CN105950721A - Applications of ITM2A gene as esophagus cancer diagnosis and treatment marker - Google Patents

Applications of ITM2A gene as esophagus cancer diagnosis and treatment marker Download PDF

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Publication number
CN105950721A
CN105950721A CN201610279223.5A CN201610279223A CN105950721A CN 105950721 A CN105950721 A CN 105950721A CN 201610279223 A CN201610279223 A CN 201610279223A CN 105950721 A CN105950721 A CN 105950721A
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itm2a
gene
product
reagent
diagnosis
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CN105950721B (en
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杨承刚
宋宏涛
王欣月
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Qingdao Yangshen Biomedical Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The invention discloses a molecular marker, namely, an ITM2A gene used for the early diagnosis of esophagus cancer and an expression product of the molecular marker. According to the gene and the expression product, the QPCR and Western blot methods are adopted for proving that the ITM2A gene has different expressions in the esophagus cancer tissue and the normal esophagus tissue, and thus the ITM2A gene can be taken as an indicator for early diagnosis of the esophagus cancer. In addition, the invention further discloses applications of the ITM2A gene and the expression product of the ITM2A gene as targets for treating the esophagus cancer, so that the research and development of new drugs can be guided.

Description

ITM2A gene is as the purposes of esophageal carcinoma diagnosis and treatment mark
Technical field
The present invention relates to biological technical field, more particularly to ITM2A gene use in the diagnosis, treatment of the esophageal carcinoma On the way.
Background technology
Gene expression profile (expression profile) refers to organism under a certain state relative to a reference The integral status of the gene expression of thing, i.e. biological integrity gene expression difference, have certain characteristic and representativeness.It is main Syllabus be by this phenotypic data disclose gene structure-function relationship under a certain particular biological state and then Disclose the essence of some biosis.
The esophageal carcinoma is one of mankind's common cancer, and its sickness rate has obvious regionality, and wherein 70% in China, Mortality rate occupies the 4th, and within 5 years, survival rate is less than 10%.The esophageal carcinoma is broadly divided into squamous cell carcinoma (Squamous by histological type Cell Carcinoma, SCC) and adenocarcinoma (Adenocarcinoma, ADC), China's scale cancer sickness rate is far above adenocarcinoma (Glickman JN,2003).The change of the gene expression pattern of research prompting in the past is to determine esophageal squamous cell carcinoma malignant phenotype Basis.
The application finds the gene expression pattern difference between tumor tissues and normal structure from transcript profile level.Wish logical Cross the research to differential gene expression to infer gene and intergenic mutual relation, in cell carcinogenesis gene " open " or The mechanism " closed "；Disclose gene and the generation of the esophageal carcinoma and the internal relation of development.In a word, the application is it is intended that find canceration During may relate to signal transduction pathway, illustrate esophageal carcinoma Carcinogenesis Mechanism, find for prevention and the candidate of in early days detection The possible target spot of molecular marker and gene therapy provides new thinking.
Summary of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of esophageal carcinoma early diagnosis of can be used for Molecular marker.Use gene marker to carry out diagnosis of esophageal cancer and there is promptness, specificity and susceptiveness, so that patient is in disease Sick early stage just can know disease risks, for risk height, takes to prevent accordingly and remedy measures.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the product detecting ITM2A gene expression application in the instrument preparing diagnosis of esophageal cancer.
Further, the product of described detection ITM2A gene expression include detect ITM2A gene mRNA levels product and/ Or the product of detection ITM2A protein level.
Further, the product of described detection ITM2A gene expression includes: by RT-PCR, real-time quantitative PCR, immunity inspection The expression of survey, in situ hybridization or chip detection ITM2A gene and expression product thereof is with the product of diagnosis of esophageal cancer.
Further, the product of described RT-PCR diagnosis of esophageal cancer at least includes drawing of a pair specific amplified ITM2A gene Thing；The product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes the primer of a pair specific amplified ITM2A gene；Described Include with the product of immune detection diagnosis of esophageal cancer: the antibody being combined with ITM2A protein-specific；Described in situ hybridization diagnoses The product of the esophageal carcinoma includes: with the probe of the nucleic acid array hybridizing of ITM2A gene；The product bag of described chip diagnosis of esophageal cancer Include: protein chip and gene chip；Wherein, protein chip includes the antibody being combined with ITM2A protein-specific, gene chip bag Include the probe of nucleic acid array hybridizing with ITM2A gene.
Drawing of a pair specific amplified ITM2A gene that the product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes Thing is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of described detection ITM2A gene expression can be the reagent of detection ITM2A gene expression, can also be to comprise The test kit of described reagent, chip, reagent paper etc., it is also possible to be the high-flux sequence platform using described reagent.
The instrument of described diagnosis of esophageal cancer includes but not limited to chip, test kit, reagent paper or high-flux sequence platform；High Flux order-checking platform is the instrument of a kind of special diagnosis of esophageal cancer, along with the development of high throughput sequencing technologies, to a people's The structure of gene expression profile will become and work the most easily.By contrast Disease and the gene expression profile of normal population, The exception easily analyzing which gene is relevant to disease.Therefore, in high-flux sequence, know exception and the food of ITM2A gene Pipe cancer is correlated with and is fallen within the purposes of ITM2A gene, equally within protection scope of the present invention.
Present invention also offers the instrument of a kind of diagnosis of esophageal cancer, described instrument includes the examination detecting ITM2A gene expression Agent；Described reagent includes primer and/or probe, the antibody of detection ITM2A albumen detecting ITM2A gene mRNA.
Described instrument includes but not limited to chip, test kit, reagent paper or high-flux sequence platform.
Wherein, described chip includes gene chip, protein chip；Described gene chip includes solid phase carrier and fixes At the oligonucleotide probe of solid phase carrier, described oligonucleotide probe include for detect ITM2A gene transcription level for The oligonucleotide probe of ITM2A gene；Described protein chip includes solid phase carrier and is fixed on the ITM2A egg of solid phase carrier White specific antibody；Multiple genes that described gene chip can be used for detecting including ITM2A gene are (such as, with esophagus Multiple genes that cancer is relevant) expression.Described protein chip can be used for the multiple eggs detecting including ITM2A albumen The expression of white matter (such as relevant to the esophageal carcinoma multiple protein).By multiple marks with the esophageal carcinoma are examined simultaneously Survey, be greatly improved the accuracy rate of esophagus cancer diagnosis.
Wherein, described test kit includes gene detecting kit and protein immunization detection kit；Described gene test tries Agent box includes the reagent for detecting ITM2A gene transcription level；Described protein immunization detection kit includes ITM2A albumen Specific antibody.Further, described reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip Reagent required during method detection ITM2A gene expression dose.Preference, described reagent includes for ITM2A gene Primer and/or probe.Nucleotide sequence information according to ITM2A gene is easily designed and be may be used for detecting ITM2A gene table Reach primer and the probe of level.
Described reagent paper includes the reagent detecting ITM2A gene expression.
Described high-flux sequence platform includes the reagent detecting ITM2A gene expression.
With the probe of the nucleic acid array hybridizing of ITM2A gene can be DNA, RNA, DNA-RNA chimera, PNA or other Derivant.The length of described probe does not limit, as long as it is specific binding with purpose nucleotide sequence to complete specific hybrid, Any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, described probe Length can be grown to 60,80,100,150,300 base pairs or longer, the most whole gene.Owing to different probe length is to miscellaneous Hand over efficiency, signal specificity to have different impacts, the length of described probe to be typically at least 14 base pairs, the longest typically do not surpass Cross 30 base pairs, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe self-complementary sequence Row are most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
Further, the specific antibody of described ITM2A albumen includes monoclonal antibody, polyclonal antibody.Described ITM2A egg White specific antibody include complete antibody molecule, any fragment of antibody or modify (such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described fragment can retain and the binding ability of ITM2A albumen.For protein level The preparation of antibody time well known to a person skilled in the art, and the present invention can use any method to prepare described antibody.
In specific embodiments of the present invention, the primer of described detection ITM2A gene mRNA include SEQ ID NO.3 and Primer pair shown in SEQ ID NO.4.
Present invention also offers the accelerator medicine in the preparation treatment esophageal carcinoma of ITM2A gene and/or its expression product In application.
Described accelerator includes the reagent promoting ITM2A gene expression and promotes the reagent of ITM2A gene expression product；Institute State and promote that the reagent of ITM2A gene expression includes promoting the reagent of genetic transcription, promotes the reagent of gene translation, promotion ITM2A The reagent of protein content；The reagent of described promotion ITM2A gene expression product includes promoting ITM2A gene expression product stability Reagent, promote ITM2A gene expression product activity reagent, promote ITM2A gene expression product function reagent.
Specifically, the reagent of described promotion ITM2A gene expression includes: reagent containing ITM2A gene, carry ITM2A The reagent that the carrier of gene or host cell are formed, the reagent containing ITM2A protein.
On the one hand the accelerator of the present invention may be used for supplementing disappearance or the deficiency of endogenic ITM2A albumen, by carrying The expression of high ITM2A albumen, thus the esophageal carcinoma that treatment causes because of ITM2A hypoproteinosis.On the other hand may be used for promoting The activity of ITM2A albumen or function, thus treat the esophageal carcinoma.
The carrier carrying gene of the present invention is various carrier known in the art, as commercially available carrier, include plasmid, Cosmid, phage, virus etc..
In the present invention, term " host cell " includes prokaryotic cell and eukaryotic cell.Conventional prokaryotic host cell Example includes escherichia coli, bacillus subtilis etc..Conventional eukaryotic host cell includes yeast cells, insect cell and mammal Cell.It is preferred that this host cell is eukaryotic cell, such as Chinese hamster ovary celI, COS cell etc..
Present invention also offers a kind of pharmaceutical composition for treating the esophageal carcinoma, described pharmaceutical composition includes institute above The ITM2A gene stated and/or the accelerator of its expression product.
Further, the medicine of the present invention also includes pharmaceutically acceptable carrier, carrier, and this kind of carrier includes (but not It is limited to): diluent, excipient such as water etc., filler such as starch, sucrose etc.；Binding agent such as cellulose derivative, alginate, bright Glue and polyvinylpyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as agar, calcium carbonate and sodium bicarbonate；Absorption enhancer quaternary ammonium Compound；Surfactant such as hexadecanol；Absorption carrier such as Kaolin and soap clay；Lubricant such as Pulvis Talci, calcium stearate With magnesium, Polyethylene Glycol etc..
The medicine of the present invention import tissue or cell mode can by be divided into external or internal in the way of.Vitro formats Import in cell including by the medicine containing ITM2A gene or the medicine containing ITM2A protein, then by cell transplantation or return It is passed to internal.Internal mode includes directly by the medicine containing ITM2A gene or the infusion of medicine body containing ITM2A protein In inner tissue.
The medicine of the present invention also can be with the drug combination of the other treatment esophageal carcinoma, and multi-medicament is used in combination and can significantly carry Success rate to treatment.
In the context of the present invention, " ITM2A gene " includes ITM2A gene and any function etc. of ITM2A gene The polynucleotide of jljl.ITM2A gene includes and ITM2A gene in the most international common core sequence databank GeneBank (NC_000023.11) DNA sequence has more than 70% homology, and coding identical function protein DNA sequence；
Preferably, the coded sequence of ITM2A gene includes any DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) the DNA sequence hybridization that limits and coding identical function protein DNA sequence；
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% homology, and encodes identical merit Can protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described ITM2A gene is shown in SEQ ID NO.1 DNA sequence.
In the context of the present invention, ITM2A gene expression product includes ITM2A albumen and the part of ITM2A albumen Peptide.The partial peptide of described ITM2A albumen contains the functional domain relevant to the esophageal carcinoma.
" ITM2A albumen " includes any function equivalent of ITM2A albumen and ITM2A albumen.Described function equivalent Including ITM2A albumen conservative variation's protein or its active fragment, or its reactive derivative, allelic variant, natural mutation Body, induced mutants, can be with the protein coded by the DNA of the DNA hybridization of ITM2A under high or low stringent condition.
Preferably, ITM2A albumen is the protein with following amino acid sequences:
(1) protein being made up of the aminoacid sequence shown in SEQ ID NO.2 in sequence table；
(2) aminoacid sequence shown in SEQ ID NO.2 through the replacement of one or several amino acid residue and/or is lacked Lose and/or add and with the aminoacid sequence shown in SEQ ID NO.2 have identical function by the ammonia shown in SEQ ID NO.2 The protein that base acid sequence is derivative.The amino acid whose number replacing, lack or adding is usually 1-50, preferably 1-30 Individual, more preferably 1-20, most preferably 1-10.
(3) with the aminoacid sequence shown in SEQ ID NO.2, there is at least 80% homology (being also called sequence iden), It is highly preferred that with the homology of the aminoacid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%, 98%, the polypeptide that the aminoacid sequence of 99% homology is constituted.
In specific embodiments of the present invention, described ITM2A albumen is to have the aminoacid sequence shown in SEQ ID NO.2 The protein of row.
It is known that, conventionally, in a protein, one or more amino acid whose modifications do not interfere with the function of protein. Those skilled in the art can approve change single amino acids or the aminoacid of little percentage ratio or aminoacid sequence indivedual are added, Lacking, inserting, replace is conservative modification, and wherein changing of protein produces the protein with identity function.Function phase is provided As amino acid whose Conservative substitution tables be well known in the art.
By adding the fusion that the example of the protein of an aminoacid or multiple Modification of amino acid residues is ITM2A albumen Albumen.Peptide or protein with ITM2A protein fusion is not limited, as long as the fusion protein of gained retains ITM2A egg White biologic activity.
The ITM2A albumen of the present invention also includes the non-conservative modification to the aminoacid sequence shown in SEQ ID NO.2, as long as Protein through modifying remains able to retain the biologic activity of ITM2A albumen.This type of modifying protein suddenlys change Amino acid number be typically 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosis of esophageal cancer " both includes judging that experimenter has suffered from the esophageal carcinoma, also Including judging whether experimenter exists and suffer from the risk of the esophageal carcinoma.
In the context of the present invention, " the treatment esophageal carcinoma " divides from the state change of disease, can include the slow of disease Solution, the healing completely of disease, also include the evaluation for disease therapeuticing effect.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that ITM2A gene expression is relevant to the esophageal carcinoma, by detection experimenter esophageal tissue The expression of ITM2A, it can be determined that whether experimenter suffers from the esophageal carcinoma or judge whether experimenter exists the wind suffering from the esophageal carcinoma Danger, thus instruct clinicist to provide prevention scheme or therapeutic scheme to experimenter.
Present invention finds a kind of new molecular marked compound-ITM2A gene, compare traditional detection means, gene diagnosis More in time, more special, sensitiveer, it is possible to realize the early diagnosis of the esophageal carcinoma.
Accompanying drawing explanation
Fig. 1 show utilize QPCR detect ITM2A gene expression in human esophageal carcinoma and normal esophageal tissue；
Fig. 2 show utilize Western blot detect ITM2A albumen table in human esophageal carcinoma and normal esophageal tissue Reach situation；
Fig. 3 shows the process LAN situation utilizing QPCR to detect ITM2A gene on transcriptional level；
Fig. 4 shows the process LAN situation utilizing Western blot detection ITM2A albumen；
Fig. 5 show utilize MTT experiment detect the ITM2A gene expression impact on esophagus carcinoma proliferation.
Detailed description of the invention
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.
The differential expression of ITM2A gene in embodiment 1 normal esophageal tissue and human esophageal carcinoma
1, experiment material:
Human esophageal carcinoma is the specimen of hospital's thoracic surgery excision, and normal esophageal is organized as under Dndoscope Laboratory mirror the mark taken out This, draw materials after the esophageal neoplasm tissue of excision is in vitro immediately, all wears disposable sterilized glove when drawing materials, and application high pressure goes out The knife blade of bacterium cuts.
All cases are all through definitive pathological diagnosis.Including human esophageal carcinoma 40 example, normal esophageal organizes 30 examples.Wherein esophageal squamous cell carcinoma 34 examples, adenocarcinoma of esophagus 6 example.With normal esophageal setup action matched group.Equal proved by pathology before all operation in patients, preoperative the most not row is put Treatment, chemotherapy etc. are treated, and without the tumor etc. at other positions.Institute the most all after Liquid nitrogen storage, moves into-80 within half an hour of performing the operation Degree refrigerator is frozen.
2, on transcriptional level, detect the differential expression of ITM2A gene
The extraction (utilizing Norgen RNA to extract test kit) of 2.1 tissue RNA
1) clear area disturbed at less RNase, uses the mortar containing appropriate liquid nitrogen to weigh in vitro tissue sample about 20mg, It is ground to powder with pestle；
2) sample is transferred to one without in the centrifuge tube of the 2mL of RNase；
3) add 300 μ L Lysis solution, be placed in homogenizer, be fully ground 1-5min；
4) 12000g, 4 DEG C, centrifugal 10min, in transfer supernatant to the centrifuge tube of new 1.5mL；
5) add 600 μ l RNase-Free Water, mix with whirlpool device；
6) adding 20 μ l E.C. 3.4.21.64s, at 55 DEG C of temperature bath 15min, continuous vortex mixes；
7) 14000g, room temperature, centrifugal 1min, make pellet cell debris bottom centrifuge tube, take supernatant and transfer to other one In the individual centrifuge tube without RNase 1.5mL；
8) adding 95% ethanol of 450 μ l, vortex mixes；
RNA adsorbs:
9) taking the 650 μ l lysate containing ethanol to be added in centrifugal column, 14000g is centrifuged 1min；
10) abandon lower floor, reset collecting pipe on post；
11) according to the capacity of lysate, 9 are repeated)～10) step；
12) adding 400 μ l Wash solution, 14000g is centrifuged 2min；
13) abandon lower floor, post is placed on a new collecting pipe；
DNase process:
14) adding 100 μ l Enzyme Incubation Buffer and 15 μ l DNase I, 14000g is centrifuged 1min；
15) solution in collecting pipe is moved in post again；
16) room temperature places 15min；
RNA washs:
17) adding 400 μ l Wash solution, 14000g is centrifuged 1min, abandons lower floor, resets collecting pipe on post；
18) adding 400 μ l Wash solution, 14000g is centrifuged 2min, abandons collecting pipe；
RNA eluting:
19) pillar is put in 1.7mL Elution pipe；
20) 30 μ l Elution Buffer are added；
21) 200g is centrifuged 2min, makes solution fully be combined with post, and then 14000g is centrifuged 1min.
2.2 reverse transcription
The Reverse Transcriptase kit utilizing TAKARA company carries out the reverse transcription of RNA.
2.3QPCR
(1) design of primers
According to the coded sequence design QPCR amplimer of ITM2A gene and GAPDH gene in Genbank, give birth in Shanghai The synthesis of work biotechnology Services Co., Ltd.Concrete primer sequence is as follows:
ITM2A gene:
Forward primer is 5 '-TTAGTAACCTTGGCATCTT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-TGTCTAATCTTCCAGCATT-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1 PCR reaction system
Reagent Volume
Forward primer 1μl
Reverse primer 1μl
SYBR Green polymerase chain reaction system 12.5μl
Template 2μl
Deionized water Supply 25 μ l
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) * 45 circulations.Using SYBR Green as Fluorescent marker, reacts at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument, true by melt curve analysis analysis and electrophoresis Determining purpose band, Δ Δ CT method carries out relative quantification.
2.4 statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD, Using SPSS13.0 statistical software to carry out statistical analysis, the difference between different groups uses t inspection, it is believed that when P is < when 0.05 There is statistical significance.
2.5 result
Result is as it is shown in figure 1, compared with normal esophageal tissue, in human esophageal carcinoma, the mRNA level in-site of ITM2A gene is notable Reducing, difference has statistical significance (P < 0.05).
3, on protein level, detect the differential expression of ITM2A gene
3.1 extract tissue total protein
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
3.2Western blot detects
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch, two anti-hatch, Colour developing.
3.3 statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by purpose informal voucher the gray value of protein band The gray value of band is normalized.Result data is all to represent in the way of mean+SD, uses SPSS13.0 statistical software carries out statistical analysis, and difference between the two uses t inspection, it is believed that when P < has system when 0.05 Meaning learned by meter.
3.4 result
Result is as in figure 2 it is shown, compared with normal esophageal tissue, in human esophageal carcinoma, ITM2A protein content reduces, and difference has Statistically significant (P < 0.05).
Embodiment 2ITM2A gene expression plasmid builds
1, the structure of ITM2A expression vector
Coded sequence (as shown in SEQ ID NO.1) design amplimer according to ITM2A gene.From becoming Human fetal spleen The coded sequence of the ITM2A gene of amplification total length in cDNA library (clontech company, article No.: 638831), by above-mentioned cDNA Sequence is inserted in eukaryotic expression vector pcDNA3.1, connects the recombinant vector pcDNA3.1-ITM2A obtained for follow-up reality Test.
2, the cultivation of esophageal cancer cell and transfection
2.1 cells are cultivated
Esophageal carcinoma cell line ECA109 is inoculated in the DMEM culture fluid containing 10% hyclone, is placed on 37 DEG C, 5%CO2Cultivate in incubator, when cell reaches 80% fusion, pass on 0.25% trypsinization.
2.2 cell transfecting
By esophageal cancer cell by 1 × 104/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO2In incubator carefully Born of the same parents cultivate 24h, transfect and transfect according to the description of lipofectamine 2000 (purchased from Invitrogen company), and making an excessive case more excessive is tested Being divided into matched group (transfection pcDNA3.1) and experimental group (transfection pcDNA3.1-ITM2A), the working concentration of transfected plasmids is 0.5 μ g/ml。
3, the effect of QPCR experiment detection plasmid transfection is utilized.
3.1 extract cell total rna utilizes conventional method to operate.
3.2 reverse transcription
Step is with embodiment 1.
3.3QPCR
Step is with embodiment 1.
3.4 statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD, Using SPSS13.0 statistical software to carry out statistical analysis, the difference between two groups uses t inspection, it is believed that when P < has when 0.05 Statistically significant.
4, Western detection
Concrete steps are with embodiment 1.
5, result
As it is shown on figure 3, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-ITM2A The mRNA level in-site of ITM2A significantly raises, and difference has statistical significance (P < 0.05)；As shown in Figure 4, empty with transfection pcDNA3.1 The cell of carrier is compared, and in the cell of transfection pcDNA3.1-ITM2A, the protein level of ITM2A significantly raises, and difference has statistics Learn meaning (P < 0.05).
The impact of embodiment 3ITM2A gene pairs esophagus carcinoma proliferation
Use MTT experiment detection ITM2A gene pairs esophagus carcinoma proliferation capacity.
1, step: trypsinization after each group cell transfecting 12h, makes single cell suspension, with the cell inoculation of 6000, every hole In 96 well culture plates, 3 time points of every component, each time point sets 6 multiple holes.After cell attachment, carry out the 1st time and detect: Every hole adds the MTT liquid 20 μ l of 5g/L, after continuing to cultivate 4h, sucks culture medium, adds DMSO 150 μ l, careful piping and druming, makes purple Blue precipitate fully dissolves, and surveys absorbance (A value) by microplate reader at 490nm wavelength.Then every 24h detects 1 time, surveys continuously 72h, totally 3 times.
2, statistical method
Experiment, all according to being repeated 3 times, uses SPSS13.0 statistical software to carry out statistical analysis, two groups Between difference use t inspection, it is believed that when P < has statistical significance when 0.05.
3, result
Result shown in Fig. 5 shows: the vitro growth rates of transfection pcDNA3.1-ITM2A group is significantly lower than transfection The vitro growth rates of pcDNA3.1 empty carrier group, difference has statistical significance (P < 0.05) the above results and shows ITM2A table Danone enough suppresses the growth of esophageal cancer cell.
The impact of embodiment 4ITM2A gene pairs Cell Cycle of Esophageal Carcinoma Cells
Using flow cytomery cell cycle, the mono-transfection reagent of PI used in the process produces purchased from U.S. company BD Product.
1, step: trypsinization after each group cell transfecting 48h, makes single cell suspension, and PBS washs 2 times, the ethanol of 70% Fix overnight.Adding corresponding reagent by operating instruction, lucifuge places 30min, and flow cytomery respectively organizes cell cycle.
2, statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD, Using SPSS13.0 statistical software to carry out statistical analysis, the difference between two groups uses t inspection, it is believed that when P < has when 0.05 Statistically significant.
3, result
Result as shown in table 2, compared with transfection pcDNA3.1 empty carrier group cell, transfects pcDNA3.1-ITM2A group Cell is in the cell showed increased of G1 phase, is in the cell of S phase and significantly reduces.The above results shows, ITM2A gene expression energy Enough suppress cell cycle.
Cell cycle change (percentage ratio) after table 2 cell transfecting
Group The G1 phase The G2 phase The S phase
Transfection pcDNA3.1 empty carrier group 23.41±0.74 15.27±0.35 61.32±0.58
Transfection pcDNA3.1-ITM2A 56.32±1.15* 14.94±1.24 28.74±1.03*
Note: compared with transfection pcDNA3.1 empty carrier group, * P < 0.05.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement And modification, these improve and modify also by the protection domain falling into the claims in the present invention.

Claims (10)

1. the product of detection ITM2A gene expression application in the instrument preparing diagnosis of esophageal cancer.
Application the most according to claim 1, it is characterised in that described product includes: by RT-PCR, real-time quantitative PCR, Immune detection, in situ hybridization chip or high-flux sequence detection of platform ITM2A gene expression are with the product of diagnosis of esophageal cancer.
Application the most according to claim 2, it is characterised in that the product of described RT-PCR diagnosis of esophageal cancer at least includes The primer of a pair specific amplified ITM2A gene；The product of described real-time quantitative PCR diagnosis of esophageal cancer at least include a pair special The primer of amplification ITM2A gene；The product of described immune detection diagnosis of esophageal cancer includes: be combined with ITM2A protein-specific Antibody；The product of described in situ hybridization diagnosis of esophageal cancer includes: with the probe of the nucleic acid array hybridizing of ITM2A gene；Institute State and include with the product of chip diagnosis of esophageal cancer: protein chip and gene chip；Wherein, protein chip includes and ITM2A albumen Specific binding antibody, gene chip includes the probe of the nucleic acid array hybridizing with ITM2A gene.
Application the most according to claim 3, it is characterised in that the product of described real-time quantitative PCR diagnosis of esophageal cancer is extremely The primer of a pair specific amplified ITM2A gene included less is as shown in SEQ ID NO.3 and SEQ ID NO.4.
5. the instrument of a diagnosis of esophageal cancer, it is characterised in that described instrument includes the reagent detecting ITM2A gene expression；Institute State reagent and include detecting primer and/or probe, the antibody of detection ITM2A albumen of ITM2A gene mRNA.
Instrument the most according to claim 5, it is characterised in that the primer of described detection ITM2A gene mRNA includes SEQ Primer pair shown in ID NO.3 and SEQ ID NO.4.
The application in the medicine of the preparation treatment esophageal carcinoma of the accelerator of 7.ITM2A gene and/or its expression product.
Application the most according to claim 7, it is characterised in that the reagent of described promotion ITM2A gene expression, promotion The reagent of ITM2A gene expression product stability, the reagent of promotion ITM2A gene expression product activity, promotion ITM2A gene table Reach the reagent of product function.
Application the most according to claim 8, it is characterised in that promote that the reagent of ITM2A gene expression includes containing ITM2A The reagent that the reagent of gene, the carrier carrying ITM2A gene or host cell are formed, the reagent containing ITM2A protein.
10. the pharmaceutical composition being used for treating the esophageal carcinoma, it is characterised in that described pharmaceutical composition includes in 7-9 arbitrary Accelerator described in Xiang.
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Citations (3)

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EP2700652A1 (en) * 2011-04-18 2014-02-26 The University of Tokyo Diagnosis and treatment of cancer using anti-itm2a antibody
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Publication number Priority date Publication date Assignee Title
EP2700652A1 (en) * 2011-04-18 2014-02-26 The University of Tokyo Diagnosis and treatment of cancer using anti-itm2a antibody
WO2015195949A3 (en) * 2014-06-18 2016-02-18 Clear Gene, Inc. Methods, compositions, and devices for rapid analysis of biological markers
CN105296656A (en) * 2015-11-27 2016-02-03 北京泱深生物信息技术有限公司 Molecular marker for nasopharynx cancer diagnosis and treatment

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