CN108034713A - Postmenopausal Osteoporosis diagnosis and treatment target spot and its application - Google Patents
Postmenopausal Osteoporosis diagnosis and treatment target spot and its application Download PDFInfo
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Abstract
The present invention relates to Postmenopausal Osteoporosis diagnosis and treatment target spot and its application, and in particular to postmenopausal osteoporosis detects target spot FBXW7 and its application.Inventor is for the still very rare present situation of postmenopausal osteoporosis molecular marked compound, postmenopausal osteoporotic patients are taken with the forward and backward peripheral blood of Liuwei Dihuang Wan and Healthy People control peripheral blood carries out high-flux sequence, pick out candidate gene FBXW7, further, experiments prove that correlation and medication correlation of the gene with postmenopausal osteoporosis, the present invention provides fundamental basis for gene diagnosis postmenopausal osteoporosis and clinical application, has good clinical value.
Description
Technical field
The present invention relates to biomedicine field, and in particular to osteoporosis diagnosis and treatment target spot and its application, more specifically relate to
And postmenopausal osteoporosis detection target spot FBXW7 and its application.
Background technology
Osteoporosis (osteoporosis) is that h and E factor is coefficient as a result, being a complexity
Multi-factor disease.In recent years, domestic and foreign scholars have carried out the related gene of osteoporosis substantial amounts of research, find sensitive
Genetic marker, the danger of osteoporosis occurs for prediction, and the research before us finds the relevant gene of some osteoporosises,
Such as IFT52 genes (CN 2015106280814), EPS8L3 genes (CN2015106280424), CERS2 genes
(CN2015106293481), MTUS1 genes (CN2015106280246) etc..Postmenopausal Osteoporosis
(postmenopausal osteoporosis, PMOP) is one kind of osteoporosis, related with aging, occurs mainly in menopause
Women afterwards, due to estrogen deficiency cause bone amount reduce and bone structure change, make bone brittleness increase be easy to fracture and by
The problems such as complication, occur in pain, textured bone caused by fracture, or even dead, severely impacts the health of the elderly
And quality of life, or even shorten service life increase country and family's financial resources and manpower burden.
At present, for treatment method mainly inclusive hormone replacement therapy, calcium agent and the dimension life of Postmenopausal Osteoporosis
The methods of plain D, diphosphonate, fluoride.In addition, the prevention for Postmenopausal Osteoporosis, necessarily it is noted that life style
With the adjustment of meals result, increase outdoor activities, the appropriate mouth that increases shines the time, and more edible meals rich in calcium, suitably carry out body
Exercise is educated, disease of the prevention with falling risk strengthens autoprotection measure, and fall prevention causes fracture etc..
TCM investigation has confirmed that the morbidity of osteoporosis and kidney deficiency are closely related in recent years, a variety of kidney-tonifying recipes, such as six
Taste Dihuang Wan, has the effect of obvious to postmenopausal osteoporosis, and does not find apparent side effect, is gradually taken seriously, such as
Li Tao researchs find that Liu Wei Di Huang Wan with Plus o n can mitigate patients with postmenopausal osteoporosis ostalgia, improve bone density, improve its life
Bioplasm amount, and without obvious adverse reaction, it shares (the Liu Wei Di Huang Wan with Plus o n treatment liver that can heighten the effect of a treatment with zoledronic acid injection
Syndrome of deficiency of kidney yin Postmenopausal Osteoporosis clinical research, medical forum's magazine, in August, 2014 the 8th phase of volume 35).
The fast development and application of high throughput sequencing technologies, provide more comprehensive for the pathogenetic research of osteoporosis
With quick analysis means, researcher utilizes bioinformatic analysis instrument, and six drugs containing rehmanniae is illustrated from the angle of gene regulation
Ball treats the molecular mechanism of postmen opausal, and Lu Yanfang et al. is shown using GEO database related data mining analysis,
Liuwei Dihuang Wan to the beneficial effect of menopause kidney-yin deficiency osteoporosis mainly by promote estrogen express and with its by
Body combine, and balanced immune response and promote the formation of bone tissue so that reach improve osteoporosis (Liuwei Dihuang Wan is to menopause
The gene expression regulation data analysis of phase kidney-yin deficiency osteoporosis, Chinese osteoporosis magazine in the March, 2017 of volume 23 the 3rd
Phase).
Postmenopausal osteoporosis molecular marked compound is still very rare at present, so, inventor dredges 3 post menopausal sclerotin
Loose patient takes peripheral blood sample before Liuwei Dihuang Wan, 3 postmenopausal osteoporotic patients take it is outer after Liuwei Dihuang Wan half a year
All blood samples and 3 Healthy People control peripheral blood samples carry out high-flux sequence, are analyzed with reference to bioinformatics method and carry out base
The screening of cause, picks out candidate gene FBXW7, further, experiments prove that gene is to postmenopausal osteoporosis related
Property, the present invention is that clinical diagnosis and treatment postmenopausal osteoporosis and medication are provided fundamental basis, and has good clinical value.
The content of the invention
Reagent it is an object of the invention to provide detection FBXW7 genes and/or FBXW7 albumen is preparing post menopausal sclerotin
Application in loose diagnostic preparation.
To achieve the above object, the present invention screens candidate's base by high-flux sequence combination bioinformatics method first
Because of FBXW7, then there is good correlation by what molecular biology method demonstrated FBXW7 and postmenopausal osteoporosis,
And FBXW7 gene expression amounts are decreased obviously after sufferers of osteoporosis face takes Liuwei Dihuang Wan, show the gene not only and menopause
Osteoporosis is related afterwards, or the action target spot of Liuwei Dihuang Wan, available for preparing postmenopausal osteoporosis diagnostic preparation and control
Target spot is treated, there is important clinical value.
Further, FBXW7 genes and/or FBXW7 the albumen high expression in postmenopausal osteoporosis sample.
Further, the table of FBXW7 genes and/or FBXW7 albumen in postmenopausal osteoporosis diagnostic preparation detection peripheral blood
Up to situation.
Further, the diagnostic preparation of the postmenopausal osteoporosis uses one or several kinds of detections in following method
The expression of FBXW7 genes：Fluorescence quantifying PCR method, method for gene chip, high-flux sequence method.
Fluorescence quantitative PCR method is the specific probe by fluorescent dye or fluorescent marker, and PCR product is marked
Tracking, real time and on line monitoring reaction process, can analyze product with reference to corresponding software, calculate sample to be tested template
Initial concentration.The appearance of quantitative fluorescent PCR, greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation.
The appearance of a variety of detecting systems, makes the selectivity of experiment stronger.Automation mechanized operation improves work efficiency, rapid reaction, repetition
The good, high sensitivity of property, high specificity, result are clear.
Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types：1) it is fixed on poly-
Nucleic acid probe or cDNA fragments on compound substrate (nylon membrane, nitrocellulose membrane etc.) surface, usually use the target of isotope marks
Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method,
By being detected with the hybridization of the target gene of fluorescent marker.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass
Array, the target gene hybridization with fluorescent marker are detected.Genetic chip is as a kind of advanced, extensive, high throughput detection
Technology, applied to the diagnosis of disease, its advantage has the following aspects：First, the sensitivity and accuracy of height；It is second, quick
It is easy；Third, a variety of diseases can be detected at the same time.
High-flux sequence (High-throughput sequencing) is also known as sequencing technologies (next of future generation
Generation sequencing) it is the change for tradition being sequenced revolution, once to hundreds of thousands to millions of DNA
Molecule carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple species and loses
The solution reading rate of communication breath, to obtain the sequence information of all mRNA, decryption mRNA collection of illustrative plates provides guarantee.High pass measures at the same time
Sequence to carry out the analysis of careful overall picture to the transcript profile and genome of species, so the depth survey that is otherwise known as
Sequence.The representative of high-flux sequence platform is 454 sequenators (Roch GSFLX sequencer) of Roche Holding Ag (Roche),
The Solexa genome analysises instrument (Illumina Genome Analyzer) of Illumina companies and the SOLiD sequenators of ABI
(ABI SOLiD sequencer)。
The product of FBXW7 genes contains a pair of of spy in the detection postmenopausal osteoporosis for fluorescence quantifying PCR method
The primer of specific amplification FBXW7 genes；The genetic chip includes the probe with the nucleic acid array hybridizing of FBXW7 genes.It is excellent
Choosing, the primer of a pair of of specific amplification FBXW7 genes, upstream primer sequence is SEQ ID NO.1, and downstream primer sequence is
SEQ ID NO.2。
Further, the diagnostic preparation of postmenopausal osteoporosis further includes the expression with immunization method detection FBXW7 albumen.It is excellent
It is western blot and/or ELISA and/glue to select FBXW7 protein expressions in the immunization method detection postmenopausal osteoporosis
Body gold method.
Enzyme-linked immunosorbent assay (ELISA) will known antigen or antibody absorption in surface of solid phase carriers, make enzyme mark
The technology that the antigen-antibody reaction of note is carried out in solid phase surface.The technology can be used for detection macromolecular antigen and specific antibody
Deng, have the advantages that quick, sensitive, easy, carrier be easy to standardization.ELISA detection kit is according to testing goal and operation
Step can be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and
The ELISA of biotin.Horseradish peroxidase (HRP) or alkaline phosphatase may be selected in chromogenic substrate in ELISA detection kit
Enzyme (AP).
Common immune colloid gold detection technique：(1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut
Piece, can be dyed with the antibody of colloid gold label, can also be strengthened with silver-colored developer solution and marked on the basis of colloid gold label,
The silver atoms for making to be reduced are deposited on marked gold grain surface, can be remarkably reinforced the sensitiveness of colloid gold label.(2) it is immunized
Colloidal gold staining method for electron microscopy can use the antibody of colloid gold label or antiantibody to be combined with negative staining Virus Sample or tissue ultra-thin section,
Then negative staining is carried out.Observation and viral diagnosis available for morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made
Carrier, first adds sample to be checked by antigen or antibody point on film after closing, corresponding with the antibody test of colloid gold label after washing
Antigen or antibody.(4) specific antigen or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, colloidal gold
Labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be checked is added in the sample pad of test strips one end
Afterwards, move forward, react to each other after dissolving the colloid gold label reagent on bonding pad through capillary action, it is fixed when being moved to
During the region of antigen or antibody, the conjugate of thing and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
Taken in detection, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.
Further, the ELISA method of the detection FBXW7 albumen is to use ELISA detection kit.In the kit
Antibody can use commercially available FBXW7 monoclonal antibodies.Further, the kit includes：It is coated with FBXW7 monoclonal antibodies
Solid phase carrier, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid
Deng.
Further, the colloidal gold method of the detection FBXW7 albumen is that antibody is using commercially available using colloidal gold strip
FBXW7 monoclonal antibodies.Further, the colloid gold test paper strip adoption colloidal gold immunochromatographimethod technology or colloidal gold percolation.
Further, detection zone (T) specking on the colloidal gold strip nitrocellulose filter has anti-FBXW7 monoclonal antibodies, Quality Control
Area (C) specking has Immunoglobulin IgG.
It is an object of the invention to provide a kind of PCR kit for fluorescence quantitative for detecting postmenopausal osteoporosis, its feature
It is, the kit detects gene FBXW7, using special sense primer and anti-sense primer, upstream primer sequence SEQ
ID NO.1, downstream primer sequence are SEQ ID NO.2.
Further, which is suitable for presently, there are all types fluorescence quantitative gene extender of in the market, spirit
Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes：Specific primer, internal control primer, quantitative fluorescent PCR
Reaction solution.The wherein described specific primer includes sense primer and anti-sense primer, and upstream primer sequence is SEQ ID NO.1,
Downstream primer sequence is SEQ ID NO.2.The internal control primer is β-actin internal control primers, and upstream primer sequence is SEQ ID
NO.3, downstream primer sequence are SEQ ID NO.4.
The kit also includes RNA extraction agents.It is preferred thatReagent carries out sample rna extraction.
Reagent it is an object of the invention to provide detection FBXW7 genes and/or FBXW7 albumen is preparing post menopausal sclerotin
Application in loose clinical application genetic test preparation.
Further, FBXW7 genes and/or the sample group of the high expression of FBXW7 albumen are applicable in Liuwei Dihuang Wan.
Further, the expression for one or several kinds of detection FBXW7 genes that preparation is used in following method is detected：Fluorescence is determined
Measure PCR method, method for gene chip, high-flux sequence method.
It is an object of the present invention to provide a kind of postmenopausal osteoporosis detection kit, detection kit detection
FBXW7 albumen.Further, the kit further includes other detection reagents.
It is an object of the present invention to provide it is a kind of detect postmenopausal osteoporosis genetic chip, the genetic chip include with
The probe of the nucleic acid array hybridizing of FBXW7 genes.
It is an object of the present invention to provide a kind of postmenopausal osteoporosis clinical application gene detecting kit, the kit inspection
The expression of FBXW7 genes and/or FBXW7 albumen is surveyed, FBXW7 genes and/or the high sample group expressed of FBXW7 albumen are applicable in Six-element
Dihuang Wan.
It is an object of the present invention to provide a kind of postmenopausal osteoporosis clinical application genetic test genetic chip, the gene core
Piece includes the probe with the nucleic acid array hybridizing of FBXW7 genes, and FBXW7 genes and/or the high sample group expressed of FBXW7 albumen are fitted
Use Liuwei Dihuang Wan.
It is an object of the invention to provide FBXW7 genes and/or its protein inhibitor to dredge in preparation treatment post menopausal sclerotin
Application in loose medicine.
Further, the treatment postmenopausal osteoporosis medicine refers to the preparation that can suppress the expression of FBXW7 genes.This
The expression of the known suppressor of field personnel can usually use one kind in following methods and/or several：By activating FBXW7
The suppressor of gene, activate FBXW7 genes inhibition of gene expression albumen, FBXW7 genes are suppressed using RNA perturbation techniques
Expression, activation promote the microRNA of FBXW7 gene mRNAs degraded, import promote the degraded of FBXW7 gene coded proteins molecule,
Suppress the factor of promotion FBXW7 gene expressions and the expression of albumen.
RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo
Selective degradation, causes the phenomenon of posttranscriptional gene silencing, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal
The expression of certain specific gene, promotes mRNA to degrade, and cells show is gone out the technology of specific gene missing phenotype.SiRNA is designed
After the completion of can use direct synthesis technique or structure SiRNA expression vector, the siRNA prepared can be coprecipitated by calcium phosphate
Mechanical Method, the cationic-liposome such as shallow lake method, electroporation, DEAE- glucans and polybrene methods, microinjection or particle gun
The approach transfectional cell such as reagent method.
Further, one kind in one sequence of siRNA target spots of FBXW7 gene expressions and/or several is suppressed：SEQ
ID NO.5、SEQ ID NO.8、SEQ ID NO.11.It is preferred that siRNA sequence is SEQ ID NO.8.
Further, one kind in one sequence of the siRNA sequence of the suppression FBXW7 gene expressions and/or several：
SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.13.It is excellent
It is SEQ ID NO.9, SEQ ID NO.10 to select siRNA sequence.
It is an object of the invention to provide one kind to treat postmenopausal osteoporosis medicine, the treatment postmenopausal osteoporosis
The expression of Drug inhibition FBXW7 genes.
Further, postmenopausal osteoporosis medicine is treated also comprising receptible carrier in pharmacy.
Receptible carrier is the carrier usually utilized in preparation in the pharmacy of the present invention, which includes
Lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbierite (sorbitol), mannitol
(mannitol), starch, acacia gum, calcium phosphate, alginates (alginate), gel (gelatin), calcium silicates, crystallite
Cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methyl are fine
Dimension plain (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy benzoic acid
Propyl ester (propyl hydroxybenzoate), talcum, magnesium stearate (stearic acid magnesium) and mineral oil
(mineral oil) etc., but it is not limited to this.
The present invention composition except mentioned component in addition to can also comprising lubricant, wetting agent, sweetener, flavouring agent,
Emulsifying agent, suspending agent, preservative etc..Receptible carrier and preparation are recorded in Lei Mingdengshi pharmacy pandects in detail in pharmacy.
The composition of the present invention can by it is oral or it is parenteral be administered, during as non-oral administration, vein can be passed through
Interior injection, intranasal injection, local injection, intraventricular injection, spinal cavity injection, is subcutaneously injected, intraperitoneal injection, percutaneous dosing etc.
Mode is administered.
The suitable dosage of the composition of the present invention is according to preparation ways, administering mode, the age of patient, body
The factor of weight, gender, morbid state, food, administration time, method of administration, drainage rate and draw property etc and can carry out
A variety of prescriptions, in general, skilled practitioner can be easily determined by and prescription is treated or prevented effectively to medicament to desirable
Amount.
The composition method that person of an ordinary skill in the technical field can easily implement according to the present invention of the present invention,
Carried out using receptible carrier in pharmacy and/or excipient it is formulation, so as in the form of unit dose prepare or
Prepared in person in multicapacity container.At this time, formulation is the solution, suspension or emulsion of oiliness or aqueous medium
Form, can also be either extract, powder agent, granule, tablet either capsule form can also include dispersant or
Stabilizer.
Unless otherwise defined, technical and scientific term all in the present invention has general with the technical field of the invention
The normally understood implication of logical technical staff institute.
Brief description of the drawings
Fig. 1 is FBXW7 genes relative expression's spirogram in postmenopausal osteoporosis peripheral blood and healthy human peripheral blood
Fig. 2 is FBXW7 genes relative expression in peripheral blood before and after postmenopausal osteoporotic patients take Liuwei Dihuang Wan
Spirogram
Fig. 3 is each group FBXW7mRNA relative expression levels figure after RNA interference：C groups：Blank control group；C1 groups：Transfect fat
Plastid group；C2 groups：Transfect nonspecific siRNA groups；S1, S2, S3 group：Transfect specific siRNA groups.
Embodiment
With reference to specific embodiment, the present invention is further explained, is only used for explaining the present invention, and it is not intended that to this
The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle of the present invention and objective
These embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental method of actual conditions is not specified in the following example, usually according to normal condition or according to the bar proposed by manufacturer
Part examinations.
1 high-flux sequence of embodiment and analysis
3 postmenopausal osteoporotic patients are collected respectively takes the forward and backward peripheral blood sample of Liuwei Dihuang Wan and 3 Healthy Peoples
Peripheral blood sample is compareed, carries out RNA extractions, agarose gel electrophoresis after RNA extractions, can be extracted from electrophoresis result with preliminary judgement
RNA sample it is up-to-standard whether, if can be used for further transcriptome analysis.And then it is divided by NanoDrop1000
Photometer detects the extraction situation of RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2.
Microarray dataset is the 2500 high-flux sequence platforms of HiSeq of Illumina companies, carries out high throughput transcript profile depth
Sequencing, we carry out total evaluation with Fast-QC softwares to the quality of sequencing data after sequencing, include the mass value point of base
Cloth, the position distribution of mass value, G/C content, PCR duplication contents, frequency of kmer etc..In differential gene table
Up to during analysis, according to obtained FPKM values, differential screening is carried out using internationally recognized algorithm EBSeq.Wherein, during screening,
LOG2FC>1 or<-1,FDR<0.05.In order to be better understood from the function of difference expression gene, we to difference expression gene into
Gene Onlogy and signal path analysis are gone, and functional annotation and protein interaction net are carried out to difference expression gene
Network analyze, in view of data above analysis as a result, we have screened difference expression gene FBXW7 with reference to document.FBXW7 is in sclerotin
Loose group high expression, and the low expression after Liuwei Dihuang Wan is taken.
FBXW7 expression conditions in 2 postmenopausal osteoporotic patients peripheral blood of embodiment and healthy human peripheral blood
First, material and method
1st, material
32 postmenopausal osteoporotic patients peripheral bloods and 29 healthy human peripheral bloods are collected, it is grouped and is compiled
Number.
2nd, method
The extraction of 2.1 postmenopausal osteoporosis peripheral bloods and healthy human peripheral blood total serum IgE
UsingReagent carries out sample rna extraction, and experimental implementation is carried out by product description.
RNA quality judging standards：The OD260/OD280 values of RNA samples are between 1.8-2.2；Total serum IgE electrophoresis pattern has clearly
Clear 28S, 18S band；Electrophoresis pattern after when 70 DEG C of water-bath insulations 1 are small and the collection of illustrative plates no significant difference before water-bath insulation.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into
Row cDNA reverse transcriptions, experimental implementation are carried out by product description, and concrete operations are as follows：
Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgEs to be separately added into following components in PCR pipe as template ribonucleic acid：
5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/l
2 μ l, 200U/ μ l MMLV of OligodT 1.25 μ l, 1 μ g of template ribonucleic acid, add aqua sterilisa to 25 μ l of total system.42 DEG C of incubations 1 are small
When, 72 DEG C 10 minutes, of short duration centrifugation.It is spare that -20 DEG C of refrigerators are put in cDNA preservations.
2.3Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instruments of ABI, the relative quantitative assay of data is carried out using 2- △ △ CT methods.
2.3.2 design of primers
Using online primer-design software, template sequence NM_001013415.1, by invitrogen after design of primers
Company synthesizes.Specific primer sequence is as follows：
FBXW7 primers：
5’-TAGTTAGTGGTTCTGATG-3’(SEQ ID NO.1)
5’-AATGATGATGTTGTCTCT-3’(SEQ ID NO.2)
Amplification length 125BP.
Β-ACTIN primers：
5’-TAATCTTCGCCTTAATACT-3’(SEQ ID NO.3)
5’-CCTTCATACATCTCAAGT-3’(SEQ ID NO.4)
Amplification length 103bp.
Operating process is as follows：
Reaction system：2×mix 10μl；Sense primer (10uM) and each 0.5 μ l of upstream and downstream primer (10uM)；2 μ l of template；
Sterile purified water filling-in is added to 25 μ l.
Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) are expanded,
Experimental implementation is carried out by product description.
Amplification program is：95 ° of 10min, (95 DEG C of 15sec, 55 DEG C of 60sec) × 35 circulations.
Sample RealTimePCR is detected：Take 2 μ l to make template after cDNA10 times of each sample is diluted, use target gene respectively
Primer and reference gene primer are expanded.At the same time solubility curve analysis is carried out at 60-95 DEG C.
2nd, experimental result
Real-time quantitative PCR the results show (is specifically shown in Fig. 1)：QRT-PCR stable amplification results, wherein FBXW7 is in post menopausal
Expression in osteoporosis peripheral blood is higher than the expression in healthy human peripheral blood, the former is about 1.57 times of the latter,
Result above demonstrates the result of high-flux sequence.
4 postmenopausal osteoporotic patients of embodiment take peripheral blood FBXW7 expression conditions before and after Liuwei Dihuang Wan
1st, material
The agreement of 22 postmenopausal osteoporotic patients is collected, initial stage is detected at it and extracts peripheral blood, take six drugs containing rehmanniae
Its peripheral blood is collected after ball half a year again, it is grouped and is numbered.
2nd, method
Specific steps are with reference to embodiment 3.
3rd, result
Real-time quantitative PCR amplification curve flex point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is put down without raising up now, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；According to the relative quantification formula of qRT-PCR, than
Compared with FBXW7 genes in peripheral blood before and after postmenopausal osteoporotic patients take Liuwei Dihuang Wan FBXW7 expressions, take six
FBXW7 gene expression amounts are measured than 2/5ths (see Fig. 2) that entire lowering before taking about compares with high pass after taste Dihuang Wan
Sequence result trend is consistent.
The culture of 5 cell line MG-63 of embodiment
First, experiment material
Cell line MG-63 is purchased from Shanghai cell research institute of the Chinese Academy of Sciences.
2nd, main solution
Cell culture fluid：+ 10% standard hyclone of DMEM culture mediums.
0.25% tryptic digestive juice：0.25g trypsase is added in 100m1 deionized waters, filter filtration sterilization, point
Equipment is used.
3rd, cell passes on
Original nutrient solution discards in 1 blake bottle that will cover with cell, adds 0.25% trypsin solution 1m1, and covering is thin
Born of the same parents' layer, bottleneck disinfection, capping；
Cellular change is observed under 2 inverted microscopes, over time, former adherent cell gradually tends to be circular, carefully
Matrix bounces back, and space between cells increases, and discards pancreatin in also non-levitating, adds the nutrient solution that 5ml contains 10% hyclone
Terminate digestion；
3 cell counts：Above-mentioned cell suspension 0.5mI is taken, is instilled after appropriate dilution in blood cell counting plate, based on leucocyte
The big lattice inner cells sum in number method number corner four, when counting, only count nucleus and the complete cell of cytoplasm, and cell in heaps is pressed
One cell calculates, and the total number of cells in 4 block plaids is pressed following formula scales into the cell number in every milliliter of cell suspension：Carefully
Big lattice total number of cells/4 × 10 of born of the same parents' sum/ml=44× extension rate；
4, according to cell counts, are further diluted to every milliliter with DMEM complete culture solutions and contain 3 × 105A cell is dense
Degree, is sub-packed in blake bottle (every bottle of 8m1/), is positioned over 37 DEG C, 5%CO2Cultivated in incubator.
Embodiment 6RNAi suppresses FBXW7 gene expressions
First, experiment material
SiRNA is built and synthesis
According to FBXW7 genes in GenBank (NCBI Reference Sequence:NM_001013415.1 sequence in)
Design corresponding siRNA.Synesis Company's synthesis is sent to after design.Nonspecific siRNA is provided by Synesis Company.
2nd, experimental method
(1) RNA perturbation techniques specificity suppresses the expression of FBXW7 genes in MG-63 cells
1st, the culture of MG-63 cells
Method and step is the same as embodiment 3.
2nd, the design and synthesis of siRNA
SiRNA expression vector pSIREN-DNR contains neomycin resistance gene and GFP green fluorescent labels, can monitor in real time
Transfection efficiency of the carrier in cell.According to purpose mRNA sequence, 3 RNA interference target sequences (table 1) are designed.For every choosing
Fixed siRNA target sequences, design siRNA positive-sense strands and antisense strand, are connected with loop (9nt), are known as shRNA (short
hairpin RNA).Synthesize two of DNA profiling of every coding shRNA it is single-stranded, annealed dna is single-stranded to obtain the DNA double of shRNA
Chain template.Template strand followed by RNA PoIyIII polymerase transcriptions stop site, while both ends separately design BamHI and
HindIII restriction enzyme sites, can be cloned between BamHI the and HindIII restriction enzyme sites of siRNA carrier multiple cloning sites.
For siRNA empty carriers with after BamHI and HindIII double digestions, 1% agarose gel electrophoresis, recycles linear carrier.Annealing
DNA profiling double-strand is connected in linear carrier.Using T4 ligases, the molar ratio of insertion and carrier is about 3:1.Connection production
Thing converts DH5 α Escherichia coli, the coated plate on LB Amp culture mediums, 37 DEG C of overnight incubations.PCR is identified；Sequencing identification.Column extracts
Positive colony carrier is simultaneously quantitative.
Table 1siRNA transcription templates sequences
3rd, cell packet and transfection
(1) cell is grouped
C groups：Blank control group；C1 groups：Transfect liposome group；C2 groups：Transfect nonspecific siRNA groups；S1, S2, S3
Group：Transfect specific siRNA groups.
(2) transfect
According to LipofectamineTMThe step of 2000Transfection Reagent are provided carries out.
1. 24h before transfection, the cell pancreatin in growth period of taking the logarithm are digested and counted, dense with DMEM culture mediums adjustment cell
Spend for 1 × 105/ ml, takes 2m1 to be inoculated in six orifice plates, is positioned over 37 DEG C, 5%CO2Cultivate in incubator, merged in cell up to 80%
When be used for transfect.The DMEM medium cultures 3-4h without serum is used before transfection.
2. prepare transfection liquid：
A liquid：250u1 serum free mediums dilute 4.0ugDNA, gentle to mix；
B liquid：250u1 serum free mediums dilute 10u1Lipofectamine, gentle to mix, and room temperature places 5min；
3. transfect：A liquid is mixed with B liquid, and compound, is directly added in every hole by incubation at room temperature 20min, is shaken
Culture plate, gently mixes.In CO2Liquid is changed after 37 DEG C of insulations 24-48h, 6h in incubator, adds the culture medium containing serum.
4th, the verification of transfection efficiency
(1) cellular morphology and transfected condition are observed under fluorescence inverted microscope
After transfecting 24h, culture plate is placed under fluorescence inverted microscope and observes cellular morphology and growth conditions, green fluorescence
Lower observation transfected condition.
(2) using the change of FBXW7 gene expressions before and after the detection transfection of Real-time PCR methods
1. the structure of standard curve：1 bottle of the MG-63 cells normally cultivated in 50mI blake bottles are chosen at, extract RNA, are surveyed
Determine RNA concentration and purity, carry out reverse transcription reaction, ten times of dilutions of DNA profiling of generation will be reacted, obtained equivalent to 104-
100The DNA profiling of copies/ul, is separately added into FBXW7 primers and internal reference actin primers, prepares 25u1 reaction systems, makes
With Real-time PCR amplification instruments, pcr amplification reaction is carried out.Obtain the standard curve of FBXW7 and actin.
2. the change of FBXW7 gene expressions before and after the detection transfection of Real-time PCR methods：The RNA of each group cell is extracted,
RNA concentration and purity are measured, carries out reverse transcription reaction, every group of DNA profiling is carried out at the same time the Real-time of FBXW7 and actin
PCR reacts, and experiment is in triplicate.
3. to PCR product into row agarose gel electrophoresis.
3rd, experimental result
Real-time PCR detect transfection efficiency.Using the standard of Real-time PCR methods structure FBXW7 and actin
Curve, linear relationship is good, meets the requirements.Compare the expression of each group FBXW7 genes with the method for double standard curves.Blank control
Group, liposome transfection group, the expression of nonspecific transfection group gene are substantially similar, no significant difference.FBXW7-
SiRNA1, FBXW7-siRNA2, FBXW7-siRNA3 play the role of suppressing FBXW7 gene expressions, the work of FBXW7-siRNA2
With becoming apparent from, suppress efficiency up to 77%, and the inhibitory action of FBXW7-siRNA1 and FBXW7-siRNA3 is respectively 31% He
33%, compared with blank control group, liposome transfection group, nonspecific transfection group, difference is statistically significant, P<0.05 (figure
2)。
The present invention filters out postmenopausal osteoporosis pathogenic related gene FBXW7 using high-flux sequence, further analysis
It has been shown that, the gene is related to Liuwei Dihuang Wan medication effect, and aspect is of great significance in terms of clinical guidance medication, inventor
Binding molecule Cell Biology Experiment is verified, it was confirmed that FBXW7 has good correlation with postmenopausal osteoporosis disease, and carries
The siRNA of interference FBXW7 gene expressions is supplied.The present invention provides new diagnosis target spot for postmenopausal osteoporosis clinic diagnosis
With medication target spot, there is good potential applicability in clinical practice.
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Claims (10)
1. application of the reagent of detection FBXW7 genes and/or FBXW7 albumen in postmenopausal osteoporosis diagnostic preparation is prepared.
2. application according to claim 1, it is characterised in that in postmenopausal osteoporosis diagnostic preparation detection peripheral blood
The expression of FBXW7 genes and/or FBXW7 albumen.
3. application according to claim 1, it is characterised in that the diagnostic preparation of postmenopausal osteoporosis uses following method
In one or several kinds of detection FBXW7 genes expression：Fluorescence quantifying PCR method, method for gene chip, high-flux sequence side
Method, it is preferred that fluorescence quantitative PCR method uses the primer of a pair of of specific amplification FBXW7 genes；Genetic chip includes and FBXW7
The probe of the nucleic acid array hybridizing of gene.
4. the reagent of detection FBXW7 genes and/or FBXW7 albumen is preparing postmenopausal osteoporosis clinical application genetic test system
Application in agent.
5. application according to claim 4, it is characterised in that FBXW7 genes and/or the sample of the high expression of FBXW7 albumen
The applicable Liuwei Dihuang Wan of group.
6.FBXW7 genes and/or its protein inhibitor are preparing the application in treating postmenopausal osteoporosis medicine.
7. application according to claim 6, it is characterised in that treatment postmenopausal osteoporosis medicine is used in following methods
One kind and/or several suppression FBXW7 genes and/or FBXW7 protein expressions：Activate suppressor, the activation of FBXW7 genes
The albumen of the inhibition of gene expression of FBXW7 genes, suppress FBXW7 gene expressions, activation promotion FBXW7 using RNA perturbation techniques
The microRNA of gene mRNA degraded, import the molecule for promoting FBXW7 gene coded proteins to degrade, suppress to promote FBXW7 genes
The factor of expression and the expression of albumen.
8. application according to claim 7, it is characterised in that FBXW7 gene expressions are suppressed using RNA perturbation techniques
One kind in one sequence of siRNA target spots and/or several：SEQ ID NO.5、SEQ IDNO.8、SEQ ID NO.11.It is excellent
It is SEQ ID NO.8 to select siRNA target sequences.
9. a kind of PCR kit for fluorescence quantitative for detecting postmenopausal osteoporosis, it is characterised in that the kit detects gene
FBXW7, using special sense primer and anti-sense primer, upstream primer sequence is SEQ IDNO.1, downstream primer sequence SEQ
ID NO.2。
10. one kind treats postmenopausal osteoporosis medicine, which contains the siRNA for the expression for suppressing FBXW7 genes, preferably
, siRNA sequence is SEQ ID NO.9, SEQ ID NO.10.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866184A (en) * | 2018-08-27 | 2018-11-23 | 北京泱深生物信息技术有限公司 | Application of the gene marker in women osteoporosis |
| CN114333990A (en) * | 2020-09-30 | 2022-04-12 | 北京瑷格干细胞科技有限公司 | Postmenopausal osteoporosis related gene screening and function analysis method |
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| WO2014054046A1 (en) * | 2012-10-04 | 2014-04-10 | Gavish-Galilee Bio Applications Ltd | Ordered reversible fibrils, aggregates and multimers of proteins and uses thereof |
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- 2017-12-29 CN CN201711470544.4A patent/CN108034713A/en active Pending
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| WO2014054046A1 (en) * | 2012-10-04 | 2014-04-10 | Gavish-Galilee Bio Applications Ltd | Ordered reversible fibrils, aggregates and multimers of proteins and uses thereof |
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| 卢严方: "六味地黄丸对绝经期肾阴虚骨质疏松症的基因表达调控数据分析", 《中国骨质疏松杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866184A (en) * | 2018-08-27 | 2018-11-23 | 北京泱深生物信息技术有限公司 | Application of the gene marker in women osteoporosis |
| CN114333990A (en) * | 2020-09-30 | 2022-04-12 | 北京瑷格干细胞科技有限公司 | Postmenopausal osteoporosis related gene screening and function analysis method |
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