CN105925533A - Anti-tumor-migration drug screening cell model A549-GFP constructed by adopting lentivirus technology and application method of anti-tumor-migration drug screening cell model A549-GFP - Google Patents
Anti-tumor-migration drug screening cell model A549-GFP constructed by adopting lentivirus technology and application method of anti-tumor-migration drug screening cell model A549-GFP Download PDFInfo
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Abstract
The invention relates to a preparation method and applications of a mammal tumor cell line A549-GFP capable of expressing green fluorescent protein. Compared with a parental cell line, the cell line provided by the invention has a similar growth curve, in a cell migration experiment, the cell migration effect caused by HGF is obvious, a micromolecular specific inhibitor has the obvious inhibiting effect for the cell migration effect caused by the HGF, and the abovementioned biological activity is consistent with that of the parental cell line. According to the prepared cell model, the GFP (green fluorescent protein) genes are inserted into the genome of the mammal tumor cell line through a lentivirus infection manner, and then the cell line steadily expressing the GFP is obtained; for the cell model, in the cell migration experiment, the experimental result can be displayed through a direct fluorescence microscope observation manner, and therefore, the steps of the application of the cell model to the high-throughout drug screening are simplified.
Description
Present disclosure relates to drug screening field, and the A549 being specifically related to a kind of expressing green fluorescent protein is thin
Born of the same parents are.
Background technology tumor has become the number one killer threatening human health at present, the research and development of antitumor drug and tumor
Clinical treatment become the focus of scientific research, hence set up effective antitumour medicaments sifting model very urgent, tumor itself is special
Some animal migrations and aggressive are the important mechanisms of tumor evasion immune response, therefore tumor migration inhibition and aggressive
Medicine enjoy people to pay close attention to.
High throughput screening drug is new technical system multiple technologies method being organically combined and being formed, and it is to divide
Based on the experimental technique of sub-level and cellular level, using microplate format as experimental tool carrier, with automation operating system
Perform experimentation, gather experimental data with sensitive quick detecting instrument, carry out point with the data that experiment is obtained by computer
Analysis processes.Normally carrying out of it needs the compound library of a high power capacity, the operating system of automatization, highly sensitive detection
System, high efficiency data handling system and the medicaments sifting model of high specific.
The gene of green fluorescent protein (Green Fluorescent Protein, be called for short GFP) be fromAequorea victoriaThe genome of Jellyfish extracts acquisition, full length protein 238AA, its 65-67 amino acid residue (serine cheese ammonia
Acid glycine) can be spontaneous formation fluorescent chromophore, under the light of blue wavelength region excites, can send green glimmering
Light, the peak-peak of absorption spectrum is 395nm, and emission spectrum peak-peak is 509nm.Nowadays GFP is as reporter gene quilt
It is widely used in the every field such as immunology, neurobiology, hereditism, organ transplantation.And the basic research being correlated with in tumor
And Field of Drug Discovery, GFP has also played great effect.
Building field at eukaryotic cell transfection and stable cell line at present, viral vector the most increasingly comes into one's own.At present
Conventional viral vector includes adenovirus vector, gland relevant viral vector, herpesvirus vector and retroviral vector etc., its
The stability that slow virus carrier in middle retrovirus is expressed because of it, bale capacity is big and is capable of polygenes coinfection
It is widely used in scientific research etc. advantage.Genes of interest can be integrated into the genome of cell by slow virus infection, so need not
It is further cultured in base adding screening pressure and just can stably express destination protein, enormously simplify experimentation, reduce cost.
Currently used slow virus packaging system is by the genome manipulation of inhibition of HIV.Most popular it is
Third generation slow virus, is a kind of three pUC pUCs, can be used for infecting the cell of nondividing phase.Uncorrelated base is eliminated by transformation
Because of sequence, and the portion gene of allos is used to replace homologous genes (such as: envelope-coding plasmid) to increase its safety
Property, the sequences such as promoter are carried out modify transformation simultaneously and improves virus titer and transfection efficiency.Obtain finally by transformation
Self inactivation type slow virus carrier (self-inactivation, SIN) has higher titre, transfection efficiency and safety.
And after virus acquisition in addition to being used for the infection of cell, can be used for gene therapy.
Pulmonary carcinoma betides bronchial mucosa epithelium, also known as bronchogenic carcinoma.The sickness rate of pulmonary carcinoma substantially increases in recent years, man
In property carcinoma patient, pulmonary carcinoma has ranked first, increases the most rapidly at women sickness rate, account for the 2nd of women common cancer or
3rd.A549 cell is the conventional cell line of research people's pulmonary carcinoma.The present invention uses slow virus system to pack green fluorescent protein
(GFP) gene, by slow virus infection A549 cell, it is thus achieved that stably express the A549 cell strain of GFP.The foundation of this cell strain
Enormously simplify the detection means of external drug screening, in terms of can apply to the medicine curative effect to animal tumor model simultaneously
Research, it is achieved tumor size and monitoring and the data collection in real time of distribution.
Summary of the invention the technical problem to be solved is to provide a kind of energy high flux screening anti-cell migration medicine
The cell model of thing.
The technical scheme solving the technology of the present invention problem is: a kind of cell model screening anti-cell migration medicine, is logical
Cross slow virus to be transferred to by green fluorescence protein gene in the genome of A549 cell line, it is thus achieved that stable expression green fluorescence egg
White A549 cell strain.
Wherein, the GFP gene in above-mentioned cell model can stably be expressed；
Wherein, the GFP sequence in above-mentioned cell model is SEQ ID NO:1.
Present invention also offers a kind of method preparing above-mentioned screening anti-cell migration drug cell model, the method includes
Following steps:
GFP gene is cloned in prrl plasmid, is built into prrl-CMV-GFP recombiant plasmid, this plasmid and slow virus are packed
The 293T cell of plasmid co-transfection logarithmic (log) phase, gathers in the crops purified virus, and virus infects logarithmic (log) phase A549 cell, through limiting dilution assay and
Fluorescent screening, it is thus achieved that cell model of the present invention.
Present invention also offers the screening technique of a kind of anti-cell migration medicine, the method comprises the following steps:
A. the above-mentioned upper room being inoculated in transwell cell for screening the A549 cell model of anti-cell migration medicine is taken,
The upper room of screening group adds the maintenance culture medium containing thing to be screened, and lower room adds the maintenance culture medium containing HGF；In positive controls
Room is for maintaining culture medium, and lower room adds the maintenance culture medium containing HGF；In negative control group, room and lower room are maintenance culture medium；
B. cultivate each group of cell, count each group of migrating cell quantity, whether determine thing to be screened according to the number of cell migration number
For need potential drug,
Further, the cell migration number of above-mentioned anti-cell migration drug screening method is the cell migrating to lower room from upper room
Quantity.
According to the first aspect of the invention, on the basis of carefully analyzing different inoculation technique, select slow virus packaging
System prepares the A549 cell strain of stable expressing green fluorescent protein.Containing being incorporated on genome in cell model of the present invention
The coded sequence of GFP gene, and this sequence can stably express.This cell model is directly carried out by detection fluorescence signal
Cell counting, simplifies step and the flow process of Cell migration assay, establishes base for realizing anti-cell migration medicament high flux screening
Plinth.
According to the second aspect of the invention, the side of the cell model preparing above-mentioned screening anti-cell migration medicine is established
Method.The method specifically can be implemented by following steps:
A549 cell is cultivated to exponential phase；
The slow virus that will be loaded with GFP gene infects A549 cell under the mediation of polybrene；
Screen through limiting dilution assay and fluorescence microscope and obtain this cell model.
According to the third aspect of the present invention, it is established that anti-cell migration medicine based on cell model of the present invention
Screening technique, step is as follows:
A. taking the above-mentioned cell model for screening and be inoculated in the little indoor of transwell, cell inoculation number is 5 × 104/
Hole.It is grouped into screening group, positive controls and negative control group.The upper room of wherein screening group adds the maintenance training containing thing to be screened
Supporting base, lower room adds the maintenance culture medium containing HGF；In positive controls, room is for maintaining culture medium, and lower room adds the maintenance containing HGF
Culture medium；In negative control group, room and lower room are maintenance culture medium；
B. cell incubation 24h, after cell is fixing, aseptic cotton carrier is wiped and is counted little chamber lower surface 4 under ventricular cell, fluorescence microscope
According to migrating cell quantity, the migrating cell sum in individual field of excursion region, determines whether thing to be screened is the potential medicine needed
Thing；
Wherein, the consumption of the HGF added in said method step a is 12.5ng/ml-200ng/ml, preferably 100ng/ml.
The regular growth related in the present invention is cultivated and the test operation of conventional molecular biological, is all the common skill in this area
Art personnel can complete with reference to the operation instruction of existing correlation test guide or instrument, reagent.
The beneficial effects of the present invention is: cell model of the present invention can directly observe migrating cell by fluorescence microscope
Number, it is possible to high flux, high-sensitive accurate screening anti-cell migration medicine；The preparation method of cell model of the present invention simply may be used
Control, with low cost；Anti-cell migration drug screening method step of the present invention is simple, with low cost, and accuracy is high, can realize high pass
The screening of amount, has good stability and reliability.Screening for anti-cell migration medicine provides new thinking, has very
Good market prospect.
Accompanying drawing explanation
The restriction enzyme digestion and electrophoresis figure of Fig. 1: prrl-CMV-GFP recombiant plasmid.
Fig. 2: A549-GFP cell line stability analysis.Wherein in 1st generation and the 30th generation, refer to 1st generation and the 30th generation respectively
A549-GFP cell line, light field refers to that cell photo under light field, fluorescence field refer to fluorescence cell photo after the match.
Fig. 3: A549-GFP cell line is with wild type A549 cell line growth curve ratio relatively.Wherein abscissa is cell growth
Time (incubation time), vertical coordinate is the photometric absorbance value (wavelength 450nm) measured under the conditions of 450nm Single wavelength.
Fig. 4: A549-GFP cell line and wild type A549 cell line cell migration result resolution under HGF effect；
A is HGF effect lower A549-GFP cell line migration photo under the conditions of light field；
B is that under fluorescence field condition, the lower A549-GFP cell line of HGF effect migrates photo, A from B be the different light of the same area after the match according to
Sheet；
C is to migrate photo without the lower A549-GFP cell line of HGF effect under fluorescence field condition；
D is wild type A549 cell line cell migration photo (crystallized purple dyeing) under HGF effect under light field；
B with A compares, and cellular resolution is significantly raised, B with D compares, and cellular resolution is not significantly different from, it was demonstrated that A549-GFP
Cell line i.e. can reach the cellular resolution after the crystallized purple dyeing of wild type A549 cell line in the case of without dyeing.
Fig. 5: A549-GFP cell line and wild type A549 cell line cell migration Benefit Transfer under HGF effect.Wherein
Abscissa is different cell line titles, and vertical coordinate is cell migration rate (mobility).A549-GFP cell line and wild type A549
Cell line compares, and under HGF effect, cell migration effect is more significantly, it was demonstrated that A549-GFP cell line has good biology
Activity, compares as without HGF matched group.
Fig. 6: A549-GFP cell line cell migration effect under variable concentrations HGF effect and cytoactive；
A is A549-GFP cell line cell migration quantity under variable concentrations HGF effect, and wherein abscissa is HGF concentration
(HGF:ng/ml), vertical coordinate is migrating cell quantity (cell quantity)；
B is A549-GFP cell line cytoactive under variable concentrations HGF effect, and wherein abscissa is HGF concentration (HGF:ng/
Ml), the photometric absorbance value (wavelength 450nm) measured under the conditions of vertical coordinate is 450nm Single wavelength.
Fig. 7: A549-GFP cell line cell migration effect under variable concentrations PF04217903 effect and cytoactive；
A is A549-GFP cell line cell migration quantity under variable concentrations PF04217903 effect, and wherein abscissa is
PF04217903 concentration (PF04217903: μM), vertical coordinate is migrating cell quantity (cell quantity), compares as without HGF,
Without PF04217903 matched group, other respectively organize the HGF all containing 100ng/ml；
B is A549-GFP cell line cytoactive under variable concentrations PF04217903 effect, and wherein abscissa is
PF04217903 concentration (PF04217903: μM), vertical coordinate is the photometric absorbance value (wavelength measured under the conditions of 450nm Single wavelength
450nm).
Fig. 8: variable concentrations DMSO cell migration effect and the impact of cytoactive；
A is the impact of variable concentrations DMSO cell migration quantity, and wherein abscissa is variable concentrations (volume ratio) DMSO
(DMSO: concentration), vertical coordinate is migrating cell quantity (cell quantity), compares as containing HGF(100ng/ml), without DMSO pair
According to group；
B is the variable concentrations DMSO impact on cytoactive, and wherein abscissa is variable concentrations DMSO(DMSO: concentration), vertical seat
The photometric absorbance value (wavelength 450nm) measured under the conditions of being designated as 450nm Single wavelength.
Detailed description of the invention
Medicine and reagent: various restriction endonuclease, T4 DNA ligase are purchased from NEB company, glue reclaims reagent
Box, mini-scale plasmid extraction agent box are purchased from MN company；Transfection reagent M40 is that laboratory is independently prepared；DMEM culture medium, antibiosis
Element, pancreatin, D-PBS is purchased from hyclone company, and hyclone is purchased from GIBCO company；HGF, PF04217903 are purchased from RD company,
CCK-8 test kit is purchased from Dojindo company；Other reagent are import and domestic analytical reagent.
Instrument: inverted fluorescence microscope Ti, NIKON.
The efficient express cell model of the present invention can stablize expressing green fluorescent protein, therefore can under fluorescence microscope directly
Observe.
The selection of case study on implementation one green fluorescent protein (GFP) genophore
The gene of green fluorescent protein (Green Fluorescent Protein, be called for short GFP) be fromAequorea victoriaThe genome of Jellyfish extracts acquisition, full length protein 238AA, its 65-67 amino acid residue (serine cheese ammonia
Acid glycine) can be spontaneous formation fluorescent chromophore, under the light of blue wavelength region excites, can send green glimmering
Light, the peak-peak of absorption spectrum is 395nm, and emission spectrum peak-peak is 509nm.Nowadays GFP is as reporter gene quilt
It is widely used in the every field such as immunology, neurobiology, hereditism, organ transplantation.And the basic research being correlated with in tumor
And Field of Drug Discovery, GFP has also played great effect.
The present invention use Lentiviral prrl-CMV use existing as the expression vector of GFP, preparation method
Recognized technology, wherein the CDNA sequence of GFP is shown in SEQ ID NO.1.
Case study on implementation two loads packaging and the purification of the slow virus of GFP gene
In eukaryotic cell transfection and field of gene, viral vector the most increasingly comes into one's own.The most conventional viral vector
Including adenovirus vector, gland relevant viral vector, herpesvirus vector and retroviral vector etc., wherein in retrovirus
The stability expressed because of it of slow virus carrier, bale capacity is big and is capable of the advantages such as polygenes coinfection and is extensively made
In scientific research.Genes of interest can be integrated into the genome of cell by slow virus infection, so need not be further cultured in base adding
Screening pressure just can stably express destination protein, enormously simplify experimentation, reduces cost.Therefore the present invention uses slow disease
Poison packaging, the method infected obtain final stable transfected cells strain.
Currently used slow virus packaging system is by the genome manipulation of inhibition of HIV.Most popular it is
Third generation slow virus, is a kind of three pUC pUCs, can be used for infecting the cell of nondividing phase.Uncorrelated base is eliminated by transformation
Because of sequence, and the portion gene of allos is used to replace homologous genes (such as: envelope-coding plasmid) to increase its safety
Property, the sequences such as promoter are carried out modify transformation simultaneously and improves virus titer and transfection efficiency.Obtain finally by transformation
Self inactivation type slow virus carrier (self-inactivation, SIN) has higher titre, transfection efficiency and safety.
And after virus acquisition in addition to being used for the infection of cell, can be used for gene therapy.
The present invention is the packaging that recipient cell carries out slow virus with people's renal epithelial cell (293T),
Use prrl-CMV-GFP for restructuring packaging plasmid.
The packaging plasmid used is pMD2.G, pMD-PRRE and pRSV-REV.
Process of the test is as follows:
(1) cell is cultivated
With the complete DMEM culture medium inoculated cell containing antibiotic and 10% serum in 35mm Tissue Culture Dish, cell concentration is 6 ×
105/ hole.After cell is the most adherent, transfect with transfection reagent M40.
(2) transfection process
DNA and the preparation of transfection reagent mixtures
Solution A:
prrl-CMV-GFP 1.7μg
Packaging plasmid mixture 1.8 μ g
Serum-free antibiotic-free DMEM culture media supplemented is to 100 μ l, concussion mixing；
B solution:
Transfection reagent 16 μ l
Serum-free antibiotic-free DMEM culture media supplemented, to 100 μ l, softly mixes；
Softly being mixed with B liquid by A liquid, room temperature stands 10min, is joined in cell by mixed liquor.37 DEG C, 5% CO2In incubator
Cultivate, be changed to contain containing serum the DMEM culture medium of antibiotic next day.
(3) collection of slow virus and purification
Collect transfection after the 4th day and the cell conditioned medium of the 5th day, by the 4th day and the 5th day collection virus liquid 4000g, room temperature from
Heart 10min；Virus liquid after Li Xin proceeds to (ufc910024, MILLIPORE) in super filter tube after 0.45 μm membrane filtration, often
Temperature, 4000g, centrifugal 15min；Collection viral concentration liquid, subpackage, frozen in-80 degree.
The foundation of case study on implementation three cell model of the present invention
Using human liver cancer cell (A549) in this experiment is recipient cell.
The slow virus used is the slow virus loading GFP gene.
Process of the test is as follows:
(1) cell is cultivated
With the complete DMEM culture medium inoculated cell containing antibiotic and 10% serum in 35mm Tissue Culture Dish, cell concentration is 6 ×
105/ hole.After cell is the most adherent, transfect with transfection reagent M40.
(2) course of infection
After slow virus is diluted to suitable multiple by complete DMEM culture medium (containing 6 μ g/ml polybrene), join supernatant discarded
A549 cell surface；After slow virus infection 24h, use the complete DMEM culture medium containing antibiotic and 10% serum instead and continue to cultivate.
(3) Cell-cloned
Take feeder cells.Experiment mice dislocation is lethal, 75% alcohol-pickled 10min, aseptic dissection, and abdominal cut skin does not damage
Hinder peritoneum, draw 6-8ml complete medium with syringe, pick up peritoneum, inserting needle with tweezers, be careful not to stamp to internal organs and fat
Fat, the fat of lower abdominal in the middle of abdominal part, can be avoided in inserting needle orientation；
Piping and druming abdominal cavity is for several times repeatedly, and period gently rubs mouse web portion with cotton ball soaked in alcohol, culture medium is inhaled when sucking-off culture medium turns yellow
Go out, standby in transferring to centrifuge tube.(peritoneal macrophage of an adult mice can meet 6-8 block 96 porocyte culture plate
Bed board need)；
Taking A549 cell after infection, digestion, counting, every 20ml culture medium (feeder cells containing respective concentration) adds 150 carefully
Born of the same parents, point 96 orifice plates, 200 μ l/ holes；
Cell has seen whether pollution after cultivating 1 day, counts cell subclone number, and keep a record after cultivating 3-5 days.Cell is trained
Support 10 days, select monoclonal cell hole to observe under fluorescence microscope, select the cell clone of fluoresced green to disappear
Change and amplification culture.
The screening of case study on implementation four positive colony cell and cell migration Establishing
Due in exogenous gene random integration to the chromosome of host cell, according to the randomness of integration site, it is understood that there may be place
Chief cell state difference, to problems such as the signaling molecule weak and destination gene expression poor stabilities of reaction.Therefore by all sun chosen
After sex clone cell expansion is cultivated, carry out cell growth curve contrast test and GFP expression stability analysis.
Test example one GFP expression stability is analyzed
It is total to screening in this experiment and obtains 4 positive colonies.These 4 positive colonies are carried out continuous passage cultivation, passes continuously
Culture testing program: when cell confluency degree reaches 80%, discard cell conditioned medium, washs one time with D-PBS, adds appropriate pancreatin
Digest, observe under inverted microscope, when most cells becomes round, use the complete DMEM having serum to have antibiotic
Culture medium terminates digestion, blows down cell, and 1000rpm is centrifuged 10 minutes, stays the cell of 1/4 to continue to cultivate in former cultivating system,
Aforesaid operations is repeated when cell confluency degree reaches 80%.
Often pass on 5 times, the ratio of fluorescence microscopy Microscopic observation fluorecyte and the fluorescence intensity of cell, Taking Pictures recording.Knot
The most as shown in Figure 2: A549-GFP cell line its fluorescence rate when 30 generation remains in that 100%, it was demonstrated that A549-GFP cell cording
There is good stability.
Test example two cell model of the present invention compares with wild-type cell growth curve
1 strain that cell growth state is best is selected, to this strain cell and wild type from the 4 strain cells that top experiment sieving obtains
A549 cell is enlarged cultivating, and carries out growth curve analysis when cell quantity reaches certain scale.Growth curve analysis tries
Proved recipe case: cell confluency degree reaches to carry out cell dissociation and counting, according to 1.5 × 10 when 80%3The cell density bed board 96 in/hole
Porocyte culture plate, often group sets up 3 multiple holes, uses and has serum to have the complete DMEM culture medium of antibiotic to cultivate, every hole
200 μ l, carry out cell viability analysis at 24h, 48h, 72h, 96h, 120h and 144h respectively, concretely comprise the following steps: discard on cell
Clearly, the addition serum-free antibiotic-free DMEM culture medium 100 μ l containing 10% CCK-8 reagent, 37 DEG C, 5% CO2
After hatching 2h in incubator, take out cell, under the conditions of microplate reader OD450, carry out reading, result is analyzed, result
As shown in Figure 3: A549-GFP cell line is basically identical with wild type A549 cell line growth curve, it was demonstrated that A549-GFP cell line
There is good growth activity.
Test example one and test example two prove that we screen the cell model GFP good stability of acquisition, and growth curve is with wild
Raw type is consistent, is good stable cell strain.
Test example three cell model of the present invention and wild-type cell cell migration Benefit Transfer under HGF effect
Whether test example three has the cell migration effect caused by HGF from biologic activity angle analysis cell model of the present invention
Should, and the comparison of this effect and wild type A549 cell.HGF is hepatocyte growth factor, and it can promote wild type
The cell migration effect of A549 cell.
Cell migration efficiency calculation formula is:
The migrating cell number in the migrating cell number ÷ not pastille hole of cell migration efficiency=dosing holes
The most the cell migration efficiency in pastille hole is not set to 1.0.
Testing program:
1) cell point hole: cell confluency degree reaches to carry out cell dissociation and counting when 80%, by cell with low serum (1% FBS)
DMEM culture medium is diluted to aimed concn, adds little indoor, 0.5ml/well, and 5 × 104Cell/well, adhere-wall culture；
2) dosing: add the HGF containing 100ng/ml concentration and use low serum (1% FBS) DMEM culture medium to cell lower floor, HGF
It is diluted, 0.75ml/well；
3) cultivate: 37 DEG C, 5% CO224h is hatched in incubator；
5) washing: take out cell, discard culture medium, PBS 0.5ml.1 time, RT；
6) fixing: little indoor addition formaldehyde, 0.5ml, 10min, RT；
7) wiping: take out cell, wipes confluent monolayer cells on cell with cotton swab, and with PBS drip washing once；
8) dyeing: violet staining liquid is placed in 24 orifice plate 0.5ml, is placed in one by cell, > 30min, RT(step 8-9 be open country
Raw type A549 cell manipulation step, cell model of the present invention is without dyeing, directly at fluorescence microscopy Microscopic observation)
9) washing: a large amount of tap waters are placed in 500ml beaker, rinse cell；
10) imaging counting: imaging under microscope, counting.
Interpretation of result
Result is as shown in Figure 4 and Figure 5:
Fig. 4 is A549-GFP cell line and wild type A549 cell line cell migration result under HGF effect,
Under the conditions of wherein figure A is light field, the lower A549-GFP cell line of HGF effect migrates photo；
Figure B is that under fluorescence field condition, the lower A549-GFP cell line of HGF effect migrates photo, and figure A is that the same area is not shared the same light with figure B
Photo after the match；
Figure C is to migrate photo without the lower A549-GFP cell line of HGF effect under fluorescence field condition；
Figure D is wild type A549 cell line cell migration photo (crystallized purple dyeing) under HGF effect under light field；
Wherein figure B compares with figure A, and cellular resolution is significantly raised, and figure B compares with figure D, and cellular resolution is not significantly different from,
After proving that A549-GFP cell line i.e. can reach the crystallized purple dyeing of wild type A549 cell line in the case of without dyeing
Cellular resolution.
Fig. 5 is A549-GFP cell line and wild type A549 cell line cell migration Benefit Transfer under HGF effect.
A549-GFP cell line compares with wild type A549 cell line, and under HGF effect, cell migration effect is more significantly, it was demonstrated that
A549-GFP cell line has good biologic activity.
Test example one and test example two prove that we screen the cell model GFP good stability of acquisition, and growth curve is with wild
Raw type cell is consistent, is good stable cell strain.Test example three has from biologic activity angle analysis cell model of the present invention
There is to the HGF sensitive cell migration effect consistent with wild type A549 cell.
Test example four A549-GFP cell line cell migration effect under variable concentrations HGF effect and cytoactive are divided
Analysis
Test example four is by adding the HGF of variable concentrations, and cell migration effect and cell that analysis variable concentrations HGF causes are lived
Property analyze.
Cell migration effect test scheme: add the HGF of variable concentrations respectively in cell bottom.
Cell activation assay testing program: cell confluency degree reaches to carry out cell dissociation and counting when 80%, according to 5 ×
103The cell density bed board 96 porocyte culture plate in/hole, often group sets up 3 multiple holes, uses the DMEM culture medium containing 1% FBS to enter
Row is cultivated, every hole 200 μ l, and next day adds the DMEM of 1% FBS containing variable concentrations HGF, carries out cell viability after dosing during 72h
Analyze, concretely comprise the following steps: discard cell conditioned medium, add the serum-free antibiotic-free DMEM culture medium 100 containing 10% CCK-8 reagent
μ l, 37 DEG C, 5% CO2After hatching 2h in incubator, take out cell, under the conditions of microplate reader 0D450, carry out reading, result is entered
Row analyze, result as shown in Figure 6:
Wherein scheming A: wherein abscissa is HGF concentration (HGF:ng/ml), vertical coordinate is migrating cell quantity (cell
Number).HGF concentration cell migration effect of A549-GFP cell line when 100ng/ml is the most notable, therefore selects this concentration
Set up anti-cell migration drug screening method；
Figure B: wherein abscissa is HGF concentration (HGF:ng/ml), vertical coordinate is that the luminosity measured under the conditions of 450nm Single wavelength is inhaled
Receipts value (OD450).A549-GFP cell line cytoactive under variable concentrations HGF effect is unchanged, it was demonstrated that HGF is to A549-
GFP cell line proliferation activity does not affect.
Test example one and test example two prove that we screen the cell model GFP good stability of acquisition, and growth curve is with wild
Raw type cell is consistent, is good stable cell strain.Test example three has from biologic activity angle analysis cell model of the present invention
There is to the HGF sensitive cell migration effect consistent with wild type A549 cell.
Test example five A549-GFP cell line cell migration effect under variable concentrations PF04217903 effect and cell
Activity analysis
Test example five, by adding HGF inhibitor PF04217903, is analyzed PF04217903 and HGF is caused cell migration effect
Depression effect.PF04217903 is a kind of selective, and ATP competitiveness c-Met inhibitor, IC50 is 4.8 nM.
Cell migration effect test scheme: add the PF04217903 of variable concentrations, cell bottom respectively on cell top
Add the HGF of 100 ng/ml.Control is without HGF, without PF04217903 matched group.
Cell activation assay testing program: with reference to test example four step, be separately added into variable concentrations PF04217903 and
The HGF of 100 ng/ml.Control is without HGF, without PF04217903 matched group.
Result is as shown in Figure 7:
Wherein scheme A: wherein abscissa is PF04217903 concentration (PF04217903: μM), and vertical coordinate is migrating cell quantity
(cell number), control is without HGF, without PF04217903 matched group.Other are respectively organized and all contain 100ng/ml's
HGF.HGF concentration when 100ng/ml, the cell migration of the A549-GFP cell line that HGF is caused by variable concentrations PF04217903
Effect completely inhibits；
Figure B: wherein abscissa is PF04217903 concentration (PF04217903: μM), under the conditions of vertical coordinate is 450nm Single wavelength
The photometric absorbance value (OD450) measured.A549-GFP cell line is total at the HGF of variable concentrations PF04217903 and 100 ng/ml
Cytoactive under same-action is unchanged, it was demonstrated that A549-GFP cell line proliferation activity is not affected by PF04217903.
The test example six variable concentrations DMSO impact on drug screening method
Anti-cell migration medicine may be dissolved in DMSO.It is therefore desirable to the detection variable concentrations DMSO shadow to drug screening method
Ring.
Cell migration effect test scheme: add the DMSO of variable concentrations respectively on cell top, cell bottom adds 100
The HGF of ng/ml.Control is without HGF, without DMSO matched group.
Cell activation assay testing program: with reference to test example four step, be separately added into the DMSO and 100 ng/ of variable concentrations
The HGF of ml.Control is without HGF, without DMSO matched group.
Result of the test is as shown in Figure 8:
The cell migration efficiency that wherein HGF is caused by the DMSO of figure A display variable concentrations has no significant effect, figure B display difference
The DMSO cell proliferation of concentration has no significant effect.
SEQUENCE LISTING
<110>Tongji University Suzhou academy
<120>a kind of antitumor using slow virus technique construction migrates drug screening cell model A549-GFP and application thereof
Method
<130> 2015
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 720
<212> DNA
<213> Aequorea victoria
<400> 1
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
Claims (7)
1. the cell model screening antitumor migration medicine, it is characterised in that: described cell model is to have cloned GFP gene
Mammalian tumor cell；Described mammalian tumor cell is optional but is not limited to HepG2, MCF-7, U-87-MG, MDA-
One in MB-231, EA, A549.
The cell model migrating medicine for screening antitumor the most according to claim 1, it is characterised in that: described GFP
Gene can stably be expressed.
The cell model migrating medicine for screening antitumor the most according to claim 1, it is characterised in that: described GFP
Gene order is SEQ ID NO:1.
The cell model migrating medicine for screening antitumor the most according to claim 1, it is characterised in that: described cell
Model has been prepared by slow virus system, comprises the following steps: is cloned in prrl plasmid by GFP gene, is built into prrl-
CMV-GFP recombiant plasmid, by this plasmid and the 293T cell of slow virus packaging plasmid cotransfection logarithmic (log) phase, gathers in the crops purified virus,
Virus infects logarithmic (log) phase cell, through limiting dilution assay and fluorescent screening, it is thus achieved that cell model of the present invention.
The cell model migrating medicine for screening antitumor the most according to claim 1, it is characterised in that thin with parent
Born of the same parents system compares has similar growth curve.
The cell model migrating medicine for screening antitumor the most according to claim 1, it is characterised in that move at cell
Moving in experiment, the cell migration effect that HGF causes is notable, the cell migration effect that HGF is caused by specific small molecule inhibitor
Depression effect is notable, and above biologic activity is consistent with parental cell line.
7. the method for a cell migration screening model, it is characterised in that comprise the following steps:
A. taking the above-mentioned cell model for screening and be inoculated in the little indoor of transwell, the upper room of wherein screening group adds and contains
The maintenance culture medium of thing to be screened, lower room adds containing the maintenance culture medium promoting cell migration medicine；
B. cell incubation 4-24h, after cell is fixing, aseptic cotton carrier wipes ventricular cell, counts cell following table under fluorescence microscope
According to migrating cell quantity, the migrating cell sum in 4 field of excursion regions, face, determines whether thing to be screened is the potential of needs
Medicine.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220284A (en) * | 2011-04-30 | 2011-10-19 | 上海交通大学 | Fluorescent cell model for screening of antitumor drugs, labeling method and application thereof |
| CN102321587A (en) * | 2011-08-25 | 2012-01-18 | 上海吉凯基因化学技术有限公司 | Construction of lung cancer drug screening cell line |
| CN105462932A (en) * | 2015-12-29 | 2016-04-06 | 同济大学苏州研究院 | Tumor metastasis resistance medicine screening cell model constructed by adopting lentivirus technology and application method thereof |
-
2016
- 2016-04-23 CN CN201610254095.9A patent/CN105925533A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220284A (en) * | 2011-04-30 | 2011-10-19 | 上海交通大学 | Fluorescent cell model for screening of antitumor drugs, labeling method and application thereof |
| CN102321587A (en) * | 2011-08-25 | 2012-01-18 | 上海吉凯基因化学技术有限公司 | Construction of lung cancer drug screening cell line |
| CN105462932A (en) * | 2015-12-29 | 2016-04-06 | 同济大学苏州研究院 | Tumor metastasis resistance medicine screening cell model constructed by adopting lentivirus technology and application method thereof |
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