CN102559578A - Cell co-culture method for improving proliferation activity of somatic cells - Google Patents
Cell co-culture method for improving proliferation activity of somatic cells Download PDFInfo
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Abstract
The invention discloses a cell co-culture method for improving the proliferation activity of somatic cells. The cell co-culture method comprises the following steps of: transfecting suicide genes into stem cells; co-culturing the somatic cells and the stem cells transfected with the suicide gene; and after the somatic cells are proliferated, starting the suicide genes in the stem cells to remove the stem cells. The stem cells carrying the suicide genes and the somatic cells are co-cultured, the proliferation activity of the somatic cells is improved at the presence of the stem cells, a small number of somatic cells can quickly grow in short time, and sufficient somatic cells are obtained; and when the number of the somatic cells is large enough, the suicide genes carried in the stem cells are started to induce the apoptosis of the stem cells, and the pure somatic cells with high proliferation activity are obtained, and do not have tumorigenicity after transplantation, so that the method is great breakthrough for cell engineering and tissue engineering, and is a new way for next-generation clinical application. The method has the advantages of reliable principle, simple steps and stable results, and is easily applied to experimental study and clinical treatment in the field of bioengineering.
Description
Technical field:
The invention belongs to cell engineering and field of tissue engineering technology, be specifically related to the active co-culture of cells method of a kind of raising somatic cell proliferation.
Background technology:
In cell engineering and the field of tissue engineering technology; Need to use a quantity of seeds cell-somatocyte; And require somatocyte that certain proliferation activity is arranged, the two 26S Proteasome Structure and Function to recovery in the bioengineering field and reconstruction tissue and organ has fundamental influence.But because the outer multiplication capacity of somatophyte is limited; A spot of somatocyte can not obtain the somatocyte of capacity through propagation; And somatocyte is after external long-time cultivation, and along with the increase of division number of times, multiplication capacity descends gradually; The proliferation activity of cell descends, and has had a strong impact on the success of cell engineering and organizational project external structure.Because the somatic cell proliferation ability receives the influence of self and many factors such as environment, through regulating these correlative factors, can improve somatic proliferation activity, and obtain the somatocyte of sufficient amount at short notice.
Present a lot of research shows, uses strong cell of proliferation activity and the weak cell co-cultivation of proliferation activity, can effectively promote the propagation performance of the cell that multiplication capacity is weak.Stem cell is the extremely strong cell of a kind of multiplication capacity, has very strong cell proliferation, the characteristic of self, and it can provide the stem cell microenvironment; The secretion soluble proteins; Cell surface molecule, and nutrition and sustenticular cell such as extracellular matrix improve the somatic cell proliferation ability.With stem cell and somatocyte co-cultivation, can provide the external microenvironment that is beneficial to cell proliferation, improve somatic proliferation activity through direct effect with indirect two aspects.At first stem cell directly promotes somatic propagation through directly contacting with somatocyte; Secondly stem cell comprises that through the various factors that help the cell growth of paracrine action secretion BDNF (GDNF), glia cell line-derived NGFF (GDNF), pHGF, transforminggrowthfactor-, Urogastron, VEGF, Thr6 PDGF BB, keratinocyte growth factor, fibroblast growth factor and pHGF etc. improve somatic proliferation activity.Through above 2 points, somatocyte mixes cultivation altogether with stem cell finally can obtain the somatocyte of the amount of satisfying the demand fast when improving the somatic cell proliferation vigor.But this method has following defective: 1. the undifferentiated embryonic stem cell that exists in the transplanted cells can form tumour in by transplant.Because stem cell has very strong multiplication capacity, and the multidirectional different cells that is divided into of ability, can form kinds of tumors after the transplanting, mainly comprise teratocarcinoma and teratoma.2. differentiation appears in stem cell easily in cell in vitro is cultivated, and can be converted into the cell of unwanted other each germinal layers, becomes to mix cell, can't remove.So behind the high reactivity somatocyte of the q.s that to need to have obtained when us, how to remove the stem cell that no longer needs, only obtain simple somatocyte, be to wait the problem that solves at present.
Summary of the invention:
The purpose of this invention is to provide and a kind ofly can avoid the influence of stem cell again, only obtain the simple active co-culture of cells method of somatic raising somatic cell proliferation in that can when keeping somatic primary characteristic, to improve somatic cell proliferation active.
The active co-culture of cells method of raising somatic cell proliferation of the present invention is characterized in that, may further comprise the steps:
A, suicide gene is transfected in the stem cell;
B, with somatocyte and transfection the stem cell of suicide gene cultivate altogether;
C, somatocyte start the suicide gene in the stem cell after accomplishing the propagation purpose, remove stem cell.
Somatocyte and transfection the stem cell of suicide gene altogether in the culturing process; After differentiation of stem cells or no longer have the time spent of doing that promotes somatic cell proliferation; Can start suicide gene through the certain drugs precursor and remove stem cell, the stem cell of suicide gene of having added fresh transfection once more, fresh transfection the stem cell of suicide gene can continue to improve somatic proliferation activity; Promote somatic fast breeding; But like this repeated several times until reaching needed somatic amount, and improves somatic proliferation activity.
Described suicide gene is meant that some can express the gene of specific enzyme, and these enzymes can be converted into the cell toxicant material with some nontoxic or hypotoxic prodrug, thereby cause carrying the apoptosis of this gene.Described suicide gene comprises thymidine kinase (Thymidine kinase; TK) gene, Isocytosine deaminase (Cytosine deaminase, CD) one or more in the suicide genes such as gene, E.coli-gpt, Cytochrome P450 (Cytochrome P450/cytochrome P450 reductase) gene, TNT nitroreductase (Nitroreductase) gene, carboxypeptidase (Carboxypeptidase G2) gene, e.coli-DeoD gene (expression purine nucleoside phosphorylase), diphtheria toxin gene (DTA gene) and FAS gene.
Described transfection method comprises the transfection method of virus or non-virus; Wherein said virus transfection method comprises utilizes retroviral vector; Adenovirus carrier, herpes simplex virus vector, Epstein-Barr virus carrier; Parvovirus vectors, in the carrier mediated transfection method such as poxvirus vector one or more; Described non-virus transfection method comprises the method for chemistry and physics, comprises cationic polymers, the coprecipitation of calcium phosphate infection protocol; The artificial liposome method; Deae dextran mediation infection protocol, direct injection, electroporation; The laser radiation method, one or more in the transfection methods such as magnetic particle method and particle gun.
Described stem cell can be embryonic stem cell, one or more in adult stem cell or other any stem cells.Can use multiple stem cell, utilize the different dry cell to somatic different supports, Nutrition, thereby promote somatic propagation.
Described somatocyte comprises various germinal layer cells in the histoorgan, specifically can be any of skin, cardiovascular, bladder, ligament, bone, nerve, liver, enteron aisle, blood, cornea, conjunctiva or sclera cell or more than one.
Described cultivation altogether is meant somatocyte is contacted cultivation with the stem cell mixing.
Described startup suicide gene method is for using the method that can cause carrying the stem cell apoptosis of this gene with the acting low toxicity of specificity enzyme of the suicide gene coding that carries in the cell or nontoxic prodrug.Described prodrug comprises acycloguanosine (acyclovir; ACV), ganciclovir (ganciclovir, GCv), among 5-flurocytosine, 6-sulfydryl xanthine, endoxan, 6-methyl purine-2-deoxyribosyl nucleosides, ara-M, BVDU, CPA, IFO, CB1954, MeP-dR, F-araA, CMDA, 6-Thiopurines, ara-C, dFdC, ifosfamide and the MTX-α-peptides one or more.Those skilled in the art can select its corresponding prodrug according to the suicide gene of transfection, again with its effect of specificity enzyme of suicide gene coding, thereby cause the stem cell apoptosis that carries this gene.Select the knowledge of prodrug to belong to the conventional knowledge of this area according to suicide gene.Can be through using one or more suicide genes; Use one or more different drug precursors to start the method for corresponding suicide gene separately respectively; When perhaps starting suicide gene; Prodrug uses the perhaps method of different access times of different concentration, reaches the purpose of time, speed and the quantity of adjustment stem cell apoptosis, finally comes to control flexibly growth and the apoptosis quantity of stem cell through regulating these parameters; Influence somatic propagation, to adapt to the demand of different cells engineering or organizational project.
The stem cell that the present invention will carry suicide gene mixes co-cultivation with somatocyte, and the existence of stem cell has improved somatic proliferation activity, thereby a spot of somatocyte can be grown at short notice fast; Obtain the somatocyte of sufficient amount, when somatic quantity is enough, start the suicide gene that stem cell is carried; The induced dry-cell apoptosis, thereby when avoiding that stem cell and somatocyte are cultivated altogether in the prior art, because the high proliferation ability of stem cell; And the multidirectional different cells that is divided into of ability, differentiation appears in the shortcoming and the stem cell that can form kinds of tumors after the transplanting easily in cell in vitro is cultivated, can be converted into the cell of unwanted other each germinal layers; Become and mix cell; The shortcoming that can't remove finally obtains merely, has the active somatocyte of high proliferation; And do not have into knurl property after transplanting, can effectively remove after perhaps transplanting.This is not only the important breakthrough of cell engineering and organizational project, and has opened up new approach for clinical application of future generation.The present invention can use one or more stem cells; Once or repeatedly add stem cell; Once or repeatedly start suicide gene, and can be through adjusting these parameters at any time flexibly, the somatic time that the adjustment acquisition needs quantity and proliferation activity are to satisfy different demands.The principle of the invention is reliable, and step is easy, and the result is stable, very easily is applied to the experimental study and the clinical treatment of various bioengineering fields.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
The concrete steps of the active co-culture of cells method of raising somatic cell proliferation of the present invention are:
A. stem cell is transfected into suicide gene: the method that adopts virus transfection or non-virus transfection; With thymidine kinase (Thymidine kinase; TK) (Cytosine deaminase is CD) in any or more than one importing stem cells in the suicide genes such as gene, E.coli-gpt, Cytochrome P450 (Cytochrome P450/cytochrome P450 reductase) gene, TNT nitroreductase (Nitroreductase) gene, carboxypeptidase (Carboxypeptidase G2) gene, E.coli-DeoD gene (expression purine nucleoside phosphorylase), diphtheria toxin gene (DTA gene) and FAS gene for gene, Isocytosine deaminase.
The stem cell of b. using can be a kind of of embryonic stem cell or adult stem cell or more than one.
C. the somatocyte that uses can be the cell in various germinal layers source.Comprise any in skin, cardiovascular, bladder, ligament, bone, liver, enteron aisle, blood, nerve, cornea, conjunctiva or the sclera cell or more than one.
The stem cell that d. will import suicide gene contacts cultivation altogether with capable directly mixing of somatocyte.
E. somatic proliferation activity raises, and obtains enough cell concentrations fast.
F. use specific prodrug, the specificity enzyme that ability and suicide gene are expressed reacts, and is the cytotoxicity product with the metabolism of avirulent specific medication precursor component, finally causes the stem cell apoptosis.This prodrug comprises acycloguanosine (acyclovir; ACV), ganciclovir (ganciclovir; GCv), 5-flurocytosine, 6-sulfydryl xanthine, endoxan, 6-methyl purine-2-deoxyribosyl nucleosides, ara-M, BVDU, CPA, IFO, CB1954, MeP-dR, F-araA, CMDA, 6-Thiopurines, ara-C, dFdC, ifosfamide and MTX-α-peptides etc. all can work with the specificity enzyme of corresponding suicide gene coding, thereby cause carrying one or more of prodrug of the stem cell apoptosis of this gene.
G. use the number of times of specific drugs precursor can be for once or once; Concentration can also can be lower concentration for high density, and purpose all is to remove the stem cell composition, keeping the simple somatic while; Improve somatic proliferation activity, and the cell concentration of quick acquisition needs.
H. described gene transfection method comprises one or more of the virus and the transfection method of non-virus.
I. wherein said virus transfection method comprises retroviral vector, adenovirus carrier, herpes simplex virus vector; The Epstein-Barr virus carrier, parvovirus vectors, one or more in the carrier mediated transfection method such as poxvirus vector; Described non-virus transfection method comprises the method for chemistry and physics, comprises cationic polymers, the coprecipitation of calcium phosphate infection protocol; The artificial liposome method, deae dextran mediation infection protocol, direct injection; Electroporation, laser radiation method, one or more in the transfection methods such as magnetic particle method and particle gun.
Embodiment 1:
The purpose of present embodiment is to obtain the high reactivity corneal epithelial cell of q.s in the short period of time [annotate: the used suicide gene of present embodiment comprises thymidine kinase (Thymidine kinase; TK) gene, Isocytosine deaminase (Cytosine deaminase; CD) one or more of any suicide genes such as gene, E.coli-gpt, Cytochrome P450 (Cytochrome P450/cytochrome P450reductase) gene, TNT nitroreductase (Nitroreductase) gene, carboxypeptidase (Carboxypeptidase G2) gene, e.coli-DeoD gene (expression purine nucleoside phosphorylase), diphtheria toxin gene (DTA gene) and FAS gene; Stem cell is one or more of embryonic stem cell or adult stem cell; The transfection way comprises one or more of the virus and the transfection method of non-virus; The time that starts suicide gene is for once or once; The medicine that starts suicide gene is acycloguanosine (acyclovir; ACV), ganciclovir (ganciclovir; GCv), 5-flurocytosine, 6-sulfydryl xanthine, endoxan, 6-methyl purine-2-deoxyribosyl nucleosides, ara-M, BVDU, CPA, IFO, CB1954, MeP-dR, F-araA, CMDA, 6-Thiopurines, ara-C, dFdC, ifosfamide and MTX-α-peptides etc. all can work with the specificity enzyme of corresponding suicide gene coding, thereby cause carrying one or more of prodrug of the stem cell apoptosis of this gene.Its effect is with following identical].
1. with mouse embryo stem cell transfection suicide gene: adopt the method for liposome transfection, thymidine nucleoside kinase gene (TK gene) is imported in the mouse embryo stem cell.
2. capable directly mixing of corneal epithelial cells of rabbit and the mouse embryo stem cell that has suicide gene contacted cultivation altogether, nutrient solution is a Mouse Embryonic Stem Cell Culture liquid.
3. the generation time of the corneal epithelial cells of rabbit cell concentration of vitro culture acquisition needs shortened to 5 days from 14 days, and the result shows that the proliferation activity of corneal epithelial cells of rabbit improves.The corneal epithelial cell cloning efficiency of wherein representing rabbit corneal epitheliosis function by 9.95% from having brought up to 34.2%; On behalf of the p63 of the low differentiation sign of corneal epithelial cells of rabbit, flow cytometer detects brought up to about 72% from 12%; Ki67 rises to 60%, cell entering S/G from 26%
2The amount of phase is increased to 32% from 14%.
4. disposable interpolation uses the acting certain drugs precursor of the specificity enzyme ganciclovir 20 μ mol/L that encode to the TK suicide gene in nutrient solution.The specificity enzyme that ganciclovir and suicide gene are expressed reacts, and is triphosphoric acid product GCT-TP with avirulent ganciclovir metabolism, produces cytotoxicity, the whole apoptosis of mouse embryo stem cell after 3 days.
5. through detecting, only keep corneal epithelial cells of rabbit in the petridish, cell concentration does not reduce; Cell-proliferation activity is high; The cell that is in the DNA synthesis phase is constant, and the low differentiation sign p63 that the immunohistochemical experiment showed cell is expressed does not reduce, and the multiplication capacity of cloning efficiency experiment confirm cell does not influence.
Embodiment 2:
The purpose of present embodiment is to obtain the high reactivity human skin epidermic cell of q.s in the short period of time [annotate: the used suicide gene of present embodiment comprises thymidine kinase (Thymidine kinase; TK) gene, Isocytosine deaminase (Cytosine deaminase; CD) one or more of any suicide genes such as gene, E.coli-gpt, Cytochrome P450 (Cytochrome P450/cytochrome P450reductase) gene, TNT nitroreductase (Nitroreductase) gene, carboxypeptidase (Carboxypeptidase G2) gene, e.coli-DeoD gene (expression purine nucleoside phosphorylase), diphtheria toxin gene (DTA gene) and FAS gene; Stem cell is one or more of embryonic stem cell or adult stem cell; The transfection way comprises one or more of the virus and the transfection method of non-virus; The time that starts suicide gene is for once or once; The medicine that starts suicide gene is acycloguanosine (acyclovir; ACV), ganciclovir (ganciclovir; GCv), 5-flurocytosine, 6-sulfydryl xanthine, endoxan, 6-methyl purine-2-deoxyribosyl nucleosides, ara-M, BVDU, CPA, IFO, CB1954, MeP-dR, F-araA, CMDA, 6-Thiopurines, ara-C, dFdC, ifosfamide and MTX-α-peptides etc. all can work with the specificity enzyme of corresponding suicide gene coding, thereby cause carrying one or more of apoptotic prodrug of this gene.Its effect is with following identical].
1. with human cord blood stem cell transfection suicide gene: adopt the method for adenovirus carrier transfection, (Cytosine deaminase, CD) gene import in the human cord blood stem cell with Isocytosine deaminase; With human embryo stem cell transfection suicide gene: use the method for liposome transfection, thymidine nucleoside kinase gene (TK gene) is imported in the human embryo stem cell.
2. the human cord blood stem cell that will have suicide gene directly mixes to contact altogether with human embryo stem cell and human skin epidermic cell to be cultivated, and its nutrient solution is the embryonic stem cell nutrient solution.
3. the vitro culture generation time that obtains first human skin epidermic cell cell concentration that needs shortened to 4 days from 11 days, and the result shows, human skin epidermal cell proliferation increased activity is represented the G of skin progenitor cell
0/ G
1The phase cell proportion brings up to 55% from 36%, and cloning efficiency brings up to 23.6% by 11.2%, and flow cytometer detects the low differentiation sign K19 that showed cell expresses and brings up to 32% from 21%.
4. add the prodrug 5-flurocytosine; This prodrug can react with the specificity enzyme that the CD suicide gene in this human cord blood stem cell is expressed; With avirulent 5-flurocytosine metabolism is 5 FU 5 fluorouracil, produces cytotoxicity, finally causes human umbilical blood stem cell whole apoptosis after 1 day.
5. the vitro culture generation time that obtains the human skin epidermic cell cell concentration of second needs shortened to 2 days from 3 days, and the result shows, human skin epidermal cell proliferation increased activity is represented the G of skin progenitor cell
0/ G
1The phase cell proportion brings up to 85% from 55%, and cloning efficiency brings up to 36.9% by 23.6%, and flow cytometer detects the low differentiation sign K19 that showed cell expresses and brings up to 41% from 32%.
6. disposable interpolation uses the acting certain drugs precursor of the specificity enzyme ganciclovir 20 μ mol/L that encode to the TK suicide gene in nutrient solution.The specificity enzyme that ganciclovir and suicide gene are expressed reacts, and is triphosphoric acid product GCT-TP with avirulent ganciclovir metabolism, produces cytotoxicity, the whole apoptosis of embryonic stem cell after 3 days.
7. through detecting, only keep the human skin epidermic cell in the petridish, do not influence its proliferation activity, represent the G of skin progenitor cell
0/ G
1The phase cell proportion is constant, and the multiplication capacity that the low differentiation sign K19 ratio that flow cytometer detection showed cell is expressed does not reduce cloning efficiency experiment confirm cell does not influence.
Embodiment 3:
The purpose of present embodiment is the high reactivity cat endothelial cell that obtains q.s in the short period of time.[annotate: the used suicide gene of present embodiment comprises thymidine kinase (Thymidine kinase; TK) gene, Isocytosine deaminase (Cytosine deaminase; CD) one or more of any suicide genes such as gene, E.coli-gpt, Cytochrome P450 (Cytochrome P450/cytochrome P450reductase) gene, TNT nitroreductase (Nitroreductase) gene, carboxypeptidase (Carboxypeptidase G2) gene, e.coli-DeoD gene (expression purine nucleoside phosphorylase), diphtheria toxin gene (DTA gene) and FAS gene; Stem cell is one or more of embryonic stem cell or adult stem cell; The transfection way comprises one or more of the virus and the transfection method of non-virus; The time that starts suicide gene is for once or once; The medicine that starts suicide gene is acycloguanosine (acyclovir; ACV), ganciclovir (ganciclovir; GCv), 5-flurocytosine, 6-sulfydryl xanthine, endoxan, 6-methyl purine-2-deoxyribosyl nucleosides, ara-M, BVDU, CPA, IFO, CB1954, MeP-dR, F-araA, CMDA, 6-Thiopurines, ara-C, dFdC, ifosfamide and MTX-α-peptides etc. all can work with the specificity enzyme of corresponding suicide gene coding, thereby cause carrying one or more of apoptotic prodrug of this gene.Its effect is with following identical].
1. human marrow mesenchymal stem cell is transfected into suicide gene: adopt the method for coprecipitation of calcium phosphate, Cytochrome P450 2B1 gene is imported in the human marrow mesenchymal stem cell.
2. the endothelial cell of cat and the human marrow mesenchymal stem cell that has suicide gene are directly mixed to contact altogether and cultivate, its nutrient solution is the mesenchymal stem cells MSCs nutrient solution.
3. the generation time of the cat endothelial cell amount of vitro culture acquisition needs shortened to 12 days from 21 days; And the result shows; Cat endothelial cell proliferation activity strengthens; Flow cytometer detects Ki67 positive cell represent cell proliferative breeds to 9.4% from 3.2%, and cloning efficiency brings up to 2.2% by 1.3%, cell entering S/G
2The cell of phase is increased to 30.9% by 16.4%.
4. disposable interpolation prodrug endoxan; The specificity enzyme of expressing with the suicide gene in the human marrow mesenchymal stem cell reacts; With avirulent endoxan metabolism is cytotoxic 4-OHCPA, finally causes human marrow mesenchymal stem cell whole apoptosis after 2 days.
5. through detecting, only keep the cat endothelial cell in the petridish, do not influence its proliferation activity, be in DNA synthesis phase and G
2The cell concentration of phase is constant, and the multiplication capacity of cloning efficiency experiment confirm cell does not influence.
Embodiment 4:
The purpose of present embodiment is to obtain the high reactivity rabbit conjunctival epithelial cell of q.s in the short period of time [annotate: the used suicide gene of present embodiment comprises thymidine kinase (Thymidine kinase; TK) gene, Isocytosine deaminase (Cytosine deaminase; CD) one or more of any suicide genes such as gene, E.coli-gpt, Cytochrome P450 (Cytochrome P450/cytochrome P450reductase) gene, TNT nitroreductase (Nitroreductase) gene, carboxypeptidase (Carboxypeptidase G2) gene, E.coli-DeoD gene (expression purine nucleoside phosphorylase), diphtheria toxin gene (DTA gene) and FAS gene; Stem cell is one or more of embryonic stem cell or adult stem cell; The transfection way comprises one or more of the virus and the transfection method of non-virus; The time that starts suicide gene is for once or once; The medicine that starts suicide gene is acycloguanosine (acyclovir; ACV), ganciclovir (ganciclovir; GCv), 5-flurocytosine, 6-sulfydryl xanthine, endoxan, 6-methyl purine-2-deoxyribosyl nucleosides, ara-M, BVDU, CPA, IFO, CB1954, MeP-dR, F-araA, CMDA, 6-Thiopurines, ara-C, dFdC, ifosfamide and MTX-α-peptides etc. all can work with the specificity enzyme of corresponding suicide gene coding, thereby cause carrying one or more of apoptotic prodrug of this gene.Its effect is with following identical].
1. with human adipose-derived stem cell transfection suicide gene: adopt the method for liposome transfection, thymidine nucleoside kinase gene (TK gene) is imported in the human adipose-derived stem cell.
2. rabbit conjunctival epithelial cell and the human adipose-derived stem cell that has suicide gene are directly mixed to contact altogether and cultivate, its nutrient solution is the human adipose-derived stem cell nutrient solution.
3. the generation time of the rabbit conjunctival epithelial cell amount of vitro culture acquisition needs shortened to 8 days from 16 days; And the result shows; Rabbit conjunctival epithelial cell proliferation activity strengthens; Represent the cloning efficiency of cell proliferation performance to bring up to 22%, represent the MTT experiment BrdU absorbancy of ability of cell proliferation to bring up to 3.65 by 1.45 by 14%.The histology showed cell is regularly arranged, and endochylema is abundant.
4. cultivate at twice and add the prodrug ganciclovir.The specificity enzyme of expressing with TK suicide gene in the stem cell reacts, and is triphosphoric acid product GCT-TP with the ganciclovir metabolism, produces the cytotoxicity product, finally causes human adipose-derived stem cell whole apoptosis after 3 days.
5. through detecting, only keep the rabbit conjunctival epithelial cell in the petridish, do not influence its proliferation activity, represent the MTT experimental result of ability of cell proliferation constant, the cloning efficiency experiment is constant, confirms the multiplication capacity of rabbit conjunctival cells, keeps high growth active.
Claims (10)
1. one kind is improved the active co-culture of cells method of somatic cell proliferation, it is characterized in that, may further comprise the steps:
A, suicide gene is transfected in the stem cell;
B, with somatocyte and transfection the stem cell of suicide gene cultivate altogether;
C, somatocyte start the suicide gene in the stem cell after accomplishing the propagation purpose, remove stem cell.
2. the active co-culture of cells method of raising somatic cell proliferation according to claim 1; It is characterized in that, somatocyte and transfection the stem cell of suicide gene altogether in the culturing process, after differentiation of stem cells or no longer have the time spent of doing that promotes somatic cell proliferation; Start suicide gene and remove stem cell; The stem cell of suicide gene of having added fresh transfection once more, but repeated several times like this are accomplished the propagation purpose until somatocyte.
3. the active co-culture of cells method of raising somatic cell proliferation according to claim 1 and 2; It is characterized in that described suicide gene is one or more in thymidine kinase gene, cytosine deaminase gene, E.coli-gpt, cytochrome P450 gene, TNT nitroreductase gene, carboxypeptidase gene, E.coli-DeoD gene, diphtheria toxin gene and the FAS gene.
4. the active co-culture of cells method of raising somatic cell proliferation according to claim 1 and 2 is characterized in that, described transfection method is the transfection method of virus or non-virus.
5. the active co-culture of cells method of raising somatic cell proliferation according to claim 4; It is characterized in that; Described virus transfection method is for utilizing retroviral vector, adenovirus carrier, herpes simplex virus vector; The Epstein-Barr virus carrier, in the transfection method of parvovirus vectors and poxvirus vector mediation one or more.
6. the active co-culture of cells method of raising somatic cell proliferation according to claim 4 is characterized in that, described non-virus transfection method is a cationic polymers; The coprecipitation of calcium phosphate infection protocol, artificial liposome method, deae dextran mediation infection protocol; Direct injection; Electroporation, laser radiation method, one or more in magnetic particle method and the particle bombardment.
7. the active co-culture of cells method of raising somatic cell proliferation according to claim 1 and 2 is characterized in that described stem cell is an embryonic stem cell, one or more in adult stem cell or other any stem cells.
8. the active co-culture of cells method of raising somatic cell proliferation according to claim 1 and 2; It is characterized in that described somatocyte is any of skin, cardiovascular, bladder, ligament, bone, nerve, liver, enteron aisle, blood, cornea, conjunctivae and selerae cell or more than one.
9. the active co-culture of cells method of raising somatic cell proliferation according to claim 1 and 2 is characterized in that, described cultivation altogether is meant somatocyte is contacted cultivation with the stem cell mixing.
10. the active co-culture of cells method of raising somatic cell proliferation according to claim 1 and 2; It is characterized in that; Described startup suicide gene is meant that the specificity enzyme that utilizes the suicide gene coding that carries in low toxicity or nontoxic prodrug and the stem cell works and causes carrying the stem cell apoptosis of this gene and remove stem cell, and described prodrug is one or more among acycloguanosine, ganciclovir, 5-flurocytosine, 6-sulfydryl xanthine, endoxan, 6-methyl purine-2-deoxyribosyl nucleosides, ara-M, BVDU, CPA, IFO, CB1954, MeP-dR, F-araA, CMDA, 6-Thiopurines, ara-C, dFdC, ifosfamide and the MTX-α-peptides.
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|---|---|---|---|---|
| CN104877954A (en) * | 2015-05-19 | 2015-09-02 | 暨南大学 | Stem cell niche as well as preparation method and application thereof |
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