CN107326076A - A kind of scoliosis early stage auxiliary detection kit and its application - Google Patents
A kind of scoliosis early stage auxiliary detection kit and its application Download PDFInfo
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- CN107326076A CN107326076A CN201710567399.5A CN201710567399A CN107326076A CN 107326076 A CN107326076 A CN 107326076A CN 201710567399 A CN201710567399 A CN 201710567399A CN 107326076 A CN107326076 A CN 107326076A
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Abstract
The invention discloses a kind of scoliosis early stage auxiliary detection kit.The invention also discloses a kind of siRNA molecule for suppressing HMGN1, P3H2 and NOLC1 gene expression, described siRNA molecule is preparing the application in treating scoliosis pharmaceutical composition.It can not only fast and effectively accomplish early diagnosis using HMGN1, P3H2 and NOLC1 genetic test scoliosis, and develop for predictive diagnosis AIS, even provide therapy target and important evidence to treat AIS in biology level from now on.
Description
Technical field
The present invention relates to biomedicine field, and in particular to a kind of scoliosis early stage aids in detection kit and its should
With.
Background technology
Scoliosis (Scoliosis), also known as scoliosis, are that a class is bent with backbone side and with Vertebral rotation
Complex three-dimensional deformity of spine.According to pathogenic factor, 3 major classes can be classified as：Congenital scoliosis, syndrome scoliosis
And idiopathic scoliosis.Congenital scoliosis is caused by vertebral column development deformity；Syndrome scoliosis is by god
Caused by muscle disease, skeletal diseases, connective tissue disease and neurofibromatosis etc.；The definite cause of disease of idiopathic scoliosis
It is unclear, correlative study think gene genetic factor, grow with hormone, connective tissue lesion, muscle systems it is abnormal,
Central nervous system exception, effect of epiphysin etc. all may be relevant with AIS morbidity.Wherein, adolescent idiopathic backbone side
Curved (Adolescent Idiopathic Scoliosis, AIS) is most commonly seen, accounts for the 80% of whole scoliosis.Teenager is special
The incidence of disease of the hair property scoliosis in -16 years old 10 years old teenagers is higher, is after eyesight abnormality, obesity, phimosis and social mentality
The fifth-largest common disease after obstacle.Although AIS does not generally cause serious pathological state in addition to deformity is showed, severe chest is curved
The decline of the heart, PFT may be caused, cause the deformity on obvious build, or even influence life-span.
Current treatment method is rescued primarily directed to deformity, including clinical observation, Brace Treatment and lateral bending is exceeded
45 ° of persons carry out operative treatment, and treatment is complicated, has often left deformity of spine and backbone moving obstacle, effect is undesirable.Meanwhile, AIS
If patient is without examination, in first go to a doctor, the curved average angle of master is 38 °, already close to the upper limit for carrying out Brace Treatment.
Therefore, early screening and diagnosis to AIS can strive for the chance of more expectant treatments for patient, reduce operation probability.At present
AIS diagnosis is with examination mainly or by the physical examination to patient's outward appearance symmetry and imageological examination, and this can make a lot
The teenager of low-risk receives unnecessary radiological examination, and this examination means specificity is poor.With medical genetics
Learn and Medical Molecular Biology research deepening continuously and improves, the research of AIS Medical Molecular Biology mechanism also successively by
Report.Some relative biomarkers are found that during molecular biology research is carried out to AIS, such as calcium adjusts egg
In vain, leptin, Serum osteopontin, solubility CD44, SH3GL1 etc. in peripheral blood, but effect and conclusion still need to further checking.
Therefore, get on to study the AIS causes of disease from gene expression dose, continually look for AIS early screenings new, that specificity is good and diagnosis
Label is significant.
The content of the invention
In order to realize the early detection of Adolescent idiopathic scoliosis, early treatment, it is an object of the invention to provide
A kind of kit for scoliosis early screening.
Another object of the present invention is to provide a kind of suppression HMGN1 genes and/or P3H2 genes and/or NOLC1 genes
The siRNA molecule of expression, and the siRNA is in the application in treatment scoliosis pharmaceutical composition is prepared.
To achieve the above object, present invention firstly provides a kind of scoliosis early stage auxiliary detection kit, it includes inspection
The primer of HMGN1 genes such as SEQ ID NO in test sample sheet:1 and SEQ ID NO:Nucleotide sequence shown in 2.
It is preferred that, the primer that the kit also includes being used to detect P3H2 genes includes such as SEQ ID NO:3 and SEQ
ID NO:Nucleotide sequence shown in 4.
It is preferred that, the primer that the kit also includes being used to detect NOLC1 genes includes such as SEQ ID NO:5 and SEQ
ID NO:Nucleotide sequence shown in 6.
It is preferred that, the kit also includes：
(1) total RNA extraction reagent in tissue samples or blood：Trizol, chloroform, isopropanol and 75% ethanol etc..
(2) Reverse Transcription：RT Buffer, reverse transcriptase, the enzyme of heat-stable DNA polymerase activity, RNase suppress
Agent, OligodT Primer and Random 6mers.
(3) quantitative PCR reagent：PCR buffer solutions, SYBR Green fluorescent dyes, the SYBR Green polymerizations of dNTPs compositions
PCR system and RNase Free H2O。
It is preferred that, the kit contains positive control and negative control.Preferably, the positive control is normal human blood
Liquid RNA or DNA, the negative control are ddH2O。
It is preferred that, the kit using HMGN1 genes and/or P3H2 genes in real-time PCR detection samples and/
Or the expression of NOLC1 genes, HMGN1, P3H2 and NOLC1 gene up-regulated expression in biological sample.
Further, the invention provides a kind of siRNA molecule of suppression HMGN1 gene expressions, the siRNA molecule is
SEQ ID NO:9 and SEQ ID NO:10.
Further, present invention also offers a kind of siRNA molecule of suppression P3H2 gene expressions, the siRNA molecule
For SEQ ID NO:11 and SEQ ID NO:12.
Further, present invention also offers a kind of siRNA molecule of suppression NOLC1 gene expressions, the siRNA molecule
For SEQ ID NO:13 and SEQ ID NO:14.
Further, the invention provides the siRNA molecule in treatment scoliosis pharmaceutical composition is prepared
Using.
It is preferred that, the medicine includes suppressing HMGN1 genes and/or P3H2 genes and/or NOLC1 bases by RNA interfering
Protein because of the double stranded RNA of expression or for suppressing HMGN1 and/or P3H2 and/or NOLC1 protein actives.
In the present invention, the RNA interference (RNA interference, RNAi) refers to be highly conserved during evolution
, induced by double-stranded RNA (double-stranded RNA, dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Make
With RNAi technology can with specific depletion or close specific gene expression, the technology have been widely used for explore gene function and
In the field of gene of many diseases.RNAi based on cell is screened in terms of functional gene research with many excellent
Gesture, RNAi methods can be used by being mainly manifested in most cell types, and be easier to up-regulation or any purpose of silence relatively
The expression of gene.
In order to ensure HMGN1 genes and/or P3H2 genes and/or NOLC1 genes can be rejected efficiently or silence, according to
The mRNA sequence of HMGN1 genes and/or P3H2 genes and/or NOLC1 genes devises siRNA specific fragments.SiRNA passes through
Online tool design is completed, and the online tool is http://sidirect2.rnai.jp/, siRNA oligonucleotides are by Shang Haiji
The chemical synthesis of agate Pharmaceutical Technology Inc..
It is preferred that, the medicine also includes pharmaceutically acceptable carrier, and this kind of carrier includes (but being not limited to)：Dilution
It is agent, buffer, supensoid agent, emulsion, granule, encapsulation agents, excipient, filler, adhesive, spray, cutaneous permeable agent, wet
Moisten agent, disintegrant, sorbefacient, surfactant, colouring agent, flavouring or absorption carrier.Preferably, the medicine can root
According to needing to be prepared into various formulations, include but is not limited to, tablet, solution, granule, patch, paste, capsule, aerosol
Or suppository.
It is preferred that, the drug combination that the medicine can also be with other scoliosis, multi-medicament is used in combination can be significantly
Improve the success rate for the treatment of.
It is preferred that, the scoliosis is Adolescent idiopathic scoliosis.
Beneficial effects of the present invention are as follows：The invention discloses a kind of gene HMGN1, the P3H2 related to scoliosis and
NOLC1, and further confirm HMGN1, P3H2 and NOLC1 gene or its expressing protein in Adolescent idiopathic scoliosis patient
Up-regulated expression in biological sample.The generaI investigation of scoliosis is carried out in teenager using HMGN1, P3H2 and NOLC1 gene, is solved
The problem of diagnostic measures Sensitivity and Specificity difference in the prior art of having determined, the infringement of x-ray radiation is decreased, can quickly be had
The early diagnosis for accomplishing AIS of effect, and developing for predictive diagnosis AIS, even to be controlled from now in biology level
Treat AIS and provide therapy target and important evidence.
Brief description of the drawings
Scoliosis patient HMGN1, P3H2 and NOLC1 gene expression are raised in embodiment 2 in Fig. 1 present invention.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means.
The experimental method of unreceipted actual conditions in embodiment, usually this area conventional method, such as according to normal condition
The condition in such as Sambrook et al., molecular cloning, laboratory manual (third edition) (Science Press, 2002), or according to
Condition proposed by reagent manufacturing firm.
Technical scheme is specifically included：20 Adolescent idiopathic scoliosis clinical samples and 15 are compareed
Sample carry out high-flux sequence, with reference to bioinformatics method carry out genescreen, pick out candidate gene HMGN1, P3H2 and
NOLC1, does not have HMGN1, P3H2 and NOLC1 report related to Adolescent idiopathic scoliosis, enters one in existing research
Step, inventor has carried out molecular biology method checking, it was confirmed that HMGN1, P3H2 and NOLC1 are in scoliosis patient's biology
Up-regulated expression in sample, its related preparations can be used for diagnosis Adolescent idiopathic scoliosis.
HMGN1, P3H2 and NOLC1 gene of the present invention is known before making the present invention, and its essential information is as follows：
Genbank accession number：HMGN1GeneID:3150, P3H2GeneID:55214, NOLC1GeneID:9221 sources
In human genome.
HMGN1 (Homo sapiens high mobility group nucleosome binding domain 1, people
High mobility group nucleosome binding domain protein 1) it is located on No. 21 chromosomes, belong to one of HMGN family members, HMGN families bag
Containing five kinds of chromosome structure albumen, i.e. HMGN1~HMGN5, they are expressed in vertebrate cells, specific tissue specificity.
Except that can be combined and influenceed with DNA, it is replicated HMGN1 and transcription is outer, is also developed in DNA damage reparation, Organ Differentiation, disease
Played a role during generation etc..
P3H2 (prolyl 3-hydroxylase 2, prolyl hydroxylase 2) is intracellular important Fe2+, -one penta
The dioxygenase oxygen receptor that diacid is relied on, mainly including PHD1, PHD2 and PHD3.PHD2 expression quantity in cartilage cell is high,
PHD2 is the Main Factors of the oxygen dependence degraded of regulation and control HIF-1 α in cartilage cell；PHD2 also participate in SDC4 in cartilage cell according to
Rely the regulation and control of HIF-1 α approach, the expression of influence polyprotein glycan and II Collagen Type VIs.
NOLC1 (nucleolar and coiled-body phosphoprotein 1, kernel and spirochetal protein 1) is
The mammalian proteins of one hyperphosphorylation, had both participated in the generation of kernel and rDNA transcription, and inflammation generation, cell are participated in again
The regulation and control of the associated signal paths such as growth, Apoptosis, tumour generation.Studies have reported that, in the generation of nasopharyngeal carcinoma and liver cancer
In development, NOLC1 plays vital effect.
The experimental method specifically studied mainly includes following components：
1. utilize blood sample HMGN1, P3H2 and NOLC1 gene of the high-flux sequence method to 20 scoliosis patients
Level and 15 check samples in carry out comparison in difference.
2.qRT-PCR verifies the expression of scoliosis patient's HMGN1, P3H2 and NOLC1 gene
Table of HMGN1, P3H2 and NOLC1 gene in scoliosis patient and control group is detected using RT-PCR method
Reach, and demonstrate the gene for scoliosis up-regulated expression gene.
HMGN1, P3H2 and NOLC1 are expressed in 3.RNAi interference scoliosis patient cartilage cells
(1) cell culture；
(2) siRNA designs and synthesis；
(3) cell transfecting；
(4) QPCR is detected.
4. the kit of the scoliosis of the present invention includes consisting of part：
(1) primer sets in embodiment 2 described in table 1；
(2) blood sample extracts total serum IgE reagent；
(3) Reverse Transcription；
(4) quantitative PCR reagent.
5. the clinical practice of kit
The kit prepared using the present inventor detects scoliosis patient to be made a definite diagnosis and compared with actual clinical is detected
Compared with so that the validity of kit is determined.Specifically include HMGN1, P3H2 and NOLC1 gene table in measure subject's blood sample
Be compared up to amount, and with HMGN1, P3H2 and NOLC1 gene expression amount in normal blood sample, be clinician quick and precisely
The morbid state and coincident with severity degree of condition of patient is grasped, takes the control prece of more personalized to provide support in time.
The nucleotides full length sequence or its fragment of HMGN1, P3H2 and NOLC1 gene of the present invention can generally be expanded with PCR
Increasing method, recombination method or artificial synthesized method are obtained.Once obtain relevant sequence, it is possible to recombination method come in large quantity
Obtain relevant sequence.
When " scoliosis " used herein is not explained, Adolescent idiopathic scoliosis is typically refered in particular to.
Terms used herein " biological sample " includes but is not limited to the samples such as blood, serum, saliva, urine, synovia, cartilage
Product.Biological sample for the present invention derives from any tissue sample (such as interverbebral disc, articular process, spinal cord, the vertebra of any subject
Other flesh or blood sample) or cell sample (such as cartilage cell, cartilage cell or blood cell samples) can be made according to the inventive method
With.Can therefrom obtain these samples and according to the inventive method using these samples subject's example including but not limited to without
The subject of symptom subject, performance or more scoliosis symptom, clinical diagnosis are the subject with scoliosis, are susceptible to suffer from
Scoliosis subject (if any the subject of scoliosis family history, have scoliosis genetic predisposition subject and
Life style makes the subject that scoliosis possibility is suffered from its susceptible scoliosis or raising), suspect with scoliosis by
Examination person, just receiving scoliosis treatment subject, with scoliosis and it is non-receive scoliosis treatment subject, opening
Doctor (such as doctor) is defined as health or subject (i.e. normally) without scoliosis, the subject for having cured scoliosis, just
Control the subject of its scoliosis and have not been diagnosed as the subject of scoliosis.
The high-flux sequence of embodiment 1 screens difference expression gene
1st, sample
Choose the scoliosis patient gone to a doctor during in October, 2012 in December, 2015 in BJ Union Hospital's orthopaedics, disease
Example group collects 20 altogether, and all patients have typical clinical manifestation, are made a definite diagnosis through x-ray inspection.Control derives from same time orthopaedics
Other diseases patient in hospital, collects 15 altogether.Patient is the teenager of -18 years old 10 years old.Gather the blood of all research objects
Liquid sample, numbers rearmounted -80 DEG C of low temperature refrigerators and preserves.All clinical samples of this research, to patient know and inform simultaneously
Pass through through this Hospital Ethical Committee.
2nd, Total RNAs extraction is carried out to blood sample
UsingLS(Invitrogen:RNA extractions, experiment 10296-010) are carried out to the blood sample of collection
Operation is carried out by product description, and the concrete operations of every group of experiment are as follows：
The new blood being collected into is taken, 3 times of volume erythrocyte cracked liquids are added, room temperature placement 10 minutes after mixing, 10,
000rpm is centrifuged 1 minute.Thoroughly inhale and abandon supernatant, collect leukocyte cell pellet.
(1) 1mLTrizol, room temperature preservation 5 minutes are added；
(2) chlorination imitates 0.2mL, uses forced oscillation centrifuge tube, fully mixes, and room temperature is placed 3-5 minutes；
(3) 12000rpm high speed centrifugations draw upper strata aqueous phase (inhaling 70%) into another new centrifuge tube pipe after 15 minutes, note
Meaning should not be drawn onto the interface between two layers of aqueous phase.New pipe is moved into, -20 DEG C of isometric pre- cold isopropanols are added, it is fully reverse mixed
It is even, it is placed in 10 minutes on ice；
(4) supernatant carefully is discarded after 12000rpm high speeds were from 15 minutes, 75% is added in 1mL/mL Trizol ratio
DEPC ethanol washes paint precipitation (4 DEG C of preservations), washes paint sediment, vibration is mixed, and 8000rpm is centrifuged 2 minutes at 4 DEG C.Discard ethanol
Liquid, places 2 minutes fully to dry precipitation at room temperature, adds the treated water dissolving precipitations of DEPC；
(5) with Nanodrop2000 ultraviolet specrophotometers measurement RNA purity and concentration, freeze in -80 DEG C.RNA mass
Criterion：The OD260/OD280 values of RNA samples is between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly 28S, 18S bar
Band；70 DEG C of water-baths be incubated 1 hour after electrophoresis pattern be incubated with water-bath before collection of illustrative plates no significant difference.
3rd, the quality analysis of RNA sample
Agarose gel electrophoresis after RNA is extracted, the RNA sample that can be extracted with preliminary judgement from electrophoresis result it is up-to-standard with
It is no, if to can be used for further transcriptome analysis.And then RNA sample is detected by NanoDrop1000 spectrophotometers
Extraction situation, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2.
4th, high-flux sequence
Microarray dataset is the high-flux sequence platforms of HiSeq 2500 of Illumina companies, carries out high flux transcript profile depth
Sequencing, we use Fast-QC (http after sequencing://www.bioinformatics.babraham.ac.uk/projects/
Fastqc/) software carries out total evaluation to the quality of sequencing data, includes the quality Distribution value of base, the position point of mass value
Cloth, G/C content, PCR duplication contents, kmer frequency etc..In differential genes expression analysis, according to obtaining
FPKM values, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, during screening, LOG2FC>1 or<- 1, FDR<
0.05.In order to be better understood from the function of difference expression gene, we difference expression gene has been carried out Gene Ontology and
Signal path is analyzed, and carries out functional annotation and protein interaction network analysis to difference expression gene, in view of above number
According to the result of analysis, with reference to document, we have screened difference expression gene HMGN1, P3H2 and NOLC1, HMGN1, P3H2 and
NOLC1 genes up-regulated expression in scoliosis blood samples of patients sample.
The qRT-PCR of embodiment 2 verifies the expression of scoliosis patient's HMGN1, P3H2 and NOLC1 gene
1st, material
Choose the scoliosis patient 15 gone to a doctor during in October, 2012 in December, 2015 in BJ Union Hospital's orthopaedics
Example, all patients have typical clinical manifestation, made a definite diagnosis through x-ray inspection.Other diseases that control is in hospital from same time orthopaedics
Patient, collects 8 altogether.Patient is the teenager of -18 years old 10 years old.Gather after the blood sample of all research objects, numbering
Put -80 DEG C of low temperature refrigerator preservations.All clinical samples of this research, to patient know and inform and entrusted through this hospital ethics
Member can pass through.
2nd, method
2.1 pairs of blood samples carry out Total RNAs extraction, the extracting method of be the same as Example 1.
2.2 reverse transcriptions synthesize cDNA
UsingRT reagent kit (TaKaRa, article No. DRR037A) carry out cDNA reverse transcriptions, real
Test operation to carry out by product description, concrete operations are as follows：
Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to 0.3 μ g total serum IgEs with RT Buffer.Using 10 μ
L reaction systems：5xPrimerScript Buffer 2μL、PrimeScript RT Enzyme Mix I 0.5μL、OligodT
The μ L of Primer 0.5, Random 6mers 0.5 μ L, RNA templates are 0.3 μ g and RNase Free dH2O is mended to 10 μ L.Obtain
CDNA preserve that to put -20 DEG C of refrigerators standby.
2.3 Real-Time PCR
Using online primer-design software, HMGN1 gene orders are with reference to NCBI:NM_004965.6, P3H2 gene order are joined
According to NCBI:NM_001134418.1, NOLC1 gene order are with reference to NCBI:NM_001284388.1, interior participation in the election GAPDH, primer is set
Synthesized after meter by invitrogen companies.Specific primer is as shown in table 1：
The primer sequence table of table 1
WithPremix Ex TaqTMII (TaKaRa, article No. DRR081A) is expanded, and experimental implementation presses product
Specification is carried out.The μ L reaction systems of quantitative fluorescent PCR 20 are as follows：Premix Ex TaqTMII：10 μ L, primer (10
μM)：Forward and reverse each 0.8 μ L, ROX Reference DyeII (50 ×) of primer：0.4 μ L, dH2O：6 μ L, template cDNA：2μL.
Response procedures are carried out on ABI7500, and amplification program is：95 DEG C of 30sec, (95 DEG C of 5sec, 60 DEG C of 34sec) × 40 circulations.
3rd, statistical analysis
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 and without raising up now, 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 qRT-PCR relative quantification formula：2-ΔΔCt× 100%, compare expression of HMGN1, P3H2 and NOLC1 gene in scoliosis patient and control group blood.
As a result show：QRT-PCR stable amplification results, wherein HMGN1, P3H2 and NOLC1 gene are in scoliosis blood samples of patients
Expression is 3.5 times, 3.9 times and 4.1 times in control group blood respectively, as shown in Figure 1.Result above demonstrates high flux
The result of confluence analysis HMGN1, P3H2 and NOLC1 gene up-regulated expression in scoliosis patient of transcript profile expression data.
Said gene is expressed in the RNAi of embodiment 3 interference scoliosis patient cartilage cells
First, cell culture
(1) scoliosis patient's ilium cartilage of acquisition is aseptically used into D-HANKS (Hua Maike, article No.
HMK03230) liquid is rinsed 3 times.
(2) ilium cartilage is shredded into (about 1mm with scissors3Size), it is placed in sterile centrifugation tube.
(3) 20min is digested in 37 DEG C using 0.25% trypsase, is shaken once per 5min.
(4) 800rpm centrifuges 5min, abandoning supernatant.
(5) 0.2% II 37 DEG C of Collagenase Types digest 4h, with 200 mesh sieve net filtrations.
(6) filtrate centrifuges 5min, abandoning supernatant with 800rpm.
(7) DMEM culture mediums are rinsed, 800rpm centrifugation 5min, are repeated 3 times.
(8) blake bottle is inoculated in after cell count, the DMEM culture mediums containing 10% volume hyclone are as nutrient solution, in 37
DEG C, 5%CO2Incubator in cultivate.
2nd, siRNA designs and synthesis
According to (the http of Photographing On-line software siDirect version 2.0://design.rnai.jp/), according to
HMGN1 gene orders are with reference to NCBI:NM_004965.6, P3H2 gene order are with reference to NCBI:NM_001134418.1, NOLC1 base
Because sequence is with reference to NCBI:NM_001284388.1, designs corresponding siRNA, and particular sequence is shown in Table 2.Company's conjunction is sent to after design
Into.
The siRNA sequence list of table 2
3rd, cell transfecting
1st, transfect
Experiment is divided into negative control group：Transfect siRNA-nc and experimental group：Transfect siRNA-HMGN1；Transfect siRNA-
P3H2；SiRNA-NOLC1 is transfected, according to LipofectamineTMThe step of 2000 Transfection Reagent are provided is entered
OK.
(1) by 5 × 104Cell is seeded on 6 orifice plates, and these are used for the cell quantity of initial vaccination, should be able to be at 24 hours
Inside cell confluency is set to reach 70%；
(2) DNA-Lipofectamine is preparedTM2000 compounds:
A. 5 μ L Lipofectamine are diluted with 250 μ L Opti-MEMTM2000, gently mix, 5 points are incubated at room temperature
Clock；
B. experiment each group takes 7.5uL siRNA to add and is diluted in 250uL Opti-MEMI respectively, and gently shake by
It is mixed；
C. after being incubated 5 minutes, by the siRNA and Lipofectamine of dilutionTM20 are incubated at room temperature after 2000 mixing
Minute.
(3) cell, culture medium and siRNA-LipofectamineTM 2000 are added in each hole in culture plate
Compound.Then culture plate is gently shaken, them are sufficiently mixed；
(4) transfection is placed on 37 DEG C, CO2It is incubated 48 hours in incubator.
2nd, the transcriptional level of HMGN1, P3H2 and NOLC1 gene is detected using QPCR
The extraction step be the same as Example 2 of 2.1 cell total rnas.
2.2 reverse transcription step be the same as Example 2.
2.3 QPCR amplification steps be the same as Examples 2.
3rd, experimental result
As a result show, siRNA-HMGN1, siRNA-P3H2 and siRNA-NOLC1 component are transfected compared with negative control group
It can not effectively suppress the expression of HMGN1, P3H2 and NOLC1 gene.
The detection kit assembling of the scoliosis of embodiment 4
The kit of the present embodiment detection scoliosis includes consisting of part：
(1) blood sample, which extracts total serum IgE reagent, includes TRizol, chloroform, isopropanol, 75% ethanol and without enzyme water；
(2) Reverse Transcription includes：5x RT Buffers, SuperRT reverse transcriptases, dNTPS, OligodT
Primer, Random 6mers and RNase Free dH2O, the reverse transcription reaction liquid is included：250mM pH8.3 Tris-
HCl, 375mM KCl, 15mM MgCl2, 50mM DTT.The consumption for carrying out 1 reverse transcription PCR is as shown in table 3, response procedures
For：42 DEG C of 30min, 85 DEG C of 5min.
The Reverse Transcription system of table 3
| Component | Addition |
| 5x RT Buffers | 4μL |
| OligodT Primer(50μM) | 0.5μL |
| Random 6mers(100μM) | 0.5μL |
| SuperRT reverse transcriptases (200U/ μ L) | 1μL |
| dNTPs(2.5mM) | 4μL |
| Total serum IgE | 1μg |
| RNaseFreedH2O | To 20 μ L |
(3) quantitative PCR reagent includes：The primer sequence of table 1 in PCR Mix reaction systems, embodiment 2.The PCR Mix
The component of reaction system includes reaction buffer, dNTPs, Mg2+, Ex TaqHS enzymes andGreen I.1 time is carried out to determine
The consumption for measuring PCR is as shown in table 4.Response procedures are 95 DEG C of 30sec, (95 DEG C of 5sec, 60 DEG C of 34sec) × 40 circulations.It is above-mentioned
Reagent can bought on the market.
The quantitative PCR system of table 4
| Component | Addition |
| PCRMix reaction systems | 10μL |
| Sense primer (10 μM) | 0.5μL |
| Anti-sense primer (10 μM) | 0.5μL |
| Template cDNA | 2.0μL |
| Add sterile purified water | To 20 μ L |
Kit also includes：Positive control is that the blood rna or DNA and negative control of normal person are ddH2O。
One kit can include the consumption that above-mentioned each composition carries out multiple PCR, such as 25 times, 50 times, it is 100 inferior, respectively
The Specific amounts of composition depending on the circumstances or the needs of the situation depending on.
RNA extractions, reverse transcription are carried out to subject's sample into cDNA, according to optimal anti-using the reagent in mentioned reagent box
System enters performing PCR reaction, the control cDNA in quantitatively being detected as QPCR using the arm's length standard product in kit, inspection with condition
The expression quantity of HMGN1, P3H2 and NOLC1 in subject's sample are surveyed with respect to HMGN1, P3H2 and NOLC1 expression quantity in normal person
Change, is analyzed testing result, compares between sample and control and examined using t, P<0.05 is significant difference, is judged to detection sample sun
Property.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Beijing Sai Erweikang biomedicines Science and Technology Ltd.
<120>A kind of scoliosis early stage auxiliary detection kit and its application
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<170> PatentIn version 3.5
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<212> DNA
<213>Artificial sequence
<400> 2
agacagtcag agcctcccat 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
aagaggggaa caccttggct 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
tccatgtgct cagggttagc 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
agcccaccct ttccgttatg 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<400> 6
tggcagactt gagccagaag 20
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<400> 7
ggagcgagat ccctccaaaa t 21
<210> 8
<211> 23
<212> DNA
<213>Artificial sequence
<400> 8
ggctgttgtc atacttctca tgg 23
<210> 9
<211> 21
<212> RNA
<213>Artificial sequence
<400> 9
acuuuugcag gagguuuagc u 21
<210> 10
<211> 21
<212> RNA
<213>Artificial sequence
<400> 10
cuaaaccucc ugcaaaagug g 21
<210> 11
<211> 21
<212> RNA
<213>Artificial sequence
<400> 11
ucacgaauac auucuuagcc a 21
<210> 12
<211> 21
<212> RNA
<213>Artificial sequence
<400> 12
gcuaagaaug uauucgugac a 21
<210> 13
<211> 21
<212> RNA
<213>Artificial sequence
<400> 13
aaaucuauaa acucuuuccg g 21
<210> 14
<211> 21
<212> RNA
<213>Artificial sequence
<400> 14
ggaaagaguu uauagauuuc c 21
<210> 15
<211> 21
<212> RNA
<213>Artificial sequence
<400> 15
auuuugcggu ggaaaugucc u 21
<210> 16
<211> 21
<212> RNA
<213>Artificial sequence
<400> 16
gacauuucca ccgcaaaaug g 21
Claims (8)
1. a kind of scoliosis early stage auxiliary detection kit, it is characterised in that it includes drawing for HMGN1 genes in detection sample
Thing such as SEQ ID NO:1 and SEQ ID NO:Nucleotide sequence shown in 2.
2. kit as claimed in claim 1, it is characterised in that the kit also includes the primer for being used to detect P3H2 genes
Including such as SEQ ID NO:3 and SEQ ID NO:Nucleotide sequence shown in 4.
3. kit as claimed in claim 1 or 2, it is characterised in that the kit also includes being used to detect NOLC1 genes
Primer includes such as SEQ ID NO:5 and SEQ ID NO:Nucleotide sequence shown in 6.
4. a kind of siRNA molecule of suppression HMGN1 gene expressions, it is characterised in that the siRNA molecule is SEQ ID NO:9
With SEQ ID NO:10.
5. a kind of siRNA molecule of suppression P3H2 gene expressions, it is characterised in that the siRNA molecule is SEQ ID NO:11
With SEQ ID NO:12.
6. a kind of siRNA molecule of suppression NOLC1 gene expressions, it is characterised in that the siRNA molecule is SEQ ID NO:13
With SEQ ID NO:14.
7. application of any described siRNA molecule of claim 4~6 in treatment scoliosis pharmaceutical composition is prepared.
8. application as claimed in claim 7, it is characterised in that the medicine includes suppressing HMGN1 genes by RNA interfering
And/or P3H2 genes and/or NOLC1 gene expressions double stranded RNA or for suppressing HMGN1 genes and/or P3H2 genes
And/or the protein of NOLC1 gene proteins activity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710567399.5A CN107326076A (en) | 2017-07-12 | 2017-07-12 | A kind of scoliosis early stage auxiliary detection kit and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710567399.5A CN107326076A (en) | 2017-07-12 | 2017-07-12 | A kind of scoliosis early stage auxiliary detection kit and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107326076A true CN107326076A (en) | 2017-11-07 |
Family
ID=60196709
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710567399.5A Withdrawn CN107326076A (en) | 2017-07-12 | 2017-07-12 | A kind of scoliosis early stage auxiliary detection kit and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107326076A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881228A (en) * | 2017-12-21 | 2018-04-06 | 中国医学科学院北京协和医院 | A kind of molecular marked compound related to adolescent idiopathic scoliosis and its application |
| CN111575361A (en) * | 2020-04-20 | 2020-08-25 | 孙欣 | Test method for treating AIS (automatic identification system) patient by melatonin pathway regulatory factor |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080131877A1 (en) * | 2006-12-02 | 2008-06-05 | Texas Scottish Rite Hospital For Children | Chd7 gene polymorphisms are associated with susceptibility to idiopathic scoliosis |
| US20100055707A1 (en) * | 2008-09-03 | 2010-03-04 | Alain Moreau | Methods of screening for compounds for use as modulators of left-right asymmetry in scoliotic subjects and for monitoring efficacy of an orthopaedic device |
| CN102186998A (en) * | 2008-08-19 | 2011-09-14 | 人类遗传学与健康缅因州研究院 | Micro rna (mirna) and neurofibromatosis type 1 : a role in diagnosis and therapy |
| CN105408744A (en) * | 2013-06-17 | 2016-03-16 | 圣-贾斯汀大学中心医院 | New markers of severe progression of idiopathic scoliosis and their use for stratifying scoliotic patients and predicting risk of developing scoliosis |
| CN106011264A (en) * | 2016-06-30 | 2016-10-12 | 西北工业大学 | Application of miR-489-3p to preparation of medicines for diagnosing and treating human osteoporosis |
| WO2016202944A1 (en) * | 2015-06-16 | 2016-12-22 | Centro De Investigación Biomédica En Red (Ciber) | Kit and method for the diagnosis/prognosis of idiopathic scoliosis |
| CN106676177A (en) * | 2017-01-18 | 2017-05-17 | 西北工业大学 | Application of lnc (long non-coding) RNA lnc-DIF |
| CN106841593A (en) * | 2017-03-24 | 2017-06-13 | 北京赛尔达生物技术有限公司 | Application of the LACC1 and FHL2 genes in scoliosis detection product is prepared |
-
2017
- 2017-07-12 CN CN201710567399.5A patent/CN107326076A/en not_active Withdrawn
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080131877A1 (en) * | 2006-12-02 | 2008-06-05 | Texas Scottish Rite Hospital For Children | Chd7 gene polymorphisms are associated with susceptibility to idiopathic scoliosis |
| CN102186998A (en) * | 2008-08-19 | 2011-09-14 | 人类遗传学与健康缅因州研究院 | Micro rna (mirna) and neurofibromatosis type 1 : a role in diagnosis and therapy |
| US20100055707A1 (en) * | 2008-09-03 | 2010-03-04 | Alain Moreau | Methods of screening for compounds for use as modulators of left-right asymmetry in scoliotic subjects and for monitoring efficacy of an orthopaedic device |
| CN105408744A (en) * | 2013-06-17 | 2016-03-16 | 圣-贾斯汀大学中心医院 | New markers of severe progression of idiopathic scoliosis and their use for stratifying scoliotic patients and predicting risk of developing scoliosis |
| WO2016202944A1 (en) * | 2015-06-16 | 2016-12-22 | Centro De Investigación Biomédica En Red (Ciber) | Kit and method for the diagnosis/prognosis of idiopathic scoliosis |
| CN106011264A (en) * | 2016-06-30 | 2016-10-12 | 西北工业大学 | Application of miR-489-3p to preparation of medicines for diagnosing and treating human osteoporosis |
| CN106676177A (en) * | 2017-01-18 | 2017-05-17 | 西北工业大学 | Application of lnc (long non-coding) RNA lnc-DIF |
| CN106841593A (en) * | 2017-03-24 | 2017-06-13 | 北京赛尔达生物技术有限公司 | Application of the LACC1 and FHL2 genes in scoliosis detection product is prepared |
Non-Patent Citations (4)
| Title |
|---|
| NORMAND E等: "Dipeptidyl Peptidase-4 and Adolescent Idiopathic Scoliosis: Expression in Osteoblasts.", 《SCI REP.》 * |
| WANG Q等: "Spot14/Spot14R expression may be involved in MSC adipogenic differentiation in patients with adolescent idiopathic scoliosis", 《MOL MED REP.》 * |
| 孙强等: "SOX9在青少年特发性脊柱侧凸患者骨髓间质干细胞的表达及意义", 《中国脊柱脊髓杂志》 * |
| 王其飞 等: "Thrsp蛋白在青少年特发性脊柱侧弯患者MSCs成脂分化过程中的差异性表达和作用", 《中国矫形外科杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881228A (en) * | 2017-12-21 | 2018-04-06 | 中国医学科学院北京协和医院 | A kind of molecular marked compound related to adolescent idiopathic scoliosis and its application |
| CN111575361A (en) * | 2020-04-20 | 2020-08-25 | 孙欣 | Test method for treating AIS (automatic identification system) patient by melatonin pathway regulatory factor |
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Application publication date: 20171107 |