CN108690860A - A kind of method of quick screening antineoplastic specificity target drug - Google Patents
A kind of method of quick screening antineoplastic specificity target drug Download PDFInfo
- Publication number
- CN108690860A CN108690860A CN201710226741.5A CN201710226741A CN108690860A CN 108690860 A CN108690860 A CN 108690860A CN 201710226741 A CN201710226741 A CN 201710226741A CN 108690860 A CN108690860 A CN 108690860A
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- China
- Prior art keywords
- cell
- target drug
- quick screening
- specificity target
- drug
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
Abstract
The invention discloses a kind of methods of quick screening antineoplastic specificity target drug.This method improves update on the basis of conventional method, shortens action time, excludes error or mistake caused by cell in apoptotic process, the screening suitable for the specific target drug that those long timeliness can work.
Description
Technical field
The present invention relates to the screening techniques of tumour-specific target drug.
Background technology
Cell colonies assay, that is, cell inoculation survival rate indicates adherent cellular activities after inoculating cell and forms clone
Quantity.Cell after adherent not necessarily each can be proliferated and be formed clone, and the cell for forming clone must be adherent and have
The cell of proliferation activity.Cloning efficiency reflects two important characters of cell colony dependence and proliferative capacity.Conventional cell
After cloning experimentation vitellophag, cell to be blown and beaten into single cell suspension, different cells are seeded in suitable density in culture dish,
Cell colony number after cell clone is grown 2-3 weeks more than 50 cells of observation statistics.The past methods experiment period is too
Long, the probability of pollution is larger, and the technology whole cycle after improvement shortens to 6 days, easy to operate for detecting ability of cell proliferation,
As a result naked eyes can be observed directly.In addition to this, the method for being conventionally used for detection ability of cell proliferation at present also has MTT, CCK-
8, MTS etc., they act on living cells, and formazan product is reduced by the dehydrogenase in cell mitochondrial, generation formazan objects
Quantity is directly proportional to the quantity of living cells, and the cell mitochondrial dehydrogenase in apoptotic process is still active, in detection process
It cannot exclude the error even mistake that these bring.
Many specificity target spot medicines act on tumour cell, by change the methylating of target spot downstream molecules such as histone,
Acetylation etc. adjusts the activity of downstream molecules, and then influences gene regulation and expression, to play antitumous effect, these drugs
Antitumor is the process of a long timeliness, and the method for improvement is suitable for the specific target drug that these long timeliness can work
Screening.
Invention content
The present invention provides a kind of methods of quick screening antineoplastic specificity target drug, and week is screened for conventional method
Phase is long or cannot exclude the disadvantage of the cell error or mistake in apoptotic process, and the technical operation after improvement is simple, cycle time
By 6 days, the screening for the specific target drug that long timeliness can work can be effectively used for.
Description of the drawings
Fig. 1 various concentration target spot specific drugs handle cell clonal formation result after 6 d of MCF-7 cells
Fig. 2 various concentration target spot specific drugs handle cell clonal formation result after 6 d of A549 cells
Fig. 3 various concentration target spot specific drugs handle cell clonal formation result after 6 d of HT-29 cells
Specific implementation mode
In conjunction with step in detail below and attached drawing, the present invention is described in further detail, and following instance is only illustrative
, protection content of the invention is not limited to this.
Experiment material and method
1. experiment material
Complete medium:RPMI1640 or DMEM high glucose mediums(It is purchased from Gibco companies)10% fetal calf serum of middle addition(It is purchased from
BI), 100 U/ml penicillin and 100 ug/ml streptomysins(It is purchased from the green skies Bioisystech Co., Ltd in Shanghai).
4% paraformaldehyde fixer:Take 40 g paraformaldehydes(It is purchased from Sinopharm Chemical Reagent Co., Ltd.), it is dissolved in
In 600-800 ml PBS, 60 °C or so are heated to, powder is continued stirring until and is completely dissolved, supplies PBS solution to 1 L, fully
Mixing, 4 °C of preservations.
Ammonium oxalate crystal violet dye liquor:Solution A:2 g crystal violets(It is purchased from Chinese medicines group chemical reagent Beijing Co., Ltd)It is molten
Solution is in 20 ml, 95% alcohol;Solution B:0.8 g ammonium oxalate(It is purchased from Chinese medicines group chemical reagent Beijing Co., Ltd)It is dissolved in
In 80 ml distilled waters.A, B solution are uniformly mixed, filter paper filtering.The rears room temperature static 4 h can be used, and room temperature is kept in dark place.
2. cell culture
The solid tumor cell of certain a variety of gene high expression, such as MCF-7, A549, HT-29(It is purchased from Cell Bank of Chinese Academy of Sciences)Deng training
It supports in complete medium, cell is placed in 37 DEG C, 5 % CO2, cultivate in saturated humidity incubator, every 2 d replaces culture medium.
When cell confluency degree is up to 85%, culture medium is discarded.2 ml PBS cleanings cell is added 2 times, discards PBS, 1 ml 0.25% is added
Trypsin digestion attached cell.When cell retracts into circle, 2 ml complete mediums are added and terminate digestion, cell is resuspended.
Cell is transferred in 15 ml centrifuge tubes and is placed in 1000 rpm, centrifuges 3 min under the conditions of 25 DEG C.Liquid is discarded supernatant, 1 ml is added
Complete medium, according to 1:4~1:10 ratio seeds cells into new culture dish, and the cell after passage is positioned over cell
It is cultivated in incubator.
3. cell clonal formation
When cell confluency degree is up to 85%, trypsin digestion attached cell is resuspended after centrifugation, and cell density is adjusted to 0.5 ~ 5
×106Cells/ml takes 20 μ l, and trypan blue solution is added, mixes well, and 37 DEG C of 3 ~ 5 min of dyeing take 10 μ l to blood cell
It is counted in tally.Cell density is adjusted, is seeded cells into respectively in the 6 orifice plates containing acute drug set by 2 times, cross is mixed
It is even, cell is placed in cell incubator and is cultivated.After 72 h, liquid is partly changed for the first time, every 48 h partly changes liquid, culture used later
Liquid is basic culture medium added with 15% FBS and drug.
Daily microscopically observation cell clonal formation situation, untreated fish group cell clone size is suitable after 6 d, discards item
Part culture medium is added 1 ml, 4% paraformaldehydes, fixes 20 min, discards fixer, and 1 ml crystal violet solutions, dyeing 30 is added
Min discards dyeing liquor, and cell is cleaned with flowing water, waits for that blank space is remained without dyestuff.
6 orifice plates are placed on blank sheet of paper, influence of the observation drug to kinds of tumor cells Clone formation.
Various concentration target spot specific drug, which acts on 6 d rear clones of MCF-7, A549, HT-29 cell formation result, to be seen
Fig. 1,2,3.
Claims (1)
1. a kind of method of quick screening antineoplastic specificity target drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710226741.5A CN108690860A (en) | 2017-04-09 | 2017-04-09 | A kind of method of quick screening antineoplastic specificity target drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710226741.5A CN108690860A (en) | 2017-04-09 | 2017-04-09 | A kind of method of quick screening antineoplastic specificity target drug |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108690860A true CN108690860A (en) | 2018-10-23 |
Family
ID=63842338
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710226741.5A Withdrawn CN108690860A (en) | 2017-04-09 | 2017-04-09 | A kind of method of quick screening antineoplastic specificity target drug |
Country Status (1)
| Country | Link |
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| CN (1) | CN108690860A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070275434A1 (en) * | 2003-11-26 | 2007-11-29 | Guillemo Mor | Apoptosis-Based Evaluation Of Chemosensitivity In Cancer Patients |
| US20110152768A1 (en) * | 2008-07-11 | 2011-06-23 | Peter Jon Nelson | Treatment of Solid Tumors with Tissue Inhibitors of Metalloproteinases (TIMPS) |
| US20130237596A1 (en) * | 2010-10-22 | 2013-09-12 | Zhengzhou University | Uses of 15-benzylidene-14-deoxy-11,12-didehydroandrographolide Derivatives in the Preparation of Antineoplastic Drugs |
-
2017
- 2017-04-09 CN CN201710226741.5A patent/CN108690860A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070275434A1 (en) * | 2003-11-26 | 2007-11-29 | Guillemo Mor | Apoptosis-Based Evaluation Of Chemosensitivity In Cancer Patients |
| US20110152768A1 (en) * | 2008-07-11 | 2011-06-23 | Peter Jon Nelson | Treatment of Solid Tumors with Tissue Inhibitors of Metalloproteinases (TIMPS) |
| US20130237596A1 (en) * | 2010-10-22 | 2013-09-12 | Zhengzhou University | Uses of 15-benzylidene-14-deoxy-11,12-didehydroandrographolide Derivatives in the Preparation of Antineoplastic Drugs |
Non-Patent Citations (1)
| Title |
|---|
| 庞露苹: "嘧啶类衍生物的设计合成及其抑制EMT和血管生成的作用机制研究" * |
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| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20181023 |