CN108441517A - A kind of preparation method of people's induced multi-potent stem cell - Google Patents
A kind of preparation method of people's induced multi-potent stem cell Download PDFInfo
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- CN108441517A CN108441517A CN201810261803.0A CN201810261803A CN108441517A CN 108441517 A CN108441517 A CN 108441517A CN 201810261803 A CN201810261803 A CN 201810261803A CN 108441517 A CN108441517 A CN 108441517A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/602—Sox-2
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/603—Oct-3/4
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/604—Klf-4
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/606—Transcription factors c-Myc
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/10041—Use of virus, viral particle or viral elements as a vector
- C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
A kind of method of human milk tooth cell induction reprogramming, including：(1) milk tooth cell is detached；(2) milk tooth cell is cultivated；(3) Oct4, Sox2, c Myc and tetra- kinds of factors of Klf4 are transferred into milk tooth cell；(3) telomerase RNA, transient expression Telomerase are transferred to；(5) versatility is identified.The milk tooth that children fall off dental transitional period is made full use of and prepares multipotential stem cell by the method, in addition, ensuring safety while extending the telomere length of cell using telomerase RNA.
Description
Technical field
The invention belongs to biotechnologies, especially Stem Cell Engineering field.It is dry more particularly to a kind of people's induced multi-potent
The preparation method of cell.
Background technology
Stem cell can not only generate or secrete a large amount of bioactie agent, and also contain abundant life in stem cell
Active substances, these stem cell biological active factors or substance can Effective Regulation body cell signal transduction, activating human body it is dry
Cell, and then the cell etc. of physiological reparation or replacement body injury, lesion and aging.Such as stem cell can generate stem cell
Growth factor (SCF), nerve growth factor (NGF), stroma cell source property growth factor (SDF), vascular endothelial cell growth because
Sub (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor
(EGF), interleukin-6 and IL-7 (IL-6 and IL-7), megakaryocyte colony stimulating factor (M-CSF), tumor necrosis factor
(TNF), the factors such as interferon (IFN), these cell factors have the function of to promote cell Proliferation, differentiation, anti-apoptotic etc.；Stem cell
Innate immunity albumen can also be generated, itself and extraneous factor can be resisted or be repaired to such as IgG, IgA, IgM, IgD, IgE etc.
The damage caused by body cell.Therefore, the biology in the bioactie agent generated using stem cell secretion or stem cell is lived
Property substance activating body stem cell, and then body injury, lesion and aging are repaired or substituted by autologous stem cells physiological
Cell has broad application prospects in disease prevention and cure and health and beauty field.
2006, Kyoto Univ Japan Yamanaka (being stretched more in mountain) taught led research group and has filtered out 4
The gene transcription factor (Oct4/Sox2/Klf4/c-Myc) of high expression in embryonic stem cell or tumour cell, and utilize reverse
These factors are transfected into l cell by record viral vectors.By these factors in recipient cell overexpression,
Induced fibroblast dedifferentes, and it is multipotential stem cell to make adult cell reprogramming, and is defined as induced multi-potent stem cell
(Induced pluripotent stem cells, i PS).Induced multi-potent stem cell function almost as embryonic stem cell,
The various surface markers for expressing embryonic stem cell, can be divided into various histocytes, not by limits such as immunological rejection and ethics
System, shows unique advantage in terms of organizational project and reparation.
Human teeth possesses two systems, has fallen deciduous teeth and has just replaced with permanent teeth, the other organ of human body is all faithful to one's husband to the end, falls off
Deciduous teeth in include abundant cellular resources.Even if medicine and biology highly developed today, the deciduous teeth to fall off are not
It makes full use of, causes great waste.In recent years, having been reported that trial, (such as dental pulp is dry for separation stem cell from the deciduous teeth to fall off
Cell (DPSC), periodontal ligament stem cell (PDLSC) and tip of a root nipple stem cell (SCAP)), for follow-up further induction differentiation
It generates tooth and provides feasibility.
Telomerase can synthesize telomere repeat sequence and add to newly synthesized RNA chain ends, to anti-using itself RNA as template
Only end duplication problem is lost and solved to end of chromosome.In 3 subunits of Telomerase, TERT is the catalysis of telomerase activation
Subunit.Due to being expressed almost without TERT in normal somatic cell, thus these cell no telomerase activities, cell with mitosis not
It is disconnected to shorten, last aging death.Although expression can extend cell survival to Telomerase in right amount, overexpression be tumour cell again without
Limit the one of the major reasons of proliferation.
Invention content
For deficiency in the prior art, technical problem to be solved by the invention is to provide one kind can make full use of people
The method that the milk tooth that class falls off prepares induced multi-potent stem cell.The method of the invention is extracted in milk tooth all thin as far as possible
Born of the same parents' ingredient, makes full use of it.In addition, present invention transient expression Telomerase by way of being transferred to telomerase RNA, extends
Cell survival.
Specifically, the present invention provides a kind of methods of 1, human milk tooth cell induction reprogramming, including：
(1) milk tooth cell is detached；
(2) milk tooth cell is cultivated
(3) carrier of coding tetra- kinds of factors of Oct4, Sox2, c-Myc and Klf4 is transferred into milk tooth cell；
(4) telomerase RNA, transient expression Telomerase are transferred to；
(5) versatility is identified.
The method of human milk tooth cell induction reprogramming of the present invention, it is characterised in that the step (1) includes separation tooth
Myelocyte and dentin cell.
The method of human milk tooth cell induction reprogramming of the present invention, it is characterised in that separation pulp cells includes, in tooth
Enamel sclerotin boundary marks the groove as deep as pulp cavity, divides tooth, and pulp tissue is taken out as Hanks liquid with pincet
In, while rinsing pulp cavity with Hanks liquid and being incorporated into the Hanks liquid containing pulp tissue, enzyme eye scissors shred, and final concentration is added
The collagenase type I of the neutral proteinase II and 0.5mg/mL of 2mg/mL handle 15min, blow and beat repeatedly, are added containing 5%BSA's
Hanks liquid terminates reaction, crosses wire-mesh screen and obtains pulp cells.
The method of human milk tooth cell induction reprogramming of the present invention, it is characterised in that the milk tooth historrhexis of dental pulp will be removed extremely
The trypsin treatment 30min of the neutral proteinase I and 0.25mg/mL of final concentration 3mg/mL is added in average grain diameter 50-200 mesh,
It blows and beats repeatedly, the Hanks liquid containing 5%BSA is added and terminates reaction, dentin cell is resuspended to obtain after taking supernatant to centrifuge.
The method of human milk tooth cell induction reprogramming of the present invention, it is characterised in that the step (2) includes the tooth that will be detached
Myelocyte and dentin cell, which are blended in the 1640 culture medium containing 10% fetal calf serum, carries out original cuiture, every to change liquid for 24 hours,
Subculture is digested after 72h.
The method of human milk tooth cell of the present invention induction reprogramming, it is characterised in that the step (3) include will carry Oct4,
The retroviral vector of tetra- kinds of factors of Sox2, c-Myc and Klf4 and viral packaging plasmid cotransfection are to primary or squamous subculture
Milk tooth cell.
The method of human milk tooth cell of the present invention induction reprogramming, it is characterised in that the step (4) includes：It will synthesize in vitro
Telomerase catalytic subunit mRNA molecules be transferred to step (3) preparation cell, make its transient expression Telomerase, be inoculated in mouse embryo
On fibroblast, human embryonic stem cell medium is added and is cultivated.
The method of human milk tooth cell induction reprogramming of the present invention, it is characterised in that the sequence root of the Telomerase mRNA molecules
It is determined according to known telomerase catalytic subunit cDNA molecules, including poly (A) knots at the Cap structures at the end of code area 5 ' and 3 ' ends
Structure.
The method of human milk tooth cell induction reprogramming of the present invention, it is characterised in that the versatility identification includes alkaline phosphatase
Enzyme dyeing is tested and teratoma forms experiment.
Compared with the prior art, the advantages of the present invention are as follows：
Firstly, since the milk tooth to fall off comes from the mankind's young stage, therefore milk tooth source cell has higher reprogramming energy
Power.To fall off, milk tooth source cell prepares induced multi-potent instead of adult fibroblast, mescenchymal stem cell etc. as the cell that sets out
Stem cell, not only hurtless measure but also inverse differentiation effect is good.
Secondly, the present invention takes full advantage of milk tooth source cell, and milk tooth pulp cells is not only utilized and also uses dentine
In cell, the cell coexisted under these native states interacts due to existing, in Process of in vitro, when coexisting
It is more advantageous to the proliferation of growth.
Finally, normal cell telomere length is insufficient, and traditional overexpression mode is easy to cause canceration, and the present invention is straight
Switch through and synthesize Telomerase into mRNA, the extension for both having realized telomere in turn avoids the risk that Telomerase overexpression is brought.
Description of the drawings
Fig. 1 MEF feeder cells
Adherent MEF feeder cells are in spindle.
IPS prepared by Fig. 2 milk tooth cells
Form embryonic stem cell sample clone ball within three weeks after induction, the cell aggregation of a large amount of subcirculars, form in cell clone
Differ markedly from MEF feeder cells adherent outside clone ball.
Fig. 3 Teratoma sections
A class neural tube tissues；B class cartilaginous tissues；C class gland tissues.
Specific implementation mode
Below in conjunction with specific embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or presses
According to the condition proposed by manufacturer.Unless otherwise defined, all professional and scientific terms used in text and this field are ripe
It is identical to practice meaning known to personnel.In addition, any method and material similar or impartial to described content all can be applied to
In the method for the present invention.The preferred methods and materials described herein are for illustrative purposes only.
The preparation of one human milk tooth cell of embodiment
It is derived from the mankind's milk tooth for so falling off or extracting, detaches pulp cells.Concrete operations are demarcated in enamel sclerotin as follows
Place marks the groove as deep as pulp cavity, divides tooth, takes out pulp tissue as in Hanks liquid with pincet, while using Hanks
Liquid rinses pulp cavity and is incorporated into the Hanks liquid containing pulp tissue, and enzyme eye scissors shred, and the neutral egg of final concentration 2mg/mL is added
The collagenase type I of white enzyme II and 0.5mg/mL handle 15min, blow and beat repeatedly, and the Hanks liquid containing 5%BSA is added and terminates reaction,
It crosses wire-mesh screen and obtains pulp cells.
By the milk tooth historrhexis for removing dental pulp to average grain diameter 50-200 mesh, the neutral protein of final concentration 3mg/mL is added
The trypsin treatment 30min of enzyme I and 0.25mg/mL, blow and beat repeatedly, and the Hanks liquid containing 5%BSA is added and terminates reaction, takes
Dentin cell is resuspended to obtain after clearing the heart.
The pulp cells of separation and dentin cell are blended in the 1640 culture medium containing 10% fetal calf serum and carried out
Original cuiture digests subculture per liquid is changed for 24 hours after 72h；Continuous squamous subculture 1-3 is for amplifying cells.
The induction of two human milk tooth cell of embodiment reprograms
By the rat embryo fibroblast cell of purchase with 1 × 104CFU/cm2Concentration be inoculated in Tissue Culture Flask, addition contains
1640 complete mediums of 10% fetal calf serum are positioned over 37 DEG C, 5%CO2It is cultivated in incubator, it is overnight to change liquid, it is inverted
Micro- sem observation, until when cell fusion degree is about 70-75% spare (Fig. 1).
According to the method stretched in mountain more, be inserted into retroviral vector tetra- kinds of Oct4, Sox2, c-Myc and Klf4 because
Recombinant virus is to generate slow virus with viral package carrier cotransfection 293T cells, packaging, collect virus by the expression cassette of son
Liquid carries out titer determination.Virus liquid is pressed 1:The human milk tooth cell in 40 MOI inoculation squamous subculture 1-3 generations, 37 DEG C, 5%CO2It incubates
After educating 30min, 1640 complete mediums containing 10% fetal calf serum are added, continues overnight incubation, cell is digested with pancreatin, weight
Be added to after outstanding it is freshly prepared, be fused in the MEF cells of 70-75%, human embryonic stem cell medium is added and continues to cultivate.
The next day change liquid, inverted microscope observation gradually forms the cell clone group (Fig. 2) of similar human embryo stem cell sample.
The verification of three human milk tooth source IP S of embodiment
Human milk tooth IPS is passed on into sub-bottle, takes mankind's induced multi-potent stem cell of one bottle of secondary culture, inhales the mankind before abandoning
Embryonic stem cell complete medium is washed 2 times with Hanks liquid, and the paraformaldehyde (PFA) for being added 4% fixes 2 minutes, after cleaning
Alkaline phosphatase staining liquid is added, after being incubated 15min, Hanks liquid washs 2 times, and microscopy is observed under inverted microscope, cell clone
Royal purple color depth dye is presented in group, and the feeder cells of cell mass peripheral fiber shape are uncolored, show the cell in cell clone group
In undifferentiated state.
Take 1 × 105It is visible after four weeks to have tumor growth on the inside of the IPS cell infusions of CFU to NOD/SCID mouse stocks, after 8 weeks
Lump is won, histotomy HE dyeing, microscopically observation, the results are shown in Figure 3.Fig. 3 histotomies are the result shows that the present invention
The IPS cells of preparation have simultaneously to be formed in, in, the abilities of outer three embryonic tissues.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Technical staff is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, and should also belong to the protection of the present invention
Range, protection scope of the present invention are subject to claims.
Claims (9)
1. a kind of method of human milk tooth cell induction reprogramming, including：
(1) milk tooth cell is detached；
(2) milk tooth cell is cultivated；
(3) carrier of coding tetra- kinds of factors of Oct4, Sox2, c-Myc and Klf4 is transferred into milk tooth cell；
(4) telomerase RNA, transient expression Telomerase are transferred to；
(5) versatility is identified.
2. the method for human milk tooth cell induction reprogramming as described in claim 1, it is characterised in that the step (1) includes separation
Pulp cells and dentin cell.
3. the method for human milk tooth cell induction reprogramming as claimed in claim 2, it is characterised in that detaching pulp cells includes,
Enamel sclerotin boundary marks the groove as deep as pulp cavity, divides tooth, and pulp tissue is taken out as Hanks liquid with pincet
In, while rinsing pulp cavity with Hanks liquid and being incorporated into the Hanks liquid containing pulp tissue, enzyme eye scissors shred, and final concentration is added
The collagenase type I of the neutral proteinase II and 0.5mg/mL of 2mg/mL handle 15min, blow and beat repeatedly, are added containing 5%BSA's
Hanks liquid terminates reaction, crosses wire-mesh screen and obtains pulp cells.
4. the method for human milk tooth cell induction reprogramming as claimed in claim 2, it is characterised in that the milk tooth group of dental pulp will be removed
It knits and is crushed to average grain diameter 50-200 mesh, be added at the trypsase of the neutral proteinase I and 0.25mg/mL of final concentration 3mg/mL
30min is managed, is blown and beaten repeatedly, the Hanks liquid containing 5%BSA is added and terminates reaction, dentin cell is resuspended to obtain after taking supernatant to centrifuge.
5. the method for human milk tooth cell induction reprogramming as described in claim 1, it is characterised in that the step (2) includes that will divide
From pulp cells and dentin cell be blended in the 1640 culture medium containing 10% fetal calf serum and carry out original cuiture, often
Liquid is changed for 24 hours, digests subculture after 72h.
6. the method for human milk tooth cell induction reprogramming as described in claim 1, it is characterised in that the step (3) includes that will take
Retroviral vector with tetra- kinds of factors of Oct4, Sox2, c-Myc and Klf4 and viral packaging plasmid cotransfection to it is primary or after
The milk tooth cell for being commissioned to train foster.
7. the method for human milk tooth cell as described in claim 1 induction reprogramming, it is characterised in that the step (4) includes：It will
The telomerase catalytic subunit mRNA molecules synthesized in vitro are transferred to the cell of step (3) preparation, make its transient expression Telomerase, are inoculated with
It is cultivated in human embryonic stem cell medium on rat embryo fibroblast cell, is added.
8. the method for human milk tooth cell induction reprogramming as claimed in claim 7, it is characterised in that the Telomerase mRNA molecules
Sequence determined according to known telomerase catalytic subunit cDNA molecules, including code area 5 ' hold Cap structures and 3 ' end
Poly (A) structure.
9. the method for human milk tooth cell induction reprogramming as described in claim 1, it is characterised in that step (5) the versatility mirror
Surely include that alkaline phosphatase staining experiment and teratoma form experiment.
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