CN106244680A - The application in diagnosis of disease of ORC1L gene and expression product thereof - Google Patents
The application in diagnosis of disease of ORC1L gene and expression product thereof Download PDFInfo
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Abstract
The invention discloses the application in diagnosis of disease of ORC1L gene and expression product thereof, the expression of detection ORC1L genes amplification is as the instruction of esophageal squamous cell carcinoma phenotype.The level of detection ORC1L gene or its expression product that the invention provides, to diagnose esophageal squamous cell carcinoma, provides simultaneously and is improved by the expression of change ORC1L or treated the disease of patient.
Description
Technical field
The invention belongs to biomedicine field, relate to the application in diagnosis of disease of ORC1L gene and expression product thereof, tool
The described disease of body is esophageal squamous cell carcinoma.
Background technology
The esophageal carcinoma is one of most common malignant tumor of the mankind.According to WHO, within 2012, global esophageal carcinoma new cases are
455,800 examples, dead 400,200 examples, the highest with east Asia, eastern Africa and southern areas sickness rate, west area, Africa
Sickness rate is minimum, sickness rate significant difference, and China is Esophageal Cancer area, and the sickness rate of annual China is 21.88/10 ten thousand,
Occupy all kinds of malignant tumor the 5th；Mortality rate is 15.85/10 ten thousand, occupies the 4th.The esophageal carcinoma is classified as China by China's Ministry of Public Health
One of big disease of characteristic ten.The main histological type of the esophageal carcinoma has two kinds, esophageal squamous cell carcinoma and adenocarcinoma of esophagus, and esophageal squamous cell carcinoma is also claimed
For esophageal squamous cell carcinoma.The Nosology types that China's esophageal carcinoma occurs is different from western countries, and more than 90% is esophageal squamous cell
Cancer, and present obvious Regional Distribution, be concentrated mainly on Taihang Mountain, the Qinling Mountains, Dabie Mountain and Chuan Bei, Guangdong, Fujian, northern Suzhou, Xinjiang and
The geographic areas such as Gansu.
Although diagnosis and treatment technology constantly improve in recent years, the prognosis of the esophageal carcinoma is the most very good, the lifes in 5 years of patient
Deposit rate still less than 14%.And cause the reason of its poor prognosis to have a lot, effectively generally investigate means, when making a definite diagnosis including lacking
Stadium is later, misses optimal operative treatment period, postoperative or recurrence after radiotherapy, lacks specificity and the higher tumor of sensitivity
Mark etc..Improving prognosis is key issue urgently to be resolved hurrily in current esophageal carcinoma therapy.And searching can the early diagnosis state of an illness
And accurately the new tumor markers of predicted treatment effect is the importance improving prognosis.
The esophageal carcinoma is a multistage Progressive symmetric erythrokeratodermia evolution, the most effected by environmental factors, especially
Relate to multiple gene, the reciprocal action of multiple path.The formation of the esophageal carcinoma is to be occurred by normal mucous membrane of esophagus epithelial cell
Atypical hyperplasia in various degree, is developing progressively cancer in situ, early invasive carcinoma afterwards, canceration finally occurs, in the meantime
Each stepping exhibition is directed to expression or the changing function of related gene.
Esophageal carcinoma early diagnosis is delayed at present, and when making a definite diagnosis, 80% is all middle and advanced stage.Treatment now rely primarily on radiotherapy and
Chemicotherapy, the survival rate treating latter 5 years only has about 10%.Therefore, find and participate in the important molecule that the esophageal carcinoma occurs to develop, right
In improving esophageal carcinoma early diagnosis and finding new therapy target, the prognosis improving patient with esophageal carcinoma has great importance.
Summary of the invention
In order to make up the deficiencies in the prior art, it is an object of the invention to provide the molecular marker of a kind of esophageal squamous cell carcinoma,
This molecular marker is applied to clinic, it is possible to achieve the morning of esophageal squamous cell carcinoma finds early treatment.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides detection ORC1L reagent preparation diagnosis esophageal squamous cell carcinoma product in application.
Further, by the expression of detection ORC1L gene, described product diagnoses whether patient suffers from esophageal squamous cell carcinoma.
Wherein ORC1L is at patients with esophageal squamous cell carcinoma up-regulated.
Further, described product include by RT-PCR, real-time quantitative PCR, immune detection, marking hybridization, in situ hybridization,
The expression of hybridization array, chip or new-generation sequencing detection ORC1L gene and expression product thereof is with diagnosis esophageal squamous cell carcinoma
Product.
The invention provides a kind of product diagnosing esophageal squamous cell carcinoma, described product can be by ORC1L base in detection sample
The expression of cause diagnoses esophageal squamous cell carcinoma.
Wherein, described " sample " not only includes organism sample, such as cell, tissue, internal organs, body fluid (blood, lymph fluid
Deng), Digestive system, expectoration, alveole tracheal branches cleanout fluid, urine, feces, also include the nucleic acid extractive obtained by these organism samples
(extracting genome DNA thing, mRNA extract, mRNA extract the cDNA prepared product prepared or cRNA prepared product etc.) or albumen
Matter extract.Alternatively, it is also possible to said sample is implemented the fixing process of formalin, the fixing process of alcohol, freezes to process or paraffin
Embedding treatment.Preferred described sample is tissue.
Further, described product includes chip or test kit；Wherein, described chip includes gene chip, protein core
Sheet；Described gene chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, described oligonucleotide probe
Including the oligonucleotide probe for ORC1L gene for detecting ORC1L gene transcription level；Described protein chip includes
Solid phase carrier and be fixed on the specific antibody of ORC1L albumen of solid phase carrier；Described test kit includes gene detection reagent
Box and protein immunization detection kit；Described gene detecting kit includes the reagent for detecting ORC1L gene transcription level；
Described protein immunization detection kit includes the specific antibody of ORC1L albumen.
Further, described reagent includes the primer for ORC1L gene and/or probe.
It is multiple that gene chip of the present invention or gene detecting kit can be used for detecting including ORC1L gene
The expression of gene (such as, relevant to esophageal squamous cell carcinoma multiple genes).Described protein chip or protein immunization detectable
Box can be used for the table of the multiple protein (such as relevant to esophageal squamous cell carcinoma multiple protein) detecting including ORC1L albumen
Reach level.Multiple marks of esophageal squamous cell carcinoma are detected simultaneously, is greatly improved the accuracy rate of oesophagus squama cancer diagnosis.
The invention provides ORC1L gene and expression product thereof answering in the pharmaceutical composition of preparation treatment esophageal squamous cell carcinoma
With.
Further, described pharmaceutical composition includes the inhibitor of ORC1L gene and/or its expression product.Described inhibitor
Material, the material of suppression ORC1L gene expression product stability and/or suppression ORC1L including suppression ORC1L gene expression
The material of gene expression product activity.
Further, described inhibitor is the siRNA for ORC1L gene and/or the antibody for ORC1L albumen.
In the detailed description of the invention of the present invention, described inhibitor is for the siRNA of ORC1L gene.In order to carry further
The effectiveness of high siRNA segment, comprehensive Design is for the siRNA segment of screening.Finally, by sequence analysis (NCBI
BLAST) siRNA sequence is filtered, to improve the specificity of siRNA segment and to reduce the effect of missing the target of RNAi interference.Preferably,
The sequence of described siRNA is as shown in SEQ ID NO.9 and SEQ ID NO.10.
Present invention also offers a kind of pharmaceutical composition treating esophageal squamous cell carcinoma, described pharmaceutical composition comprises ORC1L base
Cause and/or its expression product inhibitor.Described inhibitor includes suppressing the material of ORC1L gene expression, suppression ORC1L gene table
Reach material and/or the material of suppression ORC1L gene expression product activity of product stability.
Further, above-mentioned pharmaceutical composition also includes that pharmaceutically acceptable carrier, this kind of carrier include (but not limiting
In): diluent, excipient such as lactose, sodium chloride, glucose, carbamide, starch, water etc., filler such as starch, sucrose etc.；Bonding
Agent such as simple syrup, glucose solution, starch solution, cellulose derivative, alginate, gelatin and polyvinylpyrrolidone；Moistening
Agent such as glycerol；Disintegrating agent such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate；Absorb
Accelerator quaternary ammonium compound, sodium lauryl sulphate etc.；Surfactant such as polyoxyethylene sorbitan fatty acid ester, 12
Alkyl sodium sulfate, glycerol monostearate, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier such as starch, lactose,
Bentonite, silica gel, Kaolin and soap clay etc.；Lubricant such as Pulvis Talci, calcium stearate and magnesium, Polyethylene Glycol, boric acid powder etc..
The pharmaceutical composition of the present invention can use different additives to be prepared, such as buffer agent, stabilizer, antibacterial
Agent, isotonic agent, chelating agen, pH controlling agent and surfactant.
Buffer agent can include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali metal or alkali
Property rare earth metal salt, such as sodium salt, potassium salt, calcium salt and magnesium salt).Isotonic agent includes potassium chloride, sodium chloride, sugar and glycerol.Chelating
Agent includes sodium ethylene diamine tetracetate and citric acid.Antibacterial include but not limited to valid density (such as ＜ 1%w/v) benzylalcohol,
Phenol, metacresol, methaform, methyl parahydroxybenzoate and/or propyl p-hydroxybenzoate.Stabilizer includes serum human egg
In vain, l-amino acid, sugar and cellulose derivative.L-amino acid can also include appointing in glycine, cysteine and glutamic acid
Anticipate one.Saccharide includes monosaccharide, such as glucose, mannose, galactose, fructose etc.；Sugar alcohol, such as mannitol, inositol, wood
Sugar alcohol etc.；Disaccharide, such as sucrose, maltose, lactose etc.；Polysaccharide, such as glucosan, hydroxypropyl starch, sulfuration chrondroitin, thoroughly
Bright matter acid etc. and their derivant.Cellulose derivative includes methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxyl
Propyl cellulose, hydroxypropyl methylcellulose and Carboxymethyl cellulose sodium.Surfactant includes ion or non-ionic surface active
Agent, such as polyethylene glycol oxide Arrcostab, sorbitan monoacyl ester, fatty glyceride.
The medicine of the present invention may also include pharmaceutically acceptable coating material and includes, but is not limited to, fast decoupled coating
Material, stain, enteric polymer, plasticizer, water-soluble polymer, insoluble polymer, dyestuff, pigment, other collapse
Powder.Common fast decoupled coating material includes OPADRY；Enteric polymer includes methylacrylic acid polymer, phosphorus hydroxypropyl first
Base cellulose phthalate, hydroxypropyl methylcellulose acetas, hydroxypropyl methylcellulose succinate, hydroxyl first ethyl cellulose
Element, cellulose acetophthalate；Plasticizer includes Polyethylene Glycol (PEG), propylene glycol etc..
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 ORC1L albumen or deficiency, by improve ORC1L albumen express or strengthen ORC1L albumen function, thus treat because of
ORC1L 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.
It is various carrier known in the art that the present invention carries the carrier of gene, such as commercially available carrier, includes plasmid, viscous
Grain, phage, virus 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 ORC1L albumen includes that monoclonal antibody, polyclonal antibody, polyspecific are anti-
Body (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.
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, ORC1L gene expression product includes people's ORC1L albumen and the portion of ORC1L albumen
Divide peptide.The partial peptide of described ORC1L albumen contains the functional domain relevant to esophageal squamous cell carninomatosis.
" ORC1L albumen " includes any function equivalent of ORC1L albumen and ORC1L albumen.Described function equivalent
Including ORC1L albumen conservative variation's protein or its active fragment, or its reactive derivative or its mutant.Mutant bag
Include allelic variant, natural mutation, induced mutants, its aminoacid sequence by lack, substitute, increase and/or insert
The mutant that changes different, can be with the protein coded by the DNA of the DNA hybridization of people ORC1L under high or low stringent condition.
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 protein that the example of the protein of an aminoacid or multiple Modification of amino acid residues is ORC1L albumen.
Peptide or protein with ORC1L protein fusion is not limited, as long as the fusion protein of gained retains the life of ORC1L albumen
Thing activity.
In the present invention, " host cell " includes prokaryotic cell and eukaryotic cell.The example of conventional prokaryotic host cell
Including escherichia coli, bacillus subtilis etc..Conventional eukaryotic host cell includes that yeast cells, insect cell and mammal are thin
Born of the same parents.It is preferred that this host cell is eukaryotic cell, such as Chinese hamster ovary celI, COS cell etc..
In the present invention, term " is treated " and is referred to but be not limited to cure, slow down pathological condition that (minimizing) target or
Disease or prevent recurrence.Include but not limited to that (1) inhibitory action suppresses progression of disease to a certain extent, it include slowing down with
And completely inhibit；(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 stop completely) disease cells penetrates into adjacent peripheral organs and/or tissue；(5) suppression (i.e. reduces, slows down or hinders completely
Only) pathophoresis；(6) one or more symptoms being associated with disease are alleviated to a certain extent；(7) increase anosis after treating
The time span of performance；(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:
Present invention firstly discovers that ORC1L gene differential expression in patients with esophageal squamous cell carcinoma, ORC1L is at patients with esophageal squamous cell carcinoma
Middle high expressed, the early stage detection for esophageal squamous cell carcinoma provides a kind of new molecular marker.
The invention provides one and treat accurate medical procedure, by target ORC1L treat esophageal squamous cell carcinoma, delay or
Cure the disease of patient, it is provided that the life quality of patient.
Accompanying drawing explanation
Fig. 1 show utilize QPCR detect ORC1L gene expression in esophageal squamous cell carcinoma tissue；
Fig. 2 show utilize QPCR detect ORC1L gene expression in esophageal cells；
Fig. 3 show utilize QPCR detect the siRNA impact on ORC1L gene expression；
Fig. 4 shows that soft-agar cloning forms experiment detection ORC1L and expresses the impact of cell proliferation；
Fig. 5 shows the impact utilizing transwell cell detection ORC1L 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, ORC1L gene expression in esophageal squamous cell carcinoma tissue significantly raises.
The differential expression of embodiment 2 QPCR sequence verification ORC1L gene
1, ORC1L gene differential expression is carried out large sample QPCR checking.Select according to the sample collection mode in embodiment 1
Select normal esophageal tissue and each 50 examples of esophageal squamous cell carcinoma tissue.
2, RNA extraction step is as described in Example 1.
3, reverse transcription:
1) reaction system:
| Reagent | Volume |
| MgCl2 | 2μl |
| 10×RT Buffer | 1μl |
| Water without Rnase | 3.75μl |
| DNTP mixed liquor | 1μl |
| Rnase inhibitor | 0.25μl |
| AMV reverse transcription | 0.5μl |
| Oligomerization dT aptamer primer | 0.5μl |
| Laboratory sample | 1μl |
2) reverse transcription reaction condition
Carry out according to reverse transcription reaction condition in RNAPCR Kit (AMV) Ver.3.0.
42 DEG C～55 DEG C of 60min, 99 DEG C of 2min, 5 DEG C of 5min.
3) polymerase chain reaction
1) design of primers
According to the coded sequence design QPCR amplimer of ORC1L gene and GAPDH gene in Genebank, by Bo Maide
Biotech firm synthesizes.Concrete primer sequence is as follows:
ORC1L gene:
Forward primer is 5 '-TGCCATCCGTGATTCTGA-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGAGTAGAGGTCGCTTCAT-3 ' (SEQ ID NO.4).
The primer sequence of house-keeping gene GAPDH is:
Forward primer: 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO.5)
Reverse primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6)
2) PCR reaction system is prepared according to table 1:
Table 1 PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| Takara Ex Taq HS | 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 45s) × 30 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.
5, 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.
6, result
Result as it is shown in figure 1, compared with surrounding normal mucous membrane of esophagus tissue, ORC1L gene table in esophageal squamous cell carcinoma tissue
Reaching rise, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
Embodiment 3 ORC1L gene differential expression in esophageal carcinoma cell line
1, cell is cultivated
Human esophageal squamous cell cancer cell strain KYSE150, 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 cell total 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, ORC1L gene esophageal squamous cell carcinoma cell KYSE150,
Expressing in KYSE450 and all raise, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
The silence of embodiment 4 ORC1L 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, siRNA design
SiRNA sequence for ORC1L gene:
Negative control siRNA sequence (siRNA-NC):
Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.7),
Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.8)；
SiRNA1-ORC1L:
Positive-sense strand is 5 '-AAGCAAUUUAGCAACAUACGG-3 ' (SEQ ID NO.9),
Antisense strand is 5 '-GUAUGUUGCUAAAUUGCUUGA-3 ' (SEQ ID NO.10)；
SiRNA2-ORC1L:
Positive-sense strand is 5 '-UUUCGAAGCUGCAAUUCGGGU-3 ' (SEQ ID NO.11),
Antisense strand is 5 '-CCGAAUUGCAGCUUCGAAAAC-3 ' (SEQ ID NO.12)；
By cell by 5 × 105/ hole is inoculated in six porocyte culture plates, at 37 DEG C, 5%CO2In incubator, cell is cultivated
24h, in without dual anti-, DMEM culture medium containing 10%FBS, transfection (is purchased from according to lipofectamine 2000
Invitrogen company) description transfection, experiment is divided into matched group (KYSE 150) negative control group (siRNA-NC) and real
Test group (20nM) (siRNA1-ORC1L, siRNA2-ORC1L), wherein the sequence of negative control group siRNA and ORC1L gene without
Homology, concentration is 20nM/ hole, transfects the most respectively.
3, the transcriptional level of QPCR detection ORC1L gene
The extraction of 3.1 cell total rnas
Concrete steps are with embodiment 3.
3.2 reverse transcription step are with embodiment 2.
3.3 QPCR amplification step 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, the difference between interference ORC1L gene expression group and matched group is adopted
Check with t, it is believed that when P < has statistical significance when 0.05.
5, result
Result such as Fig. 3 shows, compares KYSE 150, unloaded siRNA-NC, siRNA2-ORC1L group of transfection, siRNA1-
ORC1L group can significantly reduce the expression of ORC1L gene, and difference has statistical significance (P < 0.05).
The embodiment 5 ORC1L apoptotic impact of gene pairs esophageal squamous cell carcinoma
Use the flow cytomery apoptotic impact of ORC1L 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 siRNA1-ORC1L group is (17.35 ± 0.016) %, transfects siRNA-NC empty carrier group
Apoptosis rate be (5.83 ± 0.013) %, above-mentioned difference has statistical significance (P < 0.05), and the above results shows,
The apoptosis of the process LAN suppression esophageal squamous cell carcinoma cell of ORC1L gene.
Embodiment 6 soft-agar cloning forms experiment
1, being in the cell of exponential phase with 0.25% trypsinization after transfecting, piping and druming makes slender gently
Born of the same parents' suspension, 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 CO as bottom-layer agar2In incubator standby.
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
As shown in Figure 4, compared with other groups, siRNA1-ORC1L group single cell clone Colony forming digital display writes fall to result
Low.
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.
10, result
Result is as it is shown in figure 5, KYSE150, siRNA1-ORC1L, siRNA-NC group cell is trained in transwell cell
After supporting 24h, under siRNA1-ORC1L group polycarbonate membrane, the cell number in face, room significantly 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.
Claims (10)
1. detection ORC1L reagent preparation diagnosis esophageal squamous cell carcinoma product in application.
Application the most according to claim 1, it is characterised in that described product is by detecting the expression of ORC1L gene
Diagnose whether patient suffers from esophageal squamous cell carcinoma.
Application the most according to claim 2, it is characterised in that described product include by RT-PCR, real-time quantitative PCR,
The hybridization of immune detection, the marking, in situ hybridization, hybridization array, chip or new-generation sequencing detection ORC1L gene and expression product thereof
Expression with diagnose esophageal squamous cell carcinoma product.
4. the product diagnosing esophageal squamous cell carcinoma, it is characterised in that described product can be by ORC1L gene in detection sample
Expression diagnoses esophageal squamous cell carcinoma.
Product the most according to claim 4, it is characterised in that described product includes chip or test kit；Wherein, described
Chip includes gene chip, protein chip；Described test kit includes gene detecting kit, protein immunization detection kit.
6.ORC1L gene and expression product application in the pharmaceutical composition of preparation treatment esophageal squamous cell carcinoma thereof.
Application the most according to claim 6, it is characterised in that described pharmaceutical composition includes ORC1L gene and/or its table
Reach the inhibitor of product.
Application the most according to claim 7, it is characterised in that described inhibitor includes the thing suppressing ORC1L gene expression
Matter, the material of suppression ORC1L gene expression product stability and/or the material of suppression ORC1L gene expression product activity.
9. according to the application described in claim 7 or 8, it is characterised in that described inhibitor be the siRNA for ORC1L and/or
Antibody.
10. the pharmaceutical composition treating esophageal squamous cell carcinoma, it is characterised in that described pharmaceutical composition includes claim 7-9
Inhibitor described in any one.
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Citations (2)
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|---|---|---|---|---|
| CN101932724A (en) * | 2007-10-05 | 2010-12-29 | 环太平洋生物技术有限公司 | The hyperplasia label and the prognosis of gastrointestinal cancer |
| US20140148357A1 (en) * | 2012-11-29 | 2014-05-29 | Vanderbilt University | Characterizing Gastro-Intestinal Disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101932724A (en) * | 2007-10-05 | 2010-12-29 | 环太平洋生物技术有限公司 | The hyperplasia label and the prognosis of gastrointestinal cancer |
| US20140148357A1 (en) * | 2012-11-29 | 2014-05-29 | Vanderbilt University | Characterizing Gastro-Intestinal Disease |
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| Title |
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| CONG-JUN LI等: "Butyrate Induced Cell Cycle Arrest in Bovine Cells through Targeting Gene Expression Relevant to DNA Replication Apparatus", 《GENE REGULATION AND SYSTEMS BIOLOGY》 * |
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