CN105670989A - Kit and method for effectively inducing somatic cell phenotype reprogramming - Google Patents
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Abstract
The invention discloses a method and kit for effectively inducing somatic cell phenotype reprogramming, and belongs to the field of a cell biology technique. According to the method and the kit, in accordance with problems in an existing somatic cell reprogramming method, a method, which is used for effectively inducing the somatic cell phenotype reprogramming into induced pluripotent stem cells by taking an extract of a natural active material, namely fish oocyte, as an inducing agent, is invented, and the method is applicable to the preparation of the animal pluripotent stem cells. The method and the kit of the invention can be used for effectively inducing the somatic cell phenotype reprogramming into the induced pluripotent stem cells; compared with other methods, the method is high in reprogramming efficiency on somatic cells, and the inducing method is simple, high in safety and relatively low in cost; and the method disclosed by the invention can solve the problem on the source of the pluripotent stem cells, and the method has broad clinical application value and prospect in such fields as regenerative medicine.
Description
Technical field
The present invention relates to the test kit of a kind of efficient inducing adult cell epigenetic reprogramming and method thereof, belong to technical field of cell biology.
Background technology
Stem cell (Stemcell) is that a class has self renewal (Self-renewal) and the cell of Multidirectional Differentiation (Multilineagedifferentiation) potential, can be used for the Mechanism Study of the generation of life entity, law of development and aging, death etc., and be widely used in regenerative medicine (Regenerativemedicine) field etc. Currently, separating the quantity obtaining pluripotent stem cell very limited from adult tissue, Chang Buneng meets treatment needs; And directly body early embryo obtains stem cell there is serious ethical issues from growing, the source of stem cell seriously limits its research clinically and application.
The source appearing as stem cell of adult cell reprogramming (Somaticcellreprogramming) technology provides new thinking and method. Current adult cell reprogramming method mainly has (the YamanakaS such as nuclear transplantation (Nucleartransfer), cell fusion (Cellfusion), transcription factor mediation (Transcription-factortransduction), micromolecular compound induction (Small-moleculecompounds)etal, Nature, 2010,465 (7299): 704-712; HouPetal, Science, 2013,341 (6146): 651,654).
The basic thought of nuclear transplantation is exactly in adult cell nuclear transplantation to non-nucleus egg mother cell, through cultivating and embryo transfer, will finally develop into one and the identical individuality of donor nuclei genome. Therefore, in a sense, the cell that the method produces is utilized to have the totipotency feature of embryonic stem cell. At present, nuclear transfer technology is utilized to clone tens species including clone sheep " how sharp " (Dollly). But owing to the method technical difficulty is big, cost is high and efficiency is low, and current the method is mainly used in the production of transgenic animal, remains a lot of problem in clinical practice.
Cell-fusion techniques can produce stem cell and be mainly based upon following theory: in most of the cases, the hybrid cell (Hybridcell) formed after two kinds of cell fusion, the cell phenotype that differentiation degree is high is dominant (Dominance) by the cell phenotype (Phenotype) that its differentiation degree is low.Therefore, utilize embryonic stem cell or pluripotent stem cell system and adult cell to co-culture, then hybrid cell can present obvious cells and characteristic of stem. Although the fused cell that produces in this way of profit has a cells and characteristic of stem, but this hybrid cell changes the composition of hereditary material, and there occurs in Growth of Cells, propagation and differentiation characteristic and significantly change. Therefore, there is obvious safety problem in this type of cell in clinical practice. The cell that profit produces in this way is mainly used in gene expression regulation research.
2006, the appearance of induced multi-potent stem cell (inducedpluripotentstemcells, iPSCs) made adult cell reprogramming research reach peak. The generation of iPSCs is to utilize to comprise transcription factorOct4WithNanogGene is in four interior channel genes to adult cell genome so that Matrix attachment region expression pattern reprogramming (TakahashiKetal,Cell,2006,126(4):663-676;ParkIHetal, Nature, 2008,451 (7175): 141-146). The result of this reprogramming ultimately results in the unlatching of stem cell versatility related gene, promotes reprogrammed cell to present and maintains the characteristic of stem cell. The induction of iPSCs successfully make stem cell avoid embryonic stem cell on source ethical issues and in treatment the immune rejection problems of allosome. But owing to the generation of iPSCs needs viral vector to mediate, meanwhile, the intervention of inducible factor changes adult cell Matrix attachment region composition, and therefore its safety in vivo is difficult to control to. Additionally, the induced efficiency of this side's method is very low, at present still in basic research and experimental stage, it is unsuitable for clinical treatment application. The problem brought to overcome transcription factor to mediate, recently, Chinese Scientists utilizes several micromolecular compound (Small-moleculecompound) to combine and successfully reprograms Mus adult cell for iPSCs(HouPetal, Science, 2013,341 (6146): 651,654), The method avoids the safety that genetic manipulation brings, but the induced efficiency of this method is still very low (about 0.2%), therefore, under existing study condition, being applied to clinic should be also still difficult.
As seen from the above, it is feasible for utilizing the induced multi-potent stem cell that adult cell is reprogrammed as having pluripotent stem cell characteristic by said method, but all there is obvious drawback in said method, therefore, seeking the adult cell reprogramming method that a kind of preparation method is more simple, clinical practice is safer, induced efficiency is highly efficient is the important topic currently faced.
Nuclear transfer technology and cell-fusion techniques show in low level of differentiation cell exist can inducing cell reprogramming material. In addition, there will be research to also indicate that and utilize lower hydrobiont Xenopus oocytes extract inducing human adult cell can maintain gene at stem cell versatilityOct4Expression raises, and people's adult cell is reprogrammed process and plays an important role by prompting Rana ovum cell extract. Owing to cell extract belongs to bioactive substance, it is lower to the extent of injury of cell in inducing cell reprogramming process, meanwhile, the reprogramming process of cell extract is not related to genes within cells and imports, and the Matrix attachment region the Nomenclature Composition and Structure of Complexes of reprogrammed cell does not also change.
Visible, lower hydrobiont oocyte extract is a kind of very excellent adult cell epigenetic reprogramming derivant, and utilizing it to prepare induced multi-potent stem cell is a kind of method more attractive than nuclear transplantation and cell fusion.
Accordingly, the present invention utilizes the raw fish oocyte extract of natural fresh water for adult cell epigenetic reprogramming derivant, by the inducing culture of adult cell to obtain induced multi-potent stem cell (inducedmultipotentstemcells, iMSCs).Through technical appraisement such as morphology, cytobiology and molecular biology, inducing cell shows that inducing cell has the biological property of pluripotent stem cell. The adult cell epigenetic reprogramming method that the present invention sets up has that abductive approach is simple, induced efficiency is high and the feature such as virus-free carrier intervention, and the inventive method can provide seed cell source for complex disease treatment and regenerative medicine engineering.
Summary of the invention
Present invention aims to the problem that existing adult cell reprogramming method exists the low grade of induced efficiency, it is proposed to for efficiently preparing the adult cell epigenetic reprogramming method of pluripotent stem cell.
Adult cell reprogramming method for preparing pluripotent stem cell provided by the present invention comprises the following steps:
(1) the adult cell reprogramming derivant that the preparation of derivant uses is oocyte extract;
(2) adult cell that the preparation of adult cell uses is fibroblast;
(3) adult cell induction uses the derivant of preparation in step (1) that the fibroblast of preparation in step (2) is induced;
(4) CHARACTERISTICS IDENTIFICATION uses the cell that induction in step (3) is produced by morphocytology, immunocyte and immunohistochemistry, cytobiology and Protocols in Molecular Biology to carry out characterized.
The oocyte used in described step (1) is fish roe blast cell, and the Fish of use are the raw Fish of fresh water, and including Carassius auratus, Cyprinus carpio and Ctenopharyngodon idellus, oocyte extract adopts multigelation method to prepare;
Fibroblast in described step (2) derives from adults skin histology;
Carrying out the culture medium that efficiently induction uses in described step (3) is DMEM culture medium, and fish oocyte extract is derivant;
The immunocyte for inducing cell characterized and the immunohistochemical method that use in described step (4) are immunostaining, identify that expressing gene is stem-cell marker geneOct4、Sox2WithNanog; Identification of cell biology method be inducing cell internal (Invivo) and external (Invitro) directional induction; Molecular biology method identifies the methylation characteristic analysis adopting stem-cell marker gene promoter region.
It is another object of the present invention to the inducing adult cell proposed for efficiently preparing induced multi-potent stem cell and reprogram test kit.
Inducing adult cell for efficiently preparing induced multi-potent stem cell reprograms test kit and includes:
(1) adult cell epigenetic reprogramming derivant;
(2) adult cell and inducing cell culture medium
Described seminal plasma fructose detection kit or reagent are prepared material and all can be commercially available by commercial biological company or Reagent Company.
The present invention has substantive distinguishing features and significance progress, the present invention is directed to the deficiency of existing adult cell reprogramming method, creatively utilize the derivant that natural fish oocyte extract reprograms as adult cell, achieving adult cell reprogramming is induced multi-potent stem cell, the problem solving source of human stem cell difficulty, avoids the dispute of ethic of embryonic stem cell clinical practice and iPSCs induced efficiency and the low problem of safety simultaneously. Adult cell reprogramming method of the present invention belongs to stem cell basis and clinical technology, this technology has that cell reprogramming method is simple, induction duration is short, Induction Process not damaging cells, preparation stem cell safety is good, the feature such as reprogramming efficiency is high, research cost is low, have a good application prospect.
Accompanying drawing explanation
Fig. 1 is efficient inducing adult cell epigenetic reprogramming flow chart;
Fig. 2 is that people's adult cell separates from skin histology;
Fig. 3 is the cultivation of application on human skin tissue fibroblast cell;
Fig. 4 is inducing adult cell stem cell marker geneOct4Detection of expression;
Fig. 5 is inducing adult cell stem cell marker geneNanogDetection of expression;
Fig. 6 is inducing adult cell stem cell marker geneSox2Detection of expression;
Fig. 7 is differentiation in vivo research (teratomatous formation) of inducing adult cell;
Fig. 8 is inducing adult cell to (class) osteoblast differentiation;
Fig. 9 is inducing adult cell to (class) Adipocyte Differentiation;
Figure 10 is that inducing adult cell breaks up to (class) neural precursor;
Figure 11 is inducing adult cellOct4Gene promoter region methylation patterns;
Figure 12 is inducing adult cellNanogGene promoter region methylation patterns;
Figure 13 is Mus inducing adult cell stem cell marker gene expression analysis;
Figure 14 is the Mus teratomatous formation of inducing adult cell;
Figure 15 is Mus inducing adult cell to other cell type differentiation.
Detailed description of the invention
Technical scheme is further illustrated below in conjunction with drawings and Examples. Embodiment is not limited to the scope of the invention only for the purposes of being best understood from the present invention.
Embodiment 1
The foundation of efficient inducing adult cell epigenetic reprogramming method.
The present embodiment selects fresh water Carassius auratus to be that material prepared by derivant, it is organized as experiment material with application on human skin, skin histology derives from Adult male healthy prepuce tissues, prepuce tissues is provided by Kunming General Hospital Chengdu Military Area Urology Surgery, patient's informed consent, and sign Informed Consent Form voluntarily, sample is only limitted to this research and uses.
Efficient inducing adult cell reprogramming basic operation flow process is shown in Fig. 1, comprises the steps:
(1) preparation of oocyte extract
Oocyte extract adopts multigelation method to prepare, concretely comprise the following steps: choose yolk and be full of the Carassius auratus one of oocyte phase, it is placed in the alcoholic solution of 75% and soaks 30 minutes, the method adopting back head impact after taking-up is lethal by Carassius auratus, then Carassius auratus abdominal part is scratched with scalpel from cloaca, aseptic taking-up fish roe is placed in 50ml sterile centrifugation tube, put into immediately in liquid nitrogen 5 minutes, it is subsequently placed under 4 DEG C of conditions and thaws (about 10 minutes), it is that the ratio of 1:1 adds normal saline repeatedly by volume after 3 times, centrifugal 10 minutes of 1000rpm after being fully ground in mortar, then after 3000rpm is centrifugal 10 minutes, supernatant is taken, 200 order molecular sieve filtrations, Coomassie Brilliant Blue measures oocyte extract total protein concentration, after being diluted to 100mg/ml concentration, 4 DEG C save backup.
(2) preparation of adult cell
The present embodiment adult cell is human skin fibroblast, and fibroblastic preparation includes fibroblastic separation and two links of fibroblastic cultivation.
Fibroblastic separation method is: aseptic collection people's prepuce tissues about 1 × 1cm2Being placed in containing 100U/ml(U is iu) ampicillin and phosphate buffered solution (Phosphatebufferedsolution that 100mg/ml streptomycin sulfate, pH value are 7.4, PBS, 1LpH be 7.4 PBS include 8.0gNaCl, 0.2gKCl, 1.44gNa2HPO4And 0.24gKH2PO4) in, after rinsing 3 times, cut and abandon fatty tissue and connective tissue. Skin is cut into about 1mm3The piece of tissue of left and right, adds in the DMEM culture medium containing collagenase 200U/ml, hyaluronidase 300U/ml, is placed in 4 DEG C overnight, and Fig. 2 shows that 4 DEG C of overnight incubation rear section fibroblasts outwards climb out of around piece of tissue.Add the PBS Digestive system containing 0.25% pancreatin and the ethylenediaminetetraacetic acid (Ethylenediaminetetraaceticacid, EDTA) of 0.02% according to the ratio of 1:1, be placed in 180rpm, 37 DEG C of air table 20min. Adding serum and terminate reaction, the centrifugal 10min of 1000rpm collects cell.
Fibroblastic cultural method is: by DMEM culture medium by after the cell eluting 2 times that separates, adjusting cell density by the DMEM culture medium containing 15% hyclone is every milliliter about 2～4 × 105Individual, then the cell after cell concentration adjustment is inoculated in culture bottle, is placed in 37 DEG C, 5%CO2, cultivate under 90% damp condition, within every two days, change a DMEM culture medium, after cultivating 48 hours, it is seen that fibroblast is paved with (Fig. 3) bottom whole culture dish substantially.
(3) adult cell induction
Cell in step (2) is carried out Secondary Culture, when reach forth generation and cell reach 80% cell fusion time, add the oocyte extract of preparation in step (1), the ratio of culture medium and oocyte extract is 100:1, inducing culturing condition is identical with fibroblastic condition of culture in step (2), inducing culture observation of cell morphological characteristic after 12 hours, and Fig. 4 A, Fig. 5 A and Fig. 6 A show, after inducing 12 hours, cell has typical fusiformis to become round or irregularly shaped.
(4) CHARACTERISTICS IDENTIFICATION
The adult cell of induction in step (3) is carried out pluripotent stem cell CHARACTERISTICS IDENTIFICATION.
1. the adult cell after gene expression analysis Fig. 4 A display induction starts to express pluripotent stem cell marker geneOct4, matched group is then negative expression (Fig. 4 B); Adult cell after Fig. 5 A display induction starts to express pluripotent stem cell marker geneNanog, matched group is then negative expression (Fig. 5 B); Adult cell after Fig. 6 A display induction starts to express pluripotent stem cell marker geneSox2, matched group is then negative expression (Fig. 6 B). Result above tentatively illustrates, inducing adult cell has been equipped with the characteristic of pluripotent stem cell.
2. differentiation in vivo research collects about 1 × 105In individual step (3), the adult cell of induction is injected in nude mice by subcutaneous tissue, after two weeks, there is teratoma in injection site, tissue constituent analysis is carried out by utilizing histopathology and immunohistochemistry after aseptic for teratoma taking-up, result such as Fig. 7 shows, the teratoma formed comprises multiple embryonic tissue such as blood vessel (Fig. 7 the picture left above), neural (Fig. 7 top right plot), muscle (Fig. 7 lower-left figure) and fat (Fig. 7 bottom-right graph), illustrates that inducing adult cell in vivo can to multiple differentiation of germinal layers. Nude mice uses ratifies through Kunming General Hospital Chengdu Military Area Ethics Committee.
3. the adult cell of induction in step (3) is added osteoblast, lipoblast and neuroblast inducing culture by vitro differentiation research respectively, after having induced, utilizes immunohistochemical method to observe the vitro differentiation characteristic of inducing cell. Fig. 8 A shows that inducing cell can produce bone tuberosity in osteoblast induction medium after cultivating, group without osteoblast induction medium then can not produce bone tuberosity, illustrates that inducing cell can be divided into (class) osteoblast (or being called bone like cell) under osteoblast induction medium is cultivated; Fig. 9 shows that inducing cell can be secreted fat under the induction of adipogenic induction culture medium and drip, matched group and non-adipogenic induction culture medium culturing group then do not produce fat and drip, and illustrate that inducing cell can be divided into (class) adipose cell (or being called fat-like cell) under adipogenic induction culture medium effect;Figure 10 A shows that inducing cell is under neuroblast inducing culture effect, it is possible to differentiate neuron. Illustrate that inducing cell can be divided into neurocyte (or being called neural-like cells) under the effect of neuroblast inducing culture. Inducing cell can be divided into specific cell type under specific inducing culture effect as can be seen from the above results.
In summary it can be seen, utilize the adult cell that fish oocyte extract is induced to have the characteristic of pluripotent stem cell, show as inducing cell and express pluripotent stem cell marker gene, and have external enwergy in vivo and be divided into multiple tissue.
4. the adult cell stem cell gene of induction in step (3) is carried out promoter region and methylates research by epigenetics analysis, and Figure 11 shows and induces stem-cell marker gene in forward and backward adult cellOct4The methylation level of four key areas of promoter, it can be seen that induce xenogenite cell from figureOct4The methylation level of gene promoter reduces, and induction xenogenite cell is describedOct4Activity of gene expression strengthens. Figure 12 shows stem-cell marker gene in the forward and backward adult cell of inductionNanogThe methylation level of four key areas of promoter, in Figure 11Oct4Gene promoter methylation pattern similarity, in induction xenogenite cellNanogThe methylation level of gene promoter reduces, and the expression activity of Nanog gene strengthens. To sum up, it can be seen that before and after induction, the promoter methylation pattern of stem cell important symbol gene changes, show as overall methylation level and reduce, illustrate that the marker gene expression activity maintaining cells and characteristic of stem in induction xenogenite cell strengthens. Methylation patterns is consistent with gene expression analysis result in this step.
To sum up, it can be seen that adult cell is after fish oocyte extract is induced, and it is positive that inducing cell all presents stem-cell marker gene expression, display fish oocyte extract can efficiently inducing adult cell reprogramming. Simultaneously as use fish roe mother's extract to carry out adult cell induction be not related to channel genes, therefore, do not change the genome sequence array structure of reprogrammed cell, only changing genomic structural modification, therefore, the reprogramming of adult cell is act as epigenetic reprogramming by fish oocyte extract.
From Fig. 2 ~ 12 result, the present embodiment reliable results.
Embodiment 2
The preparation of Mus induced multi-potent stem cell.
Efficient inducing adult cell reprogramming test kit is utilized to study. Efficient inducing adult cell reprogramming test kit comprises:
Adult cell epigenetic reprogramming derivant;
Adult cell and inducing cell culture medium.
The present embodiment is with Mus adult cell for experiment material, and adult cell type is fibroblast, and fibroblast derives from Corium Mus skin tissue. Laboratory animal uses and uses committee's approval through Kunming General Hospital Chengdu Military Area laboratory animal.
The processes such as Mus adult cell is easily separated according to step (2) in embodiment 1, cultivation are prepared; Adult cell reprogramming is carried out according to step (3) in embodiment 1; Reprogrammed cell CHARACTERISTICS IDENTIFICATION is carried out according to step (4) in embodiment 1.
Pluripotent stem cell is the treatment material that current regenerative medicine (Regenerativemedicine) field is important, including tissue injury (Tissuedamage), genetic disorder disease (Geneticdisorders) and degenerative disease (Degenerativediseases) etc. But, limited source is to limit the obstacle that its clinical practice is maximum.The appearance of adult cell reprogramming technology makes induced multi-potent stem cell be expected to become the new way of clinical source of human stem cell. But induced multi-potent stem cell is before being applied to clinic, need the security performance evaluation of the science that carries out, induced multi-potent stem cell clinical practice animal model is the mostly important method with science of security performance evaluation, and obtaining animal pattern induced multi-potent stem cell is the key setting up animal model.
Efficient inducing adult cell epigenetic reprogramming test kit is utilized to carry out the preparation of Mus induced multi-potent stem cell.
Induction result shows, utilizes the Mus adult cell that test kit is induced to express pluripotent stem cell marker gene (Figure 13); Teratoma (Figure 14) can be formed after two weeks in inducing adult cell transplantation nude mouse; Inducing adult cell is under the induction of directional induction culture medium, it is possible to be divided into specific cell type (Figure 15). Result above all shows, utilizes the Mus adult cell of efficient inducing adult cell mass color reprogramming test kit induction to have obvious pluripotent stem cell characteristic, illustrates to utilize this test kit can successfully prepare Mus pluripotent stem cell.
As can be seen from the above, use this adult cell epigenetic reprogramming scheme can under relatively low research cost, efficiently obtain induced multi-potent stem cell safer clinically, the acquisition of induced multi-potent stem cell can provide source reliably for clinical stem cell, has good using value and promotion prospect.
Claims (11)
1. efficient inducing adult cell epigenetic reprogramming method, it is characterised in that comprise the steps:
(1) preparation of derivant;
(2) preparation of adult cell;
(3) adult cell induction;
(4) CHARACTERISTICS IDENTIFICATION;
In described step (1), derivant is natural fresh water fish oocyte extract;
In described step (2), adult cell is animal skin tissue fibroblast;
In described step (3), inducing cell culture medium is DMEM culture medium;
In described step (4), inducing cell CHARACTERISTICS IDENTIFICATION includes morphocytology, immunocytochemistry, Celluar and Molecular Biology method.
2. adopt efficient inducing adult cell epigenetic reprogramming method described in claim 1, it is characterized in that, in step (1), the preparation method of derivant is: chooses yolk and is full of the Carassius auratus one of oocyte phase, it is placed in the alcoholic solution of 75% and soaks 30 minutes, the method adopting back head impact after taking-up is lethal by Carassius auratus, then Carassius auratus abdominal part is scratched with scalpel from cloaca, aseptic taking-up fish roe is placed in 50ml sterile centrifugation tube, put into immediately in liquid nitrogen 5 minutes, it is subsequently placed under 4 DEG C of conditions and thaws (about 10 minutes), it is that the ratio of 1:1 adds normal saline repeatedly by volume after 3 times, centrifugal 10 minutes of 1000rpm after being fully ground in mortar, then after 3000rpm is centrifugal 10 minutes, supernatant is taken, 200 order molecular sieve filtrations, Coomassie Brilliant Blue measures oocyte extract total protein concentration, after being diluted to 100mg/ml concentration, 4 DEG C save backup.
3. adopt efficient inducing adult cell epigenetic reprogramming method described in claim 1, it is characterized in that, in described step (2), the preparation method of adult cell includes fibroblastic separation and cultivates two links, its method is respectively as follows: (1) fibroblastic separation method: with iodine tincture, Mus skin of abdomen tissue is rotated sterilization, hair is cut off, with scalpel clip 2 ~ 3cm with operating scissors2Skin, being placed in containing 100U/ml(U is iu) ampicillin and phosphate buffered solution (Phosphatebufferedsolution that 100mg/ml streptomycin sulfate, pH value are 7.4, PBS, 1LpH be 7.4 PBS include 8.0gNaCl, 0.2gKCl, 1.44gNa2HPO4And 0.24gKH2PO4) in, after rinsing 3 times, cut and abandon fatty tissue and connective tissue, skin is cut into about 1mm3The piece of tissue of left and right, add in the DMEM culture medium containing collagenase 200U/ml, hyaluronidase 300U/ml, it is placed in 4 DEG C overnight, ethylenediaminetetraacetic acid (the Ethylenediaminetetraaceticacid containing 0.25% pancreatin and 0.02% is added according to the ratio of 1:1, EDTA) PBS Digestive system, being placed in 180rpm, 37 DEG C of air table 20min, add serum and terminate reaction, the centrifugal 10min of 1000rpm collects cell;(2) fibroblastic cultural method is: by DMEM culture medium by after the cell eluting 2 times that separates, adjusting cell density by the DMEM culture medium containing 15% hyclone is every milliliter about 2～4 × 105Individual, then the cell after cell concentration adjustment is inoculated in culture bottle, is placed in 37 DEG C, 5%CO2, cultivate under 90% damp condition, within every two days, change a DMEM culture medium, after cultivating 48 hours, it is seen that fibroblast is paved with bottom whole culture dish substantially.
4. adopt efficient inducing adult cell epigenetic reprogramming method described in claim 1, it is characterized in that, in described step (3), adult cell abductive approach is: the cell in step (2) is carried out Secondary Culture, when reach forth generation and cell reach 80% cell fusion time, add the oocyte extract of preparation in step (1), the ratio of culture medium and oocyte extract is 100:1, inducing culturing condition is identical with fibroblastic condition of culture in step (2), and the inducing culture time is 12 hours.
5. adopt efficient inducing adult cell epigenetic reprogramming method described in claim 1, it is characterized in that, the morphological observation method that in described step (4), inducing cell CHARACTERISTICS IDENTIFICATION includes is: the induction adult cell of 12 hours in step (3) is placed in observed under electron microscope.
6. adopt efficient inducing adult cell epigenetic reprogramming method described in claim 1, it is characterised in that the immunocytochemistry that in described step (4), inducing cell CHARACTERISTICS IDENTIFICATION includes is; With 4% paraformaldehyde, the cell induced in step (3) being fixed 10min respectively with cellular control unit, rinses 5min × 3 time with PBS, add 0.3%Triton-100, parked 5min, PBS rinse 5min × 3 time, add 3%H2O2Parked 5min, PBS rinse 5min × 3 time, are separately added into mouse-anti people Oct3/4, Sox-2, Nanog and C-myc antibody in the hole that labelling is good, 4 DEG C overnight, PBS rinses 5min × 3 time, and dropping biotinylation two resists, and hatches 20min for 37 DEG C, 5min × 3 time are washed with PBS, adding DAB reagent, color development at room temperature 5min～10min(Microscopic observation controls), PBS washing is placed on observed under electron microscope.
7. adopt efficient inducing adult cell epigenetic reprogramming method described in claim 1, it is characterized in that, the cell biology method that in described step (4), inducing cell CHARACTERISTICS IDENTIFICATION includes is the internal of inducing cell and vitro differentiation, and the differentiation in vivo method of inducing cell is: collect about 1 × 105In individual step (3), the adult cell of induction is injected in nude mice by subcutaneous tissue, after one week, and the teratoma that aseptic taking-up Cell differentiation inducing activity is formed, utilize histopathology and immunohistochemistry to carry out tissue constituent analysis.
8. adopt efficient inducing adult cell epigenetic reprogramming method described in claim 1, it is characterized in that, in the identification of cell biology method that in described step (4), inducing cell CHARACTERISTICS IDENTIFICATION includes, the vitro differentiation method of inducing cell includes in step (3) inducing cell and induces to osteoblast, adipose cell and neural cellular differentiation, wherein
Osteoblast abductive approach is: be 3.1 × 10 by density3/cm2In step (3), the adult cell of induction is inoculated in six orifice plates, adds complete medium and is placed on 37 DEG C, 5%CO2Incubator in cultivate 24 hours, after 24 hours change culture medium, every hole add 2ml Osteoblast Differentiation induction liquid induce, within every 2 days, change Osteoblast Differentiation complete medium once, fix cell after inducing 3 weeks, dye by alizarin red;
Adipose cell abductive approach is: be 2.1 × 10 by density4/cm2In step (3), the adult cell of induction is inoculated in six orifice plates, adds complete medium, is positioned over 37 DEG C, 5%CO2Incubator in cultivate 24 hours, after 24 hours change culture medium, hereafter, within every 3 days, change complete medium once, until cell reaches to merge completely; After cell merges completely, careful suction abandons old culture medium, and every hole adds 2ml and becomes fat induction to cultivate A completely, after three days, induction liquid is changed to into fat induction complete medium B, after 24 hours, then gain into fat induction complete medium A, after circulating 3 to 5 times, maintain 7 days with becoming fat induction complete medium B, within every 3 days, carry out changing liquid, after having induced, fix cell, dye with oil red O;
Neural cellular differentiation abductive approach is: by 5 × 105In individual step (3), the adult cell of induction is inoculated in six orifice plates, with the L-DMEM culture medium inducing culture 24 hours containing 100 μm of ol/L β-ME, 20%CS, change into containing 1 μm of ol/LRA, 100mmol/L β-ME, 10%CS L-DMEM inducing culture to after 10 days, fix with the paraformaldehyde of 4%, the expression of detection neuronal marker NSE, nestin.
9. efficient inducing adult cell epigenetic reprogramming test kit, it is characterised in that this test kit includes:
(1) adult cell epigenetic reprogramming derivant;
(2) adult cell and inducing cell culture medium.
10. adopt described in claim 9 efficiently inducing adult cell epigenetic reprogramming test kit, it is characterised in that test kit comprises in (1) fibroblast epigenetic reprogramming derivant preparation method with derivant preparation method in claim 2.
11. adopt efficient inducing adult cell epigenetic reprogramming test kit described in claim 9, it is characterised in that it is DMEM culture medium that test kit comprises adult cell and inducing cell culture medium in (2).
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| CN107267448A (en) * | 2017-07-28 | 2017-10-20 | 陈强 | A kind of induced fibroblast is the method for in vivo bioreactor |
| CN107376025A (en) * | 2017-07-16 | 2017-11-24 | 陈强 | A kind of cytoskeleton composite material and preparation method thereof and application for cartilage damage reparation |
| CN109182256A (en) * | 2018-08-31 | 2019-01-11 | 浙江省海洋水产研究所 | A kind of spotted maigre Preadipocyte In Vitro be separately cultured and its abductive approach |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479891A (en) * | 2016-12-20 | 2017-03-08 | 北京市肿瘤防治研究所 | Colorectal cancer primitive cell culture kit and its application process |
| CN107376025A (en) * | 2017-07-16 | 2017-11-24 | 陈强 | A kind of cytoskeleton composite material and preparation method thereof and application for cartilage damage reparation |
| CN107267448A (en) * | 2017-07-28 | 2017-10-20 | 陈强 | A kind of induced fibroblast is the method for in vivo bioreactor |
| CN109182256A (en) * | 2018-08-31 | 2019-01-11 | 浙江省海洋水产研究所 | A kind of spotted maigre Preadipocyte In Vitro be separately cultured and its abductive approach |
| CN109182256B (en) * | 2018-08-31 | 2020-07-17 | 浙江省海洋水产研究所 | Separation culture and induction method of precursor adipocytes of nibea albiflora |
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