CN104419672A - Congenital heart disease ventricular septal defect cell model constructed by transduced SV40LT antigen gene and cell bank thereof - Google Patents

Abstract

The invention relates to construction of an SV40LT antigen gene transduced congenital heart disease ventricular septal defect cell model and a cell bank of the SV40LT antigen gene transduced congenital heart disease ventricular septal defect cell model applied to the field of medical research. A construction method is mainly characterized by comprising the following steps: according to a conventional route, constructing an SV40LTag-pcDNA3.1(-) recombinant by using T4DNA ligase, BamHI, pcDNA3.1(-)DNA and SV40LTag DNA; and purifying the recombinant by virtue of competent escherichia coli, then transferring the recombinant to congenital heart disease ventricular septal defect cells which are subjected to collagenase II digestive in-vitro subculture and logarithmic growth to ensure that the recombinant is integrated with DNA, performing extended culture, screening the cells containing positive recombinants for cloning by virtue of G418, and screening the cells of which the cellular morphology, growth curves, chromosome karyotypes, carcinogenicity tests in nude mice, transfection cell SV40 large T gene detection, mRNA product determination and DNA sequence determination results can meet the characteristics of immortalized cells and are the same as or similar to that of primary cells as an SV40LT transduced congenital heart disease ventricular septal defect in-vitro study cell model so as to lay a foundation for studying pathogenesis of the cells in vitro from cell levels for a long time.

Description

Transduction SV40LT antigen gene builds Closure of Ventricular Septal Defects cell model and cell bank thereof

Technical field

The present invention relates to transduction SV40LT antigen gene (simian virus 40 large T antigen gene) and build Closure of Ventricular Septal Defects cell model and cell bank thereof, be mainly used in medical science cardiovascular diseases research field, the research for Closure of Ventricular Septal Defects provides cell model and preserves its Scientific Research Resource.

Background technology

Closure of Ventricular Septal Defects is one of inborn defect that sickness rate is the highest, has become serious social concern.China newly sends out congenital heart disease 11.2 ten thousand-16.0 ten thousand example every year, will cause financial loss 10,900,000,000-156 hundred million; The treatment cost of congenital heart disease, every year up to 12,000,000,000 yuan, is caused serious psychological burden and soul pain to society and family, has had a strong impact on the quality of the people of China, become the heavy burden of social development and the serious hindrance of Sustainable development.

Research and the intervention of the inborn defects such as congenital heart disease have become Chinese government's behavior, and Party Central Committee's " decision " came into effect " inborn defect Intervening Project " from 2006; President of the People's Republic of China makes " on October 27th, 1994 the 30 No. three " announces the regulations standard that China implements (inborn defect) antenatal diagnosis from June 1 nineteen ninety-five; The National People's Congress has promulgated a series of regulation such as " People's Republic of China's law on health care of mother and baby ", " People's Republic of China's law on health care of mother and baby implementing method " in succession, facilitates inborn defect antenatal diagnosis, the carrying out of Prevention Intervention; Emphasize to strengthen inborn defect Intervention density in " 11th Five-Year Plan outline of national economy and social development ", improve the health of newborns; In " National Program for Medium-to Long-term Scientific and Technological Development (2006-2020) ", the research of inborn defect is classified as preferential theme.

Think that Closure of Ventricular Septal Defects is the interactional results of complex relationship such as h and E factor at present, mainly comprise 1.. the environmental factors of fetation: poison as ill in first trimester or bacteriological infection especially rubella virus, next is Coxsackie virus, and the sickness rate of the congenital heart diseases in infants of its birth is higher.Other is as the pathology of amnion, fetus pressurized, First Trimester threatened abortion, maternal nutrition is bad, diabetes, phenylketonuria, hypercalcemia, radioactive rays and cytotoxic drug are in the application of First Trimester, and the excessive grade of maternal age all has the possibility making fetus generation congenital heart disease.2. inherited genetic factors: most congenital heart diseases is interacted by multiple gene and environmental factors formed.3. other factors: also have relation with area, sex.

In recent years, the research of the inborn defects such as congenital heart disease is mainly carried out to the investigation of epidemiology aspect for the relation of heredity, biology, physics, chemical factor and morbidity (rate).But, due to do not have can in vitro long-term cultivation, the immortality cell model that can be used for congenital heart disease Mechanism Study 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 Closure of Ventricular Septal Defects cell affects by physics, chemistry, biology, heredity etc. in vitro, thus seriously limit the further research of Closure of Ventricular Septal Defects inborn defect.

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 Closure of Ventricular Septal Defects 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 transduction (mediation) Closure of Ventricular Septal Defects cell model and cell bank thereof, another object is for providing SV40LT antigen gene to mediate Closure of Ventricular Septal Defects cell model and cell bank thereof from the pathogenesis of cell levels research Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects amniotic fluid 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 Closure of Ventricular Septal Defects amniotic fluid histocyte 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 Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects cell model built up thus and cell bank thereof, for the pathogenesis studying Closure of Ventricular Septal Defects from cell levels in vitro lays the foundation.

Accompanying drawing explanation

Fig. 1 is Closure of Ventricular Septal Defects cell model (adherent growth) figure prepared by the present invention.

As Fig. 1, passage to the 70th generation, be cultured to the 7th 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 80%, and after this along with the increase of passage number, Growth of Cells is slack-off, change is thin.

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/L EDTA) 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, 0D600 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 SorvallGS2 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 1min 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, often pipe should be made to add DNA or ligation mixture (volume≤10 μ l, DNAx≤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 20 000g, 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 Closure of Ventricular Septal Defects cell and positive colony thereof: the 1. collection of Closure of Ventricular Septal Defects cell and cultivation: be extracted in aseptic technique and do other and test or other check unnecessary, the Closure of Ventricular Septal Defects 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, 5%CO ₂24 ~ 36h cultivated by incubator, make Hemapoiesis individual layer, cellular form be fusiformis, 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/peDNA3.1 is 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 histocyte nutrient solution, 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, changing concentration is 400mg ° of L ^-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 800mg ° of L ^-1amplification of going down to posterity can be carried out by the clone of stable growth in the G418 environment of high density, in addition after Closure of Ventricular Septal Defects cell and SV40 or Epstein-Barr virus co-cultivation, infect viral DNA and the Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects cell, or by SV40 direct infection in Closure of Ventricular Septal Defects cell, with the liquid medium-selection 1wk containing 700mg/LG418, 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 ⁸the cell suspension inoculation of/L final concentration is in 24 well culture plates, and for transfection when cell grows to about 90% fusion, by the recon SV40T/pcDNA3.1 of 0.2 μ g content, adjusting volume with DMEM or RPMI1640 is 50 μ l, and room temperature places 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.Closure of Ventricular Septal Defects cell model (clone) is built with this.

5, the going down to posterity of Closure of Ventricular Septal Defects 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 10 nmol/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 70th generation, be cultured to the 7th 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 80%, and after this along with the increase of passage number, Growth of Cells is slack-off, thinning to be dredged.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 Closure of Ventricular Septal Defects cell model (clone) biological characteristics: after using SV40 to set up cell model (clone), the key issue of qualification has: one 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 diploid " 46; XX " or " 46; XY ", or be same as the primary cell of the present invention's collection, then illustrate that vicious transformation (whether occur abnormal DNA colony in available flow cytometry analysis clone simultaneously, if do not had, illustrate that knurl feature does not appear in clone yet) does not occur this clone.Chromosome karyotype analysis method is: reach 85-95% (its medium and small circular cell accounts for 10%) at cell density, to be in the culture of logarithmic phase in by 5mL nutrient solution the 250ug/ml colchicine 100ul adding preheating, mix rearmounted 37 DEG C of incubators 4 hours, blot nutrient solution, add the 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, Taq 1U, 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. be Closure of Ventricular Septal Defects cell, karyotype is diploid " 46, XX " or " 46, XY ", or is same as the primary cell 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 70th generation, be cultured to the 7th day time, rounded, fusiformis, to be paved with bottle at the bottom of, its Growth of Cells converges rate and reaches more than 80%, and after this along with the increase of passage number, Growth of Cells is slack-off, change is thin.

7, SV40LT antigen gene mediation Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects pathogenesis in vitro study, to accelerate the new way expanding Closure of Ventricular Septal Defects research, Closure of Ventricular Septal Defects cell is studied in vitro by physics from cell levels, chemistry, biological, heredity waits the transgenation of impact, genetic expression, changing function, physiological property, the mechanism such as biological conduction.

8, cell model application

1) Closure of Ventricular Septal Defects cell model is as scientific research cell

Closure of Ventricular Septal Defects cell model is made to 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 Closure of Ventricular Septal Defects cell model, normal control cells model is to the difference of the tolerance of nuisance, nuisance causes the mechanism of Closure of Ventricular Septal Defects.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 such as in situ hybridization (FISH), Northern blot, Real-time PCR, CHIP, EMSA research cell model 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; The protein function of technical study cell model in Long Term Passages and the interactions between protein such as application two-dimensional electrophoresis, MALDI-TOF Mass Spectrometric Identification, yeast two-hybrid and co-immunoprecipitation, from viable cell culturing process dynamically, the Closure of Ventricular Septal Defects cell that studies 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 Closure of Ventricular Septal Defects: make Closure of Ventricular Septal Defects 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, Closure of Ventricular Septal Defects cell model and normal cell of different incubation time, with, study bad environmental factor mechanism of action to Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects cell 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 Closure of Ventricular Septal Defects and intervention effect.

4. the method for same available congenital heart disease atrial septal defect cell model research gene therapy, substitutes some and directly does experiment with human body.

5. the genetic resources of Closure of Ventricular Septal Defects person is preserved with the form of cell model.

Claims (7)

1. the transduction SV40LT antigen gene for medical research field builds Closure of Ventricular Septal Defects cell model and cell bank thereof, its principal character is routinely with T4DNA ligase enzyme, BamHI, pcDNA3.1 (-) DNA and SV40LTagDNA 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 Closure of Ventricular Septal Defects 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 mediate Closure of Ventricular Septal Defects in vitro study cell model (alternative human body or animal straightway testing) as SV40LT antigen gene frozen in liquid nitrogen.

2. transduction SV40LT antigen gene according to claim 1 builds Closure of Ventricular Septal Defects cell model and cell bank thereof, it is characterized in that indication cell model be (1) cell be under inverted light microscope the epithelial cell sample adherent growth of fusiformis or little circular; (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) cell chromosome caryogram is diploid " 46, XX " or " 46, xY ", or is same as the initiating cell 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) passage to the 70th generation, be cultured to the 7th 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 80%, and after this along with the increase of passage number, Growth of Cells is slack-off; (8) in vitro from cell levels research environment factor cause Closure of Ventricular Septal Defects pathogenesis, study the tolerance of environment harmful and store as genetic resources.

3. transduction SV40LT antigen gene according to claim 1 builds Closure of Ventricular Septal Defects cell model and cell bank thereof, it is characterized in that taking from gathers because other experiments are required and Closure of Ventricular Septal Defects infant amniotic fluid cast-off cells that are unnecessary after other experiments, that discard build it.

4. transduction SV40LT antigen gene according to claim 1 builds Closure of Ventricular Septal Defects cell model and cell bank thereof, it 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. transduction SV40LT antigen gene according to claim 1 builds Closure of Ventricular Septal Defects cell model and cell bank thereof, it is characterized in that being used as Secondary Culture imports recon Closure of Ventricular Septal Defects 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 Closure of Ventricular Septal Defects 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. transduction SV40LT antigen gene according to claim 1 builds Closure of Ventricular Septal Defects cell model and cell bank thereof, it is characterized in that the Closure of Ventricular Septal Defects 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. transduction SV40LT antigen gene according to claim 1 builds Closure of Ventricular Septal Defects cell model and cell bank thereof, it is characterized in that the structure of cell bank comprises (1) screening and after qualification, meets immortalized cells characteristic and the SV40LT identical or close with the primary cell Closure of Ventricular Septal Defects of transduceing; (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) recyclability ground is long-term preserves SV40LT transduction Closure of Ventricular Septal Defects cell model, for making genetic resources and scientific research material.