CN108103185A - Applications of the LOC105373420 in diagnosis of Parkinson disease, treatment - Google Patents
Applications of the LOC105373420 in diagnosis of Parkinson disease, treatment Download PDFInfo
- Publication number
- CN108103185A CN108103185A CN201810167670.0A CN201810167670A CN108103185A CN 108103185 A CN108103185 A CN 108103185A CN 201810167670 A CN201810167670 A CN 201810167670A CN 108103185 A CN108103185 A CN 108103185A
- Authority
- CN
- China
- Prior art keywords
- parkinson
- disease
- genes
- cell
- pharmaceutical composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses applications of the LOC105373420 in diagnosis of Parkinson disease and treatment.The experiment proved that compared with normal person, the LOC105373420 gene expressions up-regulation in Parkinsonian's blood shows that disturbances in patients with Parkinson disease can be diagnosed by detecting the level of LOC105373420 in blood.It by PD cell models outside construct, finds the level of LOC105373420 in cell, the growth of cell can be influenced, prompt LOC105373420 that can be applied to the accurate treatment of Parkinson as target spot.
Description
Technical field
The present invention relates to biological technical field, more particularly to people LOC105373420 in diagnosis of Parkinson disease, treatment
Purposes.
Background technology
Parkinson's disease (Parkinson ' disease, PD) is the second major class neurodegeneration for being only second to Alzheimer disease
Property disease (Tysnes OB, Storstein A.Epidemiology of Parkinson ' S disease.J Neural
Transm (Vienna) 201 7), in 60 years old or more crowd, incidence is close to 1%.It is now recognized that the deterioration of its motion function
Related with nigrostriatal dopamine serotonergic neuron reduction, this is also one of its most prominent pathological characters, and PD another most
Prominent pathological characters are abnormal deposition of the alpha-synapse nucleoprotein combined in nigral cell with ubiquitin in Lewy body, are caused
The denaturation of dopaminergic cell (Zhang ZX, Roman GC, Hong Z, et a1.Parkinson ' S disease in
China：Prevalence in Beijing, Xian, and Shanghai.Lancet 2005；365：595-7.).Parkinson
The main clinical manifestation of disease includes static tremor, and myotonia is slow in action, postural balance obstacle, and non-motor symptoms include coke
(the Weiner WJ.There is no Parkinson disease.Arch Neurol 2008 such as worry, depressed and dementia；65：
705—8.).Although by positive research and effort, the treatment and clinical research of patient nevertheless suffer from diagnosis, prognosis, right
Many restrictions such as the prediction of remedy measures reaction, the tracking of progression of disease.
At present, the problem that diagnoses and treatment PD faces mainly has：Pathogenesis is unclear：The morbidity of PD is affected by many factors,
It is now recognized that mainly on the basis of aging, caused by h and E collective effect.Many is black researches show that PD patient
Matter dopaminergic (DA) neuronal degeneration may lack with mitochondria dysfunction, oxidative stress, neurotrophic factor, excitability
A series of mechanism such as toxicity, immunoregulatory abnormality, natural death of cerebral cells are related.Epidemiological investigation shows that there are about 5~10%
Parkinsonian has family history.But only 6 are proved that heredity single gene mutation can be caused, and show the hair of Parkinson's disease
Disease is closely related with inherent cause.Lack objective laboratory diagnostic method：PD is heterogeneous high, at present except image method aids in
In addition, unified Parkinson's disease marking scales (The Unified Parkinson ' S Disease Rating Scale, UPDRS)
It is the common method of PD clinical diagnosises and assessment treatment with Hoehn-Yahr grading scales.It is examined according to the Parkinson's disease of Britain's think-tank
Disconnected standard, patient has to be moved less, along with one kind in three kinds of static tremor, myotonia, posture abnormal gait symptoms, you can examine
Break as PD.But due to the complexity of pathogenesis, clinical symptoms, the sign of different patients may be different and same
Patient can also change in the state of an illness of different time, and evaluation result is usually influenced by the master of patient and doctor, objective factor,
So that result lacks comparativity, difficulty is brought to clinical diagnosis.Therefore, the relevant biomarkers of PD are filtered out for disclosing it
Pathogenesis simultaneously develops objective diagnostic method, and then the purpose of disease prevention, control and treatment is of great significance.
The content of the invention
In order to make up for the deficiencies of the prior art, one of the objects of the present invention is to provide one kind can be used for diagnosis of Parkinson disease
Molecular marker LOC105373420 genes.Compared to the diagnostic method of traditional Parkinson's disease, examined using gene marker
Disconnected Parkinson's disease has promptness, specificity and sensitivity so that patient in early stage with regard to that can know disease risks, so as to take
Corresponding measure.
The second object of the present invention is to provide a kind of molecular target available for Parkinson treatment, passes through targeted molecular target
Point changes the expression of molecular target, so as to achieve the effect that treatment or alleviate disease.
To achieve these goals, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides a kind of reagent, and the reagent can detect the level of LOC105373420.
Further, the reagent includes：
The probe of specific recognition LOC105373420；Or
The primer of specific amplification LOC105373420.
In the specific embodiment of the present invention, the primer sequence such as SEQ of the primer of specific amplification LOC105373420
Shown in ID NO.1~2.
The second aspect of the present invention provides a kind of kit, including the reagent described in first aspect present invention, the examination
Agent can detect the level of LOC105373420.
The third aspect of the present invention provides a kind of chip, including the reagent described in first aspect present invention, the reagent
The level of LOC105373420 can be detected.
The fourth aspect of the present invention provides a kind of pharmaceutical composition, the inhibitor including LOC105373420.Wherein, institute
Inhibitor is stated to be selected from：Using LOC105373420 genes as target sequence and the disturbing molecule of LOC105373420 genes can be inhibited,
Including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisensenucleic acids can be expressed or formed
The shRNA, siRNA, dsRNA, Microrna, the construction of antisensenucleic acids.
Further, the inhibitor is siRNA.
When screening effective siRNA sequence, the present inventor is optimal effective so as to find out by largely comparing analysis
Segment.A variety of siRNA sequences have been synthesized by design, and they are transfected by transfection reagent to cell line respectively and is tested
Card, selects the optimal siRNA of interference effect, and sequence is further tested as shown in SEQ ID NO.7~8 in cellular level, knot
Fruit proves that the siRNA can effectively inhibit the level of LOC105373420 genes and the multiplication of cell in cell.
Further, described pharmaceutical composition further includes and other medicine classes of the inhibitor compatibility and pharmaceutically acceptable
Carrier and/or auxiliary material.Wherein, pharmaceutically acceptable carrier and/or auxiliary material are including but not limited to such as buffer, emulsification
Agent, suspending agent, stabilizer, preservative, physiological saline etc..As buffer, phosphate, glycine, sorbic acid, mountain can be used
Pears acid clock, the partial glyceride mixtures of saturated vegetable fatty acid, water, salt or electrolyte such as potassium sulfate, disodium hydrogen phosphate, phosphorus
Potassium hydrogen phthalate, sodium chloride, zinc salt, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, polyethylene glycol, carboxymethyl cellulose
Sodium, polyacrylate, wax, polyethylene-polyoxypropylene block copolymer, polyethylene glycol and lanolin etc..It, can as emulsifier
Use gum arabic, sodium alginate, tragacanth etc..As suspending agent, glycerol monostearate, monostearate can be used
Aluminium, methylcellulose, carboxymethyl cellulose, hydroxymethyl cellulose, NaLS etc..As stabilizer, third can be used
Glycol, diethylidene sulphite, ascorbic acid etc..As preservative, sodium azide, benzalkonium chloride can be used, to oxybenzene
Formic acid, methaform etc..
The pharmaceutical composition of the present invention can also include ion-exchanger such as alumina, aluminum stearate, lecithin, gala chemical drug
Object delivery system (SEDDS) such as d alpha-tocopherols cetomacrogol 1000 succinate, the surface-active used in pharmaceutical dosage form
Agent such as tween or other similar polymeric delivery matrices, haemocyanin such as human serum albumins can also use cyclodextrin example
Derivative such as alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin or chemical modification is, for example, hydroxyalkylcyclodextrins, including 2-
Promote the delivering of the compounds of this invention with 3- hydroxypropyls-beta-cyclodextrin or other solubilized derivatives.Carrying of the present invention
The carrier of gene is various carriers known in the art, such as commercially available carrier, including plasmid, clay, bacteriophage, virus.
The pharmaceutical composition of the present invention further includes pharmaceutically acceptable excipient, filler, coagulating agent and blender, such as
Lactose hydrous or Lactis Anhydrous, starch, glucose, sucrose, mannitol, sorbierite, silicic acid, microcrystalline cellulose, hydroxylmethyl cellulose
Plain sodium, sodium starch glycol and its derivative etc..
The pharmaceutical composition of the present invention is also comprising interfacial agent, emulsifier, diffusant, antifoaming agent, coating material etc..Appoint
What is pharmaceutically or medically acceptable interfacial agent, emulsifier, diffusant, antifoaming agent etc. can all be used.
Pharmaceutical composition Orally-administrable of the present invention, parenteral administration, by suck spray delivery, it is local to
Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that oral medication or injection
Administration.Pharmaceutical composition of the present invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.In some situations
Under, medicinal acid, alkali or buffer can be used to adjust the pH of preparation to improve the stabilization of prepared compound or its form of administration
Property.Terms used herein parenteral route include subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, in breastbone,
In bringing up, in damage location and intracranial injection or infusion techniques.As long as destination organization can be reached, pharmaceutical composition of the present invention
Receptor can be given by any approach.
The drug of the present invention can also be with the drug combination of other treatment Parkinson, and multi-medicament, which is used in combination, to be carried significantly
To the success rate for the treatment of.
The fifth aspect of the present invention provides a kind of method for the candidate substances for screening treatment Parkinson, the method bag
It includes：
The cultivating system expressed or containing LOC105373420 genes is handled with substance to be screened；With
Detect the expression of LOC105373420 genes in the system；
Wherein, if substance to be screened can reduce expression or the activity of LOC105373420 genes, it is pre- to show the substance
Anti- or treatment Parkinson candidate substances.
The cultivating system includes but is not limited to cell system, subcellular fraction system, solution system, organizational framework, organ
System or animal system (animal model of such as animal model, preferably non-human mammal, such as mouse, rabbit, sheep, monkey) etc..
The sixth aspect of the present invention provides following any one of them application：
A. application of the reagent described in first aspect present invention in the product for preparing diagnosis Parkinson；
B. the kit described in second aspect of the present invention is preparing the application in examining the product of Parkinson；
C. application of the chip described in third aspect present invention in the product for preparing diagnosis Parkinson；
D. application of the pharmaceutical composition described in fourth aspect present invention in the product for preparing treatment Parkinson；
Applications of the e.LOC105373420 in the candidate substances of screening treatment Parkinson；
F. application of the method described in fifth aspect present invention in the candidate substances of screening treatment Parkinson.
The practicability of the present invention is not limited to the gene expression of any specific variants of the marker gene to the present invention
It is quantified.As nonrestrictive example, marker gene can have such as current international public nucleic acid database GeneBank
Sequence shown in middle LOC105373420 genes (NC_000002.12).
In the present invention, LOC105373420 can use multiple nucleic acids technology known to persons of ordinary skill in the art into
Row detection, these technologies include but not limited to：Nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technologies.
The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but not limited to chain terminator (Sanger) sequencing and dye
Expect terminator sequencing.Those of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment
Be more vulnerable to nuclease attack, thus before sequencing usually by RNA reverse transcriptions into DNA.
The present invention simultaneously can expand nucleic acid (for example, ncRNA) before detection or with detection.Nucleic acid amplification technologies
Exemplary, non-limitative example include but not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT-
PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence
It expands (NASBA).Those of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification
RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).
The PCR of commonly referred to as PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand
Multiple cycling, exponentially increase target nucleic acid sequence copy number；The amplification of the transcriptive intermediate of TMA is (in substantial constant
Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more
A RNA copies autocatalytically generate other copy；The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid
The two groups of complementary DNA oligonucleotides handed over；Other amplification methods are included for example：The expansion based on nucleotide sequence of commonly referred to as NASBA
Increase；Use rna replicon enzyme (the commonly referred to as Q β replicase) amplification of amplification probe molecule in itself；Amplification method based on transcription；
And the sequence amplification of self―sustaining.
In the present invention, term " probe " refers to what can be combined with the particular sequence or subsequence or other parts of another molecule
Molecule.Unless otherwise indicated, term " probe " is often referred to be matched by complementary base with another polynucleotides (often referred to as
" target polynucleotide ") combine polynucleotide probes.Lack according to the stringency of hybridization conditions, probe energy and with the probe complete
The target polynucleotide of complementarity combines.Probe can make direct or indirect mark, and scope includes primer.Crossing system,
Include, but are not limited to：Solution phase, solid phase, mixed phase or in situ hybridization measuring method.
Exemplary probe in the present invention includes PCR primer and gene specific DNA oligonucleotide probe, such as fixed
In micro probe array, the quantitative nucleic acid enzyme protection on microarray substrate examine probe, the probe that is connected with molecular barcode and
The probe being fixed on pearl.
These probes have the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ",
As long as hybridization, can not be complete complementary.These polynucleotides, which are commonly angled relative to the specific base sequence, to be had
More than 80%, preferably more than 90%, more preferable more than 95%, particularly preferred 100% homology.These probes can be DNA,
Can also be RNA, furthermore it is possible to be artificial by PNA, LNA, ENA, GNA, TNA etc. in part of it or whole nucleotides
The polynucleotides that replacement nucleic acid obtains.
In the context of the present invention, " diagnosis Parkinson's disease " had both included judging whether subject has suffered from Parkinson
Disease also includes judging that subject whether there is the risk with Parkinson's disease.
In the context of the present invention, " treatment Parkinson's disease " divides from the state change of disease, can include disease
Alleviate, the complete healing of disease；The effect played from drug is different, can include improving DOPA amine effect, promote dopamine
Synthesis and release, the degradation for blocking dopamine.
The advantages of the present invention：
Present invention firstly discovers that LOC105373420 gene expressions are related to Parkinson's disease, by detecting subject's blood
The expression of middle LOC105373420, it can be determined that whether subject suffers from Parkinson's disease or judge subject with the presence or absence of trouble
There is the risk of Parkinson's disease, so as to which clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound LOC105373420 genes, compared to traditional detection means, base
It is more timely, more special, sensitiveer because diagnosing, the early diagnosis of Parkinson's disease can be realized, so as to reduce the death of Parkinson's disease
Rate.
Description of the drawings
Fig. 1 shows the expression in Parkinsonian's blood using QPCR detection LOC105373420 genes；
Fig. 2, which is shown, utilizes influences of the QPCR detections siRNA to LOC105373420 gene expressions；
Fig. 3, which is shown, utilizes influence of the MTT detection LOC105373420 gene expressions to Parkinsonian cell growth.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.The experimental method of actual conditions is not specified in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in or according to the condition proposed by manufacturer.
Embodiment 1 is screened and the relevant gene marker of Parkinson's disease
1st, sample collection
10 normal human bloods and Parkinsonian's blood sample are respectively collected, writes sample names, number, sampling day exactly
Situations such as phase, sample process process, the acquirement of above-mentioned all samples pass through the agreement of Ethics Committee.
2nd, the preparation of RNA sample
RNA sample is extracted using the blood rna extracts kit of Invitrogen, concrete operations refer to specification.
3rd, the quality analysis of RNA sample
By the RNA of said extracted into row agarose gel electrophoresis, using Nanodrop2000 to the concentration of carried RNA and pure
Degree is detected, and agarose gel electrophoresis detection RNA integralities, Agilent2100 measures RIN values.Single requirement for construction data base RNA is total
5ug is measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.
4th, rRNA is removed
The rRNA in total serum IgE is removed using Ribo-Zero kits.
5th, construction cDNA library
The structure of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit
By specification carries out.
6th, upper machine sequencing
CDNA library is sequenced using Illumina x-ten microarray datasets, concrete operations by specification carries out.
7th, high-throughput transcript profile sequencing data analysis
Bioinformatic analysis is carried out to sequencing result, RNA-seq read positioning is carried out using TopHat v1.3.1, leads to
It crosses Cufflinks v1.0.3 and RNA-seq segment numbers is standardized to the relative abundance for calculating transcript, utilize
Cuffdiff detects differential expression, works as p value<When 0.05, it is believed that gene significant difference is expressed.
8th, result
RNA-seq is the results show that expression quantity of the LOC105373420 genes in Parkinsonian's blood is significantly higher than
Level in normal blood.
The differential expression of embodiment 2QPCR sequence verification LOC105373420 genes
1st, according to the testing result of high-flux sequence LOC105373420 genes is selected to carry out large sample QPCR verifications.According to
Sample collection mode in embodiment 1 selects disturbances in patients with Parkinson disease blood and each 60 of normal human blood.
2nd, RNA is extracted
RNA sample is extracted using the blood rna extracts kit of Invitrogen, concrete operations refer to specification.
3rd, reverse transcription：
It is operated using the reverse transcription reagent box of TAKARA companies.It is as follows：
(1) 2 μ g of total serum IgE is taken to carry out reverse transcription, add in Oligo (dT) 2 μ l, abundant mixing.70 DEG C of water-baths are stood after five minutes
I.e. ice bath 2-3 minutes.
(2) 25 μ l reaction systems are built, including 5 × RT Buffer 5 μ l, dNTP (2.5mM) 5 μ l, RNasin
40U/ μ l, M-MLV 200U/ μ l mend nuclease free water to anticipated volume.
After sixty minutes, 95 DEG C of water-baths 5 minutes are to inactivate M-MLV for (3) 42 DEG C of water-baths.
(4) -20 DEG C store for future use.
4th, QPCR is expanded
(1) design of primers
QPCR amplimers are designed according to the coded sequence of LOC105373420 genes and GAPDH genes in Genbank, by
Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthesizes.Specific primer sequence is as follows：
LOC105373420 genes：
Forward primer is 5 '-CATTGCCGATGTGATAAG-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-TATAAGGATACATTCATATTGGAT-3 ' (SEQ ID NO.2).
GAPDH genes：
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
(2) PCR reaction systems：Forward primer and each 0.6 μ l, 2 × SuperReal PreMix Plus10 μ of reverse primer
L, DNA profiling 2 μ l, ddH27.4 μ l, 50 × ROX Reference Dye of O△2 μ l, 4.8 μ l of sterile purified water.
(3) PCR reaction conditions：95 DEG C of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 Xun Huans, 95 DEG C of 15s,
60 DEG C of 60s, 95 DEG C of 15s.PCR reactions are carried out on 7300 type fluorescence quantitative PCR instruments of ABI, pass through melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT methods carry out relative quantification.
5th, statistical method
Experiment is tested using 3 repetitions, and result data is represented in a manner of mean+SD, is used
SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
6th, result
The results are shown in Figure 1, and compared with normal human blood, LOC105373420 genes are in Parkinsonian's blood
Up-regulated expression, difference have statistical significance (P<0.05) it is, consistent with RNA-sep results, LOC105373420 is prompted as life
Object marker is applied to the diagnosis of disturbances in patients with Parkinson disease.
Embodiment 3 inhibits LOC105373420 gene expressions
1st, cell culture
Dopamine neuronal cell SH-SY5Y, with contain in the DMEM culture solutions of 10% hyclone, 1%P/S (pH7.2~
7.4), in 37 DEG C, 5%CO2, relative humidity be 90% incubator in cultivate.It was changed the liquid once every 2 days, treats cell growth extremely
It is passed on during 90% contact, is made with 0.25%-EDTA Trypsin Induceds are added in after PBS cleaning under cell separates from bottle wall
Come, terminate pancreatin digestion reaction with the DMEM culture solutions containing hyclone, 1000g centrifugation 2min abandon supernatant, with the training newly configured
Nutrient solution is resuspended, with 1:3~1:4 ratios pass on, 24 it is small when after cell enter exponential phase replace culture solution, and according to experiment will
It asks and gives different interventions.
2nd, siRNA is designed
For the siRNA sequence of LOC105373420：
siRNA1：
Positive-sense strand is 5 '-UCAAAAGCAGCAAACACGCCC-3 ' (SEQ ID NO.5),
Antisense strand is 5 '-GCGUGUUUGCUGCUUUUGAAG-3 ' (SEQ ID NO.6)；
siRNA2：
Positive-sense strand is 5 '-UUCUAGAAACAUCUGAAUGGA-3 ' (SEQ ID NO.7),
Antisense strand is 5 '-CAUUCAGAUGUUUCUAGAAAA-3 ' (SEQ ID NO.8)；
siRNA3：
Positive-sense strand is 5 '-UAUCACAUCGGCAAUGACCUA-3 ' (SEQ ID NO.9),
Antisense strand is 5 '-GGUCAUUGCCGAUGUGAUAAG-3 ' (SEQ ID NO.10).
3rd, recombined adhenovirus
According to the difference of adenovirus mediated siRNA, cell is divided into five groups, SH groups：Any viral vectors is not transfected
SH-SY5Y cells, as blank control；Ad groups：The empty adenoviral plasmid groups of cells of infection, siRNA1 groups：Adenovirus mediated interference
1 infection cell group of sequence：SiRNA2 groups：Adenovirus mediated 2 infection cell group of interference sequence；SiRNA3 groups：It is adenovirus mediated dry
Disturb 3 infection cell group of sequence.
Cell is pressed 1 × 105/ hole is inoculated into 6 porocyte culture plates, per hole 2ml, in 37 DEG C, 5%CO2It is thin in incubator
Born of the same parents cultivate for 24 hours, and cell fusion density is about 50%-60% at this time；It suctions out supernatant to discard, be washed twice with serum free medium 1ml,
With each group adenovirus that the diluted MOI of 1ml serum free mediums is 50, culture plate is rocked once at interval of 20min, to increase sense
Effect is contaminated, after infecting 48h, adds concentration as 1000 μm of ol/L MPP+Complete medium, after being incubated for 24 hours.Cell is collected to be used for
Extract RNA；
3rd, QPCR detects the transcriptional level of LOC105373420 genes
The extraction of 3.1 cell total rnas
Using TRIzol Reagent (Invitrogen Cat.No.15596-018) total RNA extraction reagent, by specification
Providing method extracts the total serum IgE of SH-SY5Y cells.
3.2 reverse transcription steps are the same as embodiment 2.
3.3QPCR amplification steps are the same as embodiment 2.
4th, statistical method
Experiment is tested using 3 repetitions, and result data is represented in a manner of mean+SD, is used
SPSS18.0 statistical softwares carry out statistical analysis, disturb the difference between LOC105373420 gene expression panels and control group
It is examined using t, it is believed that work as P<There is statistical significance when 0.05.
5th, result
As a result such as Fig. 2 is shown, compared to siRNA1, siRNA3, siRNA2 can more effectively inhibit LOC105373420 levels,
Difference has statistical significance (P<0.05), therefore selection siRNA2 carries out subsequent experiment.
The influence of 4 LOC105373420 gene pairs nerve cells of embodiment
LOC105373420 gene pairs SH-SY5Y Parkinson's disease cell model ability of cell proliferation is detected using MTT experiment
Influence.
1st, cell culture and adenovirus infection step are the same as embodiment 3.
2nd, step：
SH-SY5Y cell densities are adjusted to 5 × 104/ mL, with every 100 μ l cell inoculations of hole in 96 well culture plates, according to
Embodiment 3 handles cell, is detected after treatment every 12h applications MTT, until 72h.
MTT regression analysises method detects cytoactive：Solution in hole is discarded, 100 μ l culture mediums is added in, then is added in per hole
After MTT liquid 10 the μ l, 37 DEG C of culture 4h of 5mg/mL, culture medium is sucked, DMSO100 μ l are added in per hole, concussion 10min in room makes purple
Blue precipitate fully dissolves, and absorbance (OD values) is surveyed in 490nm wavelength with microplate reader.OD values is thin as reflection SH-SY5Y
The parameter of cytoactive.
3rd, statistical method
Experiment is tested using 3 repetitions, carries out statistical analysis using SPSS18.0 statistical softwares, difference between the two
It is examined using t, it is believed that work as P<There is statistical significance when 0.05.
4th, result
The results show shown in Fig. 3：The vitro growth rates of siRNA2 groups are higher than the vitro growth rates of control group group, poor
It is different that there is statistical significance (P<0.05).The above results show that LOC105373420 overexpressions are unfavorable for the life of SH-SY5Y cells
It is long, the growth of nerve cell can be promoted by the expression for inhibiting LOC105373420 genes.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will be also fallen into the protection domain of the claims in the present invention.
Sequence table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Applications of the LOC105373420 in diagnosis of Parkinson disease, treatment
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cattgccgat gtgataag 18
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tataaggata cattcatatt ggat 24
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tttaactctg gtaaagtgga tat 23
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggtggaatca tattggaaca 20
<210> 5
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ucaaaagcag caaacacgcc c 21
<210> 6
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gcguguuugc ugcuuuugaa g 21
<210> 7
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
uucuagaaac aucugaaugg a 21
<210> 8
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
cauucagaug uuucuagaaa a 21
<210> 9
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
uaucacaucg gcaaugaccu a 21
<210> 10
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
ggucauugcc gaugugauaa g 21
Claims (10)
1. a kind of reagent, which is characterized in that the reagent can detect the level of LOC105373420.
2. reagent according to claim 1, which is characterized in that including：
The probe of specific recognition LOC105373420；
Or the primer of specific amplification LOC105373420.
3. reagent according to claim 2, which is characterized in that the primer sequence of the primer of specific amplification LOC105373420
Row are as shown in SEQ ID NO.1~2.
4. a kind of kit, which is characterized in that including claim 1-3 any one of them reagents.
5. a kind of chip, which is characterized in that including claim 1-3 any one of them reagents.
A kind of 6. pharmaceutical composition, which is characterized in that the inhibitor including LOC105373420.
7. pharmaceutical composition according to claim 6, which is characterized in that inhibitor siRNA.
8. the pharmaceutical composition according to claim 6 or 7, which is characterized in that pharmaceutical composition further includes and the inhibition
Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material of agent compatibility.
A kind of 9. method for the candidate substances for screening treatment Parkinson, which is characterized in that the described method includes：
The system expressed or containing LOC105373420 genes is handled with substance to be screened；With
Detect the expression of LOC105373420 genes in the system；
Wherein, if substance to be screened can reduce expression or the activity of LOC105373420 genes, show the substance be prevention or
Treat the candidate substances of Parkinson.
10. following any one of them application：
A. application of claim 1-3 any one of them reagent in the product for preparing diagnosis Parkinson；
B. the kit described in claim 4 is preparing the application in examining the product of Parkinson；
C. application of the chip described in claim 5 in the product for preparing diagnosis Parkinson；
D. application of claim 6-8 any one of them pharmaceutical composition in the product for preparing treatment Parkinson；
Applications of the e.LOC100507053 in the candidate substances of screening treatment Parkinson；
F. application of the method described in claim 9 in the candidate substances of screening treatment Parkinson.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810167670.0A CN108103185B (en) | 2018-02-28 | 2018-02-28 | Application of LOC105373420 in diagnosis and treatment of Parkinson's disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810167670.0A CN108103185B (en) | 2018-02-28 | 2018-02-28 | Application of LOC105373420 in diagnosis and treatment of Parkinson's disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108103185A true CN108103185A (en) | 2018-06-01 |
| CN108103185B CN108103185B (en) | 2020-06-30 |
Family
ID=62205881
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810167670.0A Active CN108103185B (en) | 2018-02-28 | 2018-02-28 | Application of LOC105373420 in diagnosis and treatment of Parkinson's disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108103185B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337975A (en) * | 2018-12-24 | 2019-02-15 | 潘伟 | Application of the gene marker in Parkinson diagnoses |
| CN109468373A (en) * | 2018-12-24 | 2019-03-15 | 潘伟 | A kind of biomarker relevant to Parkinson's occurrence and development |
| CN111549117A (en) * | 2020-05-21 | 2020-08-18 | 天津医科大学总医院 | Biomarker and application thereof in Parkinson |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016113544A1 (en) * | 2015-01-12 | 2016-07-21 | Isis Innovation Limited | Treatment of diseases associated with mitochondrial dysfunction |
-
2018
- 2018-02-28 CN CN201810167670.0A patent/CN108103185B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016113544A1 (en) * | 2015-01-12 | 2016-07-21 | Isis Innovation Limited | Treatment of diseases associated with mitochondrial dysfunction |
Non-Patent Citations (7)
| Title |
|---|
| ENSEMBL: "LOC105373420", 《ENSEMBL》 * |
| MAJIDINIA M等: "The roles of non-coding RNAs in Parkinson"s disease", 《MOL BIOL REP》 * |
| SOREQ L等: "Long non-coding RNA and alternative splicing modulations in Parkinson"s leukocytes identified by RNA sequencing", 《PLOS COMPUT BIOL》 * |
| 余元勋: "《中国分子胃癌学》", 30 April 2016, 安徽科学技术出版社 * |
| 曾溢滔: "《遗传病分子基础与基因诊断》", 31 December 2017, 上海科学技术出版社 * |
| 解洪荣: "MPP+诱导的SH-SY5Y细胞帕金森病模型的蛋白质组学研究", 《医药卫生科技辑》 * |
| 陈宏: "《基因工程原理与应用》", 31 January 2004, 中国农业出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337975A (en) * | 2018-12-24 | 2019-02-15 | 潘伟 | Application of the gene marker in Parkinson diagnoses |
| CN109468373A (en) * | 2018-12-24 | 2019-03-15 | 潘伟 | A kind of biomarker relevant to Parkinson's occurrence and development |
| CN110511996A (en) * | 2018-12-24 | 2019-11-29 | 潘伟 | A kind of biomarker relevant to Parkinson's occurrence and development |
| CN111549117A (en) * | 2020-05-21 | 2020-08-18 | 天津医科大学总医院 | Biomarker and application thereof in Parkinson |
| CN111549117B (en) * | 2020-05-21 | 2023-03-31 | 天津医科大学总医院 | Biomarker and application thereof in Parkinson |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108103185B (en) | 2020-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111910002B (en) | Kit or device for detecting esophagus cancer and detection method | |
| CN104981548A (en) | Diagnostic mirna markers for alzheimer | |
| CN106434982B (en) | The relevant molecular marked compound of cerebral arterial thrombosis and its application | |
| CN101988120A (en) | Novel technology for diagnosing liver cancer by utilizing microRNAs in serum | |
| CN108103185A (en) | Applications of the LOC105373420 in diagnosis of Parkinson disease, treatment | |
| KR101956323B1 (en) | miR6768 as a biomarker for parkinson’s disease and diagnostic kit using thereof | |
| CN108676872A (en) | A kind of and the relevant biomarker of asthma and its application | |
| CN108456726A (en) | Spinal muscular atrophy genetic test probe, primer and kit | |
| CN108374043A (en) | The relevant biomarker of Parkinson and its application | |
| CN107312852A (en) | Myocardial infarction diagnosis mark compositions | |
| CN109652529A (en) | Osteoporosis specificity miRNA, composition and its diagnostic use | |
| CN108660211A (en) | A kind of and the relevant biomarker LINC01549 of hepatocellular carcinoma and its application | |
| CN108728439A (en) | The finger-print of tiny RNA composition and its application in Diagnosis of Bladder | |
| CN107326076A (en) | A kind of scoliosis early stage auxiliary detection kit and its application | |
| EP4036233A1 (en) | Method for detecting brain tumor | |
| CN107029238B (en) | Applications of the LINC01094 in diagnosis and treatment cerebral arterial thrombosis | |
| KR20220014004A (en) | Biomarker for the diagnosis of nonalcoholic steatohepatitis using circulating microRNA combination | |
| CN109439743A (en) | A kind of biomarker of severe asthma and its application | |
| CN109182513A (en) | Application of the biomarker SCFD2 in Alzheimer diagnosis and treatment | |
| CN107164550A (en) | A kind of reagent for detecting myocardial infarction and its application | |
| CN107164546A (en) | MiRNA, composition and its application in diagnosing the illness | |
| CN108728531A (en) | Applications of the biomarker CBX8 in preeclampsia diagnosis and treatment | |
| LU102676B1 (en) | A Biomarker for Severe Asthma and Application Thereof | |
| KR102349630B1 (en) | Use of Eef1a1 for Diagnosis of Alzheimer's Disease | |
| CN112795644B (en) | Application of miRNA biomarker in diagnosis of myocardial fibrosis diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant after: Beijing Yang Shen biology information technology company limited Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Applicant before: Beijing Yang Shen biology information technology company limited |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191128 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Applicant after: Qingdao Yangshen biomedical Co., Ltd Address before: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant before: Beijing Yang Shen biology information technology company limited |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |