CN105567642B - A kind of preparation method of xeroderma pitmentosum human pluripotent stem cells - Google Patents
A kind of preparation method of xeroderma pitmentosum human pluripotent stem cells Download PDFInfo
- Publication number
- CN105567642B CN105567642B CN201610069342.8A CN201610069342A CN105567642B CN 105567642 B CN105567642 B CN 105567642B CN 201610069342 A CN201610069342 A CN 201610069342A CN 105567642 B CN105567642 B CN 105567642B
- Authority
- CN
- China
- Prior art keywords
- stem cell
- fibroblast
- cell
- culture
- medium
- 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.)
- Active
Links
Classifications
-
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
-
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5058—Neurological cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5073—Stem 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/602—Sox-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
- C12N2501/60—Transcription factors
- C12N2501/603—Oct-3/4
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/604—Klf-4
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/606—Transcription factors c-Myc
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/608—Lin28
-
- 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
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
-
- 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
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention discloses a kind of preparation methods of xeroderma pitmentosum human pluripotent stem cells.The present invention utilizes iPSC technology by the skin fibroblasts in xeroderma pitmentosum people source, make its reprogramming inducing multi-potent stem cell for its specificity by the method that nonconformity additive type plasmid electricity turns, and direct it differentiation neuroblast, these cells show the extreme sensitivity for UV in vitro, it is easier to that Apoptosis occurs, provides certain clue for the raw neurological of xeroderma pitmentosum human hair.In addition, these carry the neural stem cell of patient's gene mutations and neuron and can be used as effective platform and carry out efficiently high-throughput personalised drug screening, for disease research, exploitation disease model, study of disease pathogenesis and carry out disease treatment and lay the foundation.There is huge application prospect in personalized treatment and translational medicine.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of preparation side of xeroderma pitmentosum human pluripotent stem cells
The preparation method of the multipotential stem cell and nerve cell of method more particularly to a variety of xeroderma pitmentosum human specifics.
Background technique
Xeroderma pitmentosum (Xeroderma pigmentosum, abbreviation XP) is that DNA is caused to repair defect by gene mutation
Caused by the rare autosomal recessive disease of one kind.According to corresponding mutated gene (XPA, XPB, XPC, XPD, XPE,
XPF, XPG and XPV) difference, xeroderma pitmentosum people can be divided into following 8 kinds of different complementary types: xeroderma pitmentosum is mutual
Apotype A, xeroderma pitmentosum complementary type B, xeroderma pitmentosum complementary type C, xeroderma pitmentosum complementary type D, coloring dry hide
Sick complementary type E, xeroderma pitmentosum complementary type F, xeroderma pitmentosum complementary type G and xeroderma pitmentosum anomaly V.
The clinical phenotypes of xeroderma pitmentosum be mainly skin for ultraviolet extremely sensitive, pigment deposition, be easy to happen light
Damage the cutaneum carcinoma photo-induced with day.About the pathogenesis of xeroderma pitmentosum skin injury, there are many reports to explain at present
It states, it is related mainly to repair defect to intracorporal two kinds of DNA damages.Wherein 7 complementary types and Nucleotide Sequence Analysis
(Nucleotide Excision Repair, NER) defect is related, and 1 anomaly synthesizes (Translesion with across damage
Synthesis, TLS) defect is related.Nucleotide Sequence Analysis can be divided into full-length genome reparation (global genome repair,
GGR) and (transcription-coupled repair, TCR) is repaired in transcription coupling, is that ultraviolet (UV) irradiation generates DNA damage
The main reparation approach of wound.GGR can repair the DNA damage on full-length genome, and TCR can only be repaired in transcription
DNA damage.In GGR repair pathways, XPC and hHR23B protein complexes identify DNA damage caused by UV；It is repaired in TCR logical
CSA-CSB complex identification of damage in road.After DNA damage is all identified by compound, albumin X PA can also be recruited further
Injury site is verified, while XPB and XPD was recruited as a part of transcription factor complex TFIIH as unwindase
The DNA double chain of damage location is opened, then XPG, XPF can be sheared at the both ends of injury site, about 30 nucleotide left sides
Right segment can be removed, and the vacancy left is recombined according to base complementrity principle, and so far DNA damage caused by UV is complete
It repairs.Anomaly patient XPV has the function of normal nucleotide excision repair, but the existing defects in synthesizing across damage dna.
XPV gene encodes a kind of DNA- polymerase η, the mutation for the cyclobutane pyrimidine dimer that energy specific recognition UV is generated, in DNA
Correctly pairing base is inserted into synthesis process on the complementary strand of mutation and completes DNA replication dna.Therefore the mutation of each gene
Protein function will be caused impaired and then influence the reparation of DNA damage, cause the unstability of genome, cause patient to ultraviolet
Sensibility and uv induction cutaneum carcinoma high rate.
Although the pathogenesis about xeroderma pitmentosum application on human skin cancer is clear, at present still without good
Treatment method.Patient also can only slow down the generation of disease by reducing ultraviolet irradiation.However there are a part of coloring dry hides
Patient is also accompanied by a degree of neurodegenerative disease, including microcephaly, dull-witted, sensorineural hearing loss, week
Enclose neuropathy etc..Since, not by ultraviolet irradiation, xeroderma pitmentosum human hair gives birth to nerve degeneration inside brain
Pathogenesis at present there is no explaining well, equally to this also without effective treatment method or drug.At present for coloring
The mechanism study of property dry hide patient's neurological stays cool, and main cause is exactly to be difficult to obtain suitable experimental material.
Therefore, urgent need will establish a kind of suitable platform or model, for the generation in experiment indoor simulation neurological disease, be convenient for
Research to disease molecules mechanism.
In recent years, induce multi-potent stem cell (Induced Pluripotent Stem Cell, iPSC) technology appearance and
Development provides bright prospects for the gene therapy of a variety of rare class diseases and cell therapy.IPSC be by by specific transcription because
Sub (such as OCT4, SOX2, KLF4, cMYC etc.) imports body cell, and being transformed into it can constantly be proliferated, latent with Multidirectional Differentiation
The stem cell of energy.IPSCs cannot be only used for cell transplantation, neomorph, especially cellular damage, the serious disease of necrosis it is thin
Born of the same parents' transplantation treatment；Disease model can also be constructed, in order to the mechanism that study of disease is formed, screens new drug, and research
New treatment method.
Summary of the invention
It is an object of the present invention to provide a kind of preparation methods of the neuron of xeroderma pitmentosum human specific.
The preparation method of the neuron of xeroderma pitmentosum human specific provided by the invention includes the following steps:
(1) fibroblast in vitro xeroderma pitmentosum people source is reprogrammed, it is dry obtains carrying coloring
Skin patient's gene induces multi-potent stem cell；
(2) induction differentiation is oriented to described induce multi-potent stem cell, obtains carrying xeroderma pitmentosum people's gene
Neural stem cell；
(3) induction differentiation is oriented to the neural stem cell, obtains the nerve for carrying xeroderma pitmentosum people's gene
Member；
The nucleotides sequence of the gene OCT3/4 is classified as the sequence 1 in sequence table；
The nucleotides sequence of the gene SOX2 is classified as the sequence 2 in sequence table；
The nucleotides sequence of the gene KLF4 is classified as the sequence 3 in sequence table；
The nucleotides sequence of the gene LMyc is classified as the sequence 4 in sequence table；
The nucleotides sequence of the gene LIN28 is classified as the sequence 5 in sequence table.
In the above method,
In step (2), described pair induce multi-potent stem cell be oriented induction differentiation include the following steps:
A carry out Fiber differentiation) is induced multi-potent stem cell to described in inducing multi-potent stem cell culture medium, obtains induction training
Cell after supporting；
B differentiation for the first time) is carried out to cell after the Fiber differentiation in neural stem cell induced medium 1 to cultivate, and is obtained
Cell after to first time differentiation culture；
C) second of differentiation is carried out to cell after first time differentiation culture in neural stem cell induced medium 2 to train
It supports to get the neural stem cell for carrying xeroderma pitmentosum people's gene is arrived；
The neural stem cell induced medium 2 is that Compound E is added in the induced medium of neural stem cell basis
Obtained culture medium, concentration of the Compound E in the neural stem cell induced medium 2 are 0.1-0.5 μM；Institute
Stating concentration of the Compound E in the neural stem cell induced medium 2 is specially 0.1 μM；
The neural stem cell induced medium 1 is to add Dorsomorphin in neural stem cell induced medium 2
Obtained culture medium, concentration of the Dorsomorphin in the neural stem cell induced medium 1 are 1-3 μM；It is described
Concentration of the Dorsomorphin in the neural stem cell induced medium 1 is 2 μM；
In step (3), described be oriented to neural stem cell is induced to differentiate into neuronal differentiation medium culture institute
State neural stem cell；
The neuronal differentiation medium is that double fourth adenosine cyclophosphates, Vitamin C are added in neuron differentiation basal medium
The culture medium that acid vitamin C, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor obtain；
Concentration of the double fourth adenosine cyclophosphates in the neuronal differentiation medium is 350-450 μM, double Ding Huan
Concentration of the phosphorus adenosine in the neuronal differentiation medium is 400 μM；
Concentration of the ascorbic acid usp/bp in the neuronal differentiation medium is 150-250 μM, described anti-bad
Concentration of the hematic acid vitamin C in the neuronal differentiation medium is 200 μM；
Concentration of the brain-derived neurotrophic factor in the neuronal differentiation medium is 5-15ng/m1, described
Concentration of the brain-derived neurotrophic factor in the neuronal differentiation medium is 10ng/m1；
Concentration of the glial cell line-derived neurotrophic factor in the neuronal differentiation medium is 5-15ng/
m1；
Concentration of the glial cell line-derived neurotrophic factor in the neuronal differentiation medium is 10ng/m1.
In the above method,
Further include following steps in step C): the cell that second of differentiation culture is obtained is in the nerve cord
Secondary culture in cell base induced medium obtains the neural stem cell for carrying xeroderma pitmentosum people's gene；
The secondary culture is that passage in every 4-5 days is primary, continuously reaches the third generation.
The culture medium that induces multi-potent stem cell is that Knockout serum substitute, nonessential is added on cell culture medium
The culture medium that amino acid, GlutaMAX, penicillin/streptomycin, beta -mercaptoethanol, people FGF2 are obtained；The Knockout serum
Substitute is 20% in the volume fraction induced multi-potent stem cell in culture medium；The nonessential amino acid is in the induction
Concentration in multipotential stem cell culture medium is 0.1mM；The GlutaMAX it is described induce multi-potent stem cell it is dense in culture medium
Degree is 1mM；The penicillin or streptomysin are 1% in the volume fraction induced multi-potent stem cell in culture medium；The β-
Mercaptoethanol is 55 μM in the concentration induced multi-potent stem cell in culture medium；The people FGF2 is dry thin in the induced multi-potent
Concentration in born of the same parents' culture medium is 10ng/ml；
Neural stem cell basis induced medium be in cell culture medium add Neurobasal, N2, B27,
The culture medium that GlutaMAX, hLIF, CHIR99021 and SB431542 are obtained；The Neurobasal is in the neural stem cell
Volume fraction in basic induced medium is 50%；Volume of the N2 in the induced medium of the neural stem cell basis
Score is 1%；Volume fraction of the B27 in the induced medium of the neural stem cell basis is 2%；The GlutaMAX
Concentration in the induced medium of the neural stem cell basis is 2mM；The CHIR99021 is on the neural stem cell basis
Concentration in induced medium is 3 μM or 4 μM；The SB431542 is lured in the nerve stem cell culture medium or neural stem cell
Leading the concentration in culture medium 2 or neural stem cell induced medium 1 is 3 μM；The hLIF is lured on the neural stem cell basis
Leading the concentration in culture medium is 10ng/ml；
The neuron differentiation basal medium is the culture medium that addition N2 and B27 is obtained in cell culture medium, described
Volume fraction of the N2 in the neuronal differentiation medium is 1%；Body of the B27 in the neuronal differentiation medium
Fraction is 2%；
The cell culture medium is specially DMEM/F12.
In the above method,
Step A) in, the time of the Fiber differentiation is 1-3 days, and the specific time of the Fiber differentiation is 2 days；
Step B) in, the time of the first time differentiation culture is 1-3 days；The time of the first time differentiation culture is specific
It is 2 days；
Step C) in, the time of second of differentiation culture is 4-6 days；The time of second of differentiation culture is 5
It；
In step (3), the time of the culture is 14-21 days；The time of the culture is 18 days.
In the above method,
The reprogramming is done for gene OCT3/4, SOX2, KLF4, LMyc and LIN28 are imported in vitro coloring jointly
In the fibroblast in skin patient source；
The method imported jointly include the following steps: will to express the Episomal carrier of OCT3/4, expression SOX2 and
The Episomal carrier of KLF4, the Episomal carrier for expressing LMyc and LIN28 and the carrier for expressing marker gene import jointly
In the fibroblast in vitro xeroderma pitmentosum people source；
The Episomal carrier of the expression OCT3/4 is pCXLE-hOCT3/4；
The Episomal carrier of the expression SOX2 and KLF4 is pCXLE-hSK；
The Episomal carrier of the expression LMyc and LIN28 is pCXLE-hUL；
The marker gene is EGFP, and the nucleotides sequence of the EGFP is classified as the sequence 6 in sequence table；
The carrier of the expression marker gene is pCXLE-EGFP.
In the above method, the fibroblast in the in vitro xeroderma pitmentosum people source be XPA fibroblast,
XPB fibroblast, XPC fibroblast, XPG fibroblast or XPV fibroblast.
It is a further object to provide a kind of examinations of neuron for being used to prepare xeroderma pitmentosum human specific
Agent box.
Kit provided by the invention include it is above-mentioned induce multi-potent stem cell culture medium and/or neural stem cell basis induction
Culture medium and/or neural stem cell induced medium 1 and/or neural stem cell induced medium 2 and/or neuron differentiation culture
Base.
It is a still further object of the present invention to provide a kind of preparation sides of the neural stem cell of xeroderma pitmentosum human specific
Method.
The preparation method of the neural stem cell of xeroderma pitmentosum human specific provided by the invention include the following steps: for
Step (1)-(2) in the above method.
The neuron that the neural stem cell or the above method that the above method is prepared are prepared also belongs to of the invention
Protection scope.
Final object of the present invention is to provide the new use of the above method or above-mentioned neural stem cell or above-mentioned neuron
On the way.
The present invention provides the above method or above-mentioned neural stem cell or above-mentioned neuron to use in preparation screening and/or identification
In the clinical medicine and/or natural organic matter and/or the product of small molecule compound treated and/or prevent xeroderma pitmentosum
Application.
The present invention also provides above-mentioned neural stem cell or above-mentioned neuron as or preparation xeroderma pitmentosum it is thin
Application in born of the same parents' model.
1) multipotential stem cell that the present invention obtains has the advantages that has self-renewing in vitro and is divided into three embryos
The potential of layer, thus the cell line accumulated by disease can be directed differentiation to；2) multipotential stem cell obtained is whole without foreign gene
Close, have safety, using existing gene targeting can for autologous patient source induction type multipotential stem cell into
The genome of row gene mutation is corrected in situ, to remove endogenous pathogenic factor in gene level；3) autologous patient source
It induces multi-potent stem cell, avoids the puzzlement of distribution type and rejection.
The present invention, by the skin fibroblasts in xeroderma pitmentosum people source, is added using iPSC technology by nonconformity
The method of type plasmid electricity turn makes its reprogramming inducing multi-potent stem cell for its specificity, and directs it and be divided into nerve carefully
Born of the same parents, these cells show the extreme sensitivity for UV in vitro, are easier to that Apoptosis occurs, raw for xeroderma pitmentosum human hair
Neurological provide certain clue.In addition, these carry the neural stem cell of patient's gene mutations and neuron can be with
Efficiently high-throughput personalised drug screening is carried out as effective platform, is disease research, exploitation disease model, study of disease
Pathogenesis and carry out disease treatment and lay the foundation.There is huge application prospect in personalized treatment and translational medicine.
Detailed description of the invention
Fig. 1 is the reprogramming of the skin fibroblasts in five plants of xeroderma pitmentosum people sources.Figure 1A is five plants of colorings
Gene mutation entrained by the skin fibroblasts in dry hide patient source；Figure 1B is five plants of skin fibroblasts and reprogramming
The form that obtained correspondence induces multi-potent stem cell.
Fig. 2 is the totipotency identification of the multipotential stem cell of five plants of xeroderma pitmentosum human specifics.Fig. 2A is immunofluorescence
Detect stem cell labeling object NANOG, OCT4, SOX2；Fig. 2 B is the whole of foreign vector EBNA1 in qPCR detection multipotential stem cell
It closes；Fig. 2 C is to throw special the inducing multi-potent stem cell of five plants of patients into immunodeficient mouse subcutaneous, is divided into there are three tools
The teratoma of germinal layer, wherein TUJ1 (ectoderm marker), FOXA2 (endodermal marker object), α-SMA (mesodermal marker)；
Fig. 2 D is five kinds of karyotypings induced multi-potent stem cell.
Fig. 3 is that special the inducing multi-potent stem cell of xeroderma pitmentosum people is directed differentiation to neural stem cell.Fig. 3 A is
Induce multi-potent stem cell the schematic diagram for being directed differentiation to neural stem cell；Fig. 3 B is the label of Immunofluorescence test neural stem cell
Object NESTIN, PAX6.
Fig. 4 is to continue the special neural stem cell of xeroderma pitmentosum people to be divided into neuron.Fig. 4 A is that nerve cord is thin
Born of the same parents are divided into the schematic diagram of neuron；Fig. 4 B is the marker TUJ1, MAP2 of Immunofluorescence test neuron.
Fig. 5 is the sensitivity Detection of xeroderma pitmentosum people special neural stem cell and neuron to UV.Fig. 5 A is to exempt from
Epidemic disease fluorescence detection neural stem cell after UV is handled cyclobutane pyrimidine dimer (cyclobutane pyrimidine dimers,
CPD content)；Fig. 5 B exists for the neural stem cell of Annexin V-FITC/PI method flow cytometer detection wild type and XPA mutation
Apoptosis after UV irradiates；Fig. 5 C is the TUNEL dyeing of the neural stem cell of wild type and XPA mutation；Fig. 5 D is glimmering to be immunized
The content of light detection neuron CPD after UV is handled；Fig. 5 E is the TUNEL dyeing of the neuron of wild type and XPA mutation.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Cell culture condition in following embodiments is 37 degrees Celsius unless otherwise specified, 5%CO2。
XPA fibroblast (GM00710), XPB fibroblast (GM13026), XPC in following embodiments is at fiber
Cell (GM15709), XPG fibroblast (GM13371) and XPV fibroblast (GM03055) and wild type are at fiber finer
Born of the same parents (GM00038) buy the Coriell Cell Repository in the U.S..
The mutation position of the skin fibroblasts in the xeroderma pitmentosum people source of above-mentioned five kinds of carryings different genes mutation
Point situation is following (Figure 1A):
(a) XPA fibroblast (GM00710) from XPA patient skin fibroblasts, carry mutation be
619th site of XPA gene is mutated into T by C, and when transcription forms terminator codon, and the translation of XPA albumen is caused to shift to an earlier date eventually
Only；
(b) XPB fibroblast (GM13026) from XPB patient skin fibroblasts, carry mutation be
295th site T of XPB gene is mutated into C, and the 99th amino acid of XPB albumen is caused to be mutated into serine by phenylalanine；
(c) XPC fibroblast (GM15709) from XPC patient skin fibroblasts, carry mutation be
There are a T to become A in the third introne of XPC gene, affects the shearing of XPC mRNA；
(d) XPG fibroblast (GM13371) from XPG patient skin fibroblasts, carry mutation be
The A base in the 2171st site of XPG gene lacks, and causes the frameshit of mRNA encoder block；
(e) XPV fibroblast (GM03055) from XPV patient skin fibroblasts, carry mutation be
376th site of XPV gene is mutated into T by C, forms terminator codon in mRNA transcription, leads to the truncation of XPV albumen.
The mutation of said gene can all cause it to encode the dysfunction or missing of albumen, repair the intracorporal DNA of patient's machine
It cannot proceed normally again, and then lead to the generation of xeroderma pitmentosum.
PCXLE-hOCT3/4-shp53-F (#27077), pCXLE-hSK (#27078), pCXLE- in following embodiments
HUL (#27080) and pCXLE-EGFP (#27082) is bought in Addgene.
The antibody for immunofluorescence in following embodiments is as follows:
Anti-human OCT4 antibody (sc-5279), Santa Cruz Biotechnology.
Anti-human SOX2 antibody (sc-17320), Santa Cruz Biotechnology.
Anti-human NANOG antibody (ab21624), Abcam.
Anti-human NESTIN antibody (MAB5326), Millipore.
Anti-human PAX6 antibody (PRB-278P), Covance.
Anti-human TUJ1 antibody (T2220), Sigma.
Anti-human MAP2 antibody (4403), Sigma.
Anti-human FOXA2 antibody (8186), Cell Signaling Technology.
Anti-human-SMA antibody (A5228), Sigma.
Anti-human CPD antibody (TMD-2), Cosmo Bio.
Culture medium prescription in following embodiments is as follows:
1) fibroblast culture medium formula: 90%DMEM (Invitrogen), 10%FBS (Hyclone), 1% mould
Element/streptomysin (Invitrogen)；
2) culture medium prescription is induced multi-potent stem cell: 80%DMEM/F12 culture medium (Invitrogen), 20% (volume hundred
Divide content) Knockout serum substitute (Invitrogen), 0.1mM nonessential amino acid (Invitrogen), 1mM
GlutaMAX (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 55 μM of beta -mercaptoethanols (Invitrogen),
10ng/ml people FGF2 (Joint Protein Central)；
3) neural stem cell induced medium 1 is formulated: 50%Advanced DMEM/F12 (Invitrogen), 50%
Neurobasal (Invitroge), 1%N2 (Invitrogen), 2%B27 (Invitrogen), 2mM GlutaMAX
(Invitrogen)、10ng/ml hLIF(Millipore)、4μM CHIR99021(Cell agentech)、3μM
SB431542(Cellagentech)、0.1μM Compound E(EMD Chemicals)、2μM Dorsomorphin
(Sigma)；
4) neural stem cell induced medium 2 is formulated: 50%Advanced DMEM/F12 (Invitrogen), 50%
Neurobasal (Invitroge), 1%N2 (Invitrogen), 2%B27 (Invitrogen), 2mM GlutaMAX
(Invitrogen)、10ng/ml hLIF(Millipore)、4μM CHIR99021(Cellagentech)、3μM SB431542
(Cellagentech),0.1μM Compound E(EMD Chemicals)；
5) nerve stem cell culture medium formula: 50%Advanced DMEM/F12 (Invitrogen), 50%
Neurobasal (Invitroge), 1%N2 (Invitrogen), 2%B27 (Invitrogen), 2mM GlutaMAX
(Invitrogen)、10ng/ml hLIF(Millipore)、3μM CHIR99021(Cellagentech)、2μM SB431542
(Cellagentech)；
6) Neuronal induction culture medium prescription: 50%Advanced DMEM/F12 (Invitrogen), 1%N2
(Invitrogen), 2%B27 (Invitrogen), 400 μM of dbcAMP (double fourth adenosine cyclophosphates, Sigma), 200 μM
Ascorbic acid (ascorbic acid usp/bp, Sigma), 10ng/m1BDNF (brain-derived neurotrophic factor,
Peprotech), 10ng/ml GDNF (glial cell line-derived neurotrophic factor, Peprotech).
Cell culture processes in following embodiments are as follows:
A, fibroblastic culture
With fibroblast culture medium culture, liquid, every 3-4 days one second generations of biography are changed every other day.It is passed on when passage by 1:3 or 1:4.
B, the culture induced multi-potent stem cell
B1 coating systems) are raised: the process mitomycin (U.S. Sigma for being seeded to and completing in advance will be induced multi-potent stem cell
Products, article No.: M0503) inactivation treatment mouse embryonic fibroblasts (U.S.'s Invitrogen Products, article No.:
S1520-100 in culture plate), using culture medium and mouse embryonic fibroblasts co-incubation is induced multi-potent stem cell, often
5-7 days one second generations of biography.
B2) without raising coating systems: will induce multi-potent stem cell and be seeded in advance with extracellular matrix (Growth factor
Article No.: reduced-Matrigel, U.S.'s BD Biosciences product 354277) in coated culture plate, use mTeSR
Culture medium (U.S.'s StemCell Technologies product) culture, every 5-7 days one second generations of biography.
C, the culture of neural stem cell
Neural stem cell is seeded in advance with extracellular matrix (Growth factor reduced-Matrigel, beauty
State's BD Biosciences product, article No.: 354277) in coated culture plate, with nerve stem cell culture medium culture.Every 4-5
It wears a second generation.
The preparation method of embodiment 1, a kind of neural stem cell of xeroderma pitmentosum people specific gene mutation and neuron
One, the acquisition and identification induced multi-potent stem cell
1, the acquisition induced multi-potent stem cell
The fibroblast for carrying the xeroderma pitmentosum people source of different genes mutation is reprogrammed respectively, respectively
Obtain XPA mutation induce multi-potent stem cell, XPB mutation induce multi-potent stem cell, XPC mutation induce multi-potent stem cell,
Inducing multi-potent stem cell for XPG mutation and inducing multi-potent stem cell for XPV mutation.The specific method is as follows:
(1) cell recovery and expansion culture
Following five plants are carried respectively the skin fibroblasts in the xeroderma pitmentosum people source of different genes mutation:
XPA fibroblast (GM00710), XPB fibroblast (GM13026), XPC fibroblast (GM15709), XPG are at fibre
Dimension cell (GM13371) and XPV fibroblast (GM03055) expand culture in fibroblast culture medium.To thin
Born of the same parents' culture is digested to proper density, is counted, and five kinds of fibroblasts respectively take 1.5 × 106It is a.
(2) electricity turns the preparation of liquid
By 82ul P2primary cell solution (Lonza), 18ul supplementary 1 (Lonza),
1.5ug pCXLE-hOCT3/4-shp53-F, 1.5ug pCXLE-hSK, 1.5ug pCXLE-hUL and 1.5ug pCXLE-EGFP
It mixes, obtains electricity and turn liquid, be placed in 1.5ml EP pipe.3 reprogramming plasmids (pCXLE-hOCT3/4-shp53-F, pCXLE-
HSK and pCXLE-hUL) and 1 markup plasmid (pCXLE-EGFP) buy in Addgene, catalog number difference is as follows:
PCXLE-hOCT3/4-shp53-F (#27077), pCXLE-hSK (#27078), pCXLE-hUL (#27080), pCXLE-EGFP
(#27082)。
(3) electricity turns
With step (2) obtain electricity turn liquid respectively to step (1) obtain XPA fibroblast, XPB fibroblast,
XPC fibroblast, XPG fibroblast and XPV fibroblast are resuspended, and are transferred in electric revolving cup after resuspension, and put
Enter in electroporation (Lonza 4D nucleofector), carry out electricity and turn, electricity turns procedure selection EN150 (specific steps reference
Lonza 4D nucleofector specification), respectively obtain the cell suspension (1.5 × 10 after electricity turns6Cell/100ul).With
Wild type fibroblast is control.
(4) cell suspension after respectively turning electricity is added to the hole of six orifice plates containing fibroblast culture medium of preheating
In, it rocks uniformly, puts back in incubator and cultivate.Liquid is changed within second day, electric transfer efficient is observed.
(5) continue with fibroblast culture medium culture to the 5th day, digestive inoculation passes through mitomycin to what is completed in advance
In the culture plate of the mouse embryonic fibroblasts of inactivation treatment, persistently cultivated using culture medium is induced multi-potent stem cell.Culture
To third week, the small clone (inducing multi-potent stem cell) similar to embryonic stem cell is respectively obtained, XPA fibroblast is induced
Inducing multi-potent stem cell for obtaining is named as inducing multi-potent stem cell for XPA mutation, lures what XPB fibroblast induced
It leads multipotential stem cell and is named as inducing multi-potent stem cell for XPB mutation, the induced multi-potent that XPC fibroblast induces is done
Cell is named as inducing multi-potent stem cell for XPC mutation, induces multi-potent stem cell name for what XPG fibroblast induced
For inducing multi-potent stem cell for XPG mutation, inducing multi-potent stem cell of inducing of XPV fibroblast is named as XPV and is dashed forward
What is become induces multi-potent stem cell, and inducing multi-potent stem cell of inducing of the fibroblast of wild type is named as wild type
It induces multi-potent stem cell.The form induced multi-potent stem cell that different fibroblasts induce is as shown in Figure 1B.Respectively will
Small clone chooses into the culture plate for the mouse embryonic fibroblasts by mitomycin inactivation completed in advance to expand and cultivate.
2, the identification induced multi-potent stem cell
In order to which whether the clone's shape cell for identifying that above-mentioned steps 1 obtain is induction type multipotential stem cell, tested as follows:
(1) expression of Immunofluorescence test stem cell labeling object NANOG, OCT4, SOX2
Resulting the inducing multi-potent stem cell for gene mutation of five kinds of carryings of step 1 is done with the induced multi-potent of wild type respectively
Cell inoculation after its is adherent, is first consolidated with 4% paraformaldehyde to in the processed mouse embryonic fibroblasts of mitomycin
After fixed 30 minutes, then with phosphate buffer (PBS) rinse 3 times, then with penetrating 30 points of PBS containing 0-4%TritonX-100
Clock, PBS are washed 3 times.Then 1 hour is closed with 10% donkey serum.Primary antibody according to proper proportion (anti-human source of mouse OCT4 antibody,
Sc-5279,1:100；Anti-human sheep source SOX2 antibody, sc-17320,1:100；Anti-human rabbit source NANOG antibody, ab21624,1:
250) it is incubated overnight after diluting in 4 DEG C.Second day, then be added after washing 3 times with PBS the diluted secondary antibody of proper proportion be incubated for it is 1 small
When.Nucleus is dyed with Hoechst33342 (Invitrogen, article No.: H3569).After PBS washes 3 times, mountant is used
(Vector company, article No. H-1000) mounting.It is finally observed, is taken pictures under laser confocal microscope.
Testing result is as shown in Figure 2 A: it can be seen from the figure that the induced multi-potent of the five kinds of carrying gene mutations obtained is dry
Cell expresses OCT4, NANOG, and the marker of these three stem cells of SOX2 illustrates the induced multi-potent of five kinds of carrying gene mutations
Stem cell maintains good cell stemness.
(2) qPCR detects the integration of foreign vector EBNA1 in multipotential stem cell
The base induced multi-potent stem cell of five kinds of carrying gene mutations induced multi-potent stem cell with wild type is extracted respectively
Because of a group DNA, qPCR is carried out using following primer.Simultaneously using 4 days after electricity turn wild type fibroblasts as positive control, quotient
The H9 embryonic stem cell of product is as negative control.Plasmid pCXLE-hFbx15-cont2 is used to generate FBXO15's and EBNA-1
The standard curve of copy number and cycle threshold.
FBXO15-F:GCC AGG AGG TCT TCG CTG TA
FBXO15-R:AAT GCA CGG CTA GGG TCA AA
EBNA-1-F:ATC AGG GCC AAG ACA TAG AGA TG
EBNA-1-R:GCC AAT GCA ACT TGG ACG TT
As a result as shown in Figure 2 B: there are many copy number that electricity turns latter 4 days fibroblastic EBNA-1, and five kinds obtained
Carry gene mutation induces multi-potent stem cell, explanation extremely low with the copy number for inducing multi-potent stem cell middle EBNA-1 of wild type
Five kinds of the inducing multi-potent stem cell for gene mutation of carrying obtained and inducing multi-potent stem cell almost without foreign gene for wild type
Insertion.
(3) teratoma is tested
Respectively by the inducing multi-potent stem cell of step 1 resulting XPA mutation, the inducing multi-potent stem cell of XPB mutation, XPC
The inducing multi-potent stem cell of mutation, inducing multi-potent stem cell for XPG mutation and inducing multi-potent stem cell and being inoculated into not for XPV mutation
Have and cultivated on the coated culture plate of the Matrigel of feeder layer, covered with wait induce multi-potent stem cell, digestion counts, and with containing 20%
The mixed liquor of Matrigel, 80%mTesR are to inducing multi-potent stem cell (4 × 106It is a) it is resuspended, obtain cell suspension；And
It is subcutaneous that the cell suspension of acquisition is squeezed into immunodeficient mouse.After about 8 weeks, that is, there is the teratoma of soya bean size to grow up to.De- neck
After putting to death mouse, teratoma is taken out, fixed in 4% paraformaldehyde two angels its it is sufficiently fixed after, steep molten in 30% sucrose
It is dehydrated in liquid.After it is sufficiently dehydrated, embedded using Compound O.C.T embedding medium.Then enterprising in freezing microtome
Row slice, with a thickness of 12 μm, after histotomy dries, can carry out the expression of three germinal layer markers of Immunofluorescence test.Exempt from
Epidemic disease fluorescence operation step is the same as above-mentioned steps 1.Wherein, primary antibody include: anti-human rabbit source TUJ1 antibody (Sigma company, article No. T2220,
1:500)；Anti-human rabbit source FOXA2 antibody (Cell Signaling Technology company, article No. 8186,1:200)；Anti-human mouse
Source-SMA antibody (Sigma company, article No. A5228,1:200).
As a result as shown in Figure 2 C: the inducing multi-potent stem cell of XPA mutation, the inducing multi-potent stem cell of XPB mutation, XPC are prominent
Become induce multi-potent stem cell, XPG mutation induce multi-potent stem cell and inducing multi-potent stem cell for XPV mutation is differentiated to form
Teratoma all include it is outer, in, interior three germinal layers further demonstrate inducing multi-potent stem cell for five kinds of carrying gene mutations
Totipotency.
(4) karyotyping
Inducing multi-potent stem cell for five kinds of carrying gene mutations is handled with colchicine, handles 40 minutes, makes cell block
After mid-term, treated cell is collected；It is handled 30 minutes with 0.075M Kcl hypotonic medium at 37 degree, then with containing methanol: ice vinegar
Acid=3:1 fixer is fixed twice；The cell fixed can carry out drop piece, and cell is dripped to glass slide by suitable density
On, it is steamed 10 seconds or so on 90 degree of water-baths with steam at once, is put into 70 degree of baking ovens and bakes 3 hours；Pancreatin digests 25~45s
Afterwards, it after physiological saline rinsing, is dyed 5-10 minutes at 37 degree with Gimsa dye liquor, tap water dries.Finally with micro- sem observation.
As a result as shown in Figure 2 D, five kinds of inducing multi-potent stem cell for gene mutation of carrying of acquisition keep normal core
Type illustrates that these gene mutations do not cause chromosome abnormality.
Two, the identification that directed differentiation is neural stem cell and neural stem cell is induced multi-potent stem cell
1, inducing multi-potent stem cell directed differentiation is neural stem cell
Five kinds of inducing multi-potent stem cell for gene mutation of carrying in step 1 are done with the induced multi-potent of wild type respectively
Cell directional is divided into neural stem cell (Fig. 3 A), and neural stem cell, the nerve cord of XPB mutation for respectively obtaining XPA mutation are thin
Neural stem cell, the neural stem cell of XPV mutation and the nerve of wild type that born of the same parents, the neural stem cell of XPC mutation, XPG are mutated
Stem cell.Specific step is as follows:
(1) the small clone induced multi-potent stem cell by five kinds of carrying gene mutations that (5) in the 1 of step 1 obtain connects
Kind to in the processed mouse embryonic fibroblasts of mitomycin, with inducing multi-potent stem cell culture medium culture about 2 days,
Reach 20% or so to its density, the cell after obtaining 1 culture.
(2) cell after 1 culture is continued to cultivate in neural stem cell induced medium 1, changes liquid daily, it is lasting to cultivate
2 days, the cell after obtaining 2 cultures.
(3) cell after 2 cultures is continued to cultivate in neural stem cell induced medium 2, changes liquid daily, it is lasting to cultivate
5 days, the cell after obtaining 3 cultures.
(4) then by 3 times culture after cell with it is unicellular go to Matrigel (U.S.'s BD Biosciences product,
Article No.: 354277) being cultivated in coated culture plate with nerve stem cell culture medium, and passage in every 4-5 days is primary, is continuously reached
The neural stem cell of xeroderma pitmentosum human specific can be obtained in the third generation.
2, the identification of neural stem cell
The neural stem cell of the neural stem cell, XPB mutation that are mutated by the XPA that Immunofluorescence test step 1 obtains,
The neural stem cell of neural stem cell, XPV mutation that the neural stem cell of XPC mutation, XPG are mutated and the nerve cord of wild type are thin
The expression of born of the same parents' marker NESTIN and PAX6.(1) in the 2 of specific experiment step reference step 1.Primary antibody and thinner ratio
Under such as: anti-human NESTIN antibody (Millipore company, article No. MAB5326,1:250) and anti-human PAX6 antibody (Covance
Company, article No. PRB-278P, 1:500).
Shown in result figure 3B: the neural stem cell for the XPA mutation broken up, the neural stem cell of XPB mutation, XPC are prominent
The neural stem cell of neural stem cell, XPV mutation that the neural stem cell of change, XPG are mutated and the neural stem cell of wild type are equal
Express the specific marker object of neural stem cell.
Three, neural stem cell continues the identification for being divided into neuron and neuron
1, neural stem cell continues to be divided into neuron
The mind that the neural stem cell for the XPA mutation that step 2 is obtained respectively, the neural stem cell of XPB mutation, XPC are mutated
Neural stem cell, the neural stem cell of XPV mutation and the neural stem cell of wild type being mutated through stem cell, XPG continue to break up
At the neuron (Fig. 4 A) for carrying xeroderma pitmentosum people specific gene mutation, neuron, the XPB for respectively obtaining XPA mutation are prominent
The neuron of change, the neuron of XPC mutation, the neuron of XPG mutation, the neuron of XPV mutation and the neuron of wild type.Tool
Body method is as follows:
(1) that the neural stem cell of five kinds of xeroderma pitmentosum human specifics is inoculated into Matrigel with low-density is coated
In culture plate, with the continuous culture of Neuronal induction culture medium 18 days.
(2) Laminin ELISA Laminin (Sigma company, article No. are added in the medium in the third day that differentiation carries out
L4544), help the adherent of neuron axon and extend.
(3) culture is about after two weeks i.e. it can be seen that have long protrusion, the preferable neuron of form.
2, the identification of neuron
The neuron of the XPA mutation obtained by Immunofluorescence test step 1, the neuron of XPB mutation, XPC are mutated
The expression of the neuron marker MAP2 and Tuj1 of the neuron and wild type of neuron, XPV mutation that neuron, XPG are mutated
Situation.(1) in the 2 of specific experiment step reference step 1.Primary antibody and dilution ratio are as follows: (Sigma is public for anti-human MAP2 antibody
Department, article No. T2220,1:500) and anti-human Tuj1 antibody (Sigma company, article No. 4403,1:500).
Shown in result figure 4B: the nerve that the neuron for the XPA mutation broken up, the neuron of XPB mutation, XPC are mutated
Neuron, the neuron of XPV mutation and the neuron of wild type that member, XPG are mutated express the specific marker object of neuron.
The UV sensibility of embodiment 2, the neural stem cell for carrying xeroderma pitmentosum people specific gene mutation and neuron
Verifying
After UV irradiation, DNA mainly exists with two kinds of damage types, respectively cyclobutane pyrimidine dimer (cyclobutane
Pyrimidine dimers, CPD) and 6-4 photoproduct (6-4pyrimidine photoproducts, 6-4PPs).Both
Form places one's entire reliance upon intracellular nucleotide excision repair pathways to repair, therefore the content of CPD can be used to indicator cells nucleosides
The ability that acid shearing is repaired.The present embodiment irradiates five kinds of carrying colorings of the preparation of embodiment 1 using the UV of doses respectively
The neural stem cell and neuron of dry hide patient's specific gene mutation, and detect intracellular CPD content and cell biology it is anti-
It answers.
One, the UV sensibility verifying of the neural stem cell of xeroderma pitmentosum people specific gene mutation is carried
1, the content and cell biology reaction of the CPD of neural stem cell are detected after UV processing
The neural stem cell for five kinds of gene mutations that the step of embodiment 1 two are obtained is with 1.5 × 105The density of a cell
24 orifice plates are inoculated in, use 1J/m within second day2UV is irradiated.It is cultivated after irradiation for 24 hours, Immunofluorescence test intracellular loops butane is phonetic
The content of pyridine dimer (CPD).(1) in the 2 of the step of specific experiment step reference embodiment 1 one.Primary antibody and dilution ratio
It is as follows: anti-human CPD antibody (Cosmo Bio company, article No. TMD-2).It is control with the neural stem cell of wild type.
As a result as shown in Figure 5A, the neural stem cell that discovery carries gene mutation all has one in the ability for repairing CPD
Determine the defect of degree, the especially neural stem cell of XPA gene mutation.
2, the apoptosis situation of neural stem cell is detected after UV processing
The neural stem cell of XPA gene mutation is detected using Annexin V-FITC/PI cell apoptosis detection kit
Apoptosis situation.It is control with the neural stem cell of wild type.
As a result as shown in Figure 5 B, as can be seen from the figure: the neural stem cell (XPA neural stem cell) of XPA gene mutation
After UV irradiates Apoptosis ratio increase, the neural stem cell (XPA neural stem cell) of XPA gene mutation is easier to send out
Raw Apoptosis.
3, DNA break situation in neural stem cell is detected after UV processing
With situ end labeling (Terminal deoxynucleotidyl transferase (TdT)-mediated
DUTP nick-end labeling, TUNEL) detect DNA break situation in the neural stem cell of XPA gene mutation.With wild
The neural stem cell of type is control.
As a result as shown in Figure 5 C: it can be seen from the figure that occurring in the neural stem cell of XPA gene mutation after UV processing
DNA break situation.
The above experiment proves the neural stem cell for carrying patient's gene mutation after UV irradiation since nucleotide excision is repaired
Reactivation power is impaired to cause intracellular DNA damage to be accumulated, and wherein the DNA damage of XPA mutation accumulation is the most serious, also occurs at high proportion
Apoptosis.
Two, the UV sensibility verifying of the neuron of xeroderma pitmentosum people specific gene mutation is carried
1, the content and cell biology reaction of the CPD of neuron are detected after UV processing
The neuron for the XPA mutation that the step of embodiment 1 three are prepared is with 1.5 × 105The density of a cell is inoculated in 24
Orifice plate uses 1J/m in second day2UV is irradiated.Renewal cultivation for 24 hours, detects intracellular cyclobutane pyrimidine dimer after irradiation
(CPD) content.(1) referring to the step of embodiment 1 in the 2 of one.Induction of committed differentiation is induced multi-potent stem cell with wild type
Neuron be control.
As a result as shown in Figure 5 D, it can be seen from the figure that there are one in the ability for repairing CPD for the neuron of XPA mutation
Determine the defect of degree.
2, DNA break situation in neuronal cell is detected after UV processing
With situ end labeling (Terminal deoxynucleotidyl transferase (TdT)-mediated
DUTP nick-end labeling, TUNEL) detect DNA break situation in the neuronal cell of XPA mutation.
As a result as shown in fig. 5e: it can be seen from the figure that it is disconnected that DNA occurs in the neuronal cell that XPA is mutated after UV processing
Split situation.Also illustrate that the level of apoptosis of the neuronal cell of XPA mutation increases.
The above experiment proves to carry the neuron of patient's gene mutation due to nucleotide excision repair energy after UV irradiation
Power is impaired to cause intracellular DNA damage to be accumulated, and wherein the DNA damage of XPA mutation accumulation is the most serious, and cell also occurs at high proportion
Apoptosis.
Claims (5)
1. a kind of preparation method of the neuron of xeroderma pitmentosum human specific, includes the following steps:
(1) fibroblast in vitro xeroderma pitmentosum people source is reprogrammed, obtains carrying xeroderma pitmentosum
People's gene induces multi-potent stem cell；
The fibroblast in the in vitro xeroderma pitmentosum people source is XPA fibroblast, XPB fibroblast, XPC
Fibroblast, XPG fibroblast or XPV fibroblast；It is described reprogramming for by gene OCT3/4, SOX2, KLF4,
LMyc and LIN28 is imported jointly in the fibroblast in vitro xeroderma pitmentosum people source；
For XPA fibroblast from the skin fibroblasts of XPA patient, the mutation carried is the 619th of XPA gene
Site is mutated into T by C, and when transcription forms terminator codon, and the translation of XPA albumen is caused to terminate in advance；
For XPB fibroblast from the skin fibroblasts of XPB patient, the mutation carried is the 295th of XPB gene
Site T is mutated into C, and the 99th amino acid of XPB albumen is caused to be mutated into serine by phenylalanine；
For XPC fibroblast from the skin fibroblasts of XPC patient, the mutation carried is the third in XPC gene
There are a T to become A in a introne, affects the shearing of XPC mRNA；
For XPG fibroblast from the skin fibroblasts of XPG patient, the mutation carried is XPG gene the 2171st
The A base of point lacks, and causes the frameshit of mRNA encoder block；
For XPV fibroblast from the skin fibroblasts of XPV patient, the mutation carried is the 376th of XPV gene
Site is mutated into T by C, forms terminator codon in mRNA transcription, leads to the truncation of XPV albumen；The induced multi-potent is dry thin
The acquisition specific steps of born of the same parents are as follows:
(a) cell recovery and expansion culture
Respectively by following five plants carry different genes mutation xeroderma pitmentosum people source skin fibroblasts: XPA at
Fibrocyte, XPB fibroblast, XPC fibroblast, XPG fibroblast and XPV fibroblast are in fibroblast
It is expanded culture in culture medium；It to cell culture to proper density, digesting, counts, five kinds of fibroblasts respectively take 1.5 ×
106It is a；
(b) electricity turns the preparation of liquid
By 82ul P2 primary cell solution, 18ul supplementary 1,1.5ug pCXLE-hOCT3/4-
Shp53-F, 1.5ug pCXLE-hSK, 1.5ug pCXLE-hUL and 1.5ug pCXLE-EGFP are mixed, and are obtained electricity and are turned liquid, are placed in
In 1.5ml EP pipe；3 reprogramming plasmid pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-hUL and 1 label
Property grain pCXLE-EGFP is bought in Addgene, and catalog number difference is as follows: the production of pCXLE-hOCT3/4-shp53-F
Product catalog number (Cat.No.) is #27077, and the catalog number of pCXLE-hSK is #27078, and the catalog number of pCXLE-hUL is #27080,
The catalog number of pCXLE-EGFP is #27082；
(c) electricity turns
With step (b) obtain electricity turn liquid respectively to step (a) obtain XPA fibroblast, XPB fibroblast, XPC at
Fibrocyte, XPG fibroblast and XPV fibroblast are resuspended, and are transferred in electric revolving cup after resuspension, and are put into electricity and are turned
It in instrument, carries out electricity and turns, electricity turns procedure selection EN150, and specific steps refer to Lonza 4D nucleofector specification, respectively
Obtain the cell suspension after electricity turns;
(d) cell suspension after respectively turning electricity is added in the hole of six orifice plates containing fibroblast culture medium of preheating,
It rocks uniformly, puts back in incubator and cultivate, change within second day liquid, observe electric transfer efficient;
(e) continue with fibroblast culture medium culture to the 5th day, digestive inoculation is inactivated to what is completed in advance by mitomycin
In the culture plate of the mouse embryonic fibroblasts of processing, persistently cultivated using culture medium is induced multi-potent stem cell；Culture is to the
It three weeks, respectively obtains and induces multi-potent stem cell, inducing multi-potent stem cell of inducing of XPA fibroblast is named as XPA
Mutation induces multi-potent stem cell, and inducing multi-potent stem cell of inducing of XPB fibroblast is named as luring for XPB mutation
Multipotential stem cell is led, the induced multi-potent that inducing multi-potent stem cell of inducing of XPC fibroblast is named as XPC mutation is done
Inducing multi-potent stem cell of inducing of XPG fibroblast is named as inducing multi-potent stem cell for XPG mutation by cell, will
What XPV fibroblast induced, which induce multi-potent stem cell, is named as inducing multi-potent stem cell for XPV mutation；
(2) induction differentiation is oriented to described induce multi-potent stem cell, obtains the nerve for carrying xeroderma pitmentosum people's gene
Stem cell；Described pair induce multi-potent stem cell be oriented induction differentiation include the following steps:
A carry out Fiber differentiation) is induced multi-potent stem cell to described in inducing multi-potent stem cell culture medium, after obtaining Fiber differentiation
Cell；
B differentiation for the first time) is carried out to cell after the Fiber differentiation in neural stem cell induced medium 1 to cultivate, and obtains the
Cell after primary differentiation culture；
C) second of differentiation is carried out to cell after first time differentiation culture in neural stem cell induced medium 2 to cultivate,
The cell that second of differentiation culture is obtained secondary culture in the induced medium of the neural stem cell basis, is taken
Neural stem cell with xeroderma pitmentosum people's gene；
Neural stem cell basis induced medium be in cell culture medium add Neurobasal, N2, B27,
The culture medium that GlutaMAX, hLIF, CHIR99021 and SB431542 are obtained；
The neural stem cell induced medium 2 is to add Compound E in the induced medium of neural stem cell basis to obtain
Culture medium, concentration of the Compound E in the neural stem cell induced medium 2 be 0.1-0.5 μM；
The neural stem cell induced medium 1 is to add Dorsomorphin in neural stem cell induced medium 2 to obtain
Culture medium, concentration of the Dorsomorphin in the neural stem cell induced medium 1 be 1-3 μM；
(3) induction differentiation is oriented to the neural stem cell, obtains the neuron for carrying xeroderma pitmentosum people's gene；
Described be oriented neural stem cell is induced to differentiate into the neural stem cell described in neuronal differentiation medium culture；
The neuronal differentiation medium is that double fourth adenosine cyclophosphates, ascorbic acid dimension are added in neuron differentiation basal medium
The culture medium that raw element C, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor obtain；
Concentration of the double fourth adenosine cyclophosphates in the neuronal differentiation medium is 350-450 μM；
Concentration of the ascorbic acid usp/bp in the neuronal differentiation medium is 150-250 μM；
Concentration of the brain-derived neurotrophic factor in the neuronal differentiation medium is 5-15 ng/m1；
Concentration of the glial cell line-derived neurotrophic factor in the neuronal differentiation medium is 5-15 ng/m1；
The neuron differentiation basal medium is the culture medium that addition N2 and B27 is obtained in cell culture medium；
Step A) in, the time of the Fiber differentiation is 1-3 days；
Step B) in, the time of the first time differentiation culture is 1-3 days；
Step C) in, the time of second of differentiation culture is 4-6 days；
In step (3), the time of the culture is 14-21 days；
The pCXLE-hOCT3/4-shP53-F is the Episomal carrier for expressing OCT3/4；
The pCXLE-hSK is the Episomal carrier for expressing SOX2 and KLF4；
The pCXLE-hUL is the Episomal carrier for expressing LMyc and LIN28；
The pCXLE-EGFP is the carrier for expressing marker gene, and the marker gene is EGFP, the nucleotides sequence of the EGFP
The sequence 6 being classified as in sequence table.
2. the neuron that method described in claim 1 is prepared.
3. method described in claim 1 screens the production for treating or preventing the natural organic matter of xeroderma pitmentosum in preparation
Application in product.
4. method described in claim 1 screens the product for treating or preventing the clinical medicine of xeroderma pitmentosum in preparation
In application.
5. small molecule compound of the method described in claim 1 in preparation screening for treating or preventing xeroderma pitmentosum
Application in product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610069342.8A CN105567642B (en) | 2016-02-01 | 2016-02-01 | A kind of preparation method of xeroderma pitmentosum human pluripotent stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610069342.8A CN105567642B (en) | 2016-02-01 | 2016-02-01 | A kind of preparation method of xeroderma pitmentosum human pluripotent stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105567642A CN105567642A (en) | 2016-05-11 |
| CN105567642B true CN105567642B (en) | 2019-07-12 |
Family
ID=55878274
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610069342.8A Active CN105567642B (en) | 2016-02-01 | 2016-02-01 | A kind of preparation method of xeroderma pitmentosum human pluripotent stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105567642B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350521B (en) * | 2016-08-24 | 2019-11-12 | 中国科学院生物物理研究所 | A kind of preparation method of the patient-specific motor neuron of ALS |
| CN106834233B (en) * | 2016-09-07 | 2019-08-13 | 余爽 | A method of preparing can the real-time source tracer iPSCs nerve member |
| US20200407422A1 (en) * | 2017-09-05 | 2020-12-31 | GLAdiator Biosciences, Inc. | A method of intracellular delivery |
| CN109122581A (en) * | 2018-09-18 | 2019-01-04 | 南通市第二人民医院 | Fra-1 and application of the XPA compound in cell cycle regulating |
| CN109852587B (en) * | 2019-03-07 | 2021-04-27 | 中国科学院动物研究所 | Preparation method of human Kekan syndrome specific adult stem cells |
| CN114807141A (en) * | 2022-05-13 | 2022-07-29 | 复旦大学附属中山医院 | Endothelial cell line for inhibiting XPB expression substance and XPB low expression thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007098954A1 (en) * | 2006-03-03 | 2007-09-07 | Ciemat | A mouse model comprising an engrafted human skin having hypersensitivity to uv-light |
| CN101824398A (en) * | 2009-03-05 | 2010-09-08 | 中日友好医院 | Method for co-culturing, inducing and differentiating dopaminergic neuron by human amniotic epithelial cells and neural stem cells |
| WO2012034101A2 (en) * | 2010-09-09 | 2012-03-15 | The Regents Of The University Of California | Expandable cell source of neuronal stem cell populations and methods for obtaining and using them |
| CN103031335A (en) * | 2011-10-09 | 2013-04-10 | 李川源 | Method for generating dopamine-like neurons from human fibroblasts through direct induction and application of dopamine-like neurons |
| CN103740757A (en) * | 2014-01-21 | 2014-04-23 | 中国科学院生物物理研究所 | Method for preparing pig neural stem cells by reprogramming |
| CN104080906A (en) * | 2011-12-01 | 2014-10-01 | 纽约干细胞基金会 | Automated system for producing induced pluripotent stem cells or differentiated cells |
| CN104379731A (en) * | 2011-11-04 | 2015-02-25 | 纪念斯隆-凯特琳癌症中心 | Midbrain dopamine (DA) neurons for engraftment |
| CN104419683A (en) * | 2013-09-10 | 2015-03-18 | 中国科学院生物物理研究所 | Method for preparing autologous induced pluripotent stem cells of patient with Fanconi anemia and application of method |
| WO2015179822A1 (en) * | 2014-05-22 | 2015-11-26 | The New York Stem Cell Foundation | Functional oligodendrocytes derived from pluripotent stem cells and methods of making and using the same |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2010315166B2 (en) * | 2009-11-04 | 2015-01-22 | FUJIFILM Cellular Dynamics, Inc. | Episomal reprogramming with chemicals |
| US10155929B2 (en) * | 2012-05-13 | 2018-12-18 | Allele Biotechnology & Pharmaceuticals, Inc. | Feeder-free derivation of human-induced pluripotent stem cells with synthetic messenger RNA |
| US20140162366A1 (en) * | 2012-08-31 | 2014-06-12 | Salk Institute For Biological Studies | Generation of vascular progenitor cells |
-
2016
- 2016-02-01 CN CN201610069342.8A patent/CN105567642B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007098954A1 (en) * | 2006-03-03 | 2007-09-07 | Ciemat | A mouse model comprising an engrafted human skin having hypersensitivity to uv-light |
| CN101824398A (en) * | 2009-03-05 | 2010-09-08 | 中日友好医院 | Method for co-culturing, inducing and differentiating dopaminergic neuron by human amniotic epithelial cells and neural stem cells |
| WO2012034101A2 (en) * | 2010-09-09 | 2012-03-15 | The Regents Of The University Of California | Expandable cell source of neuronal stem cell populations and methods for obtaining and using them |
| CN103031335A (en) * | 2011-10-09 | 2013-04-10 | 李川源 | Method for generating dopamine-like neurons from human fibroblasts through direct induction and application of dopamine-like neurons |
| CN104379731A (en) * | 2011-11-04 | 2015-02-25 | 纪念斯隆-凯特琳癌症中心 | Midbrain dopamine (DA) neurons for engraftment |
| CN104080906A (en) * | 2011-12-01 | 2014-10-01 | 纽约干细胞基金会 | Automated system for producing induced pluripotent stem cells or differentiated cells |
| CN104419683A (en) * | 2013-09-10 | 2015-03-18 | 中国科学院生物物理研究所 | Method for preparing autologous induced pluripotent stem cells of patient with Fanconi anemia and application of method |
| CN103740757A (en) * | 2014-01-21 | 2014-04-23 | 中国科学院生物物理研究所 | Method for preparing pig neural stem cells by reprogramming |
| WO2015179822A1 (en) * | 2014-05-22 | 2015-11-26 | The New York Stem Cell Foundation | Functional oligodendrocytes derived from pluripotent stem cells and methods of making and using the same |
Non-Patent Citations (1)
| Title |
|---|
| 《Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells》;Nancy Stanslowsky等;《Stanslowsky et al. Stem Cell Research & Therapy》;20141231;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105567642A (en) | 2016-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105567642B (en) | A kind of preparation method of xeroderma pitmentosum human pluripotent stem cells | |
| Kirkeby et al. | Predictive markers guide differentiation to improve graft outcome in clinical translation of hESC-based therapy for Parkinson’s disease | |
| Engelhardt et al. | Adult retinal pigment epithelium cells express neural progenitor properties and the neuronal precursor protein doublecortin | |
| CN106350521B (en) | A kind of preparation method of the patient-specific motor neuron of ALS | |
| CN108883137A (en) | Neural organoid composition and application method | |
| Jeon et al. | Regeneration of the retina: toward stem cell therapy for degenerative retinal diseases | |
| JP7219618B2 (en) | Cell reprogramming method using environmental influx by physical stimulation | |
| Gill et al. | Methods of retinal ganglion cell differentiation from pluripotent stem cells | |
| CN103740757B (en) | A kind of method utilizing reprogrammed to prepare pig neural stem cell | |
| CN104195108B (en) | Purposes of the kinases inhibitor in nerve cell is prepared from non-neuronal cells | |
| TWI493034B (en) | Neuronal epithelial cells differentiated by universal stem cells and the medium used and their differentiation methods | |
| Belinsky et al. | Physiological properties of neurons derived from human embryonic stem cells using a dibutyryl cyclic AMP-based protocol | |
| CN108531453A (en) | A method of converting non-neuronal cell to neuronal cell | |
| CN105420193B (en) | Differential medium and its purposes in preparation neural stem cell | |
| Isaev et al. | In vitro differentiation of human parthenogenetic stem cells into neural lineages | |
| CN107438668A (en) | Utilize the pluripotent cell apparatus for deivation and method of energy | |
| CN106047800B (en) | Pig multipotential stem cell Induction of committed differentiation is the method and special culture media of male sex-cell | |
| CN102796696A (en) | Neurons directly induced from human skin cells and preparation method for neurons | |
| Maxwell et al. | Analysis of the development of cellular subsets present in the neural crest using cell sorting and cell culture | |
| KR102087294B1 (en) | A method for preparing the Parkinson's disease model comprising 3D intestinal organoid and 3D neuro ectodermal sphere, and use thereof | |
| McGrath et al. | Proliferation and differentiation of human fetal brain neural stem cells in vitro | |
| KR20130133598A (en) | Screening method for candidate drugs for huntington's disease using induced pluripotent stem cells derived from huntington's disease patients | |
| KR101223396B1 (en) | Method for derivation of pluripotent stem cells using embryonic stem cell extract and pluripotent stem cells produced by the method | |
| Li et al. | Directional differentiation of chicken primordial germ cells into adipocytes, neuron‐like cells, and osteoblasts | |
| Billon et al. | Generation and characterization of oligodendrocytes from lineage-selectable embryonic stem cells in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |