CN105112554B - Application of the CLEC1B gene in cholangiocarcinoma diagnosing and treating - Google Patents
Application of the CLEC1B gene in cholangiocarcinoma diagnosing and treating Download PDFInfo
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Abstract
The invention discloses application of the CLEC1B gene in cholangiocarcinoma diagnosing and treating.The present invention is screened using RNA-sep and passes through large sample RT-PCR and confirmed, CLEC1B abnormal gene expression is related to the occurrence and development of cholangiocarcinoma.Research achievement according to the present invention, CLEC1B can be also used for the drug of preparation treatment cholangiocarcinoma.The present invention substantially increases the sensibility and specificity of cholangiocarcinoma diagnosis, while new target spot is provided for the gene therapy of cholangiocarcinoma.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to use of the CLEC1B gene in cholangiocarcinoma diagnosing and treating
On the way.
Background technique
Cholangiocarcinoma system refers to the malignant tumour that biliary system lining epithelium occurs, and can be divided into stones in intrahepatic bile duct by the position occurred
Cancer and cholangiocarcinoma two major classes.Intrahepatic cholangiocarcinoma originates from stones in intrahepatic bile duct and its branches to any portion of canals of Hering tree between leaflet
The lining epithelium of position；Cholangiocarcinoma is divided into hilar cholangiocarcinoma and distal end bile duct using cystic duct and ductuli hepaticus communis point as boundary again
Cancer.Worldwide, cholangiocarcinoma accounts for the 3% of all gastroenteric tumors, is the second common liver and gallbladder tumour.Due to having discovery
Evening, transfer morning, poor prognosis, Resection Rate be low, to radiotherapy, chemotherapy is insensitive the features such as, be always surgical field problem with
The hot spot of research.Since it is insensitive to the non-operative treatments such as chemotherapy and radiotherapy, general treatment measures are at present still without clearly improving
The meaning of late result, early prevention, early detection, early treatment are the key that improve human bile duct cancer surgical radical treatment rate.
The method clinically for cholangiocarcinoma diagnosis includes: 1) clinical manifestation at present；2) blood test: red with the total gallbladder of blood
Element, bilirubin direct, alkaline phosphatase and γ-paddy amine acyltransferase are Testing index；3) serum tumor marker: cholangiocarcinoma mesh
Preceding not to be found specific tumor marker object, only CA-19, CA125, CEA have certain values；4) imageological examination, and be divided into
Ultrasonography, high-resolution spiral CT, MRI, endoscopic ultrasonography, Positron emission computed tomography (PET-CT)；5)
ERCP and PTC；6) duodenoscope；7) enteron aisle mothers and sons mirror；8) cytology and histodiagnosis.But the above method cannot be applied
In the diagnosis of cholangiocarcinoma early stage, when making a definite diagnosis in aforementioned manners, patient has been advanced tumors mostly, and surgical radical treatment rate is low, mesh
It is preceding to be directed to cholangiocarcinoma, still lack a kind of specifically diagnostic method with sensitivity.
Summary of the invention
In order to make up for the deficiencies of the prior art, it can be used for what cholangiocarcinoma early diagnosed the purpose of the present invention is to provide a kind of
Molecular marker-CLEC1B gene.Compared to the diagnostic method of traditional cholangiocarcinoma, cholangiocarcinoma tool is diagnosed using gene marker
There are timeliness, specificity and sensitivity, make patient that can know risk of cancer in cancer early stage, for risk height, takes phase
The prevention and treatment measure answered.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides a kind of application of CLEC1B gene and its expression product in the product of preparation diagnosis cholangiocarcinoma.
Further, diagnostic products mentioned above include: by RT-PCR, real-time quantitative PCR, immune detection, original position
Hybridization or chip detect the expression of CLEC1B gene and its expression product to diagnose the product of cholangiocarcinoma.
Further, the product with RT-PCR diagnosis cholangiocarcinoma includes at least drawing for a pair of of specific amplified CLEC1B gene
Object；The product with real-time quantitative PCR diagnosis cholangiocarcinoma includes at least the primer of a pair of of specific amplified CLEC1B gene；It is described
Product with immune detection diagnosis cholangiocarcinoma includes: the antibody in conjunction with CLEC1B protein-specific；It is described to be examined in situ hybridization
The product of disconnected cholangiocarcinoma includes: the probe with the nucleic acid array hybridizing of CLEC1B gene；The production with chip diagnosis cholangiocarcinoma
Product include: protein chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with CLEC1B protein-specific, gene
Chip includes the probe with the nucleic acid array hybridizing of CLEC1B gene.
The present invention provides a kind of product for diagnosing cholangiocarcinoma, the product can be by detection bile duct tissue
The expression of CLEC1B gene diagnoses cholangiocarcinoma.
Further, product recited above includes chip or kit.Wherein, the chip includes genetic chip, albumen
Matter chip；The genetic chip includes solid phase carrier and the oligonucleotide probe that is fixed on solid phase carrier, the few nucleosides
Acid probe includes the oligonucleotide probe for CLEC1B gene for detecting CLEC1B gene transcription level；The protein
Chip includes the specific antibody of solid phase carrier and the CLEC1B albumen being fixed on solid phase carrier；The kit includes base
Because of detection kit and protein immunization detection kit；The gene detecting kit includes for detecting CLEC1B genetic transcription
Horizontal reagent；The protein immunization detection kit includes the specific antibody of CLEC1B albumen.
Further, reagent described above includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip
Method detects reagent needed for CLEC1B gene expression dose process.Preferably, the reagent includes being directed to CLEC1B gene
Primer and/or probe.
Further, the genetic chip can be used for detecting multiple genes including CLEC1B gene (for example, and bile duct
The relevant multiple genes of cancer) expression.The protein-chip can be used for detecting multiple including CLEC1B albumen
The expression of protein (such as multiple protein relevant to cholangiocarcinoma).By by multiple markers with cholangiocarcinoma simultaneously
Detection is greatly improved the accuracy rate of cholangiocarcinoma diagnosis.
The present invention also provides the application of CLEC1B gene and its expression product in the drug of preparation treatment cholangiocarcinoma.
Further, the drug includes the reagent that can promote CLEC1B gene expression or enhance CLEC1B expressive function.
Further, the reagent includes: that the reagent containing CLEC1B gene, the carrier for carrying CLEC1B gene or host are thin
Born of the same parents, the reagent containing CLEC1B protein.
The present invention also provides application of the reagent recited above in the drug of preparation treatment cholangiocarcinoma.
Drug of the invention can be used for supplementing the missing or deficiency of endogenic CLEC1B albumen, by improving CLEC1B
The expression of albumen, thus cholangiocarcinoma caused by treatment is reduced because of CLEC1B albumen.
The carrier of the present invention for carrying gene is various carriers known in the art, such as commercially available carrier including plasmid,
Clay, bacteriophage, virus etc..
In the present invention, term " host cell " includes prokaryotic cell and eukaryocyte.Common prokaryotic host cell
Example includes Escherichia coli, hay bacillus etc..Common eukaryotic host cell includes yeast cells, insect cell and mammal
Cell.Preferably, the host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cell.
Further, drug of the invention further includes pharmaceutically acceptable carrier, and this kind of carrier includes (but being not limited to):
Diluent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin and poly-
Vinylpyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as agar, calcium carbonate and sodium bicarbonate；Sorbefacient quaternary ammonium compound；
Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and magnesium gather
Ethylene glycol etc..
Drug of the invention, which imports tissue or the mode of cell, can be divided into external or intracorporal mode.Vitro formats
Including the drug containing CLEC1B gene or the drug containing CLEC1B protein are imported in cell, then by cell transplantation or
It feeds back in vivo.Internal mode includes directly infusing the drug containing CLEC1B gene or the drug containing CLEC1B protein
Enter in in-vivo tissue.
Drug of the invention can also mention significantly with the drug combination of other treatment cholangiocarcinoma, a variety of Drug combinations
The success rate of height treatment.
Can be in the present invention with the probe of the nucleic acid array hybridizing of CLEC1B gene DNA, RNA, DNA-RNA chimera,
PNA or other derivatives.There is no limit as long as complete specific hybrid and purpose nucleotide sequence spy for the length of the probe
The opposite sex combines, and any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, institute
The length for stating probe can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Due to different probes
Length has different influences to hybridization efficiency, signal specificity, and the length of the probe is typically at least 14 base-pairs, longest
30 base-pairs are usually no more than, the length complementary with purpose nucleotide sequence is best with 15-25 base-pair.The probe is certainly
Body complementary series is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The specific antibody of heretofore described CLEC1B albumen includes monoclonal antibody, polyclonal antibody.It is described
The specific antibody of CLEC1B albumen includes any segment or modification of complete antibody molecule, antibody, for example, chimeric antibody,
ScFv, Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with CLEC1B albumen.For detecting
The preparation of the antibody of protein level is well known to those skilled in the art, and the present invention may use any method to prepare
The antibody.
In the context of the present invention, " CLEC1B gene " includes any of people CLEC1B gene and people's CLEC1B gene
The polynucleotides of functional equivalent.CLEC1B gene include in the public GenBank GeneBank in the current world
CLEC1B gene (NC_000012.12) DNA sequence dna has 70% or more homology, and encodes the DNA sequence of identical function protein
Column；
Preferably, the coded sequence of CLEC1B gene includes following any DNA molecular:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；
(2) hybridize under strict conditions with the DNA sequence dna that (1) limits and encode the DNA sequence dna of identical function protein；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, 90% or more homology, and encodes identical function
The DNA molecular of energy protein.
In specific embodiments of the present invention, the coded sequence of the CLEC1B gene is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, CLEC1B gene expression product includes people CLEC1B albumen and people's CLEC1B albumen
Partial peptide.The partial peptide of the CLEC1B albumen contains functional domain relevant to cholangiocarcinoma.
" CLEC1B albumen " includes any functional equivalent of CLEC1B albumen and CLEC1B albumen.The function is equivalent
Object includes CLEC1B albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural
Mutant, induced mutants, can be with the encoded albumen of DNA of the DNA hybridization of people CLEC1B under high or low stringent condition
Matter.
Preferably, CLEC1B albumen is the protein with following amino acid sequences:
(1) protein that the amino acid sequence shown in SEQ ID NO.2 in sequence table forms；
(2) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or addition and with the ammonia with the same function as shown in SEQ ID NO.2 of amino acid sequence shown in SEQ ID NO.2
Protein derived from base acid sequence.The number for the amino acid for replacing, lacking or adding is usually 1-50, preferably 1-30
It is a, more preferably 1-20, most preferably 1-10.
(3) there is at least 80% homology (also known as sequence identity) with amino acid sequence shown in SEQ ID NO.2,
Preferably, with the homology of amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, Chang Wei 96%, 97%,
98%, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the CLEC1B albumen is with amino acid shown in SEQ ID NO.2
The protein of sequence.
It is known that, conventionally, the modification of one or more amino acid will not influence the function of protein in a protein.
Those skilled in the art can approve the amino acid for changing single amino acids or small percentage or to amino acid sequence it is individual add,
Missing, insertion, replacement are conservative modifications, and wherein the change of protein generates the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by one amino acid of addition or the protein of more amino acid modification is melting for CLEC1B albumen
Hop protein.For the peptide or protein with CLEC1B protein fusion, there is no limit as long as resulting fusion protein retains
The biological activity of CLEC1B albumen.
CLEC1B albumen of the invention also includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, only
It still to be able to retain the biological activity of CLEC1B albumen by the protein of modification.It dashes forward in such modification protein
The amino acid number of change is usually 10 perhaps less such as 6 perhaps less such as 3 or less.
In the context of the present invention, " diagnosis cholangiocarcinoma " both included judge subject whether suffered from cholangiocarcinoma or
Including judging that subject whether there is the risk with cholangiocarcinoma.
In the context of the present invention, " treatment cholangiocarcinoma " includes the complete healing of the alleviation of disease, disease.
The advantages of the present invention:
Present invention firstly discovers that CLEC1B gene expression is related to cholangiocarcinoma, by detection subject's bile duct mucous membrane
The expression of CLEC1B, it can be determined that whether subject suffers from cholangiocarcinoma or judge that subject whether there is with cholangiocarcinoma
Risk, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-CLEC1B genes, compared to traditional detection means, gene diagnosis
More in time, more special, sensitiveer, it can be realized the early diagnosis of cholangiocarcinoma, to reduce the death rate of cholangiocarcinoma.
Detailed description of the invention
Fig. 1 shows the expression using QPCR detection CLEC1B gene in cholangiocarcinoma；
Fig. 2 shows the overexpression situation using QPCR detection CLEC1B gene in cholangiocarcinoma cell；
Fig. 3 shows the influence using MTT detection CLEC1B gene expression to cholangiocarcinoma cell proliferative capacity.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually 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 normal condition proposed by manufacturer.
Embodiment 1 screens gene marker relevant to cholangiocarcinoma
1, sample collection
Respectively collect 8 normal bile duct tissues and cholangiocarcinoma sample.Above-mentioned sample is that the operation of cholangiocarcinoma patients is cut off
Sample, the acquirement of above-mentioned all samples pass through the agreement of the committee, organizational ethics.
2, the preparation (being operated using the tissue RNA extracts kit of QIAGEN) of RNA sample
1) tissue extraction
In the clear area of less RNase interference, in vitro pulmonary adenocarcinoma sample is weighed about using the mortar containing appropriate liquid nitrogen
20mg, be ground to pestle it is powdered, then by sample be transferred to one without RNA enzyme 2ml centrifuge tube in.300 μ are added
L lysate, is placed in homogenizer, is fully ground 1-5min, 12000g, 4 DEG C, is centrifuged 10min, transfer supernatant to new 1.5ml
Centrifuge tube in.The water that 600 μ l are free of RNA enzyme is added, after being mixed with vortex device, 20 μ l Proteinase Ks are added, in 55 DEG C of warm bath
15min is constantly vortexed and mixes.14000g, room temperature are centrifuged 1min, make pellet cell debris in centrifugation bottom of the tube, supernatant is taken to shift
Into another 1.5ml centrifuge tube without RNA enzyme, 95% ethyl alcohol of 450 μ l is added, is vortexed and mixes.
2) RNA is adsorbed:
650 lysates of the μ l containing ethyl alcohol are taken to be added in centrifugal column, 14000g is centrifuged 1min, abandons lower layer, pillar is set again
In collecting pipe；Repetitive operation is primary, and 400 μ l cleaning solutions are then added, and 14000g is centrifuged 2min, abandons lower layer, pillar is placed in
On one new collecting pipe.
3) DNase is handled:
100 μ l Enzyme Incubation Buffer and 15 μ l DNase I, 14000g centrifugation 1min are added, will receive
Solution in collector moves into column again, is placed at room temperature for 15min.
4) RNA is washed:
400 μ l cleaning solutions are added, 14000g is centrifuged 1min, abandons lower layer, pillar is refitted in collecting pipe, is then added
400 μ l cleaning solutions, 14000g are centrifuged 2min, abandon collecting pipe.
5) RNA is eluted:
Pillar is put into 1.5ml Elution pipe, be added 30 μ l eluents, 200g be centrifuged 2min, make solution sufficiently with
Pillar combines, and then 14000g is centrifuged 1min.
3, high-throughput transcript profile sequencing
1) RNA-seq read positions
Low-quality read is removed to obtain cleaning read first, then using TopHat v1.3.1 will clean segment and
UCSC H.sapiens is matched with reference to genome (hg19), the H.sapiens UCSC hg19 editions indexes constructed in advance
It is downloaded from TopHat homepage, and as reference genome, when matching using TopHat with genome, allows each read (default
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signal
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat method.
2) transcript abundance is assessed
The read file matched is handled by Cufflinks v1.0.3, and Cufflinks v1.0.3 is by RNA-seq piece
Number of segment mesh is standardized the relative abundance for calculating transcript.FPKM value refers to being matched in every 1,000,000 sequencing fragment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.
The GTF comment file for the reference that Cufflinks is used downloads (Homo_ from Ensembl database
sapiens.GRCh37.63.gtf)。
3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF file of downloading and by the matched original document of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF file using original matching files, detects difference table
It reaches.The only q value < 0.01 in Cuffidff output, test display is more just considered as successfully differential expression.
4, result
RNA-seq is the results show that expression quantity of the CLEC1B gene in cholangiocarcinoma is substantially less than normal bile duct tissues
In expression quantity.
The differential expression of embodiment 2QPCR sequence verification CLEC1B gene
1, large sample QPCR verifying is carried out to CLEC1B gene differential expression.According to the sample collection mode in embodiment 1
Select cholangiocarcinoma and normal bile duct tissues each 80.
2, QPCR concrete operation step is as follows:
(1) RNA is extracted
It freezes to be put into the mortar being pre-chilled tissue after liquid nitrogen, taking-up after collection sample and be ground, sample to be organized
This is at powdered rear:
1) Trizol is added, is placed at room temperature for 5min；
2) chloroform 0.2ml is added to be mixed well with forced oscillation centrifuge tube, places 5-10min at room temperature；
3) 12000rpm is centrifuged 15min, and upper strata aqueous phase is moved on in another new centrifuge tube and (is careful not to be drawn onto two layers of water
Protein substance between phase), the isopropanol of -20 DEG C of isometric pre-coolings is added, is sufficiently mixed by inversion, is placed in 10min on ice；
4) 12000rpm high speed is added 75% in the ratio of 1ml/ml Trizol from supernatant is carefully discarded after 15min
DEPC ethanol washing precipitating (4 DEG C of preservations), washing precipitate, oscillation mixes, and 4 DEG C, 12000rpm is centrifuged 5min；
5) ethanol liquid is discarded, places 5min at room temperature, DEPC water dissolution precipitating is added；
6) it with Nanodrop2000 ultraviolet specrophotometer measurement RNA purity and concentration, freezes in -70 DEG C of refrigerators.
(2) 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 PCR pipe following
Component: DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μM of Oligo dT, 200U/ μ l M-MLV,
Template ribonucleic acid.42 DEG C of incubation 1h, 72 DEG C of 10min, of short duration centrifugation.
(3) QPCR amplification is examined
Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times above to protect
Demonstrate,prove the reliability of result.Prepare following reaction system: 12.5 μ l of SYBR Green polymerase chain reaction system, forward primer (5
μM) 1 μ l, reverse primer (5 μM) 1 μ l, template cDNA 2.0 μ l, no 8.5 μ l of enzyme water；Expand the forward primer sequence of CLEC1B gene
It is classified as 5 '-TGGAGATATTATGGAGATAGC-3 ' (SEQ ID NO.3), reverse primer sequences 5 '-
TTCAGGAGAGTAGCATTC-3'(SEQ ID NO.4)；The preferred GAPDH of house-keeping gene expands the forward primer sequence of the gene
For 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ IDNO.5), reverse primer sequences 5 '-
GGTGGAATCATATTGGAACA-3'(SEQ ID NO.6).Operations are carried out on ice.Amplification program are as follows: 95 DEG C
10min, (95 DEG C of 15s, 60 DEG C of 60s) × 45 circulations.It is glimmering in Light Cycler using SYBR Green as fluorescent marker
PCR reaction is carried out on light real-time PCR, determines that purpose band, Δ Δ CT method carry out by melt curve analysis analysis and electrophoresis
Relative quantification.
3, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
4, result
As a result as shown in Figure 1, compared with normal bile duct tissues, CLEC1B gene is lowered in cholangiocarcinoma, difference tool
Statistically significant (P < 0.05), it is consistent with RNA-sep result.
Embodiment 3CLEC1B gene overexpression
1, human bile duct carcinoma strain QBC939, with DMEM (high sugar) culture medium containing 10% calf serum in 37 DEG C, 5%
CO2, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, is passed on using 0.25% trypsase conventional digestion.
2, the overexpression of CLEC1B gene
The building of 2.1CLEC1B expression vector
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.1) of CLEC1B gene, primer sequence is as follows:
Forward primer is 5 '-CCGGGATCCGCCACCATGCAGGATGAAGAT-3 ' (SEQ ID NO.7), reverse primer 5 '-
CGGCTCGAGAGGTAGTTGGTCCACCTTGG-3'(SEQ ID NO.8).From the cDNA library at Human fetal spleen, (clontech is public
Department, article No.: 638831) coded sequence of the CLEC1B gene of amplification overall length in, above-mentioned cDNA sequence is through restriction enzyme
The eukaryotic expression vector through restriction enzyme BamHI and XhoI double digestion is inserted into after BamHI and XhoI double digestion
In pcDNA3.1, the recombinant vector pcDNA3.1-CLEC1B of acquisition is connected for subsequent experimental.
2.2 transfection
Cholangiocarcinoma cell strain QBC939 is divided to for two groups, respectively control group (transfection pcDNA3.1 empty carrier) and
CLEC1B overexpression group (transfection pcDNA3.1-CLEC1B).The transfection of carrier, specific transfection method are carried out using liposome 2000
Instruction to specifications carries out.The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-CLEC1B are 0.5 μ g/ml.
2.3RT-PCR detection
Specific steps are the same as embodiment 2.
3, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
4, result
As shown in Fig. 2, being transfected in the cell of pcDNA3.1-CLEC1B compared with the cell of transfection pcDNA3.1 empty carrier
The content of CLEC1B significantly raises, and difference has statistical significance (P < 0.05).
The influence of embodiment 4CLEC1B gene pairs cholangiocarcinoma cell proliferation
It is influenced using MTT experiment detection CLEC1B gene pairs cholangiocarcinoma cell proliferative capacity.
1, step: pancreatin digests after group of cells transfects 12h, single cell suspension is made, with 6000, every hole cell inoculation
In 96 well culture plates, at 7 time points of every component, each time point sets 6 multiple holes.After cell is adherent, the 1st detection is carried out:
The 20 μ l of MTT liquid of 5g/L is added in every hole, continues after cultivating 4h, sucks culture medium, and 150 μ l of DMSO is added, and carefulness piping and druming makes purple
Blue precipitate sufficiently dissolves, and surveys absorbance value (A value) in 490nm wavelength with microplate reader.Then every 12h is detected 1 time, continuous to survey
72h, totally 7 times.This experiment is repeated 3 times.
2, statistical method
Experiment is completed according to being repeated 3 times, using SPSS13.0 statistical software come for statistical analysis, two groups
Between difference using t examine, it is believed that as P < 0.05 have statistical significance.
3, result
It is shown in Fig. 3 as the result is shown: transfection pcDNA3.1-CLEC1B group vitro growth rates significantly lower than transfects
The vitro growth rates of pcDNA3.1 empty carrier group, difference have statistical significance (P < 0.05), and the above results show CLEC1B
Expression is able to suppress the growth of cholangiocarcinoma cell.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (8)
1. the reagent for detecting CLEC1B gene and its expression product expression is preparing the application in the product for diagnosing cholangiocarcinoma.
2. application according to claim 1, which is characterized in that the product include: by RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization or chip detect the expression of CLEC1B gene and its expression product to diagnose the production of cholangiocarcinoma
Product.
3. application according to claim 2, which is characterized in that the product with RT-PCR diagnosis cholangiocarcinoma includes at least
The primer of a pair of of specific amplified CLEC1B gene；The product with real-time quantitative PCR diagnosis cholangiocarcinoma includes at least a pair of special
The primer of different amplification CLEC1B gene；It is described with immune detection diagnosis cholangiocarcinoma product include: and CLEC1B protein-specific
In conjunction with antibody；The product in situ hybridization diagnosis cholangiocarcinoma includes: the spy with the nucleic acid array hybridizing of CLEC1B gene
Needle；The product with chip diagnosis cholangiocarcinoma includes: protein chip and genetic chip；Wherein, protein chip include with
The antibody that CLEC1B protein-specific combines, genetic chip includes the probe with the nucleic acid array hybridizing of CLEC1B gene.
The application of 4.CLEC1B gene and its expression product in the drug of preparation treatment cholangiocarcinoma.
5. application according to claim 4, which is characterized in that the drug include can promote CLEC1B gene expression or
Enhance the reagent of CLEC1B expressive function.
6. application according to claim 5, which is characterized in that the reagent includes: the reagent containing CLEC1B gene, takes
Carrier or host cell with CLEC1B gene, the reagent containing CLEC1B protein.
7. promoting CLEC1B gene expression or enhancing the reagent of CLEC1B expressive function in the drug of preparation treatment cholangiocarcinoma
Using.
8. application according to claim 7, which is characterized in that the reagent includes: the reagent containing CLEC1B gene, takes
Carrier or host cell with CLEC1B gene, the reagent containing CLEC1B protein.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101809168A (en) * | 2007-09-24 | 2010-08-18 | 塞诺菲-安万特股份有限公司 | Use of CLEC1B for the determination of cardiovascular and thrombotic risk |
| CN103476932A (en) * | 2010-10-14 | 2013-12-25 | 雷加多生物科学公司 | Nucleic acid modulators of CLEC-2 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101809168A (en) * | 2007-09-24 | 2010-08-18 | 塞诺菲-安万特股份有限公司 | Use of CLEC1B for the determination of cardiovascular and thrombotic risk |
| CN103476932A (en) * | 2010-10-14 | 2013-12-25 | 雷加多生物科学公司 | Nucleic acid modulators of CLEC-2 |
Non-Patent Citations (1)
| Title |
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| 胆管癌胆管胆汁中糖蛋白糖链结构变化及其酶学机制;刘厚宝等;《中国临床医学》;20061231;944-946 |
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