CN109053866A - The improved method that epidermal stem cells break up to pancreatic cell - Google Patents

The improved method that epidermal stem cells break up to pancreatic cell Download PDF

Info

Publication number
CN109053866A
CN109053866A CN201811023578.3A CN201811023578A CN109053866A CN 109053866 A CN109053866 A CN 109053866A CN 201811023578 A CN201811023578 A CN 201811023578A CN 109053866 A CN109053866 A CN 109053866A
Authority
CN
China
Prior art keywords
stem cells
cell
epidermal stem
pancreatic
differentiation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811023578.3A
Other languages
Chinese (zh)
Other versions
CN109053866B (en
Inventor
杨骏
张勇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Zhongjing high tech Biotechnology Co., Ltd
Original Assignee
Luoyang Xuan Zhi Biological Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Luoyang Xuan Zhi Biological Technology Co Ltd filed Critical Luoyang Xuan Zhi Biological Technology Co Ltd
Priority to CN201811023578.3A priority Critical patent/CN109053866B/en
Priority to CN202010808780.8A priority patent/CN111875674B/en
Priority to CN202010792168.6A priority patent/CN111848744B/en
Priority to CN202010792164.8A priority patent/CN111848743B/en
Priority to CN202010792171.8A priority patent/CN111848745B/en
Priority to CN202010808793.5A priority patent/CN111875675B/en
Publication of CN109053866A publication Critical patent/CN109053866A/en
Application granted granted Critical
Publication of CN109053866B publication Critical patent/CN109053866B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention provides the improved methods that a kind of epidermal stem cells break up to pancreatic cell, based on epidermal stem cells, by adding activity inducement peptide, so that cell differentiation becomes pancreatic cell, and divergaence time is shorter, and epidermal stem cells source is wide in range, low in cost, preparation is easy, has fabulous market application prospect.

Description

The improved method that epidermal stem cells break up to pancreatic cell
Technical field
The present invention relates to non-embryonic multipotent stem cells fields, provide pancreatic cell more particularly to non-embryonic multipotent stem cells Purposes and generation and use their method.
Background technique
Embryonic stem cell is a kind of myeloid-lymphoid stem cell, under suitable condition it can be divided into internal any tissue body it is thin Born of the same parents.Induced differentiation of embryonic stem cells can be beta Cell of islet by the method for external five one-step inducing by Lumelsky et al. report, And Shi etc. is reported in their research, they, which can be induced embryonic stem cell by three-step approach, becomes beta Cell of islet, and These cell transplantations can make the weight gain of animal, prolonged survival period, blood glucose decline after entering in diabetic rats body.But Because there is being not possible to the oncogenicity solved at present in embryonic stem cell, at present from clinical application there are also it is certain away from From.
For the stem cell in pancreas glandular tube source, Bonner-Weir etc. is reported in their research report, in pancreas There are a kind of stem cell of CK19 positive in glandular tube, such cell can largely expand in the presence of having KGF and Matrigel Increase and is divided into beta Cell of islet.For the stem cell in pancreas islet source, the report such as Zulewski, their pancreas islet from Normal Pancreas A kind of cell for expressing neural stem cell mark nestin albumen (nestin) is separately cultured out in group, such cell can be in body Outer massive amplification, and can divide under the action of exendin-4, activinA, HGF, Betacellulin and nicotiamide Turn to pancreatic endocrine cell.
Huang&amp；Tang, 2003；Zulewski et al., the pancreatic stem cells disclosed in 2001 articles are pancreas islet The adult stem cell in source.Tissue used in Zulewski etc. is the pancreatic tissue of normal people or mouse.Due to difficult in practice It can still face that source is insufficient to ask to obtain sufficient amount of normal Human Pancreas, when so the technology being applied to clinical Topic.
In the prior art, there are also other modes to obtain pancreatic cell.For example offer will be non-in CN101310012A Method of the embryonic pleuripotent stem cell to pancreas lineage.The present invention further provides non-embryonic multipotent stem cells and its derivative Filial generation, to provide pancreatic cell for subject.The method includes the cell and niacinamide or exendiM two that will express Ngn3 One of person is in contact to generate the cell of expression of insulin, under the cell of the expression Ngn3 has passed through with the two It states process preparation: (1) Oct-4 and Telomerase will be expressed and ectoderm, entoderm and mesodermal cell types can be divided into Non-embryonic is dry, non-reproduction, non-embryonic reproduction cell are in contact with a kind of first reagent and the second reagent of one kind to generate Pdx-I table Up to raised cell, wherein first reagent is activin A, second reagent inhibits Sonic hedgehog (SHH) activity；And (2) Pdx-I is expressed raised cell to be in contact with EGF and/or HGF, expresses raised cell to generate Ngn3.But the party Method preparation is complicated, and success rate need further to improve.
A kind of method that inducing embryo stem cell breaks up to pancreatic cell is disclosed in CN1847394A, this method is successively The embryoid body formed by embryonic stem cell is handled using Activin A and cis-retinoic acid, its differentiation is made to obtain pancreatic cell. But embryonic stem cell has been used in this method, since derived from embryonic stem cells is limited, and it is not suitable with large-scale promotion use, city Field application value is not high.
Therefore, it is necessary to solve the problems, such as that pancreatic tissue source is insufficient in pancreatic islets transplantation, it is also necessary to find a kind of from a wealth of sources Stem cell, and pancreatic cell can be obtained to meet the needs of social, to promote the well-being of mankind using simple induction mode.
Summary of the invention
The present invention provides compared with prior art, by the more easy epidermal stem cells in source with the shorter time The improved method that amount (7 days) and the yield (up to 97% yield) dramatically increased are divided into determining pancreatic cell.
There is provided herein the methods for the pancreatic cell for being divided into epidermal stem cells, and the method comprising the steps of:
The first step puts epidermal stem cells into the culture dish of 1%Matrigel paving quilt as follows with activity thorn Kassinin kinin induction:
After two hours, epidermal stem cells start to sprawl, and culture medium is changed to containing 10% fetal calf serum and 100ng/ at this time It is cultivated in the DMEM culture medium of ml activin A (Sigma) 24 hours, then epidermal stem cells switch to containing 10% tire ox blood Clear and 1-10mg/L activity stimulator polypeptide DMEM, which is cultivated 6-8 hours, breaks up epidermal stem cells.Later, break up epidermal stem cells Then it uses and contains 10% fetal calf serum and 1 × 10-6The DMEM culture medium of mol/L RA (Sigma) continues induction 24 hours, obtains Diabetes.
Second step, the amplification of pancreatic islet precursor cells.
The epidermal stem cells of differentiation use the DMEM containing 10% fetal calf serum and 10ng/ml bFGF (Sigma) to cultivate 3-5 It, amplification of the induction from the diabetes of epidermal stem cells.During this period, epithelium is presented in most of adherent cell Spline structure.
Third step, the maturation of pancreatic beta cell (Pancreatic β cell).
Cell is transferred to (from Gibco-BRL company, matches as directed containing N2 and B27 (Gibco-BRL) serum substitute System), 1ug/ml Laminin (Sigma), the DMEM/ of 10ng/ml bFGF and 10mmol/L nicotinamide (Sigma) Continue culture 3-5 days in F12 culture medium (Gibco-BRL), promotes beta Cell of islet mature, obtain mature pancreatic beta cell.
The concentration of the Activin A can be 50-300ng/ml culture medium, and the concentration of the cis-retinoic acid is 1 × 10-7-1×10-5Mol/L culture medium；The concentration of the Activin A is preferably 100ng/ml culture medium, the cis-retinoic acid Concentration is preferably 1 × 10-6Mol/L culture medium.
The mass percentage of the Matrigel is 1%.
The concentration of the bFGF is 8-12ng/ml；Preferably 10ng/ml.
The diabetes that amplification is obtained addition N2, B27 serum substitute, laminin (laminin), alkali Property fibroblast growth factor (bFGF) and culture medium culture 3-5 days of niacinamide (nicotinamide), obtain mature Beta Cell of islet.
Still further aspect of the present invention is most important to be to provide a kind of active stimulator polypeptide, and the activity stimulator polypeptide being capable of specificity Generate induction trend for the insulin expression related gene in epidermal stem cells, can significantly increase epidermal stem cells Break up speed and differentiation capability, enhances its differentiation capability to pancreatic cell.
Active stimulator polypeptide involved in the present invention, amino acid sequence is respectively as SEQ ID NO:1-6 is any shown.
Specific embodiment
The preparation of 1 epidermal stem cells of embodiment
It takes (people) healthy male to peritomize postoperative skin of foreskin, prepares epidermal cell and fibroblast.
1. the separation of epidermal cell: the skin for taking 2cm long, 2mm or so wide is cleaned 3 times with the PBS containing antibiotic；Set egg In white enzyme solution, 4 DEG C digest 16 hours；Skin is taken out, epidermis and skin corium are removed；Collect epidermis skin graft, set 0.25% pancreatin/ In 0.02%EDTA (1: 1) mixed liquor, 37 DEG C digest 15 minutes, terminate digestion, filter after slightly blowing and beating, and collect cell suspension, Supernatant is abandoned in centrifugation, and fresh medium is added, and cell, adjustment cell concentration to 1 × 10 is resuspended3/ml。
2. the preparation of trophocyte: the corium skin graft after collecting above-mentioned epidermis and skin corium removing is placed in 625U/ml glue Fibroblast suspension, which is collected, in original enzyme liquid, after digestion adds mitomycin C by Fibroblast cell-culture to sprawling 80% or so To final concentration of 10-6Mol/L, 37 DEG C are incubated for taking-up in 12 hours, are digested 5 minutes with 37 DEG C of 0.25% pancreatin, terminate digestion, centrifugation 5 minutes (1000r/ minutes), supernatant is abandoned, cell is resuspended, it is spare.
3. the preparation of extracellular matrix covering plate: by above-mentioned cultured epidermal cell to converging 13 days in plate；Epidermis After cell confluency, culture solution is sucked, is cleaned with sterile PBS；Ethylenediamine tetra-acetic acid (10mmol/L) and trihydroxy methyl amino is added Methane hydrochloride (25mmol/L) and Qula lead to (1% (w/v)) mixed liquor, and 37 DEG C of incubation (30 minutes) to visible cells under mirror are split Solution；It is cleaned with PBS；The bovine serum albumin(BSA) for adding 0.5mg/ml denaturation is set 37 DEG C and is incubated for 1 hour, to close non-specificity Adherency；PBS cleaning, is added serum-free medium, sets 37 DEG C of incubators to application.
4. the screening and culture of epidermal stem cells: taking the ready plate for being covered with extracellular matrix, 2ml epidermis is added Cell suspension, 37 DEG C are taken out for culture 10 minutes, are inhaled and are abandoned liquid, and PBS is cleaned to cell-free suspension, and attached cell is that epidermal stem is thin Born of the same parents.Trophocyte (4 × 10 is added3/cm2), with KSC culture solution culture, liquid is changed within the 2nd day, is changed the liquid once within latter every 3 days.Wherein KSC culture solution contains 5% fetal calf serum and 1ng/ml Basic Fibroblast Growth Factor.
5. secondary culture: above-mentioned epidermal stem cells are long to converging, with 0.25% pancreatin/0.02%EDTA (1: 1) mixed liquor, 37 DEG C digest 6 minutes, terminate digestion；It collects cell suspension and enters centrifuge tube, be centrifuged 5 minutes, abandon supernatant, fresh medium is added, Cell is resuspended, with 2 × 105The density of/ml is inoculated in culture bottle of the paving added with trophocyte in advance, sets 37 DEG C, 5%CO2 It is cultivated in incubator, later, by immunohistochemistry testing result: the expression of 1 integrin 100% of cell rejection tablet β, Keratin 19 have 97% Expression, Keratin 10, anti-vimentin are negative, and determination obtains epidermal stem cells.
The preparation of the active stimulator polypeptide of embodiment 2
Random peptide library is constructed first, wherein inventing the rondom polypeptide is one section containing 30 or less (including three Ten) random amino acid sequence.Polypeptide random library of the present invention is by corresponding DNA library (for screening the DNA of CPP Library) translation conversion obtain.Polypeptide random library method for transformation of the present invention is method for transformation commonly used in the art.Such as change Conversion method and Electroporation Transformation method are learned, is seen " molecular cloning (the Chinese second edition) " that J. Pehanorm Brooker etc. is write.To be more at random Peptide and epidermal stem cells are mixed, and basal medium is to contain 10% fetal calf serum and 100ng/ml activin A at this time (Sigma) DMEM culture medium, according to the difference of epidermal stem cells differentiation index ability, obtaining 42 has compared with strong stimulation table Skin stem cell is adopted to the stimulator polypeptide of pancreatic cell differentiation capability wherein stronger ability is polypeptide shown in SEQ ID NO:1-6 It is synthesized with chemically synthesized method, carries out subsequent experimental.
Induction of 3 epidermal stem cells of embodiment to pancreatic cell
The epidermal stem cells that embodiment 1 is prepared are put into the culture dish of 1%Matrigel paving quilt according to such as lower section Method activity stimulation inducing peptide:
After two hours, epidermal stem cells start to sprawl, and culture medium is changed to containing 10% fetal calf serum and 100ng/ at this time It is cultivated in the DMEM culture medium of ml activin A (Sigma) 24 hours, then epidermal stem cells switch to containing 10% tire ox blood Clear and 10mg/L activity stimulator polypeptide (is individually tested, with or not 6 kinds of different active peptides of SEQ ID NO:1-6 Add the active peptide as blank control) DMEM cultivate and break up epidermal stem cells in 6 hours.Later, differentiation epidermal stem is thin Born of the same parents, which then use, contains 10% fetal calf serum and 1 × 10-6The DMEM culture medium of mol/L RA (Sigma) continues induction 24 hours, obtains To diabetes.
The epidermal stem cells of aforementioned obtained initial differentiation, which are used, contains 10% fetal calf serum and 10ng/ml bFGF (Sigma) DMEM cultivate 5 days, induction from epidermal stem cells diabetes amplification.During this period, most of adherent Epithelium spline structure is presented in cell.
Aforementioned obtained adherent cell is transferred to containing N2 and B27 serum substitute, 1ug/ml Laminin (Sigma), continue to train in the DMEM/F12 culture medium of 10ng/ml bFGF and 10mmol/L nicotinamide (Sigma) It supports 5 days, promotes beta Cell of islet mature, obtain mature pancreatic beta cell.
The detection for the mature pancreatic beta cell that embodiment 4 is broken up
One, with the expression of the cytogene of RT-PCR detection differentiation
The case where in order to detect pancreatic endocrine cell, with the table of the marker gene of RT-PCR detection pancreatic endocrine cell Up to situation, specific method and result are as follows:
The total serum IgE of noble cells and control cell is extracted with RNA extracts kit.Noble cells is mature pancreatic beta cell. Control cell is not added with the cell of active stimulator polypeptide and without adding active stimulator polypeptide also without using two kinds of activin A and RA The cell that the third step of one of small molecule induction obtains.RNA is cDNA with MMLV reverse transcriptase reverse transcription.PCR Ex Taq polymerase system is completed.PCR primer is as follows:
Insulin1:TAGTGACCAGCTATAATCAGAG and ACGCCAAGGTCTGAAGGTCC (288bp)；
Hnf3 β: ACCTGAGTCCGAGTCTGACC and GGCACCTTGAGAAAGCAGTC (345bp)；
Pdx-1:CTTAGCGTGTCGCCACAGCCCTCCA and TCCAACAGCCGCCTTTCGTTATTCT (472bp)；
Glut2:GGATAAATTCGCCTGGATGA and TTCCTTTGGTTTCTGGAACT (299bp)；
Isl1:ATTTGCCACCTAGCCACAGCACC and CGCATTTGATCCCGTACAACCTG (335bp)；
β-actin:CCTGAACCCTAAGGCCAACCGTGAA
With ATACCCAAGAAGGAAGGCTGGAAAA (480bp).
As a result as shown in the table: using the expression quantity of β-actin as datum quantity.
The expression quantity of each gene of 1 noble cells of table
As can be seen from the above table, can be good at promoting together with activin A and RA using active stimulator polypeptide of the invention Differentiation of the epidermal stem cells to pancreatic cell, and activin A and RA is only used only, embryo can only be promoted as the prior art Tire stem cell breaks up as pancreatic cell, and epidermal stem cells can not be promoted to break up to pancreatic cell.And the pancreas that differentiation obtains For its gene expression amount of cell compared with normal person's pancreatic cell, difference is little, almost the same, and deduction can travel identical function substantially Energy.
Two, ELISA detects the secretory volume of insulin
By 2X104Break up obtained pancreatic cell uses the PBS solution of 20 and 5mmol/L glucose to stimulate 1h, detection respectively Amount of insulin secretion is as a result as follows:
It can be seen from the results above that pancreatic cell is extremely significant in the secretory volume of insulin to be higher than control group, and not Amount of insulin secretion difference caused by stimulating with concentration of glucose is extremely significant, it was demonstrated that islet-like cells have concentration of glucose variation Reaction.Simultaneously from the results, it was seen that the pancreatic cell that the present invention induces, the pancreas that induction generates in low glucose concentrations Island element is slightly few compared with normal pancreatic cells secretory volume, still, in high concentration glucose, but can produce than normal pancreatic cells more High amount of insulin secretion, illustrates, the cell which obtains, and has better hyperglycemia stimulate the reaction and generates ability.
Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.
Sequence table
<110>Luoyang Xuan Zhi Biotechnology Co., Ltd
<120>improved method that epidermal stem cells break up to pancreatic cell
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
Trp Ile Met Ile His Asp Ile Tyr His Trp Asn Glu Ile Ile Glu Asn
1 5 10 15
Asn Thr Ser Phe Met Asp Leu Pro Ser Arg
20 25
<210> 2
<211> 26
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
His Ile Pro Pro Trp Trp Cys Met Thr Thr Trp Pro Met Gly Gly Ala
1 5 10 15
Asp Asn Ser Cys Lys Gly Arg Gln Arg Met
20 25
<210> 3
<211> 26
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
Ser Asp His Phe Gly Arg Gln Glu Ser Asp Met Ala Asp Lys Pro Val
1 5 10 15
Thr Gly Thr Asn Ala Lys Ile Ser Asp Gln
20 25
<210> 4
<211> 26
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
Pro Pro Leu Ala Thr Ser Ser Phe Thr Glu Trp Phe Pro Met Leu Trp
1 5 10 15
Cys Gln Asn Trp Ser Gln Ser Glu Val Tyr
20 25
<210> 5
<211> 26
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
Ser Gln Asn Pro Gly Asp Pro Thr Trp Ile Pro Leu Ser Ala Pro Gln
1 5 10 15
Thr Cys Ser Ser His Trp Arg Met Val Ile
20 25
<210> 6
<211> 26
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
Tyr Pro Asn His Trp Leu Gln Ser His Ser Gln Ala Leu Met Glu Thr
1 5 10 15
Arg Arg His Trp Thr Ile Glu His Ser Met
20 25

Claims (5)

1. a kind of activity stimulator polypeptide, is used to stimulate cell differentiation.
2. a kind of activity stimulator polypeptide, sequence such as SEQ ID NO:1-6 are any shown.
3. the described in any item active stimulator polypeptides of claim 1-2 are for promoting epidermal stem cells into pancreatic cell differentiation Using.
4. a kind of method that epidermal stem cells break up to pancreatic cell, specifically includes the following steps:
The first step puts epidermal stem cells into the culture dish of 1%Matrigel paving quilt as follows with active stimulator polypeptide Induction:
After two hours, epidermal stem cells start to sprawl, and culture medium is changed to containing 10% fetal calf serum and 100ng/ml at this time It is cultivated in the DMEM culture medium of activin A 24 hours, then epidermal stem cells switch to containing 10% fetal calf serum and 1- The DMEM of the described in any item active stimulator polypeptides of 10mg/L such as claim 1-2, which is cultivated 6-8 hours, breaks up epidermal stem cells, it Afterwards, differentiation epidermal stem cells, which are then used, contains 10% fetal calf serum and 1 × 10-6The DMEM culture medium of mol/L RA continues induction 24 Hour, obtain diabetes；
Second step, the amplification of pancreatic islet precursor cells；
The epidermal stem cells of differentiation are cultivated 3-5 days with containing the DMEM of 10% fetal calf serum and 8-12ng/ml bFGF, and induction comes Derived from the amplification of the diabetes of epidermal stem cells, during this period, epithelium spline structure is presented in most of adherent cell.
Third step, the maturation of pancreatic beta cell (Pancreatic β cell).
5. method as claimed in claim 4, wherein the concentration of the bFGF is 10ng/ml.
CN201811023578.3A 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells Active CN109053866B (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CN201811023578.3A CN109053866B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010808780.8A CN111875674B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792168.6A CN111848744B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792164.8A CN111848743B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792171.8A CN111848745B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010808793.5A CN111875675B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811023578.3A CN109053866B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells

Related Child Applications (5)

Application Number Title Priority Date Filing Date
CN202010808780.8A Division CN111875674B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792171.8A Division CN111848745B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792168.6A Division CN111848744B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792164.8A Division CN111848743B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010808793.5A Division CN111875675B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells

Publications (2)

Publication Number Publication Date
CN109053866A true CN109053866A (en) 2018-12-21
CN109053866B CN109053866B (en) 2020-12-15

Family

ID=64759286

Family Applications (6)

Application Number Title Priority Date Filing Date
CN202010808793.5A Active CN111875675B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010808780.8A Active CN111875674B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792171.8A Active CN111848745B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792164.8A Active CN111848743B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN201811023578.3A Active CN109053866B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792168.6A Active CN111848744B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells

Family Applications Before (4)

Application Number Title Priority Date Filing Date
CN202010808793.5A Active CN111875675B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010808780.8A Active CN111875674B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792171.8A Active CN111848745B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells
CN202010792164.8A Active CN111848743B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN202010792168.6A Active CN111848744B (en) 2018-09-03 2018-09-03 Improved method for differentiation of epidermal stem cells into pancreatic cells

Country Status (1)

Country Link
CN (6) CN111875675B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462636A (en) * 2021-08-05 2021-10-01 合肥滴碧云生物科技有限公司 Improved method for differentiation of epidermal stem cells into liver cells

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087468A (en) * 2015-09-01 2015-11-25 东南大学 Method for effectively inducing stem cells to differentiate into pancreas islet beta cells
CN107267447A (en) * 2017-06-18 2017-10-20 广东博溪生物科技有限公司 A kind of enrichment method of epidermal stem cells

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1253558C (en) * 2003-09-02 2006-04-26 中国人民解放军第四军医大学口腔医学院 Separation and culturing method of human epidermis stem cell
CN1637137A (en) * 2004-01-06 2005-07-13 中国人民解放军军事医学科学院野战输血研究所 Method of inducing stem cell to differentiate to insulin-like cell and its application
CN1772885B (en) * 2004-11-11 2010-12-01 中国人民解放军军事医学科学院野战输血研究所 Method of inducing stem cell to differentiate into pancreatic island-like cell and application of pancreatic island-like cell
CN100425694C (en) * 2005-04-15 2008-10-15 北京大学 Method of inducing embryo stem cell to differentiate toward pancreatic cell
CN101173249A (en) * 2007-10-17 2008-05-07 暨南大学 Method for inducing human fetal liver mesenchyma stem cell to pancreatic islet beta sample cell differentiation
CN101603027A (en) * 2008-06-12 2009-12-16 西北农林科技大学 External evoked human marrow mesenchymal stem cell is divided into the method for insulin-like cell
KR101025880B1 (en) * 2008-10-23 2011-03-30 공희숙 Method for the differentiation of human adult stem cells into insulin-secreting cells
CN101580818A (en) * 2009-03-09 2009-11-18 暨南大学 Method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof
CN102311940A (en) * 2010-07-09 2012-01-11 中国人民解放军军事医学科学院野战输血研究所 Method for inducing differentiation of stem cells into islet-like cells
CN102433300A (en) * 2011-11-25 2012-05-02 厚朴生物科技（苏州）有限公司 Method for constructing pancreatic stem cell line from human insulin and differentiating to insulin secretion cell
CN102517248A (en) * 2011-12-30 2012-06-27 中日友好医院 In-vitro induced pancreas-islet-like structure forming method
CN102618500A (en) * 2012-03-21 2012-08-01 天津科技大学 Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro
CN102618491B (en) * 2012-03-21 2014-04-09 协和干细胞基因工程有限公司 Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution
CA2871795C (en) * 2012-04-30 2023-03-14 University Health Network Methods and compositions for generating pancreatic progenitors and functional beta cells from hpscs
DK3450542T3 (en) * 2012-06-08 2021-11-01 Janssen Biotech Inc DIFFERENTIATION OF HUMAN EMBRYONAL STEM CELLS TO ENDOCRINE PANCREATIC CELLS
CN103146754A (en) * 2013-02-04 2013-06-12 南通大学附属医院 Method for differentiation of polygenic-modification induced pluripotent stem (iPS) cells into insulin secretion cells
CN103194424A (en) * 2013-03-28 2013-07-10 于涛 Method for inducing embryonic stem cell into pancreatic tissue-like cells
CN103740641A (en) * 2013-12-26 2014-04-23 广州爱菲科生物科技有限公司 Inducing culture medium and inducing method thereof for inducing human adipose-derived mesenchymal stem cell to be differentiated into islet-like cell
CN103756954A (en) * 2014-01-22 2014-04-30 青岛中天干细胞工程有限公司 Inducer for inducing human adipose derived stromal cells into insulin-secretion cells, induction medium and method
CN105462913A (en) * 2014-09-05 2016-04-06 深圳市北科生物科技有限公司 Method for inducing human umbilical cord mesenchymal stem cells to be differentiated into pancreatic beta cells
CN104263697B (en) * 2014-09-18 2017-08-04 胡振波 A kind of method that inducing culture and induction human adipose mesenchymal stem cells generate insulin secretory cell
CN104726395B (en) * 2015-03-20 2018-03-23 深圳市人民医院 It is a kind of to induce the method that people's induced multi-potent stem cell directed differentiation is pancreatic cell
CN104862268A (en) * 2015-05-27 2015-08-26 贵州北科泛特尔生物科技有限公司 Culture medium for separating and inducing human adipose stem cells into pancreatic beta cells and use method of culture medium
CN104988110A (en) * 2015-07-08 2015-10-21 深圳爱生再生医学科技有限公司 Method for transforming umbilical cord mesenchymal stem cells into islet cells
CN105062952A (en) * 2015-08-06 2015-11-18 深圳爱生再生医学科技有限公司 Method for transformation of mesenchymal stem cells into islet cells
CN105670986A (en) * 2015-11-23 2016-06-15 王意忠 Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into islet-like cells and induction method therefor
CN106676056A (en) * 2016-11-08 2017-05-17 里程 Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells
CN106854638B (en) * 2017-01-19 2019-12-27 天晴干细胞股份有限公司 Method for inducing mesenchymal stem cells to differentiate into islet-like cells
CN107460158A (en) * 2017-08-10 2017-12-12 河南省银丰生物工程技术有限公司 A kind of inducing umbilical cord mesenchymal stem is divided into the method to form insulin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087468A (en) * 2015-09-01 2015-11-25 东南大学 Method for effectively inducing stem cells to differentiate into pancreas islet beta cells
CN107267447A (en) * 2017-06-18 2017-10-20 广东博溪生物科技有限公司 A kind of enrichment method of epidermal stem cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TINGXIA GUO等: "Stem cells to pancreatic β cells : New sources for diabetes cell therapy", 《ENDOCR REV》 *
袁毅君等: "上皮组织中的干细胞", 《天水师范学院学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462636A (en) * 2021-08-05 2021-10-01 合肥滴碧云生物科技有限公司 Improved method for differentiation of epidermal stem cells into liver cells
CN113462636B (en) * 2021-08-05 2023-11-14 合肥滴碧云生物科技有限公司 Improved method for differentiating epidermal stem cells into liver cells

Also Published As

Publication number Publication date
CN111875674B (en) 2021-12-10
CN111848743B (en) 2021-05-11
CN111875675A (en) 2020-11-03
CN111848745A (en) 2020-10-30
CN111848745B (en) 2022-01-18
CN111875675B (en) 2021-10-01
CN111848744B (en) 2021-07-23
CN111875674A (en) 2020-11-03
CN111848744A (en) 2020-10-30
CN111848743A (en) 2020-10-30
CN109053866B (en) 2020-12-15

Similar Documents

Publication Publication Date Title
US20060040385A1 (en) Cultured human pancreatic islets, and uses thereof
CN104428410B (en) Pancreatic hormone produces celliferous manufacture method and pancreatic hormone produces cell and induction accelerator
CN105916978A (en) Compositions and methods of preparing airway cells
CN107058389A (en) Multipotential stem cell
WO2018086319A1 (en) Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof
CN102899288B (en) Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells
CN101724602B (en) Method for the differentiation of human adult stem cells into insulin-secreting cells
WO2018220623A1 (en) Compositions and methods for providing cell replacement therapy
CN110423720A (en) A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional islets β cell
Zhou et al. Genetic modification of primate amniotic fluid-derived stem cells produces pancreatic progenitor cells in vitro
CN109053866A (en) The improved method that epidermal stem cells break up to pancreatic cell
CN102741398A (en) Methods for producing nerve cells from stem cells, nerve cells and uses thereof
CN113038830A (en) Composition for reconstructing human skin tissue having hair follicle, human skin tissue model animal, and method for producing same
CN105441381B (en) Method of the external evoked human adipose mesenchymal stem cells to functional hepatocyte differentiation
RU2430158C1 (en) Method for differentiation of adult human stem cells in insulin-secreting cells
CN102311940A (en) Method for inducing differentiation of stem cells into islet-like cells
CN109486770A (en) A kind of microRNA-181c-5p promotion source of people iPS directed differentiation is the method for beta Cell of islet
CN101914488B (en) Method for induced differentiation of human amniotic mesenchymal cells into insulin secreting cells
CN113637630B (en) Islet-like cell mass, and preparation method and application thereof
CN107058214A (en) The culture medium and cultural method of induced multi-potent stem cell directed differentiation kidney cell
CN115120600B (en) Application of diosgenin and analogues thereof in preparing medicines for preventing or treating diabetes
CN111117945B (en) Skin model containing melanin, construction method and application thereof
CN107320781B (en) Tissue engineering skin containing living cells and preparation method thereof
CN116144584A (en) Pancreatic progenitor cells differentiated from human induced pluripotent stem cells, and preparation method and application thereof
CN109431961A (en) Cosmetic material and products thereof for skin nti-freckle reparation

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information

Inventor after: Wang Ying

Inventor after: Gao Shuping

Inventor after: Yang Jun

Inventor after: Zhang Yong

Inventor before: Yang Jun

Inventor before: Zhang Yong

CB03 Change of inventor or designer information
TA01 Transfer of patent application right

Effective date of registration: 20201124

Address after: Room 02-a116, 2 / F, block B, No. 22, information road, Haidian District, Beijing 100085

Applicant after: Beijing Zhongjing high tech Biotechnology Co., Ltd

Address before: 471000 Ludu Yuefu Building 1 Unit 1-1404, South of Qianjiang Road, West of Chang Xiamen Street, Luoyang Economic and Technological Development Zone, Henan Province

Applicant before: LUOYANG XUANZHI BIOLOGICAL TECHNOLOGY Co.,Ltd.

TA01 Transfer of patent application right
GR01 Patent grant
GR01 Patent grant