CN102311940A - Method for inducing differentiation of stem cells into islet-like cells - Google Patents
Method for inducing differentiation of stem cells into islet-like cells Download PDFInfo
- Publication number
- CN102311940A CN102311940A CN2010102225444A CN201010222544A CN102311940A CN 102311940 A CN102311940 A CN 102311940A CN 2010102225444 A CN2010102225444 A CN 2010102225444A CN 201010222544 A CN201010222544 A CN 201010222544A CN 102311940 A CN102311940 A CN 102311940A
- Authority
- CN
- China
- Prior art keywords
- cell
- cells
- islet
- insulin
- stem
- 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.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of biomedicines, in particular to a method for inducing differentiation of stem cells into islet-like cells in vitro and use of islet-like cells. The invention aims to provide a method for obtaining a large amount of islet-like cells in vitro and the use of the islet-like cells differentiated under induction as seed cells in transplantation of islet cells and preparation of microencapsulated stem cell preparation and as an in-vitro pharmacokinetic model in cell medicine screening. For realizing the aim, the invention adopts a technical scheme that: 1) constructing an NRSF interfering carrier; 2) separating, culturing and amplifying stem cells; 3) inducing stem cells to differentiate into islet-like cells by two stages; and 4) identifying islet-like cells. The construction of the method has a very bright application prospect in regenerative medicine taking gene engineering, cell engineering, tissue engineering and the like as a basis and is expected to create huge social and economic benefit.
Description
Technical field
The present invention relates to biomedical sector, specifically relate to the method for external evoked stem cell to pancreatic islet like cell directed differentiation and the purposes of insulin-like cell.
Background technology
Mellitus (DM, Diabetes Mellitus) are sugar, fat and the protein metabolism disorder property diseases that relative or absolute deficiency of insulin secretion or insulin receptor cause as defective.Clinically, I type and part type ii diabetes adopt the subcutaneous injection pancreas islet usually to treat more.Cell therapy is the especially ideal treatment strategies of type i diabetes of mellitus, and for the diabetic subject who loses islet cell function, β cell or its surrogate of implanting the ability excreting insulin are optimal treat-ment.In recent years, islet cell transplantation treatment mellitus had obtained some curative effects, and deficiency, serious difficulties such as immunological rejection have limited the application of this therapy greatly but the donor's cells originates.
Stem cell has extremely strong self ability and multidirectional differentiation potential, is the desirable target cell of gene therapy.Stem cell is divided into myeloid-lymphoid stem cell (like embryonic stem cell; Can be divided into all histocytes of body), multipotential stem cell (has multidirectional differentiation potential; Can be divided into multiple histocyte; Like mescenchymal stem cell etc.) and specially can stem cell (keep the single direction self of a certain particular organization cell, as gut epithelial stem cells etc.).The characteristic that the continuous breakthrough of stem cells technology and stem cell itself are had makes the mankind might be some stem cell of vitro culture; It is divided into our needed various histocytes directional induction; Or be used for organizational project for clinical required as seed cell; Stem Cell Engineering with this end in view relates to most of difficult medical problem of nearly all vital tissue organ of human body and face of mankind, like the treatment of cardiovascular disorder, mellitus, malignant tumour, bone and cartilage defect, senile dementia, Parkinson's disease, burn, Spinal injury and hereditary defect etc.Because the multipotential stem cell in people's amniotic fluid, Cord blood, peripheral blood and the marrow; Have advantages such as multiplication capacity is strong, the source is abundant, collection is convenient, this type of stem cell transplantation has been brought into play important effect in the treatment of disease in the blood system, malignant tumour, autoimmune disorder etc.
(Neuronal restrictive silencer factor is that a part quality is the Cys2/His2 type zinc finger protein of 116kD NRSF) to neuron-restrictive silencer factor, belongs to Gli-Kruppel appearance transcription factor family.Whole molecule comprises N end to be checked structural domain RD-1, nearly N end zinc fingers, DNA calmodulin binding domain CaM (containing 7 zinc fingerses of arranging clusters), Methionin and is rich in district, proline rich domain, C end and checks structural domain RD-2 (containing a zinc fingers).The NRSF wide expression is in embryonic stem cell and become in the somatocyte only not expression in mature neuron and islet cells.NRSF can regulate and control and the pancreas islet relevant important gene of reaching maturity, and simultaneously, the glucose-insulin secretion coupling mechanism of NRSF and β cell is closely related, and NRSF crosses to express and destroys this coupling mechanism and cause cell to reduce.Therefore, the NRSF downward modulation is expressed and can be promoted that cell breaks up to islet cells.
RNA disturb (RNA interference, RNAi) be meant high conservative during evolution, by double-stranded RNA (double-stranded RNA, phenomenon that dsRNA) bring out, the efficient specificity degraded of homologous mRNA.Because use the RNAi technology can specificity to reject or close the expression of specific gene, this technology has been widely used in exploring the field of gene of gene function and communicable disease and malignant tumour.Discover that the hair clip shRNA that utilizes the plasmid vector that carries the rna plymerase iii promotor to handle a section little expresses the silence that can cause the particular target gene equally in mammalian cell.
Lentiviral vectors (lentiviral vectors) system has following characteristics: virus does not possess the self-replication ability, has self-inactivating mechanism; Host range is wide, and multiple genus polymorphic type cells such as people, mouse can both be by efficient infection; On the basis of stable integration host genome, increase efficient infection cell stationary phase, efficiently expressed foreign protein, can also escape the characteristics that methylate and suppress simultaneously.On the basis of lentiviral vectors, transform the slow virus interference vector that comes out and in cell, to produce interference effect steady in a long-term target gene.
Stem cell is carried out suitable genetic modification; Making it to become the cell of ability excreting insulin, be one of ideal strategy of treatment type i diabetes, and the NRSF in the stem cell is expressed in downward modulation; And with inducing culture it is induced, might make us obtain the cell of ability excreting insulin.Therefore; The adult stem cell that separates different tissue sources; It is used for the preparation of islet cell transplantation, microencapsulation stem cell medicine etc. at the external evoked insulin-like cell that is divided into as seed cell, induces the insulin-like cell of differentiation to can be used as external pharmacokinetic model simultaneously and be used for the cell drug screening.The foundation of this method will have splendid application prospect in the regenerative medicine that is the basis with genetically engineered, cell engineering and even organizational project etc., and will have a tremendous social and economic benefits.
Summary of the invention
Technical problem to be solved by this invention is to set up the method that external evoked differentiation of stem cells is an insulin-like cell.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:
Utilize stem cell to have the potential of multidirectional differentiation; Induce it to break up to insulin-like cell; But induce the cell excreting insulin of differentiation; For the preparation of islet cell transplantation, microencapsulation stem cell medicine provides seed cell, induce the cell of differentiation to can be used as external pharmacokinetic model simultaneously and be used for the cell drug screening.
Used induction method among the present invention is characterized in that utilizing the expression level of carrier downward modulation NRSF in stem cell, and adds nutrient substance or cytokine simultaneously, divides two stage induced dry-cells to break up to insulin-like cell.
Stem cell among the present invention comprises peripheral blood, Cord blood and other tissue-derived mescenchymal stem cell, pancreatic stem cells, liver stem cells, hemopoietic stem cell and the NSC of people and Mammals amniotic fluid, embryo, marrow, mobilization.
Carrier used among the present invention comprises lentiviral vectors, adenovirus carrier, and gland relevant viral vector, retroviral vector is expressed in stem cell through these carrier mediated downward modulation NRSF genes.
The used external evoked method of the present invention is to utilize the expression level of carrier downward modulation NRSF in stem cell, and adds nutrient substance or cytokine simultaneously, divides two stage induced dry-cells to break up to insulin-like cell.
The carrier lentiviral vectors that the present invention is used for gene transfection is the first-selected virus vector of transfection NRSF gene.
The nutrient solution that the present invention selects for use comprises Activin-A for having added nutrient substance or cytokine, bFGF, and B27, BTC, nicotinamide can induce downward modulation to express the stem cell to pancreatic islet like cell differentiation of NRSF.
Pancreas islet genes involved and albumen can be expressed by the insulin-like cell group that is obtained among the present invention, comprise glucose transporter, gk, Regular Insulin, hyperglycemic-glycogenolytic factor, Somatostatin.
Insulin-like cell ability excreting insulin among the present invention after the external evoked stem cell to pancreatic islet like cell differentiation can be used as seed cell and is used for the preparation of islet cell transplantation, microencapsulation stem cell medicine and is used for the cell drug screening as external pharmacokinetic model.
Specific embodiments is following:
1. separate the adult stem cell of different tissue sources, the preparation method is:
(1) separation of human amniotic fluid stem cell, purifying and former generation, the cultivation of going down to posterity: get amniotic fluid 5-10ml under the aseptic condition; Centrifugal 8min under the 1600r/min condition, the collecting cell deposition is cultivated: low sugar DMEM substratum (LD)+10% foetal calf serum+100U/ml green grass or young crops-Streptomycin sulphate+4ng/ml bFGF.Change nutrient solution behind the 48h, discard not attached cell, changed liquid once in later per 2～3 days.Cell reaches 80% cultivation of going down to posterity when converging.0.25%-EDTA solution peptic cell, digestion time was controlled in 5 minutes.Serum stops digestion, blows and beats at the bottom of the ware gently and collecting cell, centrifugal 5 minutes of 1100rpm.Cell precipitation is in 1: the ratio of 4-6 goes down to posterity, and counts per generation cell number and calculating cumulative cell count.
(2) separation of mesenchymal stem cells MSCs: under the aseptic condition; Through the puncture of bilateral posterior superior iliac spine, gather marrow, through the Percoll (U.S.; The Sigma Company products; Specific density 1.073g/ml) collector's BMNC after the density gradient centrifugation, adherent culture 72h removes the mesenchymal stem cells MSCs that is of adherent growth behind the suspension cell.
(3) the rats'liver stem cell separates: under the aseptic condition, cut rat abdominal cavity and expose portal vein along median line, after infusion does not have calcium magnesium Hanks liquid and becomes yellowish pink to liver, keep portal vein; Move in the aseptic plate from disconnected liver and with liver, infusion contains the Hanks liquid of type again, reclaims the collagenase liquid in the plate; Remove Glisson's capsule and blood vessel, passivity is torn hepatic tissue, dissolves with substratum; The multilayer filtered through gauze, centrifuge washing becomes single hepatocyte suspension with medium preparation more repeatedly.
(4) separation of Cord blood mononuclearcell: get blood through umbilical vein puncture under the aseptic and ACD anti-freezing liquid anti-freezing condition; Gather healthy full-term normal delivery umbilical cord blood; Successively through (U.S., the Sigma Company products) sedimentation of 0.5% methylcellulose gum and Ficoll (U.S., Sigma Company products; Specific density 1.077g/ml) collector's Cord blood mononuclearcell after the density gradient centrifugation is resuspended among the PBS after the washing.
2. make up the NRSF interference vector, design is simultaneously missed the target carrier as contrast, carries out the slow virus packing and with behind the slow virus infection stem cell, detects the interference effect of interference vector.
3. the stem cell of dividing two stages that NRSF is expressed in downward modulation is induced, and makes it be divided into insulin-like cell.
4. to the detection of institute's inductive insulin-like cell
1. according to technological method provided by the invention, can with people and Mammals amniotic fluid, embryo, marrow, peripheral blood, Cord blood and other tissue-derived mescenchymal stem cell, pancreatic stem cells, liver stem cells, hemopoietic stem cell and NSC etc. directional induction in vitro be divided into can excreting insulin insulin-like cell;
2. the insulin-like cell of the external evoked differentiation that obtains according to technological method of the present invention can be used as the preparation that seed cell is applied to islet cell transplantation and microencapsulation stem cell medicine;
3. the insulin-like cell of the external evoked differentiation that obtains according to technological method of the present invention can be used as external pharmacokinetic model and is used for the cell drug screening.
4. the foundation of method of inducing differentiation will have splendid application prospect among the present invention in the regenerative medicine that is the basis with genetically engineered, cell engineering and even organizational project etc., and will have a tremendous social and economic benefits.
Embodiment
The structure of embodiment 1, NRSF interference vector
Utilize network RNA to interfere the interference segment of segment design tool designer NRSF gene and the contrast segment of missing the target of interference; Wherein interfere the segment sequence to be: to interfere the segment sequence of missing the target to be: after two silver cracked ends are crossed annealing, T4 mononucleotide Starch phosphorylase phosphorylation; Be connected to the pSicoR carrier behind HpaI and the Xbal double digestion with the T4 ligase enzyme; After transforming the DH5a competent cell; The picking clone shakes bacterium and cultivates back extraction plasmid, with the negative contrast of pSicoR plasmid, through 2% agarose gel electrophoresis behind XhoI, the XbaI double digestion; Can cut the plasmid (seeing accompanying drawing 1) that the positive clone of the generation 400bp pulsating plasmid of size obtains by enzyme, this clone delivers the order-checking of order-checking company.The interference vector plasmid called after pSicoR-GFP-siNRSF that checks order correct, the correct vector plasmid called after pSicoR-GFP-siControl that misses the target checks order.
Cultivate 293-FT (available from Invitrogen company) slow virus packing cell, substratum is for adding 10%FBS, 0.1mmol/L NEAA; The 2mmol/L L-glutaminate; 1% green grass or young crops-Streptomycin sulphate, and the high sugared DMEM substratum of 500 μ g/mL G418 (the Sigma Company products, Cat#D5648).The slow virus interference vector pSicoR-GFP-siNRSF that makes up with step 1 and the carrier pSicoR-GFP-siControl that misses the target press after the mixed of 5ug: 4.2ug: 2ug: 2.8ug in adding 1.5mL serum-free Opti-MEM
the I substratum (available from Invitrogen company) with packaging plasmid pLP1 (available from Invitrogen company), pLP2 (available from Invitrogen company) and envelope protein plasmid pLP/VSVG (available from Invitrogen company) respectively; Mixing gently; In another aseptic 5mL pipe, 42uL lipofectamine 2000 (available from Invitrogen company) is diluted in serum-free Opti-MEM
the I substratum of 1.5mL; Room temperature left standstill 5 minutes; Mix above two kinds of liquid, incubated at room 20 minutes is to form DNA-lipofectamine 2000 mixtures.(the Gibco Company products, Cat#25200-056) the 293-FT cell of cultivating is collected in digestion, counts about 6 * 10 with 0.25% pancreatin
6Individual cell; It is resuspended to 5mL does not contain antibiotic growth medium (in sugared DMEM substratum (the Sigma Company products of height; Interpolation 10% foetal calf serum (FBS) on the basis Cat#D5648) (the Biochrom Company products, cat#S0115), 0.1mmol/L non-essential amino acid (Non-Essential Amino Acids; NEAA) (available from HyClone company) and 2mmol/L L-glutaminate) in; Again 293-FT packing cell suspension and 3mL are contained to join behind the substratum mixing of DNA-lipofectamine2000 mixture and contain in the 10cm Tissue Culture Dish that 5mL do not contain antibiotic growth medium, mixing is put in 37 ℃, 5%CO
2Cultivate in the incubator; The inferior daily perfect medium that contains the 1mmol/L Sodium.alpha.-ketopropionate (adds 10% foetal calf serum, 0.1mmol/L non-essential amino acid, 2mmol/L L-glutaminate on the basis of the sugared DMEM substratum of height; 1% green grass or young crops-Streptomycin sulphate and 1mmol/L Sodium.alpha.-ketopropionate) change the substratum contain DNA-lipofectamine 2000 mixtures and pack; And take a sample after 24,72 hours in packing respectively, to observing under fluorescent microscope through the 293-FT cell of packing, the result packs can see after 24 hours has green fluorescent protein EGFP to express (the EGFP gene is that slow virus interference vector pSicoR carries) in the 293-FT cell; The cell of expressing green fluorescent protein EGFP surpasses 95%; Show that packaging efficiency is very high, and be accompanied by VSVG expression of gene among the coating plasmid pLP/VSVG, engender the many cells synplasm of 293-FT cell; Pack that culture supernatant is faint yellow after 72 hours; Collect viral liquid supernatant this moment in the aseptic centrifuge tube of 15mL, and 4 ℃, the centrifugal 15min removal of 3000rpm cell debris are frozen subsequent use in-70 ℃ with viral supernatant.
The checking of embodiment 3, slow virus interference vector interference effect
1. the slow virus interference vector and the carrier that misses the target infect amniotic fluid stem cell
In Tissue Culture Flask, cultivate amniotic fluid stem cell, use virus infected cell when treating its length to 80% density.During infection, every hole adds 10ml virus and final concentration is the polybrene (polybrene) of 8 μ g/ml, rocks culturing bottle and makes viral fluid power touch all cells.Change substratum after the incubated overnight.Infect the cell difference called after SiNRSF-hAFSCs and the SiControl-hAFSCs of the slow virus interference vector and the carrier that misses the target.After this cultivate according to normal cell cultural method pair cell.
2.RT-PCR and real time PCR checking
With TRIZOL (invitrogen) reagent, method is extracted siNRSF-hAFSCs and siControl-hAFSCs cell total rna to specifications, and reverse transcription mRNA wherein is cDNA then, and the reverse transcription system is:
Volume (μ l)
5×AMV?reverse?buffer 4
dNTP(10mM) 2
RNAse?inhibitor 1
AMV ThermoScript II 0.5
Water 11.5
Carrying out pcr amplification goal gene NRSF again, is reference with people GAPDH gene.The PCR reaction system is:
Volume (μ l)
10×PCR?buffer 2
dNTP(2.5mM) 1.6
Primer (forward and reverse) 1
RTaq enzyme 0.2
Water 14.2
Confirm that with real time PCR interfering efficient, used instrument accurately is the opticon 2 of bio-rad company simultaneously, reaction system is:
Volume (μ l)
10×PCR?buffer 2
dNTP(2.5mM) 1.6
Primer (forward and reverse) 1
RTaq enzyme 0.2
syberGREEN 0.5
Water 13.7
React independent triplicate to eliminate systematic error.See that from rna level the interference efficient of interference vector is 70%, and do not cause the effect (seeing accompanying drawing 2) of missing the target.
3.Western blotting checking
Respectively get 10
7SiNRSF-hAFSCs and siControl-hAFSCs cell extract nucleus albumen with nucleus protein extraction test kit (pierce).Using the BCA method to measure protein concentration is 5100 μ g/ml.SiNRSF-hAFSCs and the siControl-hAFSCs nucleus albumen of getting equivalent then carry out discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE); Method with the half dry type transfer printing moves to pvdf membrane with protein transduction again,, one resists that (the rabbit anti-NRSF antibody of originating is purchased the company in santa cruz then with 5% skim-milk sealing 2h; The rabbit anti-β-actin antibody of originating is purchased in company of middle China fir Golden Bridge) 4 ℃ spend the night; TBST washes film three times, each 10 minutes, adds the even anti-rabbit that connects two of horseradish peroxidase then and resists; After the room temperature one hour, TBST washes film four times.With the ECL chemiluminescence detection kit (available from Santa Cruz company, Cat#:SC-2048) in the darkroom autography with testing goal band and interference effect.Prove that constructed NRSF slow virus interference vector can stablize the NRSF (seeing accompanying drawing 3) that interferes the endogenous expression of cell on protein level.
Stem cell behind the virus infection was induced according to two stages; Fs is the 1st to 7 day; Inducing culture is D/F12+2%FBS+2%B27+10ng/ml Activin-A+10ng/ml bFGF; Subordinate phase is the 8th to 14 day, and inducing culture is D/F12+2%FBS+2%B27+10ng/mlBTC+10mM nicotinamide, and other conditions are cultivated according to normal cell cultural method pair cell.
Embodiment 5, to inducing the evaluation of the insulin-like cell that differentiation obtains
1.RT-PCR identify
With embodiment 3 said RT-PCR methods noble cells is carried out mrna expression and identify that identified gene comprises: Sox17, Foxa2, Pdx1, Hnf4 α, Pax4, Isl-1, Nkx6.1, Insulin, Glut-2 (seeing accompanying drawing 4), primers designed is following:
| ?Gene | Forward?primer(5′to3′) | Reverse?primer(5′to3′) |
| ?Nanog | CAGAAGGCCTCAGCACCT | CTGTTCCAGGCCTGATTGTT |
| ?Oct4 | AACCTGGAGTTTGTGCCAGGGTTT | TGAACTTCACCTTCCCTCCAACCA |
| ?Rex1 | CAGATCCTAAACAGCTCGCAGAAT | GCGTACGCAAATTAAAGTCCAGA |
| ?Sox17 | GCATGACTCCGGTGTGAATCT | TCACACGTCAGGATAGTTGCAGT |
| ?Foxa2 | CTGAGCGAGATCTACCAGTGGA | CAGTCGTTGAAGGAGAGCGAGT |
| ?Hnf4a | ACTACATCAACGACCGCCAGT | ATCTGCTCGATCATCTGCCAG |
| ?Isl-1 | ATTTCCCTATGTGTTGGTTGCG | CGTTCTTGCTGAAGCCGATG |
| ?pdx1 | TGATACTGGATTGGCGTTGT | GCATCAATTTCACGGGATCT |
| ?Glut2 | GCTACCGACAGCCTATTCTA | CAAGTCCCACTGACATGAAG |
| ?Nkx6.1 | ACACGAGACCCACTTTTTCCG | TGCTGGACTTGTGCTTCTTCAAC |
| ?insulin | GCCTTTGTGAACCAACACCTG | GTTGCAGTAGTTCTCCAGCTG |
| ?Pax4 | AAGAAAGCAGCTTGCGTTGAC | GGGCGTGAGACAGAGGTATC |
| ?NRSF | ATTGAAGTTGGCTTAGTG | TATGGGTAGATTCGTTGA |
| ?GAPDH | GAGTCAACGGATTTGGTCGT | TTGATTTTGGAGGGATCTCG |
2.Immunofluorescence identify
Plant siNRSF-hAFSCs and siControl-hAFSCs cell in 96 orifice plates, cell density is 10
4Cells/well.4% Paraformaldehyde 96 fixed cell half a hour, used the 1%TritonX-100 rupture of membranes then 15 minutes, the sealing of normal goats serum; One anti-(Pdx1, Insulin c-peptide) spend the night; PBS washing three times each 5 minutes, adds fluorescently-labeled two and resists; Fluorescence (seeing accompanying drawing 5) is observed in PBS washing three times under the fluorescence inverted microscope.
3.c-the peptide secretory volume detects
(each is 3 * 10 years old to get the cell of inducing 14 days
5Individual), hatched 1 hour with KRBB buffer again with after the KRBB buffer washing three times.Hatched 1 hour with the KRBBbuffer that contains 5.5mM and 23nM glucose respectively then; Collect buffer; Albumen in the collecting cell simultaneously is with buffer and the content (see accompanying drawing 6) of intracellular protein with Ultrasensitive C-peptide ELISA kit (Mercodia) detection c-peptide.
It is insulin-like cell that the preliminary proof of experimental result can be induced differentiated stem cells through the NRSF genetic method of interfering the endogenous expression of stem cell.
Description of drawings
Fig. 1 cuts evaluation figure to the enzyme of constructed carrier
1. plasmid pSicoR-GFP-siNRSF enzyme is cut 2. plasmid pSicoR-GFP-siControl enzymes and is cut
3. skeleton plasmid pSicoR-GFP enzyme is cut 4.DL15, and 000
Fig. 2 Real Time PCR detects the interference effect of interference vector to NRSF mRNA expression level
Fig. 3 western blotting detects the interference effect of interference vector at the NRSF protein level
Fig. 4 RT-PCR detects pancreas islet Expression of Related Genes situation in the inducing cell
It is islet cells that Fig. 5 immunofluorescence detects noble cells
The detection of c-peptide and emiocytosis c-peptide content in Fig. 6 born of the same parents
Claims (9)
1. induced dry-cell is to the method for insulin-like cell differentiation; It is characterized in that utilizing stem cell to have the potential of multidirectional differentiation; Induce it to break up to insulin-like cell; But induce the cell excreting insulin of differentiation,, induce the cell of differentiation to can be used as external pharmacokinetic model simultaneously and be used for the cell drug screening for the preparation of islet cell transplantation, microencapsulation stem cell medicine provides seed cell.
2. according to the described induction method of claim 1, it is characterized in that utilizing the expression level of carrier downward modulation NRSF in stem cell, and add nutrient substance or cytokine simultaneously, divide two stage induced dry-cells to break up to insulin-like cell.
3. according to the described induction method of claim 1; It is characterized in that; Described stem cell has multidirectional differentiation potential, comprises peripheral blood, Cord blood and other tissue-derived mescenchymal stem cell, pancreatic stem cells, liver stem cells, hemopoietic stem cell and the NSC of people and Mammals amniotic fluid, embryo, marrow, mobilization.
4. according to the described induction method of claim 1, it is characterized in that described carrier comprises lentiviral vectors, adenovirus carrier, gland relevant viral vector, retroviral vector is expressed in stem cell through these carrier mediated downward modulation NRSF.
5. according to claim 1 or 4 described induction methods, it is characterized in that lentiviral vectors is the first-selected virus vector of transfection NRSF gene in the described carrier.
6. according to the described induction method of claim 1, it is characterized in that, added nutrient substance or cytokine in the nutrient solution; Comprise Activin-A, bFGF, B27; BTC, nicotinamide can induce the stem cell to pancreatic islet like cell of transfection NRSF interference vector to break up.
7. according to the described induction method of claim 1, it is characterized in that the insulin-like cell group that obtains can express pancreas islet genes involved and albumen, comprise glucose transporter, gk, Regular Insulin, hyperglycemic-glycogenolytic factor, Somatostatin.
8. the purposes of the insulin-like cell after the differentiation of external evoked stem cell to pancreatic islet like cell, but it is characterized in that inducing the cell excreting insulin after the differentiation, can be used as the preparation that seed cell is used for islet cell transplantation, microencapsulation stem cell medicine.
9. according to the described purposes of claim 8, it is characterized in that inducing cell after the differentiation to can be used as external pharmacokinetic model and be used for the cell drug screening.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102225444A CN102311940A (en) | 2010-07-09 | 2010-07-09 | Method for inducing differentiation of stem cells into islet-like cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102225444A CN102311940A (en) | 2010-07-09 | 2010-07-09 | Method for inducing differentiation of stem cells into islet-like cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102311940A true CN102311940A (en) | 2012-01-11 |
Family
ID=45425507
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102225444A Pending CN102311940A (en) | 2010-07-09 | 2010-07-09 | Method for inducing differentiation of stem cells into islet-like cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102311940A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517251A (en) * | 2012-01-12 | 2012-06-27 | 苏州大学附属第一医院 | Mesenchymal stem cells, as well as preparation method and application thereof |
| CN109336982A (en) * | 2018-11-01 | 2019-02-15 | 长春万成生物电子工程有限公司 | The stem cell of genetic modification and its application |
| CN111848744A (en) * | 2018-09-03 | 2020-10-30 | 洛阳轩智生物科技有限公司 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1536075A (en) * | 2003-04-09 | 2004-10-13 | 中国人民解放军军事医学科学院野战输 | Method for inducing bone marrow mesenchymal stem cells to differentiate into insulin-like cells |
| CN1637137A (en) * | 2004-01-06 | 2005-07-13 | 中国人民解放军军事医学科学院野战输血研究所 | Method of inducing stem cell to differentiate to insulin-like cell and its application |
| CN1772885A (en) * | 2004-11-11 | 2006-05-17 | 中国人民解放军军事医学科学院野战输血研究所 | Method of inducing stem cell to differentiate into pancreatic island-like cell and application of pancreatic island-like cell |
| CN101603027A (en) * | 2008-06-12 | 2009-12-16 | 西北农林科技大学 | Method for inducing human bone marrow mesenchymal stem cells to differentiate into islet-like cells in vitro |
-
2010
- 2010-07-09 CN CN2010102225444A patent/CN102311940A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1536075A (en) * | 2003-04-09 | 2004-10-13 | 中国人民解放军军事医学科学院野战输 | Method for inducing bone marrow mesenchymal stem cells to differentiate into insulin-like cells |
| CN1637137A (en) * | 2004-01-06 | 2005-07-13 | 中国人民解放军军事医学科学院野战输血研究所 | Method of inducing stem cell to differentiate to insulin-like cell and its application |
| CN1772885A (en) * | 2004-11-11 | 2006-05-17 | 中国人民解放军军事医学科学院野战输血研究所 | Method of inducing stem cell to differentiate into pancreatic island-like cell and application of pancreatic island-like cell |
| CN101603027A (en) * | 2008-06-12 | 2009-12-16 | 西北农林科技大学 | Method for inducing human bone marrow mesenchymal stem cells to differentiate into islet-like cells in vitro |
Non-Patent Citations (3)
| Title |
|---|
| 李俊林等: "人脐带间充质干细胞体外向胰岛样细胞诱导分化及其治疗糖尿病效果", 《中国组织工程研究与临床康复》, vol. 13, no. 14, 2 April 2009 (2009-04-02) * |
| 杨印祥: "NRSF参与间充质干细胞向神经元及胰岛细胞分化的研究", 《中国博士学位论文全文数据库》, no. 11, 14 October 2008 (2008-10-14) * |
| 谢婷等: "犬骨髓间充质干细胞体外诱导分化为胰岛样细胞的研究", 《中国糖尿病杂志》, 30 April 2010 (2010-04-30) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517251A (en) * | 2012-01-12 | 2012-06-27 | 苏州大学附属第一医院 | Mesenchymal stem cells, as well as preparation method and application thereof |
| CN111848744A (en) * | 2018-09-03 | 2020-10-30 | 洛阳轩智生物科技有限公司 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN111875675A (en) * | 2018-09-03 | 2020-11-03 | 洛阳轩智生物科技有限公司 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN111848744B (en) * | 2018-09-03 | 2021-07-23 | 宁波希诺赛生物科技有限公司 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN109336982A (en) * | 2018-11-01 | 2019-02-15 | 长春万成生物电子工程有限公司 | The stem cell of genetic modification and its application |
| CN109336982B (en) * | 2018-11-01 | 2020-10-09 | 长春万成生物电子工程有限公司 | Genetically modified stem cells and uses thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4336821B2 (en) | Induction of cardiomyocytes using mammalian bone marrow cells or cord blood-derived cells and adipose tissue | |
| ES2613930T3 (en) | Procedure of culturing cells from adipose tissue and their applications | |
| CN103930542A (en) | Brown fat cell compositions and methods | |
| TW200521234A (en) | Method of single cell culture of undifferentiated human embryonic stem cells | |
| CN101117625B (en) | Method for evoking differentiation of mesenchyma stem cell and fat stem cell into neurons and special culture medium thereof | |
| CN112608972B (en) | Application of MYOG gene as target in preparation of medicine for treating cardiovascular diseases related to myocardial apoptosis | |
| CN110713984B (en) | Method for inducing human mesenchymal stem cells to generate functional endothelial cells | |
| CN102899288B (en) | Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells | |
| CN102344906A (en) | Hair follicle stem cell separation culture method | |
| CN106591372A (en) | Method for delaying senescence due to in vitro culture of human bone marrow MSC (Mesenchymal Stem Cells) | |
| CN110747163A (en) | Method for improving adipogenic differentiation of human adipose-derived mesenchymal stem cells and special culture medium thereof | |
| CN111500578A (en) | Circ RNA-FTO for regulating and controlling osteogenic differentiation and tissue regeneration of ADSCs and application thereof | |
| Laurent et al. | Bringing safe and standardized cell therapies to industrialized processing for burns and wounds | |
| CN102311940A (en) | Method for inducing differentiation of stem cells into islet-like cells | |
| AU2009248959A1 (en) | Method of differentiating mammalian progenitor cells into insulin producing pancreatic islet cells | |
| KR101503939B1 (en) | Method for preparation of cartilage cell | |
| CN113881624A (en) | Cell induction method | |
| CN108998417A (en) | Multipotential stem cell inducer and its application | |
| KR102281535B1 (en) | Generation of liver organoid from stem cells for hemophilia A treatment | |
| CN110205287A (en) | A kind of cell preparation for treating inflammatory enteritis | |
| JP5401669B2 (en) | Cardiomyocyte differentiation induction promotion method and differentiation induction method | |
| CN1772885B (en) | Method of inducing stem cell to differentiate into pancreatic island-like cell and application of pancreatic island-like cell | |
| CN106065401B (en) | Treatment use of the lentivirus mediated CXCR7 high expression engineering endothelial progenitor cells in ischemic disease | |
| CN109486770A (en) | A kind of microRNA-181c-5p promotion source of people iPS directed differentiation is the method for beta Cell of islet | |
| KR20120111790A (en) | Chondrogenesis differentiations of adipose tissue-derived mesenchymal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Pei Xuetao Inventor after: Li Yanhua Inventor after: Wang Sihan Inventor after: Zhang Jing Inventor after: Yao Hailei Inventor after: Yue Wen Inventor after: Li Baowei Inventor after: Shi Wei Inventor before: Pei Xuetao Inventor before: Wang Sihan Inventor before: Li Baowei Inventor before: Li Yanhua Inventor before: Shi Wei Inventor before: Yue Wen |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: PEI XUETAO WANG SIHAN LI BAOWEI LI YANHUA SHI WEI YUE WEN TO: PEI XUETAO LI YANHUA WANG SIHAN ZHANG JING YAO HAILEI YUE WEN LI BAOWEI SHI WEI |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120111 |