CN101376907A - Anti-tumor metastasis medicament high flux screening model - Google Patents
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Abstract
The invention belongs to the field of medicine and discloses an anti-tumor transfer medicine high throughput screening model. The screening method of the model comprises the following steps: an MDA-MB-435 cell is inoculated to a culture plate; a chemical compound to be screened is added into culture fluid and no inhibitor is added in; the chemical compound is cultured for an appropriate time of period and repeatedly melted, to obtain cell lysis solution; the cell lysis solution is added into human thrombinogen compound solution to be incubated together; a chromogenic substrate is added in; at 405nm, absorbency is determined and inhibitory rate is calculated; the chemical compound with the inhibitory rate higher than 20 percent is a positive compound. In the invention, the human breast cancer cell MDA-MB-435 is selected to build the anti-tumor transfer medicine screening model which is quick and stable, has high sensitivity and is convenient to be operated; the model is characterized by trace quantity and accuracy, and is applicable in screening anti-tumor transfer medicine with high throughput.
Description
Technical field
The invention belongs to pharmaceutical field, relate to anti-tumor metastasis medicament high flux screening model.
Background technology
Tissue factor (tissue factor, be called for short TF), promptly thromboplastin is the strand transmembrane glycoprotein, belongs to the cytokine superfamily member, is that physiological is ended, coagulation process and the thrombotic startup factor of multiple pathologic
[1]TF is the acceptor of proconvertin (FVII) at cell surface, is again the cofactor of FVII or FVII (a).When after the blood vessel breakage or under pathologic condition, blood is in the cell surface that can express TF, acceptor TF promptly forms mixture with its part VII or VIIa, and then activate factor X and IX simultaneously, activated FXa and FIXa further activate thrombogen (FII), FIIa makes Fibrinogen become scleroproein, thereby finishes the cascade reaction of blood coagulation, causes blood coagulation.The thromboembolic states complication of many cardiovascular disordeies such as atherosclerosis (AS), acute coronary syndrome and some disease is all closely related with TF
[2]Simultaneously, the diversity of the biological action of TF is confirmed, and except its effect in coagulation process, TF has also born frizzled receptor and other non-coagulation functions, makes it in systemic inflammation
[3], septicemia, atherosclerosis, tumour blood vessel take place and disease such as transfer in all play an important role
[4], and relevant with embryo's normal development
[5]This just makes TF or TF/FVII (a) mixture become the very attractive novel targets that anti-freezing, antithrombotic and antitumor drug etc. are found.Thereby from natural compounds, seek the TF inhibitor or its lead compound may have profound significance.
The transfer of tumour is the biological procedures of rapid, a multistage tumour cell of multistep and the interactional complexity of host cell, and is closely related with the long-term effect of oncotherapy.Roughly step is: 1. the propagation of primary tumor tumour cell reaches free; 2. tumor cell invasion basement membrane of blood vessel, migration are to blood vessel; 3. adhere to the metastasis site vascular endothelial cell, and pass vascular endothelial cell, migration to blood vessel; 4. be transferred to target organ and propagation, form metastasis.The transfer of tumour relates to many-sided extracellular, intracellular signal transduction approach, and the participation of the various kinds of cell factor, bio signal molecule is arranged.
Clinical observation in recent years and experiment in vivo and vitro research all confirm
[6]: the solid tumor of high expression level TF (as the rectum cancer) cell transfer ability is strong, and is expressed the more maternal cell height of ability of TF by the subbreed that metastatic lesion is set up.Behind TF monoclonal antibody blocking-up TF function of receptors, the tumor cell number of attacking to SCID mouse lung blood vessel is reduced, the lung metastasis reduces.The K-1735 that has the scholar will hang down expression and high expression level TF is injected to respectively in the SCID mouse body, the mouse that found that injection high expression level TF cell has 86% cancer metastasis to occur, and the low mouse of expressing the TF cell of injection only 5% metastasis of cancer occurs, and this effect can be cancelled by TF antibody
[7]
Confirm that after deliberation TF can regulate the balance between tumor vascular generation and the angiogenesis inhibitor.The interior experiment of body is found the transfectional cell of high expression level TF and is compared without cells transfected, the transfectional cell fast growth of high expression level TF, big and the rich blood vessel of gross tumor volume, low expression TF person is then opposite, and its mitogen activation of tumour cell of finding high expression level TF strengthens, VEGF transcribes enhancing, and the thrombospondin (TSP) with blood vessel formation against function is transcribed and weakened
[8]Therefore, TF may be the direct inducement that promotes that tumor vessel forms, and the generation of malignant tumour induction of vascular is one of its malignant characteristics, also is the prerequisite of tumor growth and transfer.
High-flux medicaments sifting (HTS) is nearly ten years emerging technologies of rising in the international medicine company researchdevelopment process, and it has been widely used in the screening of medicine guide thing by each big pharmaceuticals of the world.By to miscellaneous synthetic screening that reaches the natural compounds work at various medicine target bodys, therefrom seek best lead compound, and then be used for the exploitation of newtype drug.The scale that is characterized in is big, speed is fast, cost is relatively low, shortens the new drug development cycle
[15,20]
Discover that TF is relevant with generation, infiltration and the transfer of tumour, but do not have at present the cell levels tissue factor high-flux medicaments sifting model of utilization to screen the report of medicine for anti transfer of tumor as yet.
Summary of the invention
The objective of the invention is to set up a kind of tissue factor inhibitor high-flux medicaments sifting model that can be used in the screening medicine for anti transfer of tumor.
The objective of the invention is to realize by following technical proposal:
A kind of anti-tumor metastasis medicament high flux screening model, the screening method of this model is:
The MDA-MB-435 cell inoculation adds compound to be sieved respectively in culture plate in the nutrient solution, be contrast not add any inhibitor, ices repeatedly behind the co-cultivation appropriate time and melts, and gets cell pyrolysis liquid;
Cell pyrolysis liquid adds and contains CaCl
2Human Factor's solution hatch jointly, add chromophoric substrate, measure absorbancy in 405nm, calculate the TF inhibiting rate, inhibiting rate is greater than the positive compound of 20% compound.
Described anti-tumor metastasis medicament high flux screening model, wherein the cell density during the MDA-MB-435 cell inoculation is 2～4 * 10
6/ ml; Substratum is the DMEM substratum that contains 10% calf serum; Prothrombin Complex Concent-concentration is at 0.008～0.012g/ml; The time that cell pyrolysis liquid adding Human Factor solution is hatched jointly is 15～20min.
Described anti-tumor metastasis medicament high flux screening model, wherein chromophoric substrate is S-2222, chromophoric substrate concentration is 0.5mM.
Described anti-tumor metastasis medicament high flux screening model, wherein the cell density during the MDA-MB-435 cell inoculation is 4 * 10
6/ ml; Prothrombin Complex Concent-concentration is at 0.008g/ml; It is 15min that cell pyrolysis liquid adds the common incubation time of Human Factor's solution.
Described anti-tumor metastasis medicament high flux screening model, the concrete screening method of this model is:
Well-grown MDA-MB-435 cell is by 4 * 10
6/ ml is inoculated in 96 well culture plates, every hole 180 μ l enchylema, it is good to be cultured to cell state, adherent about 80%, blot substratum, blank well and control group add 200 μ l fresh cultures respectively, dosing holes adds 180 μ l fresh cultures respectively and 20 μ l treat SCREENED COMPOUND, put into 37 ℃ of incubator co-cultivation 6h, multigelation 3 times is got cell pyrolysis liquid;
18 μ l cell pyrolysis liquids and 2 μ l concentration are that Human Factor's solution of 0.01g/ml is hatched 15min jointly in 384 orifice plates, under 37 ℃, it is the S-2222 of 37 ℃ of preheatings of 0.5mM that every then hole adds 20 μ l concentration, 37 ℃ of incubation 3min, measure absorbancy with the microwell plate plate reading with 405nm, calculate inhibiting rate; Not add the negative contrast of any inhibitor, primary dcreening operation compound final concentration is 1 * 10
-5M adds inhibiting rate behind the compound greater than 20%, positive compound.
Beneficial effect of the present invention:
The present invention selects for use human breast cancer cell MDA-MB-435 successfully to set up fast and stable, medicine for anti transfer of tumor screening model highly sensitive, easy and simple to handle, has trace, characteristic of accurate, is applicable to the high flux screening medicine for anti transfer of tumor.
Description of drawings
Fig. 1: well-grown 435 cells;
Fig. 2: the influence of different cell densities to measuring;
Fig. 3: the influence of Prothrombin Complex Concent-different concns to measuring;
Fig. 4: different incubation times are to the influence of experiment;
Fig. 5: Different Ca Cl
2Concentration is to the influence of experiment;
Fig. 6: different EDTA concentration are to the influence of experiment;
Fig. 7: different chromophoric substrate concentration are to the influence of experiment;
Fig. 8: nine kinds of cell TF determination of activity.
Embodiment
The invention will be further elaborated by the following examples.
Material and method:
1. instrument
2. material and reagent:
MDA-MB-435 (human breast cancer cell, basis institute of Chinese medicine research institute cell bank), IM-3 (human liver cancer cell, liver cancer research institute of middle mountain hospital), LLC cell (fluorescent mark Lewis lung cancer cell, Nanjing University model animal center), SGC-7901 (gastric carcinoma cells, basis institute of medical courses in general institute cell bank), BGC-823 (gastric carcinoma cells, basis institute of medical courses in general institute cell bank), HL-60 (human leukemia cell, basis institute of medical courses in general institute cell bank), A549 (human lung carcinoma cell, basis institute of medical courses in general institute cell bank), L-02 (human liver cell, basis institute of medical courses in general institute cell bank), Bel-7402 cell (human liver cancer cell, basis institute of medical courses in general institute cell bank)
The DMEM substratum, (GIBCO companies) such as 1640 substratum.
The conventional reagent of cell cultures (trypsinase, lot number 1013S04, AMRESCO packing, Nanjing Sheng Xing Bioisystech Co., Ltd; Penbritin, lot number 0321S05, AMRESCO packing, Nanjing Sheng Xing Bioisystech Co., Ltd; Streptomycin sulphate, lot number 0307S05CA, AMRESCO packing, Nanjing Sheng Xing Bioisystech Co., Ltd; Sodium bicarbonate, analytical pure, lot number 021010767, Nanjing chemical reagent one factory).
Newborn calf serum (GIBCO company).
Human Factor's (magnificent blue biotechnology stock company).
S-2222(Sigma)
CaCl
2, EDTA is chemical pure.
3. solution preparation
3.1: substratum
DMEM substratum: 10g DMEM dry powder is dissolved in the 900mL tri-distilled water, adds NaHCO
33.7g, penbritin 0.1183g, Streptomycin sulphate 0.1g slowly stirs 4h, regulates pH to 7.2, is diluted to 1L, and the suction filtration degerming adds 10% calf serum during packing, 4 ℃ of preservations.
1640 substratum: 10g 1640 dry powder are dissolved in the 900mL tri-distilled water, add NaHCO
32.0g, penbritin 0.1183g, Streptomycin sulphate 0.1g slowly stirs 4h, regulates pH to 7.2, is diluted to 1L, and the suction filtration degerming adds 10% calf serum during packing, 4 ℃ of preservations.
3.2：PBS(pH7.2)
Take by weighing KCl0.2g, KH
2PO
40.2g, NaCl8g, Na
2HPO
412H
2O 2.08g is dissolved in the 1000ml tri-distilled water successively, autoclaving, 4 ℃ of preservations.
3.3: Digestive system
Take by weighing trypsinase 1.25g, be dissolved among the 500ml sterilization PBS, stirring at room 4 hours, filtration sterilization gets trypsin solution.Take by weighing EDTA0.1g, be dissolved among the 500mL PBS, be stirred to dissolving, high-temperature sterilization gets EDTA solution.With EDTA solution and trypsin solution 1:1 mixing by volume, 4 ℃ of preservations.
4. experimental procedure
4.1 determining of experiment condition
4.1.1 cell density is to the influence of experiment
With the cell density is X-coordinate, OD
405Be ordinate zou, cell density is with 10
6/ ml is a unit, and selected useful range 0.5～8 is measured the absorbance under the different cell densities in this scope, chooses only cell density.
4.1.2 the concentration of Prothrombin Complex Concent-is to the influence of experiment
With Prothrombin Complex Concent-concentration is X-coordinate, OD
405Be ordinate zou, concentration is unit with g/L, and selected useful range 6～20 is measured the absorbance under the different Prothrombin Complex Concent-concentration in this scope, chooses only Prothrombin Complex Concent-concentration.
4.1.3 incubation time is to the influence of experiment
With time is X-coordinate, OD
405Be ordinate zou, incubation time is unit with min, and selected useful range 10～35min measures the absorbance under the different incubation times in this scope, chooses only incubation time.
4.1.4 CaCl
2Concentration is to the influence of experiment
With CaCl
2Concentration is X-coordinate, OD
405Be ordinate zou, concentration is unit with mmol/L, and selected useful range 50～200 is measured Different Ca Cl in this scope
2Absorbance under the concentration is chosen only CaCl
2Concentration.
4.1.5 the influence of EDTA concentration to measuring
With EDTA concentration is X-coordinate, OD
405Be ordinate zou, concentration is unit with mmol/L, and selected useful range 50～200 is measured the absorbance under the different EDTA concentration in this scope, chooses only EDTA concentration.
4.1.6 different chromophoric substrate concentration are to the influence of experiment
With S-2222 concentration is X-coordinate, OD
405Be ordinate zou, concentration is unit with mmol/L, and selected useful range 0.0625～1 is measured the absorbance under the different chromophoric substrate concentration in this scope, chooses only chromophoric substrate concentration.
4.2 the screening of cell strain
It is good to choose growth conditions, and nine kinds of cells (MDA-MB-435, IM-3, LLC, SGC-7901, BGC-823, HL-60, A549, L-02, Bel-7402) of exponentially growth are by 4 * 10
6/ ml is inoculated in 96 well culture plates, every hole 180 μ l enchylema, and it is good to be cultured to cell state, adherent about 80%, multigelation 3 times is got cell pyrolysis liquid, adopt the chromophoric substrate method of hereinafter introducing to measure the TF activity of nine kinds of cells, choose suitable cell and carry out this experiment.
4.3 the cultivation of cell
4.3.1 cell recovery
From liquid nitrogen container, take out the cell cryopreservation pipe, put into 37 ℃ of water-baths rapidly, and shake frequently, it is melted fully.Behind the frozen pipe outer wall of 75% ethanol disinfection, 1000rmin under room temperature
-1Centrifugal 5min discards upper strata liquid, and adding 1ml DMEM+10% calf serum substratum is resuspended, be transferred in the Tissue Culture Flask, add substratum (MDA-MB-435, IM-3 DMEM substratum, all the other cells 1640 substratum again, down with) about 10ml, blow even, 37 ℃, 5%CO
2Substratum is changed in culturing cell to adherent back, continues to cultivate.
4.3.2 passage
The MDA-MB-435 cell cultures is hatched in 37 ℃, 5% CO2gas incubator in the DMEM substratum that contains 10% calf serum, changes liquid once in 2～3 days.Treat that cell removes substratum when covering with at the bottom of the Tissue Culture Flask, add the Digestive system that contains 0.25% trypsinase and 0.02% EDTA, 37 ℃ digest to cell fall in flakes (the microscopically cell becomes circle by wrinkle), add again and contain the blood serum medium termination reaction, piping and druming makes cell suspension repeatedly, be transferred to fast that 10ml is aseptic in the cap centrifuge tube 1000rmin
-1Centrifugal 5min abandons upper strata liquid, adds 5ml substratum re-suspended cell, and is transferred in the culturing bottle 37 ℃, 5%CO
2Change substratum after being cultured to cell attachment, continue to cultivate.
4.3.3 the conventional cellular form of MDA-MB-435 is observed
Get the MDA-MB-435 cell that grows to fusion in the culturing bottle, change fresh culture, with inverted phase contrast microscope observation of cell growing state.
The result shows as Fig. 1, at the bottom of cell evenly is paved with bottle, and marshalling, endochylema is full, well-grown.Get the 20th～30 generation cell be used for the test.
4.4 dosing
The MDA-MB-435 cell is by 4 * 10
6/ ml is inoculated in 96 well culture plates, every hole 180 μ l enchylema, and it is good to be cultured to cell state, adherent about 80%, blot substratum, blank well and control group (not dosing of negative control) add 200 μ l fresh cultures respectively, and dosing holes adds 180 μ l fresh cultures and 20 μ l 1 * 10 respectively
-5The various SCREENED COMPOUND for the treatment of of M are put into 37 ℃ of incubator co-cultivation 6h, and multigelation 3 times is got cell pyrolysis liquid.
4.5 the mensuration of TF procoagulant activity (chromophoric substrate method)
4.5.1 ultimate principle
Xa can make chromophoric substrate (chromogenic substrate according to TF-VIIa mixture activatory, S-2222) be decomposed into polypeptide and p-Nitroaniline (pNA), the latter has the absorption peak at the 405nm place, and with the OD value at microwell plate plate reading mensuration 405nm place, this value can reflect the activity level of TF
[6]This method sensitivity, simple, quick is the active method of mensuration TF of generally acknowledging in the world.Principle is as follows:
In addition, in conjunction with reference and practical situation, the source of commercial Human Factor (PPSB) as factor VII, X adopted in this experimental design
[6,7]
4.5.2 step
(2 μ l, 0.01g/ml contains CaCl to cell pyrolysis liquid (18 μ l) with Human Factor's solution
25mmol/l) in 384 orifice plates, hatch 15min jointly under 37 ℃, every then hole adds the Chromogenic substrate (20 μ l, 0.5mM contains 25mmol/L EDTA) of 37 ℃ of preheatings, and 37 ℃ of incubation 3min measure absorbancy with the microwell plate plate reading with 405nm
[6,8](CaCl
2, EDTA is all final concentration) step is as follows:
4.6 data processing
This experiment is not to add the negative contrast of any inhibitor, and primary dcreening operation compound final concentration is 1 * 10
-5M.With adding inhibiting rate behind the compound greater than 20%, be defined as to have and suppress active, with+expression.
5. experimental result
5.1 determining of experiment condition
5.1.1 cell density is to the influence of experiment
Cell density can directly influence the feminine gender value of measurement result, influences the screening of active compound then.According to experiment, determine that suitable cell density is 2～4 * 10
6/ ml.The results are shown in Figure-2.
5.1.2 the concentration of Prothrombin Complex Concent-is to the influence of experiment
Measuring, suitable Prothrombin Complex Concent-concentration are in 0.008～0.012g/ml scope, and the concentration that this experiment is adopted is 0.008g/ml.The results are shown in Figure-3.
5.1.3 incubation time is to the influence of experiment
Incubation time can directly influence the formation of TF-VIIa mixture, the then activation of factor of influence Xa, and the time, too short reaction was incomplete, the sensitivity of model is descended, and the reaction times was oversize, also will be difficult to reflect the true effect of inhibitor.Experiment finds that the reaction times of 15～20min is comparatively suitable, sees figure-4.The experiment incubation time is 15min.
5.1.4 CaCl
2Concentration is to the influence of experiment
From figure-5, can clearly find out, work as CaCl
2In the absorbancy maximum at 405nm place, this concentration had been adopted in this experiment just when concentration was 100mmol/L.
5.1.5 the influence of EDTA concentration to measuring
From figure-6 as can be seen, EDTA concentration be 50 or the absorbance measurement value during 100mmol/l all higher, the EDTA concentration of this experiment use 50mmol/l.
5.1.6 different chromophoric substrate concentration are to the influence of experiment
From figure-7 as can be seen, the absorbance (OD when chromophoric substrate concentration is 0.5mM
405) bigger, experiment sensitivity is higher, and concentration is suitable, is difficult for causing waste, so the chromophoric substrate concentration of 0.5mM is all adopted in later experiment.
The conditions such as working concentration of pair cell density, incubation time and all ingredients of more than testing are respectively optimized, and the results are shown in Table 1.
Table 1: the condition optimizing of testing each influence factor
5.2 the screening of cell strain (nine kinds of cell TF determination of activity)
By mensuration, filter out the active height of TF, have the human breast cancer cell MDA-MB-435 of high transfer ability as the used cell strain of modeling to LLC, SGC-7901, MDA-MB-435, BGC-823, HL-60, A549, L-02, IM-3, nine kinds of cell TF of Bel-7402 procoagulant activity.(LLC is the mice lung cancer cell, so preferentially do not select) The selection result is as table-2 and scheme-8:
Table 2: nine kinds of cell TF determination of activity
Cell strain OD
405Cell strain OD
405
Blank 0.058 HL-60 0.199
LLC 0.718 A549 0.176
7901 0.337 L-02 0.173
435 0.626 IM3 0.213
BGC 0.450 7402 0.325
Adopt optimum condition that different compounds are screened by embodiment 1 method, the results are shown in Table 2,3.
Optimum condition is: cell adopts MDA-MB-435, and the cell density during cell inoculation is 4 * 10
6/ ml; Prothrombin Complex Concent-concentration is at 0.008g/ml; It is 15min that cell pyrolysis liquid adds the common incubation time of Human Factor's solution.Chromophoric substrate is S-2222, and chromophoric substrate concentration is 0.5mM, CaCl
2Optimal concentration is 100mmol/L, and the EDTA optimal concentration is 50mmol/L.
Working method: well-grown MDA-MB-435 cell is by 4 * 10
6/ ml is inoculated in 96 well culture plates, every hole 180 μ l enchylema, it is good to be cultured to cell state, adherent about 80%, blot substratum, blank well and control group add 200 μ l fresh cultures respectively, dosing holes adds 180 μ l fresh cultures respectively and 20 μ l treat SCREENED COMPOUND, put 37 ℃ of incubator co-cultivation 6h, multigelation 3 times is got cell pyrolysis liquid;
18 μ l cell pyrolysis liquids and 2 μ l concentration are that Human Factor's solution of 0.01g/ml is hatched 15min jointly in 384 orifice plates, under 37 ℃, it is the S-2222 of 37 ℃ of preheatings of 0.5mM that every then hole adds 20 μ l concentration, 37 ℃ of incubation 3min, measure absorbancy with the microwell plate plate reading with 405nm, calculate inhibiting rate; Not add the negative contrast of any inhibitor, primary dcreening operation compound final concentration is 1 * 10
-5M adds inhibiting rate behind the compound greater than 20%, positive compound.
Table 2, part primary dcreening operation result
| Sample number into spectrum | OD405 | Inhibiting rate (%) | Active | Sample number into spectrum | OD405 | Inhibiting rate (%) | Active |
| Negative control | 0.5276 | 104249 | 0.3462 | 34.4 | + | ||
| ZM105711 | 0.3915 | 25.8 | + | 103286 | 0.3604 | 31.7 | + |
| ZM211284 | 0.5892 | 0 | ZM105717 | 0.512 | 3 | ||
| ZM211300 | 0.7034 | 0 | ZM211290 | 0.6181 | 0 | ||
| 105590 | 0.4966 | 5.9 | 105597 | 0.4695 | 11 | ||
| 104816 | 0.3538 | 32.9 | + | 105596 | 0.4304 | 18.4 | |
| 104507 | 0.3929 | 25.5 | + | 104966 | 0.3773 | 28.5 | + |
| 104343 | 0.3622 | 31.4 | + | 104392 | 0.7087 | 0 | |
| 104147 | 0.4078 | 22.7 | + | 104173 | 0.3381 | 35.9 | + |
| ZM105712 | 0.4258 | 19.3 | 103288 | 0.4428 | 16.1 | ||
| ZM211285 | 0.5121 | 2.9 | ZM105718 | 0.4378 | 17 | ||
| ZM211301 | 0.4085 | 22.6 | + | ZM211291 | 0.4079 | 22.7 | + |
| 105591 | 0.4863 | 7.8 | 105598 | 0.4236 | 19.7 | ||
| 104817 | 0.5392 | 0 | 211282 | 0.45 | 14.7 | ||
| 104508 | 0.3842 | 27.2 | + | 104967 | 0.4164 | 21.1 | + |
| 104245 | 0.3676 | 30.3 | + | 104393 | 0.7004 | 0 | |
| 04148 | 0.3667 | 30.5 | + | 104174 | 0.364 | 31 | + |
| ZM105713 | 0.6406 | 0 | ZM105719 | 0.3934 | 25.4 | + | |
| ZM211286 | 0.5537 | 0 | ZM211292 | 0.5088 | 3.6 | ||
| ZM211302 | 0.395 | 25.1 | + | 105586 | 0.4238 | 19.7 | |
| 05592 | 0.4141 | 21.5 | + | 211280 | 0.4482 | 15.1 | |
| 04818 | 0.3324 | 37 | + | 104969 | 0.5521 | 0 | |
| 04517 | 0.5425 | 0 | 104331 | 0.4108 | 22.1 | + | |
| 04246 | 0.3457 | 34.5 | + | 104175 | 0.4438 | 15.9 | |
| 04149 | 0.3767 | 28.6 | + | ZM105720 | 0.4143 | 21.5 | + |
| ZM105714 | 0.3949 | 25.2 | + | ZM211293 | 0.6794 | 0 | |
| ZM211287 | 0.5401 | 0 | 05587 | 0.8701 | 0 | ||
| ZM211303 | 0.4267 | 9.1 | 211278 | 0.5382 | 0 | ||
| 05593 | 0.4398 | 6.6 | 104971 | 0.6645 | 0 | ||
| 04819 | 0.3602 | 31.7 | + | 104334 | 0.8881 | 0 | |
| 04389 | 0.4074 | 22.8 | + | 104195 | 0.4101 | 22.3 | + |
| 104247 | 0.3776 | 28.4 | + | ZM105721 | 0.4348 | 17.6 |
| 104150 | 0.3785 | 28.3 | + | ZM211298 | 0.3892 | 26.2 | + |
| ZM105715 | 0.6207 | 0 | 105588 | 0.3816 | 27.7 | + | |
| ZM211288 | 0.5489 | 0 | 211281 | 0.4545 | 13.9 | ||
| ZM211304 | 0.3713 | 29.6 | + | 104981 | 0.6375 | 0 | |
| 105594 | 0.415 | 21.3 | + | 104335 | 0.4114 | 22 | + |
| 104820 | 0.3854 | 27 | + | 104196 | 0.4129 | 21.7 | + |
| 104390 | 0.4756 | 9.9 | ZM105722 | 0.6593 | 0 | ||
| 104248 | 0.3502 | 33.6 | + | ZM211299 | 0.5132 | 2.7 | |
| 103284 | 0.3588 | 32 | + | 105589 | 0.3948 | 25.2 | + |
| ZM105716 | 0.4811 | 8.8 | 211279 | 0.4246 | 19.5 | ||
| ZM211289 | 0.4941 | 6.4 | 104503 | 0.4182 | 20.7 | + | |
| ZM211305 | 0.4088 | 22.5 | + | 104337 | 0.5261 | 0.3 | |
| 105595 | 0.5125 | 2.9 | 104146 | 0.4743 | 10.1 | ||
| 104965 | 0.492 | 6.8 | DT-13 | 0.3488 | 33.9 | + | |
| 104391 | 0.5559 | 0 | MD | 0.3776 | 23.5 | + |
Table 3, part are sieved the result again
| Sample number into spectrum | OD405 | Inhibiting rate (%) | Active | Sample number into spectrum | OD405 | Inhibiting rate (%) | Active |
| Negative control | 0.77524 | 104507 | 1.035 | 0 | |||
| ZM105711 | 0.5737 | 26 | + | 104508 | 0.7068 | 8.8 | |
| ZM105714 | 0.5072 | 34.6 | + | 104389 | 1.1984 | 0 | |
| ZM105719 | 0.5573 | 28.1 | + | 104331 | 0.685 | 11.6 | |
| ZM105720 | 0.7515 | 3.1 | 104335 | 0.6614 | 14.7 | ||
| ZM211291 | 0.8179 | 0 | 104343 | 0.7792 | 0 | ||
| ZM211298 | 0.7825 | 0 | 104245 | 0.8875 | 0 | ||
| ZM211301 | 0.5582 | 28 | + | 104246 | 0.8344 | 0 | |
| ZM211302 | 1.0985 | 0 | 104247 | 0.3732 | 51.9 | ++ | |
| ZM211304 | 0.7788 | 0 | 104248 | 0.8307 | 0 | ||
| ZM211305 | 0.6616 | 14.7 | 104249 | 0.4216 | 45.6 | + | |
| 105588 | 0.5015 | 35.3 | + | 104173 | 0.8933 | 0 | |
| 105589 | 0.8847 | 0 | 104174 | 0.397 | 48.8 | + | |
| 105594 | 0.5464 | 29.5 | + | 104196 | 0.7079 | 8.7 | |
| 104816 | 0.5467 | 29.5 | + | 104147 | 0.47 | 39.4 | + |
| 104818 | 0.5115 | 34 | + | 104148 | 1.3759 | 0 | |
| 104819 | 0.7274 | 6.2 | 104149 | 0.9856 | 0 | ||
| 104820 | 0.5846 | 24.6 | + | 104150 | 0.8292 | 0 | |
| 104966 | 1.307 | 0 | 03284 | 0.7944 | 0 | ||
| 104967 | 0.4368 | 43.7 | + | 103286 | 0.7539 | 2.8 | |
| 104503 | 0.3823 | 50.7 | ++ | 103289 | 0.5288 | 31.8 | + |
| DT-13a | 0.3325 | 57.1 | ++ | MD a | 0.4873 | 37.1 | + |
| DT-13b | 0.503 | 35.1 | + | MD b | 0.9771 | 0 | |
| DT-13c | 0.5512 | 28.9 | + | MD c | 0.495 | 36.1 | + |
| DT-13d | 0.5128 | 33.2 | + | MD d | 1.1251 | 0 |
Illustrate: a:1 x 10
-5M, b:1 x 10
-6M, c:1 x 10
-7M, d:1 x 10
-8M.
Being numbered 104967 compound is curcumine; Being numbered 104147 compound is Berberine; DT-13 is liriope muscari Baily saponin(e C; The structure and the title of remaining numbering compound are controlled by the sample supplier.
Because TF can regulate the balance between tumor vascular generation and the angiogenesis inhibitor, the tumour cell transfer ability of high expression level TF is strong, and therefore, the higher compound of TF inhibiting rate that screening obtains can be used as medicine for treating tumor metastasis and further studies.
Reference examples: be screening model screening DT13 with Bel-7402 and the results are shown in Table 4 (other experimental techniques and condition are identical), the result shows employing Bel-7402 cell modeling and adopts the MDA-MB-435 cell to compare susceptibility low, is unfavorable for the screening of medicine for anti transfer of tumor.
Table 4
| Sample number into spectrum | OD405 | Inhibiting rate (%) | Active |
| Negative control | 0.3152 | ||
| DT-13 a | 0.2521 | 20.01 | + |
| DT-13 b | 0.2836 | 10.0 | |
| DT-13 c | 0.2934 | 7 | |
| DT-13 d | 0.3201 | 0 |
a：1 x 10
-5M，b：1 x 10
-6M，c：1 x 10
-7M，d：1 x 10
-8M。
Claims (5)
1, a kind of anti-tumor metastasis medicament high flux screening model is characterized in that the screening method of this model is:
The MDA-MB-435 cell inoculation adds compound to be sieved respectively in culture plate in the nutrient solution, be contrast not add any inhibitor, ices repeatedly behind the co-cultivation appropriate time and melts, and gets cell pyrolysis liquid;
Cell pyrolysis liquid adds and contains CaCl
2Human Factor's solution hatch jointly, add chromophoric substrate, measure absorbancy in 405nm, calculate the TF inhibiting rate, inhibiting rate is greater than the positive compound of 20% compound.
2, according to the described anti-tumor metastasis medicament high flux screening model of claim 1, the cell density when it is characterized in that the MDA-MB-435 cell inoculation is 2～4 * 10
6/ ml; Substratum is the DMEM substratum that contains 10% calf serum; Prothrombin Complex Concent-concentration is at 0.008～0.012g/ml; The time that cell pyrolysis liquid adding Human Factor solution is hatched jointly is 15～20min.
3, according to the described anti-tumor metastasis medicament high flux screening model of claim 1, it is characterized in that chromophoric substrate is S-2222, chromophoric substrate concentration is 0.5mM.
4, according to the described anti-tumor metastasis medicament high flux screening model of claim 1, the cell density when it is characterized in that the MDA-MB-435 cell inoculation is 4 * 10
6/ ml; Prothrombin Complex Concent-concentration is at 0.008g/ml; It is 15min that cell pyrolysis liquid adds the common incubation time of Human Factor's solution.
5,, it is characterized in that the screening method of this model is according to the described anti-tumor metastasis medicament high flux screening model of claim 1:
Well-grown MDA-MB-435 cell is by 4 * 10
6/ ml is inoculated in 96 well culture plates, every hole 180 μ l enchylema, it is good to be cultured to cell state, adherent about 80%, blot substratum, blank well and control group add 200 μ l fresh cultures respectively, dosing holes adds 180 μ l fresh cultures respectively and 20 μ l treat SCREENED COMPOUND, put into 37 ℃ of incubator co-cultivation 6h, multigelation 3 times is got cell pyrolysis liquid;
18 μ l cell pyrolysis liquids and 2 μ l concentration are that Human Factor's solution of 0.01g/ml is hatched 15min jointly in 384 orifice plates, under 37 ℃, it is the S-2222 of 37 ℃ of preheatings of 0.5mM that every then hole adds 20 μ l concentration, 37 ℃ of incubation 3min, measure absorbancy with the microwell plate plate reading with 405nm, calculate the TF inhibiting rate; Not add the negative contrast of any inhibitor, primary dcreening operation compound final concentration is 1 * 10
-5M adds inhibiting rate behind the compound greater than 20%, positive compound.
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| CN103194528A (en) * | 2013-04-04 | 2013-07-10 | 常州亚当生物技术有限公司 | Method for screening anticancer traditional Chinese medicine through telomerase |
| CN108060204A (en) * | 2017-12-10 | 2018-05-22 | 陈哲浩 | A kind of high-flux medicaments sifting system for inhibiting the survival of macrophage-stimulating breast cancer cell |
| CN115341011A (en) * | 2022-07-22 | 2022-11-15 | 长春科技学院 | Rapid screening system for prostate hyperplasia candidate drugs based on cell level |
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|---|---|---|---|---|
| CN103194528A (en) * | 2013-04-04 | 2013-07-10 | 常州亚当生物技术有限公司 | Method for screening anticancer traditional Chinese medicine through telomerase |
| CN108060204A (en) * | 2017-12-10 | 2018-05-22 | 陈哲浩 | A kind of high-flux medicaments sifting system for inhibiting the survival of macrophage-stimulating breast cancer cell |
| CN108060204B (en) * | 2017-12-10 | 2021-11-16 | 陈哲浩 | High-throughput drug screening system for inhibiting macrophage from stimulating survival of breast cancer cells |
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