CN103116006A - Novel screening method of anticancer medicaments - Google Patents
Novel screening method of anticancer medicaments Download PDFInfo
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- CN103116006A CN103116006A CN2013100303955A CN201310030395A CN103116006A CN 103116006 A CN103116006 A CN 103116006A CN 2013100303955 A CN2013100303955 A CN 2013100303955A CN 201310030395 A CN201310030395 A CN 201310030395A CN 103116006 A CN103116006 A CN 103116006A
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Abstract
The invention discloses a novel screening method of anticancer medicaments. The novel screening method is characterized by comprising the followinh steps of: (1) preparing TTLL12 overexpressed Hep-2 cell lines; (2) selecting positive control and negative control medicaments; (3) obtaining the optimum concentration of nitrotyrosine and the optimum diluted concentrations of medicaments to be detected; and (4) screening the medicaments, finally checking readings and comparing. The novel screening method of the anticancer medicaments disclosed by the invention has the characteristics that the method is simple, a user only needs to master a cell culture and Elisa (Enzyme-linked Immuno Sorbent Assay) technology and then masters the law, the method is fast and cheap, the method is few in reagents and variation factors, stable, high in sensitivity, good in specificity, easy to popularize and capable of screening in a large scale. Furthermore, through the experiment, whether the medicaments to be detected have anti-cancer effects and anticancer effect mechanisms of the medicaments to be detected simultaneously can be obtained, so that the speed of clinically applying the medicaments to be detected is greatly accelerated, and more lives of patients are expected to be saved.
Description
Technical field
The invention belongs to technical field of molecular biology, specifically, relate to a kind of Novel screen choosing method of cancer therapy drug.
Background technology
Malignant tumour is large class serious threat human life and a healthy disease.Chemotherapy is as the treatment means of general, start from 1948 using antifol as antineoplastic, be incorporated in leukemic treatment, there is irreplaceable status in the complex treatment of malignant tumour, but because medicament categories is limited at present, and the resistance phenomenon appears, therefore the curative effect of chemotherapy is still undesirable at present, still need us to continue to find more efficiently medicine product, the screening technique of existing cancer therapy drug has many, as:
1, human body tumour cell CFA (Human Tumor Cell Cloning Assay, HTCA), the advantage of this method: sensitivity, directly estimate cell proliferation death.And its shortcoming is real: low for the clinical samples verification and measurement ratio, the cycle is long, colony count is loaded down with trivial details.
2, differential staining (Differential Staining Cytotoxicity Assay, DISC), the method is the division of dependent cells not.But this method needs the artificial counting cell, be subject to the impact of personal error.
3, the nucleic precursor mix experiment (
3h-Tdr Assay), the required cell number of this method is less so can use pin to inhale sample, but this method is only measured in sample 5%～10% cell, and thymine and normal cell all can affect measurement result, and have radioactive contamination.
4, tetrazolium salts decoration method (MTT Assay), but mtt assay carries out the analysis of a large amount of samples within a short period of time.But some factor also can affect its result: the quality of dimethyl sulfoxide, and MTT forms formazan crystal and can fade within a few hours, and some cell received a death blow still can make MTT change formazan into.
5, ATP-Bioluminescence Assay (ATP Luminescence Assay), this method utilizes fluorescein one luciferase reagent quantitative to measure ATP level in cell, thereby the kill capability of reflection chemotherapeutics to tumour cell, also high value can be detected when tumour cell quantity seldom, there is hypersensitivity.
6, total protein decoration method (Sulforhodamine B Assay, SRB), be a kind of protein bound dyestuff, can in cell, be combined by the basic amino acid in biomacromolecule, in the absorbance log reading of 515nm and cell number, is good linear relationship.
7, fluorescence method (Fluorometric Microculture Cytotoxicity Assay, FMCA), this method can be sent fluorescence after utilizing the two acetate fluoresceins of living cells hydrolysis, can reflect the number of living cells in cultivating system by the intensity of measuring fluorescence.But the control group reading is too low sometimes, causes the failure of an experiment.
8, the collagen gel drop is implanted cultivation (CD-DST), and this method has the advantages such as the cancer cell culture success ratio is high, the drug sensitive test result is accurate.But this method price is more expensive, at present only in Japanese widespread use.
But current all drug sensitive experiments only can filter out the medicine of effective inhibition tumor growth, and can't understand the mechanism of anticancer action of filtered out medicine, if do not understand mechanism of drug action, impact is applied to clinical research greatly; If understand mechanism of drug action, still need and carry out other experiments and studied, consume regular hour and funds.
And can be integrated into tubulin by tubulin tyrosine ligase according to nitration tyrosine known to document, form nitration tyrosine tubulin, this can change the structure of microtubule significantly, cause transportation obstacle in the distortion of cell, the decline of sticking ability and born of the same parents, finally can cause natural death of cerebral cells and organ dysfunction to be lost.(referring to: Fukushima N, Furuta D, Hidaka Y, Moriyama R, Tsujiuchi T.Post-translational modifications of tubulin in the nervous system[J] .JNeurochem.2009May; 109 (3): 683-93.) nitration tyrosine tubulin can suppress the growth of head and neck scale carcinoma cell, and TTLL12 can suppress the generation of nitration tyrosine tubulin, thereby the growth of adjusting head carcinoma of neck cell, TTLL12 increases, can cause the minimizing of nitration tyrosine tubulin, cause cancer cell to escape the strike of nitration tyrosine tubulin to it, can continue propagation, so TTLL12 has the function of latent carcinoma gene.(referring to: Li Yadong, the people such as Jinsong ZHANG, tubulin tyrosine ligase analog 12 is by disturbing tubulin nitrotyrosine promotion Hep-2 Growth of Cells [J], Chinese biological chemistry and molecular biosciences journal, 2012,28 (8): 30-34.)
The shortcoming of prior art: current all drug sensitive experiments only can filter out the medicine of effective inhibition tumor growth, and can't understand the mechanism of anticancer action of filtered out medicine, if do not understand mechanism of drug action, impact is applied to clinical research greatly; If understand mechanism of drug action, still need and carry out other experiments and studied, consume regular hour and funds.
Summary of the invention
For solving above technical matters, the object of the present invention is to provide a kind of energy Large-scale Screening to go out the Novel screen choosing method of cancer therapy drug effective inhibition tumor growth, that understand its mechanism of anticancer action.
The present invention seeks to realize like this:
A kind of Novel screen choosing method of cancer therapy drug, its key is to comprise the following steps:
(1) TTLL12 crosses the preparation of expressing the Hep-2 cell line
The designed, designed primer, positive-sense strand 5 '-CGGGATCCCCGATGGACTACCACGAGGA-3 ', antisense strand 5 '-CGGGATCCTAGCTACAGGACGCCCCCG-3 ', take TTLL12cDNA as template amplification TTLL12 genetic fragment, the size that obtains the genes of interest fragment is 2818bp, again the TTLL12 genetic fragment is inserted in the pSG-5 plasmid, then recombinant plasmid is transformed respectively to e. coli jm109 by chemical method, again by Transfected Recombinant Plasmid Hep-2 cell, pick out the clone of indivedual expression TTLL12, continue to cultivate, through the cultivation of repeatedly going down to posterity, finally obtain and stablized the Hep-2 cell line of expressing TTLL12,
(2) positive control and negative control medicine selects
By the 100uM taxol and thiophene third rare respectively with stablized express TTLL12 the Hep-2 cell line in being cultivated containing in the nutrient solution of nitration tyrosine, then the western that goes tests, the known taxol of result can increase nitration tyrosine tubulin, there is the effect that suppresses the TTLL12 function, can be used as positive control; And the thiophene propylene can not impel the formation of nitration tyrosine tubulin, do not there is the effect that suppresses the TTLL12 function, can be used as negative control;
(3) nitration tyrosine optimum concentration and the medicine to be measured acquisition of suitable dilute concentration
TTLL12 is crossed to expression Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, MEM nutrient solution volume is 80 μ L, is 37 ℃, 5%CO
2in incubator, cultivate 6 hours, adding respectively final concentration is 50 μ M, 100 μ M, 200 μ M, 400 μ M, 800 μ M nitration tyrosine, every hole is totally 10 μ L, and due to Elisa, than the western sensitivity, so the consumption of taxol and thiophene propylene (Thiirene) all is less than 100 μ M (western consumption), adding respectively final concentration is 5 μ M, 10 μ M, 20 μ M, 4 dilutabilitys such as 40 μ M, every hole is totally 10 μ L, dilutabilitys different from nitration tyrosine carry out the square formation titration, continue at 37 ℃ 5%CO
2in incubator, cultivate 24 hours.Afterwards, nutrient solution in sucking-off 96 orifice bores, every hole adds 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, then remove damping fluid, the PBS that every hole adds 100 μ L to contain 3.7% formaldehyde and 0.05%Tween-20 fixes 10 minutes, then remove immobile liquid, the horseradish peroxidase-labeled nitration tyrosine antibody that every hole adds 100 μ LPBS to dilute 1: 1000, at room temperature with cytosis 1 hour, then remove antibody-solutions, with the PBS200 μ L that contains 0.05%Tween-20, rinse aperture, then add the OPD (o-phenylenediamine) of 100ul-H
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilize detector at 450nm place reading, and the optimum concentration that finally obtains nitration tyrosine is 400 μ M, and the suitableeest dilute concentration of medicine to be measured is 10 μ M;
(4) screening medicine
TTLL12 is crossed to expression Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, every hole MEM nutrient solution volume is 80 μ L, is 37 ℃, 5%CO
2in incubator, cultivate 6 hours, adding respectively final concentration is nitration tyrosine optimum concentration, and every hole is totally 10 μ L, and the final concentration of medicine to be measured and taxol is 10 μ M, and every hole is totally 10 μ L, and medicine to be measured and taxol all repeat 6 holes, continues at 37 ℃ 5%CO
2in incubator, after cultivating 24 hours, nutrient solution in sucking-off 96 orifice bores, add every hole 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, remove damping fluid, the PBS that every hole adds 100 μ L to contain 3.7% formaldehyde and 0.05%Tween-20 fixes 10 minutes, then remove immobile liquid, the horseradish peroxidase-labeled nitration tyrosine antibody that every hole adds 100 μ LPBS to dilute 1: 1000, at room temperature with cytosis 1 hour, then remove antibody-solutions, rinse aperture with the PBS200 μ L that contains 0.05%Tween-20, then add the OPD (o-phenylenediamine) of 100 μ L-H
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilize detector at 450nm place reading, finally compare, if the OD450nm value is greater than 1, illustrates that this medicine has antitumaous effect, and there is the function that suppresses TTLL12, promote the combination of nitration tyrosine and α-tubulin, the mechanism of modifying by participating in tubulin, form more nitration tyrosine tubulin, causes the cancer suppressing action mechanism of cancer cell death, otherwise do not there is above-mentioned cancer suppressing action mechanism.
Above-mentioned cytoskeleton isolation buffer liquid is by the EGTA of Mes pH6.7, the 1mM of 90mM, the MgCl of 1mM
2, 10% (v/v) the Triton X-100 of glycerol and 0.5% (v/v) formulated.
Beneficial effect:
The Novel screen choosing method of a kind of cancer therapy drug of the present invention, have method simple, a GPRS cell cultivate and the elisa technology, just grasped this law; Quick and cheap; Reagent is less, variance factor is less and stable, highly sensitive, specificity good, be easy to promote and can carry out the characteristics such as extensive examination, and can obtain medicine to be measured by this experiment simultaneously and whether there are antitumaous effect and two kinds of information of its antitumaous effect mechanism, greatly accelerate medicinal application to be measured in clinical speed, be expected to retrieve more patients' life.
The accompanying drawing explanation
Fig. 1 is process flow diagram of the present invention;
The electrophoretogram that Fig. 2 is genes of interest TTLL12;
(M is 1kd DNA to the double digestion electrophoretogram that Fig. 3 is recombinant plasmid pSG-5-TTLL12; 1 double digestion that is pSG-5-TTLL12);
The expression of results figure that Fig. 4 is Hep-2 cell TTLL12 albumen after Western blot detection transfection;
Fig. 5 be immunofluorescence experiment detect Hep-2 cell TTLL12 albumen after transfection expression (* 200) as a result figure (a is for containing blank plasmid Hep-2 cell; B is for stablizing the Hep-2 cell of high expressed TTLL12);
Fig. 6 is taxol (paclitaxel) and thiophene propylene (Thiirene) figure as a result that affects on nitration tyrosine tubulin.
Embodiment
Embodiment:
A kind of Novel screen choosing method of cancer therapy drug, its key is to comprise the following steps:
(1) preparation of TTLL12 overexpressing cell strain
1) pcr amplification TTLL12 genetic fragment
The designed, designed primer, positive-sense strand 5 '-CGGGATCCCCGATGGACTACCACGAGGA-3 ', antisense strand 5 '-CGGGATCCTAGCTACAGGACGCCCCCG-3 ', take TTLL12cDNA as template amplification TTLL12 genetic fragment.The genes of interest clip size is 2818bp, and the PCR reactant liquor forms: Ex Taq enzyme (5u/ μ L) 0.15 μ L, 10 * PCR damping fluid, 2.5 μ L, MgCL
2(25mmol/L) 2 μ L, d NTP (each 2.5mmoL/L) 2 μ L, upstream primer (10moL/L) 1.0 μ L downstream primer (10moL/L) 1.0 μ L, template DNA 4.5 μ L, add sterilizing pure water to 25 μ L.PCR loop parameter: 94 ℃ of 5min, 94 ℃ of 50s, 55 ℃ of 30s, 72 ℃ of 30s, 35cycle, 72 ℃ of 8min.The PCR Product Identification: the agarose gel electrophoresis 1% separates to be identified.The results are shown in Figure 2.
2) the TTLL12 genetic fragment is inserted to the pSG-5 plasmid
Enzyme is cut TTLL12 and pSG-5 plasmid respectively, and the enzyme time of cutting is at 37 ℃ of water-bath 4h.Reaction system following (pSG-5/ANO1 2 μ g/2 μ g, 10 * buffer, 4 μ L, double digestion EcoR I/BamHI2 μ L/2 μ L, dd H
2o supplies cumulative volume 40 μ L), then respectively get 4 μ L and carry out 1% agarose gel electrophoresis, observe enzyme and cut whether fully.By the PCR Product Identification, the results are shown in Figure 3.The enzyme that will reclaim again purifying is cut the pSG-5 fragment that the genes of interest fragment cuts with enzyme and is connected.Reaction conditions: 16 ℃ of water-baths connect spends the night, 70 ℃ of water-bath 10min deactivation T
4dNA ligase.Coupled reaction system following (pSG-5/ANO11 μ g/3 μ g, 10 * buffer1 μ L, T
4dNA ligase 1 μ L, dd H
2o supplies cumulative volume 10 μ L), then above-mentioned connection product is transformed respectively to e. coli jm109 by chemical method.
3) setting up the TTLL12 that stablizes the Hep-2 cell line of high expressed TTLL12 and utilize Western to detect transfection cell strain expresses
In 37 ℃, 5%CO
2in incubator, containing 10% calf serum, 1mM Sodium Pyruvate, the 2mM glutamine, cultivate the Hep-2 cell in the Eagle's medium of the change of 40ug/mL gentamicin (modified Eagle ' s medium, MEM), when cell grows to 70～80%, abandon nutrient solution.With reference to Lipofectamine
tM2000 reagent description operation by the grouping situation empty pSG-5-purine of transfection and containing the pSG-5-purine of TTLL12 fragment respectively, at first add serum-free and antibiotic MEM nutrient solution 250ul and plasmid 4.0 μ g in the Ep pipe, mix, and room temperature is placed 5min.Add serum-free and antibiotic MEM nutrient solution 250 μ L and Lipofectamine20008.0 μ L in the Ep pipe, mix, room temperature is placed 5min simultaneously.Then the liquid of above-mentioned two Ep pipes is mixed, room temperature is diluted to 1mL by it after placing 20min, then adds culture plate, jiggles, mixes, and the uniform fold cell surface, put 37 ℃, 5%CO
2cultivate in incubator, change the MEM nutrient solution containing the 2ug/mL purine of 10%FBS concentration after 5h, the screening cell, after 18 days, is picked out the clone of indivedual expression TTLL12.Continue to cultivate, through repeatedly going down to posterity, after 2 months, finally obtain the stable cell line that TTLL12 crosses expression.
After the Hep-2 cell of transfection is cleaned to twice with PBS, the cell pyrolysis liquid that adds 400 μ L, collect the cracking afterproduct, boil sex change, with bovine serum albumin(BSA) (BSA) preparation standard protein solution, measure total protein concentration, the capable protein electrophoresis of 12%SDS-PAGE separation gel, forward to after electrophoresis on nitrocellulose filter, 5% milk sealing 1h, then add primary antibodie (anti-TTLL12, dilution in 1: 700; Anti-TBP, dilution in 1: 2000) solution is hatched 3h, TBST washing 3 times, after each 10min, with hatch 2h, TBST washing 3 times under two anti-(horseradish peroxidase-labeled mouse-anti rabbit igg antibody, dilution in 1: 1000) room temperature, after each 10min, utilize DAB chromogenic reagent box carry out chemiluminescence reaction and take pictures.The results are shown in Figure 4.
4) indirect immunofluorescence detects the TTLL12 expression of transfection cell strain
Slide is put into to 6 orifice plates, and the Hep-2 cell line that stablizing of having set up expressed to TTLL12 and the Hep-2 cell line of blank plasmid transfection add respectively 6 orifice plates to cultivate.After cell is climbed full slide, discard nutrient solution, PBS liquid rinses 3 times, drip 100% ethanol fixed cell, normal temperature 10 minutes, 1moL/L non-ionics Triton-X100 changes processing 10min thoroughly, with cold 0.01moL/L pH7.4PBS immersion bubble, rinses 3 times, finally, with distilled water flushing 1 time, prevent spontaneous fluorescence.With 0.01moL/L, the PBS of pH7.4 suitably dilutes antibody, covers slide.Build 6 orifice plate lids, it is moistening that the outsourcing l Water Paper keeps, and on 37 ℃ of insulation shaking tables, shakes 60min.Discard liquid in hole, use 0.01moL/L, the PBS of pH7.4 rinses 3 times.Suck excessive moisture, but do not make the sample drying, drip certain dilution fluorescence labeling two anti-, cover slide.Build 6 orifice plate lids, it is moistening that the outsourcing l Water Paper keeps, and on 37 ℃ of insulation shaking tables, shakes 60min; Take out slide, with filter paper, suck excessive moisture, drip a glycerine damping fluid (glycerine and pH7.4PBS liquid mix with 9: 1 ratios, and glycerine has the effect that reduces non-specific fluorescence), then coated with cover glass.Observe under the fluorescent microscope high power field, the results are shown in Figure 5.
(2) positive control and negative control medicine selects
By taxol with thiophene third is rare carries out the western test, step is as follows: by TTLL12 high expressed Hep-2 cell line with every hole 2 * 10
5individual cell, plant into 6 orifice plates, to cultivate containing the nutrient solution of 50 μ M nitration tyrosine, test group add 100 μ M taxols or thiophene third rare, control group does not add compound, every strain cell repeats 3 holes; Every 48h upgrades 1 nutrient solution, extracts total protein of cell after 60h and carries out the western test, detects N-tub, TBP and α-tubulin.The results are shown in Figure 6.
Confirm that through western taxol can increase nitration tyrosine tubulin, illustrate that it has the effect that suppresses the TTLL12 function, can be used as positive control.And the thiophene propylene does not form nitration tyrosine tubulin, illustrate that it does not have the effect that suppresses the TTLL12 function, can be used as negative control.
(3) nitration tyrosine optimum concentration and the medicine to be measured acquisition of suitable dilute concentration
1) TTLL12 is crossed to expression Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, MEM nutrient solution volume is 80 μ L, is 37 ℃, 5%CO
2in incubator, cultivate 6 hours, adding respectively final concentration is 50 μ M, 100 μ M, 200 μ M, 400 μ M, 800 μ M nitration tyrosine, every hole totally 10 μ L, due to Elisa than the western sensitivity, therefore the consumption of taxol and thiophene propylene (Thiirene) adopts and is less than 100 μ M (western consumption), and adding respectively final concentration is 5,10,4 dilutabilitys such as 20,40 μ M, every hole is totally 10 μ L, dilutabilitys different from nitration tyrosine carry out the square formation titration, continue at 37 ℃ 5%CO
2in incubator, cultivate 24 hours. afterwards, nutrient solution in sucking-off 96 orifice bores, every hole adds 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, then remove damping fluid, the PBS that every hole adds 100 μ L to contain 3.7% formaldehyde and 0.05%Tween-20 fixes 10 minutes, then remove immobile liquid, the horseradish peroxidase-labeled nitration tyrosine antibody that every hole adds 100 μ L PBS to dilute 1: 1000, at room temperature with cytosis 1 hour, then remove antibody-solutions, rinse aperture with the PBS200 μ L that contains 0.05%Tween-20, then add the OPD (o-phenylenediamine) of 100 μ L-H
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilize detector at 450nm place reading.The results are shown in Table 1 and table 2.
Table 1 is the OD450nm value
Table 2 is the P/N value
From the square formation test findings, can find out, nitration tyrosine concentration is 400 μ M, when drug concentration to be measured is 10 μ M, the OD450nm value of positive control is in 1.0 left and right, and the OD450nm value of negative control is lower, the P/N value is the highest, thus we to obtain nitration tyrosine concentration be 400 μ M, compound concentration is 10 μ M.
2) sensitivity of drug sensitivity testing in vitro method test
Taxol, respectively with 80 μ M, 40 μ M, 20 μ M, 10 μ M, 5 μ M, 2.5 μ M concentration, is dissolved in to the MEM nutrient solution, and every hole adds 10 μ L, according to the test method of above foundation, detects its OD450nm value.The results are shown in Table 3.
Table 3: the sensitivity experiments result of drug sensitivity testing in vitro method
| Concentration | 80μM | 40μM | 20μM | 10μM | 5μM | 2.5μM |
| Taxol (OD450nm) | 1.330 | 1.291 | 1.118 | 1.086 | 0.846 | 0.681 |
Result shows that the OD450nm value of taxol least concentration 2.5 μ M still is greater than the negative control OD450nm value of 10 μ M, illustrates that its sensitivity is higher.
3) specific test of drug sensitivity testing in vitro method
According to the drug sensitivity testing in vitro method of having set up, taxol is as positive control, the thiophene propylene is as negative control, utilize known to the adiaphorous effective antitumor medicine of microtubule: endoxan (cyclophosphamide, CTX), carboplatin (carboplatin), methotrexate (MTX) (methotrexate, MTX), 4 kinds of medicines to be measured of fluorouracil (5-fluorouracil, 5-Fu) are tested, and the 10uM compound is dissolved in 10uL MEM nutrient solution, add in 96 orifice bores the specificity of the outer susceptibility test methods method of detection bodies.The results are shown in Table 4.
Table 4: specific test result
| ? | Taxol | The thiophene propylene | Carboplatin | Fluorouracil | Endoxan | Methotrexate (MTX) |
| OD450nm | 1.102 | 0.157 | 0.108 | 0.179 | 0.207 | 0.251 |
Utilize this measuring some through the anticancer effective medicine of clinical confirmation, but their anticancer mechanism is all irrelevant with microtubule, that is to say, they to microtubule all without influence, result shows that their OD450nm all is less than the positive control value, show that this experiment specificity is good, only screen the effective cancer therapy drug that those affect microtubule; In addition because this test testing sample is compound, containing other protein matter, not can with nitration tyrosine antibody response, so its specificity is higher.
4) replica test of drug sensitivity testing in vitro method
Choose taxol and other 4 kinds of medicines to be measured, be respectively negative control thiophene propylene, carboplatin, fluorouracil, endoxan.Parallel 6 repetitions, the revision test in criticizing of establishing of every duplicate samples in a collection of test.In addition 4 different tests day replication taxol and other 4 kinds of compounds, every duplicate samples is parallel establishes 6 repetitions, revision test between criticize, calculate its coefficient of variation, the coefficient of variation=standard deviation/on average * 100%.The results are shown in Table 5.
Table 5: batch in, batch between the replica test result
Result shows that the variation within batch coefficient is between 3.5%～7.87%, and interassay coefficient of variation, between 2.86%～6.99%, all is less than 10%, has repeatability preferably.
(4) screening medicine
TTLL12 is crossed to expression Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, every hole MEM nutrient solution volume is 80 μ L, in 37 ℃, and 5%CO
2in incubator, cultivate 6 hours, adding respectively final concentration is 400 μ M nitration tyrosine, and every hole is totally 10 μ L, with the positive contrast of taxol, the final concentration of medicine to be measured and taxol is 10 μ M, and every hole is totally 10 μ L, medicine to be measured and taxol all repeat 6 holes, continue at 37 ℃ 5%CO
2in incubator, cultivate 24 hours. afterwards, nutrient solution in sucking-off 96 orifice bores, add every hole 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, remove damping fluid, the PBS that every hole adds 100 μ L to contain 3.7% formaldehyde and 0.05%Tween-20 fixes 10 minutes, then remove immobile liquid, the horseradish peroxidase-labeled nitration tyrosine antibody that every hole adds 100 μ LPBS to dilute 1: 1000, at room temperature with cytosis, after 1 hour, remove antibody-solutions, rinse aperture with the PBS200 μ L that contains 0.05%Tween-20, then add the OPD (o-phenylenediamine) of 100 μ l-H
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilize detector at 450nm place reading, the results are shown in Table 6.
Table 6: each sample finally detects the OD450nm value that nitration tyrosine tubulin obtains
| Sample | OD450nm | Sample | OD450nm | Sample | OD450nm | Sample | OD450nm | Sample | OD450nm |
| Taxol | 1.091 | 1 | 0.132 | 2 | 0.147 | 3 | 0.151 | 4 | 0.129 |
| 5 | 0.136 | 6 | 0.158 | 7 | 0.235 | 8 | 0.321 | 9 | 0.351 |
| 10 | 0.358 | 11 | 0.247 | 12 | 0.241 | 13 | 0.411 | 14 | 1.003 |
| 15 | 0.412 | 16 | 0.314 | 17 | 0.452 | 18 | 0.189 | 19 | 0.338 |
| 20 | 0.371 | 21 | 1.133 | 22 | 0.365 | 23 | 0.146 | 24 | 0.352 |
| 25 | 0.347 | 26 | 0.122 | 27 | 1.057 | 28 | 0.163 | 29 | 0.221 |
| 30 | 0.289 | 31 | 0.113 | 32 | 0.167 | 33 | 0.148 | 34 | 0.128 |
| 35 | 0.168 | 36 | 0.278 | 37 | 0.263 | 38 | 0.410 | 39 | 0.247 |
| 40 | 0.162 | ? | ? | ? | ? | ? | ? | ? | ? |
The sample title: 1 is yohimbane (yohimban); 2 is ergotaman (ergotaman); 3 is yaruru rice fixed (aspidospermi dine); 4 is 1H-pyrrolizine (1H-pyrrolizine); 5 is ergot woods (ergoline); 6 is 1H-benzazepine (1H-benzazepine); 7 Wei Pi (picene); 8 is carbazole (carbazole); 9 is fluorenes (fluorene); 10 is Dencentan (acridine); 11 is folder thianthrene (thianthrene); 12 is 9H-folder oxygen anthracene (9H-Xanthene); 13 is anthracene (anthracene); 14 is nocodazole (nocodazole); 15 is spiropentane (spiropentane); 16 is 8-azaspiro [4.5] decane (8-azaspiro[4.5] decane); 17 is Isosorbide-5-Nitrae-dioxo spiro [4.5] decane (Isosorbide-5-Nitrae-dioxaspiro[4.5] decane); 18 be 2,7,9-triazaphenathrene (2,7,9-triazaphenanthrene); 19 be 1,7-phenanthroline (1,7-phenanthroline); 20 is phenanthridines (phenanthridine); 21 is epothilone B (patupilone); 22 is inosine (inosine); 23 is cytidine (cytidine); 24 is adenosine (adenosine); 25 Wei guans (coronene); 26 Wei perylenes (perylene); 27 is Docetaxel (docetaxel); 28 is pyrene (pyrene); 29 is acenaphthene (acenaphthylene); 30 is oxygen cyclobutane (Oxetine); 31 Wei Evil propylene (Oxirene); 32 is 1-azirine (Azirine); 33 is thiirane (Thiirane); 34 is oxirane (Oxirane); 35 is azirane (Aziridine); 36 is trimethylene (cyclopropane); 37 is thiophene fourth ring (Thietane); 38 Wei Evil fourth rings (Oxetane); 39 is azetidine (Azetidine); 40 is cyclo-butane (Cyclobutane)
Visible according to test findings, the OD450nm value of most of sample all is less than 0.452, wherein the OD450nm value of No. 14 samples is greater than 1, be that 1.003, No. 14 sample chemical names are called nocodazole nocodazole, By consulting literatures, learn that the nocodazole can promote microtubule polymerization, suppress microtubule depolymerization, extend mitosis, cause Apoptosis.(Li?P，Zhou?L，Dai?Z，Jin?X，Liu?X，Matsumoto?Y，Furusawa?Y，Li?Q.High?LET?radiation?enhances?nocodazole?Induced?cell?death?in?HeLa?cells?through?mitotic?catastrophe?and?apoptosis.J?Radiat?Res.2011；52(4)：481-9.)。
The OD450nm value of No. 21 samples is greater than 1, be 1.133, No. 21 the sample chemical name is called epothilone B (patupilone), and By consulting literatures learns that epothilone B (patupilone) Main Function is to promote tubulin polymerization to form microtubule and stable microtubule; Suppress cell and form normal Spindle, disturb the growth of tumour cell, even induce its death.(Cheng Yihua, Chen Xiuhua, the progress of Epothilones series antineoplastic medicament, world's clinical medicine, 2011,32 (10): 619-623).
The OD450nm value of No. 27 samples is greater than 1, is that 1.057, No. 27 sample chemical names are called Docetaxel (docetaxel), and By consulting literatures, learn that Docetaxel (docetaxel) is the paclitaxel analogs obtained by semisynthesis.By promoting microtubule polymerization, inhibition microtubule depolymerization to affect the mitosis of cell, thereby bring into play its cytotoxicity.(Guo Ruiling, Wu Guoming hide the good fortune cloud, Huang Guijun, Chen Weizhong, the Primary Study of docetaxel on human lung adenocarcinoma multidrug resistance cell A549/CDDP and parental cell mechanism of action thereof, Third Military Medical University's journal, 2005,27 (7): 610-613).
According to above result, our known this method can filter out affects the effective antitumor of microtubule medicine, and in this method, 1 96 orifice plate can detect 16 kinds of medicines, if a plurality of 96 orifice plates of while continuous detecting, just can detect hundreds of medicine in 32 hours, this high throughput test, be suitable for carrying out extensive examination.
Claims (2)
1. the Novel screen choosing method of a cancer therapy drug is characterized in that comprising the following steps:
(1) TTLL12 crosses the preparation of expressing the Hep-2 cell line
The designed, designed primer, positive-sense strand 5 '-CGGGATCCCCGATGGACTACCACGAGGA-3 ', antisense strand 5 '-CGGGATCCTAGCTACAGGACGCCCCCG-3 ', take TTLL12cDNA as template amplification TTLL12 genetic fragment, the size that obtains the genes of interest fragment is 2818bp, again the TTLL12 genetic fragment is inserted in the pSG-5 plasmid, then recombinant plasmid is transformed respectively to e. coli jm109 by chemical method, again by Transfected Recombinant Plasmid Hep-2 cell, pick out the clone of indivedual expression TTLL12, continue to cultivate, through the cultivation of repeatedly going down to posterity, finally obtain and stablized the Hep-2 cell line of expressing TTLL12,
(2) positive control and negative control medicine selects
By the 100uM taxol and thiophene third rare respectively with stablized express TTLL12 the Hep-2 cell line in being cultivated containing in the nutrient solution of nitration tyrosine, then the western that goes tests, the known taxol of result can increase nitration tyrosine tubulin, there is the effect that suppresses the TTLL12 function, can be used as positive control; And the thiophene propylene can not impel the formation of nitration tyrosine tubulin, do not there is the effect that suppresses the TTLL12 function, can be used as negative control;
(3) nitration tyrosine optimum concentration and the medicine to be measured acquisition of suitable dilute concentration
TTLL12 is crossed to expression Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, MEM nutrient solution volume is 80 μ L, is 37 ℃, 5%CO
2in incubator, cultivate 6 hours, adding respectively final concentration is 50 μ M, 100 μ M, 200 μ M, 400 μ M, 800 μ M nitration tyrosine, every hole totally 10 μ L, due to Elisa than the western sensitivity, therefore the consumption of taxol and thiophene propylene all is less than 100 μ M, and adding respectively final concentration is 5 μ M, 10 μ M, 20 μ M, 4 dilutabilitys such as 40 μ M, every hole is totally 10 μ L, dilutabilitys different from nitration tyrosine carry out the square formation titration, continue at 37 ℃ 5%CO
2in incubator, after cultivating 24 hours, nutrient solution in sucking-off 96 orifice bores, every hole adds 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, then remove damping fluid, the PBS that every hole adds 100 μ L to contain 3.7% formaldehyde and 0.05%Tween-20 fixes 10 minutes, then remove immobile liquid, the horseradish peroxidase-labeled nitration tyrosine antibody that every hole adds 100 μ L PBS to dilute 1: 1000, at room temperature with cytosis 1 hour, then remove antibody-solutions, rinse aperture with the PBS 200 μ L that contain 0.05%Tween-20, then the OPD-H that adds 100 μ L
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilize detector at 450nm place reading, and the optimum concentration that finally obtains nitration tyrosine is 400 μ M, and the suitableeest dilute concentration of medicine to be measured is 10 μ M,
(4) screening medicine
TTLL12 is crossed to expression Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, every hole MEM nutrient solution volume is 80 μ L, is 37 ℃, 5%CO
2in incubator, cultivate 6 hours, adding respectively final concentration is 400 μ M nitration tyrosine, and every hole is totally 10 μ L, and the final concentration of medicine to be measured and taxol is 10 μ M, and every hole is totally 10 μ L, and medicine to be measured and taxol all repeat 6 holes, continues at 37 ℃ 5%CO
2in incubator, cultivate 24 hours. afterwards, nutrient solution in sucking-off 96 orifice bores, add every hole 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, remove damping fluid, the PBS that every hole adds 100 μ L to contain 3.7% formaldehyde and 0.05%Tween-20 fixes 10 minutes, then remove immobile liquid, the horseradish peroxidase-labeled nitration tyrosine antibody that every hole adds 100 μ LPBS to dilute 1: 1000, at room temperature with cytosis 1 hour, then remove antibody-solutions, rinse aperture with the PBS200 μ L that contains 0.05%Tween-20, then the OPD-H that adds 100 μ L
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilize detector at 450nm place reading, finally compare, if the OD450nm value is greater than 1, illustrates that this medicine has antitumaous effect, and there is the function that suppresses TTLL12, promote the combination of nitration tyrosine and α-tubulin, the mechanism of modifying by participating in tubulin, form more nitration tyrosine tubulin, causes the cancer suppressing action mechanism of cancer cell death, otherwise do not there is above-mentioned cancer suppressing action mechanism.
2. a kind of Novel screen choosing method of cancer therapy drug according to claim 1, it is characterized in that: described cytoskeleton isolation buffer liquid is by the EGTA of Mes pH6.7, the 1mM of 90mM, the MgCl of 1mM
2, 10%glycerol and 0.5%Triton X-100 formulated.
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