CN104419727A - Construction of neural tube defect cell model and cell bank thereof under induction of hTERT recombinant - Google Patents

Abstract

The invention discloses construction of a neural tube defect cell model and a cell bank thereof under induction of an hTERT recombinant and relates to medical field. The construction is characterized in that incision enzymes EcoRI and XhoI are used for carrying out double-enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo, and Ligation Mix is used for connecting hTERT and pLXSNneo enzyme-digested products subjected to PCR amplification and gel electrophoresis separation to construct a pLXSNneo-hTERT recombinant; DH5a competent cells are transformed to purify, amplify and extract plasmids; the neural tube defect cells in logarithmic growth during in-vitro passage are subjected to liposome transfection so that the recombinant and the cell DNA are integrated and subjected to enlarged cultivation; clones containing positive recombinants are subjected to G418 screening; the cells, of which cellular morphology, growth curves, karyotype, nude mouse tumorigenesis tests, cell telomerase activity, hTERT mRNA expression, immunohistochemistry, and cell generation cycles and apoptosis rate meet immortal cell characteristics, and which are the same or similar to primary cells, are screened to be used as the neural tube defect cell model constructed under induction of hTERT to be frozen in liquid nitrogen. Therefore, the foundation is laid for long-time in-vitro researches on pathogenesis of related diseases from cellular level.

Description

The induction of hTERT recon builds neural tube defect cell model and cell bank thereof

Technical field

The present invention relates to hTERT (reverse transcriptase of telomere catalytic subunit) recon induction (mediation) and build neural tube defect cell model and cell bank thereof, be mainly used in human reproduction's health research field, the in vitro study for neural tube defect provides cell model and preserves its Scientific Research Resource.

Background technology

Neural tube defect has another name called neural tube defects, is a kind of common and serious birth defect.Neurocele is the central nervous system of fetus, start in the 15-17 day of embryo, neural system germinates, and to embryo about 22 days, the both sides of neural fold started to draw close mutually, form 1 pipeline, be called neurocele, its front end is called the front hole of neurocele, and tail end is called neurocele metapore, embryo was when 24-25 day and 26 days, and front hole and metapore are closed in succession.Fetal neurotubules malformation main manifestations is anencephalus, Naoning tablet, meningocele, hemirachischisis, harelip and cleft palate, spinal bifida aperta, closed spina bifida etc.In 2,300 ten thousand newborn infants that China is born every year, have nearly 400,000 to suffer from various congenital disorders, wherein the incidence of neural tube defects is 0.2%-0.4%.

There are some researches show, human brain is grown and is formed on neurocele, and as risen to 4 months in pregnant 3-4 week, pregnant woman lacks folic acid, just may cause fetus appearance neural system congenital abnormality in various degree.The expression of numerous species gene or sudden change and nervous system development, neural tube defects are relevant, and these genes are transcription factors, comprise 1. Regulatory genes and transcription factor genoid.2. proto-oncogene and cancer suppressor gene.3. somatomedin and acceptor gene thereof.4. protein kinase C genes involved.5. homocysteine metabolism genes involved.6. other genes: cytoskeleton class, cell connect genoid etc.The important factor that neural tube defects occurs lacks folic acid in pregnancy duration pregnant woman body, and therefore pregnant woman is in pregnant first 1 month to pregnant latter 4 months, and every day, oral 0.4 milligram, 1 folic acid, just can make Fetal neurotubules malformation incidence reduce by 70%.However, the pathogenesis of neural tube defects, how to prevent better, intervene or treat, need further to study.

But, due to do not have can in vitro long-term cultivation, the immortality cell model that can be used for neural tube defects 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 neural tube defect cell affects by physics, chemistry, biology, heredity etc. in vitro, thus more seriously limit the further research of neural tube 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.

But studies have found that recently, the cell of SV40Tag transfection is along with the loss of DNA damage repair mechanism, the unstable of caryogram and a few cell neoplastic transformation.In addition; long-term in vitro is cultivated and is found that the cell of part transfection SV40Tag just extends the life-span; or only tided over senescence phase (MI phase), most cell finally can enter climacteric (M2 phase) and dead, and cell being not immortalized is described.Simultaneously SV40 virus has tumorigenicity, and with the generation of some tumour, develop relevant, it is restricted that the cell of therefore SV40Tag transfection also exists to a certain degree to some research.

Foreign study shows, imports exogenous hTERT and cell can be made to keep normal phenotype and differentiating characteristic.Utilized hTERT successfully to establish the immortalized cell line of some cell in recent years, substantially kept that chromosome stabilityX, differentiation are normal, contact inhibition, growth characteristics without relative " normally " such as tumorigenicity.Can be used for the foundation of some cell model of human and animal, to the characteristic studying initiating cell by means of research transgenosis immortalized cells model, thus study its pathogenesis, have great importance.

In stomatology field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establishes immortal human Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number reaches more than 150 times, cell all shows original biological characteristics, can express the associated protein of derived cell after inducing culture.The transfection hTERT such as Kitagawa establish people cementoblast system, and cell multiplication reaches more than 200 times, and cytodifferentiation mark such as alkaline phosphatase, type i collagen etc. are expressed stable.Because of the needs of research work, almost often kind of disease has 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 neural tube defect and cell bank thereof from cell levels about building with hTERT, also cannot carry out the research of relevant item.In order to solve the problem, present inventors have proposed the present invention.

Summary of the invention

The object of the invention is to provide hTERT (reverse transcriptase of telomere catalytic subunit) recon to induce (mediation) to build the method for neural tube defect cell model and cell bank thereof, another object is for providing hTERT to mediate neural tube defect cell model and cell bank thereof from the pathogenesis of cell levels research neural tube defect in vitro.

The object of the present invention is achieved like this: with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo-hTERT and carrier pLXSNneo, connect through pcr amplification with Ligation Mix, hTERT and the pLXSNneo digestion products that gel electrophoresis is separated, build pLXSNneo-hTERT recon, transform DH5a competent cell with amplification, purifying the picking bacterium colony of resistance to penbritin extracting plasmid, the neural tube defect cell gone down to posterity in logarithmic growth is imported in vitro with lipofection, the DNA of recon and cell is integrated, and the clone of positive recombinant that enlarged culturing is screened through G418, screening cellular form, growth curve, karyotype, nude mice tumorigenesis is tested, transfectional cell telomerase activation, hTERT mrna expression product, immunohistochemical staining, cell generation cycle and apoptosis rate meet immortalized cells characteristic and identical with primary cell or close person to mediate neural tube defect in vitro study cell model as hTERT frozen in liquid nitrogen, for the pathogenesis of the neural tube defect that studies for a long period of time in vitro from cell levels lays the foundation.

The present invention is with pcr amplification, agarose gel electrophoresis purification hTERT, import neural tube defect cell through genophore pLXSNneo and lipofection and build 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 hTERT 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; In addition; compared with SV40; building cell model with hTERT more can make cell keep normal phenotype and differentiating characteristic; more can keep that chromosome stabilityX, differentiation are normal, contact inhibition, without tumorigenicity and tide over the growth characteristics such as M2 climacteric, to the pathomechanism studying neural tube defect from cell levels in vitro, there is larger meaning.

Accompanying drawing explanation

Fig. 1 is neural tube defect cell model (adherent growth) figure prepared by the present invention.

As Fig. 1, passage to the 89th generation, be cultured to the 13rd 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 81%, and after this along with the increase of passage number, Growth of Cells is slack-off, thinning to be dredged.

Embodiment

1, the extraction of hTERT: 1. enzyme cuts pClneo-hTERT:hTERT between EcoRI and the SalI site of plasmid pClneo-hTERT, and pLXSNneo vector multiple cloning site (MCS) is containing EcoRI and XhoI restriction enzyme site.Commercially available purchase pCIneo-hTERT plasmid, is dissolved in appropriate ultra-clean H ₂in O or TE damping fluid, add 2uL10 × enzyme cutting buffering liquid and 18uL H ₂o, adds restriction enzyme EcoR I and each 0.5ul of Xho I, 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, adds 5uL electrophoresis sample loading buffer (also by adding 0.5mol/L EDTA) termination reaction, routinely after PCR method amplification hTERT, collect amplified material in order to electrophoresis.2. hTERT 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 the about 1mm of gel surface, prepare hTERT enzyme with 10 appropriate × sample loading buffer and cut sample, then with pipettor, sample is added in sample well, and do suitable standard control thing simultaneously, connect electrode, hTERT anode is moved, then under the voltage of 1-10V/cm (80V) gel electrophoresis to (30min) during the distance of enough separation hTERT fragments, powered-down.3. hTERT purifying and recovery: be separated hTERT band from agarose: under long wave ultraviolet light source, the gel-tape containing target hTERT fragment is cut and load in dialysis tubing, 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 hTERT 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 hTERT, 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 hTERT precipitation, abandon supernatant, dry ethanol is abandoned after adding 70% washing with alcohol 2 times, add 50 μ l TE and dissolve hTERT.In addition, also object hTERT fragment is separated, is purified by available low melting-point agarose gel method, hTERT filter membrane inserted sheet method etc. from gel.

2, the connection of hTERT and pLXSNneo carrier: get the hTERT composition (0.1-5 μ g) of the above-mentioned purifying of 9 μ l, 1 μ l10mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15 DEG C of incubation 24h, build pLXSNneo-hTERT recon.

3, the purifying of pLXSNneo-hTERT recon, amplification, qualification: 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 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, and now, rapidly cell can be distributed into aliquot, freezing in liquid nitrogen ,-70 DEG C of storages are for subsequent use.2. with competence intestinal bacteria purifying, amplification pLXSNneo-hTERT recon: get 200 μ l with the sterile pipette tip cooled from every kind of competent cell suspension and transfer in aseptic Eppendorf tube, often pipe adds DNA or ligation mixture (volume≤10 μ l, 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 (every 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.3. screen, increase recon: 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).4. the qualification of recombinant plasmid and amplification: the single bacterium colony on picking plate, be inoculated in 3ml containing in 100ug/ml penbritin LB substratum, 37 DEG C, cultivate in 250r/min shaking table, culture is collected after 14h, 4 DEG C, the centrifugal 5min of 10000r/min, extract and purification of Recombinant plasmid in a small amount by test kit specification sheets; With EcoRI and HindIII double digestion recombinant plasmid reaction system: each 0.5ul of restriction enzyme, 10 × buffer2ul, recombinant plasmid 10ul, adding water complements to 20ul, 37 DEG C of enzymes cut 1h.Digestion products carries out 0.8% agarose electrophoresis under 80V voltage conditions, time 30min, and gel imaging system is taken pictures; Measure the sequence of recombinant plasmid routinely; Recombinant plasmid is cut through enzyme, after qualification accurately of checking order, by the microbionation containing this plasmid in LB nutrient solution, amplification cultivation, carries out heavy dose of plasmid extraction purifying by heavy dose of plasmid extraction test kit specification sheets, and ultraviolet spectrophotometer is for subsequent use after measuring plasmid concentration and purity.

4, pLXSNneo-hTERT recon imports the screening of neural tube defect cell and positive colony thereof: the 1. collection of neural tube defect cell and cultivation: be extracted in aseptic technique and do other and test or other check unnecessary, the neural tube defect 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 collagenase II liquid or the pancreatin (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 pLXSNneo-hTERT recon: 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, transfection recon pLXSNneo-hTERT, in 1.5ml Eppendorf tube, prepare following solutions: pipe A, pLXSNneo-hTERT recon 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-pLXSNneo-hTERT 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.Method 2: draw 1/10-1/40 cell suspension, requires that 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 just entering logarithmic phase, adopt liposome transfection method, by recon pLXSNneo-hTERT transfection in neural tube defect 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 reach about 90% merge time for transfection, by the recon pLXSNneo-hTERT 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, cell DMEM in 24 orifice plates is washed 3 times, every hole adds nutrient solution 100 μ l, adds Lipofectamine-pLXSNneo-hTERT 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.Have monoclonal cell colony to grow after about 10d, picking is also placed in continuation cultivation in 24 orifice plates, maintains and stabilize with the G418 of 300mg/L the amplification cultivation that goes down to posterity.Setting up with this can the immortalised neural defective tube cell model of Secondary Culture repeatedly in vitro.

5, the going down to posterity of pLXSNneo-hTERT transfection neural tube defect cell model, increase: collect the above-mentioned positive monoclonal cell colony importing pLXSNneo-hTERT 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, grow to cell attachment, converge rate and reach 80 ~ 85%, collecting cell when being in the early stage of logarithmic phase, again by above-mentioned steps Secondary Culture, inoculation of repeatedly going down to posterity like this, cultivate, record algebraically and observation of cell growth characteristic, as Fig. 1, passage is to the 89th generation, when being cultured to the 13rd day, in logarithmic growth, circular, fusiformis, at the bottom of being paved with bottle, its Growth of Cells converges rate and reaches more than 81%, after this along with the increase of passage number, Growth of Cells is slack-off, thinning 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 neural tube defect cell model biological characteristics: after using pLXSNneo-hTERT to set up cell model (clone), the key issue of qualification has: is that this cell of requirement has lasting multiplication capacity; 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.This clone goes down to posterity the cellular form of amplification cultivation and Normal human fibroblast's form no significant difference; 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 ", 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 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 cell 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 immortalized cells of this experiment can not form clone in soft agar, meets immortalization feature; 5. nude mice tumorigenesis test: by immortalized cells 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. transfectional cell hTERT mrna expression product measures: do pcr amplification rear electrophoresis by test kit, detect hTERT mRNA band.As with Immunohistochemical detection, in the nucleus of hTERT transfection, the visible a large amount of brown particle of dyeing, shows that hTERT has been integrated in cell; 7. transfectional cell telomerase activation: collecting cell system, illustrates by test kit and prepares lysate, does pcr amplification and electrophoretic analysis, with 302nm UV-light observations, visible in Telomerase positive band.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 hTERT integrates, expresses; In addition with Flow cytometry cell generation cycle and apoptosis rate, the Proliferating antigen Ki67 of this clone comparatively before and after transfection without obviously changing, Transfected cells apoptosis rate comparatively obviously reduces before transfection.

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 neural tube defect 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 hTERT gene in the DNA of 6. cell, express the mRNA product of hTERT gene; 7. passage to the 89th generation, be cultured to the 13rd 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 81%, and after this along with the increase of passage number, Growth of Cells is slack-off, thinning to be dredged; 8. for preserving genetic resources, and study from cell levels the mechanism such as transgenation, genetic expression, changing function, physiological property, biology conduction that neural tube defect cell affects by physics, chemistry, biology, heredity etc. in vitro.

7, hTERT mediates neural tube defect 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 packing, mark, 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 immortalised neural defective tube 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 neural tube defect pathogenesis in vitro study, to accelerate the new way expanding neural tube defect research, study from cell levels the mechanism such as transgenation, genetic expression, changing function, physiological property, biology conduction that neural tube defect cell affects by physics, chemistry, biology, heredity etc. in vitro.

8, cell model is applied: neural tube defect cell model is as scientific research cell: make neural tube defect 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 neural tube defect cell model, normal control cells model is to the difference of the tolerance of nuisance, nuisance causes the mechanism of neural tube defect.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, 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, neural tube defect 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 neural tube defect: make neural tube defect 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, neural tube defect cell model and normal cell of different incubation time, with, study bad environmental factor mechanism of action to neural tube defect 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 neural tube defect 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 neural tube defect and intervention effect.4. the method for same available neural tube defect cell model research gene therapy, substitutes some and directly does experiment with human body.5. the genetic resources of neural tube defect person is preserved with the form of cell model.

Claims (7)

1. the method for the induction of the hTERT recon for a medical field structure neural tube defect cell model and cell bank thereof, its principal character is with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo-hTERT and carrier pLXSNneo, connect through pcr amplification with Ligation Mix, hTERT and the pLXSNneo digestion products that gel electrophoresis is separated, build pLXSNneo-hTERT recon, transform DH5a competent cell with purifying amplification also extracting plasmid, to go down to posterity in vitro the neural tube defect cell in logarithmic growth with liposome transfection, recon and cell DNA are integrated, and enlarged culturing is cloned containing positive recombinant through G418 screening, screening cellular form, growth curve, karyotype, nude mice tumorigenesis is tested, Cell Telomerase Activity, hTERT mrna expression, immunohistochemical methods, cell generation cycle and apoptosis rate thereof meet immortality cell characteristic and identical with primary cell or close person to mediate neural tube defect in vitro study cell model as hTERT frozen in liquid nitrogen, for laying the foundation from cell levels (substituting human body or animal straightway testing) its pathogenesis that studies for a long period of time in vitro.

2. hTERT recon according to claim 1 induction builds the method for neural tube defect 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) be neural tube defect 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; (6) incorporate hTERT gene in the DNA of cell, express the mRNA product of hTERT gene; (7) passage to the 89th generation, be cultured to the 13rd 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 81%, and after this along with the increase of passage number, Growth of Cells is slack-off, thinning to be dredged; (8) for preserving genetic resources, and study from cell levels the mechanism such as transgenation, genetic expression, changing function, physiological property, biology conduction that neural tube defect cell affects by physics, chemistry, biology, heredity etc. in vitro.

3. hTERT recon according to claim 1 induction builds the method for neural tube defect cell model and cell bank thereof, it is characterized in that taking from gathers because other experiments are required and neural tube defect infant amniotic fluid cast-off cells that are unnecessary after other experiments, that discard build it.

4. hTERT recon induction according to claim 1 builds the method for neural tube defect 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. hTERT recon induction according to claim 1 builds the method for neural tube defect cell model and cell bank thereof, it is characterized in that being used as Secondary Culture imports recon neural tube defect 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 neural tube defect 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. hTERT recon induction according to claim 1 builds the method for neural tube defect cell model and cell bank thereof, it is characterized in that the neural tube defect 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. hTERT recon according to claim 1 induction builds the method for neural tube defect 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 hTERT recon identical or close with the primary cell neural tube defect cell of inducing; (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 neural tube defect cell model preserving the induction of hTERT recon in recyclability ground, for making genetic resources and scientific research material.