction is pancreatic stem cells, it is by culture medium A, culture medium B and culture medium C composition, wherein
Described culture medium A is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 1.00ml/L-20.00ml/L hyclone, 1.00ng/mL-15.00ng/mL Activin A and 10-200ng/mL wnt3a;
Described culture medium B is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 1.00ml/L-20.00ml/L hyclone and 1.00ng/mL-15.00ng/mL activin A;
Described culture medium C is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 0.50 × 10-5mol/L-2.0×10-5Mol/L retinoic acid, 10ng/ml-30ng/ml EGF、10ng/ml-100ng/ml bFGF、20ng/ml-200ng/ml Dkk1、 0.2mmol/L-10mmol/L glutamine, volume ratio are the non essential amino acid of 0.2%-5% and optional body Long-pending than the B27 for 0.5%-5%;And wherein said zooblast basal medium is DMEM in high glucose culture medium Or DMEM/F12 culture medium.
The most according to claim 1 by the test kit that mescenchymal stem cell induction is pancreatic stem cells, wherein
Described culture medium A is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 4.75ml/L-5.25ml/L hyclone, 4.75ng/mL-5.25ng/mL Activin A and 40-60ng/mL wnt3a;
Described culture medium B is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 4.75ml/L-5.25ml/L hyclone and 4.75ng/mL-5.25ng/mL activin A;
Described culture medium C is made up of zooblast basal medium and solute;Described solute and at zooblast Concentration in basal medium is as follows: 0.95 × 10-5mol/L-1.05×10-5Mol/L retinoic acid, 19ng/ml-21ng/ml EGF、32ng/ml-64ng/ml bFGF、80ng/ml-120ng/ml Dkk1、 1.9mmol/L-2.1mmol/L the non essential amino acid that glutamine, volume ratio are 0.95%-1.05% and optional The B27 that volume ratio is 1.9%-2.1%.
The most according to claim 2 by the test kit that mescenchymal stem cell induction is pancreatic stem cells, it is special Levy and be:
Described culture medium A is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium A In concentration as follows: 5ml/L hyclone, 5ng/mL activin A and 60ng/mL wnt3a;
Described culture medium B is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium A In concentration as follows: 5ml/L hyclone and 5ng/mL activin A;
Described culture medium C is made up of DMEM/F12 culture medium and solute;Described solute and in culture medium B Concentration as follows: 10-5Mol/L retinoic acid, 20ng/ml EGF, 60ng/ml bFGF, 100ng/ml Dkk1, 2mmol/L glutamine, volume ratio be 1% non essential amino acid and optional volume ratio be the B27 of 2%.
4. according to the reagent that being induced by mescenchymal stem cell described in any one of claim 1-3 is pancreatic stem cells Box, it is characterised in that it also includes culture medium D or E, wherein
Culture medium D is made up of DMEM in high glucose culture medium and solute;Described solute and in culture medium D Concentration is as follows: 5ml/L hyclone, 20ng/ml exendin-4,10ng/ml activin A, 10mmol/L Buddhist nun Gram amide, volume ratio be 2% B27 and volume ratio be the N2 of 1%;
Culture medium E is made up of DMEM in high glucose culture medium and solute;Described solute and dense in culture medium E thereof Spend as follows: 5ml/L hyclone, 20ng/ml exendin-4,15ng/ml FGF7,10mmol/L niacin amide, Volume ratio be 2% B27 and volume ratio be the N2 of 1%.
5. according to the reagent that being induced by mescenchymal stem cell described in any one of claim 1-3 is pancreatic stem cells Box, it is characterised in that described mescenchymal stem cell is fat mesenchymal stem cell.
The most according to claim 5 by the test kit that mescenchymal stem cell induction is pancreatic stem cells, it is special Levy and be that described mescenchymal stem cell is the 3rd generation mescenchymal stem cell.
7., by the method that mescenchymal stem cell induction is pancreatic stem cells, it includes step: done by mesenchyme Cell is successively by culture medium A, culture medium B and the culture medium C in the test kit described in any one of claim 1-3 Cultivating, obtain pancreatic stem cells, wherein said mescenchymal stem cell uses conventional method to obtain.
The most according to claim 7 by the method that mescenchymal stem cell induction is pancreatic stem cells, its feature It is: described method comprises the steps:
1) mescenchymal stem cell that conventional method obtains is inoculated in described culture medium A, cultivates 1d;
2) step 1 will be completed) cell transfer in described culture medium B, cultivate 4-5d;
3) step 2 will be completed) cell transfer in described culture medium C, cultivate 4-6d, obtain pancreatic stem thin Born of the same parents.
The most according to claim 8 by the method that mescenchymal stem cell induction is pancreatic stem cells, its feature It being: in described step (1), described mescenchymal stem cell initial concentration in described culture medium A is 2 × 105Individual/ml-1 × 106Individual/ml;In described method, the condition of culture of whole incubation is: at 37 DEG C, 50ml/L CO2, relative air humidity be 95% incubator in cultivate.
10. according to the side that being induced by mescenchymal stem cell described in any one of claim 7-9 is pancreatic stem cells Method, it also comprises the steps:
The pancreatic stem cells obtained is separately added into culture medium D in test kit as claimed in claim 4 or training Supporting base E, every hole 2000 μ L, at 37 DEG C, 50ml/L CO2, relative air humidity be 95% incubator in train Support 8d, every 2d suction abandon supernatant and add 2000 culture medium D new for μ L or E.
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