CN101392284A - Construction and application of anticancer natural drug screening model - Google Patents
Construction and application of anticancer natural drug screening model Download PDFInfo
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- CN101392284A CN101392284A CNA2008100513882A CN200810051388A CN101392284A CN 101392284 A CN101392284 A CN 101392284A CN A2008100513882 A CNA2008100513882 A CN A2008100513882A CN 200810051388 A CN200810051388 A CN 200810051388A CN 101392284 A CN101392284 A CN 101392284A
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Abstract
The invention relates to an antioncogene promoter activator screening model which is used for screening antineoplastic natural medicines , in particular a screening method of a p53 gene promoter activator screening model. The screening method adopts that: the p53 gene promoters are connected with reporter genes; recombinant plasmids after connection are transfected with human embryonic kidney HEK293T cells to form stable cell lines; and then traditional Chinese medicine extracts to be screened are added for measuring the expression level of the reporter genes; simultaneously, a positive control group and a negative control group are established so as to evaluate the biological activity of the traditional Chinese medicines; and then the traditional Chinese medicine extracts with high biological activity are separated into monomeric compounds so as to obtain the p53 gene promoter activator. The activator is used as a candidate medicine for antineoplastic medicines and pharmacological experiments are carried out to develop a new natural antineoplastic medicine with precise curative effect. The method is a sensitive and effective screening method with relatively clear mechanism and can be applicable to new natural antineoplastic medicines with high flux.
Description
Technical field
The present invention relates to novel method, the especially screening method of p53 gene promoter cell model that a kind of screening model that utilizes the cancer suppressor gene promotor to set up screens antitumor natural drug, belong to the molecular pharmacology technical field.
Background technology
Cancer is the killer of serious threat human health and life, and World Health Organization's prediction: the year two thousand twenty cancer patients will reach 1,500 ten thousand every year.According to estimates, there were 7,600,000 people to die from cancer in 2005, and if hold fire, at 10 years from now on 8,400 ten thousand people's death will be arranged, the annual New Development cancer patients of China is more than 2,500,000, annually controlling patient down 6,000,000, and medical expense is above 150,000,000,000 yuan.
Cancer is a kind of genopathy, is under the human body cell environmental factors effect outside, inherent multiple before oncogene be activated and the process of the multistage long term evolution of cancer suppressor gene inactivation.The main treatment means of cancer is operation, radiation and chemotherapy at present, although physicians constantly improve this 3 big treatment means, but many cancer patientss still are difficult to obtain cure, chemical synthetic drug curative effect on the disease of these cause of disease complexity of treatment cancer is unsatisfactory, and toxic side effect is big, and the limitation of modern medicine and pharmacology begins to reveal gradually.Chinese traditional medicine is little because of its side effect, have defective and deficiency that dual regulation often can replenish Western medicine.The research and development of natural drug cancer therapy drug are international heat subjects, yet the medication direction in past is directly to suppress or kill cancer cell with various medicines, the mechanism of action is clear and definite inadequately, the drug target tropism is poor, toxic side effect is poor, medicine has only the effect of treatment cancer, can not preventing cancer and diseases related generation thereof.The major subjects of cancer research forefront, the world is to repair and activate intravital cancer suppressor gene now, by repairing and activating intravital cancer suppressor gene and treat cancer.
The p53 gene is a kind of important cancer suppressor gene, is to study the most thoroughly so far, and function is the most powerful, with the highest cancer suppressor gene of human cancer dependency.Wild type p53 mainly is to play a crucial role at aspects such as cell cycle regulating cell growth inhibiting, cancer cell specific induction of apoptosis.Studies show that it and human 50% related to cancer, for example: liver cancer, mammary cancer, bladder cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, soft tissue sarcoma, ovarian cancer, brain tumor, lymphocyte cancer, esophagus cancer, lung cancer, osteogenic sarcoma etc.The human p53 assignment of genes gene mapping is in 17P13, and total length 16～20kb has 10 introns and 11 exons, and wherein first exon is not encoded, and there is promotor P1 at 400bp place, its upstream, and there is promotor P2 at 1kb place in downstream, and the two is a transcripting start point.Normal p53 gene claims wild type p53 gene (wild type p53 again, wt-p53), the phosphocarnic acid protein relevant that its encoded protein matter is made up of 393 amino acid (aa) with cell division cycle, Mr53000, be called p53 albumen, this product is a kind of transcriptional regulator, can come cell death inducing or cell-cycle arrest by the target gene of regulating the downstream and anticancer is grown in external, body.
To have a material usage few because of it for the medicaments sifting model of molecule and cell levels, and mechanism of drug action is clear and definite, can realize characteristics such as high flux screening, becomes present searching and find the main method of novel drugs.Studies show that utilizing the promotor activator to select the cancer suppressor gene of inactivation in activation or the reparation cancer cells is a kind of feasible and effective means of treatment cancer.The present invention has set up a kind of new natural anti-cancer drugs screening model-cancer suppressor gene p53 promotor activator screening model, and it is a kind of sensitivity, special efficacy, the clear and definite novel method that is applicable to the screening of high-throughput natural anti-cancer original new drug of drug target tropism.The p53 gene is the ubiquitous cancer suppressor gene of human body, and the medicine of exploitation both can be used for treating the generation that cancer also can be used for preventing cancer.
Summary of the invention
The object of the present invention is to provide a kind of sensitivity, directly, the drug screening cell model of rapid screening natural anti-cancer drugs.The method of this modelling is as follows:
At first, clone p53 gene promoter (p53 promoter, p53p) sequence: the dna sequence dna according to known human P 53 gene finds its promoter region, design comprises the primer of this promotor full sequence, and at upstream primer introducing KpnI restriction enzyme site, downstream primer is introduced the XhoI restriction enzyme site, is that template is carried out pcr amplification with the human genome, obtain the promoter sequence of p53 gene, then the promotor of the p53 gene that obtains is connected to pMD-18 and goes up and check order.Sequence and known array that order-checking obtains are compared, be the p53 gene promoter sequence of wanting required for the present invention to confirm this sequence.
Make up pGL-p53p luciferase expression carrier then: above-mentioned clone's p53 gene promoter fragment is inserted in the pGL2 reporter plasmid, cut by enzyme and identify and gene sequencing, prove that the pGL-p53p luciferase expression plasmid successfully constructs.
Set up p53 activator drug screening cell model at last and carry out drug screening: utilize the plasmalogen transfection method to be transfected into Human Embryonic Kidney HEK 293T clone the pGL-p53p plasmid that makes up, carry out the pressure screening, determine stable rotaring redyeing system with G418.The cell inoculation of the P53 promotor of stable transfection in 96 orifice plates, is added different Chinese medical extracts, detect the activity of luciferase, establish control group, screening P53 gene promoter activator by Chemiluminescence Apparatus.
Medicaments sifting model provided by the invention is to have cloned the promoter sequence of human tumor suppressor gene p53 and the cultured cell in vitro of luciferase reporter gene.Wherein, the promoter sequence of p53 gene is the molecular target of drug screening.The promotor of described p53 gene is to have cloned the P2 in the P1 of first exon upstream of p53 gene or downstream or connected a placed in-line sequence of promotor.Selected cultured cell in vitro is Human Embryonic Kidney HEK 293T clone preferably, and the cells of mamma animals of vitro culture also can.
Beneficial effect of the present invention:
The present invention relates to the method, the especially screening method of p53 gene promoter activator that utilize cancer suppressor gene promotor activator medicaments sifting model to screen antitumor natural drug.Screening method of the present invention is taked p53 gene promoter and report base are connect, gene transfection Human Embryonic Kidney HEK 293T cell after will connecting then forms stable cell line, add Chinese medical extract to be screened subsequently, measure the expression level of reporter gene, the biological activity of assessment Chinese medicine, the Chinese medical extract that biological activity is high is separated into monomeric compound, obtain p53 gene promoter activator, thereby obtain the candidate medicine of cancer therapy drug, provide a kind of novel method for screening has the antitumor natural drug of definite curative effect.It is a kind of sensitivity, effectively and molecule mechanism is clear and definite relatively, the screening method that can be applicable to high throughput chemical and natural antitumor original new drug, especially the p53 gene is the ubiquitous cancer suppressor gene of human body again, and the medicine of exploitation not only can be used for treating the generation that cancer can also be used for preventing cancer.
Embodiment
Clone related among all embodiment is all available from heredity institute of the Chinese Academy of Sciences, Superfect TransfectionReagent is available from QIAGEN company, all cells is cultivated reagent all available from Gibco company, liposome Gene SHUTTLE-20 is available from Quantum biotechnologies company, the immunocytochemistry test kit is available from doctor's moral company, polymerase chain reaction (PCR) Amplification Kit, DNA Ligation Kit, RNAPCR Kit (AMV) be all available from TaKaRa company, and all experiments are all by the routine operation of molecular biology experiment.
Embodiment 1:p53 gene promoter (p53 promoter, clone p53p)
Human genome DNA's extraction:
This experiment cell pyrolysis liquid lysing cell film, collecting cell nuclear, add the SDS nuclear membrane that breaks, make nucleoprotein be degraded into small segment and disintegrate down with Proteinase K from DNA, remove protein through phenol, chloroform extracting, dehydrated alcohol deposit D NA, 75% washing with alcohol DNA precipitation, after the vacuum-drying, be dissolved in and promptly obtain high-molecular weight DNA among the TE.
1. get 100 μ l anticoagulated whole bloods and place 1.5ml Eppendorf centrifuge tube, add 100 μ l lysates and 20 μ lRNaseA/ Proteinase K liquid again, blow and beat mixing back and forth with pipettor, bathed 35 minutes in 55 ℃ of temperature the chamber, put upside down centrifuge tube back and forth several times therebetween, be water-soluble shape until solution.
2. the NaAc (pH4.8) that adds 10 μ l 3M adds 1250 μ l binding buffer liquid subsequently, the mixing that fully vibrates, 12, centrifugal 30 seconds of 000rpm.
3. shift supernatant with pipettor Tip and join in the centrifugal adsorption column (packaging collection tube), placed 1 minute, 12, centrifugal 30 seconds of 000rpm outwells the waste liquid in the collection tube.
Attention: honest and upright and thrifty 1300 μ l to be transferred, the about 650 μ l of centrifugal adsorption column capacity, thus need each supernatant 650 μ l that shift in centrifugal adsorption column, centrifugal, outwell the waste liquid in the collection tube, repeated experiments operation steps 3.
4. add 600 μ l lavation buffer solutions (washing buffer) again in centrifugal adsorption column (packaging collection tube), 12, centrifugal 30 seconds of 000rpm.
Repeating step 4 once, 12, centrifugal 3 minutes of 000rpm is fully to remove lavation buffer solution.
6. carefully take out centrifugal adsorption column, abandon collection tube, centrifugal adsorption column is inserted in a clean 1.5ml Eppendorf centrifuge tube, and add 50 μ l elution buffers (elution buffer) (elution buffer must be added in the center of centrifugal adsorption column) in centrifugal adsorption column, placed 1 minute, in 12, centrifugal 30 seconds of 000rpm.Taking out the Eppendorf centrifuge tube, wherein is the genomic dna that extracts.
7. get 8～10 μ l genomic dnas, and add 2 μ l sample-loading buffers, mixing, application of sample in 1% sepharose point sample hole, electrophoresis detection.
The p53 gene promoter (p53 promoter, clone p53p):
At first design a pair of primer according to known human p53 gene promoter sequence, and introduce Kpn I restriction enzyme site at upstream primer, downstream primer is introduced Xho I restriction enzyme site.Be template with human genome DNA then, carry out pcr amplification.The PCR system is: 10 * PCR damping fluid, 5 μ l, 15mM MgCl
24 μ l, 10mMdNTP mix 1 μ l, 10 μ M Primer1,2 each 2 μ l, template DNA 20～200pg, Taq archaeal dna polymerase (5U/ μ l) 0.5 μ l, moisturizing to 50 μ l.Reaction conditions is: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are prolonged 60 seconds; 30 circulations.
The PCR product reclaims:
1. the Solution BS mixing that in the PCR product, adds 4 times of volumes;
2. mixed solution is transferred in the 3S post, room temperature is placed 2min; 10, the centrifugal 1min of 000rpm;
3. outwell the waste liquid in the collection tube, the 3S post is put into same collection tube, add 600 μ l Wash Solution, 10, the centrifugal 1min of 000rpm;
4. repeating step 3 once;
5. outwell the waste liquid in the collection tube, the 3S post is put into same collection tube, 10, the centrifugal 2min of 000rpm;
6. the 3S post is put into a new centrifuge tube, added 30 μ lTE, uncap and place 2min in room temperature in the central authorities of 3S post film;
7.10, the centrifugal 1min of 000rpm, the liquid in the centrifuge tube is the dna fragmentation of recovery.
Ligation:
The fragment that reclaims is directly carried out ligation, the insertion fragment and the carrier segments that add the 3:1 ratio in the 10 μ l reaction systems, 1 T4DNA of unit ligase enzyme is selected different temperature of reaction and time for use according to the reagent of different company, and general 16 ℃ connect 4h or 4 ℃ of connections of spending the night.
Colibacillary conversion:
1. get 100 μ l competent cells and add 5 μ l connection product, mixing is iced and was put 30 minutes gently;
2. heat shock 90 seconds in 42 ℃ of water placed cooled on ice rapidly 1～2 minute;
3. add 800 μ l LB liquid nutrient mediums, 37 ℃ of water-baths or gentle shaking culture 45 minutes to 1 hour;
4. get 50～200 μ l bacterium liquid and evenly coat and contain on the suitable antibiotic LB flat board, be inverted overnight incubation for 37 ℃.
The structure of embodiment 2:pGL-p53p luciferase expression plasmid
Alkaline lysis prepares plasmid DNA in a small amount:
1. picking list colony inoculation is in containing suitable antibiotic LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night;
2. get 1.5ml bacterium liquid in little centrifuge tube, 10, centrifugal 30 seconds of 000rpm;
3. bacterial sediment is resuspended in the 100 μ l solution I, the vibration mixing;
4. add 200 μ l solution II of new configuration, flick mixing, room temperature was placed 4 minutes, and is more clear to solution;
5. add 150 μ l solution III, flick mixing, room temperature was placed 6 minutes;
6. add 450 μ l phenol: chloroform: primary isoamyl alcohol (25:24:1), behind the mixing 12, centrifugal 5 minutes of 000rpm;
7. get supernatant, add the equal-volume Virahol, left standstill 5 minutes;
8.12, centrifugal 4 minutes of 000rpm, liquid in the evacuation pipe;
9. add 200 μ l ddH2O dissolution precipitations, add 100 μ l 7.5M ammonium acetates afterwards, left standstill on ice 5 minutes, 12, centrifugal 4 minutes of 000rpm;
10. get supernatant, be transferred in another EP pipe, add 2 times of volume dehydrated alcohols, placed 5 minutes for-20 ℃;
11.12 centrifugal 5 minutes of 000rpm abandons supernatant, with 70% ethanol washing and precipitating, and 12, centrifugal 3 minutes of 000rpm, supernatant discarded, seasoning or drain gained DNA is dissolved in the suitable water or with dry powder form and preserves.
The plasmid enzyme restriction reaction:
PGL2 carrier and p53p plasmid are used Kpn I and Xho I double digestion respectively: in 30 μ l reaction systems, add 10 * enzyme cutting buffering liquid, 3 μ l, DNA1～5 μ g, restriction enzyme 0.5 μ l (5U), add aseptic double-distilled water and mend to 30 μ l, 37 ℃ were reacted 1～2 hour.Agarose gel electrophoresis detects enzyme and cuts the result, reclaims the purpose band, with the directed pGL2 carrier that connects of p53p.Cut and the gene sequencing evaluation by enzyme, prove that the pGL-p53p luciferase expression plasmid successfully constructs.
The foundation and the application of embodiment 3:p53 promotor activator drug screening cell model
Human Embryonic Kidney HEK 293T cell cultures is contained the DMEM culture medium culturing of 10% foetal calf serum, placing 37 ℃, 5%CO2 incubator to hatch.
Plasmid transfection HEK293T cell: (Superfect Transfection Reagent test kit)
1. with 3 μ g DNA (carrier: pcDNA3=5:1) in the aseptic EP pipe of 1.5ml, mix cumulative volume 100 μ l with the DMEM substratum that serum-free does not have antibody.
2. add 10 μ l transfection reagents, place 10min under the room temperature behind the mixing.
3. discard the nutrient solution in six orifice plates, 3ml PBS flushing 3 times adds and contains serum and two anti-DMEM nutrient solution 1.5ml.
4. in the EP pipe that contains the transfection reagent mixture, add and contain serum and two anti-DMEM nutrient solution 0.6ml, all add in six orifice plates behind the mixing.
5. rock six orifice plates gently, mixture is evenly distributed, 37 ℃, cultivate the 48h harvested cell among the 5%CO2.
The stable strain screening:
After the transfection, go down to posterity by 1:10, change to cultivating the row filter of going forward side by side in the complete culture solution of the G418 that contains 0.8g/L, the G418 that uses 0.6g/L when non-transfected cells is all dead instead keeps screening, changes liquid in per three days once and go out downright bad cell debris.Obtain the G418 resistance clone, amplification cultivation after screening for 2 weeks.The blank carrier of transfection simultaneously is as negative control group and only add the blank group that liposome does not add plasmid.
The application of p53 promotor activator medicaments sifting model
With the cell inoculation of the P53 promotor of stable transfection in 96 orifice plates, add different Chinese medical extracts, measure the expression level of reporter gene, if control group, assess the biological activity of Chinese medicine with this, the Chinese medical extract that biological activity is high is separated into individualized compound, obtains p53 gene promoter activator, thereby obtains the candidate medicine of cancer therapy drug.
Claims (5)
1, a kind of p53 of utilization gene promoter activator screening model novel method of screening antitumor natural drug, it may further comprise the steps:
A) the p53 gene promoter is connected with reporter gene;
B) recombinant plasmid transfection Human Embryonic Kidney HEK 293T cell is formed stable cell line;
C) in the stabilized cell model that filters out, add sample to be screened, measure the expression level of reporter gene, the anti-tumor biological of assessment natural drug;
D) biological activity is high Chinese medical extract is separated into monomeric compound, obtains p53 gene promoter activator;
E) the p53 gene promoter activator that obtains is carried out antineoplastic pharmacological experiment as antitumor candidate medicine, to filter out antineoplastic original new drug.
2, p53 gene promoter activator screening model according to claim 1, it is characterized in that: reporter gene is a luciferase gene.
3, p53 gene promoter activator screening model according to claim 1 is characterized in that: what be connected with reporter gene is the p53 gene promoter.
4, p53 gene promoter activator screening model according to claim 1, it is characterized in that: mammalian cell is a human embryo kidney (HEK) HEK293T cell, also can be other mammalian cells.
5, the anticancer natural drug of screening comprises according to claim 1: the activeconstituents of plant, animal and mineral, extract, processed product, processed goods, through any or multiple products such as processing or the product through concocting, efficient part, monomers.
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| CNA2008100513882A CN101392284A (en) | 2008-11-06 | 2008-11-06 | Construction and application of anticancer natural drug screening model |
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| CNA2008100513882A CN101392284A (en) | 2008-11-06 | 2008-11-06 | Construction and application of anticancer natural drug screening model |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103116006A (en) * | 2013-01-25 | 2013-05-22 | 重庆医科大学附属第一医院 | Novel screening method of anticancer medicaments |
| WO2020000453A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Novel human indo gene-targeted rnai interference fragment, rnai carrier and preparation method therefor and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1590562A (en) * | 2004-04-08 | 2005-03-09 | 华东师范大学 | Screening method of klothe gene promotor activator |
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2008
- 2008-11-06 CN CNA2008100513882A patent/CN101392284A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1590562A (en) * | 2004-04-08 | 2005-03-09 | 华东师范大学 | Screening method of klothe gene promotor activator |
Non-Patent Citations (3)
| Title |
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| D. C. NEW ET AL.: "Reporter Gene Assays and their Applications to Bioassays of Natural Products", 《PHYTOTHERAPY RESEARCH》 * |
| HUAIXING LI ET AL.: "p53 Promoter-based Reporter Gene in vitro Assays for Quick Assessment of Agents with Genotoxic Potential", 《ACTA BIOCHIMICA ET BIOPHYSICA SINICA》 * |
| WENGE WANG ET AL.: "Small-molecule modulators of p53 family signaling and antitumor effects in p53-deficient human colon tumor xenografts", 《PNAS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103116006A (en) * | 2013-01-25 | 2013-05-22 | 重庆医科大学附属第一医院 | Novel screening method of anticancer medicaments |
| CN103116006B (en) * | 2013-01-25 | 2015-09-30 | 重庆医科大学附属第一医院 | A kind of Novel screen choosing method of cancer therapy drug |
| WO2020000453A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Novel human indo gene-targeted rnai interference fragment, rnai carrier and preparation method therefor and application thereof |
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