CN110423720A - A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional islets β cell - Google Patents

A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional islets β cell Download PDF

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CN110423720A
CN110423720A CN201910665356.XA CN201910665356A CN110423720A CN 110423720 A CN110423720 A CN 110423720A CN 201910665356 A CN201910665356 A CN 201910665356A CN 110423720 A CN110423720 A CN 110423720A
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cell
concentration
stem cell
amnioic epithelium
epithelium stem
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辛洪波
李婧嫄
柳全文
刘倩玉
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Nanchang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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    • C12N5/0676—Pancreatic cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C12N2506/025—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells from extra-embryonic cells, e.g. trophoblast, placenta

Abstract

The invention discloses a kind of methods for being induced to differentiate into the beta Cell of islet with biological function using people amnioic epithelium stem cell, comprising the following steps: 1) separation, culture, amplification, the identification of amnioic epithelium stem cell；2) Differentiation Induction in vitro: experimental selection the 2nd~3 generation amnioic epithelium stem cell carries out induction differentiation with the culture medium containing niacinamide.Broken up within external 14 days and obtains to express beta Cell of islet Specific marker and the insulin secretory cell with normal islets β cell function；3) transplanting in vivo: the insulin secretory cell that induction generates is transplanted in type 1 diabetes Mice Body, can obviously relieve mouse hyperglycemia state.

Description

A kind of amnioic epithelium stem cell be induced to differentiate into functional islets β cell method and It is applied
Technical field
The present invention relates to a kind of methods using amnioic epithelium stem cell induction differentiated islet β cell.
Background technique
With the raising of living standards of the people and the reasons such as reduction of amount of exercise, in recent years, on diabetes morbidity is continuous It rises.According to statistics in 2013, the number that diabetes are suffered from the whole world alreadyd exceed 3.82 hundred million, and the diabetic in China is up to 1.14 hundred million People, total bit rank first in the world.Among the urban population in our country, adult's diabetes morbidity is close to 12%, and also with every The speed of year 3%-5% increases sharply.Therefore, diabetes are just endangering the life of many patients as a global disease Life.The pathogenic factor of diabetes is sufficiently complex, related with the factors such as environment, heredity, so far its unclear pathogenesis. The reason of diabetes are mainly shown as the metabolic disorder characterized by chronic hyperglycemia, and blood glucose increases has insulin secretion or work Defect, or both exists simultaneously.Diabetes are clinically divided into 1 type, 2 types, other specific types and gestational period glycosuria Disease etc..Type 1 diabetes are a kind of autoimmune diseases, in pancreas islet the β cell of excreting insulin due to autoimmunity attack and It is permanently damaged, leads to hypoinsulinism, type 1 diabetes account for about the 5% of diabetic's sum.Diabetes B is Resistant function is generated to normal level or high-caliber insulin since pancreas islet generates insufficient insulin or bodily tissue, leads to pancreas Island element is opposite to be lacked.In the late stage of diabetes B, beta Cell of islet quantity reduces about 50%.Domestic and foreign scholars are to diabetes Have conducted extensive research work, inquire into its pathogenesis, find effective remedy measures, however so far to the definite cause of disease of diabetes and Pathogenesis there is no satisfied elaboration, in treatment also due to there are many bottlenecks without safe and effective measure.Injection of insulin is at present The main method of clinical treatment diabetes can control blood glucose in a short time.Shortcoming is to need frequently monitoring blood glucose and note Insulin is penetrated, and the generation of the complication such as renal failure caused by diabetes, heart disease, eyeground pathological changes, gangrene cannot be prevented And development.The ideal method for treating diabetes is the quantity for restoring function beta Cell of islet.Transplantation of pancreas in recent years and islet cells Cell transplantation for diabetes has made great progress, and can be effectively improved the secretion of patient's endogenous insulin, improves minimal invasive treatment's shape State, it is often more important that can be reduced and improve the complication of diabetes.The problem of pancreatic islets transplantation maximum is exactly that pancreas islet donor source is tight The problems such as weight is insufficient and can not solve the immunological rejection and side effect that occur after transplanting.Therefore, finding one kind being capable of mass production The method for applying to clinical islet cells is extremely urgent.Traditional concept has been broken in the discovery of stem cell, to cure diabetes etc. Refractory disease brings new hope.So far had via through hemocytoblast, umbilical cord blood mesenchymal stem cells, embryonic stem cell and The report that inducing pluripotent stem cells etc. break up to beta Cell of islet direction.However menses and derived from cord blood is limited, embryonic stem cell There are ethics problem and oncogenicity, inducing pluripotent stem cells, there are oncogenicity, so that these cells are difficult really for clinic.
2005, the researchs such as Toshio confirmed that human amnion membrane meets the standard of stem cell.Express embryonic stem cell Marker such as SSEA-3, SSEA-4, TRA1-60, TRA1-81, SOX2, Oct-4, Nanog etc. express mescenchymal stem cell mark Will object such as CD29, CD73, CD105 etc., while expressing epithelial stem cell marker CK7, E-cadherin etc.；But part mesenchyma It is feminine gender if stem cell markers (CD90) and hemopoietic stem cell surface marker (such as CD34 and CD45).Amnioic epithelium stem cell In vitro can long-term cultivation and keep caryotype stablize, Proliferation, Differentiation potential be apparently higher than bleeding of the umbilicus, marrow and fat etc. come The mescenchymal stem cell in source.Amnioic epithelium stem cell not expression of HLA-DR (human leukecyte antigen-antigen D Related), low expression HLA-ABC (human leukocyte antigen classI), NOD-SCI (nonobese Diabetic severe combined immunodeficient) in Mice Body transplanting do not generate teratoma, illustrate that it is immune Originality is low, without oncogenicity, this provides certain guarantee for the safety of cell transplantation.Amnioic epithelium stem cell can by it is non-enter Invading property means obtain easily, and amplification and directed differentiation result well can be reached using the laboratory condition of standard.
Currently, existing research report amnioic epithelium stem cell has the potential to three differentiation of germinal layers.The researchs such as Gao discovery Amnioic epithelium stem cell in vitro is orientable to be divided into the nerve cell with biological function, these cell transplantations are entered spinal cord damage Damage can be repaired in wound model.The discovery amnioic epithelium stem cell such as TAN can secrete a variety of factors, have powerful immune regulative Can, the polarity and activity of macrophage can be influenced, the LXA4 of secretion plays a significant role in acute lung injury；Mary etc. It was found that amnioic epithelium stem cell can directly adjust the microglia of brain, trophic factors is discharged, repairs cerebral injury；The hair such as Miki Existing amnioic epithelium stem cell can be divided into bile duct epithelial cell, can treat chronic bile obstruction after transplanting in vivo.Amnioic epithelium is dry Cell not expression of HLA-DR, low expression HLA-ABC, illustrate its immunogenicity it is low, this to reduce or solving variant cell transplanting after Generated rejection provides important cell origin.
Amnioic epithelium stem cell is one kind of newly discovered adult stem cell, belongs to epithelial stem cell.In amnion tissue It was found that there are epithelial stem cell abundant, is quickly separated on the amnion that can be discarded after delivery of fetus, be a kind of noninvasive, safety Source of human stem cell mode.In cell marker molecules expression and differentiation capability, amnioic epithelium stem cell and embryonic stem cell are most It is similar.
Summary of the invention
Functional pancreas is induced to differentiate into using amnioic epithelium stem cell the technical problem to be solved in the present invention is to provide a kind of The method of island β cell.
In order to solve the above technical problems, being induced to differentiate into pancreas islet β using amnioic epithelium stem cell the present invention provides a kind of The method of cell, comprising the following steps:
In order to solve the above technical problems, being induced to differentiate into pancreas islet β using amnioic epithelium stem cell the present invention provides a kind of The method of cell, comprising the following steps:
A. the good amnioic epithelium stem cell of the 2nd~3 generation growth conditions is digested with pancreatin, when seeing that cell is under microscope It is cultivated when single round with the DMEM containing 10% fetal calf serum of volumetric concentration (FBS, Thermo Fisher, 10099-141) Base (Thermo Fisher, 10569044) terminates digestion, centrifugation, and gained cell precipitation is cleaned 1 time with PBS；
B. cell precipitation obtained in above-mentioned steps A is inoculated in 10cm culture dish and carries out Fiber differentiation, inoculating cell Density is 1 × 106/ ware, every ware are added 10ml induction culture medium I, are subsequently placed in 5%CO2, 95% humidity, 37 DEG C culture It is cultivated in case；It is inhaled after 2 days and abandons induced medium I, be changed to the induced medium II of 10ml, change liquid within every 2 days, totally 12 days；Daily Cell state is observed under inverted microscope, induction time is 14 days, obtains having the insulin secretion of islet beta cell function thin Born of the same parents；
Induced medium I's the preparation method comprises the following steps: in 34.32ml DMDM (Dulbecco ' s Modified Dulbecco's Medium, Thermo Fisher, 12440053) 4mlFBS (10%) is added in, 0.4ml concentration is 100mM Nonessential amino acid (NEAA, Thermo Fisher, 11140050), the L-Glutamine (L- that 0.4ml concentration is 200mM Glutaminne, Thermo Fisher, 21051024), the 2 mercapto ethanol (2- that 0.04 μ l concentration is 55mM Mercaptoethanol, Sigma, M6250), 0.4ml is dual anti-(receive with 10,000 μ g/ml chain by the parasiticin containing 10,000U/ml Mycin), 400ng hEGF (EGF, peprotech, AF-100-15-100), 0.4ml concentration be 1M Buddhist nun gram acyl Amine (nicotinamide, Sigma, N0636)；
The induced medium II the preparation method comprises the following steps: that 4ml fetal calf serum, 0.4ml are added in 34.36ml DMDM is dense Spend the 2- mercapto that the nonessential amino acid for being 100mM, the L-Glutamine that 0.4ml concentration is 200mM, 0.04 μ l concentration are 55mM The niacinamide that base ethyl alcohol, 0.4ml are dual anti-, 0.4ml concentration is 1M.
People's amnioic epithelium stem cell preparation method are as follows:
A, amnion is collected:
The amnion that will be removed from placenta is put into the PBS containing antibiotic, is handled 1-2 hours, is washed at room temperature with PBS Amnion 3 times, amnion is transferred in the beaker containing fresh PBS every time；
B, originally culture
1) amnion obtained in step A is transferred in 50ml centrifuge tube, pancreatin/EDTA of the fresh preheating of 30ml is added (0.25%, Thermo Fisher, 25200056) is incubated in 37 DEG C of water-baths, every shaking 1 time on 10 minutes oscillators； The DMEM culture medium containing 10%FBS that 2 times of volumes are added after forty minutes terminates digestion；
2) supernatant is removed into the centrifugation of cell suspension obtained in step 1), 7-8ml amnioic epithelium stem cell media is added, connects Kind is placed in 5%CO in 10cm Tissue Culture Dish2, 95% humidity, cultivate in 37 DEG C of incubators；
D, cell secondary culture:
When the cell of step B) originally culture grows to 70%-80% convergence degree, that is, use pancreatin had digestive transfer culture, tool Steps are as follows for body:
1) denier step B) cell of originally culture when growing to 70%-80% convergence degree, removes culture medium,
Then it is washed with the PBS without calcium ions and magnesium ions；
2) 1~2ml pancreatin is added, is placed in 5%CO2, 95% humidity, be incubated for 2~3 minutes in 37 DEG C of incubators；
3) 2~4ml, which is added, inactivates pancreatin containing the DMEM culture medium that volumetric concentration is 10% fetal calf serum；
4) soft piping and druming, makes attached cell fall off into unicellular, and supernatant is removed in centrifugation；
5) 3ml amnioic epithelium stem cell collective media is added, is passed in the ratio of 1:3；
6) the amnioic epithelium stem cell collective media of 7-8ml is added in the cell suspension of every 1ml, it is thin to be inoculated in 10cm In born of the same parents' culture dish, it is placed in 5%CO2, 95% humidity, cultivate in 37 DEG C of incubators.
The antibiotic bactericidal liquid configuration is as follows: Hank ' the s balanced salt solution system of 20ml contains following component And following concentration: vancomycin 60-100 μ g/ml, cefalexin 150-300 μ g/ml, kanamycins 50-150 μ g/ml, celebrating are big Toxin 80-160 μ g/ml, amphotericin B 2-3 μ g/m and 300-500 units of heparin sodium.
The configuration method of the amnioic epithelium stem cell media is as follows: 430ml DMEM- being added in sterile chamber The nonessential ammonia that benzenealkonic acids sodium that high glucose, 5ml concentration are 100mM, 50ml fetal calf serum, 5ml concentration are 100mM Base acid, 5ml concentration be 200mM L-Glutamine, 500 μ l concentration be 55mM 2 mercapto ethanol, 5ml it is dual anti-, contain 10, 000U/ml parasiticin is received and 10,000 μ g/ml streptomysins, 5 μ g hEGFs；It mixes well, 4 DEG C save for use.
It is specific as follows using the culture medium of people amnioic epithelium stem cell induction differentiation function beta Cell of islet:
Induced medium I's the preparation method comprises the following steps: in 34.32ml DMDM be added 4ml FBS (10%), 0.4ml concentration Nonessential amino acid (NEAA, Thermo Fisher, 11140050), the L- paddy ammonia that 0.4 ml concentration is 200mM for 100mM Amide (L-glutaminne, Thermo Fisher, 21051024), the 2 mercapto ethanol (2- that 0.04 μ l concentration is 55mM Mercaptoethanol, Sigma, M6250), 0.4 ml is dual anti-(receive with 10,000 μ g/ml chain by the parasiticin containing 10,000U/ml Mycin), 400ng hEGF (EGF, peprotech, AF-100-15-100), 0.4ml concentration be 1M Buddhist nun gram acyl Amine (nicotinamide, Sigma, N0636)；
Induced medium II the preparation method comprises the following steps: 4ml fetal calf serum, 0.4 ml concentration be added in 34.36ml DMDM being The 2- sulfydryl second that L-Glutamine that the nonessential amino acid of 100mM, 0.4ml concentration are 200mM, 0.04 μ l concentration are 55mM The niacinamide that alcohol, 0.4ml are dual anti-, 0.4ml concentration is 1M.
Beneficial effects of the present invention are that 1, cell origin is abundant, are separately cultured technology and have grasped, and are provided a kind of novel Adult stem cell source；
2, amnioic epithelium stem cell is divided by functional islets β cell by directional induction in vitro, provided a kind of big The method that amount obtains functional islets β cell；
3, by transplanting in vivo, discovery can be improved by the functional islets β cell that the induction of amnioic epithelium stem cell generates Diabetic mice hyperglycemia state improves mouse survival quality, provides a kind of new selection for the treatment of clinical diabetes.
In conclusion the present invention is using the people amnioic epithelium stem cell that is separately cultured, using ex vivo stem cell inductive technology, The versatility and its directed differentiation of studying amnioic epithelium stem cell are the potential of functional islets β cell, and are moved by kidney peplos Plant the beta Cell of islet of differentiation is implanted into type 1 diabetes Mice Body, it was demonstrated that its be applied to treating diabetes feasibility, be The stem-cell therapy of diabetes provides a kind of new approach.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is the result figure of amnioic epithelium stem cell separation and identification:
(A) i is the primary cell colony that culture was formed to the 7th day in the culture medium without EGF, and ii is to contain EGF Culture medium obtained in cell, be in oval, typical epithelioid cell, monolayer growth；
(B) RT-PCR detection shows that amnioic epithelium stem cell expresses embryonic stem cell marker gene Nanog and Oct4；Table Up to epithelial stem cell marker gene E-cadherin and CK7；Mescenchymal stem cell marker gene CD73 and CD105 is expressed, not table Up to mescenchymal stem cell marker gene CD90；
(C) Flow cytometry show amnioic epithelium stem cell expression mescenchymal stem cell marker protein CD29, CD73, CD105 and low expression ajor histocompatibility class Ⅰ antigens HLA-ABC；Mescenchymal stem cell marker protein is not expressed CD90, candidate stem cell mark marker protein CD34, CD45 and ajor histocompatibility class Ⅱ antigens HLA-DR；
(D) RT-PCR detection shows that amnioic epithelium stem cell does not express ajor histocompatibility class Ⅱ antigens HLA-DR, low Expression ajor histocompatibility class Ⅰ antigens HLA-ABC, and different algebra amnioic epithelium stem cell HLA-DR and HLA-ABC Express no significant difference；
(E) Immunofluorescence test show amnioic epithelium stem cell expression embryonic stem cell marker protein Oct-4, Nanog, SSEA-4 and epithelial stem cell marker protein E-cadherin.
Fig. 2 is that amnioic epithelium stem cell (hAESCs) is external and the testing result figure of internal oncogenicity；
(A) hAESCs cannot be proliferated on soft agar, grow, not formed colony, and control group hepatocarcinoma cell line HEPG2 energy nothing Limit breeding, forms macroscopic larger colony group.Illustrate hAESCs in vitro without oncogenicity；
(B) hAESCs transplanting enters in NOD-SCID Mice Body, until having no within 6th month that tumour occurs.And embryonal vaccination is dry The control group of cell (Embryonic stem cells, ESCs) occurred as soon as tumour at 2nd month.The result shows that amnioic epithelium is dry Without oncogenicity in cell body.
Fig. 3 is that amnioic epithelium stem cell (hAESCs) induction is functional islets β cell differentiation testing result figure；
(A) amnioic epithelium stem cell cellular morphology under induced medium effect gradually changes, by original oval It is that cellular morphology (D14) that is round and mutually assembling is thin similar to normal islets β that shape epithelioid cell form (D0), which gradually develops, The form of born of the same parents；
(B) RT-PCR detects amnioic epithelium stem cell 3 days, 7 days and 14 days entoderm specific genes of differentiation and pancreas islet β is thin The expression of born of the same parents' gene.The result shows that amnioic epithelium stem cell induction differentiation 3 days after express entoderm marker gene FOXA2, Islet progenitor cells marker gene PDX1, NKX6.1 is expressed in SOX17, induction differentiation after 7 days, expression beta Cell of islet is special after differentiation 14 Anisotropic marker gene insulin；
Undifferentiated HAESCs does negative control within D0 days；
(C) Immunofluorescence test expresses beta Cell of islet marker protein after showing the induction of amnioic epithelium stem cell differentiation 14 days insulin、c-peptide、PDX1、NKX6.1。
(D) Immunofluorescence test expresses beta Cell of islet marker protein after showing the induction of amnioic epithelium stem cell differentiation 14 days Insulin and c-peptide.
Fig. 4 is the inside and outside Function detection figure for the insulin secretory cell that the induction of amnioic epithelium stem cell generates；
(A) ELISA detects insulin content in different differentiation number of days culture mediums, the results showed that induction differentiation 7 days and 14 days Culture medium in can be detected actrapid monotard.
(B) sugared stimulation test is carried out to the cell after induction differentiation 14 days, ELISA is the result shows that hAESCs induction generates β cell can response glucose stimulate excreting insulin.
(C) kidney peplos of the insulin secretory cell of hAESCs induction differentiation transplant picture.
(D) mouse blood sugar variation diagram after kidney peplos transplanting.It is obviously obtained the result shows that mouse blood sugar is horizontal after transplanting in vivo Improve.
Specific embodiment
Embodiment 1
Separation, culture, the amplification of amnioic epithelium stem cell (HAESCs):
Under the premise of obtaining relatives of newborn agreement, isolated fresh amnion.After antibiotic treatment 2h, carry out micro- Biological detection and infectious disease pathogens safety detection.Amnion adds after digesting 1h in (0.25%) 37 DEG C of water-bath of pancreatin/EDTA Enter terminate liquid.It is centrifuged, removes supernatant, be placed in 5%CO2,95% humidity, 37 DEG C of incubators with amnioic epithelium stem cell media Middle culture.Liquid is changed after 2-3 days and removes non-attached cell, then changes liquid within every 2 days.When cell confluency degree reaches 80%, using pancreas Enzyme/EDTA had digestive transfer culture.Using immunofluorescence and Flow Cytometry to amnioic epithelium stem cell carry out OCT4, Nanog, The molecular phenotypes such as SSEA-4, E-cadherin, CD29, CD73, CD90, CD105, HLA-ABC, HLA-DR, CD34, CD45 mirror It is fixed.
Concrete operations regulation is as follows:
One, the separation, culture and amplification of amnioic epithelium stem cell:
The pregnant woman for recruiting different age group, contributes amnion under the premise of completely voluntary.
Concrete operations regulation is as follows:
1, amnion sample collection (cell separation)
500ml indigo plant lid bottle is sent to delivery room.Blue lid bottle is pre-loaded with 200mlHBSS (Hank ' s balanced salt solution, Hanks' Balanced Salt Solution) collection liquid, wherein contain vancomycin 60-100 μ g/ml, cefalexin 150-300 μ G/ml, kanamycins 50-150 μ g/ml, gentamycin 80-160 μ g/ml, amphotericin B 2-3 μ g/m and 300-500 unit liver Plain sodium；In this, as antibiotic bactericidal liquid
Above-mentioned HBSS (Hank ' s balanced salt solution) collection liquid the preparation method is as follows:
Following component is added in Hank ' the s balanced salt solution of 200ml to following concentration: vancomycin 60-100 μ g/ Ml, cefalexin 150-300 μ g/ml, kanamycins 50-150 μ g/ml, gentamycin 80-160 μ g/ml, amphotericin B 2-3 μ G/m and 300-500 units of heparin sodium；Then it sterilizes under high temperature according still further to conventional program.
After pregnant woman childbirth, placenta is put into above-mentioned collecting bottle.In the case where obtaining amnion contributor's informed consent It cultivates the cells.Sample must be saved before being sent to laboratory under the conditions of low temperature (0-4 DEG C), and (storage life is general For 24-48h).
1) amnion is removed from placenta, enters in the above-mentioned beaker equipped with antibiotic bactericidal liquid, handles 1-2 at room temperature Hour；It is washed amnion 3 times with PBS, amnion is transferred in the beaker containing fresh PBS every time；
2) above-mentioned amnion is transferred in the 50ml centrifuge tube containing 30ml preheating pancreatin/EDTA (0.25%), 37 DEG C of water It is incubated in bath, every being shaken 1 time on 10 minutes oscillators；The 10% tire ox containing volumetric concentration of 2 times of volumes is added after forty minutes The DMEM culture medium of serum terminates digestion；
3) Centrifuge A sample 5 minutes under the revolving speed of 1000rpm remove supernatant.
Above-mentioned supernatant can carry out microorganism detection and infectious disease pathogens safety detection in a conventional manner, generally to normal See virus such as AIDS virus (HIV, human immunodeficiency virus), hepatitis B (HBV, hepatitis Bvirus), hepatitis C virus (HCV, hepatitis Cvirus) and syphilis pathogen (Syphilis pathogen) are examined It surveys.If positive findings, then terminate being separately cultured and expanding for entire amnioic epithelium stem cell.Testing goal is to guarantee The use peace of the amnioic epithelium stem cell (not doing the amnioic epithelium stem cell for transplantation treatment of induction processing referred to) obtained Quan Xing.
2, originally culture
Culture medium configuration-amnioic epithelium stem cell collective media ingredient is as follows:
1) 430ml DMEM-high glucose culture medium；
2) 50ml fetal calf serum (10%v/v)；
3) 5ml concentration is the benzenealkonic acids sodium (1%v/v) of 100mM；
4) 5ml concentration is the nonessential amino acid (1%v/v) of 100mM；
5) 5ml concentration is the L-Glutamine (1%v/v) of 200mM；
6) 500 μ l concentration are the 2 mercapto ethanol (0.1%v/v) of 55mM；
7) 5ml dual anti-(parasiticin containing 10,000U/ml is received with 10,000 μ g/ml streptomysin), (1%v/v)；
8) 5 μ g hEGF (final concentration of 10ng/ml).
The configuration method of amnioic epithelium stem cell collective media is as follows: 430ml DMEM- being added in sterile chamber The nonessential ammonia that benzenealkonic acids sodium that high glucose, 5ml concentration are 100mM, 50ml fetal calf serum, 5ml concentration are 100mM Base acid, 5ml concentration be 200mM L-Glutamine, 500 μ l concentration be 55mM 2 mercapto ethanol, 5ml it is dual anti-(contain 10, 000U/ml parasiticin is received and 10,000 μ g/ml streptomysins), 5 μ g hEGFs；Mix well, 4 DEG C save to With.
1) 7ml amnioic epithelium stem cell collective media is taken, the amnioic epithelium stem-like cell for having been removed supernatant is added to In product (i.e. the gains of step 1), soft piping and druming is mixed, and obtains cell suspension,
2) a 10cm ware is taken, the resulting whole cell suspensions of step 1) are transferred in the culture dish；
3) it is put into 5%CO2, 95% humidity, cultivate in 37 DEG C of incubators；
4) after cultivating 2-3 days, full dose changes liquid (changing 7ml amnioic epithelium stem cell collective media)；
5) 5ml epithelial stem cell collective media is taken, slowly instills, softly rocks, rinse cellular layer in culture dish side wall, Culture medium is removed with pipette；
6) 7ml amnioic epithelium stem cell collective media is taken, is slowly instilled in culture dish side wall；
7) under the microscope, there is more attached cell.It is put into 5%CO2, 95% humidity, continue to cultivate in 37 DEG C of incubators. During the cultivation process, it changes the liquid once within every 3 days, continues to cultivate.When cell confluency degree reaches 70-80%, passed on.
3, cell secondary culture
1) culture solution in above-mentioned originally culture step 7) gains is removed；
2) it is washed with the PBS cleaning solution of no calcium and magnesium；
The PBS cleaning solution of above-mentioned no calcium ions and magnesium ions is formulated as follows:
It is settled to 1L and is put into 4 DEG C of refrigerators preservations after high pressure steam sterilization (conventional program) for use.
3) 1ml pancreatin, 37 DEG C of incubation (5%CO are added2, 95% humidity) 5 minutes；
4) 2ml amnioic epithelium stem cell collective media is added inactivates pancreatin；
5) soft piping and druming, makes attached cell fall off and forms unicellular；
6) it passes in the ratio of 1:3: from the 10cm culture dish for taking 1ml to one new in the cell suspension of 3ml, adding The amnioic epithelium stem cell collective media of 6ml, shakes up；
7) it is put into 5%CO2, 95% humidity, cultivate in 37 DEG C of incubators；
8) every 3-4 days passage cells are primary (i.e. from step 1) to step 7)).
4, amnioic epithelium stem cell surface molecular marker is identified:
One, RT-PCR detects cell sign gene
1) cell is collected when cell reached for 3 generation, it is outstanding that cell is made in pancreatin/EDTA digestive juice (0.25%) vitellophag Liquid, adjustment cell density to 1 × 106A/ml；
2) RNA is extracted: taking 1ml cell suspension, 1000rpm is centrifuged 5 minutes, abandons supernatant.1ml TRIzol is added, until cell It is transferred to after being completely dissolved in 1.5mlEP pipe；200 μ L chloroforms are added, shakes vigorously and mix well, is placed at room temperature for 5min；12 at 4 DEG C, 000rpm is centrifuged 15min, draws the upper water 300 μ L that make an appointment and enters in new 1.5mlEP pipe；Isometric isopropanol is added, overturns It mixes, is stored at room temperature 10min；12,000rpm is centrifuged 10min at 4 DEG C, abandons supernatant；It is precipitated with 75% ethanol washing, at 4 DEG C 12,000rpm centrifugation 5min, abandon supernatant；After drying at room temperature 2-5min, appropriate DEPC water dissolution is added；
3) the first chain of cDNA synthesizes: total serum IgE sample is handled with dnase digestion and removes remaining contaminating genomic DNA, is used Ultraviolet specrophotometer measures (wavelength 260nm) RNA concentration.Each sample takes 2 μ g total serum IgEs (0.5-1 μ L) for the first chain Synthesis.Specific step is as follows: 2 μ g total serum IgEs (0.5-1 μ L), 1 μ Loligo (dT), 18 primer and 1 μ L random primer, ddH2O is mended To 12 μ L of total volume, 65 DEG C of reaction 5min are set；5 × buffer, 4 μ L, 2 dNTP μ L, ribolockTM RNase is added 1 μ L and RevertAidTM M-MulV reverse transcriptase of inhibitor, 1 μ L, reaction total volume are 20 μ L, 25 DEG C of 5min → 42 DEG C 60min→70℃5min.The first chain of cDNA of synthesis is reacted for next step PCR.
4) PCR reacts: the cDNA of above-mentioned preparation is expanded with PCR respectively.Primer needed for PCR see the table below:
1 amnioic epithelium stem-cell marker gene RT-PCR primer sequence of table and fragment amplification length
PCR amplification system are as follows:
2 μ L of cDNA, 1 μ L of 2mM dNTP, 10 μM of upstream and downstream primer (GAPDH, Nanog, OCT4, E-cadherin, CK7, CD105, CD73, CD90, HLA-ABC and HLA-DR etc.) each 1 μ L, 10 × buffer (contain Mg2+) 5 μ L and TaqDNA polymerization 1 μ L of enzyme, adds H2O is mended to 50 μ L of total volume.PCR program are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 53 DEG C~62 DEG C are annealed 30s, 72 DEG C of extension 30s, 35 circulations, 72 DEG C of extension 7min, annealing temperature and recurring number are selected according to the concrete condition of primer It selects.It takes 5 μ L amplified productions to add 1 μ L bromjophenol blue indicator after the reaction was completed, carries out gel electrophoresis with 1.2% agarose.Electrophoresis After, with Labworks image acquisition and analysis software observed result, take pictures.
Acquired results are as follows: Nanog, OCT4, E-cadherin, CK7, CD105, CD73, HLA-ABC be the positive, CD90 and HLA-DR is feminine gender.
Two, Flow cytometry cell surface antigen
1) cell is collected when cell reached for 3 generation, it is outstanding that cell is made in pancreatin/EDTA digestive juice (0.25%) vitellophag Liquid, adjustment cell density to 1 × 106A/ml；
2) required amount of 1.5ml EP is taken to manage respectively, be separately added into mouse monoclonal anti-human's antibody (CD29, CD90, CD73, CD105, CD34, CD45, HLA-DR, HLA-ABC and Isotype control) 20 μ l；
3) it is separately added into pipe obtained by cell sample (above-mentioned steps 1)) 50 μ l, it is protected from light 4 DEG C of incubation 30min；
4) 1000rpm is centrifuged 5 minutes；
5) supernatant is abandoned, the PBS1ml for containing 1% (mass concentration) human serum is added, mixes well, 1000rpm is centrifuged 5 points Clock；
6) supernatant is abandoned, PBS adjustment sample size is added to 100 μ l, flow cytometer is analyzed.
Acquired results are as follows: CD29, CD105, CD73, HLA-ABC are the positive, and CD90, CD34, CD45 and HLA-DR are yin Property.
Three, Immunofluorescence test cell sign albumen
1) cell is collected when cell reached for 3 generation, it is outstanding that cell is made in pancreatin/EDTA digestive juice (0.25%) vitellophag Liquid, adjustment cell density to 1 × 106A/ml；
2) burnt slide will be copolymerized to be put into 12 orifice plates, cell sample is seeded on slide, it is adherent carry out being immunized for 24 hours afterwards it is glimmering Light detection；
3) fixed cell 15 minutes of 4% paraformaldehyde, pre-cooling PBS are cleaned twice of cell；
4) PBS containing 0.25%Triton-X100 is handled 10 minutes and is carried out rupture of membranes processing to cell, and PBS cleans 3 times, often All over 5 minutes；
5) 30 minutes are incubated for close non-specific egg through the PBST (PBS containing 0.1%Tween-20) containing 1%BSA again White binding site；
6) primary antibody (OCT-4, SSEA-4, Nanog and E-cadherin) is diluted with the PBST containing 1%BSA, in 4 DEG C of wet box Incubated cell is stayed overnight.
7) PBS is cleaned 3 times, every all over 5 minutes；Secondary antibody is diluted with the PBS containing 1%BSA, at room temperature incubated cell 1 hour；
8) secondary antibody is sucked out, addition DAPI is redyed 1 minute after cell is cleaned multiple times in PBS, is observed under laser confocal microscope.
Acquired results are as follows: OCT-4, SSEA-4, Nanog and E-cadherin are the positive.
The general phenotypic criteria that above-mentioned detection determines that gained cell meets amnioic epithelium stem cell (is amnion obtained by determining Epithelial stem cell).
Embodiment 2
Oncogenicity detects inside and outside amnioic epithelium stem cell body
1, oncogenicity detects inside and outside amnioic epithelium stem cell body
1) the good amniotic epithelial cells of upgrowth situation for taking the culture third generation, show as growing that vigorous, cell space is big, karyon Clearly, endochylema is abundant, the strong cell of refractivity under microscope, micro- with trypsase-EDTA digestive juice (0.25%) digestion See under mirror and digestion is terminated with the DMEM culture medium containing 10% (volumetric concentration) fetal calf serum when cell becomes single round, softly It is centrifuged after piping and druming, obtains cell precipitation, PBS (the PBS cleaning solutions of no calcium ions and magnesium ions) washes 2 times, and (purpose is to wash away tryptose The substances such as enzyme, fetal calf serum)；
2) amnioic epithelium stem cell in step 1) is seeded on soft agar, after culture 30 days, observes Colony forming feelings Condition；
3) use PBS dilution step 1) in amnioic epithelium stem cell, adjustment density is to 2.5 × 107A/ml.Take 200 μ l (cell number is 5 × 106It is a), respectively NOD-SCID back of mice is subcutaneous and leg muscle injection；Mouse is put into SPF grades and moves Object room continues to raise, and mouse tumor formational situation is detected after 2 months；
According to this as a result, we learn that amnioic epithelium stem cell in vitro cannot be proliferated on soft agar, grow, not formed collection Fall (arrow meaning is single suspension cell)；Transplanting does not generate teratoma in vivo.Show amnion stem cell safety, without oncogenicity.
2, control group is set
Following change is done relative to " oncogenicity detects inside and outside amnioic epithelium stem cell body " above, remaining is the same as " amnion Step 1)~step 3) of epithelial stem cell inside and outside oncogenicity detection "
Amnioic epithelium stem cell in step 2) is changed to hepatocarcinoma cell line HEPG2, the sun as the detection of external oncogenicity Property control group；
Amnioic epithelium stem cell in step 3) is changed to embryonic stem cell, the positive as the detection of internal oncogenicity is right According to group；
It as a result is that the external energy Immortalization of hepatocarcinoma cell line HEPG2 forms macroscopic larger colony group；Embryo is dry thin Born of the same parents can produce tumour in NOD-SCID Mice Body.
Embodiment 3
Amnioic epithelium stem cell is induced to differentiate into insulin secretory cell and its identification
Concrete operations regulation is as follows:
1, directional induction in vitro amnioic epithelium stem cell is divided into insulin secretory cell
1) the good cell of upgrowth situation for taking the culture third generation, is disappeared with trypsase-EDTA digestive juice (0.25%) Change, sees under microscope and disappeared when cell becomes single round with the DMEM culture medium termination containing 10% (volumetric concentration) fetal calf serum Change, is centrifuged after soft piping and druming, obtains cell precipitation, PBS (the PBS cleaning solutions of no calcium ions and magnesium ions) washes 2 times, and (purpose is to wash away The substances such as trypsase, fetal calf serum)；
2) cell precipitation obtained in above-mentioned steps is inoculated in 10cm culture dish and carries out Fiber differentiation, inoculating cell is close Degree is 1 × 106/ ware, every ware are added 10ml induction culture medium I, are subsequently placed in 5%CO2, 95% humidity, 37 DEG C of incubators Middle culture；It is inhaled after 2 days and abandons induced medium I, be changed to the induced medium II of 10ml, change liquid within every 2 days, totally 12 days.Then press Step 3) executes operation.
Induced medium I's the preparation method comprises the following steps: 4mlFBS (10%), 0.4ml concentration are added in 34.32mlDMDM being The 2- sulfydryl second that L-Glutamine that the nonessential amino acid of 100mM, 0.4ml concentration are 200mM, 0.04 μ l concentration are 55mM Alcohol, 0.4ml be dual anti-, 400ng hEGF (EGF), the niacinamide that 0.4ml concentration is 1M.
The induced medium II the preparation method comprises the following steps: that 4ml fetal calf serum, 0.4ml are added in 34.36mlDMDM is dense Spend the 2- mercapto that the nonessential amino acid for being 100mM, the L-Glutamine that 0.4ml concentration is 200mM, 0.04 μ l concentration are 55mM The niacinamide that base ethyl alcohol, 0.4ml are dual anti-, 0.4ml concentration is 1M.
3) cell state is observed under inverted microscope daily, induction time is 14 days, is obtained with islet beta cell function Insulin secretory cell.
2, control group is set:
Following change is done relative to " directional induction in vitro amnioic epithelium stem cell is divided into insulin secretory cell ", remaining With " directional induction in vitro amnioic epithelium stem cell is divided into insulin secretory cell " step 1)~step 3).
The Buddhist nun gram acyl that 400ng hEGF (EGF), 0.4ml concentration in cancellation " induced medium I " are 1M Amine；The niacinamide that 0.4ml concentration in " induced medium II " is 1M, the control group as induction system.
As a result are as follows: the cellular morphology of control group does not change.
3, the insulin secretory cell identification of vitro differentiation:
1) morphological observation of amnioic epithelium stem cell
Cell regular its form, cytoplasmic granule and fine structure under the microscope after induction.
As a result as shown in Fig. 3 (A), according to the result, we can be concluded that amnioic epithelium stem cell is trained in induction It supports base and acts on lower cellular morphology and gradually change, gradually develop that the nucleocytoplasmic ratio for polygon is biggish to gather by original oval Collect the cellular morphology of shape, until cellular morphology that is round and mutually assembling is presented within the 14th day, with normal human islets' β cellular morphology phase Seemingly.
2) RT-PCR detects the expression of beta Cell of islet specific proteins mRNA
The 3rd day, the 7th day and the 14th after taking the 2nd generation amnioic epithelium stem cell (hAESCs) (D0) without induction and inducing It cell counts about 1 × 106A, Trizol method extracts total serum IgE.Reverse transcription simultaneously carries out amplification in vitro.Primer needed for PCR is shown in Following table:
2 beta Cell of islet specificity marker gene RT-PCR primer sequence of table and fragment amplification length
Amplification system are as follows:
Amplification condition are as follows: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 53 DEG C~62 DEG C annealing 30s, 72 DEG C of extension 30s, 35 circulations, 72 DEG C of extension 7min, annealing temperature and recurring number are selected according to the concrete condition of primer.5 μ are taken after the reaction was completed L amplified production adds 1 μ L bromjophenol blue indicator, carries out gel electrophoresis with 1.2% agarose.After electrophoresis, gel imaging is used Analysis system observed result, takes pictures.
As a result as shown in Fig. 3 (B), according to the result, we can be concluded that RT-PCR detection at beta Cell of islet point Change the result shows that entoderm marker gene FOXA2, SOX17, induction differentiation 7 are expressed in the induction of amnioic epithelium stem cell after differentiation 3 days Islet progenitor cells marker gene PDX1, NKX6.1 is expressed after it, expresses beta Cell of islet specificity marker gene after differentiation 14 insulin；Undifferentiated amnioic epithelium stem cell cannot then express the gene of most of beta Cell of islet.
3) expression of Immunofluorescence test beta Cell of islet marker protein PDX1, NKX6.1, insulin, c-peptide
A) amnioic epithelium stem cell is seeded on slide, after Differentiation Induction in vitro 14 days；Burnt slide will be copolymerized and be put into 12 holes Immunofluorescence test is carried out in plate；
B) fixed cell 15 minutes of 4% paraformaldehyde, pre-cooling PBS are cleaned twice of cell；
C) PBS containing 0.25%Triton-X100 is handled 10 minutes and is carried out rupture of membranes processing to cell, and PBS cleans 3 times, often All over 5 minutes；
D 30 minutes) are incubated for through the PBST (PBS containing 0.1%Tween-20) containing 1%BSA to close nonspecific proteins Binding site；
E primary antibody) is diluted with the PBST containing 1%BSA, incubated cell is stayed overnight in 4 DEG C of wet box.
F) PBS is cleaned 3 times, every all over 5 minutes；Secondary antibody is diluted with the PBS containing 1%BSA, at room temperature incubated cell 1 hour；
F secondary antibody) is sucked out, addition DAPI is redyed 1 minute after cell is cleaned multiple times in PBS, is observed under laser confocal microscope.
Acquired results are as shown in Fig. 3 (C): after the stem cell external evoked differentiation of amnioic epithelium 14 days, insulin, c- Peptide is the positive.
Above-mentioned detection is divided into insulin secretory cell after determining amnioic epithelium stem cell external evoked 14 days.
Embodiment 4
The insulin secretory cell inside and outside Function detection of amnioic epithelium stem cell induction differentiation
1, the external Function detection of insulin secretory cell of amnioic epithelium stem cell induction differentiation
One, insulin content in detection differentiation culture supernatant
1) amnioic epithelium stem cell induction the 0th day, the 3rd day, the 7th day and the 14th day cells and supernatant of differentiation is taken respectively, 1000rpm is centrifuged 5min, takes supernatant that can be detected.
2) ELISA detects insulin content: operating process is according to ELISA detection kit (Enzyme Linked Immunosorbent Assay Kit For Insulin, Cloud-Clone Corp, CEA448Hu) specification progress.As a result As Fig. 4 (A) shows that actrapid monotard can be detected in the culture medium of induction differentiation 7 days and 14 days.
Two, glucose stimulation test
In order to further detect insulin releasing of the insulin secretory cell of induction under glucose stimulation, we distinguish Induced insulin emission levels are detected in the case where concentration of glucose is 5.5mM and 25mM condition of culture.Cell Krebs-Ringer Buffer prewashing 2 times, it is then incubated for 2h in the Krebs-Ringer buffer of the glucose containing 5.5mM, collects supernatant；Buffer It washes 2 times, the buffer for changing the glucose containing 25mM into is incubated for 2h, collects supernatant, detects insulin using ELISA detection kit Content.
As a result as Fig. 4 (B) shows that the β cell that hAESCs induction generates can response glucose stimulation excreting insulin.
Three, control group is set:
Following change is done relative to " glucose stimulation test ", remaining is the same as " glucose stimulation test " step.By differentiation Insulin secretory cell changes undifferentiated amnioic epithelium stem cell into.
The result shows that undifferentiated amnioic epithelium stem cell is not responding to sugared stimulation, also not excreting insulin.
2, transplanting and its treatment type 1 diabetes potential in the insulin secretory cell body that the induction of amnioic epithelium stem cell generates Detection
One, the building of type 1 diabetes mouse model
Choose 8 week old C57BL/6J mouse (purchased from Hunan SJA Laboratory Animal Co. , Ltd) 24, random point For Normal group and type 1 diabetes group, euglycemia, weight are measured；Normal group intraperitoneal injection citrate buffer solution, glycosuria Disease group intraperitoneal injection STZ (streptozocin, Sigma, S0130), 50mg/kg/ days, and same time injection in continuous 5 days, Mouse gives chow diet, sterile water is fed；And with blood glucose meter, (easy type is surely selected by Johnson & Johnson in having beaten the 3rd, 7,10,14,21,28 day Blood glucose meter) detection mouse blood sugar, positive group > 16.9mmol/l, i.e. model construction success.
The preparation of STZ:
0.1M citrate buffer (PH4.5): (0.21014g citric acid is dissolved in 10ml distillation to 0.1M citron acid solution 4.5ml Dissolved in water)+0.1M sodium citrate liquid 5.5ml (0.29412g sodium citrate is dissolved in 10ml distilled water)；
Sterile STZ solution: STZ concentration is 7.5mg/ml, takes 37.5mgSTZ to be dissolved in 5ml citrate buffer, in super-clean bench The filtering of 0.22 filter, packing are stored in -20 DEG C.(attention is protected from light)
Following group is set:
A, citrate buffer control group (n=8): intraperitoneal injection citrate buffer solution
B, type 1 diabetes group (n=8): continuous 5 days intraperitoneal injection STZ solution of mouse
C, type 1 diabetes+insulin secretory cell transplantation group (n=8): after continuous 5 days intraperitoneal injection STZ solution of mouse, Blood glucose is continued to monitor, 2 times/week, after mouse blood sugar is stablized (continuous 2 blood glucose > 16.9mmol/l), transplants sheep by kidney peplos The insulin secretory cell that the induction of film epithelial stem cell generates enters in type 1 diabetes Mice Body, transplanting amount are as follows: 2-3 × 106Carefully Born of the same parents/only.
Two, transplanting in cell body: after amnioic epithelium stem cell induces 14 days in conditioned medium, inhaling and abandon culture medium, PBS is washed 3 times；Trypsase-EDTA digestive juice (0.25%) digests, and is seen when cell becomes single round under microscope i.e. to contain The DMEM culture medium of 10% (volumetric concentration) fetal calf serum terminates digestion；1000rpm is centrifuged 5 minutes, removes supernatant；It is clear that PBS is added It washes 1 time, and counts.It is centrifuged again, removes supernatant, kidney peplos transplanting is carried out with the cell precipitation that this is obtained, is transplanted such as Fig. 4 (C) institute Show.
Three, mouse blood sugar monitors: observing mouse survival situation after cell transplantation for three days on end, and continues mousetail weekly Blood is taken, mouse blood sugar is detected.As a result as shown in Fig. 4 (D), according to the result, we can be concluded that mouse transplants 2 weeks left sides Hyperglycemia situation is obviously improved when right, and shows persistently hypoglycemic state.Show that insulin secretory cell can be in body It is interior to play hypoglycemic effect.
There are early period three reports to show that amnioic epithelium stem cell in vitro can induce and is divided into insulin secretory cell. Bernard Okere etc. utilizes 3D cultivating system, and process is divided into three phases and is induced.First amnioic epithelium stem cell is existed It is cultivated in SFM 5 days and forms the bead of certain size, then it is broken up 4 in the SFM containing 100ng/mL Activin A in advance It；Then the nicotinamide that 10mM is added in the medium continues induction 4 days；Finally culture medium is changed into containing 10mM's The SFM culture medium of nicotinamide and 2uM Retinoic Acid continues induction 10 days, finally obtains expression insulin With the insulin secretory cell of c-peptide.Amnioic epithelium stem cell is being contained DMEM, 10mM by Lin Peng et al. It induces 14 days, obtains in the culture medium of nicotinamide and 10ng/mL betacellulin and N-2 Supplement Secretion insulin and the cell mass for responding sugar stimulation.Dilli Ram Bhandari et al. first uses 1mM sodium Butyrate and 50 ng/mL Activin A processing cell for 24 hours, is then used and contains 0.1-0.2%bovine serum albumin It is induced 2 days with the culture medium of 50 ng/mL Activin A；Finally with the culture medium of KGF/FGF7 containing 50ng/mL and 1%BSA Further differentiation.Finally obtain the cell of expression insulin and c-peptide.But early period, this three researchs did not all detect Whether the cell of differentiation has hypoglycemic effect in vivo.Differentiation is divided by we on the basis of comprehensive forefathers' induction scheme In two stages, 2 are induced in the culture medium containing DMEM, 10%FBS, 10ng/mlEGF, 10mM nicotinamide first It, then continues induction 12 days in the culture medium of the nicotinamide containing 10mM, finally obtains expression insulin and can feel By the insulin secretory cell of sugar stimulation.And we demonstrate for the first time is moved by the insulin secretory cell that this induction scheme obtains Mouse hyperglycemia can be reduced in implantation diabetic mice body, alleviates diabetes mice symptom.Compared with the induction scheme of forefathers, Our induction scheme significantly reduces induction time and demonstrates the pancreas that the induction of amnioic epithelium stem cell generates in vivo for the first time Island element secretory cell has function.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair Bright to be not limited to above embodiments, acceptable there are many accommodation, amnioic epithelium stem cell that is such as induction and not inducing can be used for The treatment of diabetes including diabetes B.Those skilled in the art can be direct from present disclosure All accommodations for exporting or associating, are considered as protection scope of the present invention.

Claims (6)

1. utilizing the method for people's amnioic epithelium stem cell induction differentiation function beta Cell of islet, which is characterized in that including following step It is rapid:
A. the good amnioic epithelium stem cell of the 2nd~3 generation growth conditions is digested with pancreatin, when seeing that cell is single under microscope Digestion is terminated with the DMEM culture medium of 10% fetal calf serum containing volumetric concentration when round, is centrifuged, gained cell precipitation is clear with PBS It washes 1 time；
B. cell precipitation obtained in above-mentioned steps A is inoculated in 10cm culture dish and carries out Fiber differentiation, inoculating cell density It is 1 × 106/ ware, every ware are added 10ml induction culture medium I, are subsequently placed in 5%CO2, 95% humidity, train in 37 DEG C of incubators It supports；It is inhaled after 2 days and abandons induced medium I, be changed to the induced medium II of 10ml, change liquid within every 2 days, totally 12 days；It is being inverted daily Microscopically observation cell state, induction time are 14 days, obtain the insulin secretory cell with islet beta cell function；
Induced medium I's the preparation method comprises the following steps: 4ml FBS (10%), 0.4ml concentration are added in 34.32ml DMDM being The 2- sulfydryl second that L-Glutamine that the nonessential amino acid of 100mM, 0.4ml concentration are 200mM, 0.04 μ l concentration are 55mM Alcohol, 0.4ml are dual anti-, described dual anti-to receive containing 10,000 U/ml parasiticins with 10,000 μ g/ml streptomysin, 400ng people's epidermis Growth factor, the niacinamide that 0.4ml concentration is 1M；
The induced medium II the preparation method comprises the following steps: 4ml fetal calf serum, 0.4ml concentration be added in 34.36ml DMDM being The 2- sulfydryl second that L-Glutamine that the nonessential amino acid of 100mM, 0.4ml concentration are 200mM, 0.04 μ l concentration are 55mM The niacinamide that alcohol, 0.4ml are dual anti-, 0.4ml concentration is 1M.
2. the method according to claim 1 using amnioic epithelium stem cell induction differentiated islet β cell, it is characterized in that institute State people's amnioic epithelium stem cell preparation method are as follows:
A, amnion is collected:
The amnion that will be removed from placenta is put into the PBS containing antibiotic, is handled 1-2 hours at room temperature, is washed amnion 3 with PBS It is secondary, amnion is transferred in the beaker containing fresh PBS every time；
B, originally culture
1) amnion obtained in step A is transferred in 50ml centrifuge tube, pancreatin/EDTA of the addition fresh preheating of 30ml, 37 DEG C It is incubated in water-bath, every being shaken 1 time on 10 minutes oscillators；The DMEM containing 10%FBS of 2 times of volumes is added after forty minutes Culture medium terminates digestion；
2) supernatant is removed into the centrifugation of cell suspension obtained in step 1), 7-8ml amnioic epithelium stem cell media is added, is inoculated in In 10cm Tissue Culture Dish, it is placed in 5%CO2, 95% humidity, cultivate in 37 DEG C of incubators；
C, cell secondary culture:
When the cell of step B) originally culture grows to 70%-80% convergence degree, that is, pancreatin had digestive transfer culture is used, it is specific to walk It is rapid as follows:
1) denier step B) cell of originally culture when growing to 70%-80% convergence degree, removes culture medium, then with not calcium-magnesium-containing The PBS of ion is washed；
2) 1~2ml pancreatin is added, is placed in 5%CO2, 95% humidity, be incubated for 2~3 minutes in 37 DEG C of incubators；
3) 2~4ml, which is added, inactivates pancreatin containing the DMEM culture medium that volumetric concentration is 10% fetal calf serum；
4) soft piping and druming, makes attached cell fall off into unicellular, and supernatant is removed in centrifugation；
5) 3ml amnioic epithelium stem cell collective media is added, is passed in the ratio of 1:3；
6) the amnioic epithelium stem cell collective media of 7-8ml is added in the cell suspension of every 1ml, is inoculated in the training of 10cm cell It supports in ware, is placed in 5%CO2, 95% humidity, cultivate in 37 DEG C of incubators.
3. according to the method described in claim 2, it is characterized by:
The antibiotic bactericidal liquid configuration is as follows: Hank ' the s balanced salt solution system of 20ml, containing following component and with Lower concentration: vancomycin 60-100 μ g/ml, cefalexin 150-300 μ g/ml, kanamycins 50-150 μ g/ml, gentamycin 80-160 μ g/ml, amphotericin B 2-3 μ g/m and 300-500 units of heparin sodium.
4. according to the method described in claim 2, it is characterized by:
The configuration method of the amnioic epithelium stem cell media is as follows: 430ml DMEM-high being added in sterile chamber Nonessential amino acid that benzenealkonic acids sodium that glucose, 5ml concentration are 100mM, 50ml fetal calf serum, 5ml concentration are 100mM, 2 mercapto ethanol that L-Glutamine that 5ml concentration is 200mM, 500 μ l concentration are 55mM, 5ml are dual anti-, contain 10,000 U/ml Parasiticin is received and 10,000 μ g/ml streptomysins, 5 μ g hEGFs；It mixes well, 4 DEG C save for use.
5. the culture medium according to claim 1 using people amnioic epithelium stem cell induction differentiation function beta Cell of islet, It is characterized in that, specific as follows:
Induced medium I's the preparation method comprises the following steps: 4ml FBS (10%), 0.4ml concentration are added in 34.32ml DMDM being The 2- sulfydryl second that L-Glutamine that the nonessential amino acid of 100mM, 0.4ml concentration are 200mM, 0.04 μ l concentration are 55mM Alcohol, 0.4ml are dual anti-, and dual anti-10, the 000 U/ml parasiticin that contains is received with 10, and 000 μ g/ml streptomysin, 400ng people's epidermis are raw The long factor, the niacinamide that 0.4ml concentration is 1M；
Induced medium II the preparation method comprises the following steps: in 34.36ml DMDM be added 4ml fetal calf serum, 0.4ml concentration be 100mM Nonessential amino acid, 0.4ml concentration be 200mM L-Glutamine, 0.04 μ l concentration be 55mM 2 mercapto ethanol, The niacinamide that 0.4ml is dual anti-, 0.4ml concentration is 1M.
6. utilizing people amnioic epithelium stem cell induction differentiation function beta Cell of islet answering on preparation treatment diabetes medicament With.
CN201910665356.XA 2019-07-23 2019-07-23 A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional islets β cell Pending CN110423720A (en)

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