CN105400888B - A kind of molecular marker of diagnosis and treatment intracranial aneurysm - Google Patents
A kind of molecular marker of diagnosis and treatment intracranial aneurysm Download PDFInfo
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Abstract
The invention discloses the molecular markers that DMBX1 gene can be used as diagnosis of intracranial aneurysms, the experiment proves that: it is compared with normal people, DMBX1 gene expression quantity in intracranial aneurysm patients is high.The invention also discloses the drug that DMBX1 gene can be used for preparing treatment intracranial aneurysm, cell apoptosis assay proves that DMBX1 gene overexpression can promote vascular smooth muscle cell apoptosis.Research achievement of the invention provides a kind of new intracranial aneurysm methods for clinical diagnosis, while providing a kind of drug new target drone for treating intracranial aneurysm.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to people DMBX1 gene in the diagnosis, treatment of intracranial aneurysm
Purposes.
Background technique
Intracranial aneurysm, which is called, does cerebral artery vessel tumor, refers to that the localized abnormality expansion of cerebral artery inner cavity causes arterial wall
A kind of warty it is prominent.On the basis of intracranial aneurysm mostly increases because of the birth defects and cavity pressure of cerebral artery tube wall part
Cause capsule bulging, is the first cause of disease for causing subarachnoid hemorrhage.Past people are referred to as congenital cerebral aneurysm, true
Upper congenital cerebral aneurysm accounts for the 70%~80% of cerebral aneurysm.Since the course of disease is hidden, onset is unexpected, once morbidity, dead, residual rate
It is high, thus " the not timing bomb " of referred to as encephalic, it is most dangerous one of cerebrovascular disease.It is only secondary in cerebrovascular accident
In cerebral thrombosis and hypertensive cerebral hemorrhage, third is occupied.Since the annual morbidity of the disease is up to 1~2/10000, China increases newly every year
Aneurysm patient up to 200,000, and intracranial aneurysm is to threaten the most common major disease of human life and health.
The specific mechanism about the formation of intracranial aneurysm and rupture is also imperfectly understood at present, thus aneurysm rupture it
Before there is no effective screening technique and satisfactory control measure.The pathogenesis of intracranial aneurysm is inquired into molecular level,
It will be helpful to the molecular marked compound it is found that aneurysmal early diagnosis, provide new molecular target for the prevention and treatment of intracranial aneurysm
Point.
Summary of the invention
In order to make up for the deficiencies of the prior art, can be used for intracranial aneurysm the purpose of the present invention is to provide one kind to examine in early days
Disconnected molecular marker.Compared to the diagnostic method of traditional intracranial aneurysm, carry out Diagnosis of intracranial aneurysms using gene marker
Have timeliness, specificity and sensitivity, thus make patient disease early stage can know disease risks, for risk height
It is low, take corresponding prevention and treatment measure.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides product the answering in the tool for preparing Diagnosis of intracranial aneurysms of detection DMBX1 gene expression
With.
Further, it is described detection DMBX1 gene expression product include detect DMBX1 gene mRNA levels product and/
Or the product of detection DMBX1 protein level.
Further, the product of the detection DMBX1 gene expression includes: by RT-PCR, real-time quantitative PCR, immune inspection
It surveys, in situ hybridization or chip detection DMBX1 gene expression are with the product of Diagnosis of intracranial aneurysms.
Further, the product with RT-PCR Diagnosis of intracranial aneurysms includes at least a pair of of specific amplified DMBX1 gene
Primer；The product with real-time quantitative PCR Diagnosis of intracranial aneurysms includes at least drawing for a pair of of specific amplified DMBX1 gene
Object；The product with immune detection Diagnosis of intracranial aneurysms includes: the antibody in conjunction with DMBX1 protein-specific；The use
The product of in situ hybridization Diagnosis of intracranial aneurysms includes: the probe with the nucleic acid array hybridizing of DMBX1 gene；It is described to be examined with chip
The product of disconnected intracranial aneurysm includes: protein chip and genetic chip；Wherein, protein chip includes and DMBX1 protein-specific
In conjunction with antibody, genetic chip includes the probe with the nucleic acid array hybridizing of DMBX1 gene.
A pair of of specific amplified DMBX1 gene that the product with real-time quantitative PCR Diagnosis of intracranial aneurysms includes at least
Primer as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of the detection DMBX1 gene expression, which can be the reagent of detection DMBX1 gene expression, be also possible to, includes
Kit, chip, test paper of the reagent etc. are also possible to the high-flux sequence platform using the reagent.
The tool of the Diagnosis of intracranial aneurysms includes but is not limited to that chip, kit, test paper or high-flux sequence are flat
Platform；High-flux sequence platform is a kind of tool of special Diagnosis of intracranial aneurysms, right with the development of high throughput sequencing technologies
The building of the gene expression profile of one people will become very easily work.By the gene for comparing Disease and normal population
Express spectra, the exception for being easy to analyze which gene are related to disease.Therefore, DMBX1 gene is known in high-flux sequence
The abnormal purposes for also belonging to DMBX1 gene related to intracranial aneurysm, equally within protection scope of the present invention.
The present invention also provides a kind of tool of Diagnosis of intracranial aneurysms, the tool includes detection DMBX1 gene expression
Reagent；The reagent includes the antibody for detecting the primer and/or probe, and/or detection DMBX1 albumen of DMBX1 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 DMBX1 gene transcription level
The oligonucleotide probe of DMBX1 gene；The protein-chip includes solid phase carrier and the DMBX1 egg for being fixed on solid phase carrier
White specific antibody；The genetic chip can be used for detecting multiple genes including DMBX1 gene (for example, and encephalic
The relevant multiple genes of aneurysm) expression.The protein-chip can be used for detecting more including DMBX1 albumen
The expression of a protein (such as multiple protein relevant to intracranial aneurysm).By will be multiple with intracranial aneurysm
Marker detects simultaneously, is greatly improved the accuracy rate of diagnosis of intracranial aneurysms.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting DMBX1 gene transcription level；The protein immunization detection kit includes DMBX1 albumen
Specific antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip
Method detects reagent needed for DMBX1 gene expression dose process.Preference, the reagent include for DMBX1 gene
Primer and/or probe.It is easy to design according to the nucleotide sequence information of DMBX1 gene and can be used for detecting DMBX1 gene table
Up to horizontal primer and probe.
The test paper includes the reagent for detecting DMBX1 gene expression.
The high-flux sequence platform includes the reagent for detecting DMBX1 gene expression.
It can be DNA, RNA, DNA-RNA chimera, PNA or other with the probe of the nucleic acid array hybridizing of DMBX1 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 DMBX1 albumen includes monoclonal antibody, polyclonal antibody.The DMBX1 egg
White specific antibody 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 DMBX1 albumen.For protein level
Antibody preparation when well known to a person skilled in the art and the present invention may use any method to prepare the antibody.
In specific embodiments of the present invention, it is described detection DMBX1 gene mRNA primer include SEQ ID NO.3 and
Primer pair shown in SEQ ID NO.4.
The present invention also provides the inhibitor of DMBX1 gene and/or its expression product in preparation treatment intracranial aneurysm
Application in drug.The inhibitor includes the reagent, and/or inhibition DMBX1 gene expression product for inhibiting DMBX1 gene expression
Reagent.
Further, the reagent for inhibiting DMBX1 gene expression includes the reagent of suppressor transcription, suppressor translation
Reagent；The reagent for inhibiting DMBX1 gene expression product includes the reagent for inhibiting DMBX1 gene mRNA, inhibits DMBX1 egg
White reagent.The reagent for inhibiting DMBX1 gene mRNA includes the reagent for inhibiting mRNA stability, inhibits mRNA translation activity
Reagent.The reagent for inhibiting DMBX1 albumen includes the reagent for inhibiting DMBX1 protein stability, inhibits DMBX1 protein active
Reagent, inhibit DMBX1 protein function reagent.
Further, the reagent for inhibiting DMBX1 gene mRNA includes the double stranded RNA for being directed to DMBX1 gene mRNA；Suppression
The reagent of DMBX1 protein function processed includes the tumor vaccine of DMBX1 antigen protein, the antibody for inhibiting DMBX1 protein function.It is described
Antibody can be polyclonal antibody or monoclonal antibody.
The double stranded RNA for DMBX1 gene mRNA can be siRNA.In order to ensure DMBX1 gene can
It is efficiently rejected or silencing, siRNA specific fragment is designed according to the mRNA sequence of DMBX1 gene.The design of siRNA is according to
General design principle (Elbashir et.al 2001, the Schwarz et.al 2003, Khvorova et.al delivered
2003, Reynolds et.al 2004, Hsieh et.al 2004, Ui-Tei et.al 2004), it is completed by online tool
Design, the online tool are as follows: siRNASelectionProgram of Whitehead Institute (BingbingYuan
Et.al 2004, http://jura.wi.mit.edu/bioc/siRNAext/) and BLOCK-iTTM RNAi
DesignerofINVITROGEN(winner of the 2004Frost&Sullivan Excellence in Research
Award, https: //rnaidesigner.invitrogen.com/sirna/).In order to further increase having for siRNA segment
The advantages of effect property, comprehensive two Photographing On-line tools, is designed for the siRNA segment of screening.Finally, passing through sequence analysis
(NCBI BLAST) filters siRNA sequence, to improve the specificity of siRNA segment and reduce the undershooting-effect of RNAi interference.
The present invention also provides a kind of for treating the pharmaceutical composition of intracranial aneurysm, and described pharmaceutical composition includes upper
The inhibitor of DMBX1 gene and/or its expression product described in face.
Pharmaceutical composition of the invention further includes pharmaceutically acceptable carrier, carrier, and this kind of carrier includes (but and unlimited
In): diluent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin
And polyvinylpyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as agar, calcium carbonate and sodium bicarbonate；Sorbefacient is quaternized
Close object；Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and
Magnesium, polyethylene glycol etc..
Pharmaceutical composition of the invention can also be with the drug combination of other treatment intracranial aneurysm, a variety of Drug combinations
The success rate for the treatment of can be mentioned significantly.
In the context of the present invention, " DMBX1 gene " includes any function of people DMBX1 gene and people's DMBX1 gene
The polynucleotides of energy equivalent.DMBX1 gene includes and DMBX1 base in the public GenBank GeneBank in the current world
Because (NC_000001.11) DNA sequence dna has 70% or more homology, and encode the DNA sequence dna of identical function protein；
Preferably, the coded sequence of DMBX1 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 DMBX1 gene is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, DMBX1 gene expression product includes people DMBX1 albumen and people's DMBX1 albumen
Partial peptide.The partial peptide of the DMBX1 albumen contains functional domain relevant to intracranial aneurysm.
" DMBX1 albumen " includes any functional equivalent of people DMBX1 albumen and people's DMBX1 albumen.Described function etc.
Jljl includes people DMBX1 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, day
Right mutant, induced mutants, can be with the encoded albumen of DNA of the DNA hybridization of people DMBX1 under high or low stringent condition
Matter.
Preferably, DMBX1 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, 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 DMBX1 albumen is with amino acid sequence shown in SEQ ID NO.2
The protein of column.
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 the fusion of DMBX1 albumen
Albumen.For the peptide or protein with DMBX1 protein fusion, there is no limit as long as resulting fusion protein retains DMBX1 egg
White biological activity.
DMBX1 albumen of the invention also includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, as long as
Protein by modification still is able to retain the biological activity of DMBX1 albumen.It is mutated in such modification protein
Amino acid number be usually 10 perhaps less such as 6 perhaps less such as 3 or less.
In the context of the present invention, " Diagnosis of intracranial aneurysms " had both included judging whether subject is dynamic with encephalic
Arteries and veins tumor also includes the risk that judges subject and whether there is with intracranial aneurysm.
In the context of the present invention, " treatment intracranial aneurysm " divides from the state change of disease, may include disease
Alleviation, disease complete healing.
The treatment that the present invention studies DMBX1 gene pairs intracranial aneurysm using the vascular smooth muscle cells of in vitro culture is made
With.Skilled person will appreciate that the important physiological characteristic that intracranial aneurysm occurs is vascular smooth muscle cells
Apoptosis aggravation, therefore the present invention is tested using the apoptosis of vascular smooth muscle cells and is controlled to study DMBX1 gene with intracranial aneurysm
The relationship for the treatment of.
The advantages of the present invention:
Present invention firstly discovers that DMBX1 gene expression is related to intracranial aneurysm, pass through the table of detection subject DMBX1
It reaches, it can be determined that whether subject suffers from intracranial aneurysm or judge that subject whether there is the wind with intracranial aneurysm
Danger, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-DMBX1 genes, compared to traditional detection means, gene diagnosis
More in time, more special, sensitiveer, it can be realized the early diagnosis of intracranial aneurysm, to reduce the death rate of intracranial aneurysm.
Detailed description of the invention
Fig. 1 shows the expression using high-flux sequence detection DMBX1 gene in intracranial aneurysm tissue；
Fig. 2 shows the expression using QPCR detection DMBX1 gene in intracranial aneurysm tissue；
Fig. 3 shows the overexpression situation using QPCR detection DMBX1 gene.
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 DMBX1 gene in 1 normal control tissue of embodiment and intracranial aneurysm tissue
1, sample collection
8 intracranial aneurysm patients are collected, operation folder cuts off aneurysm sample after closing tumor neck, removes adhesion organization and attached wall
Thrombus puts into liquid nitrogen save immediately.The superficial temporal artery of 5 patients with cerebral injury is taken, puts into liquid nitrogen and saves as normal control
Tissue.It is age 40-60 years old, preoperative without special treatment.Patient is obtained when sample collection and its family members agree to.
2, the extraction of total tissue RNA
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, room temperature preservation 5min；
2. chlorination imitates 0.2ml, with forced oscillation centrifuge tube, mixes well, place 5min-10min at room temperature；
3. drawing upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15min into another new centrifuge tube, pay attention to not
The protein substance being drawn between two layers of water phase.New pipe is moved into, the pre- cold isopropanol of isometric -20 DEG C is added, it is sufficiently reverse mixed
It is even, it is placed in 10min on ice；
4. 12000rpm high speed is added 75% from supernatant is carefully discarded after 15min, in the ratio of 1ml/ml Trizol
DEPC ethanol washing precipitating (4 DEG C of preservations), washing precipitate, oscillation mixes, 12000rpm high speed centrifugation 5min at 4 DEG C；
5. discarding ethanol liquid, 5min is placed at room temperature sufficiently to dry precipitating, it is heavy that the processed water dissolution of DEPC is added
It forms sediment；
6. measuring RNA purity and concentration with Nanodrop2000 ultraviolet specrophotometer, freeze in -70 DEG C.
3, the quality analysis (NanoDrop1000 spectrophotometer) of RNA sample
NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq sequencing, and: OD260/OD280 is
1.8-2.2。
4, the quality analysis (2100 Bioanalyzer of Agilent Technologies) of RNA sample
Agilent Technologies 2100Bioanalyzer detects RNA sample quality, observes 28S rRNA and 18S
RRNA master tape is obvious, cDNA library structure is sequenced without degradation, the RNA-seq that meets that RNA Perfection Index is qualified, concentration reaches requirement
The requirement built can be used for library construction and sequencing.
5, 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.
6, result
RNA-seq as the result is shown (as shown in Figure 1), compared with normal control tissue, DMBX1 base in intracranial aneurysm tissue
The mRNA level in-site of cause dramatically increases, and difference has statistical significance (P < 0.05).
2 large sample of embodiment verifies difference expression gene
1, sample collection
Collect 40 intracranial aneurysm patients, operation folder cuts off aneurysm sample after closing tumor neck, removes adhesion organization and attached
Wall thrombus puts into liquid nitrogen save immediately.A shallow artery of 30 patients with cerebral injury is taken, puts into liquid nitrogen and saves as normal right
According to tissue.It is age 40-60 years old, preoperative without special treatment.Patient is obtained when sample collection and its family members agree to.
2, the differential expression of DMBX1 gene is detected on transcriptional level
2.1 extract total tissue RNA
Step is the same as embodiment 1.
2.2 reverse transcription
The reverse transcription of RNA is carried out using the Reverse Transcriptase kit of TAKARA company.
2.3QPCR
(1) design of primers
QPCR amplimer is designed according to the coded sequence of DMBX1 gene and GAPDH gene in Genbank, is given birth to by Shanghai
The synthesis of work biotechnology Services Co., Ltd.Specific primer sequence is as follows:
DMBX1 gene:
Forward primer is 5 '-GCAGGTGTGGTTCAAGAA-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-TTCTGGAGCTGTTCCTTCT-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-AAAGGGTCATCATCTCTG-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GCTGTTGTCATACTTCTC-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
1 PCR reaction system of table
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 10s, 60 DEG C of 60s) * 45 circulations.Using SYBR Green as glimmering
Signal object carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, is determined by melt curve analysis analysis and electrophoresis
Purpose band, Δ Δ CT method carry out relative quantification.
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, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
2.5 result
As a result as shown in Fig. 2, compared with normal control tissue, DMBX1 gene expression amount is significant in intracranial aneurysm tissue
Increase, difference has statistical significance (P < 0.05).
Embodiment 3DMBX1 gene overexpression
1, the building of DMBX1 expression vector
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.1) of DMBX1 gene, primer sequence is as follows:
Forward primer is 5 '-ATGCAGCACTACGGGGTGAA-3 ' (SEQ ID NO.7), reverse primer 5 '-
TCAGTTGGGCAGCGTATCGA-3'(SEQ ID NO.8).From at Human fetal spleen cDNA library (clontech company, article No.:
638831) coded sequence of the DMBX1 gene of amplification overall length in, above-mentioned cDNA sequence is through restriction enzyme BamHI and XhoI
It is inserted into after double digestion in the eukaryotic expression vector pcDNA3.1 through restriction enzyme BamHI and XhoI double digestion, even
The recombinant vector pcDNA3.1-DMBX1 obtained is used for subsequent experimental.
2, cell culture
Vascular smooth muscle cells HA-VSMC is taken, is based on 37 DEG C, concentration with 1640 culture of height sugar of 10% calf serum of concentration
5%CO2Cell incubator in cultivate, every 2d is changed liquid 1 time, and 3-8 is taken to be tested for cell.
3, cell transfecting
Vascular smooth muscle cells are pressed 1.5 × 104/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO2Culture
Cell culture for 24 hours, in sugared 1640 culture mediums of height without double antibody, containing 10%FBS, is transfected according to lipofectamine in case
The specification transfection of 2000 (being purchased from Invitrogen company), experiment are divided into control group (transfection empty carrier pcDNA3.1) and mistake
Expression group (transfection pcDNA3.1-DMBX1), the working concentration of pcDNA3.1 empty carrier and pcDNA3.1-DMBX1 are 0.5 μ g/
ml。
4, detection DMBX1 gene overexpression situation is tested using QPCR
4.1 extract cell total rna
Cell total rna is extracted using the tissue/cell RNA extracts kit of QIAGEN company.
4.2 reverse transcriptions and QPCR
Step is the same as embodiment 2.
4.3 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 DMBX1 gene overexpression group and control group uses t
It examines, it is believed that there is statistical significance as P < 0.05.
4.4 result
As a result such as Fig. 3 is shown, compared with the control group, transfects DMBX1 gene expression liter in the cell of pcDNA3.1-DMBX1
Height, difference have statistical significance (P < 0.05).
The influence of 4 DMBX1 gene pairs vascular smooth muscle cell apoptosis of embodiment
1, cell culture step is the same as embodiment 3.
2, cell transfecting vascular smooth muscle cells suspension 3.0 × 104/ ml is inoculated in the 6 orifice plates of preset coverslip, presses later
It is transfected according to the step of embodiment 3.
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 0.023 ± 0.004, transfects the Apoptosis of pcDNA3.1-DMBX1
Index is 0.114 ± 0.003, and difference has statistical significance (P < 0.05), and the above results show the high expression of DMBX1 gene
It can promote the apoptosis of vascular smooth muscle cells.
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 (1)
1. detecting application of the product of DMBX1 gene expression in the tool for preparing Diagnosis of intracranial aneurysms, which is characterized in that institute
Stating product is to detect DMBX1 gene expression by real-time quantitative PCR with the product of Diagnosis of intracranial aneurysms；It is described by fixed in real time
The product for measuring PCR Diagnosis of intracranial aneurysms includes at least the primer of a pair of of specific amplified DMBX1 gene；It is described to pass through real-time quantitative
The primer such as SEQ ID NO.3 for a pair of of specific amplified DMBX1 gene that the product of PCR Diagnosis of intracranial aneurysms includes at least and
Shown in SEQ ID NO.4.
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| Title |
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| Functional analysis of transcriptional repressor Otx3/Dmbx1;Keita Kimura et al;《GEBS Letters》;20051231;第579卷;第2926-2932页 |
| 蛋白质组学技术筛选破裂颅内动脉瘤的血清标志物;石忠松等;《中华神经外科杂志》;20071231;第23卷(第11期);第850-855页 |
| 颅内动脉瘤相关糖蛋白标志物的蛋白质组学研究;许璟等;《2014浙江省神经外科学学术年会论文汇编》;20141231;第142-143页 |
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