CN102424818B - Caspase-1-ES cell line induced by mifepristone - Google Patents
Caspase-1-ES cell line induced by mifepristone Download PDFInfo
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Abstract
The invention relates to a caspase-1-ES cell line induced by mifepristone. The collection number of the caspase-1-ES cell line is CCTCC No.C201193. The invention also provides a preparation method and application of the cell line. The invention has the advantages that: caspase-1 is transferred into an embryonic stem (ES) cell for the first time, and the differential potential of the cell is not influenced; the caspase-1 is highly expressed in the ES cell for the first time, it is proved that activated caspase-1 initiates DNA damage to cause death of undifferentiated caspase-1-ES cells in vitro, and a risk of nodule formation caused by ES cell transplantation is reduced; it is discovered that the highly expressed caspase-1 in the differentiated ES cell does not obviously influence the cell for the first time, and the treatment effect of the ES cell is maintained; and the safety of embryonic stem cell transplantation is improved, the effect of treating parkinson disease and other neurologic diseases is remained, and the caspase-1-ES cell line can be widely applied to stem cell transplantation.
Description
Technical field
The present invention relates to a kind of clone, specifically, is a kind of mifepristone caspase-1-ES clone of inducing and its preparation method and application.
Background technology
Parkinson's disease (Parkinson ' s disease, PD) be that sickness rate occupies deputy common nervous system degeneration disease, the loss that its pathological characters main manifestations is substantia nigra dopaminergic neuron and the formation of Lewy body.Therapeutic strategy commonly used only is confined to symptomatic treatment, alleviates patient's clinical symptom as far as possible, and this class treatment means can not fundamentally reverse generation and the development of PD.Embryonic stem cell (Embryonic stem cells, ES cell) is the cell with multiple differentiation potential, can induce differentiation to become specific cell type, and this specific character has broad prospects it aspect cell replacement therapy.The ES Transplanted cells is expected to be applied to treat PD, but sneaks into not breaking up on a small quantity the ES cell and may forming in vivo tumour of graft, and this is very large threat to the patient.
Tumour is the common risk of ES Transplanted cells, and this is that the juvenile cell of sneaking in transplanted cells causes.Modal suicide gene therapy is that to cell self, the gene of harmless enzyme proceeds to cell by one, kills this cell but this enzyme can change nontoxic prodrug into poisonous form, for example the HSVtk/GCV system.The ES cell that proceeds to HSVtk can be induced death by GCV.But consider cellulotoxic effect and bystander effect, still can not confirm that the cell of differentiation and maturation required when GCV exists can be survived.With the HSVtk/GCV system, compare, caspase-1 is intrinsic gene in cell, and it is to be confined to specific cell type that caspase-1 expresses the necrocytosis effect caused.In different cell types, caspase-1 expresses the effect difference produced.The activation of caspase-1 can cause necrocytosis in tumour cell, but at epithelial cell, CHO(Chinese hamster ovary cell, Chinese hamster ovary) contribute on the contrary cell survival in cell and Hela cell.But about caspase-1 being proceeded to the ES cell, both maintained the curative effect of ES treatment, and reduced again the ES Transplanted cells and become the risk of knurl to yet there are no report.
Summary of the invention
The caspase-1-ES clone that provides a kind of mifepristone to induce is provided for deficiency of the prior art.
One purpose more of the present invention is, the preparation method of the caspase-1-ES clone that a kind of mifepristone induces is provided.
Another purpose of the present invention is, the application of the caspase-1-ES clone that a kind of mifepristone induces is provided.
For achieving the above object, the technical scheme that the present invention takes is: the caspase-1-ES clone that a kind of mifepristone is induced, the preserving number of described caspase-1-ES clone is CCTCC C201193.
For realizing above-mentioned second purpose, the technical scheme that the present invention takes is: the preparation method of the caspase-1-ES clone that a kind of mifepristone is induced, and described preparation method comprises the following steps:
Be transfected into the CGR8 cell with Lipofectamine 2000 after a, pSwitch plasmid linearization, filter out pSwitch with Hygromycin and surely turn strain;
The full-length cDNA of b, PCR reaction amplification caspase-1;
C, caspase-1 cDNA and pGene carrier are carried out to double digestion, enzyme is cut after product connects with Solution I and is transformed into intestinal bacteria;
D, from intestinal bacteria the extracting plasmid, be transfected into pSwitch with Lipofectamine 2000 after linearizing and surely turn strain, surely turn strain with Zeosin screening;
E, choose mono-clonal and increased, identify with RT-PCR and western blot.
In described step b, the full length cDNA sequence of caspase-1 is the described nucleotide sequence of SEQ ID NO.3.
In described step b, the full-length cDNA primer sequence used of PCR reaction amplification caspase-1 is SEQ ID NO. 1 and the described nucleotide sequence of SEQ ID NO.2.
Described ES cell is mouse ES cells.
For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is: the application of described caspase-1-ES clone in preparation treatment Parkinson's disease medicine.
The application of described caspase-1-ES clone in stem cell transplantation.
The application of described caspase-1-ES clone in stem cell transplantation refers to that caspase-1-ES clone can adopt the application in the disease medicament that stem cell transplantation treats in preparation treatment, and the described disease that can adopt stem cell transplantation to treat refers to brain paralysis, Spinal injury, motor neuron, hematencephalon, cerebral infarction sequela, cerebral trauma sequela, diabetes, dermatomyositis, myasthenia, vascular lesion, sclerosis, leukemia, hepatopathy, liver cirrhosis, necrosis of femoral head.
The invention has the advantages that:
1, the present invention proceeds to caspase-1 the ES cell first, and does not affect this cytodifferentiation potential.High expression level caspase-1 in the ES cell, confirm to cause external undifferentiated caspase-1-ES necrocytosis by causing DNA damage after caspase-1 activates first, can reduce the risk that the ES Transplanted cells becomes knurl;
2, the present invention finds that high expression level caspase-1 does not make significant difference to cell in the ES cell of differentiation first, contributes to maintain the curative effect of ES cell therapy;
3, the caspase-1-ES clone that the present invention sets up has not only improved the security of embryo stem cell transplantation but also retained the Parkinsonian effect for the treatment of, can widespread use in stem cell transplantation.
The accompanying drawing explanation
Fig. 1 is the diagram of GeneSwtich System Operation mechanism.
Fig. 2 is that mifepristone is the dosage dependence to the effect of inducing pSwitch-ES cell GFP to express.
Fig. 3 is that mifepristone is induced caspase-1 mRNA high expression level in the caspase-1-ES cell, and is dosage dependence (n=3).
Fig. 4 is that mifepristone induces caspase-1 at the intracellular high expression level of caspase-1-ES.
Fig. 5 is that mifepristone is induced the caspase-1-ES necrocytosis.
Fig. 6 is that mifepristone is induced the caspase-1-ES necrocytosis, and is time-dependent (n=10).
Fig. 7 is that mifepristone is induced the caspase-1-ES necrocytosis, and is dosage dependence (n=10).
Fig. 8 is that mifepristone induces the caspase-1-ES cell to occur that DNA shears.
Fig. 9 processes the plastidogenetic gross tumor volume of caspase-1-ES through 3 weeks mifepristones obviously to dwindle.
Figure 10 obviously reduces after the plastidogenetic gross tumor volume injection of caspase-1-ES mifepristone, and the gross tumor volume that wild-type cell generates is not affected by mifepristone.
Figure 11 is that tissue slice shows tumour internal memory entoderm, mesoderm and ectoderm in early days, confirms that teratoma forms.
Figure 12 is the plastidogenetic death of neoplastic cells of caspase-1-ES after 3 weeks mifepristones of injection.
Figure 13 is that the injection mifepristone makes caspase-1 expression amount phenomenal growth in the plastidogenetic tumour of caspase-1-ES.
Figure 14 is that the tumour cell that mifepristone induces caspase-1-ES to form occurs that DNA shears.
Figure 15 is without the plastidogenetic tumour cell survival of caspase-1-ES after the injection mifepristone.
Figure 16 is that the caspase-1-ES cytodifferentiation is dopaminergic neuron.
Figure 17 be mifepristone process 6 days afterwards the caspase-1-ES cell of differentiation without considerable change.
Figure 18 is that mifepristone is processed the caspase-1-ES cell break up afterwards in 2-6 days without considerable change.
No matter Figure 19 has or not mifepristone, and the canspase-1-ES cell of differentiation does not all occur that DNA shears.
Figure 20 is that the mifepristone processing makes caspase-1 p10 in the caspase-1-ES cell express increase.
Figure 21 is the MAP2 of differentiation after mifepristone is processed
+/ TH
+The caspase-1-ES cell still survive.
Figure 22 is MAP2
+Or TH
+Clone's ratio is not affected by mifepristone.
Figure 23 is that undifferentiated wild-type and caspase-1-ES cell are Oct-4
+, and be mixed with Oct-4 in the caspase-1-ES cell of differentiation
+Cell.
Figure 24 is that the injection mifepristone has extended the survival time of mice (n=4) that the caspase-1-ES cell is not broken up in transplanting.
No matter Figure 25 has or not mifepristone all in brain, to form tumour after wild-type ES Transplanted cells, and transplants differentiation and do not break up the interior tumour that forms of mouse brain that the caspase-1-ES cell only has injection ethanol.
Figure 26 is that wild-type ES cell and caspase-1-ES cell lysing agent group all can form tumour.
No matter whether Figure 27 inject mifepristone, SRY
+/ TH
+/ MAP2
+Cell can be survived in the caspase-1-ES cell mouse brain of having transplanted differentiation.
Figure 28 is that the caspase-1 high expression level that mifepristone is induced produces different impacts to differentiation and undifferentiated caspase-1-ES cell.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
The caspase-1 that embodiment 1 is induced by mifepristone expresses and has removed the tumour that embryonic stem cell forms
One, experiment purpose
Caspase-1 belongs to one of inflammation caspase, after activation, can cause inflammatory cell death.Cross in vivo to express and also can cause death of neoplastic cells.Caspase-1 can induce plasma membrane to form pore and make water enter cell, cause osmotic lysis after activating; Cause the release of proinflammatory entocyte and the caspase-1 relevant cell factors such as IL-1 β and IL-18; Also can activate certain unknown Nucleotide restriction endonuclease and shear DNA.Therefore we select this gene as killing and wounding gene.
By introduce the GeneSwitch technology platform in the ES cell, build the caspase-1 expression system of mifepristone regulation and control, and observe with mifepristone, process after the impact of caspase-1 high expression level on the tumour cell that do not break up ES cell and generation in situation in vitro and in vivo.
Two, materials and methods
1. main reagent and material
BSA, 5 * albumen sample-loading buffer, 4 * Tris-HCL/SDS (pH6.8), 4 * Tris-HCL/SDS (pH 8.8), Totomycin, DMSO, MTT(Shanghai Sheng Gong bio-engineering corporation), the anti-caspase-1 antibody of rabbit (German Santa Cruz company), Ex Taq archaeal dna polymerase, medical x-ray film (Japanese TAKARA company), mouse-anti β-actin antibody (U.S. Sigma company), liposome 2000 transfection reagent boxes, Superscript III cDNA reverse transcription test kit, Trizol RNA extractant, penicillin/streptomycin, hot deactivation is without endotoxic foetal calf serum (FBS), mifepristone, the GeneSwitch test kit, 0.05% pancreatin, GMEM, L-Glutamine, Sodium.alpha.-ketopropionate, non-essential amino acid, Knockout Serum Replacement, 2 mercapto ethanol (cell cultures is used), the special-purpose FBS(American I of ES cell nvitrogen company), cell apoptosis detection kit (the green skies, Haimen, Jiangsu company), DAB test kit (U.S. Vector company), SuperSignal West detection test kit (U.S. PIERCE company), RIPA lysate (Shanghai betting office), Immobilon pvdf membrane (U.S. Millipore company), Tissue Culture Plate (Corning) (Shanghai Bo Yun company), nude mice (Chinese Academy of Sciences's Shanghai Experimental Animal Center), cell culture incubator (German HERA company), microplate reader (U.S. Phenix Research company), inverted fluorescence microscope (IX 81) (Japanese Olympus company), the PCR instrument, horizontal cataphoresis apparatus, the gel imaging instrument, Western Vertial electrophorestic tank (U.S. Bio-Rad company), Zeocin(Shanghai Pu Fei company), the Matrigel(U.S. company BD).
Cell cultures
The screening of the cultivation of mouse ES cells CGR8 and stable transfected cells strain
Mouse ES cells (19-30 generation), the nutrient solution composition is GMEM, containing the special-purpose FBS of 10%ES cell, 1% Sodium.alpha.-ketopropionate, 1% non-essential amino acid, 1% penicillin streptomycin, 1% glutamine and 1 ‰ 2 mercapto ethanols, with fresh 1/10000 the LIF(10 added
7U/ml) keep the undifferentiated state of CGR8.Tissue Culture Dish is coated with 30 minutes with 0.2% gelatin, 0.05% trysinization 3 minutes for passage.
The screening step of stable transfected cells strain is: CGR8 is inoculated in the coated 3.5cm culture dish of gelatin, in the time of Growth of Cells to 30% area, required plasmid is transfected into to cell with Lipofectamine 2000, cultivates after 48 hours and add corresponding microbiotic to be screened.The pSwitch plasmid surely turns hygromycin(750 μ g/ml for strain), the pGene plasmid surely turns Zeosin(80 μ g/ml for strain) screen after two weeks cell dissociation to unicellular, 100-500 cell inoculated in the 10cm culture dish, choose mono-clonal until Single cell culture after one week and increased, finally being identified with the method for RT-PCR and western blot.
Plasmid construction
With the total RNA of Trizol extracting mouse spleen, ultraviolet spectrometer detects RNA concentration, then 1mg RNA reverse transcription is become to cDNA, usings this cDNA as masterplate; Primer sequence: positive-sense strand: 5 '-CGAGGTACCATGGCTGACAAGATCCTGA-3 ' (SEQ ID NO.1), antisense strand: 5 '-CAGGCCGCTTAATGTCCCGGGAAGAG-3 ' (SEQ ID NO.2).Carry out the full-length cDNA of PCR reaction amplification caspase-1, program is: 94 ℃ of sex change 30 seconds, anneal 56 ℃ 30 seconds, extend 72 ℃ 2 minutes, 35 circulations, finally extend 10 minutes.The PCR product cloning is entered in pUcm-T vector to the caspase-1cDNA(SEQ ID NO.3 by sequencing analysis to building) with database sequence (NM_001043585), compare.Respectively caspase-1 cDNA and pGene carrier are carried out to double digestion with Kpn I and two restriction enzyme sites of Not I, enzyme is cut after product connects with Solution I and is transformed into intestinal bacteria, and order-checking is carried out the follow-up experiments such as transfection after identifying correctly.
Analyze
With mtt assay, cell viability is analyzed.Cell is inoculated in 96 orifice plate dosings after 1 day.Mifepristone is dissolved in ethanol, and storage liquid concentration is 10 μ M.Add nutrient solution during use, final concentration is 10nM, and control group adds same volume ethanol, changes every other day liquid.Discard nutrient solution after 2-6 days, every hole adds the MTT PBS solution 100 μ l of 0.5mg/ml, hatches 3 hours for 37 ℃, and supernatant discarded, add the DMSO dissolution precipitation.Read the 570nM light absorption ratio by microplate reader.The data of mifepristone group obtain respectively the per-cent of cell survival divided by the data of control group.
Extraction
Cell discards nutrient solution after treated 1 day, and PBS washes 1 time, and the 6 every holes of porocyte culture plate add 200 μ l TRIzol reagent, put room temperature 5 minutes, draws lysate and enters the 1.5ml centrifuge tube, and every pipe adds chloroform 200 μ l, shake, and room temperature is placed 2-3 minute.4 ℃ centrifugal, and the upper strata water is carefully drawn in 12000g * 5 minute, moves to another centrifuge tube.Add Virahol 500 μ l, standing 10 minutes of room temperature.4 ℃ centrifugal, and 12000g * 10 minute, discard Virahol; Add 75% ethanol 1ml, shake, washing precipitation, 4 ℃ are centrifugal, and 10000g * 5 minute, discard ethanol, dries.Every pipe adds the water that removes in right amount RNase, gets spectrophotometric instrumentation OD260/280 ratio for 1 μ l, calculates RNA purity and concentration, for reverse transcription.
Reverse transcription
Get each 1 μ g of RNA of said extracted, the row two-step approach is carried out reverse transcription, adds Oligo dT 1 μ l, then to add without RNase water to final volume be 13.5 μ l, 65 ℃ of reactions 5 minutes; Add respectively afterwards 5 * RT damping fluid, 4 μ l, dNTP 0.5 μ l, RNasin 1 μ l, M-MLV reversed transcriptive enzyme 1 μ l, 37 ℃ are reacted 60 minutes, and cDNA is in-80 ℃ of Refrigerator stores.Get reverse transcription product 1 μ l while being PCR as template.Primer sequence: positive-sense strand: 5 '-CGAGGTACCATGGCTGACAAGATCCTGA-3 ' (SEQ ID NO.1), antisense strand: 5 '-CAGGCCGCTTAATGTCCCGGGAAGAG-3 ' (SEQ ID NO.2).
Gene expression detection
Reaction system is 20 μ l, with reference to the Ex Taq specification sheets of TAKARA company, carries out, and primer sequence is the same, and the PCR condition is the same, 25 circulations.
Transplant
20 of the female nude mices of body weight 18-22g, be divided into 4 groups, 5 every group.With 0.05% pancreatin, the ES cell dissociation is got off, blow and reach individual cells, with 70 μ m strainer filterings.With PBS, wash centrifugal twice, counting.By 1 * 10
6Individual ES cell suspension, at 100 μ l matrigel, is expelled under the right ribbed hide of nude mice.Inject tumour after 1～2 week and occur, for the effect of estimating that mifepristone is eliminated in-vivo tumour, use every other day the length of vernier caliper measurement Subcutaneous tumor and wide, cubature formula is: volume=1/2 * length * wide
2.When gross tumor volume reaches 200mm
3The time, mifepristone or isopyknic ethanol of subcutaneous injection every day 1mg/kg body weight.Injectable drug is craned one after 3 weeks and is put to death mouse, and tumor tissues shreds to be cultivated 2 weeks afterwards with the ES nutrient solution, observes and has or not clone formation.
Immunoblotting assay
7.1 the preparation of protein sample
The ES cell is after drug treating, and PBS washes 1 time, adds the cracking of ice RIPA (1%PMSF) lysate.Use cell to scrape cell and collect centrifuge tube, 30 times/ml of pipettor piping and druming, standing 30 minutes on ice.4 ℃, 20000g * 30 are minute centrifugal, collect supernatant ,-80 ℃ of preservations.
After drug injection 2 days, the mouse execution of craning one, the Subcutaneous tumor tissue that takes a morsel, take weight, by 15 μ l/ μ g, adds the RIPA lysate, uses the Ultrasonic Cell Disruptor fragmentation.4 ℃, 20000g * 30 are minute centrifugal, collect supernatant ,-80 ℃ of preservations.
Electrophoresis, transferring film and sealing
Configure 12% separation gel and 5% concentrated glue, the glue damping fluid is run in configuration, and compound method is shown in 7.3.Get 20 μ g protein samples and sample-loading buffer mixed, 100 ℃ of water-baths are boiled 5 minutes, centrifugal, loading.First 80V electrophoresis 20 minutes, then under 120V, electrophoresis (got final product with tetrabromophenol sulfonphthalein band electrophoresis to the nearly base of separation gel) about 1 hour.Prepare pvdf membrane, with methyl alcohol, soak and within 1 minute, make the film activation, after be immersed in the transferring film damping fluid 10 minutes make its balance together with the SDS-PAGE running gel.Make transferring film " sandwich ", order is followed successively by: negative pole face (black side)-fiber mat-3 metafiltration paper-separation gel-pvdf membrane-3 metafiltration paper-fiber mat-positive pole-face (red face).The 250mA transferring film, transferring film 45 minutes.Take out film after finishing, make marks, albumen transferring film situation is observed in ponceau dyeing in 1 minute.After washing the film damping fluid and washing, configure 5% skim-milk or 5%BSA, room temperature sealing pvdf membrane 1 hour.
Antibody incubation
After sealing, with 5% skim-milk, (mouse-anti β-actin antibody, 1:4000) or the anti-caspase-1 antibody of 5%BSA(rabbit, 1:500) confining liquid configuration primary antibodie Incubating Solution, be added on film 4 ℃ of overnight incubation.Wash film damping fluid rinsing 3 * 15 minutes, with horseradish peroxidase two anti-incubated at room 2 hours (goat anti-rabbit igg, or goat anti-mouse IgG, 1:2000).Wash film damping fluid rinsing 3 * 15 minutes, hang angle and dry, add the agent of SuperSignal ECL fluorescence display, the darkroom exposure, the time shutter is depending on fluorescence intensity.After development, photographic fixing, punching is dried.Scanner for figure (BIORAD GS-800) reads, with Quantity one software analysis fluorescence intensity.
The preparation of partially liq:
Run the glue damping fluid: 25mmol/l Tris-base, 0.2mol/l glycine, 0.1%SDS;
Transferring film damping fluid: 25mmol/l Tris-base, 0.2mol/l glycine, 20% methyl alcohol;
Wash the film damping fluid: 0.242% Tris base, 0.8% NaCl; Adjusting pH with 1N HCl is 7.6, with the Tween-20 that front adds 0.1%;
Confining liquid: 5% skim-milk or 5%BSA, with washing the preparation of film damping fluid.
Shear and detect
With fixing cell climbing sheet or the frozen section of PBS washing 4%PFA, with 0.1%Triton-100 PBS solution ice bath 2 minutes, PBS washing, by 0.3% hydrogen peroxide methanol solution incubated at room 20 minutes, PBS washed.Add biotin labeling liquid, hatch 60 minutes for 37 ℃, PBS washing, the reaction terminating liquid of labelling, incubated at room 10 minutes, PBS washing.Add the Streptavidin-HRP working fluid, incubated at room 30 minutes, PBS washing.Add DAB nitrite ion colour developing 5 minutes, the PBS washing.Hematorylin is redyed 10 seconds, and flowing water rinses, dehydration, resin mounting.
Dyeing
Paraffin embedding after tumor tissues is fixing with PFA, slice thickness 5 μ m.After dewaxing, Hematorylin is hatched 5 minutes, and flowing water rinses and returns indigo plant, and hatch 1 minute in Yihong, washing, dehydration, resin mounting.
Tumor cell culture
After drug injection 3 weeks, the execution of craning one of the mouse of transplanting the caspase-1-ES cell.The Subcutaneous tumor tissue that takes a morsel, add the ES cell culture fluid to cultivate 1 week.
Statistics
Other the data variance analyses and two tail t check, take P<0.05 as significant difference.All data mean (Means ± SD) with mean ± standard variance.
Three, experimental result
1. the GeneSwitch platform based on embryonic stem cell
The Principles of Regulation of GeneSwitch platform as shown in Figure 1, it sets up the screening based on twice stable transfected cells strain, screening for the first time will be regulated carrier pSwitch and be transiently transfected in mouse ES cells CGR8, with antibiotic hygromycin with the concentration screening of 80 μ g/ml after two weeks, the picking mono-clonal is identified.We utilize the inductor mifepristone to process after 1 day, in its intensity protein induced to GFP of fluorescence microscopy Microscopic observation by the pGene-GFP carrier is transiently transfected in the strain of pSwitch-ES stable transfected cells.As can be seen from Figure 2, along with the concentration increase of the mifepristone added, in cell, the abduction delivering of GFP obviously increases.According to the test-results in this laboratory and other laboratories, mifepristone does not exert an influence to the differentiation of ES cell.
Induce the foundation of the stable transfected cells strain caspase-1-ES of lower caspase-1 high expression level at mifepristone
But utilize the GeneSwtich abduction delivering platform of setting up, we are stably transfected into the pGene-caspase-1 plasmid in the strain of pSwitch-ES stable transfected cells, with zeosin, carry out the screening of stable transfection for the second time.We use the method for western immunoblotting assay and RT-PCR to detect the expression of caspase-1, and the monoclonal cell of picking is identified, obtain caspase-1-ES clone.The preserving number of described caspase-1-ES clone is CCTCC C201193.The result demonstration of RT-PCR, the caspase-1 that mifepristone is induced expresses and is the dosage dependence.When the mifepristone final concentration, during higher than 0.1nM, the expression of caspase-1 gene increases 6-7 doubly, and when final concentration is 0.001nM, the expression of caspase-1 gene only increases 30%(Fig. 3).The caspase-1-ES cell is collected as sample and is tested after 1 day with ethanol or mifepristone processing.The RT-PCR result shows that mifepristone is at extremely low concentration (0.1 nM & 10 nM) induce caspase-1 mRNA high expression level (p<0.01), the mifepristone amplification of lower concentration (0.001 nM that diminished; P<0.05).* mean p<0.05, * * means that p<0.01(compares with mifepristone 0 nM).
As can be seen from Figure 4, the result of western immunoblotting assay shows the high expression level that mifepristone can significantly be induced caspase-1 p10, and wild-type ES cell is unaffected.No matter whether add mifepristone, wild-type (CGR8) ES cell does not all detect caspase-1 p10(n=3).The caspase-1 precursor generates caspase-1 subunit p20 and the p10 of function after proteolysis.So the level that we detect caspase-1 p10 with Western blot represents the level that caspase-1 activates in wild-type and caspase-1-ES cell.With ethanol or mifepristone (10 nM) process the ES cell after 1 day collecting cell detected.In wild-type ES cell, no matter have or not mifepristone caspase-1 all not detected.A small amount of caspase-1 background can be detected while there is no mifepristone in the caspase-1-ES cell and express, and the mifepristone processing can make caspase-1 p10 express significantly increase (p<0.01).* means p<0.01. WT: wild-type.
The caspase-1 high expression level that mifepristone is induced causes the caspase-1-ES necrocytosis
In order to observe the impact of mifepristone on the caspase-1-ES cell viability, we have done the MTT analysis.It is to be dosage dependence and time dependent that mifepristone is induced to the quantity of caspase-1-ES necrocytosis.Mifepristone is on the not impact of wild-type ES cell.Add mifepristone (10 nM) after 2 days, the caspase-1-ES cell quantity significantly descends.Along with time lapse, cell quantity descends rapidly, and 6 days all dead afterwards.Although removed mifepristone after 6 days, continued to cultivate the cell of also not observing survival after 2 days.(Fig. 5 and 6).Yet while there is no mifepristone, the caspase-1-ES cell proliferation rate is not slower than wild-type.* means that p<0.01(and wild-type group of solvents are relatively),
##Mean that p<0.01(and caspase-1-ES group of solvents are relatively).After processing 2 days with 0.1 nM and 10 nM mifepristones, the caspase-1-ES cell quantity obviously reduces, and the effect of 0.001 nM mifepristone much smaller (Fig. 7), * * means p<0.01, * * * means that p<0.001(compares with mifepristone 0nM).As can be seen here, the caspase-1 high expression level that mifepristone is induced causes the caspase-1-ES necrocytosis.Therefore, we select 10 nM final concentration mifepristones.
The caspase-1 activated induces the caspase-1-ES necrocytosis by shearing DNA
The mechanism that causes the caspase-1-ES necrocytosis in order to understand mifepristone, we have done TUNEL dyeing.With the caspase-1-ES cell of mifepristone processing after 1 day, the phenomenon (Fig. 8) that DNA shears has appearred.It is that mifepristone is induced the caspase-1 high expression level that the results show mifepristone before adding causes the mechanism of caspase-1-ES necrocytosis, causes the damage of DNA.
3 weeks interior gross tumor volumes of injection mifepristone group constantly dwindle
Whether can cause the caspase-1-ES death of neoplastic cells equally in vivo in order to test mifepristone, 1 * 106 undifferentiated wild-type or caspase-1-ES cell suspension, in matrigel, are expelled under the right ribbed hide of nude mice.As negative control, left ribbed hide hemostasis matrigel, have no tumour and generate.Use every other day the vernier caliper measurement tumor size.When gross tumor volume reaches about 200cm
3The time subcutaneous injection every day mifepristone.(1 mg/kg) or ethanol.Inject once a day 3 weeks until mouse is condemned to death (n=5).Do not inject mifepristone and transplanted the Subcutaneous tumor that the mouse of caspase-1-ES cell has generated large.And 3 weeks rear gross tumor volumes of injection mifepristone obviously dwindle.After 3 weeks, mifepristone group gross tumor volume is significantly less than control group (Fig. 9).According to the Continuous Observation to tumor size, mifepristone is injected latter 8 days caspase-1-ES gross tumor volumes and is significantly dwindled, and still continued growth of the plastidogenetic tumour of caspase-1-ES (p<0.01) in the Mice Body of injection ethanol.As time goes on mifepristone group and group of solvents gross tumor volume gap are increasing, half (Figure 10) while being contracted to the treatment beginning after 3 weeks.The plastidogenetic tumour of wild-type ES is not affected by mifepristone, and its speed of growth is significantly faster than the plastidogenetic tumour of caspase-1-ES.No matter whether inject 2 weeks rear gross tumor volumes of mifepristone and can increase by 5 times.* means that p<0.01(compares with the wild-type group of solvents).# means p<0.05, and ## means that p<0.01(compares with the caspase-1-ES group of solvents).
Cell forms teratoma in vivo
The control group mice of transplanting caspase-1-ES cell is injected after 3 weeks and is condemned to death, and after tumor tissues is fixing, makes paraffin section.HE dyeing shows the weave construction that comprises the triploblastica source in tumor tissues.Keratin pearl belongs to ectoderm.Fatty tissue belongs to mesoderm.Ciliated epithelium belongs to entoderm.Confirm that the caspase-1-ES cell can form teratoma (Figure 11).This proof proceeds to the not impact of totipotency of GeneSwitch system and this ES cell of caspase-1 gene pairs, for next step differentiation research lays the foundation.
Under mifepristone is induced, the plastidogenetic death of neoplastic cells of caspase-1-ES
Injected in mice mifepristone or the ethanol of transplanting the caspase-1-ES cell were condemned to death after 3 weeks.HE dyeing shows after mifepristone is processed 3 weeks, injects the ethanol tumour that after 3 weeks, multipotential stem cell forms and continues growth.Caspase-1-ES tumor tissues inner tissue structure disappears, the substitute is fibrous tissue.And the group of solvents teratoma structure (Figure 12) that still visible stem cell forms.
Under mifepristone is induced, caspase-1 is high expression level in the plastidogenetic tumour of caspase-1-ES
The injection mifepristone took out tumor tissues after 2 days, with the expression amount of western immunoblotting assay casapase-1 p10.As shown in figure 13, under mifepristone is induced, caspase-1 expresses and increases to 8 times in the plastidogenetic tumour of caspase-1-ES.In conjunction with the variation of each group gross tumor volume, can confirm mifepristone group caspase-1-ES gross tumor volume less be high expression level due to caspase-1.The expression of caspase-1 p10 do not detected in the plastidogenetic tumour of wild-type ES.* means that p<0.01(compares with the caspase-1-ES group of solvents).
The death of neoplastic cells that the caspase-1 activated induces caspase-1-ES to form by shearing DNA
In order to understand mifepristone, induce caspase-1 to cross the mechanism that expression causes death of neoplastic cells, we have done TUNEL dyeing.The phenomenon (Figure 14) that DNA shears has appearred in the caspase-1-ES tumour cell of injection mifepristone after 2 days.Experimental result before considering, it is that mifepristone induces caspase-1 to cross expression that this proof mifepristone causes the mechanism of caspase-1-ES death of neoplastic cells in vivo, causes the damage of DNA.
The caspase-1-ES tumor tissues of processing 3 weeks with mifepristone can not be cultivated the generation tumor cell line
The caspase-1-ES tumor tissues of mifepristone group and control group is shredded, cultivate with the ES cell culture fluid.Control group is easy to set up tumor cell line, and the mifepristone group can not be set up clone (Figure 15).After this explanation mifepristone is processed 3 weeks, tumour cell is really all dead.
Caspase-1 is Mammals apoptosis genes involved, it is proceeded in body and will activate certain unknown Nucleotide restriction endonuclease shearing DNA, thereby cause death of neoplastic cells.We insert caspase-1 the expression system of mifepristone regulation and control of Geneswitch system made, thereby are based upon the ES cell that mifepristone is induced lower high expression level caspase-1.Experiment in vitro shows, adds caspase-1 high expression level after mifepristone, DNA damage occurs simultaneously, undifferentiated caspase-1-ES cell mortality subsequently.In body, experiment also shows, after the injection mifepristone, the caspase-1 high expression level, DNA damage occurs simultaneously, subsequently the plastidogenetic death of neoplastic cells of caspase-1-ES.As can be seen here, the present invention has reached first aim: when the ES cell of transplanting forms tumour, can be eliminated.
The caspase-1 expression that embodiment 2 is induced by mifepristone has been removed the tumour that in the brain, embryonic stem cell forms and has not been removed the dopaminergic neuron of transplanting
One, experiment purpose
Research and utilization is subject to the caspase-1-ES clone of mifepristone regulation and control to retain at the plastidogenetic tumour cell of elimination ES the dopaminergic neuron that the ES cytodifferentiation forms simultaneously.
Two, materials and methods
1. main reagent and material
The anti-Oct-4 antibody of rabbit (U.S. Cell Signaling company), the anti-tyrosine hydroxylase of rabbit (TH) antibody, mouse-anti nestin antibody (U.S. Chemicon company), mouse-anti SRY antibody (Britain Abcam company), mouse-anti MAP2 antibody, mouse-anti microtubule-associated protein 2(MAP 2) antibody (U.S. Sigma company), the anti-MAP2 antibody of goat (German Santa Cruz company), Prolong gold antifade reagent, the Alpha-MEM nutrient solution, the anti-goat antibody of Alexa Fluor 350 donkey (American I nvitrogen company), laser confocal microscope (LCM510) (German Zeiss company), PA6 clone (Japanese Riken cell bank), stereotaxic instrument (U.S. Benchmark company), Cy2 mark goat anti-rabbit antibodies, Cy3 mark goat anti-mouse antibody (U.S. Jackson ImmunoResearch company).
Cell cultures
2.1 marrow stromal cell PA6 cell cultures:
Take out the PA6 cell cryopreservation tube in liquid nitrogen, be placed in 37 ℃ of water-bath rapid rewarmings, proceed to the centrifuge tube (alpha-MEM containing the equal-volume complete culture solution, 10%FBS, penicillin/streptomycin 50U/ml), centrifugal 5 minutes of 1200rpm, remove supernatant, precipitation is resuspended in complete culture solution, and pipettor dispels, with 5x10
4/ cm
2Be inoculated in diameter 10cm Tissue Culture Dish.Be placed in 5%CO
2, cultivate 3-5 days in 37 ℃ of constant temperature cell culture incubators, during change liquid once, wash twice with PBS when cell attachment length causes the 80-90% area, change differentiation culture liquid, be the PA6 conditioned medium after one day.Differentiation culture liquid composition is GMEM, containing 10% serum replacement (Knockout Serum Replacement, KSR), and 1% Sodium.alpha.-ketopropionate, 1% non-essential amino acid, 1% penicillin streptomycin, 1% glutamine and 1 ‰ 2 mercapto ethanols.First with PBS, wash twice when cell dissociation goes down to posterity, then within 5 minutes, gone down to posterity with 0.05% trysinization.
The cultivation of cell and differentiation
The same first part of ES cell cultures, slightly.The ES cell is as follows to the step of dopaminergic neuron differentiation: by the ES cell with 3 * 10
5The concentration of/ml is inoculated in 6 cm Micro-Organism Culture Dishs suspension culture and within 4 days, forms embryoid (Embryoid Body, EB), during change nutrient solution in second day, the nutrient solution composition is containing LIF.EB was sucked to 15 ml centrifuge tubes precipitation after 15 minutes, supernatant discarded with the resuspended EB of PBS, redeposition 15 minutes, supernatant discarded, finally resuspended with the PA6 conditioned medium, be inoculated in 5%matrigel coated cell culture dish.Change every other day liquid.
Analyze (with reference to embodiment 1)
4. transplant
20 of the female nude mices of body weight 18-22g, be divided into 4 groups, 4 every group.By the ES cell or induce the differentiation cell dissociation of 4 days to get off, blow and beat into individual cells, with 70 μ m strainer filterings with 0.05% pancreatin.With PBS, wash centrifugal twice, counting.By 1 * 10
5The concentration of/μ l in nutrient solution, is expelled to cell suspension in the nude mice brain.0.1ml 10% chloral hydrate anesthesia for nude mice, be fixed on stereotaxic instrument.Iodine disinfection, cut open skin of head, with 30% hydroperoxide dissolution periosteum, exposes operative region, at surgery location, holes.Injection position: take bregma as 0 point, forward 1.0,2.0, downward 3.0 to the right.Now with microsyringe, draw cell, record initial coordinate, with descend 1.0 speed of per minute, enter brain, arrive after specified location with the speed injection of per minute 1 μ l, after injection, let the acupuncture needle remain at a certain point 2 minutes, exits at the same rate brain.With the two anti-solution-treated wounds of penicillin/streptomycin stitching.Inject mifepristone or isopyknic ethanol of subcutaneous injection every day 1mg/kg body weight after 2 weeks, record the dead mouse situation.Injectable drug is excessive anesthetized mice after 10 weeks, with PBS and 4%PFA, carries out total body perfusion, takes out cerebral tissue and continues to fix 2 days with PFA, with the heavy sugar of 15% and 30% sucrose, spend the night, and the OCT embedding, frozen section, thickness is 10 μ m.
Immunoblotting assay
5.1 the preparation of protein sample (with reference to embodiment 1)
5.2 SDS-PAGE electrophoresis, transferring film and sealing (with reference to embodiment 1)
5.3. antibody incubation (with reference to embodiment 1)
6. immunofluorescence dyeing
The cell be seeded on creep plate is fixed 30 minutes through 4%PFA, and PBS washes 3 * 5 minutes, with 3%BSA sealing 30 minutes, adds afterwards 4 ℃ of overnight incubation of primary antibodie.Primary antibodie comprises: the anti-Oct-4 antibody of rabbit (1:200), the anti-TH antibody of rabbit (1:500), mouse-anti MAP2 antibody (1:2000), mouse-anti nestin antibody (1:500) mouse-anti SRY, the anti-MAP2 antibody of antibody (1:200) and goat (1:20).Remove the primary antibodie Incubating Solution, PBS washes 3 * 5 minutes, add Cy2 mark goat anti-rabbit antibodies (1:500), two anti-mixing of Cy3 mark goat anti-mouse antibody (1:500) and the Alexa Fluor anti-goat antibody of 350 donkey (1:200), under room temperature, lucifuge is hatched 2 hours.Remove two anti-Incubating Solutions, PBS washes 3 * 5 minutes, dries, and adds anti-fluorescence quenching mounting, uses the laser microscope video picture.
Shear and detect (with reference to embodiment 1)
8. add up
Survival analysis is used Kaplan – Meier method and chi square test.Other the data variance analyses and two tail t check, take P<0.05 as significant difference.All data mean (Means ± SD) with mean ± standard variance.
Three, experimental result
1. the ES cell is to the technology platform of dopaminergic neuron differentiation
The ES cell has five one-step inducing methods and stroma cell (PA6) to be total to two kinds of culture methods to the main method of dopaminergic neuron differentiation, wherein five one-step inducing method times were grown (needing more than 20 days), need during this time inducing of several cytokines, cost is higher, and efficiency lower (10% left and right).Therefore we have adopted induction time shorter (about 14 days), efficiency higher (10-30%), and the method for lower-cost co-culture of cells.By the ES cell is formed to EB, then with the PA6 conditioned medium, cultivate EB, change every other day liquid, can be divided into MAP2
+/ TH
+Dopaminergic neuron (Figure 16).Caspase-1-ES cell suspension culture in the petri culture dish forms EB, then EB adherent differentiation in the PA6 conditioned medium in 4 days.Break up and MAP2 occurred after 10 days
+/ TH
+Cell, i.e. dopaminergic neuron.Scale length: 50 μ m.In the time of can also avoiding collecting cell with the PA6 conditioned medium, there is the PA6 cell to sneak into, more be applicable to Transplanted cells.
Add mifepristone to have no significant effect the caspase-1-ES cell quantity of differentiation
EB adds mifepristone to process 6 days with the differentiation of PA6 conditioned medium after 10 days.Figure 17 show mifepristone process 6 days afterwards the wild-type of differentiation and caspase-1-ES cellular form without noticeable change.In order to understand the impact of mifepristone on the caspase-1-ES cell quantity of differentiation, we have done the MTT analysis.Figure 18 shows that mifepristone processing 2-6 days wild-type and caspase-1-ES cell quantities to differentiation have no significant effect.Obviously mifepristone induce the caspase-1-ES cell effect of not differentiation and maturation and differentiation and maturation different.
DNA damage does not appear in the caspase-1-ES cell of the differentiation of processing with mifepristone
Add mifepristone with the differentiation of PA6 conditioned medium after 10 days, carry out TUNEL dyeing after 2 days.Figure 19 shows that DNA damage does not appear in the caspase-1-ES cell of the differentiation of processing with mifepristone, and this further confirms the not effect of caspase-1-ES cell to differentiation of inducing of mifepristone.Scale length: 10 μ m.
In mifepristone is induced the caspase-1-ES cell of lower differentiation, the expression of caspase-1 significantly increases
For which step in the process of the induce → caspase-1 high expression level → DNA damage → necrocytosis of understanding mifepristone has been interrupted, we with the western immunoblotting assay expression of function subunit caspase-1 p10.The expression that Figure 20 is presented at the interior caspase-1 p10 of caspase-1-ES cell of differentiation significantly increases.And the wild-type cell of differentiation does not detect caspase-1 p10.* mean that p<0.05(compares with the caspase-1-ES group of solvents).That is to say that the high expression level of caspase-1 does not cause shearing and the necrocytosis of DNA in the caspase-1-ES cell of differentiation.
The dopaminergic neuron that after the mifepristone processing, the caspase-1-ES cytodifferentiation becomes is still survived
Cultivate the caspase-1-ES cell with the PA6 conditioned medium and add mifepristone after 10 days, dyeed after 4 days.50 μ m), and MAP2 Figure 21 shows MAP2/TH positive cell (being dopaminergic neuron) survival (scale length:
+/ TH
+Clone's ratio is not subject to mifepristone to affect (Figure 22, (scale length: 500 μ m)).From the above, we think that caspase-1 crosses expression and optionally causes and do not break up the aspase-1-ES necrocytosis, but invalid to the cell of dopaminergic neuron and other differentiation.
A large amount of neural precursors are arranged in transplanted cells
In order to verify that can mifepristone remove the plastidogenetic tumour of caspase-1-ES in brain, the dopaminergic neuron that differentiation produces simultaneously also can be survived, and we are transplanted to the nude mice striatum to the ES cell that does not break up and induced.Undifferentiated wild-type and caspase-1-ES cell are Oct-4
+.The caspase-1-ES cell produces a large amount of nestin with the cultivation of PA6 conditioned medium after 4 days
+Cell, i.e. neural precursor.But wherein also be mixed with a small amount of Oct-4
+Cell, show the possibility (Figure 23) that forms tumour.Can in order to check the caspase-1-ES cell be divided in vivo dopaminergic neuron survival, we have transplanted 1 * 10
5Individual cell is to female nude mice striatum.EB forms the rear caspase-1-ES cell that produces the differentiation for transplanting in 4 days of cultivating with the PA6 conditioned medium.Be Oct-4 for undifferentiated wild-type and the caspase-1-ES cell of transplanting
+.The caspase-1-ES cell of differentiation mostly is nestin
+, show that it is neural precursor.The caspase-1-ES cell of great majority differentiation is nestin
+, a cell is Oct-4
+.A small amount of Oct-4
+Cell shows undifferentiated caspase-1-ES cell and sneaks into transplanted cells, may produce tumour.Scale length: 10 μ m.
After Transplanted cells, tumour does not all appear in the mifepristone group, and tumour all appears in control group
Transplant cells in the nude mice brain and start the subcutaneous injection mifepristone after 2 weeks, injected dose is the same.2 time-of-weeks enough allow neural precursor be divided into dopaminergic neuron in brain.Table 1 shows that caspase-1-ES cell that induced or undifferentiated all can not form tumour after mifepristone is processed, and group of solvents has tumour to form.Wild-type ES cell has or not mifepristone all can form tumour.
Table
Accept after Transplanted cells to form in mouse brain tumour
| The transplanted cells kind | Treatment | Form tumour ratio (n=4) | |
| Do not break up wild- | Solvent | 100% | |
| Do not break up wild- | Mifepristone | 100% | |
| Do not break up Caspase-1-ES | Solvent | 75% | |
| Do not break up Caspase-1- | Mifepristone | 0% | |
| Differentiation Caspase-1-ES | Solvent | 25% | |
| Differentiation Caspase-1- | Mifepristone | 0% |
Differentiation and undifferentiated cell are digested single-cell suspension in nutrient solution, and are transplanted to the right brain striatum of mouse.Transplant after 2 weeks and start the injected in mice medicine.We have then observed mouse 8 weeks.No matter have or not mifepristone, all mouse of having transplanted wild-type ES cell have all produced tumour.Do not inject the caspase-1-ES cell mouse generation tumour that has 25% transplanting to break up in the mouse of mifepristone, 75% transplants undifferentiated caspase-1-ES cell mouse produces tumour.Otherwise, in the mouse of injection mifepristone, all do not produce tumour.
The injection mifepristone has extended to transplant and has not broken up caspase-1-ES cell mouse lifetime
Transplant after the injection mifepristone and do not break up the caspase-1-ES cell mouse and all survive and be condemned to death by 70 days, and the mouse of injection ethanol has 75% to die from 58-67 days.Figure 24 shows that mifepristone has extended to transplant and does not break up caspase-1-ES cell mouse lifetime.
We have observed after mouse is accepted to transplant the situation of surviving.No matter have or not mifepristone, transplant the mouse that does not break up wild-type ES cell and all die from tumour.Produce blastomogenic mouse in brain and all die in 67 days, all the other mouse all survive and were condemned to death after 70 days.Mifepristone has extended transplanting and has not broken up caspase-1-ES cell mouse (χ lifetime
2=4.213, p<0.05).After the injection mifepristone, we have observed mouse 8 weeks, transplant differentiation and none death of mouse of not breaking up the caspase-1-ES cell.
The mouse of accepting to transplant dies from tumour in brain
Transplant dead mouse in latter 67 days and dissect tumour in the rear visible brain of naked eyes, all occupy right side (being the Transplanted cells side) cranial cavity.After putting to death the mouse dissection after 70 days, naked eyes are showed no tumour in brain.After wild-type ES Transplanted cells, no matter have or not mifepristone all in brain, to form tumour, dissect visible tumour and occupied the right side cranial cavity.And transplant differentiation and do not break up the interior tumour that forms of mouse brain that the caspase-1-ES cell only has injection ethanol, the mifepristone group does not all form tumour (Figure 25).The mouse that dies from tumour in brain all shows excited in one week in dead precontract, and beating heart jumps, anorexia.3-4 dyskinesia occurs after day, can not diet, become thin.Can not initiatively stand up after a few days, judge dead.In conjunction with anatomical results and symptom judgement, mouse should die from tumour in brain.
Tumor tissues all derives from transplanted cells
HE dyeing confirms that in the nude mice brain, tumour all derives from transplanted cells, and does not all find tumor tissues (Figure 26) in the mouse brain lived.Tissue slice shows that in brain, tumour derives from wild-type ES cell, injects the differentiation of ethanol and does not break up the caspase-1-ES cell.Inject the differentiation of mifepristone and do not break up the caspase-1-ES cell and do not form tumour.Scale length: 50 μ m.In sum, mifepristone also can prevent the tumour that the caspase-1-ES stem cell forms in brain.
The caspase-1-ES cell of inducing is divided into dopaminergic neuron in mouse brain, and by mifepristone, is not removed
We identify the mouse of having transplanted the caspase-1-ES cell broken up.Sry gene is present on Y chromosome, is sex determining gene, and expresses in mouse TH positive neuron.Therefore we use SRY, TH and MAP2 antibody test the brain tissue slice of these mouse, survival and the differentiation situation of observation transplanted cells.No matter Figure 27 is mifepristone group or control group if showing, the cell of the SRY/TH/MAP2 positive is all arranged in mouse brain.With coming to the same thing of experiment in vitro, the caspase-1-ES cell of differentiation is transplanted in mouse brain and can be divided into dopaminergic neuron, and by mifepristone, is not removed.But the plastidogenetic tumour of caspase-1-ES can not be survived.In order to understand the impact of mifepristone on male transplanted cells differentiation and survival in vivo, we have detected SRY, TH and the MAP2 expression in female transplanting mouse brain.The male decision gene of Y linkage SRY is at male rodents TH
+In neurone, express.The brain sheet dyed at high power Microscopic observation three, the no matter equal more visible SRY of mifepristone group or group of solvents mouse
+/ TH
+/ MAP2
+Cell.Arrow means SRY
+/ TH
+/ MAP2
+The caspase-1-ES cell.Scale length: 10 μ m.
Four, conclusion
The method that the ES cytodifferentiation is dopaminergic neuron has many kinds, and commonly used have five one-step inducing methods and stroma cell is total to culture method at present.These two kinds of methods are current the most frequently used methods in the world, the method that the present invention has adopted stroma cell PA6 to cultivate altogether, and this method has been carried out to certain improvement.The present invention first forms EB by the ES cell, then with the PA6 conditioned medium, cultivates.Directly the ES cell is seeded on PA6 and compares with other investigator, method of the present invention can obtain more transplanted cells on the one hand, makes on the other hand the PA6 cell can not sneak into transplanted cells.
Tumour is the common risk of ES Transplanted cells, this be have in transplanted cells, sneak into juvenile cell cause.Result of the present invention also shows, transplants and sneaks into Oct-4
+The differentiation caspase-1-ES cell of cell has 25% probability to produce tumour.The present invention has built one and has induced the ES clone of lower high expression level caspase-1 only under the state at tumorigenesis, to cause stem cell death at mifepristone.The result demonstration, the caspase-1 high expression level causes undifferentiated caspase-1-ES necrocytosis in vitro.Subcutaneous and tumour encephalic also can be removed by low dose of mifepristone.Yet, the caspase-1-ES cell not obviously impact of caspase-1 high expression level on differentiation and maturation in vitro.In brain, mifepristone induces the caspase-1 high expression level also can not affect the survival of dopaminergic neuron.Because the cell of differentiation and maturation can be survived, this therapy can not only be used for removing the tumour of having found, can also be used for preventing potential tumour to form.In the present invention, we find that the activation of caspase-1 does not does not kill and wound the dopaminergic neuron of differentiation in mouse brain, but can cause DNA damage and necrocytosis (Figure 28) to undifferentiated caspase-1-ES cell.
The present invention, by introduce the GeneSwitch technology platform in the ES cell, builds the caspase-1 expression system of mifepristone regulation and control, has set up caspase-1-ES clone.Transplant after this cell under the inducing of mifepristone the death of neoplastic cells that the dopaminergic neuron of caspase-1-ES cytodifferentiation can be survived and be formed simultaneously.
The present invention proceeds to caspase-1 the ES cell first, and does not affect this cytodifferentiation potential.High expression level caspase-1 in the ES cell, confirm to cause external undifferentiated caspase-1-ES necrocytosis by causing DNA damage after caspase-1 activates first.In subcutaneous and brain, caspase-1 also causes the plastidogenetic death of neoplastic cells of caspase-1-ES after activating.This method contributes to reduce the risk that the ES Transplanted cells becomes knurl.
The present invention finds that high expression level caspase-1 does not make significant difference to cell in the ES cell of differentiation first.In vitro, the caspase-1 high expression level does not make significant difference to the caspase-1-ES cell quantity of differentiation, and the ratio of dopaminergic neuron is not also made significant difference.In brain, the caspase-1 high expression level is not removed the dopaminergic neuron of transplanted cells differentiation when removing the tumour cell formed.This contributes to maintain the curative effect of ES treatment.
In sum, the caspase-1-ES clone that the present invention sets up has not only improved the security of embryo stem cell transplantation but also retained the Parkinsonian effect for the treatment of.Present method forms teratomatous risk when in addition, also can be used for reducing other specific cells shortage diseases of ES cell therapy.
Described caspase-1-ES clone can widespread use in stem cell transplantation.Be used for the treatment of nervous system disorders as brain paralysis, Spinal injury, motor neuron, Parkinson's disease, hematencephalon, cerebral infarction sequela, cerebral trauma sequela etc.; Be used for the treatment of disease of immune system as diabetes, dermatomyositis, myasthenia, vascular lesion, sclerosis, leukemia etc.; Be used for the treatment of other diseases: as hepatopathy, liver cirrhosis, necrosis of femoral head etc.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.
SEQUENCE LISTING
<110 > Ruijin Hospital, Shanghai Jiao Tong University School of Medicine
<120 > the caspase-1-ES clone that a kind of mifepristone is induced
<130> /
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213 > artificial sequence
<400> 1
cgaggtacca tggctgacaa gatcctga 28
<210> 2
<211> 26
<212> DNA
<213 > artificial sequence
<400> 2
caggccgctt aatgtcccgg gaagag 26
<210> 3
<211> 1209
<212> DNA
<213 > artificial sequence
<400> 3
atggctgaca agatcctgag ggcaaagagg aagcaattta tcaactcagt gagtataggg 60
acaataaatg gattgttgga tgaactttta gagaagagag tgctgaatca ggaagaaatg 120
gataaaataa aacttgcaaa cattactgct atggacaagg cacgggacct atgtgatcat 180
gtctctaaaa aagggcccca ggcaagccaa atctttatca cttacatttg taatgaagac 240
tgctacctgg caggaattct ggagcttcaa tcagctccat cagctgaaac atttgttgct 300
acagaagatt ctaaaggagg acatccttca tcctcagaaa caaaggaaga acagaacaaa 360
gaagatggca catttccagg actgactggg accctcaagt tttgcccttt agaaaaagcc 420
cagaagttat ggaaagaaaa tccttcagag atttatccaa taatgaatac aaccactcgt 480
acacgtcttg ccctcattat ctgcaacaca gagtttcaac atctttctcc gagggttgga 540
gctcaagttg acctcagaga aatgaagttg ctgctggagg atctggggta taccgtgaaa 600
gtgaaagaaa atctcacagc tctggagatg gtgaaagagg tgaaagaatt tgctgcctgc 660
ccagagcaca agacttctga cagtactttc cttgtattca tgtctcatgg tatccaggag 720
ggaatatgtg ggaccacata ctctaatgaa gtttcagata ttttaaaggt tgacacaatc 780
tttcagatga tgaacacttt gaagtgccca agcttgaaag acaagcccaa ggtgatcatt 840
attcaggcat gccgtggaga gaaacaagga gtggtgttgt taaaagattc agtaagagac 900
tctgaagagg atttcttaac ggatgcaatt tttgaagatg atggcattaa gaaggcccat 960
atagagaaag attttattgc tttctgctct tcaacaccag ataatgtgtc ttggagacat 1020
cctgtcaggg gctcactttt cattgagtca ctcatcaaac acatgaaaga atatgcctgg 1080
tcttgtgact tggaggacat tttcagaaag gttcgatttt catttgaaca accagaattt 1140
aggctacaga tgcccactgc tgatagggtg accctgacaa aacgtttcta cctcttcccg 1200
ggacattaa 1209
Claims (3)
1. the caspase-1-ES clone that mifepristone is induced, is characterized in that, the preserving number of described caspase-1-ES clone is CCTCC C201193.
2. the preparation method of caspase-1-ES clone according to claim 1, is characterized in that, described preparation method comprises the following steps:
Be transfected into the CGR8 cell with Lipofectamine 2000 after a, pSwitch plasmid linearization, filter out pSwitch with Hygromycin and surely turn strain;
The full-length cDNA of b, PCR reaction amplification caspase-1, the full length cDNA sequence of described caspase-1 is as described in SEQ ID NO.3, and the full-length cDNA of described PCR reaction amplification caspase-1 primer sequence used is as described in SEQ ID NO. 1 and SEQ ID NO.2;
C, caspase-1 cDNA and pGene carrier are carried out to double digestion, enzyme is cut after product connects with Solution I and is transformed into intestinal bacteria;
D, from intestinal bacteria the extracting plasmid, be transfected into pSwitch with Lipofectamine 2000 after linearizing and surely turn strain, surely turn strain with Zeosin screening;
E, choose mono-clonal and increased, identify with RT-PCR and western blot;
Described ES cell is mouse ES cells.
3. the application of caspase-1-ES clone according to claim 1 in preparation treatment Parkinson's disease medicine.
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| CN108486155A (en) * | 2018-04-27 | 2018-09-04 | 中国医学科学院输血研究所 | Build double inducible expression carriers with fluorescent protein marker gene that Doxycycline/Mifepristone inductions are overexpressed |
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Non-Patent Citations (7)
| Title |
|---|
| Bjorklund L.M.,et al..Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model.《PNAS》.2002,第99卷(第4期),第2344–2349页. |
| Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model;Bjorklund L.M.,et al.;《PNAS》;20020219;第99卷(第4期);第2344–2349页 * |
| Mifepristone-Inducible Caspase-1 Expression in Mouse Embryonic Stem Cells Eliminates Tumor Formation but Spares Differentiated Cells In Vitro and In Vivo;Wang Y, et al.;《Stem Cells》;20111130;第30卷;第169-179页 * |
| Wang Y, et al..Mifepristone-Inducible Caspase-1 Expression in Mouse Embryonic Stem Cells Eliminates Tumor Formation but Spares Differentiated Cells In Vitro and In Vivo.《Stem Cells》.2011,第30卷第169-179页. |
| 王晶等.胚胎干细胞移植治疗帕金森病的研究进展.《基础医学与临床》.2007,第27卷(第8期),第944-947页. |
| 王颐等.通过诱导caspase-x 表达清除胚胎干细胞形成的肿瘤但不影响分化的多巴胺能神经元.《2011中国（威海）干细胞与组织工程治疗前沿论坛论文集》.2011,第18页. * |
| 胚胎干细胞移植治疗帕金森病的研究进展;王晶等;《基础医学与临床》;20070831;第27卷(第8期);第944-947页 * |
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