CN105671158B - Purposes of the FAM63B gene in the product of preparation diagnosis and treatment fibroid - Google Patents
Purposes of the FAM63B gene in the product of preparation diagnosis and treatment fibroid Download PDFInfo
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- CN105671158B CN105671158B CN201610112986.0A CN201610112986A CN105671158B CN 105671158 B CN105671158 B CN 105671158B CN 201610112986 A CN201610112986 A CN 201610112986A CN 105671158 B CN105671158 B CN 105671158B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57442—Specifically defined cancers of the uterus and endometrial
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
Abstract
The invention discloses a kind of molecular markers for fibroid diagnosis.The present invention proves that FAM63B gene is significantly lowered in Human Leiomyma compared with the expression in normal tissue using QPCR and Western blot method, since there are above-mentioned correlations with fibroid for FAM63B gene, it can diagnose whether subject suffers from fibroid by the detection of FAM63B gene expression.In addition, the experiment proves that the proliferation of Leiomyoma Cell can be inhibited while deposit into Apoptosis by increasing FAM63B gene expression, therefore FAM63B gene and its expression product can be used for preparing the drug for the treatment of fibroid, clinically apply extensively.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to FAM63B gene in the diagnosis, treatment of fibroid
Purposes.
Background technique
Fibroid (alias: uterus benign tumour English: myoma of uterus), is that female sex organs are most common
A kind of benign tumour.Mostly asymptomatic, minority shows as colporrhagia, and abdomen touches swollen object and pressure symptom etc..With multiple
Fibroid is common, and the definite etiology unknown of this disease, modern Western medicine takes sex hormone or operative treatment, other ideals there is no to treat
Method is apt to occur in 30~45 years old more vigorous women of ovarian function, until after patient 50 years old, since ovarian function obviously declines
It moves back, myomata voluntarily reduces mostly.
Fibroid diagnosis can carry out preliminary judgement by the symptom of some fibroids, then do further
Inspection, but cannot only be made a definite diagnosis by gynecologial examination.Firstly, may feel that abdominal pain with fibroid, next women's
Menstruation can also change, or even can cause infertile or multiple miscarriage.Country's ultrasound diagnosis is more universal and common at present
The diagnostic method of fibroid can relatively be explicitly shown myomata size and position.Laparoscope, hysteroscope, diagnostic curettage help
In the diagnosis of fibroid.But above-mentioned physical method haves the defects that sensitivity difference or keeps the patient feels uncomfortable, therefore the phase
Appearance to new diagnostic method.
Summary of the invention
For overcome the deficiencies in the prior art, the purpose of the present invention is to provide one kind can be used for fibroid early diagnosis
Molecular marker.Fibroid is diagnosed using gene marker with timeliness, specificity and sensitivity, to make patient
Disease risks can be known in disease early stage, for risk height, take corresponding prevention and treatment measure.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides application of the product of detection FAM63B gene expression in the tool of preparation diagnosis fibroid.
Further, it is described detection FAM63B gene expression product include detect FAM63B gene mRNA levels product,
And/or the product of detection FAM63B protein level.
Further, the product of the detection FAM63B gene expression includes: by RT-PCR, real-time quantitative PCR, immune inspection
It surveys, in situ hybridization or chip detect the expression of FAM63B gene and its expression product to diagnose the product of fibroid.
Further, the product with RT-PCR diagnosis fibroid includes at least a pair of of specific amplified FAM63B gene
Primer；The product with real-time quantitative PCR diagnosis fibroid includes at least the primer of a pair of of specific amplified FAM63B gene；
The product with immune detection diagnosis fibroid includes: the antibody in conjunction with FAM63B protein-specific；The original position
The product of hybridization diagnosis fibroid includes: the probe with the nucleic acid array hybridizing of FAM63B gene；It is described to diagnose son with chip
The product of palace myomata includes: protein chip and genetic chip；Wherein, protein chip includes in conjunction with FAM63B protein-specific
Antibody, genetic chip include the probe with the nucleic acid array hybridizing of FAM63B gene.
A pair of of specific amplified FAM63B gene that the product with real-time quantitative PCR diagnosis fibroid includes at least
Primer is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of the detection FAM63B gene expression can be the reagent of detection FAM63B gene expression, be also possible to wrap
Kit, chip, test paper containing the reagent etc. are also possible to the high-flux sequence platform using the reagent.
The tool of the diagnosis fibroid includes but is not limited to chip, kit, test paper or high-flux sequence platform；
High-flux sequence platform is a kind of tool of special diagnosis fibroid, with the development of high throughput sequencing technologies, to one
The building of the gene expression profile of people will become very easily work.By the gene expression for comparing Disease and normal population
Spectrum, the exception for being easy to analyze which gene are related to disease.Therefore, the exception of FAM63B gene is known in high-flux sequence
The purposes for also belonging to FAM63B gene related to fibroid, equally within protection scope of the present invention.
The present invention also provides a kind of tool for diagnosing fibroid, the tool includes detection FAM63B gene expression
Reagent；The reagent includes the primer and/or probe, the antibody for detecting FAM63B albumen for detecting FAM63B gene mRNA.
The tool includes but is not limited to chip, kit, test paper or high-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes for detecting being directed to for FAM63B gene transcription level
The oligonucleotide probe of FAM63B gene；The protein-chip includes solid phase carrier and the FAM63B for being fixed on solid phase carrier
The specific antibody of albumen；The genetic chip can be used for detecting multiple genes including FAM63B gene (for example, with son
The relevant multiple genes of palace myomata) expression.The protein-chip can be used for detecting including FAM63B albumen
The expression of multiple protein (such as multiple protein relevant to fibroid).By by multiple with fibroid mark
Will object detects simultaneously, is greatly improved the accuracy rate of fibroid diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting FAM63B gene transcription level；The protein immunization detection kit includes FAM63B albumen
Specific antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or core
Piece method detects reagent needed for FAM63B gene expression dose process.Preference, the reagent include being directed to FAM63B base
The primer and/or probe of cause.Being easy to design according to the nucleotide sequence information of FAM63B gene can be used for detecting FAM63B
The primer and probe of gene expression dose.
The test paper includes the reagent for detecting FAM63B gene expression.
The high-flux sequence platform includes the reagent for detecting FAM63B gene expression.
It can be DNA, RNA, DNA-RNA chimera, PNA or other with the probe of the nucleic acid array hybridizing of FAM63B gene
Derivative.There is no limit for the length of the probe, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over to have different influences, the length of the probe is typically at least 14 base-pairs, and longest does not surpass generally
30 base-pairs are crossed, the length complementary with purpose nucleotide sequence is best with 15-25 base-pair.The probe self-complementary sequence
Column are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the FAM63B albumen includes monoclonal antibody, polyclonal antibody.The FAM63B
The specific antibody of albumen include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with FAM63B albumen.For protein water
When the preparation of flat antibody well known to a person skilled in the art, and the present invention may use any method it is described anti-to prepare
Body.
In specific embodiments of the present invention, the primer of the detection FAM63B gene mRNA includes SEQ ID NO.3
With primer pair shown in SEQ ID NO.4.
The present invention also provides the promotors of FAM63B gene and/or its expression product in the medicine for preparing treatment fibroid
Application in object.
The promotor includes the reagent for promoting FAM63B gene expression and the reagent for promoting FAM63B gene expression product；
The reagent for promoting FAM63B gene expression includes the reagent for promoting genetic transcription, the reagent for promoting gene translation, promotes
The reagent of FAM63B protein content；The reagent for promoting FAM63B gene expression product includes that FAM63B gene expression is promoted to produce
The reagent of object stability promotes the active reagent of FAM63B gene expression product, promotes FAM63B gene expression product function
Reagent.
Specifically, the reagent for promoting FAM63B gene expression includes: the reagent containing FAM63B gene, carries
The carrier or host cell of FAM63B gene are formed by reagent, the reagent containing FAM63B protein.
On the one hand promotor of the invention can be used for supplementing the missing or deficiency of endogenic FAM63B albumen, by mentioning
The expression of high FAM63B albumen, thus fibroid caused by treating because of FAM63B hypoproteinosis.On the other hand can be used for promoting
Into the activity or function of FAM63B albumen, to treat fibroid.
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.
The present invention also provides a kind of for treating the pharmaceutical composition of fibroid, and described pharmaceutical composition includes above
The promotor of the FAM63B gene and/or its expression product.
Further, drug of the invention further includes pharmaceutically acceptable carrier, carrier, and this kind of carrier includes (but not
It is limited to): diluent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, bright
Glue and polyvinylpyrrolidone；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
With magnesium, polyethylene 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 FAM63B gene or the drug containing FAM63B protein are imported in cell, then by cell transplantation or
It feeds back in vivo.Internal mode includes directly infusing the drug containing FAM63B gene or the drug containing FAM63B protein
Enter in in-vivo tissue.
Drug of the invention can also be with the drug combination of other treatment fibroid, and a variety of Drug combinations can be significantly
Mention the success rate for the treatment of.
In the context of the present invention, " FAM63B gene " includes any function of FAM63B gene and FAM63B gene
The polynucleotides of equivalent.FAM63B gene includes and FAM63B base in the public GenBank GeneBank in the current world
Because (NC_000015.10) DNA sequence dna has 70% or more homology, and encode the DNA sequence dna of identical function protein；
Preferably, the coded sequence of FAM63B gene includes following any DNA molecular:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) DNA sequence dna that limits hybridizes and encodes 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 FAM63B gene is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, FAM63B gene expression product includes the portion of FAM63B albumen and FAM63B albumen
Divide peptide.The partial peptide of the FAM63B albumen contains functional domain relevant to fibroid.
" FAM63B albumen " includes any functional equivalent of FAM63B albumen and FAM63B albumen.The function is equivalent
Object includes FAM63B albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural
Mutant, induced mutants, can be with the encoded protein of DNA of the DNA hybridization of FAM63B under high or low stringent condition.
Preferably, FAM63B 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,
It is highly preferred that the homology with amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%,
98%, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the FAM63B 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 FAM63B albumen
Hop protein.For the peptide or protein with FAM63B protein fusion, there is no limit as long as resulting fusion protein retains
The biological activity of FAM63B albumen.
FAM63B 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 FAM63B 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 fibroid " both includes judging whether subject has suffered from uterus muscle
Tumor also includes the risk that judges subject and whether there is with fibroid.
In the context of the present invention, " treatment fibroid " divides from the state change of disease, may include disease
Alleviate, the complete healing of disease.
The advantages of the present invention:
Present invention firstly discovers that FAM63B gene expression is related to fibroid, by detection subject uterine tissue
The expression of FAM63B, it can be determined that whether subject suffers from fibroid or judge that subject whether there is with uterus muscle
The risk of tumor, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-FAM63B genes, compared to traditional detection means, gene diagnosis
More in time, more special, sensitiveer, it can be realized the early diagnosis of fibroid.
Detailed description of the invention
Fig. 1 shows the expression using QPCR detection FAM63B gene in uterine tissue；
Fig. 2 shows the expression using Western blot detection FAM63B albumen in uterine tissue；
Fig. 3 shows the overexpression situation for detecting FAM63B gene on transcriptional level using QPCR；
Fig. 4 shows the overexpression situation using Western blot detection FAM63B albumen；
Fig. 5 shows influence of the FAM63B gene expression to proliferation of human uterine leiomyoma cells.
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.
The differential expression of FAM63B gene in the normal muscle layer tissue of embodiment 1 and Human Leiomyma
1, experimental material:
The sterile Human Leiomyma taken because of fibroid row panhysterectomy patient and neighbouring normal muscle layer tissue suffer from
Person did not received hormone therapy in the preoperative in 3 months, postoperative is fibroid through definitive pathological diagnosis, this experimental material is derived from 40
Patients with uterine myoma, patient age is between 25-45 years old.
2, the differential expression of FAM63B gene is detected on transcriptional level
The extraction of 2.1 tissue RNA
Using Trizol one-step method extract tissue in total serum IgE, by Nanodrop ND-1000 read 260nm and
The purity of absorbance value (A) measurement RNA solution at 280nm.Through 1% denaturing formaldehyde agarose gel electrophoresis, ultraviolet transmission light
Lower observation, detects the integrality of RNA.
2.2 reverse transcription
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgE with RT Buffer.Using 25 μ l reaction systems, each sample
It takes 1 μ g total serum IgE as template ribonucleic acid, following components is separately added into PCR pipe: DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
2.3QPCR
Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample.Prepare following reaction system: SYBR Green is poly-
12.5 μ l of polymerase chain reaction system, 1 μ l of forward primer (5 μM), reverse primer (5 μM) 2.0 μ l of 1 μ l, template cDNA, no enzyme
8.5 μ l of water；The forward primer sequence for expanding FAM63B gene is 5 '-TCTTATGGCATTATCTCTA-3 ' (SEQ ID NO.3),
Reverse primer sequences are 5 '-TGTTCCTGATAGTATTGA-3 ' (SEQ ID NO.4)；Expand the forward primer sequence of GAPDH gene
It is classified as 5 '-AAAGGGTCATCATCTCTG-3 ' (SEQ ID NO.5), reverse primer sequences 5 '-
GCTGTTGTCATACTTCTC-3 ' (SEQ ID NO.6), operations are carried out on ice.Amplification program are as follows: 95 DEG C of 5min,
(95 DEG C of 10s, 60 DEG C of 60s) * 45 circulations.It is real-time in Light Cycler fluorescence using SYBR Green as fluorescent marker
PCR reaction is carried out on quantitative PCR apparatus, purpose band is determined by melt curve analysis analysis and electrophoresis, it is relatively fixed that Δ Δ CT method carries out
Amount.
2.4 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, the difference between different groups is examined using t, it is believed that as P < 0.05
With statistical significance.
2.5 result
As a result as shown in Figure 1, compared with normal muscle layer tissue, the mRNA level in-site of FAM63B gene is aobvious in Human Leiomyma
Writing reduces, and difference has statistical significance (P < 0.05).
3, the differential expression of FAM63B gene is detected on protein level
3.1 step
Extraction, the quantitative and separation of protein are carried out referring to " Molecular Cloning:A Laboratory guide ".After transferring film successively with 1% degreasing
The secondary antibody incubation of milk powder, rabbit-anti people FAM63B polyclonal antibody, peroxidase conjugate, with ECL Western blot kit
Colour developing.
3.2 statistical procedures
The gray value of protein band is analyzed using Image J software, using β-actin as internal reference, by purpose informal voucher
The gray value of band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical software is next for statistical analysis, and difference between the two is examined using t, it is believed that has system as P < 0.05
Meter learns meaning.
3.3 result
As a result as shown in Fig. 2, compared with normal muscle layer tissue, FAM63B protein content is reduced in Human Leiomyma, poor
It is different that there is statistical significance (P < 0.05).
The building of 2 FAM63B gene expression plasmid of embodiment
1, the building of FAM63B expression vector
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.1) of FAM63B gene.From at Human fetal spleen
CDNA library (clontech company, article No.: 638831) coded sequence of the FAM63B gene of amplification overall length in, by above-mentioned cDNA
Sequence is inserted into eukaryotic expression vector pcDNA3.1, connects the recombinant vector pcDNA3.1-FAM63B of acquisition for subsequent
Experiment.
2, the culture and transfection of Leiomyoma Cell
After operation excision fibroid, Human Leiomyma 1cm is aseptically taken immediately, it is dual anti-to be placed in 10mL 3%
In the PBS of (penicillin and streptomycin), closed ice bath is sent into cell culture chamber as early as possible.Human Leiomyma is floated with 3% dual anti-PBS liquid
It washes, is trimmed to 1mm in the plate containing DMEM culture solution；The tissue block of size.The tissue fritter elbow straw that will be shredded
It is sent into culture bottle, every fritter spacing 2.0-3.0mm or so.After tissue block is set, culture solution is sucked out, gently turns over culture bottle
Turn, allow bottom of bottle upward, injection contains the DMEM culture solution 2mL of 15% fetal calf serum into bottle, covers bottle cap, and culture bottle inversion is put
In 37 DEG C, 5%CO2In incubator.After 4h, culture bottle is slowly overturn and is laid flat, stationary culture.Every 2-3d is changed liquid 1 time.To cell
Up to when 80% fusion, passed on 0.25% trypsin digestion.
Take 3-4 for Leiomyoma Cell by 1 × 104/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO2Training
Supporting cell culture in case, for 24 hours, transfection turns according to the specification of lipofectamine 2000 (being purchased from Invitrogen company)
Dye, experiment are divided into control group (transfection pcDNA3.1) and experimental group (transfection pcDNA3.1-FAM63B), and the work of transfected plasmids is dense
Degree is 0.5 μ g/ml.
3, the effect of detection plasmid transfection is tested using QPCR.
3.1 extraction cell total rnas are operated using conventional method.
3.2 reverse transcription
Step is the same as embodiment 1.
3.3QPCR
Step is the same as embodiment 1.
3.4 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, the difference between two groups is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
4, Western is detected
The specific steps are the same as those in embodiment 1.
5, result
As shown in figure 3, being transfected in the cell of pcDNA3.1-FAM63B compared with the cell of transfection pcDNA3.1 empty carrier
The mRNA level in-site of FAM63B significantly raises, and difference has statistical significance (P < 0.05)；As shown in figure 4, with transfection pcDNA3.1
The cell of empty carrier is compared, and the protein level for transfecting FAM63B in the cell of pcDNA3.1-FAM63B significantly raises, and difference has
Statistical significance (P < 0.05).
The influence of 3 FAM63B gene pairs proliferation of human uterine leiomyoma cells of embodiment
1, according to the culture and transfection for carrying out cell the step of embodiment 2
2,3H-TdR (1 hole μ Ci/) is added after transfecting 24 hours, is further cultured for 24 hours, collects cell, adds liquid scintillation solution,
β calculating instrument detects cpm value.
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, the difference between FAM63B gene expression panel and control group uses t
It examines, it is believed that there is statistical significance as P < 0.05.
4, result
It is shown in fig. 5 as the result is shown: transfection pcDNA3.1-FAM63B group vitro growth rates significantly lower than transfects
There are statistical significance (P < 0.05) the above results to show FAM63B base for the vitro growth rates of pcDNA3.1 empty carrier group, difference
Because high expression is able to suppress the growth of Leiomyoma Cell.
The influence of 4 FAM63B gene pairs Leiomyoma Cell apoptosis of embodiment
1, cell culture step is the same as embodiment 2.
2, cell transfecting uterus myomata cell suspension 3.0 × 104/ ml is inoculated in the 6 orifice plates of preset coverslip, later according to
The step of embodiment 2, is transfected.
3, TUNEL method in situ detection Apoptosis: after transfection 48h, 4% paraformaldehyde of coverslip Fresh is taken out
It is fixed, it is operated by TUNEL kit (being purchased from Wuhan Boster Biological Technology Co., Ltd.) specification, BLIP/NBT is aobvious
Color, core fast red are redyed, glycerol mounting.It replaces TUNEL dyeing liquor to compare group with PBS, counts 300 cells under high power lens.
TUNEL apoptotic index calculation formula are as follows: positive cell number/total cell number.
4, 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.
5, result:
The apoptotic index for transfecting pcDNA3.1 is 0.047 ± 0.009, and the cell for transfecting pcDNA3.1-FAM63B withers
Dying index is 0.142 ± 0.005, and difference has statistical significance (P < 0.05), and the above results show the high table of FAM63B gene
Up to the apoptosis that can promote Leiomyoma 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 (7)
1. detecting application of the product of FAM63B gene expression in the tool of preparation diagnosis fibroid, which is characterized in that institute
The coded sequence for stating FAM63B gene is DNA sequence shown in SEQ ID NO.1.
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, chip or high-flux sequence detection of platform FAM63B gene expression are to diagnose the product of fibroid.
3. application according to claim 2, which is characterized in that the product with RT-PCR diagnosis fibroid at least wraps
Include the primer of a pair of of specific amplified FAM63B gene；The product with real-time quantitative PCR diagnosis fibroid includes at least one
To the primer of specific amplified FAM63B gene；It is described with immune detection diagnosis fibroid product include: and FAM63B albumen
The antibody of specific binding；The product in situ hybridization diagnosis fibroid includes: the nucleic acid sequence with FAM63B gene
The probe of hybridization；The product with chip diagnosis fibroid includes: protein chip and genetic chip；Wherein, protein chip
Including the antibody in conjunction with FAM63B protein-specific, genetic chip includes the spy with the nucleic acid array hybridizing of FAM63B gene
Needle.
4. application according to claim 3, which is characterized in that the product with real-time quantitative PCR diagnosis fibroid
Including at least the primer of a pair of of specific amplified FAM63B gene, sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.
Application of the promotor of 5.FAM63B gene and/or its expression product in the drug of preparation treatment fibroid, it is special
Sign is that the coded sequence of the FAM63B gene is DNA sequence shown in SEQ ID NO.1.
6. application according to claim 5, which is characterized in that the promotor includes the examination for promoting FAM63B gene expression
Agent, the reagent for promoting FAM63B gene expression product stability promote the active reagent of FAM63B gene expression product, promotion
The reagent of FAM63B gene expression product function.
7. application according to claim 6, which is characterized in that the reagent for promoting FAM63B gene expression includes containing
The reagent of FAM63B gene, the carrier for carrying FAM63B gene or host cell are formed by reagent, contain FAM63B protein
Reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610112986.0A CN105671158B (en) | 2016-02-29 | 2016-02-29 | Purposes of the FAM63B gene in the product of preparation diagnosis and treatment fibroid |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610112986.0A CN105671158B (en) | 2016-02-29 | 2016-02-29 | Purposes of the FAM63B gene in the product of preparation diagnosis and treatment fibroid |
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| CN101273144A (en) * | 2005-07-27 | 2008-09-24 | 肿瘤疗法科学股份有限公司 | Method of diagnosing esophageal cancer |
| CN101541977A (en) * | 2006-09-19 | 2009-09-23 | 诺瓦提斯公司 | Biomarkers of target modulation, efficacy, diagnosis and/or prognosis for RAF inhibitors |
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| CN105671158A (en) | 2016-06-15 |
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