A kind of molecular target of esophageal squamous cell carcinoma
Technical field
The invention belongs to biomedicine field, relate to the molecular target of a kind of esophageal squamous cell carcinoma, described molecular target is
AGXT2L1。
Background technology
The esophageal carcinoma is one of common malignant gastrointestinal tumors, and M & M occupies pernicious respectively in the world
The 8th and the 6th, tumor.Its main histological type is divided into two big classes, esophageal squamous cell carcinoma and adenocarcinoma of esophagus, esophagus squameous
Cell carcinoma is also referred to as esophageal squamous cell carcinoma.The morbidity of the esophageal carcinoma has obvious areal variation, and adenocarcinoma of esophagus is mainly distributed on west state
Family, and over nearly 20 years, also show the trend that sickness rate rises.Esophageal squamous cell carcinoma is distributed mainly on developing country, and in
Sub-and Caspian Sea region sickness rate is concentrated very much.China is based on esophageal squamous cell carcinoma, and the mortality rate of the domestic esophageal carcinoma is 15.21/10
Ten thousand, occupy the 4th of malignant tumor, be only second to pulmonary carcinoma, hepatocarcinoma and gastric cancer, male is higher than women, and rural area is higher than city.Whole nation ether
Row mountain, Da Bie Mountain area, boundary, Guangdong, Fujian and Northern Sichuan Province are district occurred frequently, and Jiangsu Province is with Along North Jiangsu and raises medium ground sickness rate relatively
High.
Research shows, the esophageal carcinoma is by including that the multifactor functionings such as heredity, environment, life style cause.Induction esophagus
The risk factor of cancer has, genetic predisposition, smoking, drinks, likes and scald the living habit such as food, and nitrosamine compound is taken in, low water
Flat social and economic condition, precancerous lesion etc..Primary disease onset is hidden, and has stronger invasive ability, when most of patients is made a definite diagnosis
Having been enter into middle and advanced stage, treatment is in the way of operation, chemotherapy, radiotherapy combine, and within 5 years, survival rate is only 15%-25%, prognosis
Situation depends on early diagnosis.Studying display, the development that occurs of the esophageal carcinoma may be with the swashing of the oncogene such as c-myc, H-ras in the past
Living, the antioncogene such as Rb, P53 inactivation is relevant, but concrete pathogenesis illustrates the most completely.
Along with sequencing technologies and the development of correlation molecule biology techniques, people are more and more deeper to the understanding of genome
Carving, prior art finds that the development that occurs of esophageal squamous cell carcinoma has important relation with the change of gene or its expression.But mesh
Before do not have effective mark to be applied to clinic.Give farther insight into the pathogeny of esophageal squamous cell carcinoma, searching esophageal squamous cell carcinoma is examined
Disconnected molecular marker, determine the drug target of research and development clinical treatment and formulate effective therapeutic scheme there is important clinical meaning
Justice.
Summary of the invention
In order to make up the deficiencies in the prior art, it is an object of the invention to provide a kind of mark for esophageal squamous cell carcinoma diagnosis and treatment
Thing AGXT2L1.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the reagent detecting AGXT2L1 gene application in the product of preparation diagnosis esophageal squamous cell carcinoma.
Further, by the expression of AGXT2L1 gene in detection sample, described product judges whether patient suffers from
Esophageal squamous cell carcinoma.Wherein, AGXT2L1 gene down-regulated expression in patients with esophageal squamous cell carcinoma.
Described " sample " includes that cell, tissue, internal organs, body fluid (blood, lymph fluid etc.), Digestive system, expectoration, alveole prop up gas
Pipe cleanout fluid, urine, feces etc..Preferably, described sample is tissue, blood.In the detailed description of the invention of the present invention, described sample
This is tissue.
Further, described product includes: by RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization, chip or height
The expression of flux order-checking detection of platform AGXT2L1 gene is to diagnose esophageal squamous cell carcinoma.
Wherein, the product of described RT-PCR diagnosis esophageal squamous cell carcinoma at least includes a pair specific amplified AGXT2L1 gene
Primer;The product of described real-time quantitative PCR diagnosis esophageal squamous cell carcinoma at least includes drawing of a pair specific amplified AGXT2L1 gene
Thing;The product of described immune detection diagnosis esophageal squamous cell carcinoma includes: the antibody being combined with AGXT2L1 protein-specific;Described use
The product of in situ hybridization diagnosis esophageal squamous cell carcinoma includes: with the probe of the nucleic acid array hybridizing of AGXT2L1 gene;Described chip is examined
The product of disconnected esophageal squamous cell carcinoma includes: protein chip and gene chip;Wherein protein chip includes tying with AGXT2L1 protein-specific
The antibody closed, gene chip includes the probe of the nucleic acid array hybridizing with AGXT2L1 gene.
Further, the product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes a pair specific amplification AGXT2L1
The primer of gene, described primer is as shown in SEQ ID NO.5 and SEQ ID NO.6.
The invention provides a kind of product diagnosing esophageal squamous cell carcinoma, described product can be by AGXT2L1 in detection sample
The expression of gene diagnoses esophageal squamous cell carcinoma.
Further, described product includes chip or test kit;Wherein, described chip includes gene chip, protein core
Sheet;Described test kit includes gene detecting kit, protein immunization detection kit.Described gene chip include solid phase carrier with
And it being fixed on the oligonucleotide probe of solid phase carrier, described oligonucleotide probe includes for detecting AGXT2L1 genetic transcription water
The flat oligonucleotide probe for AGXT2L1 gene;Described protein chip includes solid phase carrier and is fixed on solid phase load
The specific antibody of the AGXT2L1 albumen of body;Described gene detecting kit includes for detecting AGXT2L1 gene transcription level
Reagent;Described protein immunization detection kit includes the specific antibody of AGXT2L1 albumen.
In the present invention, gene detecting kit or gene chip can be used for the multiple bases detecting including AGXT2L1 gene
Expression because of (such as, relevant to esophageal squamous cell carcinoma multiple genes).Described protein immunization detection kit or protein core
Multiple protein (such as relevant to esophageal squamous cell carcinoma multiple protein) that sheet can be used for detecting including AGXT2L1 albumen
Expression.Multiple marks of esophageal squamous cell carcinoma are detected simultaneously, is greatly improved the accuracy rate of oesophagus squama cancer diagnosis.
The invention provides the application in the pharmaceutical composition of preparation treatment esophageal squamous cell carcinoma of the AGXT2L1 gene.
Further, described pharmaceutical composition include increase AGXT2L1 gene expression, strengthen AGXT2L1 expressive function and/
Or strengthen the reagent of AGXT2L1 expression product activity.
Further, described reagent includes: containing the energy reagent of nucleic acid of encoding function AGXT2L1 albumen, AGXT2L1 egg
White activator, the reagent containing AGXT2L1 protein.
Wherein, the reagent of the described nucleic acid containing energy encoding function AGXT2L1 albumen can be to turn under advantage
Being translated into single-chain nucleic acid (such as mRNA) or the double-strandednucleic acid (such as DNA) of the AGXT2L1 albumen of activity form, described nucleic acid can connect
It is connected on expression vector or recombinates in host cell, as long as activity AGXT2L1 albumen can be encoded into, any AGXT2L1
The carrying mode of gene.Described AGXT2L1 protein activator refers to stimulate AGXT2L1 protein active, increase AGXT2L1
Protein active, promote AGXT2L1 protein active, strengthen AGXT2L1 protein activation, make AGXT2L1 protein active sensitization or on
Adjust AGXT2L1 protein active reagent, as specific in demethylation reagent, AGXT2L1 promoter and/or enhancer transcribe sharp
Agent alive, the agonist (as activated antibody) etc. of AGXT2L1 albumen.
The invention provides a kind of pharmaceutical composition treating esophageal squamous cell carcinoma, described pharmaceutical composition includes increasing
AGXT2L1 gene expression, enhancing AGXT2L1 expressive function and/or the reagent of enhancing AGXT2L1 expression product activity.Wherein,
Described reagent includes but not limited to containing the energy reagent of nucleic acid of encoding function AGXT2L1 albumen, the activation of AGXT2L1 albumen
Agent, reagent containing AGXT2L1 protein.
Further, described pharmaceutical composition also includes pharmaceutically acceptable carrier, as buffer agent, emulsifying agent, suspending agent,
Stabilizer, preservative, physiological saline etc..As buffer agent, it is possible to use phosphate, glycine, sorbic acid, sorbic acid clock, satisfy
With the partial glyceride mixtures of vegetable fatty acid, water, salt or electrolyte such as potassium sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, chlorine
Change sodium, zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, material based on cellulose, Polyethylene Glycol, carboxylic first
Base sodium cellulosate, polyacrylate, wax, polyethylene-polyoxypropylene block copolymer, Polyethylene Glycol and lanoline etc..As breast
Agent, it is possible to use Radix Acaciae senegalis, sodium alginate, tragacanth etc..As suspending agent, it is possible to use glycerol monostearate,
Aluminum monostearate, methylcellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate etc..As stabilizer, energy
Enough use propylene glycol, diethylidene sulphite, ascorbic acid etc..As preservative, it is possible to use Hydrazoic acid,sodium salt, benzene to prick chlorine
Ammonium, p-hydroxybenzoic acid, methaform etc..The pharmaceutical composition of the present invention can also include ion-exchanger such as Alumina, aluminium stearate,
Lecithin, semi-emulsifying drug delivery system (SEDDS) such as d α mono-tocopherol cetomacrogol 1000 succinate, at pharmaceutical dosage form
The surfactant of middle use such as tween or other similar polymeric delivery matrices, serum albumin such as human serum albumin, also
Cyclodextrin such as alpha-cyclodextrin, beta-schardinger dextrin-and gamma-cyclodextrin, or the derivant of chemical modification e.g. hydroxyl can be used
Alkyl cyclodextrins, promotes passing of the compounds of this invention including 2-and 3-hydroxypropyl-beta-schardinger dextrin-or other solubilising derivant
Send.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..
The pharmaceutical composition of the present invention also includes pharmaceutically acceptable excipient, filler, coagulating agent and blender, as
Lactose hydrous or Lactis Anhydrous, starch, glucose, sucrose, mannitol, sorbitol, silicic acid, microcrystalline Cellulose, hydroxylmethyl cellulose
Element sodium, sodium starch glycol and derivant thereof etc..
The pharmaceutical composition of the present invention also comprises interfacial agent, emulsifying agent, diffusant, defoamer etc..Any pharmaceutically
Or medically acceptable interfacial agent, emulsifying agent, diffusant, defoamer etc. all can be used.
The pharmaceutical composition of the present invention also includes that pharmaceutically acceptable coating material includes, but is not limited to, fast decoupled
Coating material, stain, enteric polymer, plasticizer, water-soluble polymer, insoluble polymer, dyestuff, pigment, its
His disintegrating agent.Common fast decoupled coating material includes OPADRY;Enteric polymer includes methylacrylic acid polymer, phosphorus hydroxyl
Third methylcellulose phthalic acid ester, hydroxypropyl methylcellulose acetas, hydroxypropyl methylcellulose succinate, hydroxyl the first and second base are fine
Dimension element, cellulose acetophthalate;Plasticizer includes Polyethylene Glycol (PEG), propylene glycol etc..
In the present invention, " probe " refer to be combined with the particular sequence of another molecule or subsequence or other parts point
Son.Unless otherwise noted, term " probe " is often referred to be matched and another polynucleotide (often referred to as " target by complementary base
Polynucleotide ") polynucleotide probes that combines.According to the preciseness of hybridization conditions, probe energy and lack complete sequence with this probe
The target polynucleotide combination that row are complementary.Probe can make direct or indirect labelling, and its scope includes primer.Crossing system, bag
Include, but be not limited to: solution phase, solid phase, mixed phase or in situ hybridization algoscopy.
Described probe has the base sequence of the specific base sequence complementary with target gene.Here, so-called " complementary ",
As long as hybridize, can not be complete complementary.These polynucleotide are commonly angled relative to this specific base sequence to be had
More than 80%, preferably more than 90%, more preferably more than 95%, the homology of particularly preferred 100%.These probes can be DNA,
Can also be RNA, furthermore it is possible to be to pass through PNA (Polyamide nucleic at one part or whole nucleotide
Acid, peptide nucleic acid(PNA)), LNA (registered trade mark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core
Acid), ENA (registered trade mark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol
Nucleic acid, glycerol nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains
Polynucleotide.
The specific antibody of heretofore described AGXT2L1 albumen includes monoclonal antibody, polyclonal antibody, polyspecific
Antibody (such as bi-specific antibody) and antibody fragment, combinatorial antibody etc., as long as they show desired biologic activity.
In the present invention, " monoclonal antibody " refers to the antibody obtained from the antibody of a group substantially homogeneity, i.e. constitutes each of colony
Individual antibody is identical and/or combines identical epi-position, during producing monoclonal antibody in addition to issuable possible variant, this
Class variant is typically with indivisible existence.This type of monoclonal antibody is typically include the antibody comprising the peptide sequence combining target,
Wherein target Binding peptide sequence is by selecting including single target Binding peptide sequence in many peptide sequences of comforming
Process obtains.
Clonal antibody the most clearly includes " being fitted together to " antibody, and wherein a part for heavy chain and/or light chain is with derivative
Identical or the homology from individually defined thing species or genus corresponding sequence in the antibody of specific antibodies classification or subclass, and the remainder of chain
With derived from another species or belong to another antibody isotype or subclass antibody in corresponding sequence is identical or homology, and this type of
The fragment of antibody, as long as they show desired biologic activity.
" complete antibody " refers to comprise two antigen binding domains and the antibody in Fc district in the present invention.Preferably, completely resist
Body has functional Fc district.
" antibody fragment " comprises a part for complete antibody, preferably comprises its antigen binding domain.The example bag of antibody fragment
Include Fab, Fab ', F (ab ')2With Fv fragment;Double antibody;Linear antibodies;Single-chain antibody molecules;And formed many by antibody fragment
Specific antibody.
The present invention detects the antibody of AGXT2L1 gene expression product, suitable known method can be selected to prepare,
Manufacture method etc. such as monoclonal antibodies such as hybridoma, recombinant DNA method, phage methods.Use and be combined with the anti-of mark substance
During body, by detecting this labelling, it is possible to directly detection target point protein matter.As mark substance, as long as can with antibodies,
The material that can detect, is not particularly limited, for example, it is possible to be peroxidase, beta-D-galactosidase, micro-peroxidating
Thing enzyme, horseradish peroxidase (HRP), Fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase
Enzyme, biotin and radioactive substance.It addition, be combined with, except using, the method that the antibody of mark substance directly detects target point protein matter
In addition, it is also possible to utilize use to be combined with the secondary antibodies of mark substance, protein G or a-protein etc. and indirectly detect target spot
Method of protein.
Pharmaceutical composition Orally-administrable of the present invention, parenteral administration, by suck spray delivery, local give
The administration of medicine, rectally, nasal administration, cheek, vagina administration or the storage medicine device passing through to implant are administered.Preferred oral is administered or injection
It is administered.Pharmaceutical composition of the present invention can be containing any commonly employed nontoxic pharmaceutically suitable carrier, adjuvant or excipient.In some situation
Under, medicinal acid, alkali or buffer agent can be used to the pH regulating preparation to improve stablizing of the compound prepared or its form of administration
Property.In terms used herein parenteral route includes subcutaneous, Intradermal, intravenous, intramuscular, intraarticular, intra-arterial, intrasynovial, breastbone,
In bringing up interior, damage location and intracranial injection or infusion techniques.As long as destination organization can be reached, pharmaceutical composition of the present invention
Receptor can be given by any approach.
Pharmaceutical composition of the present invention can include but not limited to capsule, sheet with the form oral administration of any peroral dosage form
Agent, Emulsion and water slurry, dispersant and solution.For oral tablet, common carrier includes lactose and corn starch.The most also
Add lubricant such as magnesium stearate.In order to be administered with capsules per os, the diluent being suitable for includes lactose and anhydrous Semen Maydis
Starch.When Orally administered water slurry and/or emulsion, active component can be suspended or dissolved in oil phase, and and emulsifying agent
And/or suspending agent merges.If necessary, some sweeting agents and/or correctives and/or coloring agent can be added.Time suitably, can
The dosage unit preparations bag microcapsule of oral administration will be used for.Such as, by polymer, wax etc. by particulate matter coating or bag
Bury, it is possible to prepare described preparation and extended or maintained release.The pharmaceutical composition of the present invention may be used for supplementary endogenic
The disappearance of AGXT2L1 albumen or deficiency, by improving the function expressing or strengthening AGXT2L1 albumen of AGXT2L1 albumen, thus
Treat because AGXT2L1 albumen reduces the esophageal squamous cell carcinoma caused.
The medicine of the present invention also can be with the drug combination of other treatment esophageal squamous cell carcinoma, and other treatment compound can be with master
The active component wanted is administered simultaneously, and is even administered simultaneously in same compositions.Can also with single compositions or with mainly
The different dosage form of active component individually give other therapeutic compound.The Fractional of main component can be with other
Therapeutic compound is administered simultaneously, and other dosage can be individually dosed.Over the course for the treatment of, can be according to the serious journey of symptom
Degree, the frequency of recurrence and the physiologic response of therapeutic scheme, adjust the dosage of pharmaceutical composition of the present invention.
Pharmaceutical composition of the present invention can also Liposomal delivery systems form be administered, such as little unilamellar vesicle, the most single
Layer vesicle and multilamellar vesicle.Liposome can have multiple phospholipid to be formed, such as cholesterol, stearic amine or phosphatidylcholine.
Pharmaceutical composition of the present invention can be configured to ointment, ointment, suspendible with the pharmaceutical composition of topical
Agent, lotion, powder, solution, paste, gel, spray, aerosol or oil preparation.
It would be recognized by those skilled in the art that the practicality of the present invention is not limited to the marker gene to the present invention
The gene expression of any specific variants is carried out quantitatively.As nonrestrictive example, marker gene can have SEQ ID NO.1
Or the coded sequence specified of SEQ ID NO.2 or aminoacid sequence.In some embodiments, it has with listed sequence extremely
Few 85% same or analogous cDNA sequence or aminoacid sequence, the most above-mentioned listed sequence at least 90%, 91%, 92%,
93%, the same or analogous cDNA sequence of 94%, 95%, 96%, 97%, 98% or at least 99% or aminoacid sequence.
In the context of the present invention, AGXT2L1 gene expression product includes people's AGXT2L1 albumen and AGXT2L1 egg
White partial peptide.The partial peptide of described AGXT2L1 albumen contains the functional domain relevant to esophageal squamous cell carninomatosis.
" AGXT2L1 albumen " includes any function equivalent of AGXT2L1 albumen and AGXT2L1 albumen.Described function
Equivalent includes AGXT2L1 albumen conservative variation's protein or its active fragment, or its reactive derivative or its mutant.
Mutant include allelic variant, natural mutation, induced mutants, its aminoacid sequence by lacking, substitute, increase and/
Or insert morph mutant, under high or low stringent condition can with the DNA of the DNA hybridization of people AGXT2L1 coded by
Protein.
Generally, in a protein, one or more amino acid whose modifications do not interfere with the function of protein.This area skill
Art personnel can approve change single amino acids or the aminoacid of little percentage ratio or to the adding individually of aminoacid sequence, lack, insert
Entering, replacing is conservative modification, and wherein changing of protein produces the protein with identity function.Intimate amino is provided
The Conservative substitution tables of acid is well known in the art.
The modification of aminoacid sequence is modified after can being derived from spontaneous mutation or heredity, it is also possible to artificial induction's natural gene produces
Raw.By adding the fusion egg that the example of the protein of an aminoacid or multiple Modification of amino acid residues is AGXT2L1 albumen
In vain.Peptide or protein with AGXT2L1 protein fusion is not limited, as long as the fusion protein of gained retains AGXT2L1
The biologic activity of albumen.
The pharmaceutical composition of the present invention can be administered by amount effective on pharmaceutics, the term " pharmaceutics of the present invention
Upper effective amount " refer to be applicable to reasonably the receiving benefits of therapeutic treatment or prevention/risk-benefit risks and be enough to treat or prevent
The amount of disease, can be according to including the order of severity of disease, the activity of medicine, the age of patient, body weight, health, sex, patient couple
The sensitivity of medicine, the administration time of the present composition used, route of administration and discharge ratio, treatment time and institute
The compositions of the present invention used coordinates or known in the key element of medicine that simultaneously uses and other medical domain usually to determine
Determine effective dose level.The pharmaceutical composition of the present invention can be administered as single therapeutic agent, or with other therapeutic agent
And land used is administered, in turn or simultaneously can be administered with conventional therapeutic agent.Additionally, can enter single or multiple
Row is administered.It is essential that need above-mentioned key element is all taken in, and maximum effect can be obtained with the minimum amount having no side effect
The amount of fruit is administered.
In the present invention, term " chip " or " array ", " microarray " are that hybridised arrays original paper is ordered in substrate,
Described hybridised arrays original paper such as polynucleotide probe (such as oligonucleotide) or bonding agent (such as antibody).Described substrate is permissible
It is solid matrix, such as, glass or silicon dioxide slide, pearl, fibre optics binding agent or semi-solid matrix, such as cellulose nitrate
Element film.Nucleotide sequence can be any arrangement of DNA, RNA or therein.
Term " is treated " and is referred to not for the purpose of curing, but slows down pathological condition or disease that (minimizing) target or prevent
Recurrence.Including but not limited to that (1) inhibitory action suppresses progression of disease to a certain extent, it includes slowing down and pressing down completely
System;(2) seizure of disease and/or the quantity of symptom are reduced;(3) focal size is reduced;(4) suppression (i.e. reduces, slows down or hinders completely
Only) disease cells penetrates into adjacent peripheral organs and/or tissue;(5) suppression (i.e. reduce, slow down or stop completely) disease passes
Broadcast;(6) one or more symptoms being associated with disease are alleviated to a certain extent;(7) treatment after increase anosis performance time
Between length;(8) some preset time after the treatment reduces mortality rate;And/or have no side effect after (9) treatment.
Advantages of the present invention and beneficial effect:
The invention provides the molecular marker of a kind of esophageal squamous cell carcinoma, for study mechanism and the clinical practice of esophageal squamous cell carcinoma
Provide fundamental basis.
Present invention firstly discovers that AGXT2L1 differential expression in patients with esophageal squamous cell carcinoma tissue, by detection AGXT2L1's
Expression may determine that whether patient suffers from the height of esophageal squamous cell carcinoma or risk.
The method that the invention provides personalized treatment esophageal squamous cell carcinoma, by improving the expression of AGXT2L1, to esophagus
Squamous cell carcinoma patients is treated.
Accompanying drawing explanation
Fig. 1 show utilize QPCR detect AGXT2L1 gene expression in esophageal squamous cell carcinoma tissue;
Fig. 2 show utilize QPCR detect AGXT2L1 gene expression in esophageal cells;
Fig. 3 shows that utilizing QPCR to detect transfects AGXT2L1 gene transfection efficiency in esophageal squamous cell carcinoma cell;
Fig. 4 shows that utilization utilizes soft-agar cloning to form experiment detection AGXT2L1 gene expression and increases esophageal squamous cell carcinoma cell
Grow the impact of ability;
Fig. 5 shows the impact utilizing transwell cell detection AGXT2L1 gene pairs esophageal squamous cell carcinoma cell invasion.
Specific embodiment
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 gene marker that embodiment 1 screening is relevant to esophageal squamous cell carcinoma
1, sample collection
Each collection 6 example surrounding normal mucous membrane of esophagus tissue and esophageal squamous cell carcinoma tissue, the equal informed consent of patient, above-mentioned all marks
This acquirement is all by the agreement of committee of organizational ethics.
2, the preparation (the tissue RNA utilizing QIAGEN extracts test kit and operates) of RNA sample
Take out frozen tissue samples in liquid nitrogen, tissue samples is put in the mortar of pre-cooling and be ground, according to
Description in test kit is extracted and is separated RNA.Specific as follows:
1) adding Trizol, room temperature places 5min;
2) adding chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5-10min;
3) 12000rpm is centrifuged 15min, moves on to upper water mutually (be careful not to be drawn onto two-layer water in another new centrifuge tube
Protein substance between Xiang), add the isopropanol of isopyknic-20 DEG C of pre-coolings, the most reverse mixing, it is placed in 10min on ice;
4) 12000rpm carefully discards supernatant at a high speed after 15min, adds 75% in the ratio of 1ml/ml Trizol
DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate, vibration mixing, 4 DEG C, 12000rpm is centrifuged 5min;
5) discarding ethanol liquid, ambient temperatare puts 5min, adds DEPC water dissolution precipitation;
6) RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C of refrigerators.
3, reverse transcription and labelling
With Low RNA Input Linear Amplification Kit, mRNA reverse transcription is become cDNA, use Cy3 simultaneously
Labelling experiment group and matched group respectively.
4, hybridization
Gene chip uses people's full-length genome chip of expression spectrum of Aglient company, and every chip includes 45015 few cores
Thuja acid, wherein has 43376 people's gene probes and 1639 experiment control probes.Carry out by the step of chip operation instructions,
Temperature, at 65 DEG C, rolls hybridization through 17h 10r/min, develops a film for 37 DEG C.
5, data process
Chip Agilent scanner scanning after hybridization, resolution is 5 μm, and scanner is automatically with 100% and 10%PMT
Each scanning 1 time, 2 times result Agilent software merges automatically.Scan image data uses at Feature Extraction
Reason is analyzed, and the initial data application Bioconductor program bag obtained carries out follow-up data process.Last Ratio value is experiment
Group and matched group.Differential gene screening criteria: ratio >=4 are up-regulated gene, ratio≤0.25 is down-regulated gene.
6, result
Compared with normal esophageal mucosal tissue, AGXT2L1 gene expression in esophageal squamous cell carcinoma tissue is significantly lowered.
The differential expression of embodiment 2 QPCR sequence verification AGXT2L1 gene
1, AGXT2L1 gene differential expression is carried out large sample QPCR checking.According to the sample collection mode in embodiment 1
Select surrounding normal mucous membrane of esophagus tissue and each 50 examples of esophageal squamous cell carcinoma tissue.
2, RNA extraction step is with embodiment 1.
3, reverse transcription:
Using 25 μ l reaction systems, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into following in PCR pipe
Component: DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μMs of Oligo dT, 200U/ μ l M-MLV,
Template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
(3) QPCR amplification inspection
Design of primers
The primer sequence of house-keeping gene GAPDH is:
Forward primer: 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO.3)
Reverse primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4)
The primer sequence of AGXT2L1 gene is:
Forward primer: 5 '-ACCAACTCCAGATACTTAC-3 ' (SEQ ID NO.5)
Reverse primer: 5 '-CTTCCTTCCACTGTTATGA-3 ' (SEQ ID NO.6)
Using 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to protect
The reliability of card result.
Prepare following reaction system: SYBR Green polymerase chain reaction system 12.5 μ l, forward and reverse primer (5 μMs)
Each 1 μ l, template cDNA 2.0 μ l, without enzyme water 8.5 μ l.Operations is all carried out on ice.
Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 45s) × 30 circulations.
Using SYBR Green as fluorescent marker, anti-at the Light Cycler enterprising performing PCR of fluorescence real-time quantitative PCR instrument
Should, determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
3, 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 SPSS18.0 statistical software to carry out statistical analysis, difference between the two uses t inspection, it is believed that when P < has when 0.05
Statistically significant.
4, result
Result is as it is shown in figure 1, compared with surrounding normal mucous membrane of esophagus tissue, AGXT2L1 gene is in esophageal squamous cell carcinoma tissue
Down-regulated expression, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
Embodiment 3 AGXT2L1 gene differential expression in esophageal carcinoma cell line
1, cell is cultivated
Human esophageal squamous cell cancer cell strain KYSE 150, KYSE450 are purchased from institute of oncology of the Chinese Academy of Sciences, normal esophageal epithelial cell
Strain Het-1a is purchased from Guangzhou Ji Niou company.With culture medium DMEM containing 10% hyclone and 1%P/S 37 DEG C, 5%
CO2, relative humidity be 90% incubator in cultivate.Within 2-3 days, change liquid 1 time, use 0.25% trypsin containing EDTA conventional
Had digestive transfer culture.
2, the extraction of RNA
1) trypsinization attached cell, piping and druming obtain cell by centrifugation, resuspended, clean after, with 1640 culture medium (10%
Calf serum) resuspended;
2) resuspended cell being transferred to 6 orifice plates (/ hole), interpolation culture medium is to 2m1/ hole, and jog 6 orifice plate makes cell uniform
Resuspended;
3) cell attachment growth 48h, goes culture medium;
4) with 1ml Trizol reagent cell lysis, repeatedly blow and beat 6 orifice plate walls, make cell crack completely as far as possible;
5), in the EP pipe that transfer cell pyrolysis liquid processed to 1.5ml DEPC, it is placed on ice.Add 0.2m1 chloroform, surplus
Remaining operating procedure extracts process with RNA in tissue.
3, reverse transcription
Concrete steps are with embodiment 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 SPSS18.0 statistical software to carry out statistical analysis, difference between the two uses t inspection, it is believed that when P < has when 0.05
Statistically significant.
5, result
Result as in figure 2 it is shown, compared with esophageal epithelial cell, AGXT2L1 gene esophageal squamous cell carcinoma cell KYSE150,
Expressing in KYSE450 and all lower, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
The process LAN of embodiment 4 AGXT2L1 gene
1, cell is cultivated
Human esophageal squamous cell cancer cell strain KYSE 150, with culture medium DMEM containing 10% hyclone and 1%P/S 37 DEG C,
5%CO2, relative humidity be 90% incubator in cultivate.Within 2-3 days, change liquid 1 time, use 0.25% trypsin containing EDTA normal
Rule had digestive transfer culture.
2, the process LAN of AGXT2L1 gene
The structure of 2.1AGXT2L1 expression vector
Coded sequence (as shown in SEQ ID NO.1) design amplimer according to AGXT2L1 gene, primer sequence is such as
Under:
Forward primer: 5 '-CCGAAGCTTGCCACCATGTGCGAGCTGTACAGTA-3 ' (SEQ ID NO.7)
Reverse primer: 5 '-CGGCTCGAGTGTCTTGAGCCTCTTACTGAGCAG-3 ' (SEQ ID NO.8)
The AGXT2L1 gene of amplification total length from the cDNA library (clontech company, article No.: 638831) becoming Human fetal spleen
Coded sequence, above-mentioned cDNA sequence is inserted into through restriction enzyme after restricted enzyme HindIII and XhoI double digestion
In the eukaryotic expression vector pcDNA3.1 of enzyme HindIII and XhoI double digestion, connect the recombinant vector pcDNA3.1-obtained
AGXT2L1 is used for subsequent experimental.
2.2 transfection
Esophageal squamous cell carcinoma cell is divided into two groups, and respectively matched group (transfection pcDNA3.1 empty carrier) and AGXT2L1 crosses table
Reach group (transfection pcDNA3.1-AGXT2L1).Using liposome 2000 to carry out the transfection of carrier, concrete transfection method is according to explanation
The instruction of book is carried out.The transfection concentrations of pcDNA3.1 empty carrier and pcDNA3.1-AGXT2L1 is 0.5 μ g/ml.
2.3RT-PCR detection
Concrete steps are with embodiment 2.
3, 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 SPSS18.0 statistical software to carry out statistical analysis, difference between the two uses t inspection, it is believed that when P < has when 0.05
Statistically significant.
4, 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-AGXT2L1
The content of AGXT2L1 significantly raises, and difference has statistical significance (P < 0.05).
The embodiment 5 AGXT2L1 apoptotic impact of gene pairs esophageal squamous cell carcinoma
Use the flow cytomery apoptotic impact of AGXT2L1 gene pairs.
1, cell culture step is with embodiment 3.
2, cell transfecting step is with embodiment 3.
3, step
After cell transfecting 72h, use pre-cooling PBS washed cell, then use 0.25% trypsin digestion cell, stop digestion,
Use PBS resuspended in the cell of centrifugal collection, be 1 × 10 by cell quantification6Individual/ml, takes the 200 above-mentioned cell suspension of μ l and is placed into
In Eppendorf pipe, adding 10 μ l Annexin-V-FITC mixings, dyeing 15min is hatched in room temperature dark place, and before upper machine, 5min adds
Enter 10mg/L propidium iodide (PI) to dye 5 μ l.The cell of untransfected siRNA is used for Annexin-V-FITC and PI dyeing respectively
Standard quantitative.Two Colour Fluorescence cell cytometry, observing apoptosis cell percentages is carried out with FACS flow cytometer.
3, statistical method
Experiment is all according to being repeated 3 times, and result data is all to represent in the way of mean+SD,
SPSS18.0 statistical software is used to carry out statistical analysis, the t inspection that difference between the two uses, it is believed that when P is < when 0.05
There is statistical significance.
4, result:
The apoptosis rate of transfection pcDNA3.1-AGXT2L1 group is (15.14 ± 0.021) %, and transfection pcDNA3.1 is unloaded
The apoptosis rate of body group is (6.21 ± 0.011) %, and above-mentioned difference has statistical significance (P < 0.05), the above results table
Bright, the process LAN of AGXT2L1 gene promotes the apoptosis of esophageal squamous cell carcinoma cell.
Embodiment 6 soft-agar cloning forms experiment
1, be in the cell of exponential phase with 0.25% trypsinization, piping and druming makes unicellular outstanding gently
Liquid, centrifugal collecting cell precipitates.
2, resuspended with the DMEM complete medium containing 20% hyclone, suitably count after dilution, adjusting cell concentration is 5
×103Individual/ml.
3, two concentration of preparation are respectively the LMP agar sugar liquid of 1.2% and 0.7%, after autoclaving, maintain 40
In DEG C water-bath.
4, agarose and 2 × DMEM culture medium 1:1 of 1.2% mixes, and adds 2 × antibiotic and the calf serum of 20%,
Take 3ml mixed liquor and inject placement 5min cooled and solidified in diameter 6cm plate, be placed in CO2 incubator standby as bottom-layer agar.
5, in sterile test tube, 1:1 mixes agarose and 2 × DMEM culture medium of 0.7%, then it is dense to add 0.2ml in pipe
Degree is 5 × 103The stable infection cell suspension of individual/ml, fully mixes, injects in above-mentioned plate, gradually forms double agar layer, often
Individual experimental group repeats 4 samples.
6, after top-layer agar solidifies, 37 DEG C of 5%CO are inserted2Incubator is cultivated, within every 3 days, adds culture medium 1.5ml.
7, culture dish is taken out after cultivating 14 days, with the Gentian Violet dyeing 90min that 1ml concentration is 0.005%.Plate is placed
Observing under inverted microscope, often group cell randomly selects 10 low power fields, the number of cell clones that under mirror, technology is formed.
8, result
Result as shown in Figure 4, compared with other groups, transfects the groups of cells single cell clone colony of pcDNA3.1-AGXT2L1
Formation number significantly reduces.
Embodiment 7 Transwell cells in vitro Matrigel
1, the serum-free medium Matrigel adding 100 μ l in the upper hole of Transwell cell soaks 30min.
2, respectively organizing cell be in the stable infection of exponential phase with trypsinization after, PBS cell 3 times, with containing
The culture fluid re-suspended cell of 10% serum, adjusting cell concentration is 1 × 105/ml。
3, in the upper room of Transwell cell being coated with Matrigel, cell suspension 200 μ l is added.
4, the lower room of Transwell cell adds the 600 μ l DMEM culture fluid containing 20%FBS.
5,37 DEG C, 5%CO224h is cultivated in incubator.
6, the culture fluid in room and lower room in removing, strikes off the Matrigel in upper room and the cell retained with cotton swab, uses
PBS 2 times.
7, with 1% violet staining liquid dyeing 45min, PBS 1 time.
8, randomly select 4 100 times of visuals field, the most respectively counting invasion and attack cell number, every kind different types of carefully
Born of the same parents set 3 multiple holes, are repeated 3 times altogether.
9, data process
With SPSS18.0 software, data are carried out statistical analysis.Measurement data mean ± standard deviation represents.Multiple samples
This mean compares employing one factor analysis of variance, and P < 0.05 is that difference is statistically significant, and P < 0.01 is significant difference.
10, result
Result is as it is shown in figure 5, KYSE150, pcDNA3.1 are unloaded, pcDNA3.1-AGXT2L1 group cell is at transwell
After cultivating 24h in cell, under pcDNA3.1-AGXT2L1 group polycarbonate membrane, the cell number in face, room substantially reduces.
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.