CN105200137B - The diagnosis and treatment target of CERS2 gene and its expression product as osteoporosis - Google Patents
The diagnosis and treatment target of CERS2 gene and its expression product as osteoporosis Download PDFInfo
- Publication number
- CN105200137B CN105200137B CN201510629348.1A CN201510629348A CN105200137B CN 105200137 B CN105200137 B CN 105200137B CN 201510629348 A CN201510629348 A CN 201510629348A CN 105200137 B CN105200137 B CN 105200137B
- Authority
- CN
- China
- Prior art keywords
- cers2
- gene
- osteoporosis
- product
- diagnosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
-
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/108—Osteoporosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the molecular markers that CERS2 gene and its expression product can be used as osteoporosis diagnosis and treatment.May determine that whether subject suffers from osteoporosis or diagnose subject by the content of CERS2 gene and its expression product in detection bone tissue whether there is the risk with osteoporosis.The present invention has found that the expression inhibiting of CERS2 gene can promote osteoblastic proliferation by the proliferative conditions of the osteoblast of research in vitro culture, and by inhibiting the function of CERS2 albumen that can equally promote the proliferation of osteoblast, the studies above is the result shows that CERS2 gene and its expression product are a potential drug targets for treating osteoporosis.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to CERS2 gene in the diagnosis, treatment of osteoporosis
Purposes.
Background technique
Osteoporosis (osteoporosis) is a kind of systemic osteopathy, it is characterized in that the fine knot of bone amount decline and bone
Structure destroys, and the brittleness for showing as bone increases, thus the risk fractured greatly increases, even slight wound or atraumatic
In the case of be also easy to happen fracture.Vertebral compression fracture is unconsciously usually occurring, it can also be because of cough, sneezing, slight
Wound etc. induces vertebral fracture.Osteoporosis is a kind of multifactor caused chronic disease.Before fracture occurs, usual nothing
Special Clinical Manifestation.Disease women is more than male, but each period at age can fall ill, and be common in postmenopausal women and the elderly.
With the increase of China's elderly population, developing osteoporosis rate is in ascendant trend, is all a value in China or even the whole world
The health problem that must be paid close attention to.
The harmfulness of osteoporosis essentially consists in the fracture caused by it, can betide any position of whole body, be most commonly in
Lumbar vertebrae, hip region and radius.The discovery of investigation in 1999,60 years old Chinese or more osteoporosis incidence, lumbar vertebrae 2~4：Men and women
Difference 11% and 21%；Neck of femur：Respectively 11% and 27%.There are osteoporosis for the 1/3~1/2 of postmenopausal women.?
The U.S., the disease about make the raw fracture of 1,500,000 human hairs every year, and medical expense related with this is more than 10,000,000,000 dollars.Over 65 years old woman
1/3 will occur vertebral fracture in female, and when the age is bigger, Hip Fracture will occur for 1/3 women and 1/6 male, wherein 20%
Die of various complication caused by fracture, the residential care for separately there are 30% needs long-term.It is dredged although China still lacks sclerotin at present
The definite incidence data of Song Bingyou fracture, but since elderly population are numerous, estimate considerable.Osteoporosis is Chinese same
Sample is not only a medical care problem and a serious public health and social concern.
The detection of osteoporosis includes the detection and auxiliary detection of laboratory checking index.
Laboratory checking index：
Patients with osteoporosis part serum studentization index can convert (including bon e formation and bone resorption) shape with reactive bone
State, under the high transition status (such as I type osteoporosis) of bone, these indexs can be increased, it can also be used to monitor treatment
Early reaction.But its clinical meaning in osteoporosis is still up for further studying.These Biochemistry measurement indexs include：
The special alkaline phosphatase of bone (Bone-specific alkaline phosphatase reacts bon e formation), anti-tartaric acid acid
Acid phosphatase (tartrated resistant acid phosphatse, react to bone resorption), osteocalcin (Osteocalcin,
React bon e formation), I type virgin rubber former peptide (Type I procollagenpeptidase, react to bon e formation), Pyridinoline
(Urinary pyridinoline) and Deoxypyridinoline (Urinary deoxypyridinoline reacts bone resorption), I type
The crosslinking of the end N-C- peptide (cross-linked N-and C-telopeptide of type I collagen, the reaction of collagen
Bone resorption).As previously noted, the accuracy using biochemical indicator detection osteoporosis is inadequate.
Auxiliary examination：Including bone imageological examination and Bone mineral density.The object of auxiliary examination is typically all osteoporosis
The patients with terminal of disease, it is impossible to be used in the screening of early stage patients with osteoporosis.
Based on the limitation for the means for detecting osteoporosis in the prior art, find it is a kind of effectively can be in early days i.e.
The method that diagnosable osteoporosis occurs is a problem to be solved.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide one kind can be used for osteoporosis
The molecular marker of (Osterarthritis, OA) early diagnosis.Compared to the diagnostic method of traditional osteoporosis, base is used
Because marker come Diagnosis of osteoporosis have timeliness, specificity and sensitivity, thus make patient disease early stage energy
Know disease risks, for risk height, takes corresponding prevention and treatment measure.
To achieve the goals above, the present invention adopts the following technical scheme that：
The present invention provides product the answering in the tool for preparing Diagnosis of osteoporosis of detection CERS2 gene expression
With.
Further, it is described detection CERS2 gene expression product include detect CERS2 gene mRNA levels product and/
Or the product of detection CERS2 protein level.
Further, the product of the detection CERS2 gene expression includes：Pass through RT-PCR, real-time quantitative PCR, immune inspection
It surveys, in situ hybridization or chip detection CERS2 gene expression are with the product of Diagnosis of osteoporosis.
Further, the product with RT-PCR Diagnosis of osteoporosis includes at least a pair of of specific amplified CERS2 gene
Primer；The product with real-time quantitative PCR Diagnosis of osteoporosis includes at least drawing for a pair of of specific amplified CERS2 gene
Object；The product with immune detection Diagnosis of osteoporosis includes：Antibody in conjunction with CERS2 protein-specific；The use
The product of in situ hybridization Diagnosis of osteoporosis includes：With the probe of the nucleic acid array hybridizing of CERS2 gene；It is described to be examined with chip
The product of disconnected osteoporosis includes：Protein chip and genetic chip；Wherein, protein chip includes and CERS2 protein-specific
In conjunction with antibody, genetic chip includes the probe with the nucleic acid array hybridizing of CERS2 gene.
A pair of of specific amplified CERS2 gene that the product with real-time quantitative PCR Diagnosis of osteoporosis includes at least
Primer as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of the detection CERS2 gene expression, which can be the reagent of detection CERS2 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 osteoporosis 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 osteoporosis, 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, CERS2 gene is known in high-flux sequence
The abnormal purposes for also belonging to CERS2 gene related to osteoporosis, equally within protection scope of the present invention.
The present invention also provides a kind of tool of Diagnosis of osteoporosis, the tool includes detection CERS2 gene expression
Reagent；The reagent includes the primer and/or probe, the antibody for detecting CERS2 albumen for detecting CERS2 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 CERS2 gene transcription level
The oligonucleotide probe of CERS2 gene；The protein-chip includes solid phase carrier and the CERS2 egg for being fixed on solid phase carrier
White specific antibody；The genetic chip can be used for detecting multiple genes including CERS2 gene (for example, and sclerotin
The relevant multiple genes of osteoporosis) expression.The protein-chip can be used for detecting more including CERS2 albumen
The expression of a protein (such as multiple protein relevant to osteoporosis).By will be multiple with osteoporosis
Marker detects simultaneously, is greatly improved the accuracy rate of diagnosis of osteoporosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting CERS2 gene transcription level；The protein immunization detection kit includes CERS2 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 CERS2 gene expression dose process.Preference, the reagent include for CERS2 gene
Primer and/or probe.It is easy to design according to the nucleotide sequence information of CERS2 gene and can be used for detecting CERS2 gene table
Up to horizontal primer and probe.
The test paper includes the reagent for detecting CERS2 gene expression.
The high-flux sequence platform includes the reagent for detecting CERS2 gene expression.
It can be DNA, RNA, DNA-RNA chimera, PNA or other with the probe of the nucleic acid array hybridizing of CERS2 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 CERS2 albumen includes monoclonal antibody, polyclonal antibody.The CERS2 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 CERS2 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 CERS2 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 CERS2 gene and/or its expression product in preparation treatment osteoporosis
Application in drug.The inhibitor includes the reagent, and/or inhibition CERS2 gene expression product for inhibiting CERS2 gene expression
Reagent.
Further, the reagent for inhibiting CERS2 gene expression includes the reagent of suppressor transcription, suppressor translation
Reagent；The reagent for inhibiting CERS2 gene expression product includes the reagent for inhibiting CERS2 gene mRNA, inhibits CERS2 egg
White reagent.The reagent for inhibiting CERS2 gene mRNA includes the reagent for inhibiting mRNA stability, inhibits mRNA translation activity
Reagent.The reagent for inhibiting CERS2 albumen includes the reagent for inhibiting CERS2 protein stability, inhibits CERS2 protein active
Reagent, inhibit CERS2 protein function reagent.
Further, the reagent for inhibiting CERS2 gene mRNA includes the double stranded RNA for being directed to CERS2 gene mRNA；Suppression
The reagent of CERS2 protein function processed includes the tumor vaccine of CERS2 antigen protein, the antibody for inhibiting CERS2 protein function.It is described
Antibody can be polyclonal antibody or monoclonal antibody.
In specific embodiments of the present invention, the double stranded RNA for CERS2 gene mRNA is siRNA.
In order to ensure CERS2 gene can be rejected efficiently or silencing, it is special that siRNA is devised according to the mRNA sequence of CERS2 gene
Property segment.The design of siRNA is according to general design principle (Elbashir et.al 2001, the Schwarz et.al delivered
2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004, Ui-Tei et.al
2004), by online tool complete design, which is：siRNASelectionProgram of Whitehead
Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/siRNAext/) and
BLOCK-iTTM RNAi Designer ofINVITROGEN(winner of the 2004Frost&Sullivan
Excellence in Research Award, https://rnaidesigner.invitrogen.com/sirna/).In order to
It further increases the validity of siRNA segment, the siRNA piece of screening is designed for the advantages of comprehensive two Photographing On-line tools
It is disconnected.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve the specificity of siRNA segment and subtract
The undershooting-effect of few RNAi interference.
Preferably, the sequence of the siRNA is as shown in SEQ ID NO.7 and SEQ ID NO.8.
The present invention also provides a kind of for treating the pharmaceutical composition of osteoporosis, and described pharmaceutical composition includes upper
The inhibitor of CERS2 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 osteoporosis, a variety of Drug combinations
The success rate for the treatment of can be mentioned significantly.
In the context of the present invention, " CERS2 gene " includes any function etc. of CERS2 gene and CERS2 gene
The polynucleotides of jljl.CERS2 gene includes and CERS2 gene in the public GenBank GeneBank in the current world
(NC_000001.11) DNA sequence dna has 70% or more homology, and encodes the DNA sequence dna of identical function protein；
Preferably, the coded sequence of CERS2 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 CERS2 gene is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, CERS2 gene expression product includes the part of CERS2 albumen and CERS2 albumen
Peptide.The partial peptide of the CERS2 albumen contains functional domain relevant to osteoporosis.
" CERS2 albumen " includes any functional equivalent of CERS2 albumen and CERS2 albumen.The functional equivalent
Including CERS2 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural mutation
Body, induced mutants, can be with the encoded protein of DNA of the DNA hybridization of CERS2 under high or low stringent condition.
Preferably, CERS2 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 CERS2 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 CERS2 albumen
Albumen.For the peptide or protein with CERS2 protein fusion, there is no limit as long as resulting fusion protein retains CERS2 egg
White biological activity.
CERS2 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 CERS2 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 osteoporosis " had both included judging whether subject has dredged with sclerotin
Loose disease also includes the risk that judges subject and whether there is with osteoporosis.
In the context of the present invention, " treatment osteoporosis " divides from the state change of disease, may include disease
Alleviation, disease complete healing.
The advantages of the present invention：
Present invention firstly discovers that CERS2 gene expression is related to osteoporosis, by detection subject bone tissue
The expression of CERS2, it can be determined that whether subject suffers from osteoporosis or judge that subject whether there is is dredged with sclerotin
The risk of loose disease, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-CERS2 genes, compared to traditional detection means, gene diagnosis
More in time, more special, sensitiveer, it can be realized the early diagnosis of osteoporosis, to reduce the death rate of osteoporosis.
Detailed description of the invention
Fig. 1, which is shown, utilizes expression of the genechip detection CERS2 gene in bone tissue；
Fig. 2 shows the expression using Western blot detection CERS2 albumen in bone tissue；
Fig. 3 shows the jamming effectiveness using QPCR detection siRNA to CERS2 gene；
Fig. 4 shows the influence using Western blot detection siRNA to CERS2 protein expression；
Fig. 5 display inhibits influence of the CERS2 protein function to osteoblastic proliferation.
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 1 CERS2 gene of embodiment
1, research object：
Random selection will carry out the patient of marrow joint replacement, detect bone density, select patients with osteoporosis 50,
Normal bone density control group (for exogenous injury, detecting without osteoporosis) 50, the age 52-68 years old.Way of questionnaires investigation by
Situations such as examination person's life style and health status.
Patients with osteoporosis is included in standard：(1) meet diagnosis of osteoporosis standard person, reference《Chinese's sclerotin
Osteoporosis suggests diagnostic criteria (the second original text)；(2) the equal informed consent of patient.
The exclusion criteria of patients with osteoporosis：Secondary osteoporosis person.
2, the acquisition of bone tissue RNA
From the human femur head that marrow joint replacement is removed, hard bone 1g is taken, is immediately placed in liquid nitrogen and saves.In laboratory
The total serum IgE in people's bone tissue is extracted using Trizol one-step method, is read at 260nm and 280nm by Nanodrop ND-1000
Absorbance value (A) measurement RNA solution purity.Through 1% denaturing formaldehyde agarose gel electrophoresis, observed under ultraviolet transmission light,
Detect the integrality of RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit
Purifying, carries out fragmentation processing to the cRNAs marked with the RNA Fragmentation Reagents of Amhion.Using beauty
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company of state, 65 DEG C of hybridization 17h in chip hybridization furnace, then
Elution, dyeing, finally use Agilent DNA MicroarrayScanner scanner scanning.
4, chip data processing and analysis
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point
Natural logrithm absolute value be greater than 2.0 or the gene less than 0.5 as difference expression gene.
5, statistical procedures
Data analysis is carried out using 13.0 statistical software of SPSS, group difference compares using one-way analysis of variance method, P<
0.05 difference has significant.
6, result
(as shown in Figure 1) as the result is shown, is compared with normal people, CERS2 gene in patients with osteoporosis bone tissue
MRNA level in-site dramatically increases, and difference has statistical significance (P<0.05).
The differential expression of 2 CERS2 albumen of embodiment
1, research object is the same as embodiment 1.
1, the materials and culture of human osteoblast cell
Cancellous bone is taken from the human femur head that marrow joint replacement is removed, and rejects soft tissue, physiological saline repeated flushing bone
Matter after flushing liquor clarification, is swayed 3 times with the flushing of PBS liquid, is cut into 1mm3Fragment, separated using enzyme digestion and purify skeletonization
Cell.(the suitable DMEM-F12 (1 of culture bottle addition is inoculated in the culture bottle of 30ml：1) culture medium and 10% tire ox blood
Clearly), it is placed in the CO of 37 DEG C, 5%2It is cultivated in constant incubator.Liquid is changed after 2d and removes not adherent cell, and later every 3d changes liquid
It 1 time, observes under inverted microscope.After primary cell is fused into single layer, with pancreatin digestion, the passage of 2.5g/L.
2, the extraction of total protein of cell
The well-grown osteoblast of logarithmic phase is taken to carry out the extraction of total protein of cell, according to the full cell extraction of EpiQuik
The specification of kit carries out the operation of protein extraction.
3, Western blot is detected
The protein quantification of extraction is subjected to SDS-PAGE electrophoresis, carry out later transferring film, closing, primary antibody is incubated for, secondary antibody is incubated for,
Colour developing.
4, statistical procedures
The gray value of protein band is analyzed using Image J software, using β-actin as internal reference, by CERS2 albumen
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 works as P<There is system when 0.05
Meter learns meaning.
5, result
As a result as shown in Fig. 2, being compared with normal people, the expression of CERS2 albumen in patients with osteoporosis bone tissue
It dramatically increases, difference has statistical significance (P<0.05).
Embodiment 3 inhibits CERS2 gene expression
1, siRNA design synthesis
For the siRNA sequence of CERS2：
siRNA1-CERS2：
Positive-sense strand is 5 '-AACUUUCUUCAUGUCAUAGAA-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-CUAUGACAUGAAGAAAGUUUG-3 ' (SEQ ID NO.8),
siRNA2-CERS2：
Positive-sense strand is 5 '-UCAUGUAGUACCAAUACUGGG-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-CAGUAUUGGUACUACAUGAUU-3 ' (SEQ ID NO.10),
siRNA3-CERS2：
Positive-sense strand is 5 '-AAUCCUUUCGCUUGACAUCAG-3 ' (SEQ ID NO.11)；
Antisense strand is 5 '-GAUGUCAAGCGAAAGGAUUUC-3 ' (SEQ ID NO.12)
Negative control siRNA sequence (siRNA-NC)：
Positive-sense strand is 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO.13)；
Antisense strand is 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO.14).
The in vitro culture of osteoporosis osteoblast, the skeletonization of logarithmic growth phase are carried out according to the method for embodiment 2
Cell presses 1 × 104/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO2For 24 hours, transfection is pressed for cell culture in incubator
According to the specification transfection of lipofectamine 2000 (be purchased from Invitrogen company), experiment is divided into, negative control group and
Experimental group (20nM), wherein the sequence of negative control group siRNA and CERS2 gene is without homology, and concentration is the hole 20nM/, simultaneously
It transfects respectively.
2, the jamming effectiveness of detection siRNA is tested using QPCR.
2.1 extraction cell total rnas are operated using conventional method.
2.2 reverse transcription
The reverse transcription of RNA is carried out using the Reverse Transcriptase kit of TAKARA company.
2.3 QPCR
(1) design of primers
QPCR amplimer is designed according to the coded sequence of CERS2 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：
CERS2 gene：
Forward primer is 5 '-GATTACCTGCTGGAGTCA-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-AGACGATGAAGATGTTGTTG-3 ' (SEQ ID NO.4),
GAPDH gene：
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-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 12min, (95 DEG C of 15s, 60 DEG C of 50s) * 42 circulations.Using SYBR Green as
Fluorescent marker carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT method carries 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, the difference between CERS2 gene expression panel and control group is interfered to adopt
It is examined with t, it is believed that work as P<There is statistical significance when 0.05.
2.5 result
As a result as shown in figure 3, compared with siRNA2-CERS2, siRNA3-CERS2, siRNA1-CERS2 can be more effective
Inhibition CERS2 gene expression, difference have statistical significance (P<0.05), subsequent reality is carried out using siRNA1-CERS2
It tests.
3, the jamming effectiveness of Western blot experiment detection siRNA1-CERS2
Step is the same as embodiment 2.
As a result as shown in figure 4, transfecting CERS2 albumen in the cell of siRNA1-CERS2 compared with transfecting siRNA-NC group
Content be substantially reduced, difference have statistical significance (P<0.05).
Measurement of the expression of 4 CERS2 gene of embodiment to osteoblastic proliferation ability
It is used to detect the detection of osteoblastic proliferation using Cell Counting kit-8 (cck-8) kit
1, step
The culture and transfection of osteoblast are carried out according to the method for preceding embodiment, cell is divided into three experimental groups：
Group 1：The osteoblast transfection siRNA-NC (normal+siRNA-NC) in normal bone dense tissue source；
Group 2：The osteoblast of sufferers of osteoporosis face transfects siRNA-NC (osteoporosis+siRNA-NC)；
Group 3：The osteoblast of sufferers of osteoporosis face transfects siRNA-CERS2 (osteoporosis+siRNA-CERS2)
After transfection for 24 hours, with 2 × 105/ ml density is inoculated in 96 porocyte culture plates, and each experimental group designs three wells,
Every 100 μ l of hole, is placed in 37 DEG C, 5%CO2It is incubated in incubator, after cell is adherent for 24 hours, respectively in the culture hole of required detection
10 μ l CK-8 solution are added in interior every hole, continue to be incubated for 1h in cell incubator, measure each hole absorbance value (OD at 450nm
Value)
2, 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 works as P<Have when 0.05
It is statistically significant.
3, result
The results are shown in Table 2, and compared with normal bone density people, the osteoblastic proliferation of sufferers of osteoporosis face is slow, sclerotin
After loose patient interferes CERS2 gene expression, the proliferation of osteoblast is accelerated, and difference has statistical significance (P<0.05).On
State the experimental results showed that, the CERS2 gene expression inhibition proliferation of sufferers of osteoporosis face osteoblast uses and inhibits CERS2 base
Because the inhibited proliferation of osteoblast after the reagent of expression releases.
2 osteoblast OD value of table
| Experimental group | OD value (optical density) |
| Normally+siRNA-NC | 0.1792±0.005 |
| Osteoporosis+siRNA-NC | 0.0831±0.007 |
| Osteoporosis+siRNA-CERS2 | 0.1624±0.002 |
In 5 osteoblast antibody of embodiment and test
1, step：
Osteoblast is inoculated in 96 porocyte culture plates, every hole 2x103The hole cells//200 μ l, cell are adherent laggard
The following processing of row：
Experimental group 1：Unrelated monoclonal antibody (1 is added in the osteoblast in normal bone dense tissue source:30), as a control group；
Experimental group 2：Unrelated monoclonal antibody (1 is added in the osteoblast of sufferers of osteoporosis face:30) (osteoporosis+unrelated list
It is anti-)；
Experimental group 3：Anti-human CERS2 monoclonal antibody (1 is added in the osteoblast of sufferers of osteoporosis face:30) (osteoporosis+
CERS2 monoclonal antibody)
By cell in 37 DEG C, 5%CO2After incubator is incubated for 24 hours, it is added3H-TdR (1 hole μ Ci/), it is small to be further cultured for 24
When, cell is collected, liquid scintillation solution is added, β calculating instrument detects cpm value.
2, 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 works as P<Have when 0.05
It is statistically significant.
3, result
As a result as shown in figure 5, compared to control group, it is slack-off that (osteoporosis+unrelated monoclonal antibody) organizes cell Proliferation, and is added
The proliferation of (osteoporosis+CERS2 monoclonal antibody) group cell of CERS2 monoclonal antibody is restored.It is above-mentioned the experimental results showed that, inhibit
The function of CERS2 albumen can promote osteoblastic proliferation.
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 (9)
1. detecting application of the product of CERS2 gene expression in the tool for preparing Diagnosis of osteoporosis.
2. application according to claim 1, which is characterized in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform CERS2 gene expression are with the production of Diagnosis of osteoporosis
Product；The product with RT-PCR Diagnosis of osteoporosis includes at least the primer of a pair of of specific amplified CERS2 gene；The use
The product of real-time quantitative PCR Diagnosis of osteoporosis includes at least the primer of a pair of of specific amplified CERS2 gene；Described use is immunized
The product of checkout and diagnosis osteoporosis includes：Antibody in conjunction with CERS2 protein-specific；It is described to diagnose bone in situ hybridization
The product of matter osteoporosis includes：With the probe of the nucleic acid array hybridizing of CERS2 gene；It is described with chip Diagnosis of osteoporosis
Product includes：Protein chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with CERS2 protein-specific, gene
Chip includes the probe with the nucleic acid array hybridizing of CERS2 gene.
3. application according to claim 2, which is characterized in that the production with real-time quantitative PCR Diagnosis of osteoporosis
The primer for a pair of of specific amplified CERS2 gene that product include at least is as shown in SEQ ID NO.3 and SEQID NO.4.
4. application according to claim 1, which is characterized in that the tool includes the reagent for detecting CERS2 gene expression；
The reagent includes the primer and/or probe, the antibody for detecting CERS2 albumen for detecting CERS2 gene mRNA.
5. application according to claim 4, which is characterized in that the primer of the detection CERS2 gene mRNA includes SEQ
Primer pair shown in ID NO.3 and SEQ ID NO.4.
Application of the inhibitor of 6.CERS2 gene and/or its expression product in the drug of preparation treatment osteoporosis.
7. application according to claim 6, which is characterized in that the inhibitor includes the examination for inhibiting CERS2 gene expression
Agent, and/or the reagent for inhibiting CERS2 gene expression product.
8. application according to claim 7, which is characterized in that it is described inhibit CERS2 gene expression product reagent include
Inhibit the antibody of the siRNA, and/or CERS2 albumen for CERS2 gene.
9. application according to claim 8, which is characterized in that such as SEQ ID of the siRNA sequence for CERS2 gene
Shown in NO.7 and SEQ ID NO.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510629348.1A CN105200137B (en) | 2015-09-28 | 2015-09-28 | The diagnosis and treatment target of CERS2 gene and its expression product as osteoporosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510629348.1A CN105200137B (en) | 2015-09-28 | 2015-09-28 | The diagnosis and treatment target of CERS2 gene and its expression product as osteoporosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105200137A CN105200137A (en) | 2015-12-30 |
| CN105200137B true CN105200137B (en) | 2018-11-30 |
Family
ID=54948091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510629348.1A Active CN105200137B (en) | 2015-09-28 | 2015-09-28 | The diagnosis and treatment target of CERS2 gene and its expression product as osteoporosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105200137B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506171A (en) * | 2016-03-01 | 2016-04-20 | 北京泱深生物信息技术有限公司 | LYRM2 (LYR motif-containing 2) gene and new use of expression product thereof |
| CN108866184B (en) * | 2018-08-27 | 2019-07-12 | 北京泱深生物信息技术有限公司 | Application of the gene marker in women osteoporosis |
| CN109486935A (en) * | 2018-10-18 | 2019-03-19 | 中国医学科学院北京协和医院 | AKNAD1 gene as osteoporosis diagnosis and treatment target |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101999002A (en) * | 2008-02-04 | 2011-03-30 | 彼帕科学公司 | Methods of diagnosing and treating PARP-mediated diseases |
-
2015
- 2015-09-28 CN CN201510629348.1A patent/CN105200137B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101999002A (en) * | 2008-02-04 | 2011-03-30 | 彼帕科学公司 | Methods of diagnosing and treating PARP-mediated diseases |
Non-Patent Citations (3)
| Title |
|---|
| Differential expression of (dihydro)ceramide synthases in mouse brain: oligodendrocyte-speciWc expression of CerS2/Lass2;Ivonne Becker，et al;《Histochem Cell Biol》;20070928(第129期);233-241页 * |
| Disruption of ceramide synthesis by CerS2 down-regulation leads to autophagy and the unfolded protein response;Stefka D. SPASSIEVA，et al;《Biochem. J.》;20091231(第424期);273-283页 * |
| 骨质疏松症的诊断与鉴别诊断;李梅等;《中国临床医生》;20131231;第41卷(第6期);5-7页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105200137A (en) | 2015-12-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105132575B (en) | Molecular marker for osteoporosis and application thereof | |
| CN105200137B (en) | The diagnosis and treatment target of CERS2 gene and its expression product as osteoporosis | |
| CN105112552B (en) | Application of the IFT52 genes in diagnosis of osteoporosis | |
| CN109517898A (en) | A kind of detection of the cancer of the esophagus, diagnosis or prognostic evaluation preparation, treat the application of the drug and RND2 gene of the cancer of the esophagus | |
| CN105132550B (en) | Detect application of the reagent of MUC21 gene expressions in preparing osteoarthritis diagnosis and treatment product | |
| CN105296623B (en) | A kind of molecular marker of diagnosis and treatment osteoarthritis | |
| CN106947809B (en) | C6orf58 gene is preparing the application in Dendritic cell diagnosis and treatment product | |
| CN105886625B (en) | Application of the CHKA gene in preparation cancer of the esophagus diagnosis and treatment product | |
| CN105648076B (en) | The diagnosis and treatment target of NUDT11 gene and its expression product as fibroid | |
| CN105603085B (en) | The diagnosis and treatment target of intervertebral disk retrogression lesion | |
| CN105132574B (en) | Osteoarthritis diagnosis and treatment marker and its application | |
| CN105112550B (en) | MTUS1 genes as osteoporosis diagnosis and treatment target | |
| CN105400894B (en) | marker for diagnosis and treatment of intervertebral disc degenerative disease | |
| CN105838797B (en) | A kind of molecular marker of the diagnosis and treatment cancer of the esophagus | |
| CN105087821B (en) | A kind of molecular marker of diagnosis and treatment osteoporosis | |
| CN105154540B (en) | The diagnosis and treatment target of PAEP gene and its expression product as osteoarthritis | |
| CN105039577B (en) | Application of the CYP2F1 genes in osteoarthritis diagnosis and treatment | |
| CN105506169B (en) | Fibroid diagnosis and treatment marker | |
| CN105648103B (en) | Purposes of the VSIG10L gene as lung squamous cancer transfer diagnosis and treatment marker | |
| CN105861741B (en) | The diagnosis and treatment target of SENP3 gene and its expression product as the cancer of the esophagus | |
| CN105296622B (en) | The diagnosis and treatment target of LMNB2 gene and its expression product as nasopharyngeal carcinoma | |
| CN105755154A (en) | Molecular marker differentiating metastatic squamous cell lung carcinoma from non-metastatic squamous cell lung carcinoma | |
| CN105400895B (en) | Purposes of the SHISA4 gene as intervertebral disk retrogression lesion diagnosis and treatment marker | |
| CN105755155B (en) | KRT71 gene expression product is preparing the application in lung squamous cancer transfer diagnosis and treatment product | |
| CN105506170B (en) | Purposes of the SAV1 gene as fibroid diagnosis and treatment marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Dong Lin Inventor after: Li Shuguang Inventor after: Yang Chenggang Inventor before: Li Shuguang Inventor before: Yang Chenggang |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210401 Address after: 266000 room 2503, Qianshan building, D2, Qingdao International Innovation Park Phase II, No.1 Keyuan Weiyi Road, Laoshan District, Qingdao City, Shandong Province Patentee after: Qingdao Yangshen biomedical Co.,Ltd. Address before: 100080 room 3103, cube court building, 1 good luck street, Haidian District, Beijing. Patentee before: BEIJING MEDINTELL BIOMED INFORMATION TECHNOLOGY Co.,Ltd. |