CN105738607A - Kit for screening antitumor drugs, and use method thereof - Google Patents
Kit for screening antitumor drugs, and use method thereof Download PDFInfo
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- CN105738607A CN105738607A CN201410752690.6A CN201410752690A CN105738607A CN 105738607 A CN105738607 A CN 105738607A CN 201410752690 A CN201410752690 A CN 201410752690A CN 105738607 A CN105738607 A CN 105738607A
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- caenorhabditis elegans
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Abstract
The invention discloses a kit for screening antitumor drugs, and a use method thereof, and belongs to the technical field of biological detection. The kit for screening the antitumor drugs comprises Caenorhabditis elegans and Escherichia coli, can be used for screening the antitumor drugs, and is suitable for being massively promoted in the field of antitumor drug screening.
Description
Technical field
The present invention relates to the test kit of a kind of antitumor medicine screening and using method thereof, belong to technical field of biological.
Background technology
At present, malignant tumor has become as the number one killer of the mankind, the serious health endangering the mankind, the data of World Health Organization (WHO) shows, the morbidity of annual whole world tumor is 10,000,000 people, and death is 7,000,000 people about, the annual new cases of the annual cancer of China are 2,000,000, it is about 1,400,000 because of cancer mortality, in China's death 5 people, namely has 1 people to die from cancer.
Present stage, the model that domestic and international screening anti-tumor medicine is selected mainly has cell, fruit bat, nematicide, mice and rat.Cell is the drug screening of model is external medicine Effect study, and efficiency is high but often dependency is poor；The life pattern of fruit bat limits its application in high-flux medicaments sifting；The modeling experiment operating technology of muroid requires height and because its condition of culture height causes that screening efficiency is low, research cost is high.And Caenorhabditis elegans condition of culture is simple, it is possible on a lbmc agar plate or fluid medium takes food antibacterial (E.coli)；Generation cycle is short, is generally about 3 days；Life-span is short, is generally about 3 weeks；Caenorhabditis elegans experimental population easily amplifies, and can obtain the Caenorhabditis elegans of synchronization (growth period is in same stage) in a large number.Caenorhabditis elegans is short because of life cycle and easily realizes high-throughout advantage, becomes the ideal model of tumor research.But present stage uses Caenorhabditis elegans to yet suffer from certain difficulty, primarily with respect to common people, Caenorhabditis elegans modeling, cultivation, inspection etc. remain a need for technical training and the practice of system, just can complete drug screening, therefore, everybody is badly in need of the test kit of a kind of antitumor medicine screening based on above-mentioned Caenorhabditis elegans model, for this area research staff antitumor medicine screening efficiently and accurately.
To this, spy of the present invention provides the test kit of a kind of antitumor medicine screening based on above-mentioned Caenorhabditis elegans model, use this test kit can quickly, antitumor medicine screening accurately, easily, present invention simultaneously provides the using method of mentioned reagent box.
Summary of the invention
It is an object of the invention to provide the test kit of a kind of antitumor medicine screening, use this test kit can filter out antitumor drug.
It is a further object to provide the using method of the test kit of above-mentioned antitumor medicine screening.
The present invention provides the test kit of a kind of antitumor medicine screening, and described test kit includes following reagent:
1. reagent A: Caenorhabditis elegans；
2. reagent B: escherichia coli OP50；
Described reagent B is the lyophilized powder containing escherichia coli OP50 viable bacteria or bacterium solution.
Wherein, the concentration of described Caenorhabditis elegans is 80-100 bar/20 μ l, and the concentration of escherichia coli OP50 is 10mg/ml.
Described Caenorhabditis elegans is synchronization nematicide, and the synchronized step of Caenorhabditis elegans is as follows: swept away by the Caenorhabditis elegans on flat board with M9 buffer, flushing liquor is sucked centrifuge tube；4000rpm/min is centrifuged 3min, removes supernatant；Add the lysate of final concentration of 3.2%NaClO and 1MNaOH；Vortex instrument shakes 7min, makes the polypide of Caenorhabditis elegans break release ovum；4000rpm/min is centrifuged 3min, removes supernatant, collects precipitation and obtains substantial amounts of Caenorhabditis elegans ovum；M9 wash buffer twice, adds 1mlM9 buffer, is placed in 20 DEG C of incubators and hatches 48 hours.
The present invention also provides for the using method of the test kit of a kind of antitumor medicine screening, i.e. the using method of mentioned reagent box:
Using 96 porocyte culture plates, the final volume in every hole is 200 μ l；Every hole adds the medicinal liquid to be measured of 160 μ l, adds 20 μ l Caenorhabditis elegans liquid, adds 20 μ l escherichia coli OP50 bacterium solution, and each process sets three repetitions, is placed in 20 DEG C of biochemical cultivation cases and cultivates；After being cultured to adult, under inverted microscope, observe Caenorhabditis elegans wild type and mutant number；Wild type %=100 × wild type nematicide number/line insect number (that is: wild type number+mutant number)；Compared with blank, the effect of the more high expression drugs against tumor of WT ratio is more good.
The beneficial effects of the present invention is: the present invention provides the test kit of a kind of antitumor medicine screening based on Caenorhabditis elegans model, this test kit testing result is accurate, and using method is simple, is adapted at the field large-scale promotion of antitumor medicine screening.
Specific embodiment presented below is to realize the test kit of antitumor medicine screening of the present invention, but is not limited to these embodiments.
Accompanying drawing explanation
The effect to RAS excessive activation let60/ras (gf) many vaginal orifices mutant MT2124 of Fig. 1 Sorafenib
The effect to transcription factor lin-31 (gf) many vaginal orifices mutant MT301 in RAF downstream of Fig. 2 Sorafenib
The effect to RAS excessive activation let60/ras (gf) many vaginal orifices mutant MT2124 of Fig. 3 etoposide
The effect to transcription factor lin-31 (gf) many vaginal orifices mutant MT301 in RAF downstream of Fig. 4 etoposide
Detailed description of the invention
1. bioorganism:
Caenorhabditis elegans MT2124: many vaginal orifices phenotype, genotype: let-60 (n1046sd, gf) IV, buy in TheCaenorhabditisGeneticsCenter (CGC).
Caenorhabditis elegans MT301: many vaginal orifices phenotype, genotype: lin-31 (n301) II, buy in TheCaenorhabditisGeneticsCenter (CGC).
Bacterial strain: uracil leaky mutant escherichia coli OP50, buys in TheCaenorhabditisGeneticsCenter (CGC).
2. culture medium:
NGM culture medium: NaCl1.5g, K2HPO41.3g, KH2PO48.5g, peptone 1.4g, agar powder 8.5g, distilled water 500mL.The 500 μ L cholesterol (5mg/ml, dehydrated alcohol is prepared) of filtration sterilization, 500 μ L1mol/LMgSO of high pressure steam sterilization are added after sterilizing4, 500 μ L1mol/LCaCl2。
M9 buffer: NaCl1.98g, Na2HPO42.38g, KH2PO41.20g, MgSO40.048g, distilled water 400mL.
LB fluid medium: NaCl2g, peptone 2g, yeast extract 1g, distilled water 200mL.Regulating pH with 1MNaOH after dissolving is 7.0, high pressure steam sterilization.
3. reagent:
Sorafenib: purity >=98%, purchased from Dalian U.S. logical sequence；
Etoposide injection: Jiangsu perseverance is auspicious, lot number: vp101202, is purchased from No.1 Hospital of Lanzhou University.
4. instrument:
High-pressure sterilizing pot (Shenan Medical Appliances Factory, Shanghai)
Single one side super-clean bench (Suzhou Decontamination Equipment Plant)
Biochemical cultivation case (south of the River, Ningbo instrument plant)
Water isolation type constant incubator (Shanghai Yiheng Scientific Instruments Co., Ltd)
Zoom-stereo microscope (Shanghai Yiheng Scientific Instruments Co., Ltd)
Centrifuge (CT15E, Hitachi)
Electronic balance (Beijing Sai Duolisi instrument system company limited)
The synchronization of embodiment 1 Caenorhabditis elegans
The synchronized step of Caenorhabditis elegans is as follows: swept away by the Caenorhabditis elegans on flat board with M9 buffer, flushing liquor is sucked centrifuge tube；4000rpm/min is centrifuged 3min, removes supernatant；Add the lysate of final concentration of 3.2%NaClO and 1MNaOH；Vortex instrument shakes 7min, makes the polypide of Caenorhabditis elegans break release ovum；4000rpm/min is centrifuged 3min, removes supernatant, collects precipitation and obtains substantial amounts of Caenorhabditis elegans ovum；M9 wash buffer twice, adds 1mlM9 buffer, is placed in 20 DEG C of incubators and hatches 48 hours.
The assembling of embodiment 2 test kit
The test kit of a kind of antitumor medicine screening of the present invention, this test kit includes following reagent:
1., reagent A: Caenorhabditis elegans；
2., reagent B: escherichia coli OP50；
Wherein, described reagent B is the lyophilized powder containing escherichia coli OP50 viable bacteria or bacterium solution.
The concentration of described Caenorhabditis elegans is 80-100 bar/20 μ l, and the concentration of escherichia coli OP50 is 10mg/ml.
Those skilled in the art according to concrete research contents, can select similar strain, and be not limited to the Caenorhabditis elegans strain described in the embodiment of the present invention.
The using method of embodiment 3 test kit
Using 96 porocyte culture plates, the final volume in every hole is 200 μ l；Every hole adds the medicinal liquid to be measured of 160 μ l, adds 20 μ l Caenorhabditis elegans liquid, adds 20 μ l escherichia coli OP50 bacterium solution, and each process sets three repetitions, is placed in 20 DEG C of biochemical cultivation cases and cultivates；After being cultured to adult, under inverted microscope, observe Caenorhabditis elegans wild type and mutant number；Wild type %=100 × wild type nematicide number/line insect number (that is: wild type number+mutant number)；Compared with blank, the effect of the more high expression drugs against tumor of WT ratio is more good.
Embodiment 4 test kit compliance test result is tested
Selected conventional anticancer drugs has been carried out the compliance test result of antitumor medicine efficacy screening by this test kit.
Experimental procedure:
1. contain the preparation of the solution of medicine to be measured
Sorafenib, etoposide injection
By above-mentioned medicine dissolution to be measured, elimination impurity, filtrate is diluted to each Concentraton gradient stand-by.This area research worker can according to experiment needs, from Row sum-equal matrix concentration range.
2. antitumor pharmacodynamic analysis experiment
(1) Sorafenib antitumor pharmacodynamic analysis
Sorafenib, is the biological targeting new drug of a kind of Mutiple Targets that Bayer and Onyx company develops jointly, and preclinical study and clinical trial prompting Sorafenib have antitumor action widely.Research shows: observed the anti-tumor activity of Sorafenib in the tumor with RAS or B-RAF gene mutation, Sorafenib can pass through to suppress RAF activity thus having blocked RAF/MEK/ERK signal transduction pathway, suppress the propagation of tumor cell, directly suppress tumor growth.
Therefore, apply anti-tumor agent comprising salmosin box provided by the invention and evaluate the Sorafenib impact on transcription factor lin-31 (gf) many vaginal orifices mutant MT301 in RAS excessive activation let60/ras (gf) many vaginal orifices mutant MT2124 and RAF downstream.Result shows: Sorafenib can substantially reverse the phenotype of RAS excessive activation let60/ras (gf) many vaginal orifices mutant MT2124, and in dose-dependence (Fig. 1, p < 0.01).But, almost without affecting (Fig. 2, p > 0.05), this is consistent with previous studied Sorafenib targeting RAF/MEK/ERK result on the phenotype of transcription factor lin-31 (gf) many vaginal orifices mutant MT301 in RAF downstream for Sorafenib.
(2) etoposide antitumor pharmacodynamic analysis
Etoposide is cell cycle specific antitumor drug (Meleyetal., 2010), and its mechanism of action is mainly suppression TOP-001, thus disturbing DNA structure and function.It is the same that it is not as Sorafenib, is a signal path targeted drug, but a cytotoxic cancer therapy drug, therefore, the mechanism of anticancer action of this medicine is not relying on cell-signaling pathways.
Apply the anti-tumor agent comprising salmosin box provided by the invention detection etoposide impact on transcription factor lin-31 (gf) many vaginal orifices mutant MT301 in RAS excessive activation let60/ras (gf) many vaginal orifices mutant MT2124 and RAF downstream.Result shows: etoposide has substantially reversed many vaginal orifices phenotype of two plant mutant bodies, and in drug dose dependence (Fig. 3 and 4, p < 0.01).
Can being drawn by above-mentioned experimental data, using test kit disclosed by the invention that existing antitumor drug is detected, the intended result of its antitumous effect is consistent with existing document conclusion.Thus proving that the test kit of the present invention can be used to antitumor medicine screening, and result is accurate, using method is easy, it is easy to grasps, is adapted at antitumor medicine screening field large-scale promotion.
Claims (5)
1. the test kit of an antitumor medicine screening, it is characterised in that described test kit includes following reagent:
1. reagent A: Caenorhabditis elegans；
2. reagent B: escherichia coli OP50.
2. the test kit of a kind of antitumor medicine screening according to claim 1, it is characterised in that: described reagent B is the lyophilized powder containing escherichia coli OP50 viable bacteria or bacterium solution.
3. the test kit of a kind of antitumor medicine screening according to claim 1, it is characterised in that the concentration of described Caenorhabditis elegans is 80-100 bar/20 μ l, and the concentration of escherichia coli OP50 is 10mg/ml.
4. the test kit of a kind of antitumor medicine screening according to claim 1, it is characterized in that, described Caenorhabditis elegans is synchronization nematicide, and the synchronized step of Caenorhabditis elegans is as follows: swept away by the Caenorhabditis elegans on flat board with M9 buffer, flushing liquor is sucked centrifuge tube；4000rpm/min is centrifuged 3min, removes supernatant；Add the lysate of final concentration of 3.2%NaClO and 1MNaOH；Vortex instrument shakes 7min, makes the polypide of Caenorhabditis elegans break release ovum；4000rpm/min is centrifuged 3min, removes supernatant, collects precipitation and obtains substantial amounts of Caenorhabditis elegans ovum；M9 wash buffer twice, adds 1mlM9 buffer, is placed in 20 DEG C of incubators and hatches 48 hours.
5. the using method of the test kit of the antitumor medicine screening described in a claim 1, it is characterised in that the using method of described test kit is as follows:
Using 96 porocyte culture plates, the final volume in every hole is 200 μ l；Every hole adds the medicinal liquid to be measured of 160 μ l, adds 20 μ l Caenorhabditis elegans liquid, adds 20 μ l escherichia coli OP50 bacterium solution, and each process sets three repetitions, is placed in 20 DEG C of biochemical cultivation cases and cultivates；After being cultured to adult, under inverted microscope, observe Caenorhabditis elegans wild type and mutant number；Wild type %=100 × wild type nematicide number/line insect number, compared with blank, the effect of the more high expression drugs against tumor of WT ratio is more good.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107875374A (en) * | 2017-10-13 | 2018-04-06 | 西安电子科技大学 | The method that Caenorhabditis elegans infection model is precisely quickly established based on orthogonal optimization |
| CN108478809A (en) * | 2018-03-22 | 2018-09-04 | 复旦大学 | A kind of natural small molecule HEC-23 purposes in the drug for preparing the cell death for promoting lysosome to mediate |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107875374A (en) * | 2017-10-13 | 2018-04-06 | 西安电子科技大学 | The method that Caenorhabditis elegans infection model is precisely quickly established based on orthogonal optimization |
| CN108478809A (en) * | 2018-03-22 | 2018-09-04 | 复旦大学 | A kind of natural small molecule HEC-23 purposes in the drug for preparing the cell death for promoting lysosome to mediate |
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