CN103116006B - A kind of Novel screen choosing method of cancer therapy drug - Google Patents
A kind of Novel screen choosing method of cancer therapy drug Download PDFInfo
- Publication number
- CN103116006B CN103116006B CN201310030395.5A CN201310030395A CN103116006B CN 103116006 B CN103116006 B CN 103116006B CN 201310030395 A CN201310030395 A CN 201310030395A CN 103116006 B CN103116006 B CN 103116006B
- Authority
- CN
- China
- Prior art keywords
- μms
- nitration
- ttll12
- tyrosine
- hole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of Novel screen choosing method of cancer therapy drug, its key is to comprise the following steps: the preparation of (1) TTLL12 process LAN Hep-2 cell line; (2) positive control and positive control agent is selected; (3) acquisition of nitration tyrosine optimum concentration and the suitableeest dilute concentration of medicine to be measured; (4) screen medicine, finally detect reading and compare.The invention discloses a kind of Novel screen choosing method of cancer therapy drug, have method simple, a GPRS cell chulture and Elisa technology, just grasped this law; Fast cheap; Reagent is less, variance factor is less and stable, highly sensitive, specificity good, be easy to promote and can the features such as extensive examination be carried out, and medicine to be measured can be obtained by this experiment simultaneously whether there are antitumaous effect and its antitumaous effect mechanism two kinds of information, greatly accelerate medicinal application to be measured in clinical speed, be expected to the life retrieving more patients.
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 a large class serious threat human life and healthy disease.Chemotherapy is as the treatment means of general, start from 1948 using antifol as antineoplastic, be incorporated in leukemic treatment, in the complex treatment of malignant tumour, there is irreplaceable status, but due to current medicament categories limited, and there is resistance phenomenon, 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: responsive, directly evaluates cell proliferation dead.And its shortcoming is real: low for 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 division of the method not dependent cells.But this method needs artificial counting cell, be subject to the impact of personal error.
3, nucleic precursor mix experiment (
3h-Tdr Assay), sample inhaled by the less pin that therefore can use of cell number needed for this method, but this method only measures the cell of in sample 5% ~ 10%, and thymine and normal cell all can affect measurement result, and there is radioactive contamination.
4, tetrazolium salts decoration method (MTT Assay), mtt assay can carry out the analysis of a large amount of sample within a short period of time.But some factor also can affect its result: the quality of dimethyl sulfoxide, 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 measurement intracellular ATP levels, thus reflection chemotherapeutics is to the kill capability of tumour cell, also can high value be detected when tumour cell quantity is little, there is hypersensitivity.
6, total protein decoration method (Sulforhodamine B Assay, SRB), is a kind of protein bound dyestuff, can be combined by the basic amino acid in the large molecule of intracellular biological, is good linear relationship at the absorbance log reading of 5l5nm and cell number.
7, fluorescence method (Fluorometric Microculture Cytotoxicity Assay, FMCA), this method can send fluorescence after utilizing living cells to be hydrolyzed two acetate fluorescein, can be reflected the number of living cells in cultivating system by the intensity measuring fluorescence.But control group reading is too low sometimes, causes the failure of an experiment.
8, collagen gel drop implants cultivation (CD-DST), and this method has the advantages such as cancer cell culture success ratio is high, 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 Tumor suppression growth, and cannot understand the mechanism of anticancer action of filtered out medicine, if do not understand mechanism of drug action, then impact is applied to clinical research greatly; To understand mechanism of drug action, still needing and carrying out other experiments and study, consumption regular hour and funds.
And be integrated into tubulin according to nitration tyrosine known to document by tubulin tyrosine ligase, form nitration tyrosine tubulin, this can change the structure of microtubule significantly, cause the distortion of cell, the decline of Adhering capacity and transmitter loss obstacle, natural death of cerebral cells and organ dysfunction finally can be caused to lose.(see: Fukushima N, Furuta D, Hidaka Y, Moriyama R, Tsujiuchi T.Post-translational modifications of tubulin in the nervous system [J] .JNeurochem.2009 May; 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, thus the growth of adjusting head carcinoma of neck cell, TTLL12 increases, the minimizing of nitration tyrosine tubulin can be caused, the nitration tyrosine tubulin that caused cancer cell to be escaped is to its strike, and can continue propagation, therefore TTLL12 has the function of latent carcinoma gene.(see: Li Yadong, the people such as Jinsong ZHANG, tubulin tyrosine ligase analog 12 promotes Hep-2 Growth of Cells [J] by interference tubulin nitrotyrosine, 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 Tumor suppression growth, and cannot understand the mechanism of anticancer action of filtered out medicine, if do not understand mechanism of drug action, then impact is applied to clinical research greatly; To understand mechanism of drug action, still needing and carrying out other experiments and study, consumption regular hour and funds.
Summary of the invention
For solving above technical matters, the object of the present invention is to provide the Novel screen choosing method of cancer therapy drug that a kind of energy Large-scale Screening goes out the growth of effective Tumor suppression, that understand its mechanism of anticancer action.
The present invention seeks to realize like this:
A Novel screen choosing method for cancer therapy drug, its key is to comprise the following steps:
(1) preparation of TTLL12 process LAN Hep-2 cell line
Designed, designed primer, positive-sense strand 5 '-CGGGATCCCCGATGGACTACCACGAGGA-3 ', antisense strand 5 '-CGGGATCCTAGCTACAGGACGCCCCCG-3 ', take TTLL12cDNA as template amplification TTLL12 genetic fragment, the size obtaining genes of interest fragment is 2818bp, again TTLL12 genetic fragment is inserted in pSG-5 plasmid, then recombinant plasmid is chemically distinguished transformation of E. coli JM109, again by Transfected Recombinant Plasmid Hep-2 cell, pick out indivedual clone expressing TTLL12, continue to cultivate, through repeatedly Secondary Culture, finally obtain the Hep-2 cell line stablizing process LAN TTLL12,
(2) the selecting of positive control and positive control agent
100uM taxol and thiophene propylene are cultivated in containing the nutrient solution of nitration tyrosine with the Hep-2 cell line of stable process LAN TTLL12 respectively, then row western tests, the known taxol of result can increase nitration tyrosine tubulin, there is the effect suppressing TTLL12 function, can be used as positive control; And thiophene propylene can not impel the formation of nitration tyrosine tubulin, not there is the effect suppressing TTLL12 function, can be used as negative control;
(3) acquisition of nitration tyrosine optimum concentration and the suitableeest dilute concentration of medicine to be measured
By TTLL12 process LAN Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, MEM nutrient solution volume is 80 μ L, is 37 DEG C, 5%CO
2in incubator, cultivate 6 hours, adding final concentration is respectively 50 μMs, 100 μMs, 200 μMs, 400 μMs, 800 μMs of nitration tyrosine, every hole is totally 10 μ L, and due to Elisa, comparatively western is responsive, and therefore the consumption of taxol and thiophene propylene (Thiirene) is all less than 100 μMs (western consumptions), adding final concentration is respectively 5 μMs, 10 μMs, 20 μMs, 4 dilutabilitys such as 40 μMs of grades, every hole is totally 10 μ L, dilutability different from nitration tyrosine carries out square formation titration, continues at 37 DEG C, 5%CO
2in incubator, cultivate 24 hours.Afterwards, nutrient solution in sucking-off 96 orifice bore, every hole adds 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, then damping fluid is removed, every hole adds the PBS that 100 μ L contain 3.7% formaldehyde and 0.05%Tween-20 and fixes 10 minutes, then immobile liquid is removed, every hole adds the horseradish peroxidase-labeled nitration phosphotyrosine antibody 1:1000 that 100 μ L PBS dilute, at room temperature with cytosis 1 hour, then remove antibody-solutions, rinse aperture with the PBS200 μ L containing 0.05%Tween-20, then add OPD (the o-phenylenediamine)-H of 100ul
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilizes detector at 450nm place reading, and the optimum concentration finally obtaining nitration tyrosine is 400 μMs, and the suitableeest dilute concentration of medicine to be measured is 10 μMs;
(4) medicine is screened
By TTLL12 process LAN 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 DEG C, 5%CO
2in incubator, cultivate 6 hours, adding final concentration is respectively 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 μMs, and every hole is totally 10 μ L, and medicine to be measured and taxol all repeat 6 holes, continues at 37 DEG C, 5%CO
2in incubator, after cultivating 24 hours, nutrient solution in sucking-off 96 orifice bore, add every hole 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, remove damping fluid, every hole adds the PBS that 100 μ L contain 3.7% formaldehyde and 0.05%Tween-20 and fixes 10 minutes, then immobile liquid is removed, every hole adds the horseradish peroxidase-labeled nitration phosphotyrosine antibody 1:1000 that 100 μ LPBS dilute, at room temperature with cytosis 1 hour, then antibody-solutions is removed, aperture is rinsed with the PBS 200 μ L containing 0.05%Tween-20, then OPD (the o-phenylenediamine)-H of 100 μ L is added
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 OD450nm value is greater than 1, then illustrate that this medicine has antitumaous effect, and there is the function suppressing TTLL12, promote the combination of nitration tyrosine and α-tubulin, by participating in the mechanism that tubulin is modified, forming more nitration tyrosine tubulin, causing the cancer suppressing action mechanism of cancer cell death, otherwise then not there is above-mentioned cancer suppressing action mechanism.
Above-mentioned cytoskeleton isolation buffer liquid is by the MgCl of the Mes pH 6.7 of 90mM, EGTA, 1mM 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, and a GPRS cell chulture and elisa technology, just grasped this law; Fast cheap; Reagent is less, variance factor is less and stable, highly sensitive, specificity good, be easy to promote and can the features such as extensive examination be carried out, and medicine to be measured can be obtained by this experiment simultaneously whether there are antitumaous effect and its antitumaous effect mechanism two kinds of information, greatly accelerate medicinal application to be measured in clinical speed, be expected to the life retrieving more patients.
Accompanying drawing explanation
Fig. 1 is process flow diagram of the present invention;
The electrophoretogram of gene TTLL12 for the purpose of Fig. 2;
Fig. 3 is that (M is 1kd DNA for the double digestion electrophoretogram of recombinant plasmid pSG-5-TTLL12; 1 is the double digestion of pSG-5-TTLL12);
Fig. 4 is the expression of results figure of Hep-2 cell TTLL12 albumen after Western blot detection transfection;
Fig. 5 is that immunofluorescence experiment detects expression (× 200) the result figure of Hep-2 cell TTLL12 albumen after transfection (a is for containing empty plasmid Hep-2 cell; B is the Hep-2 cell of stable high expressed TTLL12);
Fig. 6 is that taxol (paclitaxel) and thiophene propylene (Thiirene) affect result figure to nitration tyrosine tubulin.
Embodiment
Embodiment:
A Novel screen choosing method for cancer therapy drug, its key is to comprise the following steps:
(1) preparation of TTLL12 overexpressing cell strain
1) pcr amplification TTLL12 genetic fragment
Designed, designed primer, positive-sense strand 5 '-CGGGATCCCCGATGGACTACCACGAGGA-3 ', antisense strand 5 '-CGGGATCCTAGCTACAGGACGCCCCCG-3 ' take TTLL12cDNA as template amplification TTLL12 genetic fragment.Genes of interest clip size is 2818bp, PCR reactant liquor composition: 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, adds sterilizing pure water to 25 μ L.PCR loop parameter: 94 DEG C of 5min, 94 DEG C of 50s, 55 DEG C of 30s, 72 DEG C of 30s, 35cycle, 72 DEG C of 8min.PCR primer is identified: the agarose gel electrophoresis 1% is separated qualification.The results are shown in Figure 2.
2) TTLL12 genetic fragment is inserted pSG-5 plasmid
Enzyme cuts TTLL12 and pSG-5 plasmid respectively, and the enzyme time of cutting is at 37 DEG C 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/BamH I 2 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 completely.Identified by PCR primer, the results are shown in Figure 3.Again the enzyme reclaiming purifying is cut genes of interest fragment to be connected with the pSG-5 fragment that enzyme is cut.Reaction conditions: 16 DEG C of water-baths connect spends the night, 70 DEG C of water-bath 10min deactivation T
4dNA ligase.Coupled reaction system following (pSG-5/ANO1 1 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 chemically distinguished transformation of E. coli JM109.
3) set up the Hep-2 cell line stablizing high expressed TTLL12 and utilize Western to detect the TTLL12 expression of transfection cell strain
In 37 DEG C, 5%CO
2in incubator, containing 10% calf serum, 1mM Sodium Pyruvate, 2mM glutamine, cultivates Hep-2 cell in the Eagle's medium (modified Eagle ' s medium, MEM) of the change of 40ug/mL gentamicin, 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 the pSG-5-purine containing TTLL12 fragment respectively, in Ep pipe, first add serum-free and antibiotic MEM nutrient solution 250ul and plasmid 4.0 μ g, mixing, room temperature places 5min.In Ep pipe, add serum-free and antibiotic MEM nutrient solution 250 μ L and Lipofectamine2000 8.0 μ L, mixing, room temperature places 5min simultaneously.Then mixed by the liquid of above-mentioned two Ep pipes, room temperature is diluted to 1mL, is then added culture plate, jiggled, mixed, uniform fold cell surface, put 37 DEG C, 5%CO after placing 20min
2cultivate in incubator, change the MEM nutrient solution containing 2ug/mL purine of 10%FBS concentration after 5h, screening cell, after 18 days, picks out indivedual clone expressing TTLL12.Continue to cultivate, through repeatedly going down to posterity, after 2 months, the final stable cell line obtaining TTLL12 process LAN.
By the Hep-2 cell of transfection with after PBS cleaning twice, add the cell pyrolysis liquid of 400 μ L, collect cracking afterproduct, denatured by boiling, prepare standard protein solution with bovine serum albumin(BSA) (BSA), measure total protein concentration, the capable protein electrophoresis of 12%SDS-PAGE separation gel, forwards to after electrophoresis on nitrocellulose filter, and 1h closed by the milk of 5%, then add primary antibodie (anti-TTLL12,1 ︰ 700 dilutes; Anti-TBP, 1 ︰ 2000 dilutes) solution hatches 3h, TBST washs 3 times, after each 10min, 3 times are washed with two anti-(horseradish peroxidase-labeled mouse-anti rabbit igg antibody, 1 ︰ 1000 dilutes) incubated at room temperature 2h, TBST, after each 10min, DAB chromogenic reagent box is utilized to 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 6 orifice plates, the Hep-2 cell line of stable process LAN TTLL12 set up and the Hep-2 cell line of empty plasmid transfection are added respectively 6 orifice plates and cultivate.Climb after full slide until cell, discard nutrient solution, PBS liquid rinses 3 times, drip 100% ethanol fixed cell, normal temperature 10 minutes, 1moL/L non-ionics Triton-X100 changes process 10min thoroughly, with cold 0.01moL/L pH7.4PBS liquid soaking flushing 3 times, finally with distilled water flushing 1 time, prevent spontaneous fluorescence.Suitably dilute antibody with the PBS of 0.01moL/L, pH7.4, cover slide.Build 6 orifice plate lids, outsourcing l Water Paper keeps moistening, and 37 DEG C of insulation shaking tables shake 60min.Discard liquid in hole, rinse 3 times with the PBS of 0.01moL/L, pH7.4.Suck excessive moisture, but do not make sample dry, drip certain dilution fluorescence labeling two and resist, cover slide.Build 6 orifice plate lids, outsourcing l Water Paper keeps moistening, and 37 DEG C of insulation shaking tables shake 60min; Take out slide, suck excessive moisture with filter paper, drip a glycerol buffer (glycerine and pH7.4PBS liquid mix with 9:1 ratio, and glycerine has the effect reducing non-specific fluorescence), then coated with cover glass.Observe under fluorescent microscope high power field, the results are shown in Figure 5.
(2) the selecting of positive control and positive control agent
Taxol and thiophene propylene are carried out western test, and step is as follows: by TTLL12 high expressed Hep-2 cell line with every hole 2 × 10
5individual cell, plants into 6 orifice plates, and cultivate with the nutrient solution containing 50 μMs of nitration tyrosine, test group adds 100 μMs of taxols or thiophene propylene, and control group does not add compound, and every strain cell repeats 3 holes; Every 48h upgrades 1 nutrient solution, extracts total protein of cell and carry out western test after 60h, detects N-tub, TBP and α-tubulin.The results are shown in Figure 6.
Confirm that taxol can increase nitration tyrosine tubulin through western, illustrate that it has the effect suppressing TTLL12 function, can be used as positive control.And thiophene propylene does not form nitration tyrosine tubulin, illustrate that it does not have the effect suppressing TTLL12 function, can be used as negative control.
(3) acquisition of nitration tyrosine optimum concentration and the suitableeest dilute concentration of medicine to be measured
1) by TTLL12 process LAN Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, MEM nutrient solution volume is 80 μ L, is 37 DEG C, 5%CO
2in incubator, cultivate 6 hours, adding final concentration is respectively 50 μMs, 100 μMs, 200 μMs, 400 μMs, 800 μMs of nitration tyrosine, every hole is totally 10 μ L, and due to Elisa, comparatively western is responsive, and therefore the consumption of taxol and thiophene propylene (Thiirene) adopts and is less than 100 μMs (western consumptions), adding final concentration is respectively 5,10,20,4 dilutabilitys such as 40 μMs of grades, every hole is totally 10 μ L, dilutability different from nitration tyrosine carries out square formation titration, continues at 37 DEG C, 5%CO
2in incubator, cultivate 24 hours. afterwards, nutrient solution in sucking-off 96 orifice bore, every hole adds 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, then damping fluid is removed, every hole adds the PBS that 100 μ L contain 3.7% formaldehyde and 0.05%Tween-20 and fixes 10 minutes, then immobile liquid is removed, every hole adds the horseradish peroxidase-labeled nitration phosphotyrosine antibody 1:1000 that 100 μ L PBS dilute, at room temperature with cytosis 1 hour, then antibody-solutions is removed, aperture is rinsed with the PBS 200 μ L containing 0.05%Tween-20, then OPD (the o-phenylenediamine)-H of 100 μ L is added
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilizes detector at 450nm place reading.The results are shown in Table 1 and table 2.
Table 1 is OD450nm value
Table 2 is P/N value
As can be seen from square formation test findings, nitration tyrosine concentration is 400 μMs, when drug concentration to be measured is 10 μMs, the OD450nm value of positive control is about 1.0, and the OD450nm value of negative control is lower, P/N value is the highest, and therefore we obtain nitration tyrosine concentration is 400 μMs, and compound concentration is 10 μMs.
2) the sensitivity test of drug sensitivity testing in vitro method
By taxol respectively with 80 μMs, 40 μMs, 20 μMs, 10 μMs, 5 μMs, 2.5 μMs concentration, be dissolved in MEM nutrient solution, every hole adds 10 μ L, detects its OD 450nm value according to the above test method set up.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 |
The OD450nm value of result display taxol least concentration 2.5 μMs is still greater than the negative control OD450nm value of 10 μMs, 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 set up, taxol is as positive control, 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), fluorouracil (5-fluorouracil, 5-Fu) 4 kinds of medicines to be measured are tested, and are dissolved in by 10uM compound 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 | 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 all has nothing to do with microtubule, that is, they to microtubule all without influence, their OD450nm of result display is all less than positive control value, show that this experimental specificity is good, only screen the effective cancer therapy drug that those affect microtubule; In addition because this test testing sample is compound, not containing other protein matter, can not react with nitration phosphotyrosine antibody, therefore 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.Every part of sample parallel establishes 6 repetitions in a collection of test, revision test in carrying out batch.In addition 4 different tests day replication taxol and other 4 kinds of compounds, every part of sample parallel establishes 6 repetition, and revision test between carrying out batch, calculates 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 replica test result
Result display variation within batch coefficient is between 3.5% ~ 7.87%, and interassay coefficient of variation, between 2.86% ~ 6.99%, is all less than 10%, has good repeatability.
(4) medicine is screened
By TTLL12 process LAN 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 DEG C, and 5%CO
2in incubator, cultivate 6 hours, adding final concentration is respectively 400 μMs of nitration tyrosine, and every hole is totally 10 μ L, take taxol as positive control, the final concentration of medicine to be measured and taxol is 10 μMs, and every hole is totally 10 μ L, medicine to be measured and taxol all repeat 6 holes, continue at 37 DEG C, 5%CO
2in incubator, cultivate 24 hours. afterwards, nutrient solution in sucking-off 96 orifice bore, add every hole 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, remove damping fluid, every hole adds the PBS that 100 μ L contain 3.7% formaldehyde and 0.05%Tween-20 and fixes 10 minutes, then immobile liquid is removed, every hole adds the horseradish peroxidase-labeled nitration phosphotyrosine antibody 1:1000 that 100 μ L PBS dilute, at room temperature remove antibody-solutions with cytosis after 1 hour, aperture is rinsed with the PBS200 μ L containing 0.05%Tween-20, then OPD (the o-phenylenediamine)-H of 100 μ l is added
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilizes 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 |
Sample ID: 1 is yohimbane (yohimban); 2 is ergotaman (ergotaman); 3 is yaruru rice fixed (aspidospermidine); 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 is 2,7,9-triazaphenathrene (2,7,9-triazaphenanthrene); 19 is 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 guan (coronene); 26 Wei perylene (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 ring (Oxetane); 39 is azetidine (Azetidine); 40 is cyclo-butane (Cyclobutane)
Visible according to test findings, the OD450nm value of major part sample is all 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 nocodazole can promote microtubule polymerization, suppress microtubule depolymerization, extend mitosis, cause Apoptosis.(Li P,Zhou L,Dai Z,Jin X,Liu X,Matsumoto Y,FurusawaY,Li Q.High LET radiation enhances nocodazole Induced cell death inHeLa 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 sample chemical names are called epothilone B (patupilone), By consulting literatures, learn that epothilone B (patupilone) Main Function promotes that tubulin polymerization forms microtubule and stable microtubule; T suppression cell forms normal Spindle, the growth of interference tumour cell, even induces it dead.(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, be 1.057, No. 27 sample chemical names are called Docetaxel (docetaxel), By consulting literatures, learn that Docetaxel (docetaxel) is the paclitaxel analogs obtained by semisynthesis.By promoting microtubule polymerization, suppressing microtubule depolymerization to affect the mitosis of cell, thus play its cytotoxicity.(Guo Ruiling, Wu Guoming, hide 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 the effective antitumor medicine affecting microtubule, and in this method, 1 96 orifice plate can detect 16 kinds of medicines, if multiple 96 orifice plates of continuous detecting simultaneously, in 32 hours, just can detect hundreds of medicine, this high throughput test, be suitable for carrying out extensive examination.
Claims (1)
1. a screening technique for cancer therapy drug, is characterized in that comprising the following steps:
(1) preparation of TTLL12 process LAN Hep-2 cell line
Designed, designed primer, positive-sense strand 5 '-CGGGATCCCCGATGGACTACCACGAGGA-3 ', antisense strand 5 '-CGGGATCCTAGCTACAGGACGCCCCCG-3 ', take TTLL12cDNA as template amplification TTLL12 genetic fragment, the size obtaining genes of interest fragment is 2818bp, again TTLL12 genetic fragment is inserted in pSG-5 plasmid, then recombinant plasmid is chemically distinguished transformation of E. coli JM109, again by Transfected Recombinant Plasmid Hep-2 cell, pick out indivedual clone expressing TTLL12, continue to cultivate, through repeatedly Secondary Culture, finally obtain the Hep-2 cell line stablizing process LAN TTLL12,
(2) the selecting of positive control and positive control agent
100uM taxol and thiophene propylene are cultivated in containing the nutrient solution of nitration tyrosine with the Hep-2 cell line of stable process LAN TTLL12 respectively, then row western tests, the known taxol of result can increase nitration tyrosine tubulin, there is the effect suppressing TTLL12 function, can be used as positive control; And thiophene propylene can not impel the formation of nitration tyrosine tubulin, not there is the effect suppressing TTLL12 function, can be used as negative control;
(3) acquisition of nitration tyrosine optimum concentration and the suitableeest dilute concentration of medicine to be measured
By TTLL12 process LAN Hep-2 cell line with 10000, every hole, be inoculated in 96 orifice plates, MEM nutrient solution volume is 80 μ L, is 37 DEG C, 5%CO
2in incubator, cultivate 6 hours, adding final concentration is respectively 50 μMs, 100 μMs, 200 μMs, 400 μMs, 800 μMs of nitration tyrosine, every hole is totally 10 μ L, and due to Elisa, comparatively western is responsive, therefore the consumption of taxol and thiophene propylene is all less than 100 μMs, and adding final concentration is respectively 5 μMs, 10 μMs, 20 μMs, 40 μMs of 4 dilutabilitys, every hole is totally 10 μ L, dilutability different from nitration tyrosine carries out square formation titration, continues at 37 DEG C, 5%CO
2in incubator, after cultivating 24 hours, nutrient solution in sucking-off 96 orifice bore, every hole adds 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, then damping fluid is removed, every hole adds the PBS that 100 μ L contain 3.7% formaldehyde and 0.05%Tween-20 and fixes 10 minutes, then immobile liquid is removed, every hole adds the horseradish peroxidase-labeled nitration phosphotyrosine antibody 1:1000 that 100 μ L PBS dilute, at room temperature with cytosis 1 hour, then antibody-solutions is removed, aperture is rinsed with the PBS 200 μ L containing 0.05%Tween-20, then the OPD-H of 100 μ L is added
2o
2develop the color 3 minutes, each hole adds the 2moL/L H of 50 μ L
2sO
4cessation reaction, utilizes detector at 450nm place reading, and the optimum concentration finally obtaining nitration tyrosine is 400 μMs, and the suitableeest dilute concentration of medicine to be measured is 10 μMs,
(4) medicine is screened
By TTLL12 process LAN 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 DEG C, 5%CO
2in incubator, cultivate 6 hours, adding final concentration is respectively 400 μMs of nitration tyrosine, and every hole is totally 10 μ L, and the final concentration of medicine to be measured and taxol is 10 μMs, and every hole is totally 10 μ L, and medicine to be measured and taxol all repeat 6 holes, continues at 37 DEG C, 5%CO
2in incubator, cultivate 24 hours. afterwards, nutrient solution in sucking-off 96 orifice bore, add every hole 100 μ L cytoskeleton isolation buffer liquid, cultivate 3 minutes, remove damping fluid, every hole adds the PBS that 100 μ L contain 3.7% formaldehyde and 0.05%Tween-20 and fixes 10 minutes, then immobile liquid is removed, every hole adds the horseradish peroxidase-labeled nitration phosphotyrosine antibody 1:1000 that 100 μ LPBS dilute, at room temperature with cytosis 1 hour, then antibody-solutions is removed, aperture is rinsed with the PBS 200 μ L containing 0.05%Tween-20, then the OPD-H of 100 μ L is added
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 OD450nm value is greater than 1, then illustrate that this medicine has antitumaous effect, and there is the function suppressing TTLL12, promote the combination of nitration tyrosine and α-tubulin, by participating in the mechanism that tubulin is modified, forming more nitration tyrosine tubulin, causing the cancer suppressing action mechanism of cancer cell death, otherwise then not there is above-mentioned cancer suppressing action mechanism,
Described cytoskeleton isolation buffer liquid is by the MgCl of the Mes pH 6.7 of 90mM, EGTA, 1mM of 1mM
2, 10%glycerol and 0.5%Triton X-100 is formulated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310030395.5A CN103116006B (en) | 2013-01-25 | 2013-01-25 | A kind of Novel screen choosing method of cancer therapy drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310030395.5A CN103116006B (en) | 2013-01-25 | 2013-01-25 | A kind of Novel screen choosing method of cancer therapy drug |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103116006A CN103116006A (en) | 2013-05-22 |
| CN103116006B true CN103116006B (en) | 2015-09-30 |
Family
ID=48414433
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310030395.5A Expired - Fee Related CN103116006B (en) | 2013-01-25 | 2013-01-25 | A kind of Novel screen choosing method of cancer therapy drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103116006B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755263A (en) * | 2016-12-05 | 2017-05-31 | 中山大学 | One kind is set up for detecting the dynamic (dynamical) method of taxol subcellular proteomics and model |
| CN106868144A (en) * | 2017-03-06 | 2017-06-20 | 广州金域医学检验中心有限公司 | The primer and method of a kind of detection new transcript of TTLL12 genes |
| CN106884049A (en) * | 2017-03-06 | 2017-06-23 | 广州金域医学检验中心有限公司 | The identification primer and its method of a kind of new transcript of TTLL12 genes |
| CN113717941B (en) * | 2020-05-26 | 2023-11-14 | 中科院合肥技术创新工程院 | Culture medium for laryngeal carcinoma epithelial cells, culture method and application of culture medium |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343633A (en) * | 2008-09-05 | 2009-01-14 | 北京大学 | HER2 stable expression cell line and application in medicament sifting motion |
| CN101392284A (en) * | 2008-11-06 | 2009-03-25 | 长春康彼达科技有限公司 | Construction and application of anticancer natural drug screening model |
| CN102115762A (en) * | 2009-12-30 | 2011-07-06 | 上海中医药大学附属普陀医院 | Method for establishment of recombinant human colon cancer cell line for high-level expression of human cyclooxygenase-2, and application thereof |
| CN102337247A (en) * | 2011-09-13 | 2012-02-01 | 西安交通大学 | Recombinant HEK293 cell with high FGFR4 expression and application thereof |
-
2013
- 2013-01-25 CN CN201310030395.5A patent/CN103116006B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343633A (en) * | 2008-09-05 | 2009-01-14 | 北京大学 | HER2 stable expression cell line and application in medicament sifting motion |
| CN101392284A (en) * | 2008-11-06 | 2009-03-25 | 长春康彼达科技有限公司 | Construction and application of anticancer natural drug screening model |
| CN102115762A (en) * | 2009-12-30 | 2011-07-06 | 上海中医药大学附属普陀医院 | Method for establishment of recombinant human colon cancer cell line for high-level expression of human cyclooxygenase-2, and application thereof |
| CN102337247A (en) * | 2011-09-13 | 2012-02-01 | 西安交通大学 | Recombinant HEK293 cell with high FGFR4 expression and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| Tubulin tyrosine ligase like 12 links to prostate cancer through tubulin posttranslational modification and chromosome ploidy;Wasylyk C等;《International Journal of Cancer》;20101130;第127卷(第11期);全文 * |
| Tubulin tyrosine like 12, a TTLL family member with SET- and TTL-like domains and roles in histone and tubulin modifications and mitosis;Jan Brants 等;《Plos One》;20121231;第7卷(第12期);全文 * |
| 微管蛋白的分离鉴定及其在抗癌药物筛选中的应用;李占荣等;《药学学报》;19860930;第21卷(第9期);全文 * |
| 微管蛋白酪氨酸连接酶类似物12和硝基化酪氨酸对前列腺癌细胞生长的影响;李雅冬等;《第二军医大学学报》;20120131;第33卷(第1期);全文 * |
| 微管蛋白酪氨酸连接酶类似物12通过干扰微管蛋白酪氨酸硝基化促进Hep-2细胞生长;李雅冬等;《中国生物化学与分子生物学报》;20120831;第28卷(第8期);摘要第6~10行，第729~731页第1.4~1.6、2.3和2.4节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103116006A (en) | 2013-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101478978B (en) | Delta-tocotrienol treatment and prevention of pancreatic cancer | |
| CN103116006B (en) | A kind of Novel screen choosing method of cancer therapy drug | |
| CN103648505B (en) | The material relevant to microRNA-21, mispairing reparation and colorectal carcinoma and method | |
| Fiorillo et al. | Bedaquiline, an FDA-approved drug, inhibits mitochondrial ATP production and metastasis in vivo, by targeting the gamma subunit (ATP5F1C) of the ATP synthase | |
| CN104017868B (en) | SETD4 is the purposes in preparation treatment pancreatic cancer drug in the purposes prepared in diagnosis of pancreatic cancer and/or prognosis kit and SETD4 blocker | |
| CN103179968A (en) | Aryl hydrocarbon receptor (ahr) modifiers as novel cancer therapeutics | |
| CN105979780A (en) | Glucocorticoid inhibitors for treatment of prostate cancer | |
| Byun et al. | Antitumor activity of pulvomycin via targeting activated-STAT3 signaling in docetaxel-resistant triple-negative breast cancer cells | |
| Pustovalova et al. | The CD44high subpopulation of multifraction irradiation-surviving NSCLC cells exhibits partial EMT-program activation and DNA damage response depending on their p53 status | |
| Lin et al. | Oxypalmatine regulates proliferation and apoptosis of breast cancer cells by inhibiting PI3K/AKT signaling and its efficacy against breast cancer organoids | |
| Wang et al. | Glutamine deprivation impairs function of infiltrating CD8+ T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis | |
| CN103966169A (en) | Chinese people tongue squamous epithelial cell cancer cell line CTSC-1 and establishment method thereof | |
| CN105943522B (en) | Clofoctol is used to prepare the purposes of the drug for the treatment of human glioma | |
| CN111235218B (en) | Third-generation EGFR-TKI drug-resistant cell strain and application thereof | |
| CN108998495B (en) | A kind of high-throughput screening method of anti-breast cancer medicines | |
| CN104981697B (en) | Promote purposes of the chromatin transcription complex (FACT) in cancer | |
| Randhawa et al. | Viral drug sensitivity testing using quantitative PCR: effect of tyrosine kinase inhibitors on polyomavirus BK replication | |
| CN103751221B (en) | The application in the medicine of preparation treatment liver cancer of the SIRT1 inhibitor associating Sorafenib | |
| CN114164176B (en) | Cisplatin-resistant cell strain for human bladder cancer and application thereof | |
| CN102329889A (en) | Primer and probe and method for detecting poxvirus muris | |
| CN107998396A (en) | A kind of target spot of oncotherapy and its application | |
| CN108472306A (en) | Adjust the composition of IRAK1 | |
| CN107095876B (en) | Diphenyl joins application of the alkenyl phosphine oxide compound in preparation treatment lung-cancer medicament | |
| CN104958306A (en) | Application of compound Hu-17 alone or in combination with platinum drugs in preparing drugs for treating ovarian cancer | |
| CN104403996A (en) | Human gastric cancer cell line with 5-fluorouracil resistance and establishing method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150930 Termination date: 20160125 |
|
| EXPY | Termination of patent right or utility model |