CN104419669A

CN104419669A - Building of klinefelter syndrome cell model and cell bank of klinefelter syndrome cell by virtue of recombinant SV40 gene

Info

Publication number: CN104419669A
Application number: CN201310404034.2A
Authority: CN
Inventors: 翁炳焕; 黄荷凤; 吕时铭; 朱瑞建; 王爱飞; 刘蓓
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-09-01
Filing date: 2013-09-01
Publication date: 2015-03-18

Abstract

The invention relates to building of an SV40LT antigen gene-mediated klinefelter syndrome cell model and a cell bank of the klinefelter syndrome cell model, applied to the field of medical research. The building method is mainly characterized by comprising the following processes: building an SV40LTag-pcDNA3.1(1) recombinant by using T4DNA ligase, BamHI, pcDNA3.1(1) DNA and SV40LTag DNA according to a conventional method; purifying the recombinant by using competent escherichia coli, introducing the recombinant into an in vitro passage cell which is digested by collagenase II, or klinefelter syndrome histocyte which is in logarithmic growth, and integrating the recombinant with DNA; carrying out enlarging cultivation, screening cells containing positive recombinants by G418, and cloning; and screening a cell of which the cell morphology, the growth curve, karyotype, soft agar colony growth, nude mice tumor experiment result and transfection cell SV40 big T gene detection result, mRNA product measuring result and DNA sequencing result are in fit with the characteristics of an immortalized cell and are the same as or similar to that of a primary cell to be taken as an in vitro research cell model for the SV40LT-mediated klinefelter syndrome; and a foundation is laid for in vitro long-term research of pathogenesis of the klinefelter syndrome from the cellular level.

Description

The restructuring gene constructed klinefelter syndrome cell model of SV40 and cell bank thereof

Technical field

The present invention relates to the structure of SV40 gene (simian virus 40 large T antigen gene or SV40LT antigen gene) restructuring (mediation) klinefelter syndrome cell model and cell bank thereof, be mainly used in human reproduction's health research field, the research for klinefelter syndrome provides cell model and preserves its Scientific Research Resource.

Background technology

Klinefelter syndrome is one of karyomit(e) inborn defect, is also called klinefelter's syndrome or convoluted seminiferous tubule dysplasia, is a kind of comparatively common male sex chromosome abnormal diseases.The feature of this disease is numerical abnormalities of chromosomes, many X chromosomes.Modal caryogram is 47, XXY.Some patients have plural X, are called the anomaly of Klinefelter, as: 48, XXXY, 49, xXXXY, general X is more, and clinical symptom is more serious.The sickness rate of this disease is 0.1%.Preadolescence, general non-evident sympton.Pubescence, symptom was obvious gradually, it is poor that vulva is grown, microcaulia, and testis is little and hard, the male sex second is raw, and development delay is not even grown, skin is tenderly white, and it is poor that whole body chaeta is grown, pubes, beard are rare and armpit hair often lacks as, Adam's apple is not obvious, body is just high, and lower body is longer than upper body, and nearly half patient's chest is grown in gynecoid type breast.Patient's hyposexuality, sexual function badness, is almost azoospermia and can not gives birth to except few patients.Because of sexual function badness and the reason such as can not to give birth to, and general treatment is invalid, the birth of this patient, cause serious psychological burden and soul pain must to sufferers themselves and family.

Klinefelter syndrome main because smart ovum insemination in conjunction with time, ploidy level causes.There is bibliographical information, cause the reason of ploidy level relevant with factors such as physics, chemistry, biology, heredity.But, due to do not have can in vitro long-term cultivation, the immortality cell model that can be used for klinefelter syndrome study of incident mechanism and cell bank thereof, just be difficult to study from cell levels the mechanism such as transgenation, genetic expression, changing function, physiological property, biology conduction that klinefelter syndrome affects by physics, chemistry, biology, heredity etc. in vitro, thus seriously limit the further research of klinefelter syndrome.

Foreign literature is reported, simian virus 40 (SV40) can make some human cell that immortalization occurs.The research such as Poulin DL, Kung AL and Sullivan CS shows, the importing of SV40T antigen gene can accelerate the growth velocity of transformant, after immortalized cells repeatedly goes down to posterity in vitro, still there is metastable multiplication characteristic and functional status, also can retain many phenotypic differentiations of its initiating cell simultaneously, may be used for the foundation of the cell model of transgenic animal model and some kind of human and animal, the characteristic of initiating cell can be studied accordingly by means of research transgenosis immortalized cells and animal model in vitro, thus study its pathogenesis.

According to bibliographical information, vascular smooth muscle cell strain is set up in Reilly simian virus (SV40) large T antigen gene transformation, builds cell model to study the restraining effect mechanism of heparin for vascular unstriated muscle.Su etc. utilize the superficial cell strain transformed through SV40, build the regulating and controlling effect that cell model analyzes the synthesis of epithelial cell internal protein.The superficial cell strain that Miquel etc. transform with SV40, as the cell adhesion that cell model research ln 5 mediates.Webber etc. study physiological function and the secreting function of prostate epithelial cell as cell model with the prostate epithelial cell strain transformed through SV40.Racusen etc. transform with through Ad12-SV40 damage and the disease that proximal convoluted tubule studied by renal cells model.Hougton etc. transform with SV40 and set up Bone marrow Stromal cell as cell model to study under certain culture condition, and cell has the potential to adipocyte and osteoblastic two-way differentiation, further the osteoporotic mechanism of research.

Because of the needs of research work, almost often kind of disease all establishes respective cell model.As diabetes cell model, cancer cell model, transgenic cell model, climacteric syndrome cell model, endometrial cell model, epilepsy cell model, E-Cell models, alcoholic dementia cell model, cerebral edema cell model.Etc..

But, there is not yet so far and be used in the external bibliographical information studying the pathogenetic immortality cell model of klinefelter syndrome and cell bank thereof from cell levels about building, also cannot carry out relevant research project.In order to solve the problem, present inventors have proposed the present invention.

Summary of the invention

The object of the invention is the construction process that will provide with SV40LT antigen gene mediation klinefelter syndrome cell model and cell bank thereof, another object is for providing SV40LT antigen gene to mediate klinefelter syndrome cell model and cell bank thereof from the pathogenesis of cell levels research klinefelter syndrome in vitro.

The object of the present invention is achieved like this: connect pcDNA3.1 (-) DNA and pcr amplification that cut through BamHI enzyme with T4DNA ligase enzyme simultaneously, the SV40LTag DNA that agarose gel electrophoresis is separated, build SV40LTag-pcDNA3.1 (-) recombinant plasmid, transform DH5a competent escherichia coli cell with amplification, purifying picking amp-R bacterium colony extracting plasmid, the klinefelter syndrome suspension histocyte or containing 20% foetal calf serum of going down to posterity digested through collagenase II is imported in vitro with lipofection, be in the nutrient solution of 5 ~ 10nmol/L Regular Insulin and will enter or just enter logarithmic phase, spindle cell accounts for more than 90%, converge the adherent growth klinefelter syndrome cell that rate reaches 55% ~ 65%, the DNA of recon and cell is integrated, with the cell containing positive recombinant of G418 screening, go down to posterity, enlarged culturing, screening cellular form, cell growth curve, karyotype, soft agarose growth test, nude mice tumorigenesis is tested, the large T gene test of SV40 in transfectional cell DNA, mrna expression product measures and determined dna sequence result meets immortalized cells characteristic and identical with primary cell or close person is frozen in liquid nitrogen as cell model, SV40LT antigen gene mediation klinefelter syndrome cell model and cell bank thereof is built with this.

The SV40 large T antigen gene that the present invention purifies with pcr amplification, agarose gel electrophoresis imports klinefelter syndrome cell through genophore pcDNA3.1 and lipofection and builds its cell model, its histocyte digest with 0.01% collagenase II and need not be conventional tryptic digestion, digested or make cell suspension with the collagen only making to cause cytoadherence, the protein decreasing cell walls is digested and injure cell, makes the success ratio of cell cultures bring up to more than 95% by general about 85%; Select the defined medium containing 20mL/L foetal calf serum, 5 ~ 10nmol/L Regular Insulin, make cell can not grow too fast and affect the integration of SV40 large T antigen gene, also can not make because lacking nutrition or Growth of Cells stimulating factor cell do not meet the requirements of converge rate, do not enter logarithmic phase before with regard to premature death, or logarithmic phase shortens; Clone recovery cultivation after frozen 1 month in-196 DEG C of liquid nitrogen of preparation, all can grow attached cell; When making clone Chromosome Identification, the consumption of colchicine and action time are conventional 5 ~ 10 times, and chromosome separation is increased mutually, are enough to counting and analyze; The klinefelter syndrome cell model built up thus and cell bank thereof, for the pathogenesis studying klinefelter syndrome from cell levels in vitro lays the foundation.

Accompanying drawing explanation

Fig. 1 is klinefelter syndrome cell model (adherent growth) figure prepared by the present invention.

As Fig. 1, passage to the 80th generation, be cultured to the 10th day time, in logarithmic growth, circle, fusiformis, to be paved with bottle at the bottom of, its Growth of Cells converges rate and reaches more than 95%, and after this along with the increase of passage number, Growth of Cells is slack-off, become sparse.

Embodiment

1, the extraction of SV40 large T antigen DNA: 1. SV40DNA enzyme is cut: the SV40 freeze dried powder or the SV40 plasmid that contain large T antigen gene from commercially available purchase, be dissolved in appropriate H ₂in O or TE damping fluid, add 2uL10 × enzyme cutting buffering liquid and 18uL H ₂o, adds restriction enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, and inactivator, adds 5uL electrophoresis sample loading buffer (also by adding 0.5mol/LEDTA) termination reaction in order to electrophoresis.2. SV40DNA electrophoresis: power taking swimming level agarose is made into 10% sepharose with electrophoretic buffer, pour on the gel casting platform sealed, plug sample comb, from glue platform removing envelope band after gelling is solid, extract comb, put into the electrophoresis chamber being added with enough electrophoretic buffers, damping fluid exceeds gel surface and is about 1mm, DNA sample is prepared with 10 appropriate × sample loading buffer, then with pipettor, sample is added in sample well, and do suitable DNA molecular amount standard control thing simultaneously, connect electrode, DNA anode is moved, when under the voltage of 1-10V/cm gel, electrophoresis is to the distance of enough isolation of DNA fragments, powered-down.3. from agarose, about 2600bp SV40 large T antigen DNA is separated: under 300-360nm long wave ultraviolet light source, (use long wave ultraviolet light source to prevent DNA damage) cut in loading dialysis tubing by the gel-tape containing target DNA fragments, 2ml electrophoretic buffer is added in dialysis tubing, make it submergence gel, and emptying steam bubble, dialysis tubing level is put into electrophoresis chamber (length direction is parallel with electrophoresis), add appropriate amount of buffer solution by dialysis tubing submergence (about 6-7mm), switch on power, 150 volts of electricity are washed, observe under ultraviolet lamp and treat that DNA all shifts out gel, change direction of an electric field and continue energising 1 minute, from dialysis tubing, sucking-off damping fluid is in 1-5ml Eppendorf pipe, add 1.5 times of volume propyl carbinols, EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, suck upper strata butanol solution, repetition secondary like this, equal-volume phenol chloroform (2) extracting 2 times is added in the solution of lower floor speech DNA, supernatant proceeds in another Eppendorf pipe and adds 1/10 times of volume 3M NaAc, 2 times of volume precooling dehydrated alcohols, spend the night in 20 DEG C, 12000g, at 4 DEG C centrifugal 10 minutes, obtain DNA precipitation, abandon supernatant, dry ethanol is abandoned after adding 70% washing with alcohol 2 times, add 50 μ l TE dissolving DNAs.In addition, also target DNA fragment is separated, is purified by available low melting-point agarose gel method, DNA filter membrane inserted sheet method etc. from gel.

2, the connection of SV40 large T antigen DNA and pcDNA3.1 genophore: get the above-mentioned DNA composition of 9 μ l (0.1-5 μ g), 10 μ l2 × connection damping fluids, 1 μ l10mmol/L ATP, T4DNA ligase enzyme (20 ~ 500 sticky end unit) or e. coli dna ligase, pcDNA3.1 empty carrier mixes, 15 DEG C of incubation 24h, are built into SV40T/pcDNA3.1 recon.

3, the amplification of SV40T/pcDNA3.1 recon, Isolation and ldentification: the 1. preparation of E. coli competent: its basic skills is ice-cold CaCl ₂or the process such as multiple divalent positively charged ion bacterium, making it to enter competence is transformed, and uses CaCl ₂prepare fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence bacterium, this law is applicable to most of coli strain, operating process is summarized as follows: cultivate picking single bacterium colony (as bacillus coli DH 5 2) the fresh plate of 16 ~ 20h from 37 DEG C, or 16 ~ 20h overnight culture that 1ml is fresh, forward one to containing in 1L or the 500ml flask of 100mlLB substratum, about 2 ~ 3h (rotary shaker 200 ~ 300r/min) is cultivated in 37 DEG C of violent joltings, OD600 value ≈ 0.4 is measured every 20 ~ 30min, aseptically bacterium is transferred to one, in ice-cold 50ml polypropylene centrifuge tube, 10 ~ 20min is placed on ice, in 4 DEG C with Sorval lGS2 rotary head (or the rotary head matched with its centrifuge tube) with the centrifugal 10min of 4000r/min, to reclaim cell, nutrient solution reciprocal, pipe is inverted 1min to flow to end to make the trace nutrient solution of final residual, with the ice-cold 0.1mMCaCl of 10ml ₂resuspended every part of precipitation, be put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding rotary head) with the centrifugal 10min of 4000r/min, to reclaim cell, pour out nutrient solution, pipe is inverted lmin to flow to end to make the trace nutrient solution of final residual, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice precooling ₂resuspended every part is sunk calmly, now, rapidly cell can be distributed into aliquot, freezing in liquid nitrogen,-70 DEG C of storages are for subsequent use, from every kind of competent cell suspension, getting 200 μ l with the sterile pipette tip of cooling transfers in aseptic Eppendorf tube, DNA or ligation mixture (volume≤10 μ l is added in every pipe, DNA≤50ng), rotate gently to mix content, 30min is placed in ice, centrifuge tube is put into pre-heating on the test-tube stand in the circulator bath of 40 DEG C, place 90s ~ 2min, do not shake test tube, fast pipe is transferred in ice bath, cell is made to cool 1 ~ 2min, every centrifuge tube adds 800 μ lSOC substratum, with water-bath, substratum is warmed to 37 DEG C, then pipe is transferred on 37 DEG C of shaking tables, incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, the competent cell that proper volume (each 90mm flat board can reach 200 μ l) has transformed is transferred to containing on 200mmol/LMgSO4 and corresponding antibiotic SOB substratum, flat board is placed in room temperature to be absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, bacterium colony can be there is after 12 ~ 16h.2. the screening of recon, amplification and extraction: select single colony inoculation in the aseptic LB substratum of 5mL or rich medium (as super broth or TB super broth substratum) with sterile toothpick or disinfection inoculation pin, after overnight incubation, join 500mL again containing in the 2L flask of LB substratum (containing suitable microbiotic), then be cultured to state of saturation (OD in 37 DEG C ₆₀₀≈ 4, for improving output, the flask of the comparatively large and band traverse baffle of surface-area should be adopted to increase venting quality as far as possible, jolting speed should be greater than 400r/min), in 4 DEG C, the centrifugal 10min of 6000g, by the resuspended precipitation of 4mL GTL solution, and transfer to (bacterial precipitation can be preserved at-20 DEG C or-70 DEG C of indefinitely) in the high speed centrifugation pipe of a volume>=20mL, add the GTE solution containing 25mg/mL N,O-Diacetylmuramidase that 1mL newly joins, resuspended precipitation, 10min is placed in room temperature, add 10mL and newly join NaOH/SDS solution, and mixing is until liquid becomes homogeneous gently, limpid and thickness, in placing 10min on ice, add 7.5mL acetic acid solution, stir until viscosity declines and forms large precipitation gently with suction pipe, in placing 10min on ice, in 4 DEG C, the centrifugal 10min of 20000g, supernatant is poured into gently in another clean centrifuge tube, if there is visible drift can by several layers of filtered through gauze, add the Virahol of 0.6 times of volume, put upside down mixing, room temperature places 5 ~ 10min, in room temperature, the centrifugal 10min of 1500g, add the washing precipitation gently of 2mL70% ethanol, then of short duration centrifugal fast, suck ethanol, and vacuum-drying (precipitation can be preserved for a long time at 4 DEG C).3. the qualification of recon: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competence intestinal bacteria, the same method restriction enzyme BamH I carries out enzyme and cuts, 10g/L agarose gel electrophoresis is identified, obtain 2 bands that size is about 2600bp and 5600bp, the former meets the size of SV40T fragment in GenBank.

4, SV40T/pcDNA3.1 recon imports screening and the amplification of klinefelter syndrome cell and positive colony thereof: the 1. collection of klinefelter syndrome cell and cultivation: be extracted in aseptic technique and do other and test or other check unnecessary, the klinefelter syndrome infant amniotic fluid cast-off cells discarded afterwards, be about 1 × 10 with final concentration of cells ⁵/ mL is for subsequent use, the cell got above-mentioned cell in single dispersion or become single dispersion is after treatment inoculated in containing 5 ~ 10nmol/L Regular Insulin, in the RPMI1640 liquid of 20% foetal calf serum or be inoculated in containing 20% foetal calf serum, in the low sugar DMEM cell culture medium of 5 ~ 10nmol/L Regular Insulin, be placed in 37 DEG C, in volume fraction 5%CO2 incubator, cell attachment is cultivated about 3 ~ 4 days, cellular form is fusiformis, little circular cell is less than 10%, the rate of converging of attached cell reaches 75% ~ 85%, be in when just entering logarithmic phase, namely consider to collect culturing cell, first blot the nutrient solution in clean culturing bottle, add the collagenase II liquid (can cover at the bottom of whole bottle with Digestive system and be as the criterion) of 1-2ml0.01%, leave standstill 2-10min (under microscope dynamic monitoring), suck collagenase II liquid, add low sugar DMEM or RPMI1640 nutrient solution, culture in glassware liquid is drawn with suction pipe, repeatedly blow and beat bottle parietal cell, form cell suspension, proceed to centrifuge tube centrifugation, remove supernatant liquor, after cell precipitation cleans 2 times with above-mentioned nutrient solution, make suspension, import as recon.2. the importing of SV40T/pcDNA3.1: method 1: in upper method Isolated cells, select lipofection, by above-mentioned 2 × 10 ⁵individual cell is inoculated in 35mm culture dish, at 37 DEG C of CO ₂24 ~ 36h cultivated by incubator, make Hemapoiesis individual layer, cellular form be fusiformis, there is not yet or be rarely seenly less than 10% little circular cell, the rate of converging of attached cell reaches 55% ~ 65%, is in when entering or just entered logarithmic phase, namely transfection recon SV40T/pcDNA3.1 should be considered, in 1.5ml Eppendorf tube, prepare following solutions: pipe A, SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free mediums; Pipe B, 20 μ l Lipofectamine are dissolved in 80 μ l serum-free mediums, mixed by pipe A and pipe B, the underlying 45min of room temperature, after sucking cell culture fluid, with serum-free medium washed cell 2 times, in Lipofectamine-SV40T/pcDNA3.1 mixture, add 1ml serum-free medium, mix gently, then drop in Tissue Culture Dish, then 1ml serum-free medium is added, at CO ₂10h cultivated by incubator, sucking-off transfection liquid, and add 4ml complete culture solution and continue to cultivate 16h, discard nutrient solution, replacing concentration is 400mgL ^-1g418 nutrient solution continue to cultivate, within 8 ~ 10 days, select individual cells colony to carry out subclone afterwards, strengthen G418 concentration after enlarged culturing again to 800mgL ^-1, in the G418 environment of high density, amplification of going down to posterity can be carried out by the clone of stable growth, in addition after klinefelter syndrome cell and SV40 or Epstein-Barr virus co-cultivation, infect viral DNA and the klinefelter syndrome cell occurring to integrate carries out amplification of going down to posterity.Method 2: draw 1/10-1/40 cell suspension, being mixed with final concentration is 1 × 10 ⁵/ mL cell suspension, be inoculated in culturing bottle, select low sugar DMEM or RPMI1640 substratum, wherein containing 20mL/L foetal calf serum, 10nmol/L Regular Insulin, after cultivating 48h, make Hemapoiesis individual layer, cellular form is fusiformis, there is not yet or rarely seenly be less than 10% little circular cell, attached cell converges rate and reaches 55% ~ 65%, be in when will enter or just enter logarithmic phase, adopt the method for liposome transfection, by recon SV40T/pcDNA3.1 transfection in klinefelter syndrome cell, or by SV40 direct infection in klinefelter syndrome cell, with the liquid medium-selection 1wk containing 700mg/L G418, change G418 concentration into 300mg/L, to occurring positive colony, transfer them to new culturing bottle to expand, Secondary Culture, method 3: above-mentioned cell suspension is made into 5 × 10 with low sugar DMEM or RPMI1640 nutrient solution ⁸the cell suspension inoculation of/L final concentration in 24 well culture plates, until cell grow to about 90% merge time for transfection, by the recon SV40T/pcDNA3.1 of 0.2 μ g content, with DMEM or RPMI1640 adjust volume be 50 μ l, room temperature place 5min, liposome 6 μ l, adjusting volume with DMEM is 50 μ l, room temperature places 5min, two kinds of reagent mix gently, room temperature places 20min, after cell DMEM in 24 orifice plates being washed 3 times simultaneously, add nutrient solution 100 μ l in every hole again, add liposome-SV40T/pcDNA3.1 mixed solution, gently cell surface paving evenly, 6h is placed in 37 DEG C of CO2 incubators, use 0.01g/L collagenase by cell dissociation subsequently, proceed in 6 orifice plates, add perfect medium, adding G418 microbiotic next day makes final concentration be 500mg/L, until there is monoclonal cell to grow.There is monoclonal cell colony to grow after about 10d, choose to be placed in 24 orifice plates and continue to cultivate, maintain and stabilize with the G418 of 300mg/L the amplification cultivation that goes down to posterity.Klinefelter syndrome cell model (clone) is built with this.

5, the going down to posterity of klinefelter syndrome cell model (clone), increase: collect the above-mentioned positive monoclonal cell colony importing SV40 large T antigen gene through lipofection, be made into about 1 × 10 with low sugar DMEM or RPMI1640 substratum ⁵the cell suspension of/mL, is inoculated in several bottles of 20 ~ 50cm ²in culturing bottle, add 5mL containing 20mL/L foetal calf serum, the low sugar DMEM of 10nmol/L Regular Insulin or RPMI1640 substratum, to cell attachment growth, converge rate reach 80 ~ 85%, be in the early stage of logarithmic phase time collecting cell, again by above-mentioned steps Secondary Culture, inoculation of repeatedly going down to posterity like this, cultivation, record algebraically and observation of cell growth characteristic.As Fig. 1, passage to the 80th generation, be cultured to the 10th day time, rounded, fusiformis, to be paved with bottle at the bottom of, its Growth of Cells converges rate and reaches more than 95%, and after this along with the increase of passage number, Growth of Cells is slack-off, change is thin.Wherein logarithmic growth refers to high cell growth speed, and the increase of cell quantity and incubation time are multiplication relation, when usually cultivating by kind of a cell routinely, cultivates the individual cells that just can find growth for 1 ~ 2 day under the microscope; Visible cell colony when 4 ~ 5 days, at the bottom of adherent growth cell bedding culturing bottle 1/3, at the bottom of visible adherent growth cell bedding culturing bottle more than 2/3 when cultivating 7 ~ 13 days, even cell overlap, interspace hardly (converge rate and reach 95 ~ 100%), cell light, without particle, without catabiosis such as harsh feelings, also can judge according to the relation of cell counting and incubation time; Dead cell is few, and cause death and floating cell are less than 10% [differentiate dead cell and viable cell by trypan blue staining, because normal viable cell, after birth structural integrity, can repel, and trypan blue can not be entered in born of the same parents weekly; And the cell of loss of activity, the permeability of after birth increases, blueness can be dyed by trypan blue, can be judged as that cell is dead, method draws weekly a certain amount of suspension culture, mixes rearmounted room temperature 5 ~ 10 minutes, then make cell sheet with Trypan Blue agent, count 1000 total cellular score under the microscope, calculate the per-cent of painted dead cell and non-staining viable cell].

6, the qualification of klinefelter syndrome cell model biological characteristics: after using SV40 to set up cell model (clone), the key issue of qualification has: is that this cell of requirement has lasting multiplication capacity, i.e. T antigen stably express in clone; Two are its forms of requirement, basic physiological function isophenous remains unchanged.Whether 1. observation of cell form: every day, whether observation of cell was in typical epithelial cell sample adherent growth under inverted light microscope is fusiformis or the growth of little circular; 2. observation of cell growth curve: get the good transfectional cell of growth, adopts 0.25 trypsin solution digestion, makes cell suspension, through counting, get 1.4 × 10 respectively ⁴cell is inoculated in 30 containing 15FBS low sugar DMEM culture medium culturing bottle.Get 2 bottles of cells every day to count, computation of mean values, Continuous Observation, until cell quantity obviously declines, is cultivated after 3 days and was changed liquid every 2 days to the cell of no count, adopts same method to observe the growing state of transfectional cell in hepato ZYME-SFM serum free medium.Result take incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), be depicted in semi-logarithmic scale to make after curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof "; 3. karyomit(e) is checked: by analyzing karyotype, if karyotype is " 47; XXY ", or be same as the primary cell of the present invention's collection, then illustrate that this clone vicious transformation does not occur and (whether occurs abnormal DNA colony in available flow cytometry analysis clone simultaneously, if no, also illustrate that knurl feature does not appear in clone).Chromosome karyotype analysis method is: reach 85-95% (its medium and small circular cell accounts for 10%) at cell density, be in the 250ug/ml colchicine 100ul adding preheating in the culture of logarithmic phase by 5mL nutrient solution, mix rearmounted 37 DEG C of incubators 4 hours, blot nutrient solution, add 2-3mL EDTA-trysinization liquid of preheating, 37 DEG C act on 5 minutes, stop digestion, wash lower attached cell, through centrifugal, hypotonic, fixing, film-making, G aobvious band post analysis karyotype; 4. soft agarose growth test: by the cell of immortalization after SV40T transfection with 5 × 10 ⁴it is in 35mm soft agar plate that/ml is inoculated in diameter, after observing three weeks, does not find Clone formation, illustrates that the SV40T immortalization chromosome abnormalty amniocyte of this experiment can not form clone in soft agar, meets immortalization feature; 5. nude mice tumorigenesis test: by the cell of SV40T immortalization with 3 × 10 ⁷inoculation nude mice dorsal sc, after 2 months, 4 nude mices are showed no tumour and are formed, and prove that this cell is non-malign cells; 6. the large T gene test of SV40 in transfectional cell DNA: as with Immunohistochemical detection, in the nucleus of SV40 transfection, the visible a large amount of brown particle of dyeing, shows that SV40T antigen has been integrated in cell; Also the expression of T antigen in cell can be detected by RT-PCR method, the wherein primer of T antigen: upstream primer (A4239) 5 '-GTT ATG ATT ATA ACT GTT ATG-3 ', downstream primer (S4496) 5 '-GAA ATG CCA TCT AGT GAT-3 '; Amplified production length is 268bp, and amplification condition is 94 DEG C, 5min, that is: and (94 DEG C, 1min; 55 DEG C, 1min ,-0.5 DEG C/circulation; 72 DEG C, 1min) × 30, (94 DEG C, 30S; 40 DEG C, 30S, 72 DEG C, 30S) × 15, amplification system is 50 μ l:[Mg ²⁺] 2mmol/L, dNTPs200 μm ol/L, primer concentration 0.4 μm of ol/L, Taq1U, template 5 μ l; Experimental group with the 19th generation cell cDNA for template (with reference to commercially available cDNA first chain synthetic agent box carry out cDNA first chain synthesis, product-20 DEG C preservation); Negative control establishes two, template is done respectively with the cDNA of sterilized water, primary cell, positive control is that template (extracts SV40DNA with reference to SDS-proteinase-K pathway with SV40DNA, because SV40 virus is without coating, do not use SDS rupture of membranes, get 5 μ l and carry out 1.5% agarose gel electrophoresis detection, all the other-20 DEG C save backup); 7. mrna expression product measures: T antigen mRNA RT-PCR product checks order: the amplified production getting 100 μ l systems, test kit (Takara is reclaimed with gel, Japan) reclaim product, get 2 μ lDNA solution dilution 100 times, survey concentration, remaining DNA and each 10 μ l of upstream and downstream primer checks order.8. Flow cytometry: the cell proportion detecting synthesis in the 19th continuous cell line, division, if its multiplication capacity obviously strengthens than not building the normal cell being, explanation is the result that SV40 large T antigen is integrated, expressed; 9. determined dna sequence: sequenator detects routinely, display SV40 large T antigen DNA sequence dna.

So cell model of the present invention is that 1. cell is the epithelial cell sample adherent growth of fusiformis or little circular under inverted light microscope; 2. the growth curve of cell is as taken incubation time as transverse axis, and cell quantity is the longitudinal axis, is that " S " feature or " arched roof " are formed in hepato ZYME-SFM serum free medium; 3. karyotype is " 47, XXY ", or is same as the original klinefelter syndrome cell caryogram of the present invention's collection; 4. cell can not grow (forming clone) in soft agar; 5. cell is non-malign cells, nude mice tumorigenesis negative; Incorporate SV40 large T antigen gene in the DNA of 6. cell, express SV40 large T antigen mRNA product; 7. passage to the 80th generation, be cultured to the 10th day time, in logarithmic growth, circle, fusiformis, to be paved with bottle at the bottom of, its Growth of Cells converges rate and reaches more than 95%, and after this along with the increase of passage number, Growth of Cells is slack-off, thinning to be dredged.

7, SV40LT antigen gene mediation klinefelter syndrome cell model and cell bank thereof: screen and continue to go down to posterity, enlarged culturing meets immortalized cells characteristic and the cell identical or close with primary cell after above-mentioned qualification, get the attached cell that growth conditions is good, be in the different generations of logarithmic phase, through digestion, stop and centrifugation (1200r/min, 6min), with the frozen storing liquid 0.5 ~ 1ml re-suspended cell containing methyl-sulphoxide, cell density is 5 × 10 ⁵individual/ml, adds cryopreservation tube, through 4 DEG C, and 0.5h;-20 DEG C, 2h;-70 DEG C, spend the night, enter-196 DEG C of liquid nitrogen cryopreservations, build the stable immortalization klinefelter syndrome cell model of biological characteristics and cell bank thereof in this way, with concentrated preservation genetic resources, for other researchs provide scientific research material, again can directly as the cell model of klinefelter syndrome pathogenesis in vitro study, to accelerate the new way expanding klinefelter syndrome research, study from cell levels the mechanism such as transgenation, genetic expression, changing function, physiological property, biology conduction that klinefelter syndrome cell affects by physics, chemistry, biology, heredity etc. in vitro.

8, cell model application

1) klinefelter syndrome cell model is as scientific research cell: make klinefelter syndrome cell model be in the artificial nuisance with different content or concentration manufactured as physics (as X-ray), chemistry is (as formaldehyde, gasoline, plumbous, mercury), biological (as rubella virus, cytomegalovirus, simplexvirus) condition under cultivate, then the viable cell of the different cycles of external Long Term Passages is got, the apoptotic cell of succeeding generations, nutrient solution containing the meta-bolites produced in Secondary Culture from different perspectives and level, comparative study klinefelter syndrome cell model, normal control cells model is to the difference of the tolerance of nuisance, nuisance causes the mechanism of klinefelter syndrome.Such as apply ordinary method as the gene of the screening differences such as gene chip, miRNA chip, comparative genomic hybridization hybrid chip (CGH), differential methylation hybridization hybrid chip (DMH) and SNPs and polymorphism, methylation level, the gene rate of rotation in the technology for detection gene place research cell models such as in situ hybridization (FISH), Northern blot, Real-time PGR, CHIP, EMSA is used to express, locate and regulation and control, utilize the meta-bolites of protein metabolism process and key in enzyme reaction, metabonomic technology identification of cell model Long Term Passages, application two-dimensional electrophoresis, MALDI-TOF Mass Spectrometric Identification, the protein function of the technical study such as yeast two-hybrid and co-immunoprecipitation cell model in Long Term Passages and the interaction between protein, dynamic from viable cell culturing process, klinefelter syndrome cell study for a long period of time to the tolerance of bad environmental factor and adaptability, such as: 1. common in environment toxicant benzopyrene causes the research of klinefelter syndrome: make klinefelter syndrome cell model and compared with control cells contain 0.1 respectively, 1.0, 5.0, 10.0, cultivate in the nutrient solution of 20.0pmol/L benzopyrene, detect cultivation 2 weeks, 4 weeks, 6 weeks, 8 weeks, the apoptosis that can be used as cytotoxicity index of the different incubation time such as 16 weeks, downright bad, dikaryocyte rate and the micronuclear rates that can be used as genetoxic index, caryoplasm bridge rate, core bud rate, namely the micronucleus number in 10000 dikaryocytes is counted under an optical microscope, caryoplasm bridge number and core bud number, necrosis and apoptosis cell count in 500 viable cell, dikaryocyte number, and detection cell survival rate, namely tetrazolium-based colorimetric assay (mtt assay) is applied, after sucking original fluid, 96 orifice plates add the serum-free medium of the 5mg/mlMTT of 20%, continue to cultivate 4h, supernatant liquor in hole is abandoned in careful suction, every hole adds 150 μ L methyl-sulphoxides, vibration 10min, purple crystal thing is fully dissolved, and this microplate reader measures the absorbance in each hole with 490nm, calculates cell survival rate.Other normal experiment methods can also detect containing toxic substance and not containing the transgenation, proteomics, emiocytosis function, chromosome aberration, cell survival rate (life-span) etc. of respectively organizing cell in toxic substance, klinefelter syndrome cell model and normal cell of different incubation time, with, study bad environmental factor effect to klinefelter syndrome dynamic from cell levels from viable cell culturing process.2. replace toxicant benzopyrene with certain sex pheromone and do same research, can from viable cell culturing process from cell levels dynamically, the klinefelter syndrome that studies for a long period of time is to the tolerance of biotic factor and adaptability and mechanism of action.3. by the medicine being made into concentration gradient be made into the sex pheromone of concentration gradient or poisonous chemical substance and cell model co-cultivation, detect cell cultivation process life-span, transgenation, various molecule changes etc., can from viable cell culturing process from cell levels dynamically, the medicine that studies for a long period of time is to the result for the treatment of of klinefelter syndrome and intervention effect.4. the method for same available klinefelter syndrome cell model research gene therapy, substitutes some and directly does experiment with human body.5. klinefelter syndrome genetic resources is preserved with the form of cell model.

2) cell model is as Quality Control cell: the quality control in laboratory is the access system of carrying out laboratory diagnosis project, becomes action by government.Klinefelter syndrome cell model can be used as Quality Control cell, comments for Internal Quality Control and room interstitial.Such as under the prerequisite of chromosomal disorder laboratory diagnostic methods being synchronized with clinical samples, regular growth cultivation, karyomit(e) preparation, chromosome karyotype analysis is made with cell model, to examine the reagent quality, instrument performance, working method etc. of experimentation whether reliable, be equivalent to the positive, the negative control in testing; By cell model after the image that routine chromosome preparation, chromosome karyotype analysis obtain arranges, each laboratory can be sent to, to examine each room to the accuracy of the diagnosis of various chromosome abnormalty image; Cell model can be sent to each laboratory, Result after each room makes regular growth cultivation, chromosome sectioning, chromosome karyotype analysis voluntarily, External quality evaluation is done to the diagnostic result of each room, by appraising through comparison the accuracy of each room, to find associated problem, deal with problems, to improve quality of diagnosis; Cell model can be mixed in the kind required for Quality Control and ratio with various normal dyeing somatocyte, make the mixing Quality Control cell containing different sorts and ratio, numerical abnormalities of chromosomes Internal Quality Control cell is detected and room interstitial comments cell as fluorescence in situ hybridization (FISH), if certain laboratory is being done not detect different types of chromosome abnormalty of (failing to pinpoint a disease in diagnosis) Quality Control cell in FISH, accurately do not detect the ratio (%) of different types of chromosome abnormalty, then there are quality problems in illustrative experiment method, and reason should be looked for correct.Now be described as follows with the example that has particular application as of Quality Control cell in fluorescence in situ hybridization room interstitial is commented of preparation after several chromosome abnormalty cell model mixing: 1. prepare Quality Control cell: get 1 example or numerical example klinefelter syndrome cell model, mix Quality Control cell in the example required for Quality Control time or ratio, the Quality Control cell of mixing also can use through amplification after discharge.If mixed with equal proportion cell count with karyomit(e) normal cell with 1 routine klinefelter syndrome cell model, then often the cell of kind of numerical abnormalities of chromosomes accounts for 50%.Then Quality Control cell is sent to the External quality evaluation that each study subject does fluorescence in situ hybridization detection numerical abnormalities of chromosomes.2. fluorescence in situ hybridization (FISH) detects Quality Control cell chromosome number and result judgement: after each study subject receives Quality Control cell, and can going down to posterity to Quality Control cell strain as one sees fit, amplification is rear to be tested, to increase cell count.Reagent adopts the AneuVysion test kit of Abbott GmbH. & Co. Kg of Switzerland, complete 2 chromosomal number analyses, method gets X and Y chromosome probe, and get Quality Control cell, be synchronized with the operation of the tested sample in laboratory, through the centrifugal 10min of 2000r/min, remove supernatant, stay precipitation 0.2ml, in centrifuge tube, add 4ml collagenase 37 DEG C temperature bath 30min, the centrifugal 10min of 2000r/min, remove supernatant, then add the 15mol/L KC1 of the pre-temperature of 5ml, hypotonic 25min in 37 water-baths, the centrifugal 10min of 2000r/min, removes supernatant.Add the fixing 10min of stationary liquid (methyl alcohol: Glacial acetic acid=3:1) of new preparation, centrifugal 2000r/min is centrifugal, and 10min removes supernatant, twice repeatedly.Drip sheet, seasoning is aging.Taken out in refrigerator-freezer by the sample slice prepared, put successively in people 70%, 85%, 100% ethanol and dewater, drying at room temperature, the slide that pre-treatment is good puts 73 DEG C, 5min in 70% methane amide.Two kinds of each 10 μ 1 of probe mixed solution are added in the target region of slide in darkroom, covered, avoid producing bubble, cover glass is sealed by mounting glue, put 42 DEG C of hybridized overnight in dark wet box, cleaning, at high power Microscopic observation crossbreeding effect, every routine sample counting 60 ~ 100 cells, record hybridization signal number also gathers image, require that nuclear membrane must be complete, hybridization signal zero lap limpid in sight in core, signal disperse and faintly all not take statistics.3. test result: after probe hybridization, the point that in cell, the display of X (women) karyomit(e) is green, every bar X chromosome shows 1 green point, 2 points that then display 2 is green, 3 points that then display 3 is green; The point that in cell, the display of Y (male sex) karyomit(e) is red, every bar Y chromosome shows 1 red point, 2 points that then display 2 is red, 3 points that then display 3 is red.If certain laboratory in an experiment, do not detect the signal of X chromosome, or X chromosome signal ambiguity, or X chromosome signal increases or reduces, or its proportional jitter is large, do not meet the numerical value of Quality Control cell (model), then the existing problems such as the operational condition in this laboratory, technology, instrument, reagent are described, should go the measures such as calibration.

Claims

1. the gene constructed klinefelter syndrome cell model of the restructuring SV40 for medical research field and cell bank thereof, its principal character is routinely with T4DNA ligase enzyme, BamHI, pcDNA3.1 (-) DNA and SV40LTag DNA builds SV40LTag-pcDNA3.1 (-) recon, with competence intestinal bacteria purification of Recombinant, imported through going down to posterity of digesting of collagenase II or in the klinefelter syndrome histocyte of logarithmic growth in vitro with lipofection, the DNA of recon and cell is integrated, and enlarged culturing is through the cell clone of G418 screening containing positive recombinant, screening cellular form, growth curve, karyotype, soft agarose grows, nude mice tumorigenesis is tested, the large T gene test of SV40 in transfectional cell DNA, mrna expression product measure and determined dna sequence result meet immortalized cells characteristic and identical with primary cell or close person to mediate klinefelter syndrome in vitro study cell model as SV40LT antigen gene frozen in liquid nitrogen, for laying the foundation from the pathogenesis of cell levels (substituting human body or the animal straightway testing) klinefelter syndrome that studies for a long period of time in vitro.

2. the gene constructed klinefelter syndrome cell model of restructuring SV40 according to claim 1 and cell bank thereof, is characterized in that described cell model is the epithelial cell sample adherent growth of (1) cell in fusiformis or little circular under inverted light microscope; (2) growth curve of cell is as taken incubation time as transverse axis, and cell quantity is the longitudinal axis, is that " S " feature or " arched roof " are formed in hepato ZYME-SFM serum free medium; (3) karyotype is " 47, XXY ", or is same as the primary klinefelter syndrome cell caryogram of the present invention's collection; (4) cell can not grow (forming clone) in soft agar; (5) cell is non-malign cells, nude mice tumorigenesis negative; (6) incorporate SV40 large T antigen gene in the DNA of cell, express SV40 large T antigen mRNA product; (7) when passage to the 80th generation, be cultured to the 10th day time, cell is logarithmic growth, circle, fusiformis, to be paved with bottle at the bottom of, converge rate and reach more than 95%, after this along with the increase of passage number, Growth of Cells is slack-off, thinning to be dredged; (8) be used in vitro from the pathogenesis of cell levels research klinefelter syndrome, genetic resources storage, experiment contrast and Quality Control cell as cell model.

3. the gene constructed klinefelter syndrome cell model of restructuring SV40 according to claim 1 and cell bank thereof, it is characterized in that taking from gathers because other experiments are required and klinefelter syndrome infant amniotic fluid cast-off cells that are unnecessary after other experiments, that discard build it.

4. the gene constructed klinefelter syndrome cell model of restructuring SV40 according to claim 1 and cell bank thereof, is characterized in that nutrient solution used is the RPMI1640 containing 20% foetal calf serum, 5 ~ 10nmol/L Regular Insulin or the low sugar DMEM nutrient solution containing 20mL/L foetal calf serum, 5 ~ 10nmol/L Regular Insulin.

5. the gene constructed klinefelter syndrome cell model of restructuring SV40 according to claim 1 and cell bank thereof, it is characterized in that being used as Secondary Culture imports recon klinefelter syndrome histocyte in order to lipofection, in primary amplification cultivation, its collect index be cell attachment cultivate about 3 ~ 4 days, cellular form is fusiformis, little circular cell less than 10%, the rate of converging of attached cell reaches 75% ~ 85%, is in collecting cell when just entering logarithmic phase; Import the klinefelter syndrome histocyte that goes down to posterity of recon as lipofection, the index of its choose opportunities is cellular form be fusiformis, there is not yet or be rarely seenly less than 10% little circular cell, the rate of converging of attached cell reaches 55% ~ 65%, is in culturing cell when entering or just entered logarithmic phase; The index that continuous cell line is collected be select cell attachment growth converge that rate reaches 80 ~ 85%, collecting cell when being in the early stage of logarithmic phase; The cell harvesting index of chromosome karyotype analysis is that cell density reaches 85-95%, little circular cell accounts for more than 10%, is in the cell of logarithmic phase.

6. the gene constructed klinefelter syndrome cell model of restructuring SV40 according to claim 1 and cell bank thereof, it is characterized in that the klinefelter syndrome histocyte digesting adherent growth with 0.01% collagenase II, when making chromosome karyotype analysis, add 250ug/ml colchicine 100ul in every 5mL nutrient solution, mix rearmounted 37 DEG C of incubators 4 hours.

7. the gene constructed klinefelter syndrome cell model of restructuring SV40 according to claim 1 and cell bank thereof, is characterized in that the structure of cell bank comprises (1) screening and after qualification, meets immortalized cells characteristic and the klinefelter syndrome cell that mediates of the SV40LT identical or close with primary cell; (2) go down to posterity, enlarged culturing, get the attached cell that growth conditions is good, be in the different generations of logarithmic phase; (3) through digestion, termination, centrifugal (1200r/min, 6min) step harvested cell; (4) preparing density with the frozen storing liquid containing methyl-sulphoxide is 5 × 10 ⁵the cell suspension of individual/ml; (5) according to 4 DEG C, 0.5h;-20 DEG C, 2h;-70 DEG C, spend the night; Enter the program freeze-stored cell of-196 DEG C of liquid nitrogen; (6) the long-term SV40LT of preservation in recyclability ground mediates klinefelter syndrome cell model, for making genetic resources and scientific research material.