CN102586174A - Pluripotent cell derived from cataract mouse model, its preparation method and application - Google Patents
Pluripotent cell derived from cataract mouse model, its preparation method and application Download PDFInfo
- Publication number
- CN102586174A CN102586174A CN2012100442309A CN201210044230A CN102586174A CN 102586174 A CN102586174 A CN 102586174A CN 2012100442309 A CN2012100442309 A CN 2012100442309A CN 201210044230 A CN201210044230 A CN 201210044230A CN 102586174 A CN102586174 A CN 102586174A
- Authority
- CN
- China
- Prior art keywords
- mouse
- cell
- cataract
- multipotential cell
- multipotential
- 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)
Abstract
The invention relates to a pluripotent cell derived from genetic cataract mouse. Tests show that the cell has embryonic stem cell characteristic. The cell can be used for studying pathogenic mechanism of cataract, screening drugs for treating cataract, providing research carrier for genetic treatment means, and providing new materials and methods for cataract research.
Description
Technical field
The invention belongs to the zooblast field, be specifically related to a kind of multipotential cell such as embryonic stem cell (ES) Its Preparation Method And Use that derives from the cataract disease mice.
Background technology
Cataract is the major disease that has a strong impact on public health.According to World Health Organization's statistics, the whole world has 4,500 ten thousand people blind approximately, wherein has half to be caused by cataract.Cataract is that to be caused by multiple reasons such as h and E Hazard Factor is multistress imbalance ophthalmic diseases (the Mccarty C A of cardinal symptom with the phacoscotasmus; Taylor H R.The genetics of cataract [J] .Invest Ophthalmol Vis Sci; 2001,42 (8): 1677-8.).Cataractous mode of inheritance comprises autosomal dominant inheritance, autosomal recessive inheritance and x linked recessive heredity.Hereditary cataract still is all very complicated on the hereditary property from clinical manifestation, and many transgenations relevant with lens all can cause cataractous formation.Because being exactly the simple cataract of research heredity, the complicacy of cataract disease, effective means come further to understand in depth all genes and the pathogenesis that can cause and influence the lens pathology.
People begin from animal model the understanding of cataract inherited genetic factors the earliest, mainly are mouse model (Graw J.Mouse models of cataract [J] .J Genet, 2009,88 (4): 469-86.).This is because phacoscotasmus can relatively easily detect in mouse model, and can observe cataractous mode of inheritance simultaneously.The animal model of disease is the strong tool of pathogenesis, pathologic process of this disease of in vitro study etc.Mouse model plays an important role in the cataract research field.Up to now, the hereditary cataract mouse model of having found both at home and abroad has more than 140 approximately, and these mouse models mainly are through spontaneous mutation, bring out sudden change, knock out and suddenly change or modes such as transgenic obtain.Utilize mouse model; Can overcome that the cataract incidence and development is slow, latent period is long, pathogenic factor is various and often with factor interferential problems such as various other diseases; Can be through the single cause of disease; Copy typical animal disease model at short notice, therefore, the cataract mouse model is very important means and instrument for research cataractous generation, the rule of development and medical diagnosis on disease treatment etc.
Along with the reach of science; Had more than 20 gene locus to be proved to be relevant with congenital cataract, wherein 17 genes are cloned (He W, Li S.Congenitalcataracts:gene mapping [J] .Hum Genet fully; 2000; 106 (1): 1-13.), people have also had certain understanding gradually to cataractous mechanism, but still also have many families to receive the cataractous influence that the unknown gene sudden change causes.In the cataract mouse mutantion line of having found, also have the part mutator gene in the not clearly definition as yet of its molecular level, even those known genes relevant with cataract, its definite mechanism is not yet understood fully yet.Though along with the continuous progress of medical skill, the diagnosis of congenital cataract and the non-difficult matter of treatment, current diagnosis still is diagnosed as the master with symptomology, and treatment is main with operation, and has certain complication behind the operative treatment.Perhaps can imitate the way of other heredopathias; Through to methods such as fetus period disease gene screenings; Medical diagnosis on disease is advanceed to fetus period, for efficacious therapy provides opportunity and foundation, means through gene therapy again; Hereditary cataract is controlled at before cataract do not form as yet or be not completed into as yet, to be implemented in the control of gene level hereditary cataract.Gene therapy is operated at cell levels usually, and the embryonic stem cell with versatility is undoubtedly optimal selection.
Embryonic stem cell (embryonic stem cell; ES) be one type of cell that the clone comes out from the body early embryo inner cell mass; Under the ideal condition, cultivate; This type cell can be kept its unlimited self proliferation potential, and can be divided into the broad variety daughter cell that comprises sexual cell, has ability (Martin G R.Isolation of a pluripotentcell line from early mouse embryos cultured in medium conditioned byteratocarcinoma stem cells [J] the .Proc Natl Acad Sci USA that develops into complete living individual; 1981,78 (12): 7634-8; Mastui Y, Zsebo K, Hongan B L.Derivation ofpluripotential embryonic stem cells from murine primordial germ cellsin culture [J] .Cell, 1992,70 (5): 841-7.).The purposes of embryonic stem cell is a lot, in the every field of life science and medical science very important and far-reaching influence is arranged all.The totipotency genetic manipulation property of embryonic stem cell and the ability that infinitely increases make it become good material and the method for studying extremely early stage incident in the ontogenetic process on cell and the molecular level.Learn the application of biotechnologys such as research gene chip, gene trap along with human genome; Relatively ES cell and the cell of different developmental phases and the genetic transcription and the expression of noble cells, can confirm fetal development and cytodifferentiation molecular mechanism, find new gene.In conjunction with gene targeting, can find the function of different genes in vital movement etc.Embryonic stem cell also provides the research means of the cell levels of researchs such as pharmacological effect, toxicity and the drug metabolism of novel drugs; Can significantly reduce the quantity of the required laboratory animal of drug study; The ES cell also can be used to zoologize mechanism and the development with human diseases, and process is so that find effective and persistent treat-ment.Pass through ES cell and gene therapy technology in addition, also might the rectification of defects gene.
1981; Evans etc. adopt the STO cell as feeder layer; In the nutrient solution that has added serum, successfully from blastaea ICM, cultivated ES cells (Evans M J; Kaufman M H.Establishment in culture of pluripotential cells from mouse embryos [J] .Nature, 1981,292 (5819): 154-156.).Martin GR with the immunosurgery method shell blastaea inner cell mass (ICM) and being placed on the STO cell feeder layer; And cultivate with mouse PSA21-ES cell conditioned medium; Obtain ES cells (Marin G R.Isolation of apluripotent cell line from early mouse embryos cultured in mediumconditioned by teratocarcinoma stem cells [J] .Proc Natl Acad Sci USA; 1981,78 (12): 7634-8.).Dhhaise etc. are digested to single blastomere with 8-cell mouse embryo and are incubated on the mouse inoblast feeder layer of former generation; To have added the DMEM/F12 culture medium culturing of foetal calf serum; Set up an ES clone MSB1, this cell line cell has the ability that forms chimeric mouse.Above-mentioned investigator uses classical feeder layer increase serum culture system to cultivate the acquisition ES cells.Though scientist uses this individual system successfully to set up the ES clone of multiple mouse species, still there are a lot of deficiencies in this culture system.Do not have a resistance like feeder layer cells; Therefore can't be used for the embryonic stem cell screening of transfection allogenic gene; And dead feeder layer cells may cause the sudden change of ES cell and influence maintenance (the Suemori H of normal karyotype; Nakatsuji N.Establishment of the Embryo-derived Stem (ES) Cell Linesfrom mouse blastocysts:effects of the feeder cell layers [J] .DevelopmentGrowth & Differentiation; 1987,29 (2): 133-9.).It is animal derived to the most important thing is that feeder layer and serum all have, and complicated component, and mechanism of action is unclear, has uncertainty and uncontrollability, thereby has limited the more successful foundation of polyembryony tire stem cell line.
2003; People such as Ying find; Acting composition is BMP4 in the serum, and utilizes the serum-free of LIF+BMP4 not have the feeder layer culture system and successfully set up ES cells system (YingQ L, Nichols J; Chambers I; Et al.BMP induction of Id proteinssuppresses differentiation and sustains embryonic stem cell self-renewalin collaboration with STAT3 [J] .Cell, 2003,115 (3): 281-92.).2007, Ying etc. more obtained pluripotent cell (comprising ES cells) in the serum free medium that contains mek inhibitor, GSK3 suppressor factor and FGF receptor antagonist, and kept self state (number of patent application 200780011232.5) therein.The advantage of this culture system is not add from arbitrary stimulation renewal in feeder layer and the serum keeps the not composition of differentiation state of ES cell; The embryonic stem cell line of more and more mouse species utilizes this culture system to be able to build up; Like (Ohta H such as NOD mouse ES system, C57BL/6N mouse ES system and non-obese diabetic ES cells systems; Ohinata Y; Ikawa M; Et al.Male germline and embryonic stem cell linesfrom NOD mice:efficient derivation of Gscells from a nonpermissivestrain for ES cell derivation [J] .Biology of reproduction, 2009,81 (6): 1147; Kiyonari H; Kaneko M; Abe S, et al.Three inhibitors ofFGF receptor, ERK; And GSK3establishes germline-competentembryonic stem cells of C57BL/6N mouse strain with high efficiencyand stability.Genesis, 48 (5): 317-27; Nichols J, Jone K, Phillips J, et al.Validated germline-competent embryonic stem cell lines from nonobesediabetic mice.Nature medicine, 2009,15 (7): 814-8.).
But so far, both at home and abroad still not about the report of the embryonic stem cell that successfully obtains to derive from cataract animal model.The embryonic stem cell of cataract mouse is badly in need of obtaining in this area simultaneously, on cell levels, for research cataract disease, like pathogenesis research, drug screening and gene therapy etc. new material and method is provided.Compare with animal model, the advantage of embryonic stem cell be can be more convenient carry out large-scale genetic manipulation, screening.
Summary of the invention
For this reason; The inventor carries out many-sided research; Finally be starting material with the cataract mouse, cultivate from the body early embryo of cataract mouse successfully that to have obtained the embryonic stem cell of cataract mouse and to have verified with the cataract mouse serve as that the method that the source can successfully obtain to have the embryonic stem cell of cataract genetic information is feasible.
Particularly, the present invention includes following several:
First aspect of the present invention relates to a kind of multipotential cell, and it separates from cataract disease model mouse,
Preferably, described model mice is a hereditary cataract disease model mouse,
Preferably, described hereditary cataract disease model mouse is selected from:
A: heredity BALB/c
Cat/CatThe cataract mouse;
The b:Philly mouse;
The c:Crybal mutant mouse;
D: dominance cataract mouse Hif;
Further be preferably heredity BALB/c
Cat/CatThe cataract mouse;
Preferably, described multipotential cell is an embryonic stem cell.
The described multipotential cell of first aspect present invention, it separates the body early embryo from cataract disease model mouse.
The described multipotential cell of first aspect present invention, it is expressed SEAP, SSEA1 gene and/or is selected from one or more genes among Oct4, Nanog, Sox2, Eras, Esg1, Fgf4, Utf1, Cripto, Rex1, the Gdf3;
Preferably, this multipotential cell is expressed Oct4, Nanog and Sox2;
More preferably, this multipotential cell is expressed Oct4, Nanog, Sox2 and Eras;
Further preferably, this multipotential cell is expressed SEAP, SSEA1 simultaneously and is selected from one or more genes among Oct4, Nanog, Sox2, Eras, Esg1, Fgf4, Utf1, Cripto, Rex1, the Gdf3;
Further preferably, this multipotential cell is expressed SEAP, SSEA1, Oct4, Nanog and Sox2 simultaneously.
The described multipotential cell of first aspect present invention, it can form teratoma or teratocarcinoma, preferably, contains the noble cells that comes from ectoderm, mesoderm and three germinal layers of entoderm in formed teratoma or the teratocarcinoma.
The described multipotential cell of first aspect present invention, it can be used as individual cells growth and/or propagation in substratum.
The described multipotential cell of first aspect present invention, it can form mosaic;
Preferably, all cells in the said mosaic and said multipotential cell come from same parent;
Further preferably, described mosaic is chimeric mouse;
Further preferably, described chimeric mouse suffers from the cataract disease.
Second aspect of the present invention relates to a kind of multipotential cell, and it is that preserving number is CCTCCC201217, is deposited in the heredity BALB/c that the address is the Chinese typical culture collection center of Chinese Wuhan University on February 15th, 2012
Cat/CatThe embryonic stem cell line of cataract mouse.
The third aspect of the invention relates to a kind of chimeric mouse, behind aforementioned each multipotential cell and recipient embryo polymerization, cultivates and obtains.
Fourth aspect of the present invention relates to a kind of multipotential cell crowd, and it comprises aforementioned each described multipotential cell;
Preferably, it comprises at least 95% described multipotential cell.
The 5th aspect of the present invention relates to a kind of method of separating multipotential cell, and this method is separated the acquisition multipotential cell from cataract disease model mouse, preferably, from the body early embryo of cataract disease model mouse, separates obtaining multipotential cell.
Preferably, this method may further comprise the steps:
(1) makes female mouse of cataract mouse and the public mouse mating of cataract mouse, female mouse is become pregnant;
(2) from the uterus of female mouse of becoming pregnant, obtain blastaea;
(3) blastaea from step (2) obtains inner cell mass (ICM, inner cell mass: the totipotent cell mass that has that is positioned at segmentation cavity one side in the mammals blastaea);
(4) randomly, the one or more cell cultures of inner cell mass sorting from step (3) become colony; Preferably with the inner cell mass cultivation of going down to posterity after with trysinization;
(5) randomly, the colony from step (4) obtains mono-clonal, the cultivation of going down to posterity.
In a concrete embodiment, described method may further comprise the steps:
Select that the female mouse of sexually matured described cataract mouse becomes pregnant female mouse with the public mouse mating of growing up more than 6 ages in week;
From the cataract disease model Mouse Uterus of becoming pregnant, dash and get blastaea, cultivate with nutrient solution and obtain inner cell mass, dissociate inner cell mass, be digested to unicellular after, the cultivation of going down to posterity obtains the described multipotential cell that can infinitely go down to posterity;
Preferably, described cataract disease model mouse of becoming pregnant is the mouse of 3.5dpc of becoming pregnant;
Preferably, described nutrient solution is the N2B27 nutrient solution that has added GSK3 inhibitor C HIR99021 and kinases inhibitor PD0325901;
Preferably, the final concentration of GSK3 inhibitor C HIR99021 is 1～5 μ M in the described nutrient solution, is preferably 2～4 μ M, 3 μ M more preferably, and the final concentration of kinases inhibitor PD0325901 is 0.5～3.5 μ M, is preferably 1～3 μ M, more preferably 2 μ M;
Preferably, what use during digestion is trypsinase, and concentration is: 0.25%, 37 ℃ of digestion 1～2min.
The 6th aspect of the present invention relates to aforementioned each multipotential cell or chimeric mouse or multipotential cell crowd's method of use, and it is included in and under appropriate condition, uses said multipotential cell or chimeric mouse or multipotential cell crowd in drug screening, the research of cataract mechanism, gene therapy cell, the animal model;
Preferably, said use comprises with medicine and contacts, imports target spot as contrast and gene.
Preferably, described medicine is the medicine of treatment cataract disease.
The 7th aspect of the present invention relates to aforementioned each multipotential cell or chimeric mouse or multipotential cell crowd, is used for the purposes of screening of medicaments, perhaps is used to prepare the purposes of gene therapy medicament;
Preferably, described medicine is the medicine of treatment cataract disease.
In sum, the cataract mouse multipotential cell that the present invention relates to has the embryonic stem cell characteristic, and its alkaline phosphatase expression of enzymes is positive; Embryo's cell surface in period specific antigens SSEA-1 expresses positive; Express the multiple genes relevant such as Oct4, Nanog and Sox2 with pluripotency; The special generation that has contains the teratomatous ability of three primary germ layers (ectoderm, entoderm and mesoderm) noble cells and helps form chimeric ability.
In a concrete embodiment, described multipotential cell from the hereditary cataract mouse, its principal character is:
(1) express the relevant gene of pluripotencies such as SEAP, SSEA1 and Oct4, Nanog, Sox2, Eras, Esg1, Fgf4, Utf1, Cripto, Rex1, Gdf3 simultaneously, these these cell strains of expression of gene proof are in undifferentiated state;
(2) can, not cultivate serum-free in having the nutrient solution of feeder layer;
(3) can go down to posterity for a long time and increase external;
(4) be differentiated to form embryoid body naturally after, ectoderm Nestin (+), primitive ectoderm GATA4 (+), Sox17 (+), entoderm marker gene AFP (+), mesoderm Flk1 (+);
(5) form teratoma behind the injection nude mice, teratoma has ectoderm, mesoderm and endoblastic all noble cellss;
(6) can form chimeric mouse, and chimeric mouse suffers from the cataract disease.
The invention provides the thinking of vitro culture cataract animal model embryonic stem cell; The multipotential cell that is obtained can be used for the screening of cataract Study on Pathogenesis, treatment cataract medicine and the research carrier of gene therapy means is provided, the new material and the method that provide for the cataract disease research.
Description of drawings
Fig. 1 is that cataract mouse inner cell mass is hatched the form of coming out after adherent from zona pellucida.
Fig. 2 is the cellular form of cataract mouse ES after repeatedly going down to posterity.
Fig. 3 is a cataract mouse ES alkaline phosphatase activities detected result.
Fig. 4 is that Reverse Transcription-PCR detects the electrophorogram that cataract mouse ES does not break up the marker expression, and from left to right swimming lane is positive control 46CES cell, 129 mousetail inoblasts, cataract ES cells strain 4, cataract ES cells strain 5, cataract ES cells strain 6 successively.
Fig. 5 is that immunofluorescence dyeing detects the relevant marker expression of cataract mouse ES stem cell, and wherein A is the SSEA1 coloration result, and B is the Oct4 coloration result, and C is the Nanog coloration result, and D is the Sox2 coloration result.
Fig. 6 is that Reverse Transcription-PCR detects cataract mouse ES at external differentiation potential to three germinal layers, and from left to right swimming lane is the cataract ES cells successively, 5 days embryoid body, the embryoid body that breaks up naturally after 10 days.
Fig. 7 is that cataract mouse ES teratoma forms situation, and wherein A is nervous tissue (ectoderm), and B is muscle tissue (mesoderm), and C is epithelium (entoderm).
Fig. 8 is the chimeric mouse with cataract disease that cataract mouse ES participates in formation.
Embodiment
To combine embodiment that embodiment of the present invention are described in detail below, but it will be understood to those of skill in the art that the following example only is used to explain the present invention, and should not be regarded as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be through the conventional products of commercial acquisition.
In following examples, the substratum of using has:
The embryonic stem cell substratum is the 2i substratum; Specifically consist of: 49%DMEM/F12 (available from GIBCO company); 49%Neurobasal Medium (available from GIBCO company), 0.5 * L-glutaminate (available from GIBCO company), 1 * beta-mercaptoethanol (available from GIBCO company), 1 * N2 (available from GIBCO company), 1 * B27 (available from GIBCO company), 3 μ M CHIR99021 (available from Stemgent company), 2 μ M PD0325901 (available from Stemgent company).This substratum is used to obtain and keep the ES cell that comes from the cataract mouse.
The concrete composition of embryoid body substratum is: 79%DMEM/F12 (available from GIBCO company); 20%FBS (available from PAA company); 1% nonessential amino acid (available from GIBCO company), 0.5 * L-glutaminate (available from GIBCO company), 1 * beta-mercaptoethanol (available from GIBCO company).
Obtaining of embodiment 1, cataract mouse blastaea
Get the 6-8 heredity BALB/c in age in week
Cat/CatThe female mouse (available from Chinese Academy of Sciences's Shanghai Experimental Animal Center) of cataract; In about 14:00, abdominal injection PMSG (pregnant mare serum gonadotrop(h)in (PMSG), Pregnant Mare Serum Gonadotrophin) (10IU/ is only); HCG injection (pregnancy urine extract behind the 48h; Human chorionic gonadotrophin) (10IU/ only), it is empirical with the public mouse (available from Chinese Academy of Sciences's Shanghai Experimental Animal Center) of strain cataract to have selected mating, and in public mouse: 2: 1 ratios of female mouse mate.See that bolt is designated as conceived 0.5d morning next day.See bolt the 4th day (conceived 3.5d), disconnected neck is put to death female mouse, 75% ethanol disinfection; Under gnotobasis, expose the uterus, take out the uterus with tweezers and place sterile petri dish.Contain 10% foetal calf serum (Fetal Bovine Serum with the suction of 1ml asepsis injector; PAA company) DMEM/F12 (GIBCO company) is as towards ovum liquid, carries out from horn of uterus end inserting needle that (concrete steps see also: Andras, Marina towards embryo; Christina; Deng. mice embryonic experimental implementation handbook [M]. Sun Qingyuan, Chen Dayuan master translates. Beijing: Chemical Industry Press, 2005.).Under three-dimensional dissecting microscope, seek the embryo who is in blastula stage, collect with getting ovum pin (be purchased, also can make by oneself,, can draw the embryo and get final product) as long as pin oral pore footpath size is big slightly than embryo's size.
Towards embryo previous day, will be layered in 96 well culture plates with 300 cells/well through the MEF (mouse embryofibroblast, the primary culture of MEC, the 3rd generation) of MTC C deactivation and prepare feeder cell.
The blastula embryo of collecting is placed on the above-mentioned feeder cell; 1 blastaea in every hole; Add 100 microlitres and added the N2B27 nutrient solution of GSK3 inhibitor C HIR99021 (Stemgent company, final concentration 3 μ M) and kinases inhibitor PD0325901 (Stemgent company, final concentration 2 μ M); Place 37 ℃, the cell culture incubator of 5%CO2, saturated humidity to cultivate, changed a nutrient solution in 2-3 days.
The blastula embryo that obtains among the embodiment 1 is after cultivating 1～2 day under the described culture condition, and inner cell mass (ICM) begins outside zona pellucida, to hatch, and continues to cultivate after 2 days, and the inner cell mass of hatching (ICM) is clone's bulk adherent growth (referring to Fig. 1).Nutrient solution is removed in suction, and 37 ℃ of digestion of the pancreatin with 0.25%, 1～2min adds FBS (foetal calf serum) and stops digestion; Blow and beat gently with pipettor, make inner cell mass be dissociated into single or small cell cluster, the centrifugal 2min of 1000rpm; Abandon supernatant, with having added GSK3 inhibitor C HIR99021 (Stemgent company, final concentration 3 μ M) and kinases inhibitor PD0325901 (Stemgent company; Final concentration 2 μ M) N2B27 nutrient solution is resuspended; Be passaged to (cell in former one 96 hole still directly passes in one 96 hole after the digestion and cultivates) in 96 orifice plates that are covered with the MEF feeder cell 37 ℃, 5%CO
2, saturated humidity cell culture incubator in cultivate.
Trysinization is gone down to posterity and was cultivated for 2～3 generations as stated above, and cell proliferation this moment is very fast, can pass a generation (ratio that goes down to posterity this moment look actual clone's number and decide) in per afterwards three days.
Cellular form microscopy result after going down to posterity is as shown in Figure 2, can be found out that by figure the similar embryonic stem cell of cell growthhabit of acquisition is the growth of clone's shape, edge clear, surface smoothing, compact structure.
3.1 alkaline phosphatase expression of enzymes
Get among the embodiment 2 stem cell that obtains, with the fixing 10min of 4% Paraformaldehyde 96 (PAF), wash 3 times with PBS, each used time is respectively 5min, 10min, 15min, removes Paraformaldehyde 96 as much as possible.Add the 0.5ml naphthyl alcohol: Tris base: Fast red TR=1: mix dye liquor at 4: 5, incubated at room 5～10min inhales and removes dye liquor, with PBS rinsing termination reaction.Microscopic examination, the result is as shown in Figure 3.
Can find out that by Fig. 3 cell is dyed redness, and red expression has alkaline phosphatase activities; Thereby explain that the cell that embodiment 2 obtains has very strong alkaline phosphatase activities, further says; Cultured cells has the embryonic stem cell characteristic, promptly expresses SEAP.
3.2 undifferentiated state detects
3.2.1Reverse the cell that Transcription-PCR method check and analysis embodiment 2 obtains is the differentiation gene expression level not
Because the specific expressed following gene of undifferentiated cell meeting; Comprise Oct4, Nanog, Sox2, Eras, Esg1, Fgf4, Utf1, Cripto, Rex1, Gdf3, so can learn the undifferentiated state of the cell that is obtained among the embodiment 2 through detecting these expression of gene situation.
It is as shown in table 1 not break up marker primer sequence design, wherein with Eif4g2 as confidential reference items, primer is by Beijing Liuhe Huada Genomics Technology Co., Ltd's synthetic (wherein F representes forward primer, and R representes reverse primer).
Table 1
Extract total mRNA with PureLinkTMRNA Mini Kit test kit (TIANGEN company), the process for extracting by specification carries out, and measures the concentration of RNA.
Get the above-mentioned RNA of 1 μ g as template, with Quantscript RT Kit (the Quant cDNA first chain synthetic agent box) reverse transcription (TIANGEN company), reverse transcription system and response procedures are all undertaken by the test kit specification sheets.
The reverse transcription system:
| 10×RT?mix | 2μl |
| The dNTP mixed solution | 2μl |
| Oligo-dT | 2μl |
| Quant?Reverse?Transcriptase | 1μl |
| Template ribonucleic acid (1 μ g) | xμl |
| RNase-Free water | Complement to 20 μ l |
Response procedures: 25 ℃, 10min → 42 ℃, 1h → 95 ℃, 5min → 4 ℃, forever
The reverse transcription product that obtains carries out pcr amplification with Taq enzyme (Shanghai Shenergy Biocolor BioScience & Technology Company) under following condition.
PCR reaction system (total system 20 μ l) is: template 1 μ l, 10 * Buffer, 2 μ l, dNTP (2mM) 1 μ l, primer (10 μ M) 1 μ l, Taq enzyme 0.5 μ l, ddH
2O 13.5 μ l, wherein template is the product that the cell extraction RNA reverse transcription of acquisition obtains.
Reaction conditions: 94 ℃, 5min; 94 ℃, 45s; Annealing temperature, 45s; 72 ℃, 1min; Circulate 30 times; 72 ℃, 10min.Wherein annealing temperature is set according to primer Tm value: the annealing temperature of Eras, Fgf4, Cripto, Rex1 is 58 ℃; The annealing temperature of Esg1, Sox2, Nanog is 54 ℃; The annealing temperature of Utf1 is 59 ℃; The annealing temperature of Gdf3, Oct4, Eif4g2 is 60 ℃.
The PCR product is carried out sepharose (gel strength is 1.2%) electrophoresis, and the result is as shown in Figure 4.Wherein the template taking 46CES cell of positive control (Sox1-GFP knockin (46C) mouse embryo stem cell, by Dr.Austin Smith (University of Edinburgh, Edinburgh, U.K.,
Http:// www.ed.ac.uk) provide, this clone has confirmed to have all characteristics of embryonic stem cell, and this cell is at Ying QL; Nichols J; Evans EP, Smith AG.Changing potency by spontaneous fusion [J] .NATURE, 416 (6880): mention among the 545-548.); The template of negative control is mousetail inoblast (primary cultured cell, the 3rd generation).
Can know from the result of Fig. 4; The cell that obtains is the same with positive control 46CES; The marker that it is relevant with stem cell; Be Oct4, Nanog, Sox2, Eras, Esg1, Fgf4, Utf1, Cripto, Rex1, Gdf3, have high level expression, form obviously contrast with fibroblastic the expression hardly of negative control mousetail.
Whether do not express (comprising SSEA1, Oct4, Nanog and Sox2) 3.2.2 break up relevant marker through immunofluorescence detection embryonic stem cell
The antibody relevant information is following:
One is anti-: and SSEA-1 (MC-480) Antibody (Santa Cruz, SC-101462), Oct-3/4 (C-10) Antibody (Santa Cruz; SC-5279); Sox-2 (Y-17) Antibody (SantaCruz, SC-17320), Nanog (M-17) Antibody (Santa Cruz; SC-30329), dilute the back as reaction solution with 1: 100 ratio with 0.1%BSA.
Two is anti-: (the green skies, A0568), (Tianjin China source of students science and technology BA1032), is diluted the back as reaction solution with 1: 500 ratio with 0.1%BSA to fluorescence (Cy3) mark goat anti-rabbit igg to FITC mark goat anti-mouse IgG (H+L).
The practical implementation step:
Get the cell that 4 groups of embodiment 2 are obtained respectively, remove nutrient solution after, use the PBS rinsing, add fixedly 20min of 4%PFA; Room temperature PBS rinsing 3 times, each 10min is to remove PFA as much as possible; Then with containing the penetrating cell of 0.1%BSA of 0.3%TritionX-100 and sealing nonspecific binding site 40min; PBS rinsing 3 times, each 10min; Adding an anti-reaction solution (SSEA1, Oct4, Nanog and Sox2) incubated at room 1h or 4 ℃ respectively spends the night; Remove an anti-reaction solution, PBS rinsing 3 times, each 10min; Add corresponding two anti-reaction solutions, the room temperature lucifuge is hatched 1～2h; Remove two anti-reaction solutions, the PBS rinsing is inferior, each 10min; Add 1 μ g/ml DAPI reaction solution, the room temperature lucifuge is hatched 10min; The DAPI reaction solution is removed in suction, PBS rinsing 3 times, each 10min; Mounting, fluorescence microscope.
Immunofluorescence result is as shown in Figure 5.
As can beappreciated from fig. 5; The fluorescence microscopy detects the relevant marker SSEA1 of cell expressing stem cell, Oct4, Nanog and the Sox2 that embodiment 2 obtains; Explain that the cell that obtains still can self after continuing to go down to posterity, keep undifferentiated state, have the characteristic of embryonic stem cell self.
3.3 embryoid body is to the potential (vitro differentiation ability) of three germinal layer differentiation
In order to verify that embodiment 2 obtains the versatility of cell, we have done embryoid body and have formed experiment.
Then embryoid body is transplanted in 24 orifice plates, is carried out adherent culture, the differentiation of giving free rein to the DMEM/F12 substratum that contains 15%FBS.
Use the method for relative quantification PCR, detect the expression of the different marker gene of three germinal layers in atomization:
The design of the marker gene of three germinal layers choosing and primer sequence thereof is as shown in table 2, wherein with mouse Eif4g2 as confidential reference items, primer is by Beijing Liuhe Huada Genomics Technology Co., Ltd's synthetic (wherein F representes forward primer, and R representes reverse primer).
Table 2
Collect 5 days embryoid body of above-mentioned cultivation and differentiation cell of (embryoid body experiment 10 days) after 5 days naturally respectively with trypsin digestion; Extract total mRNA with PureLinkTMRNA Mini Kit test kit (TIANGEN company); The process for extracting by specification carries out, and measures the concentration of RNA.
Get the above-mentioned RNA of 1 μ g as template, with Quantscript RT Kit (the Quant cDNA first chain synthetic agent box) reverse transcription (TIANGEN company), reverse transcription system and response procedures are all undertaken by the test kit specification sheets.Reverse transcription system and response procedures are with embodiment 3.2.1.
The reverse transcription product that obtains carries out pcr amplification with Taq enzyme (Shanghai Shenergy Biocolor BioScience & Technology Company) under following condition.
PCR reaction system (total system 20 μ l) is: template 1 μ l, 10 * Buffer, 2 μ l, dNTP (2mM) 1 μ l, primer (10 μ M) 1 μ l, Taq enzyme 0.5 μ l, ddH
2O 13.5 μ l, wherein template is the cDNA that the cell extraction RNA reverse transcription of acquisition obtains.
Reaction conditions: 94 ℃, 5min; 94 ℃, 45s; 60 ℃, 45s; 72 ℃, 1min; Circulate 30 times; 72 ℃, 5min.
The PCR product of gained carries out sepharose (gel strength is 1.2%) electrophoresis, and electrophoresis result is as shown in Figure 6.
Can know that by Fig. 6 the cell of acquisition proves that at the external differentiation gene that can express ectoderm (Nestin), primitive endoderm (GATA4 and Sox17), entoderm (AFP) and mesoderm (FLK1) cell that obtains can be differentiated to form all three germinal layers in the embryoid body.
Above result proves, from embodiment 2, cultivates the cell that obtains in the cataract mouse inner cell mass and has the characteristic of embryonic stem cell, has the potential that vitro differentiation becomes the different tissues cell.
3.4 the neoplastic detection of monster
In order to verify that the clone who obtains has the differentiation capability of many tissues in vivo, the cell preparation that embodiment 2 is obtained becomes single cell suspension, gets 5 * 10
6Individual injection cell is subcutaneous to nude mice, the teratoma of about about 1 month about 1.5cm of the subcutaneous formation diameter of nude mice.Teratoma is stripped out, fix with 4%PFA, carry out paraffin embedding, section then, hematoxylin-eosin staining shows three germinal layer histocytes.
HE (hematoxylin-eosin) staining procedure: paraffin section is washed 3 times with the YLENE dewaxing, each 10min, absolute ethyl alcohol is washed 2 times, each 10min; 90%, 80%, 75% ethanol washed 10 minutes successively, fully washed with tap water again, and tap water fully washed after phenodin dyed 15min, 1% acidic alcohol differentiation, 5～30s; The tap water flushing, 60 ℃ are returned blue 2min, and the tap water flushing is newly joined Yihong then and is dyed 45s; The tap water flushing, natural air drying, resinene mounting.
At the microscopically microscopy, the result is as shown in Figure 7 with slide.
Can know that from Fig. 7 the cell that embodiment 2 obtains can be divided into three germinal layer cells in vivo, comprises entoderm intestines appearance epithelial cell, mesoderm muscle cell and ectoderm neurocyte.
Prove further that by above result the cell that embodiment 2 obtains has the embryonic stem cell characteristic, has the potential that is divided into the different tissues cell in the body.
3.5 form chimeric mouse
For whether the cell that proves acquisition has totipotency, promptly can form complete individuality, the cell that embodiment 2 obtains is processed small cell cluster with trypsin digestion; (dpc refers to the fate of post-coitum, and (the concrete operations step is seen Andras, Marina in dayspostcoitum) recipient embryo polymerization with the 2.5dpc from the 129/J mouse to select cell count and be 8～15 small cell cluster; Christina, etc. mice embryonic experimental implementation handbook [M]. Sun Qingyuan, Chen Dayuan master translates. Beijing: Chemical Industry Press; 2005.); Be transplanted in the white female mouse uterine cavity of the false pregnancy elder brother who sees bolt 2.5～3dpc, postoperative is placed on mouse in the clean mouse cage, with the bulb irradiation insulation of a 50W; Cover in the mouse eyes simultaneously, revive until mouse.Then mouse is sent back in the Animal House, the normal nursing is until its production.In the offspring mouse of observation output whether chimeric mouse is arranged.
The result sees Fig. 8.Can see that by Fig. 8 the mouse hair color of output is chimeric, and the cataract symptom occur that the cell that illustrative embodiment 2 obtains has the new individual ability that forms that participates in.
In sum; The present invention has turned out one type of multipotential cell from the hereditary cataract mice embryonic; This type cell meets all characteristics of embryonic stem cell; Have alkaline phosphatase activities and express stem cell surface specific marker (SSEA1, Oct4, Nanog and Sox2), simultaneously have embryoid body to the potential of three germinal layers differentiation, can form teratoma and chimeric mouse, confirm its stem cell characteristic.
The cell that embodiment 2 is obtained is preserved in Chinese typical culture collection center (CCTCC) (Chinese Wuhan, Wuhan University) on February 15th, 2012, and preserving number is CCTCCC201217.
The present invention helps advancing the process of the research of cataract pathogenesis, new medicament screen and the exploitation of gene therapy research means for the cataract disease research provides new material and instrument.
Claims (13)
1. multipotential cell, it separates from cataract disease model mouse,
Preferably, described model mice is a hereditary cataract disease model mouse,
Preferably, described hereditary cataract disease model mouse is selected from:
A: heredity BALB/c
Cat/CatThe cataract mouse;
The b:Philly mouse;
The c:Crybal mutant mouse;
D: dominance cataract mouse Hif;
Further be preferably heredity BALB/c
Cat/CatThe cataract mouse;
Preferably, described multipotential cell is an embryonic stem cell.
2. the multipotential cell of claim 1, it separates the body early embryo from cataract disease model mouse.
3. the multipotential cell of claim 1, it is expressed SEAP, SSEA1 gene and/or is selected from one or more genes among Oct4, Nanog, Sox2, Eras, Esg1, Fgf4, Utf1, Cripto, Rex1, the Gdf3;
Preferably, this multipotential cell is expressed Oct4, Nanog and Sox2;
More preferably, this multipotential cell is expressed Oct4, Nanog, Sox2 and Eras;
Further preferably, this multipotential cell is expressed SEAP, SSEA1 simultaneously and is selected from one or more genes among Oct4, Nanog, Sox2, Eras, Esg1, Fgf4, Utf1, Cripto, Rex1, the Gdf3;
Further preferably, this multipotential cell is expressed SEAP, SSEA1, Oct4, Nanog and Sox2 simultaneously.
4. the multipotential cell of claim 1, it can form teratoma or teratocarcinoma, preferably, contains the noble cells that comes from ectoderm, mesoderm and three germinal layers of entoderm in formed teratoma or the teratocarcinoma.
5. the multipotential cell of claim 1, it can be used as individual cells growth and/or propagation in substratum.
6. the multipotential cell of claim 1, it can form mosaic;
Preferably, all cells in the said mosaic and said multipotential cell come from same parent;
Further preferably, described mosaic is chimeric mouse;
Further preferably, described chimeric mouse suffers from the cataract disease.
7. multipotential cell, it is that preserving number is CCTCC C201217, is deposited in the embryonic stem cell that the address is the Chinese typical culture collection center of Chinese Wuhan University on February 15th, 2012.
8. chimeric mouse behind each multipotential cell and recipient embryo polymerization of claim 1 to 7, is cultivated and obtains.
9. multipotential cell crowd, it comprises each described multipotential cell of claim 1 to 7;
Preferably, it comprises each described multipotential cell of claim 1 to 7 of at least 95%.
10. method of separating multipotential cell, this method are separated from cataract disease model mouse and are obtained multipotential cell, preferably, from the body early embryo of cataract disease model mouse, separate obtaining multipotential cell;
Preferably, this method may further comprise the steps:
(1) makes female mouse of cataract mouse and the public mouse mating of cataract mouse, female mouse is become pregnant;
(2) from the uterus of female mouse of becoming pregnant, obtain blastaea;
(3) blastaea from step (2) obtains inner cell mass;
(4) randomly, the one or more cell cultures of inner cell mass sorting from step (3) become colony; Preferably with the inner cell mass cultivation of going down to posterity after with trysinization;
(5) randomly, the colony from step (4) obtains mono-clonal, the cultivation of going down to posterity.
11. the method for claim 10 may further comprise the steps:
Select that the female mouse of the described cataract mouse of sexually matured claim 1 becomes pregnant female mouse with the public mouse mating of growing up more than 6 ages in week;
From the cataract disease model Mouse Uterus of becoming pregnant, dash and get blastaea, cultivate with nutrient solution and obtain inner cell mass, dissociate inner cell mass, be digested to unicellular after, the cultivation of going down to posterity obtains the described multipotential cell that can infinitely go down to posterity;
Preferably, described cataract disease model mouse of becoming pregnant is the mouse of 3.5dpc of becoming pregnant;
Preferably, described nutrient solution is the N2B27 nutrient solution that has added GSK3 inhibitor C HIR99021 and kinases inhibitor PD0325901;
Preferably, the final concentration of GSK3 inhibitor C HIR99021 is 1～5 μ M in the described nutrient solution, is preferably 2～4 μ M, 3 μ M more preferably, and the final concentration of kinases inhibitor PD0325901 is 0.5～3.5 μ M, is preferably 1～3 μ M, more preferably 2 μ M;
Preferably, what use during digestion is trypsinase, and concentration is: 0.25%, 37 ℃ of digestion 1～2min.
12. each multipotential cell crowd's the method for use of chimeric mouse or claim 9 of multipotential cell or claim 8 of claim 1 to 7, it is included in and under appropriate condition, uses said multipotential cell or chimeric mouse or multipotential cell crowd in drug screening, the research of cataract mechanism, gene therapy cell, the animal model;
Preferably, said use comprises with medicine and contacts, imports target spot as contrast and gene;
Preferably, described medicine is the medicine of treatment cataract disease.
13. each multipotential cell or the chimeric mouse of claim 8 or the multipotential cell crowd of claim 9 of claim 1 to 7 is used for the purposes of screening of medicaments, perhaps is used to prepare the purposes of gene therapy medicament;
Preferably, described medicine is the medicine of treatment cataract disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100442309A CN102586174A (en) | 2012-02-24 | 2012-02-24 | Pluripotent cell derived from cataract mouse model, its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100442309A CN102586174A (en) | 2012-02-24 | 2012-02-24 | Pluripotent cell derived from cataract mouse model, its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102586174A true CN102586174A (en) | 2012-07-18 |
Family
ID=46475411
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100442309A Pending CN102586174A (en) | 2012-02-24 | 2012-02-24 | Pluripotent cell derived from cataract mouse model, its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102586174A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116041477A (en) * | 2022-10-13 | 2023-05-02 | 呈诺再生医学科技(北京)有限公司 | Application of TDGF1 gene in preparing medicine for treating senility related diseases or reversing cell senility |
-
2012
- 2012-02-24 CN CN2012100442309A patent/CN102586174A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| YANG C ET AL: "Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions", 《FASEB J》 * |
| 付玉华等: "诱导多潜能干细胞(iPSCs)的研究与应用进展", 《生物化学与生物物理进展》 * |
| 赵国际等: "疑似白内障小鼠小鼠模型培育初报", 《实验动物与比较医学》 * |
| 赵国际等: "遗传性BALB/c白内障小鼠模型培育初报", 《中国比较医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116041477A (en) * | 2022-10-13 | 2023-05-02 | 呈诺再生医学科技(北京)有限公司 | Application of TDGF1 gene in preparing medicine for treating senility related diseases or reversing cell senility |
| CN116041477B (en) * | 2022-10-13 | 2023-08-08 | 呈诺再生医学科技(北京)有限公司 | Application of TDGF1 gene in preparing medicine for treating senility related diseases or reversing cell senility |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Isolation and culture of embryonic stem cells from porcine blastocysts | |
| CN100422315C (en) | Isolated homozygous stem cells differentiated cells derived therefrom and materials and methods for making and using same | |
| US10501723B2 (en) | Methods of reprogramming animal somatic cells | |
| Piedrahita et al. | On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos | |
| CN100497598C (en) | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues | |
| Woods et al. | The next (re) generation of ovarian biology and fertility in women: is current science tomorrow's practice? | |
| Artus et al. | Generation of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos | |
| Brevini et al. | Derivation and characterization of pluripotent cell lines from pig embryos of different origins | |
| KR20110032989A (en) | Method for derivation of pluripotent stem cells from adult somatic cells and pluripotent stem cells produced by thereof | |
| Intawicha et al. | Characterization of embryonic stem cell lines derived from New Zealand white rabbit embryos | |
| CN104024404A (en) | Haploid cells | |
| CN101984050B (en) | Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof | |
| WO2002034890A2 (en) | Pluripotential stem cells | |
| CN106754729B (en) | Method for preparing induced pluripotent stem cells by using Xist Tale inhibitory transcription factor | |
| CN104371971A (en) | Method for obtaining amniotic epithelial cells through separation | |
| CN101525592A (en) | Human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof | |
| CN107937445A (en) | The method that gene knockout dog is prepared using somatic cell clone technique | |
| CN102586174A (en) | Pluripotent cell derived from cataract mouse model, its preparation method and application | |
| CN107723273A (en) | A kind of preparation method of the induction goat multipotential stem cell of micromolecular compound completely | |
| CN103224908A (en) | Tissue engineering material construction method based on amniotic mesenchymal stem cells | |
| CN102533764A (en) | Figla gene promoter sequence, marking carrier built by same and application | |
| WO2013159313A1 (en) | Animal embryonic stem cell line, and preparation method and application thereof | |
| CN106282097A (en) | Induced multi-potent stem cell, the method preparing induced multi-potent stem cell | |
| Fong et al. | Unsuccessful derivation of human embryonic stem cell lines from pairs of human blastomeres | |
| Bieniasz et al. | Stem Cells–general characteristic and sources |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120718 |