CN108179181A - Application of the RDX genes in clinical application - Google Patents
Application of the RDX genes in clinical application Download PDFInfo
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Abstract
The present invention relates to application of the RDX genes in clinical application, more particularly relate to new opplication of the RDX genes in postmenopausal osteoporosis clinical application and detection postmenopausal osteoporosis.Inventor, to the molecular mechanisms of action of osteoporosis, is illustrated Liuwei Dihuang Wan mechanism of action from gene angle, picks out candidate gene RDX and verify using high throughput sequencing technologies research Liuwei Dihuang Wan.The present invention provides molecular theory basis for postmenopausal osteoporosis and clinical application, has good clinical value.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to application of the RDX genes in clinical application more particularly relates to
New opplication of the RDX genes in postmenopausal osteoporosis clinical application and detection postmenopausal osteoporosis.
Background technology
Radixin (RDX) connects albumen for cell membrane-cytoskeleton, is the ERM connected between cell membrane and cytoskeleton
(ezrin/radixin/moesin) one of family member, is expressed in liver cell, kidney cell etc., participate in cellular morphology adjust,
A variety of vital movements such as signal transduction, cell movement.RDX is mainly made of 3 structural domains：What is connect with cell membrane is highly conserved
Spherical amino terminal, intermediate region is alpha helical region, and threonine residues are contained in positively charged carboxyl terminal, the region, can
Phosphoric acid is phosphorylated and gone, is the major regulatory site of RDX.It is existing research shows that RDX maintain liver cell polarity in terms of send out
Wave important function (Re-defining ERM function in lymphocyte activation and migration,
Immunol Rev, 2013,256 (1):63-79).
Osteoporosis (osteoporosis) is that h and E factor is coefficient as a result, being a complexity
Multi-factor disease.In recent years, with the further investigation of accurate medical treatment, it has been found that part related molecule sign, we it
Preceding research is it has also been found that the relevant gene of some osteoporosises, such as IFT52 genes (CN2015106280814), EPS8L3 genes
(CN2015106280424), CERS2 genes (CN2015106293481), MTUS1 genes (CN 2015106280246) etc..Absolutely
Osteoporosis (postmenopausal osteoporosis, PMOP) is one kind of osteoporosis after, related with aging,
Postmenopausal women is occurred mainly in, since estrogen deficiency leads to bone amount reduction and bone structure variation, increases bone brittleness
The problems such as being easy to fracture and the pain as caused by fracture, textured bone, complication occur or even is dead, severely impacts old
The health and quality of life of year people or even shorten the service life and increase country and family's financial resources and manpower burden.
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 apparent, and do not find apparent side effect to postmenopausal osteoporosis, is gradually taken seriously.Hair
A person of good sense studies molecular mechanisms of action of the Liuwei Dihuang Wan to osteoporosis using high throughput sequencing technologies, to postmenopausal osteoporosis
Patient takes the forward and backward peripheral blood sample of Liuwei Dihuang Wan and Healthy People control peripheral blood sample carries out sequencing analysis, from gene angle
Degree illustrates its mechanism of action, picks out candidate gene RDX.The present invention provides molecule reason for postmenopausal osteoporosis and clinical application
By basis, there is good clinical value.
Invention content
Reagent the purpose of the present invention is to provide detection RDX genes and/or RDX albumen is preparing postmenopausal osteoporosis
Application in clinical application genetic test preparation.
To achieve the above object, the present invention screens candidate base by high-flux sequence combination bioinformatics method first
Because of RDX, it is bright that RDX gene expression amounts after sufferers of osteoporosis face takes Liuwei Dihuang Wan are then demonstrated by molecular biology method
It is aobvious to decline, the gene not only action target spot related to postmenopausal osteoporosis or Liuwei Dihuang Wan is shown, available for preparing
Postmenopausal osteoporosis diagnostic preparation and therapy target have important clinical value.
Further, RDX genes and/or the sample group of RDX albumen height expression are applicable in Liuwei Dihuang Wan.
Further, detection preparation is using one or several kinds of expression for detecting RDX genes in following method：Fluorescent quantitation
PCR method, method for gene chip, high-flux sequence method.
Reagent the purpose of the present invention is to provide detection RDX genes and/or RDX albumen is preparing postmenopausal osteoporosis
Application in diagnostic preparation.
Further, RDX genes and/or RDX the albumen high expression in postmenopausal osteoporosis sample.
Further, the expression feelings of RDX genes and/or RDX albumen in postmenopausal osteoporosis diagnostic preparation detection peripheral blood
Condition.
Further, the diagnostic preparation of the postmenopausal osteoporosis is using one or several kinds of detections in following method
The expression of RDX genes：Fluorescence quantifying PCR method, method for gene chip, high-flux sequence method.
Fluorescence quantitative PCR method is the probe of the specificity 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, the selectivity for making experiment are 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 (DNAmicroarray), can be divided into three kinds of main Types：1) it is fixed on poly-
The nucleic acid probe or cDNA segments on object substrate (nylon membrane, nitrocellulose membrane etc.) surface are closed, usually uses the target of isotope labelling
Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method,
Hybridized by the target gene with fluorescent marker and be detected.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass
Array hybridizes with the target gene of fluorescent marker and is detected.Genetic chip is as a kind of advanced, extensive, high-throughput detection
Technology, applied to the diagnosis of disease, advantage has the following aspects：First, the sensitivity and accuracy of height；It is second is that quick
It is easy；Third, a variety of diseases can be detected simultaneously.
High-flux sequence (High-throughput sequencing) is also known as next-generation sequencing technologies (next
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 simultaneously
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 analysis instrument (Illumina Genome Analyzer) of Illumina companies and the SOLiD sequenators of ABI
(ABI SOLiD sequencer)。
The product of RDX genes contains a pair of special in the detection postmenopausal osteoporosis for fluorescence quantifying PCR method
Property amplification RDX genes primer；The genetic chip includes the probe with the nucleic acid array hybridizing of RDX genes.Preferably, one
To the primer of specific amplification RDX 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 RDX albumen.It is preferred that
RDX protein expressions are western blot and/or ELISA and/colloidal gold in the immunization method detection postmenopausal osteoporosis
Method.
Enzyme-linked immunosorbent assay (ELISA) adsorbs known antigen or antibody in surface of solid phase carriers, makes 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 many advantages, such as 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 enhanced with silver-colored developer solution and marked on the basis of colloid gold label,
The silver atoms being reduced is made to be deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(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
Antigen or antibody point are first added sample to be checked by carrier on film after closing, corresponding with the antibody test of colloid gold label after washing
Antigen or antibody.(4) antigen of specificity or antibody are fixed on ribbon on film by colloidal gold immunity chromatography, 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, it moves forward, reacts 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 object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
It is 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 RDX albumen is uses ELISA detection kit.It is anti-in the kit
Commercially available RDX monoclonal antibodies can be used in body.Further, the kit includes：The solid phase for being coated with RDX monoclonal antibodies carries
Body, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid etc..
Further, the colloidal gold method of the detection RDX albumen is using colloidal gold strip, and antibody uses commercially available RDX
Monoclonal antibody.Further, the colloid gold test paper strip adoption colloidal gold immunochromatographimethod technology or colloidal gold percolation.Into one
It walks, detection zone (T) specking on the colloidal gold strip nitrocellulose filter has anti-RDX monoclonal antibodies, quality control region (C) spray
Point has Immunoglobulin IgG.
The purpose of the present invention is to provide a kind of PCR kit for fluorescence quantitative for detecting postmenopausal osteoporosis, features
It is, the kit detects gene RDX, and using special sense primer and downstream primer, upstream primer sequence is 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 in the market, clever
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 downstream 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.
It is an object of the present invention to provide a kind of postmenopausal osteoporosis detection kit, detection kit detection RDX
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 RDX 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 RDX genes and/or RDX albumen is surveyed, the sample group that RDX genes and/or RDX albumen height are expressed is applicable in Liuwei 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 cores
Piece includes the probe with the nucleic acid array hybridizing of RDX genes, and the sample group that RDX genes and/or RDX albumen height are expressed is applicable in Six-element
Dihuang Wan.
The purpose of the present invention is to provide RDX genes and/or its protein inhibitor to prepare treatment postmenopausal osteoporosis
Application in drug.
Further, the treatment postmenopausal osteoporosis drug refers to inhibit the preparation of the expression of RDX genes.Ability
One kind in following methods and/or several usually may be used in the expression of the known suppressor of domain personnel：By activating RDX genes
Suppressor, activate RDX genes inhibition of gene expression albumen, RDX gene expressions are inhibited using RNA perturbation techniques, are swashed
The microRNA of promotion RDX gene mRNAs degradation living, the molecule for importing promotion RDX gene coded protein degradations, inhibition promote RDX
The factor of gene expression 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, the phenomenon that leading to 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 made to go out the technology of specific gene missing phenotype.SiRNA is designed
Direct synthesis technique or structure SiRNA expression vector may be used after the completion, the siRNA prepared can be coprecipitated by calcium phosphate
Mechanical Methods, 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, inhibit one kind in following sequence of siRNA target spots of RDX gene expressions and/or several：SEQ ID
NO.5、SEQ ID NO.8、SEQ ID NO.11.It is preferred that siRNA sequence is SEQ ID NO.5.
Further, described one kind in following sequence of siRNA sequence for inhibiting RDX 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.6, SEQ ID NO.7 to select siRNA sequence.
Postmenopausal osteoporosis drug, the treatment postmenopausal osteoporosis are treated the purpose of the present invention is to provide a kind of
The expression of Drug inhibition RDX genes.
Further, treatment postmenopausal osteoporosis drug is 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 in addition to mentioned component other than can also comprising lubricant, wetting agent, sweetener, flavouring agent,
Emulsifier, 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 non-oral be administered, during as non-oral administration, vein can be passed through
Interior injection, intranasal injection, local injection, intraventricular injection, spinal cavity injection are 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, administration route, 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 effective to desirable treatment or prevention to medicament
Amount.
The method that the composition of the present invention can easily be implemented according to general technical staff of the technical field of the invention,
Carried out using carrier receptible in pharmacy and/or excipient it is formulation, so as in the form of unit dose prepare or
It is prepared in multicapacity container in person.At this point, dosage form is solution, suspension or emulsion in 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 meaning of logical technical staff institute.
Description of the drawings
Fig. 1 is that RDX genes relative expression's spirogram Fig. 2 in postmenopausal osteoporosis peripheral blood and healthy human peripheral blood is
RDX genes relative expression's spirogram in peripheral blood before and after postmenopausal osteoporotic patients take Liuwei Dihuang Wan
Fig. 3 is each group RDX mRNA relative expression levels figure after RNA interference：C groups：Blank control group；C1 groups：Transfect lipid
Body group；C2 groups：Transfect nonspecific siRNA groups；S1, S2, S3 group：Transfect the siRNA groups of specificity.
Specific 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 this
The limitation of invention.It will be understood by those skilled in the art that：It 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.Test method without specific conditions in the following example, usually according to normal condition or according to the item 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, the mass value including base point with Fast-QC softwares to the quality of sequencing data after sequencing
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
It has gone Gene Onlogy and signal path analysis, and functional annotation and protein interaction net is carried out to difference expression gene
Network analyze, in view of data above analysis as a result, we have screened difference expression gene RDX with reference to document.RDX is in osteoporosis
The high expression of group, and the low expression after Liuwei Dihuang Wan is taken.
2 postmenopausal osteoporotic patients of embodiment take peripheral blood RDX expression conditions one, material before and after Liuwei Dihuang Wan
Material and method
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
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；70 DEG C of water-baths keep the temperature 1 hour after electrophoresis pattern and the collection of illustrative plates no significant difference before water-bath heat preservation.
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 in 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.
2.3 Real-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_001260492.1, by invitrogen after design of primers
Company synthesizes.Specific primer sequence is as follows：
RDX primers：
5’-agtcattcctccaacaga-3’(SEQ ID NO.1)
5’-gttcttcctcgcttctatg-3’(SEQ ID NO.2)
Amplification length 123bp.
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 in 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, 58 DEG C of 60sec) × 35 cycles.
Sample RealTimePCR is detected：2 μ l is taken to make template after cDNA10 times of each sample is diluted, use target gene respectively
Primer and reference gene primer are expanded.Simultaneously solubility curve analysis is carried out at 60-95 DEG C.2nd, experimental result
Real-time quantitative PCR result shows and (is specifically shown in Fig. 1)：Compare RDX genes and take six in postmenopausal osteoporotic patients
RDX expressions in peripheral blood before and after taste Dihuang Wan, RDX gene expression amounts ratio takes preceding entire lowering after taking Liuwei Dihuang Wan
1/3rd or so, it is with high-flux sequence result trend consistent.
RDX expression conditions in 4 postmenopausal osteoporotic patients peripheral blood of embodiment and healthy human peripheral blood
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
Specific steps are with reference to embodiment 3.
3rd, result
Real-time quantitative PCR amplification curve inflection 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,
The expression of qRT-PCR stable amplification results, wherein RDX in postmenopausal osteoporosis peripheral blood is higher than healthy human peripheral blood
In expression, the former is about 1.26 times (see Fig. 2) of the latter, and result above demonstrates the result of high-flux sequence.
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 fetal calf serum 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
1 discards culture solution original in the culture bottle for covering with cell, adds in 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 round, carefully
Matrix bounces back, and space between cells increases, and discards pancreatin in also non-levitating, adds in the culture solution that 5ml contains 10% fetal calf serum
Terminate digestion；
3 cell counts：Above-mentioned cell suspension 0.5mI is taken, is instilled in blood cell counting plate after appropriate dilution, based on leucocyte
Four big lattice inner cells sum of number method number quadrangle, 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 are 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, every milliliter are further diluted to containing 3 × 10 with DMEM complete culture solutions5A cell is dense
Degree, is sub-packed in culture bottle (every bottle of 8m1/), is positioned over 37 DEG C, 5%CO2It is cultivated in incubator.
6 RNAi of embodiment inhibits RDX gene expressions
First, experiment material
SiRNA is built and synthesis
According to RDX genes in GenBank (NCBI Reference Sequence:NM_001260492.1 sequence is set in)
Count 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 inhibits the expression of RDX 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), referred to as shRNA (short
hairpin RNA).Synthesize the DNA profiling of every coding shRNA two are single-stranded, and annealed dna is single-stranded to obtain the DNA double of shRNA
Chain template.Template strand followed by RNAPoIyIII 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.
After siRNA empty carriers 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
Object 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.
1 siRNA transcription templates sequences of table
3rd, cell grouping 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 the siRNA groups of specificity.
(2) it transfects
According to LipofectamineTMThe step of 2000Transfection Reagent are provided carries out.
1. before transfection for 24 hours, the cell pancreatin in growth period of taking the logarithm is digested and is counted, dense with DMEM culture mediums adjustment cell
Spend is 1 × 105/ ml takes 2m1 to be inoculated in six orifice plates, is positioned over 37 DEG C, 5%CO2It cultivates in incubator, is merged in cell up to 80%
When for transfecting.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, mild mixing；
B liquid：250u1 serum free mediums dilute 10u1Lipofectamine, and mild mixing is placed at room temperature for 5min；
3. it transfects：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 mixing.In CO2Liquid is changed after 37 DEG C of heat preservations 24-48h, 6h in incubator, adds in the culture medium containing serum.
4th, the verification of transfection efficiency
(1) cellular morphology and transfected condition are observed under fluorescence inverted microscope
After transfection for 24 hours, culture plate is placed under fluorescence inverted microscope and observes cellular morphology and growth conditions, green fluorescence
Lower observation transfected condition.
(2) using the variation of RDX 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 culture 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, obtain being equivalent to 104-
The DNA profiling of 100copies/ul is separately added into RDX primers and internal reference actin primers, prepares 25u1 reaction systems, uses
Real-time PCR amplification instruments carry out pcr amplification reaction.Obtain the standard curve of RDX and actin.
2. the variation of RDX gene expressions before and after the detection transfection of Real-time PCR methods：The RNA of each group cell is extracted, is surveyed
Determine RNA concentration and purity, carry out reverse transcription reaction, the Real-time PCR that every group of DNA profiling is carried out at the same time RDX and actin are anti-
Should, experiment is in triplicate.
3. to PCR product into row agarose gel electrophoresis.
3rd, experimental result
Real-time PCR detect transfection efficiency.Standard using Real-time PCR methods structure RDX and actin is bent
Line, linear relationship is good, meets the requirements.Compare the expression of each group RDX 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.RDX-siRNA1,
RDX-siRNA2, RDX-siRNA3 play the role of inhibiting RDX gene expressions, and the effect of RDX-siRNA1 becomes apparent from, and inhibit effect
Rate is up to 75%, and the inhibiting effect of RDX-siRNA2 and RDX-siRNA3 is respectively 21% and 32%, with blank control group, lipid
Body transfection group, nonspecific transfection group are compared, and difference is statistically significant, P<0.05 (Fig. 2).
The present invention filters out postmenopausal osteoporosis pathogenic related gene RDX using high-flux sequence, and further analysis is aobvious
Show, the gene is related to Liuwei Dihuang Wan medication effect, and aspect is of great significance in terms of clinical guidance medication, inventor's knot
Close molecular cytobiology experimental verification, it was confirmed that RDX has good correlation with postmenopausal osteoporosis disease, and provides
Interfere the siRNA of RDX gene expressions.The present invention provides new diagnosis target spot and medication for postmenopausal osteoporosis clinic diagnosis
Target spot has good potential applicability in clinical practice.
Sequence table
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<120>Application of the RDX genes in clinical application
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Claims (10)
1. the reagent of detection RDX genes and/or RDX albumen is in postmenopausal osteoporosis clinical application genetic test preparation is prepared
Application.
2. application according to claim 1, which is characterized in that RDX genes and/or the sample group of RDX albumen height expression are fitted
Use Liuwei Dihuang Wan.
3. application of the reagent of detection RDX genes and/or RDX albumen in postmenopausal osteoporosis diagnostic preparation is prepared.
4. application according to claim 3, which is characterized in that in postmenopausal osteoporosis diagnostic preparation detection peripheral blood
The expression of RDX genes and/or RDX albumen.
5. application according to claim 3, which is characterized in that the diagnostic preparation of postmenopausal osteoporosis uses following method
In one or several kinds of detection RDX 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 RDX genes；Genetic chip includes and RDX genes
Nucleic acid array hybridizing probe.
6.RDX genes and/or its protein inhibitor are preparing the application in treating postmenopausal osteoporosis drug.
7. application according to claim 6, which is characterized in that treatment postmenopausal osteoporosis drug is using in following methods
One kind and/or several inhibition RDX genes and/or RDX protein expressions：Activate suppressor, the activation RDX genes of RDX genes
Inhibition of gene expression albumen, RDX gene expressions, activation is inhibited to promote the degradation of RDX gene mRNAs using RNA perturbation techniques
MicroRNA, the molecule for promoting the degradation of RDX gene coded proteins, the factor of inhibition promotion RDX gene expressions and albumen are imported
Expression.
8. application according to claim 7, which is characterized in that RDX gene expressions are inhibited using RNA perturbation techniques
One kind in following sequence of siRNA target spots and/or several：SEQ ID NO.5、SEQ ID NO.8、SEQ ID NO.11.
It is preferred that siRNA target sequences are SEQ ID NO.5.
9. a kind of PCR kit for fluorescence quantitative for detecting postmenopausal osteoporosis, which is characterized in that the kit detects gene
RDX, using special sense primer and downstream primer, upstream primer sequence is SEQ ID NO.1, downstream primer sequence SEQ
ID NO.2。
10. a kind of treat postmenopausal osteoporosis drug, which contains the siRNA for the expression for inhibiting RDX genes, it is preferred that
SiRNA sequence is SEQ ID NO.6, SEQ ID NO.7.
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Citations (2)
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| US20110070237A1 (en) * | 2003-03-04 | 2011-03-24 | Calcimedica, Inc. | Methods of modulating and identifying agents that modulate intracellular calcium |
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