CN105112550A - MTUSI gene serving as osterarthritis diagnosis target - Google Patents

MTUSI gene serving as osterarthritis diagnosis target Download PDF

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CN105112550A
CN105112550A CN201510628024.6A CN201510628024A CN105112550A CN 105112550 A CN105112550 A CN 105112550A CN 201510628024 A CN201510628024 A CN 201510628024A CN 105112550 A CN105112550 A CN 105112550A
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mtus1
gene
osteoporosis
product
diagnosis
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CN105112550B (en
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董东
杨承刚
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Qingdao Yangshen Biomedical Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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

Abstract

The invention discloses an osterarthritis diagnosis marker which refers to MTUSI gene and expression products thereof. According to gene chip and protein immunization tests, expressions of the MTUSI gene and expression products thereof are lowered in blood and bone tissue of osterarthritis patients as compared with those of the normal; thus, products for diagnosis of osterarthritis can be developed and can be applied to testing expression level of the MTUSI gene or expression products thereof. According to experiment in vitro, bone cell proliferation can be improved by over-expression of the MTUSI gene, and accordingly the MTUSI gene and the expression products thereof can be applied to development of medicines for treating osterarthritis.

Description

As the MTUS1 gene of osteoporosis diagnosis and treatment target
Technical field
The present invention relates to biological technical field, relate to the purposes of MTUS1 gene in the diagnosis, treatment of osteoporosis particularly.
Background technology
Osteoporosis and osteoporosis, be one group of osteopathy that many reasons causes, osseous tissue has normal calcification, and calcium salt and matrix are normal rates, and the metabolic osteopathy being reduced to feature with unit volume inner bone tissues amount becomes.In most osteoporosis, caused by the minimizing of osseous tissue increases mainly due to bone absorption.With skeleton pain, be easy to fracture for feature.
Epidemiologic data shows, and the U.S. about 8 million people suffers from osteoporosis, and 2,000 ten thousand people's bone amount reduce, and has 1,500,000 patients to fracture because osteoporosis causes every year.Post menopausal pink toes osteoporosis sickness rate is 17%, and Black women's sickness rate is 6%.Age is greater than in 50 years old crowd, and nearly 50% women and 25% male sex exist the potential risk of osteoporotic fracture.China is the country that elderly population absolute number quantity is maximum, " osteoporosis prevents and treats Chinese white paper " was according to national Chinese Han Population sample survey results display in 2006, in more than the 50 years old crowd in the estimation whole nation, about there is 6,944 ten thousand people (man 1,534 ten thousand, female 5,410 ten thousand) suffer from osteoporosis, there are 2.1 hundred million people to suffer from Low BMD, there is osteoporosis risk, expect the year two thousand twenty China osteoporosis and Low BMD patient will be increased to 2.8 hundred million.
Current diagnosis osteoporosis mainly relies on bone density iconography detection method, and bone densitometry method comprises following several: x-ray optical density method, single photon absorptiometry, two-photon absorption method, dual intensity x line absorption method, QCT, By Naa, using ultrasonic quantitative measurement, magnetic resonance measurement, quantitative MR imaging, high resolving power nuclear magnetic resonance.What the main limitation of aforesaid method was can not to occur in osteoporosis provides reliable judgement in early days.
In order to improve the treated effect of osteoporosis, reduce the economical load of patient and country, finding a kind of method for osteoporosis early diagnosis is problem demanding prompt solution.
Summary of the invention
In order to make up the deficiencies in the prior art, one is the object of the present invention is to provide to can be used for the molecular marker of osteoporosis (Osterarthritis, OA) early diagnosis.Compare the diagnostic method of traditional osteoporosis, what use gene marker to carry out Diagnosis of osteoporosis has promptness, specificity and susceptibility, thus make patient just can know disease risks in early days in disease, for risk height, take corresponding prevention and therapy measure.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides a kind of MTUS1 gene and the application of expression product in the product preparing Diagnosis of osteoporosis thereof.
Further, the diagnostic products mentioned above comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection MTUS1 gene and expression product thereof with the product of Diagnosis of osteoporosis.
Further, the product of described RT-PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified MTUS1 gene; The product of described real-time quantitative PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified MTUS1 gene; The product of described immunodetection Diagnosis of osteoporosis comprises: the antibody be combined with MTUS1 protein-specific; The product of described in situ hybridization Diagnosis of osteoporosis comprises: with the probe of the nucleic acid array hybridizing of MTUS1 gene; The product of described chip Diagnosis of osteoporosis comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with MTUS1 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of MTUS1 gene.
Preferably, described product comprises chip, test kit.
Present invention also offers the application of MTUS1 gene in high-flux sequence platform.Along with the development of high throughput sequencing technologies, will become the structure of the gene expression profile of a people and work very easily.By contrasting the gene expression profile of Disease and normal population, easily analyze exception and the disease-related of which gene.Therefore, in high-flux sequence, the exception of the MTUS1 gene purposes that also belong to MTUS1 gene relevant to osteoporosis is known, equally within protection scope of the present invention.
Present invention also offers MTUS1 gene and the application of expression product in the medicine of preparation treatment osteoporosis thereof.
Further, described medicine comprises: the medicine that the medicine containing MTUS1 gene, the carrier carrying MTUS1 gene or host cell are formed, MTUS1 pharmaceutical grade protein or other can promote the medicine of MTUS1 genetic expression.Medicine of the present invention may be used for disappearance or the deficiency of supplementary endogenic MTUS1 albumen, by improving the expression of MTUS1 albumen, thus the osteoporosis that treatment causes because of MTUS1 hypoproteinosis.
The carrier carrying gene of the present invention is various carrier known in the art, as commercially available carrier, comprises plasmid, clay, phage, virus etc.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of conventional prokaryotic host cell comprises intestinal bacteria, Bacillus subtilus etc.Conventional eukaryotic host cell comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
Further, medicine of the present invention also comprises pharmaceutically acceptable carrier, carrier, and this kind of carrier comprises (but being not limited to): thinner, vehicle are if water etc., weighting agent are as starch, sucrose etc.; Tackiness agent is as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone; Wetting agent is as glycerine; Disintegrating agent is as agar, calcium carbonate and sodium bicarbonate; Absorption enhancer quaternary ammonium compound; Tensio-active agent is as cetyl alcohol; Absorption carrier is as kaolin and soap clay; Lubricant is as talcum powder, calcium stearate and magnesium, polyoxyethylene glycol etc.
The mode of drugs delivery tissue of the present invention or cell can be divided into the mode in external or body.Vitro formats comprises by the medicine containing MTUS1 gene or containing in the drugs delivery cell of MTUS1 protein, then by Transplanted cells or feed back in body.In body, mode comprises directly by the medicine containing MTUS1 gene or containing in the infusion of medicine in-vivo tissue of MTUS1 protein.
Medicine of the present invention also can with the drug combination of other treatment osteoporosis, multi-medicament conbined usage can mention the success ratio for the treatment of greatly.
Present invention also offers a kind of product of Diagnosis of osteoporosis, described product can be chip, test kit or high-flux sequence platform.Described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for MTUS1 gene for detecting MTUS1 gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of MTUS1 albumen of solid phase carrier.
Further, described gene chip can be used for detecting the expression level of the multiple genes (such as, relevant to osteoporosis multiple genes) comprising MTUS1 gene.Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to osteoporosis multiple protein) comprising MTUS1 albumen.By being detected by multiple mark with osteoporosis simultaneously, the accuracy rate of diagnosis of osteoporosis greatly can be improved.
Test kit for Diagnosis of osteoporosis provided by the invention, described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting MTUS1 gene transcription level; Described protein immunization detection kit comprises the specific antibody of MTUS1 albumen.
Further, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection MTUS1 gene expression dose process.Preference, described reagent comprises primer for MTUS1 gene and/or probe.The primer and probe that may be used for detecting MTUS1 gene expression dose is easily designed according to the nucleotide sequence of MTUS1 gene.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of MTUS1 gene.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
Further, the specific antibody of described MTUS1 albumen comprises monoclonal antibody, polyclonal antibody.The specific antibody of described MTUS1 albumen comprise complete antibody molecule, any fragment of antibody or modification (such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with MTUS1 albumen.Well known to a person skilled in the art during preparation for the antibody of protein level, and the present invention can use any method to prepare described antibody.
Present invention also offers a kind of medicine being used for the treatment of osteoporosis, described medicine includes but not limited to: the medicine that the medicine containing MTUS1 gene, the carrier carrying MTUS1 gene or host cell are formed, MTUS1 pharmaceutical grade protein or other can promote the medicine of MTUS1 genetic expression.
In the context of the present invention, " MTUS1 gene " comprises the polynucleotide of any function equivalent of MTUS1 gene and MTUS1 gene.MTUS1 gene comprises and has more than 70% homology with MTUS1 gene (NC_000008.11) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence.
Preferably, the encoding sequence of MTUS1 gene comprises any DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1;
(2) under strict conditions with 1) DNA sequence dna that limits hybridizes and identical function protein DNA sequence of encoding;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described MTUS1 gene is the DNA sequence dna shown in SEQIDNO.1.
In the context of the present invention, MTUS1 gene expression product comprises the partial peptide of MTUS1 albumen and MTUS1 albumen.The partial peptide of described MTUS1 albumen contains the functional domain relevant to osteoporosis.
" MTUS1 albumen " comprises any function equivalent of MTUS1 albumen and MTUS1 albumen.Described function equivalent comprises MTUS1 albumen conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of MTUS1 under high or low stringent condition.
Preferably, MTUS1 albumen is the protein with following amino acid sequences:
(1) protein be made up of the aminoacid sequence shown in SEQ ID NO.2;
(2) aminoacid sequence shown in SEQIDNO.2 had the protein derivative by the aminoacid sequence shown in SEQIDNO.2 of identical function with the aminoacid sequence shown in SEQIDNO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), more preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.
In specific embodiment of the invention scheme, described MTUS1 albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Usually, it is known that in a protein one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with identity function.Intimate amino acid whose Conservative substitution tables is provided to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is MTUS1 albumen.Peptide or protein with MTUS1 protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of MTUS1 albumen.
MTUS1 albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying still can retain the biologic activity of MTUS1 albumen.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " Diagnosis of osteoporosis " had both comprised and had judged whether experimenter has suffered from osteoporosis, also comprised and judge whether experimenter exists the risk suffering from osteoporosis.
In the context of the present invention, " treatment osteoporosis " divides from the change of state of disease, can comprise the healing completely of the alleviation of disease, disease.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention MTUS1 genetic expression is relevant to osteoporosis, by detecting the expression of MTUS1 in experimenter's blood or osseous tissue, can judge whether experimenter suffers from osteoporosis or judge whether experimenter exists the risk suffering from osteoporosis, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-MTUS1 gene, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, the early diagnosis of osteoporosis can be realized, thus reduce the mortality ratio of osteoporosis.
Accompanying drawing explanation
Fig. 1 display utilizes genechip detection MTUS1 gene expression in blood;
Fig. 2 display utilizes the expression of genechip detection MTUS1 gene in osseous tissue;
Fig. 3 display utilizes Westernblot to detect the expression of MTUS1 albumen in osseous tissue;
Fig. 4 display utilizes QPCR to detect the change of mRNA level in-site after MTUS1 gene overexpression;
Fig. 5 display utilizes Westernblot to detect the change of protein level after MTUS1 process LAN.
Concrete embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Difference expression gene relevant to osteoporosis in embodiment 1 blood
1, research object:
Osteoporosis group: randomly draw the 50 routine Osteoporosis that hospital orthopedics is accepted for medical treatment, man, each 5 examples of female, minimum 50 years old of age, maximum 75 years old.Inclusive criteria: meet " Chinese's osteoporosis suggestion Case definition " (the second original text).Cause the various endocrinopathys of secondary osteoporosis without the obvious heart, liver, kidney, pulmonary insufficiency, nothing, get rid of other serious diseases such as tumour, diabetes interference bone metabolism person.
Normal group: choose age 50-75 year healthy volunteer 50 example, men and women each 5 example.
Between two groups, age, gender difference not statistically significant (P>0.10), have comparability.
All research objects are all known the inside story to this research and be endorsed Informed Consent Form.
2, the extraction of total serum IgE in blood
(1) fresh whole blood, erythrocyte cracked liquid (1:1), puts upside down mixing for several times, leaves standstill 5min.10000g，4℃，10min。The now liquid of visible leukocyte cell pellet and upper strata shiny red.
(2) TRIzol (10 is added 6-10 7cell adds 500 μ l), repeatedly aspirate, until there is a large amount of foam to produce (one of mark of lysis), normal temperature hatches 5min.
(3) add chloroform (chloroform: TRIzol=1:5), acutely mix 15s, room temperature leaves standstill 10min.
(4) centrifugal, 12,000g15min, 4 DEG C.
(5) careful Aspirate supernatant, transfers in new EP pipe, adds Virahol (Virahol: TRIzol=1:2), fully mixing (8-10 time), incubated at room 10min.
(6) centrifugal, 12,000g10min, 4 DEG C.
(7) there is gelatinous precipitate, abandoning supernatant at the bottom of visible pipe, add 75% ethanol (ethanol: TRIzol=1:1), gentle mixing, 7500g, 5min.
(8) abandon most supernatant liquor, tip upside down on filter paper and (be placed in glass dish) drying at room temperature 5min (not parching, when namely RNA occurs transparent slightly), add 60 μ lDEPC water dissolution precipitations.
3, the mass analysis (NanoDrop1000 spectrophotometer) of RNA sample
NanoDrop1000 spectrophotometer detects RNA sample, and OD260/OD280 is 1.8-2.2.
4, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP marks, and the cRNAs after fluorescent mark adopts RNEASYMiniKit purifying, carries out fragmentation process with the RNAFragmentationReagents of Amhion to the cRNAs marked.Adopt people's full genome chip of expression spectrum (4x44K gene) of Agilent company of the U.S., in chip hybridization stove, 65 DEG C of hybridization 17h, then wash-out, dyeing, finally use AgilentDNAMicroarrayScanner scanner scanning.
5, chip data treatment and analyses
Chip after hybridization after chip scanner reading of data point, by data importing analysis software, the natural logarithm absolute value for two groups of ratios be greater than 2.0 or be less than 0.5 gene as difference expression gene.
6, statistical procedures
Adopt SPSS13.0 statistical software to carry out data analysis, group difference compares employing one-way analysis of variance method, and P<0.05 difference has significant.
7, result
Result display (as shown in Figure 1), compared with normal people, in patients with osteoporosis blood, the mRNA level in-site of MTUS1 gene significantly reduces, and difference has statistical significance (P<0.05).
The difference expression gene that in embodiment 2 osseous tissue, osteoporosis is relevant
1, research object:
Stochastic choice is about to the patient carrying out marrow joint replacement, detects bone density, selects patients with osteoporosis 40 example, and normal bone density control group (be exogenous injury, detect without osteoporosis) 50 is routine, age 50-70 year.Situation such as way of questionnaires investigation experimenter's mode of life and healthy state etc.
The inclusive criteria of patients with osteoporosis: (1) meets diagnosis of osteoporosis standard person, with reference to " Chinese's osteoporosis suggestion Case definition (the second original text); (2) the equal informed consent of patient.
The exclusion standard of patients with osteoporosis: secondary osteoporosis person.
2, the acquisition of osseous tissue RNA
From the human femur head that marrow joint replacement takes off, get hard bone 1g, be placed in rapidly liquid nitrogen and preserve.Use the total serum IgE in Trizol single stage method extraction people osseous tissue in laboratory, measured the purity of RNA solution by the absorbance (A) at NanodropND-1000 reading 260nm and 280nm place.Through 1% denaturing formaldehyde agarose gel electrophoresis, observe under ultraviolet transmission light, detect the integrity of RNA.
3, the mass analysis (NanoDrop1000 spectrophotometer) of RNA sample
It is 1.8-2.2 that NanoDrop1000 spectrophotometer detects RNA sample: OD260/OD280.
4, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP marks, and the cRNAs after fluorescent mark adopts RNEASYMiniKit purifying, carries out fragmentation process with the RNAFragmentationReagents of Amhion to the cRNAs marked.Adopt people's full genome chip of expression spectrum (4x44K gene) of Agilent company of the U.S., in chip hybridization stove, 65 DEG C of hybridization 17h, then wash-out, dyeing, finally use AgilentDNAMicroarrayScanner scanner scanning.
5, chip data treatment and analyses
Chip after hybridization after chip scanner reading of data point, by data importing analysis software, the natural logarithm absolute value for two groups of ratios be greater than 2.0 or be less than 0.5 gene as difference expression gene.
6, statistical procedures
Adopt SPSS13.0 statistical software to carry out data analysis, group difference compares employing one-way analysis of variance method, and P<0.05 difference has significant.
7, result
Result display (as shown in Figure 2), compared with normal people, in patients with osteoporosis osseous tissue, the mRNA level in-site of MTUS1 gene significantly reduces, and difference has statistical significance (P<0.05).
The differential expression of MTUS1 albumen in embodiment 3 scleroblast
1, research object is with embodiment 2.
2, the drawing materials and cultivating of human osteoblast cell
From the human femur head that marrow joint replacement takes off, get spongy bone, reject soft tissue, physiological saline rinses sclerotin repeatedly, after washing fluid clarification, rinses and sways 3 times, be cut into 1mm with PBS liquid 3fragment, adopt enzyme digestion be separated and purifying scleroblast.Be inoculated in (culturing bottle adds appropriate DMEM-F12 (1:1) substratum and 10% foetal calf serum) in the culturing bottle of 30ml, be placed in 37 DEG C, the CO of 5% 2cultivate in constant incubator.Change liquid after 2d and remove not adherent cell, every 3d changes liquid 1 time later, observes under inverted microscope.Be fused into after individual layer until primary cell, with the trysinization of 2.5g/L, go down to posterity.
3, the extraction of total protein of cell
Phase of taking the logarithm, well-grown scleroblast carried out the extraction of total protein of cell, carried out the operation of protein extraction according to the specification sheets of the full cell extraction test kit of EpiQuik.
4, Westernblot detects
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carries out transferring film afterwards, close, primary antibodie hatches, two anti-ly to hatch, develop the color.
5, data processing
Use ImageJ software to analyze the gray-scale value of protein band, with β-actin for internal reference, the gray-scale value of MTUS1 protein band is normalized.Result data is all represent in the mode of mean+SD, adopts SPSS13.0 statistical software to carry out statistical study, and difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
6, result
As shown in Figure 3, compared with normal people, in patients with osteoporosis scleroblast, the expression level of MTUS1 albumen significantly reduces result, and difference has statistical significance (P<0.05).
Embodiment 4MTUS1 gene expression plasmid builds
1, the process LAN of MTUS1 gene
The structure of 1.1MTUS1 expression vector
According to encoding sequence (as shown in the SEQIDNO.1) design of amplification primers of MTUS1 gene, primer sequence is as follows: forward primer is 5 '-GACTGATGATAATTCAGATGA-3 ' (SEQIDNO.3), and reverse primer is 5 '-AAATGCTGGGGCTAGGGAA-3 ' (SEQIDNO.4).From cDNA library (the clontech company becoming Human fetal spleen, article No.: the encoding sequence of the MTUS1 gene of amplification total length 638831), above-mentioned cDNA sequence is inserted in eukaryotic expression vector pcDNA3.1, connects the recombinant vectors pcDNA3.1-MTUS1 obtained and is used for subsequent experimental.
Carry out osteoblastic vitro culture according to the method for embodiment 3, the scleroblast in vegetative period of taking the logarithm is by 1 × 10 4/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO 2cell cultures 24h in incubator, is divided into two experimental group by cell, be respectively control group (transfection pcDNA3.1 empty carrier) and MTUS1 process LAN group (transfection pcDNA3.1-MTUS1).Use liposome 2000 to carry out the transfection of carrier, the instruction to specifications of concrete transfection method is carried out.The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-MTUS1 is 0.5 μ g/ml.
2, utilize QPCR to test to detect the level of mRNA after MTUS1 process LAN
Ordinary method is utilized to operate 2.1 extract cell total rna.
2.2 reverse transcription
With RT Buffer, reverse transcription synthesis cDNA is carried out to l μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, adds following component respectively: DEPC water in PCR pipe, 5 × RT Buffer, 10mmol/LdNTP, 0.1mmol/lDTT, 30 μm of mol/lOligodT, 200U/ μ lM-MLV, template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
2.3QPCR
Adopt 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to ensure the reliability of result all in triplicate.Prepare following reaction system: SYBRGreen polymerase chain reaction system 12.5 μ l, forward primer (5 μMs/μ l) 1 μ l, reverse primer (5 μMs/μ l) 1 μ l, template cDNA2.0 μ l, without enzyme water 8.5 μ l; The forward primer sequence of amplification MTUS1 gene is 5 '-CCAATAGCGAACCACATT-3 ' (SEQIDNO.5), and reverse primer sequences is 5 '-TCATCATCAATAAGACAATAGGA-3 ' (SEQIDNO.6); The forward primer sequence of amplification GAPDH gene is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQIDNO.7), reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQIDNO.8), and operations is all in carrying out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 10s, 60 DEG C of 60s) * 45 circulation.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler fluorescence real-time quantitative PCR instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification,
2.4 statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, the difference between two groups adopts t inspection, thinks to have statistical significance as P<0.05.
3, Westernblot is utilized to detect the expression level of albumen after MTUS1 process LAN
Step is with embodiment 3.
4, result
As shown in Figure 4, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-MTUS1, the mRNA level in-site of MTUS1 significantly raises result, and difference has statistical significance (P<0.05); As shown in Figure 5, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-MTUS1, the protein level of MTUS1 significantly raises, and difference has statistical significance (P<0.05).
The process LAN of embodiment 5MTUS1 gene is to the mensuration of osteoblastic proliferation ability
Use CellCountingkit-8 (cck-8) test kit for being detected as the detection of bone cell proliferation
1, step
Carry out osteoblastic cultivation and transfection according to the method for preceding embodiment, cell is divided into three experimental group:
Group 1: the scleroblast transfection pcDNA3.1 (normal+pcDNA3.1) in normal bone dense tissue source;
Group 2: the scleroblast transfection pcDNA3.1 (osteoporosis+pcDNA3.1) of sufferers of osteoporosis face;
Group 3: the scleroblast transfection pcDNA3.1-MTUS1 (osteoporosis+pcDNA3.1-MTUS1) of sufferers of osteoporosis face.
After transfection 24h, with 2 × 10 5/ ml density is inoculated in 96 porocyte culture plates, and each experimental group design three wells, every hole 100 μ l, is positioned over 37 DEG C, 5%CO 2hatch in incubator, after 24h cell attachment, in the culture hole of required detection, every hole adds 10 μ lCK-8 solution respectively, continues to hatch 1h in cell culture incubator, measures each hole, 450nm place absorbance (OD value).
2, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
3, result
Result is as shown in table 2, compared with normal bone density people, the osteoblastic proliferation of sufferers of osteoporosis face is slow, after sufferers of osteoporosis face MTUS1 gene overexpression, osteoblastic propagation is accelerated, and difference has statistical significance (P<0.05).Above-mentioned experimental result shows, MTUS1 genetic expression process LAN can accelerate the osteoblastic propagation of sufferers of osteoporosis face.
Table 2 scleroblast OD value
Experimental group OD value (optical density(OD))
Normally+pcDNA3.1 0.1704±0.003
Osteoporosis+pcDNA3.1 0.0956±0.004
Osteoporosis+pcDNA3.1-MTUS1 0.1529±0.003
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.

Claims (10)

1. detect the application of product in the instrument preparing Diagnosis of osteoporosis of MTUS1 genetic expression.
2. application according to claim 1, is characterized in that, described product comprises: by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization chip or high-flux sequence detection of platform MTUS1 genetic expression with the product of Diagnosis of osteoporosis.
3. application according to claim 2, is characterized in that, the product of described RT-PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified MTUS1 gene; The product of described real-time quantitative PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified MTUS1 gene; The product of described immunodetection Diagnosis of osteoporosis comprises: the antibody be combined with MTUS1 protein-specific; The product of described in situ hybridization Diagnosis of osteoporosis comprises: with the probe of the nucleic acid array hybridizing of MTUS1 gene; The product of described chip Diagnosis of osteoporosis comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with MTUS1 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of MTUS1 gene.
4. application according to claim 3, is characterized in that, the primer of a pair specific amplified MTUS1 gene that the product of described real-time quantitative PCR Diagnosis of osteoporosis at least comprises is as shown in SEQIDNO.5 and SEQIDNO.6.
5. an instrument for Diagnosis of osteoporosis, is characterized in that, described instrument comprises the reagent detecting MTUS1 genetic expression; Described reagent comprises the antibody of the primer that detects MTUS1 gene mRNA and/or probe, detection MTUS1 albumen.
6. instrument according to claim 5, is characterized in that, the primer of described detection MTUS1 gene mRNA comprises the primer pair shown in SEQIDNO.5 and SEQIDNO.6.
The application of promotor in the medicine of preparation treatment osteoporosis of 7.MTUS1 gene and/or its expression product.
8. application according to claim 7, it is characterized in that, the reagent of the reagent of described promotion MTUS1 genetic expression, the reagent promoting MTUS1 gene expression product stability, promotion MTUS1 gene expression product activity, the reagent of promotion MTUS1 gene expression product function.
9. application according to claim 8, is characterized in that, the reagent promoting the reagent of MTUS1 genetic expression to comprise the reagent containing MTUS1 gene, the carrier carrying MTUS1 gene or host cell to be formed, the reagent containing MTUS1 protein.
10. be used for the treatment of a pharmaceutical composition for osteoporosis, it is characterized in that, described pharmaceutical composition comprises the promotor according to any one of 7-9.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148520A (en) * 2016-07-06 2016-11-23 西北工业大学 MACF1 gene and expression product application in preparation diagnosis and treatment osteoporosis agents thereof
CN109266736A (en) * 2018-10-18 2019-01-25 中国医学科学院北京协和医院 Molecular marked compound and its application for osteoporosis early screening
CN109486934A (en) * 2018-10-18 2019-03-19 中国医学科学院北京协和医院 CWF19L2 gene is preparing the application in Diagnosis of osteoporosis product

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KAWAKAMI YET.AL: "Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes.", 《ARTHRITIS》 *
NAGHMEH VARGHAYEE ET.AL: "Function and expression of ATIP and its variants in cardiomyoblast cell line H9c2", 《JOURNAL OF THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM》 *
XIAOFENGZHOU ET.AL: "MTUS1(mitochondrialtumorsuppressor 1)", 《ATLASDATABASE》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148520A (en) * 2016-07-06 2016-11-23 西北工业大学 MACF1 gene and expression product application in preparation diagnosis and treatment osteoporosis agents thereof
CN109266736A (en) * 2018-10-18 2019-01-25 中国医学科学院北京协和医院 Molecular marked compound and its application for osteoporosis early screening
CN109486934A (en) * 2018-10-18 2019-03-19 中国医学科学院北京协和医院 CWF19L2 gene is preparing the application in Diagnosis of osteoporosis product

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