CN103937872A - Application of Crm1 to stomach cancer diagnosis and treatment - Google Patents

Application of Crm1 to stomach cancer diagnosis and treatment Download PDF

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CN103937872A
CN103937872A CN201310025332.0A CN201310025332A CN103937872A CN 103937872 A CN103937872 A CN 103937872A CN 201310025332 A CN201310025332 A CN 201310025332A CN 103937872 A CN103937872 A CN 103937872A
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crm1
cancer
stomach
cell
gene
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高勇
李砚东
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Shanghai East Hospital
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Abstract

The invention discloses application of Crm1 gene and an expression product thereof to prepare products for stomach cancer diagnosis and treatment. The provided Crm1 gene and the expression product thereof can be used as a specific marker gene for diagnosing stomach cancer, and help stomach cancer diagnosis to be relatively accurate and rapid; and the Crm1 gene and the expression product thereof also can be used as a target gene for preparing stomach cancer treatment medicines, and help to provide a new stomach cancer treatment approach.

Description

The application of Crm1 in diagnosing gastric cancer and treatment
Technical field
The present invention relates to oncology.More specifically, the present invention relates to the application in cancer of the stomach context of detection of Crm1 gene and albumen thereof.The invention still further relates to the application of Crm1 inhibitor in curing gastric cancer.
Background technology
China is the High Risk For Gastric Cancer country, and its M & M is in respectively the 3rd and the 2nd of whole malignant tumours.Due to the concealment of cancer of the stomach onset, early stage many non-evident symptons, subtle, make the early discovery of cancer of the stomach very difficult.Particularly in China, exceed 80% cancer of the stomach in the time making a definite diagnosis just in progressive stage, prognosis is very poor, people's life and health (Leung WK in serious threat, et al.Lancet Oncol.2008, Yang L. et al.World J Gastroenterol 2006).
At present, except imaging examination and endoscopy, the detection of biomacromolecule tumor markers also can be used for the diagnosis of cancer of the stomach.Conventional biomacromolecule tumor markers can improve the particularly recall rate of gastrointestinal cancer of malignant tumour in body as carcinomebryonic antigen (CEA), glycoprotein analog antigen (CA19-9, CA125) clinically, but these marks can not be accomplished early stage highly sensitive, diagnosis of gastric cancer with high specificity.Due to the one-tenth knurl reason of each Organ Differentiation source, tumour in body and the tumour factor of tumour secretion all not identical, therefore will be specifically, early detection goes out a certain specific tumors with sensitivity, is still a very huge problem.
In addition, the treatment of cancer of the stomach makes great progress in 20 years in the past, the fastest with chemotherapy progress at present.Chemotherapeutics comprises fluorouracil, mitomycin, anthracycline, platinum class, cytosine arabinoside and nitrosourea etc.Although this type of medicine has certain effect to the treatment of cancer of the stomach, its severe side effect is also brought a lot of injuries to cancer patient simultaneously, such as gastrointestinal side effect is as nausea,vomiting,diarrhea, or oligoleukocythemia etc., all make the application of chemotherapeutic be restricted.
As can be seen here, at present in the urgent need to finding the cancer of the stomach early diagnosis marker with high specific and validity, and exploitation can targeting in cancer of the stomach, reduce the novel drugs of the injury of normal tissue and human body.
Summary of the invention
The invention provides the reagent of specific diagnosis cancer of the stomach and medicine and the method for the treatment of cancer of the stomach.
A first aspect of the present invention, provides the purposes of a kind of Crm1 gene or albumen, for the preparation of the reagent or the test kit that detect cancer of the stomach.
In another preference, described test kit comprises: the reagent and corresponding label or the specification sheets that Crm1 are carried out to detection by quantitative.
In another preference, in described label or specification sheets, record following operation instruction: Crm1 is carried out to detection by quantitative, and detected result is used for characterizing cancer of the stomach probability height.
In another preference, described reagent comprises the Auele Specific Primer of Crm1, probe and chip.
In another preference, described Crm1 gene source, in Mammals, preferably derives from people, mouse, rat.More preferably, derive from people.
In another preference, described Auele Specific Primer comprises upstream primer and downstream primer: described upstream primer sequence has sequence or its complementary sequence as shown in SEQ ID NO:1, and described downstream primer sequence has sequence or its complementary sequence as shown in SEQ ID NO:2.
In another preference, described chip comprises nucleic acid chip and protein chip.
In another preference, described nucleic acid chip comprises substrate and the point sample specific oligonucleotide probe at on-chip cancer related gene, and the specific oligonucleotide probe of described cancer related gene comprises the probe with the specific binding of Crm1.
In another preference, described protein chip comprises substrate and the point sample specific antibody in on-chip cancer of the stomach associated protein, and the specific antibody of described cancer of the stomach associated protein comprises the specific antibody of anti-Crm1.
A second aspect of the present invention, provides a kind of diagnostic kit for detection of cancer of the stomach, and described test kit contains a container, contains the detection reagent that detects Crm1 in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detection of cancer of the stomach.
In another preference, described detection reagent comprises: specific antibody and/or Auele Specific Primer.
In another preference, in described label or specification sheets, indicate following content:
As the ratio >1 of the mrna expression amount of the mrna expression amount of the relative beta-actin of the Crm1 of detected object and the relative beta-actin of Crm1 of cancer beside organism, point out the cancered probability of this detected object higher than general population.
In another preference, described test kit is for detection of people's stomach-tissue sample or blood sample.
A third aspect of the present invention, provides a kind of purposes of Crm1 inhibitor, described inhibitor to be used to the medicine that preparation suppresses Growth of Gastric or propagation, or for the preparation of the medicine for the treatment of cancer of the stomach.
In another preference, described inhibitor comprises: the activity inhibitor of the antibody of Crm1, sense-rna, siRNA, shRNA and the Crm1 of Crm1 nucleic acid.
In another preference, described inhibitor is siRNA or shRNA.
In another preference, described siRNA positive-sense strand has the sequence as shown in SEQ ID NO:7, and the antisense strand of described siRNA has the sequence as shown in SEQ ID NO:8; And/or
Described shRNA comprises sh-NC and sh-Crm1-1; Wherein, the positive-sense strand of described sh-NC has the sequence as shown in SEQ ID NO:11, and antisense strand has the sequence as shown in SEQ ID NO:12; The positive-sense strand of described sh-Crm1-1 has the sequence as shown in SEQ ID NO:13, and antisense strand has the sequence as shown in SEQ ID NO:14.
A fourth aspect of the present invention, provides a kind of pharmaceutical composition, comprises Crm1 inhibitor and pharmaceutically acceptable carrier.
In another preference, described Crm1 inhibitor comprises the activity inhibitor of sense-rna, siRNA, shRNA and the Crm1 of antibody, the Crm1 nucleic acid of Crm1.
A fifth aspect of the present invention, provides a kind of inhibition Growth of Gastric of external non-therapeutic or the method for propagation, comprises step: under Crm1 inhibitor exists, cultivate cancer cells, thus anticancer growth or propagation.
In another preference, described method comprises in the culture system of cancer cells adds Crm1 inhibitor, thereby suppresses anticancer growth or propagation.
A sixth aspect of the present invention, provides a kind of method of screening the candidate compound for the treatment of cancer of the stomach, and described method comprises step:
(a) in test group, in the culture system of cell, add test compounds, and observe expression amount and/or the activity of Crm1 in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of Crm1 in the described cell of control group;
Wherein, if the expression amount of the Crm1 of cell and/or activity are less than control group in test group, just show that this test compounds is expression and/or the active candidate compound that has inhibiting treatment cancer of the stomach to Crm1.
In another preference, the expression amount of described Crm1 is detected and is drawn by RT-PCR.
In another preference, described method also comprises step:
(b) for the candidate compound obtaining in step (a), further test its restraining effect to Growth of Gastric or propagation; And/or further test it and whether Crm1 gene is had to the effect of downward.
In another preference, comprise step in step (b): in test group, in the culture system of stomach cancer cell, add test compounds, and observe quantity and/or the growing state of stomach cancer cell; In control group, in the culture system of stomach cancer cell, do not add test compounds, and observe quantity and/or the growing state of stomach cancer cell; Wherein, if the quantity of stomach cancer cell or the speed of growth are less than control group in test group, just show that this test compounds is the candidate compound that growth to stomach cancer cell or propagation have inhibiting treatment cancer of the stomach.
A seventh aspect of the present invention, provides a kind of method that suppresses or treat cancer of the stomach, comprises step: the Crm1 inhibitor of using safe and effective amount to the object (Mammals) of needs treatment.
In another preference, described inhibitor comprises: the activity inhibitor of the antibody of Crm1 albumen, the sense-rna of Crm1, siRNA, shRNA and Crm1.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tire out and state no longer one by one at this.
Brief description of the drawings
Fig. 1 has shown the expression schematic diagram of RT-PCR checking Crm1 gene in 21 routine Patients with Gastric Cancer cancerous tissues and cancer beside organism in embodiment 1.Wherein, " N " refers to cancer beside organism, and " C " refers to stomach organization.Result shows in 13 examples (61.9%) patient's cancerous tissue that Crm1 gene expression amount is higher than corresponding cancer beside organism.
Fig. 2 has shown that in embodiment 2, real-time quantitative PCR detects the expression schematic diagram of Crm1 gene in Human Stomach Tissue and cancer beside organism.Result shows that in 10 examples (66.7%) patient's cancerous tissue, Crm1 gene expression amount is higher than corresponding cancer beside organism, similar to the result of RT-PCR in embodiment 1.
Fig. 3 has shown and in embodiment 3, in stomach cancer cell BCG823 and AGS, has disturbed Crm1 genetic expression and analysis of cells propagation situation schematic diagram.Wherein, Fig. 3 A represents that Western Blot detects siRNA to Crm1 Gene silencing efficacy; Fig. 3 B is the data analysis of cell proliferation.The siRNA of Crm1 represents with si-1 and si-2 respectively, and NC represents contrast.Result shows: specificity disturbs Crm1 genetic expression can suppress the multiplication capacity of stomach cancer cell BCG823 and AGS, shows that the downward of people Crm1 genetic expression can suppress the growing multiplication of stomach cancer cell to a certain extent.
Fig. 4 disturbs Crm1 genetic expression in stomach cancer cell BCG823 and AGS in embodiment 4, detect cell soft-agar cloning and form ability schematic diagram.Wherein, the siRNA in two kinds of disturbance sites of Crm1 represents with si-1 and si-2 respectively, and NC represents contrast.Result shows and is subject to that siRNA disturbs and the stomach cancer cell of Crm1 down-regulated expression forms in experiment and suppressed significantly at soft-agar cloning.
Fig. 5 disturbs Crm1 genetic expression in stomach cancer cell BCG823 and MKN-28 in embodiment 5, detect cell and become knurl ability schematic diagram at nude mice by subcutaneous.The wherein same si-1 of si-Crm1 sequence.Result shows and is subject to that siRNA disturbs and ability that the stomach cancer cell of Crm1 down-regulated expression forms tumour at nude mice by subcutaneous reduces.
Fig. 6 represents for the Crm1 shRNA(Lv-shCrm1 with lentivirus mediated in embodiment 7) treatment nude mice by subcutaneous becomes the situation of knurl body.Result shows that the Lv-shCrm1 of lentivirus mediated can obviously suppress the growth of knurl body, and this shRNA of prompting Crm1 can be used as the target spot for the treatment of cancer of the stomach.
Embodiment
The inventor, through extensive and deep research, is surprised to find that first, and Crm1 expresses higher than cancer beside organism or healthy tissues in gastric carcinoma cell lines, the mark that can be used as cancer of the stomach detection for detection of or complementary detection cancer of the stomach.In addition the growth that, the inhibitor of Crm1 can anticancer.Complete on this basis the present invention.
Experiment also proves, the special inhibitor of application Crm1 can obviously suppress growth and the clonality of stomach cancer cell in vitro as RNA interfering (siRNA), shRNA, and can affect the one-tenth knurl of in-vivo tumour.Therefore, suppress the function of Crm1 with Crm1 inhibitor, can suppress the growth of the stomach cancer cell of in vitro and in vivo, thereby provide new approach for the treatment of cancer of the stomach.
Crm1 albumen and polynucleotide
The full name of Crm1 is that chromosome region maintenance 1 protein(chromosomal region maintains albumen 1), claim again Exportin-1(Xpo1), its coded product mediation has is rich in leucic nuclear export signal albumen from nucleus to cytoplasmic transhipment, it also can suppress the core of Rev and U snRNAs and export (Maarten F, et al.Cell.1997).Crm1 participates in the activity of various kinds of cell biology, and its mode of action is mainly transported out core from nucleopore with RAN GTPase together with transporter formation ternary complex.In addition, Crm1 can also exercise the function that ripe microRNAs caryoplasm shuttles back and forth, and this function plays effect (Daniela C.et al.PNAS.2009) to the function of microRNAs performance regulate gene expression.Have been reported and show Crm1 up-regulated (Yao Y, et al.Oncol Rep.2009, Noske A.et al.Cancer.2008) in osteosarcoma and ovarian cancer.
In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " Crm1 albumen " are used interchangeably, and refer to Crm1 albumen.Should be understood that described term also comprises active fragments and the derivative of Crm1.
In the present invention, " gene of the present invention ", " polynucleotide of the present invention ", " Crm1 gene " refer to the to encode nucleotide sequence of Crm1 albumen or its active fragments and derivative, comprises justice and antisense nucleic acid.
In the present invention, term " Crm1 albumen ", " Crm1 polypeptide " or " cancer of the stomach mark Crm1 " are used interchangeably, and all refer to have albumen or the polypeptide of Crm1 aminoacid sequence.
The nucleotide sequence of Crm1 as shown in SEQ ID NO.:15, its GenBank Accession:NM_003400.3, Entrez Gene ID:7514, this nucleotide sequence coded aminoacid sequence is as shown in SEQ ID NO.:16.
As used herein, " separation " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.
As used herein, " Crm1 albumen or the polypeptide of separation " refers to that Crm1 albumen does not basically contain natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be purified Crm1 albumen with the purified technology of protein of standard.Substantially pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferably recombinant polypeptide.
Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.
The polynucleotide of the mature polypeptide of coding Crm1 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence (with optional additional code sequence) and the non-coding sequence of mature polypeptide.Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and can be also the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or the fragment of polypeptide, analogue and derivative with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of its coded polypeptide.
The invention still further relates to and the nucleic acid fragment of above-mentioned sequence hybridization, comprise the nucleic acid fragment of justice and antisense.As used herein, the length of " nucleic acid fragment ", containing 15 Nucleotide, is at least better at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding Crm1 albumen.
People Crm1 Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be according to published relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.In the time that sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment amplifying for each time is stitched together by proper order.
Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by ordinary method.
In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic multiple small segments, and then connect and can obtain the fragment that sequence is very long.
The method of application round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.Primer for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment increasing by gel electrophoresis separation and purifying.
The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell producing through genetically engineered with carrier of the present invention or Crm1 albumen coded sequence, and produce the method for polypeptide of the present invention through recombinant technology.
By conventional recombinant DNA technology, can utilize polynucleotide sequence of the present invention to can be used to the Crm1 albumen of expression or Restruction.In general there are following steps:
(1) with the polynucleotide (or varient) of encoding human Crm1 albumen of the present invention, or transform or the suitable host cell of transduceing with the recombinant expression vector that contains these polynucleotide;
(2) host cell of cultivating in suitable substratum;
(3) separation, protein purification from substratum or cell.
Method well-known to those having ordinary skill in the art can be used for building containing people Crm1 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of the host cell transforming, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of use, or for colibacillary tsiklomitsin or amicillin resistance.
Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, the bacterial cell of streptomyces; Fungal cell is as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or 293 cells etc.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl 2method processing, step used is well-known in this area.Another kind method is to use MgCl 2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packaging etc.
The transformant obtaining can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promotor of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell for some time again.
Extracellular can be expressed or be secreted into recombinant polypeptide in the above methods in cell or on cytolemma.If needed, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processing, with the combination of protein precipitant processing (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Antibody
The present invention also comprises that people Crm1 albumen is had to specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into people Crm1 gene product or fragment.Preferably, refer to that those can be combined with people Crm1 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people Crm1 gene product of purification or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, the cell of expression people's Crm1 albumen or its antigen also can be used for animal to cause immunity and produce antibody.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) 2fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered scFv molecule; Or chimeric antibody.
Antibody of the present invention comprises the antibody that can suppress Crm1 function, can be also the antibody that does not affect people Crm1 function.Each antibody-like can cause immunity by the fragment to people Crm1 gene product or functional domain and produce, and people Crm1 gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the antibody that the Crm1 of non-modified form gene product is combined, can utilize the gene product producing in prokaryotic cell prokaryocyte (as E.coli) carry out immune animal and obtain.With the antibody that posttranslational modification form is combined as glycosylation or phosphorylation Crm1 albumen or polypeptide, can utilize the gene product producing in as yeast or insect cell at eukaryotic cell carry out immune animal and obtain.
Can be used for Crm1 antibody of the present invention can be anti-human Crm1 protein antibodies.Anti-human Crm1 protein antibodies of the present invention can be used in immunohistochemistry technology, detects the people Crm1 albumen in biopsy specimen.
Inhibitor and pharmaceutical composition
Utilize albumen of the present invention, by various conventional screening methods, can filter out with Crm1 albumen interactional material occurs, especially inhibitor etc.
The inhibitor (comprising antibody, antisense nucleic acid and other inhibitor) of Crm1 albumen of the present invention, when use (administration) in treatment time, expression and/or the activity of Crm1 albumen be can suppress, and then growth or the propagation of stomach cancer cell suppressed.Conventionally, but these materials are formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably pH is about 6-8, although pH value can change to some extent with being formulated the character of material and illness to be treated.The pharmaceutical composition preparing can carry out administration by conventional route, comprising (but being not limited to): in knurl, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Can be used for inhibitor of the present invention comprises: the activity inhibitor of the antibody of Crm1, sense-rna, siRNA, shRNA and the Crm1 of Crm1 nucleic acid.Wherein, typical Crm1 inhibitor is siRNA and shRNA.
Can be used for siRNA of the present invention and comprise si-1 and the si-2 with disturbance site, wherein, the positive-sense strand of si-1 has the sequence as shown in SEQ ID NO:7, and antisense strand has the sequence as shown in SEQ ID NO:8; The positive-sense strand of si-2 has the sequence as shown in SEQ ID NO:17, and antisense strand has the sequence as shown in SEQ ID NO:18.
Can be used for shRNA of the present invention, comprise sh-NC and sh-Crm1-1, the positive-sense strand of described sh-NC has the sequence as shown in SEQ ID NO:11, and antisense strand has the sequence as shown in SEQ ID NO:12; The positive-sense strand of described sh-Crm1-1 has the sequence as shown in SEQ ID NO:13, and antisense strand has the sequence as shown in SEQ ID NO:14.
Typically, Crm1 gene is comprised to following scheme as the technical scheme of the target spot of preparing curing gastric cancer medicine:
1. chemosynthesis double stranded ribonucleic acid molecule, its sequence-specific is for Crm1 gene order, utilize liposome to be delivered to the expression of stomach cancer cell internal interference Crm1 gene, observe the change of the characteristics of cell biology such as soft-agar cloning formation ability, cell proliferation.Can utilize the method for this area routine to design and synthetic specificity for the nucleotide sequence (as siRNA) of Crm1.
2. utilize various carriers, comprise that DNA vector, lentiviral vectors disturb the expression of Crm1 gene, reach the effect of body internal interference Crm1 gene, detect them and nude mice by subcutaneous is become to the result for the treatment of of knurl body, thereby realize the object that suppresses cancer of the stomach propagation.
3. obtain the polypeptide, the monoclonal antibody that can specificity suppress Crm1 gene delivery activity, reach the object that suppresses Crm1 activity, thereby reality suppresses the object of stomach cancer cell proliferation in vivo.
The present invention also provides a kind of pharmaceutical composition, Crm1 inhibitor of the present invention (as antibody, antisense sequences (as siRNA) or inhibitor) and pharmaceutically acceptable carrier or vehicle that it contains safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, for example, be prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as Tablet and Capsula, can be prepared by ordinary method.Pharmaceutical composition should be manufactured as injection, solution, Tablet and Capsula under aseptic condition.The dosage of activeconstituents be treatment significant quantity, for example every day approximately 1 microgram-10 mg/kg body weight.
Detection method and test kit
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people Crm1 protein level or mRNA level.These tests are known in the art.The people Crm1 protein level detecting in test, can be for diagnosis of gastric cancer.
A kind ofly detect that in sample, whether to have the method for Crm1 albumen be to utilize the specific antibody of Crm1 albumen to detect, it comprises: sample is contacted with Crm1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in sample Crm1 albumen.
Crm1 albumen or its polynucleotide can be used for diagnosis and the treatment of Crm1 protein related diseases.Part or all of polynucleotide of the present invention can be used as probe and is fixed on microarray or DNA chip, for analyzing Differential expression analysis and the gene diagnosis of tissue gene.The antibody of anti-Crm1 can be fixed on protein chip, for detection of the Crm1 albumen in sample.
The present invention also provides a kind of test kit that detects cancer of the stomach, the primer pair that it contains specific amplification Crm1 and/or Crm1 specific antibody and label or specification sheets.
Wherein, described label or specification sheets indicate following content: as the ratio >1 of the mrna expression amount of the mrna expression amount of the relative beta-actin of the Crm1 of detected object and the relative beta-actin of Crm1 of cancer beside organism, point out the cancered probability of this detected object higher than general population.
Described test kit can be used for detecting people's stomach-tissue sample or blood sample.
Screening method
The present invention also provides the method for carrying out drug screening based on Crm1.One method is first to screen impact (inhibition) Crm1 expression or active compound, then the compound filtering out is further tested to its restraining effect to cancer cells.
Wherein, representational cancer cells is included as stomach cancer cell.
A kind of preferred screening method can be based on Crm1 the expression level of mRNA.Concrete steps are as follows:
(a) in test group, in the culture system of cell, add test compounds, and observe expression amount and/or the activity of Crm1 in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of Crm1 in the described cell of control group;
If the expression amount of the Crm1 of cell and/or activity are less than control group in test group, just show that this test compounds is expression and/or the active candidate compound that has inhibiting treatment cancer of the stomach to Crm1.
Wherein, the expression amount of described Crm1 is detected and is drawn by RT-PCR.
This screening method also can comprise step:
(b) for the candidate compound obtaining in step (a), further test its restraining effect to Growth of Gastric or propagation; And/or further test it and whether Crm1 gene is had to the effect of downward.
In test group, in the culture system of stomach cancer cell, add test compounds, and observe quantity and/or the growing state of stomach cancer cell; In control group, in the culture system of stomach cancer cell, do not add test compounds, and observe quantity and/or the growing state of stomach cancer cell; Wherein, if the quantity of stomach cancer cell or the speed of growth are less than control group in test group, just show that this test compounds is the candidate compound that growth to stomach cancer cell or propagation have inhibiting treatment cancer of the stomach.
The method of screening treatment cancer of the stomach medicine can also detect by active oxygen.Active oxygen (ROS) detects the method for the detection intracellular reactive oxygen level that is a kind of routine, can determine by detecting ROS the activity of Crm1.The activity of Crm1 is higher, and ROS is lower.
Active oxygen (ROS) detects available commercially available detection kit to carry out.A kind of active oxygen detection kit (Reactive Oxygen Species Assay Kit) is to utilize fluorescent probe DCFH-DA to carry out the test kit of active oxygen detection.Itself does not have fluorescence DCFHDA, can pass freely through cytolemma, after entering in cell, can be generated DCFH by intracellular esterase hydrolyzed.And DCFH can not penetrating cytolemma, thereby make probe be easy to be loaded onto in cell.Intracellular active oxygen can be oxidized non-blooming DCFH and generate the DCF that has fluorescence.The fluorescence that detects DCF just can be known the level of reactive oxygen species.
In the present invention the results show specificity for the small ribonucleic acid molecules of Crm1 gene can suppress the external propagation of stomach cancer cell with pernicious and suppress stomach cancer cell nude mice by subcutaneous become knurl performance, illustrate that Crm1 gene can be used as preparing the target gene of curing gastric cancer medicine.
Beneficial effect of the present invention
The expression level of 1.Crm1 and activity level can be used as the mark that cancer of the stomach detects: Crm1 can be used as the mark of diagnosis of gastric cancer at stomach organization or intraserous high expression level, its specificity, highly sensitive is easy and simple to handle.
2.Crm1 inhibitor can effectively suppress proliferation of human gastric cancer cell, or as the medicine for the treatment of cancer of the stomach.
Following examples are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Universal method:
1. separate tissue and total RNA extracting
1.1 separate tissue
Test with tissue-derived in the operation patients of cancer of the stomach.The lump of excision is once in vitro, cuts rapidly focus and 5 centimeters of outer cancer beside organisms around, puts into liquid nitrogen (80 DEG C) and preserves.The diagnosis on cancer and cancer side is all taking pathological diagnosis as final foundation.
1.2 total RNA extractings
Adopt the Trizol reagent of Invitrogen company, according to conventional extraction steps, extracting total RNA (RNA).This Trizol reagent is produced based on acidic phenol one step extraction process.Extracting vessel used and water all carry out rnase inactivation treatment, to ensure the environment of deoxyribonuclease in experiment.
The general step that Yeast Nucleic Acid (RNA) extracts is: broken tissue → isolation of RNA → precipitated rna → washing RNA → melt RNA → preservation RNA.Broken tissue and deactivation RNA enzyme can synchronously carry out, and can use the broken tissue such as Guanidinium hydrochloride, guanidine thiocyanate, NP-40, SDS, Proteinase K, add β-ME can suppress RNA enzymic activity.Isolation of RNA is the organic solvent such as phenol, chloroform for half, adds a small amount of primary isoamyl alcohol, and through this step, centrifugal, RNA is generally distributed in upper strata, separates with egg white layer.Precipitated rna is generally used ethanol, 3M NaAc(pH-5.2) or Virahol.Washing RNA uses 70% washing with alcohol.Melt RNA and generally use TE.Preserve the RNA low temperature of should trying one's best.
2. reverse transcription
2.1 ice bath centrifuge tube the insides add template ribonucleic acid 2 μ g, primer (10pmol) 2 μ L, and deionized water polishing to 15 μ L, mixes, centrifugal 3～5 seconds.
2.2 70 DEG C of water-baths 5 minutes, ice bath 30 seconds.
2.3 add 5 × M-MLV reaction buffer, 5 μ L, and RNase inhibitor (25units/ μ L) 1 μ L, dNTP mixture (concentration is 10mM separately) 1.25 μ l, mix.
2.4 37 DEG C of water-baths 5 minutes, add 1 μ L AMV-RT ThermoScript II (200units/ μ L), mix.
2.5 37 DEG C of water-baths 1 hour (this step is transcriptive process,reversed).
2.6 70 DEG C, 10 minutes finish reaction, and product is put and carried out next step PCR experiment on ice ,-70 DEG C of remaining preservations.
3.RT-PCR and real-time quantitative PCR
3.1 RT-PCR and design of primers thereof
RT-PCR(reverse transcription-polymerase chain reaction) refer to reverse transcription (Reverse Transcription; RT) reaction and PCR (Polymerase Chain Reaction) react the method for combining.RT-PCR combines with PCR synthetic the cDNA taking RNA as template, and a kind of method of rapid sensitive of analyzing gene expression is provided.The template of RT-PCR can be total RNA or poly (A) selectivity RNA.Reverse transcription reaction can use reversed transcriptive enzyme, initial with the primer (GSP) of random primer, oligo (dT) or gene specific.
The reaction conditions of PCR is as follows: 94 DEG C, and 5 minutes denaturations; 94 DEG C, sex change in 30 seconds; 55 DEG C, annealing in 30 seconds; 72 DEG C, within 30 seconds, extend; 35 circulations, electrophoresis detection pcr amplification product.
The primer of design is specific as follows:
Crm1:(F) as shown in SEQ ID NO:1; (R) as shown in SEQ ID NO:2;
β-actin:(F) as shown in SEQ ID NO:3; β-actin(R) as shown in SEQ ID NO:4.
Using house-keeping gene β-actin as internal reference, PCR reaction system is: 10 × Taq Buffer, 2 μ l, dNTP mixture (concentration is 10mM separately) 0.5 μ l, 10 μ M forward primer 0.5 μ l, 10 μ M reverse primer 0.5 μ l, template cDNA 2 μ l, Taq enzyme (5U/ μ l, TaKaRa) 0.5 μ l, ddH 2o 14 μ l, total system 20 μ l.
3.2 real-time quantitative PCR
Adopt relative quantification method to detect the differential expression of Crm1 gene in cancer of the stomach sample.Use TP800 type real-time fluorescence quantitative PCR instrument (the Thermal Cycler Dice of Japanese Takara company tMreal Time System) and premix Ex Taq tMreagent.The use of instrument is by the description operation of producer.
Quantitative fluorescent PCR (real-time PCR) reaction system is as follows: cumulative volume 20 μ L, SYBR Premix Ex Taq 10 μ L, the each 0.4 μ L of upstream and downstream primer (10 μ mol/L), cDNA 1 μ L, ddH2O 8.2 μ L, mix reagent, putting into PCR instrument after centrifugal carries out amplified reaction.
Reaction conditions: 95 DEG C, 30 seconds denaturations; 95 DEG C, sex change in 5 seconds, 60 DEG C, annealing in 30 seconds/extend totally 40 circulations.Primer is specific as follows:
Crm1 the primer is consistent with sxemiquantitative RT-PCR Crm1 primer used, and its upstream primer is as shown in SEQ ID NO:1; Downstream primer is as shown in SEQ ID NO:2.
House-keeping gene β-actin is as internal reference, and its upstream primer is as shown in SEQ ID NO:5; Downstream primer is as shown in SEQ ID NO:6.
4. antigen-antibody preparation
The acquisition of 4.1 antigen proteins
The cDNA sequence that obtains people Crm1 gene from Genebank database, obtains encoder block by pcr amplification, inserts in prokaryotic organism or eukaryote expression vector, expresses Crm1 albumen, and presses the purification system purifying protein of gene engineering expression product.
The preparation of 4.2 antibody
Can adopt following several method Dispersal risk:
4.2.1 cytogamy method: with the Crm1 protein immune animal (comprising rabbit, goat etc.) of above-mentioned preparation, obtain spleen cell, then merge with myeloma cell, and monoclonal antibody technology of preparing is prepared monoclonal antibody routinely.
4.2.2 utilize phage display storehouse, the spleen IgG variable region of clone immune animal is also expressed as gene engineering monoclonal antibody.
4.2.3 utilize the protein immune animal of purifying, prepare polyvalent antibody.
5.RNA interference experiment
Ability of cell proliferation experiment after 5.1 siRNA disturb
The Cell Counting Kit-8 reagent that adopts Dojindo Laboratories, carries out according to product description:
5.1.1 transfection the day before yesterday, by a certain amount of cell with the culture medium inoculated of antibiotic-free in culture dish (quantity of inoculation depends on the speed of growth of different cells), while making it to transfection, can reach the density of 30 – 50%.
5.1.2 prepare siRNA-Lipofectamine tM2000 transfection composites, comprising: A pipe: Lipofectamine tM2000 with mix, room temperature leaves standstill 5min; B pipe: siRNA with mix, room temperature leaves standstill 5min.
5.1.3 mix A pipe and B pipe, incubated at room 20min.
5.1.4 cell sucks nutrient solution, is changed to Free(serum-free, antibiotic-free) nutrient solution, add transfection composite (final concentration of siRNA is 33nM), mix.
5.1.5 37 DEG C, 5%CO 2cultivate after 4 – 6h, replacing nutrient solution is complete culture solution (containing 10%FBS).
5.1.6 peptic cell after transfection 24 – 72h, is inoculated in 96 well culture plates, and every hole adds 100ul to contain the nutrient solution of 3000 cells, and 3, each sample is answered holes.
5.1.7 every other day detect with CCK8 reagent, continuously 5-7 days.Concrete grammar is for to add respectively 10ul CCK8 reagent in every hole, cultivates 2 hours for 37 DEG C, detects A450 by microplate reader.
5.1.8 cell transfecting is after 24 hours, and cell is reclaimed in digestion.
After 5.2 siRNA disturb, soft-agar cloning formation ability detects
5.2.1 the 2%Agarose of 37 ° of C insulations and 2.5xDMEM equal-volume are mixed, be added in 24 orifice plates, every hole 0.5ml, is placed in 4 ° of C refrigerators, after solidifying, uses.
5.2.2 BCG823 and AGS are inoculated in deep bid with 80% density,, to cell, cultivate after 24-36 hour containing the DMED condition of 10%FBS with the method transfection siRNA of liposome or negative control siRNA, digestion, counting, is diluted to same concentrations.
5.2.3 cell suspension is mixed with 1% Agarose equal-volume of 37 ° of C insulations, be added in 24 orifice plates of completing lower floor's glue, every hole 0.5ml, is placed in 4 ° of C refrigerators 10 minutes.
5.2.4 on the soft agar solidifying, add the nutrient solution of 0.2ml, be placed in 37 ° of C, 5%CO 2incubator, continues to cultivate 2-3 week.
After 5.3 siRNA disturb, nude mice by subcutaneous becomes knurl ability to detect
5.3.1 with 80% density, BCG823 and Mkn-28 are inoculated in deep bid, to cell, cultivate after 24-36 hour digestion, counting with the method transfection siRNA of liposome or negative control containing the DMED condition of 10%FBS.
5.3.2 inject respectively 2 × 10 6individual cell is subcutaneous to nude mice belly left side and right side, and the 1-2 month is cultivated by SPF culturing room.
5.3.3 about one week after growing knurl body, every the 2 days length by vernier caliper measurement tumor and wide, utilize formula V=long × wide 2/ 2 calculate tumor volume.
5.3.4 after one month, kill nude mice and take out knurl body, weigh and take pictures.
After 5.4 shRNA disturb, nude mice by subcutaneous becomes knurl ability to detect
5.4.1 by the BCG823 cell 1 × 10 of normally cultivating 6individual cell/only, be injected into nude mice subcutaneous abdomen, is divided into 2 groups at random;
5.4.2 after becoming knurl, when the about 5mm diameter of tumorous size, start to multi-point injection in knurl body, two component cloth injection shRNA and negative control slow viruss, per injection 5 × 10 7the slow virus amount of copy, injects once totally 5 times for every 3 days.
5.4.3 after one and a half months, kill nude mice, take out knurl body, weigh.
Embodiment 1 RT-PCR experiment detects the expression of Crm1 gene in stomach organization
To the total RNA of cancer of the stomach sample collecting in universal method 1, adopt the RT-PCR of universal method 2 and 3.1 to test detection, measure the expression of Crm1 gene in stomach organization.
As shown in Figure 1, the expression level of Crm1 gene in stomach organization is apparently higher than the expression level in cancer beside organism: in 21 routine cancer of the stomach cases, in 13 routine cancerous tissues, the expression amount of Crm1 gene, higher than cancer beside organism, accounts for 61.9% ratio for experimental result.Wherein, " N " refers to cancer beside organism, and " C " refers to stomach organization.
The present embodiment presentation of results, can be by RT-PCR diagnosis of gastric cancer: design the PCR primer of Crm1 gene, detect the content of Crm1 gene RNA in tumor tissues, rna content height illustrates get a cancer of the stomach possible high, otherwise low.
Embodiment 2 real-time quantitative PCRs detect the expression of Crm1 gene in stomach organization
Sample is with embodiment 1, and difference is to adopt universal method 3.2 real-time quantitative PCRs to detect.
As shown in Figure 2, the expression level of Crm1 gene in stomach organization is apparently higher than the expression level in cancer beside organism: in 15 pairs of samples, have 10 pairs of Crm1 up-regulateds (seeing Fig. 2) for experimental result.
The present embodiment presentation of results, can pass through real-time quantitative PCR diagnosis of gastric cancer: the PCR primer of design Crm1 gene, the content of Crm1 gene RNA in detection tumor tissues, rna content height illustrates that the possibility getting a cancer of the stomach is high, otherwise low.
Ability of cell proliferation test after the small interference ribonucleic acid (siRNA) of embodiment 3Crm1 gene and interference thereof
For Crm1 gene mRNA sequence, design and external chemosynthesis siRNA(si-1, si-2), its corresponding target sequence is as follows:
Si-1: positive-sense strand is as shown in SEQ ID NO:7; Antisense strand is as shown in SEQ ID NO:8;
Si-2: positive-sense strand is as shown in SEQ ID NO:1 7; Antisense strand is as shown in SEQ ID NO:18;
Irrelevant sequence is set in addition as negative control siRNA, its sequence is as follows:
Positive-sense strand is as shown in SEQ ID NO:9; Antisense strand is as shown in SEQ ID NO:10.
Adopting universal method 5.1 to carry out siRNA disturbs rear ability of cell proliferation to detect
Experimental result is as shown in Figure 3: specificity disturbs Crm1 genetic expression (si-1 and si-2) can suppress the multiplication capacity of stomach cancer cell BCG823 and AGS, shows that the downward of people Crm1 genetic expression can suppress the growing multiplication of stomach cancer cell to a certain extent.
After embodiment 4siRNA disturbs, soft-agar cloning formation ability detects
Adopt siCrm1 sequence and universal method 5.2 in embodiment 3 to carry out the rear soft-agar cloning formation ability detection of siRNA interference.
Experimental result is as shown in Figure 4: transfection siCrm1(si-1, si-2) after, the cell strain that tumour cell forms on soft agar is obviously less than negative control group.
The present embodiment presentation of results, siRNA expression specificity disturbs after Crm1, can effectively limit the clonality of stomach cancer cell line on soft agar, indirectly shows that Crm1 lowers the grade malignancy that has reduced stomach cancer cell.
After embodiment 5siRNA disturbs, nude mice by subcutaneous becomes knurl ability to detect
SiCrm1 sequence in employing embodiment 4 and universal method 5.3 are carried out siRNA and are disturbed rear nude mice by subcutaneous to become knurl ability to detect.
Experimental result is as shown in Figure 5: the stomach cancer cell of transfection siCrm1 becomes knurl volume and weight all little than transfection siNC control group.The present embodiment presentation of results, the downward of Crm1 has effectively suppressed the growth of stomach cancer cell at nude mice by subcutaneous.
Embodiment 6 is for the synthetic shRNA of Crm1 gene design
By synthetic DNA fragmentation sh-NC and the sh-Crm1-1 with hairpin structure of Shanghai Ying Jun Bioisystech Co., Ltd, sequence is as follows:
Sh-NC: shown in positive-sense strand SEQ ID NO:11; Antisense strand is as shown in SEQ ID NO:12;
Sh-Crm1-1 positive-sense strand is as shown in SEQ ID NO:13; Antisense strand is as shown in SEQ ID NO:14.
PSUPER carrier (Oligoengine, Seattle, WA, USA) total length 3176bp, selects restriction endonuclease sites BglII and HindIII as DNA fragmentation insertion point, and vector construction step is as follows:
(1) ddH 2it is 10 μ M that O dissolves synthetic DNA powder, gets the each 1 μ l of positive-sense strand and antisense strand, adds in the annealing buffer of 48 μ l, through 95 ° of C, 4 minutes; 70 ° of C, within 10 minutes, hatch rear room temperature naturally place cooling, annealing form double-stranded DNA;
(2) terminal phosphate of double-stranded DNA: reaction system is as follows: the double-stranded DNA that 2 μ l have annealed, 1 μ lT4 PNK damping fluid, 1 μ l ATP(1mM), 1 μ l T4 PNK, 5 μ l ddH 2o; 37 ° of C, hatch for 30 minutes; 70 ° of C, 10 minutes heat inactivation T4 PNK;
(3) BglII and HindIII double digestion pSUPER:20 μ l system are as follows: 2 μ l 10xK buffer, 1 μ l BglII, 1 μ l HindIII, 2 μ g pSUPER plasmids, ddH 2o mends to 20 μ l, and 37 ° of C react 1 hour; Use the linearizing pSUPER of alkaline phosphatase treatment, remove the phosphate group of end, prevent certainly connecting of plasmid;
(4) connect: the phosphorylation double-stranded DNA oligos of 2 μ l annealing, 1 μ l ligase enzyme buffer, the pSUPER plasmid after 1 μ l double digestion, 1 μ l ligase enzyme, 5 μ l ddH 2o, 16 ° of C react 1 hour;
(5) transform;
(6) the little plasmid of taking out is also cut qualification with EcoRI-HindIII enzyme, the fragment of positive colony big or small about 360bp after enzyme is cut, and last positive colony is entirely true through sequence verification insertion sequence, and-20 ° of C preserve, for subsequent experimental.
In embodiment 7 knurls, multi-point injection detects the therapeutic action of shCrm1-1 slow virus to knurl body
Adopt the sh-Crm1-1 sequence in embodiment 6, be responsible for packing slow virus LV-shCrm1-1 and contrast slow virus LV-shNC by Shanghai Ji Ma Bioisystech Co., Ltd.
Experimental result is as shown in Figure 6: tumor volume and the weight of injection LV-Crm1-1 (representing with Lv-shCrm1) slow virus are significantly less than control group, and result has statistical significance.
The present embodiment presentation of results, the slow virus interference carrier of Crm1 has positive effect to the treatment of cancer of the stomach.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. a purposes for Crm1 gene or albumen, is characterized in that, for the preparation of the reagent or the test kit that detect cancer of the stomach.
2. purposes as claimed in claim 1, is characterized in that, described test kit comprises: the reagent and corresponding label or the specification sheets that Crm1 are carried out to detection by quantitative.
3. purposes as claimed in claim 1, is characterized in that, described reagent comprises the Auele Specific Primer of Crm1, probe and chip.
4. for detection of a diagnostic kit for cancer of the stomach, it is characterized in that, described test kit contains a container, contains the detection reagent that detects Crm1 in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detection of cancer of the stomach.
5. a purposes for Crm1 inhibitor, is characterized in that, described inhibitor is used to the medicine that preparation suppresses Growth of Gastric or propagation, or for the preparation of the medicine for the treatment of cancer of the stomach.
6. purposes as claimed in claim 5, is characterized in that, described inhibitor comprises: the activity inhibitor of the antibody of Crm1, sense-rna, siRNA, shRNA and the Crm1 of Crm1 nucleic acid.
7. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises Crm1 inhibitor and pharmaceutically acceptable carrier.
8. the inhibition Growth of Gastric of external non-therapeutic or a method for propagation, is characterized in that, comprises step: under Crm1 inhibitor exists, cultivate cancer cells, thus anticancer growth or propagation.
9. a method of screening the candidate compound for the treatment of cancer of the stomach, is characterized in that, described method comprises step:
(a) in test group, in the culture system of cell, add test compounds, and observe expression amount and/or the activity of Crm1 in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of Crm1 in the described cell of control group;
Wherein, if the expression amount of the Crm1 of cell and/or activity are less than control group in test group, just show that this test compounds is expression and/or the active candidate compound that has inhibiting treatment cancer of the stomach to Crm1.
10. method as claimed in claim 9, is characterized in that, described method also comprises step:
(b) for the candidate compound obtaining in step (a), further test its restraining effect to Growth of Gastric or propagation; And/or further test it and whether Crm1 gene is had to the effect of downward.
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CN110205384A (en) * 2019-05-21 2019-09-06 张鹏 The purposes of SPZ1 and its purposes of inhibitor and drug screening method

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