CN108611413A - A kind of Parkinson's associated biomarkers and its application - Google Patents
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Abstract
The invention discloses a kind of Parkinson's associated biomarkers and its applications, the experiment proved that FGFBP2 up-regulated expressions in the blood of disturbances in patients with Parkinson disease, prompt FGFBP2 that can be applied to the clinical diagnosis of Parkinson as Testing index；The present invention discloses the proliferation that the expression of regulation and control FGFBP2 can influence nerve cell, prompt FGFBP2 that can be applied to as drug targets in the personalized treatment of disturbances in patients with Parkinson disease.
Description
Technical field
The present invention relates to biotechnology, it is related to a kind of Parkinson's associated biomarkers and its application, specifically gives birth to
Object marker is FGFBP2.
Background technology
Neurodegenerative disease seriously endangers the general level of the health of the elderly, and the World Health Organization announces with aging of population
Phenomenon becomes increasingly conspicuous, and will become second largest after angiocardiopathy by the year two thousand forty neurodegenerative disease endangers the mankind
Disease (.Valera E, the Masliah E.Therapeutic approaches in Parkinson's disease of health
and related disorders.Journal of neurochemistry,(2016)).Parkinson's disease (Parkinson's
Disease, PD) it is a kind of second largest common neurodegenerative disease, further recognize the pathomechanism of PD and researches and develops effective
Therapy it is very urgent.PD major pathologic features are substantia nigra of midbrain compact part dopamine (Dopamine, DA) serotonergic neuron
Progressive is lost, and clinicopathological study shows that the early stage of the PD courses of disease may occur in which the phenomenon that DA serotonergic neurons are lost and with fortune
The impaired DA serotonergic neurons for moving the appearance of the typical motor symptoms such as slow, splinting, and occurring in early stage be can be by
It repairs.However, with the progress of the PD courses of disease, intracerebral nucleus ceruleus, basal nuclei, nuclei pontis, dorsal nucleus of vagus nerve, amygdaloid nucleus and sea
The brain areas such as horse also will appear the symptom of neuron loss, to the clinical manifestations such as depression, dementia (Dickson occur
DW.Parkinson's disease and parkinsonism:neuropathology.Cold Spring Harbor
perspectives in medicine,(2012),2(8)).The exception of a- synapse nucleoproteins (a-synuclein) in neuron
The deposition of aggregation and Lewy body (Lewy body, LB), Louis neural process (Lewy neuritis, LN) is PD another big important
Pathological characters.Fully realize change (CN201510463617.1, CN of Biological indicators in PD pathogenesis
2015107135273) and PD pathologies are illustrated, improves the level of prevention and treatment PD, delays PD pathogenesis, is PD
Researchers' critical issue urgently to be resolved hurrily.
PD is a kind of neurodegenerative disease caused jointly by a variety of composite factors, and principle pathological mechanism includes：It loses
Biography factor, aging, environmental factor, misfolded protein accumulation, mitochondria dysfunction, lysosomal dysfunction, oxidative stress
With neuroinflamation etc., these pathomechanisms participate in jointly DA serotonergic neuron retrogressions pathologic process (Kalia LU,
LangAE.Parkinson's disease.Lancet,(2015),386(9996):896-912.).Single neuroprotective agent
PD symptoms not can effectively improve, thus according to different risk factor and pathogenesis, carry out individuation analysis, to make yet
The therapeutic scheme of targeting tropism regulating strategy and drug combination is most important to delaying PD processes.
Invention content
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide one kind can be used for Parkinson's disease early diagnosis
And the molecular marker FGFBP2 genes for the treatment of.Compared to the diagnostic and therapeutic method of traditional Parkinson's disease, gene mark is used
Will object has promptness, specificity and sensitivity to diagnose Parkinson's disease, to formulate targeting regulating strategy and medicine for patient
Scheme associated with object treats PD.
To achieve the goals above, the present invention adopts the following technical scheme that：
The present invention provides application of the reagent of detection FGFBP2 levels in the product for preparing diagnosis Parkinson's disease.
Further, diagnostic products mentioned above include：Pass through RT-PCR, real-time quantitative PCR, immune detection, original position
Hybridization or the expression of chip detection FGFBP2 genes and its expression product are to diagnose the product of Parkinson's disease.
Further, the product that Parkinson's disease is diagnosed with RT-PCR includes at least a pair of of specific amplified FGFBP2 genes
Primer；The product that Parkinson's disease is diagnosed with real-time quantitative PCR includes at least the primer of a pair of of specific amplified FGFBP2 genes；
It is described with immune detection diagnose Parkinson's disease product include：The antibody combined with FGFBP2 protein-specifics；The original position
Hybridization diagnosis Parkinson's disease product include：With the probe of the nucleic acid array hybridizing of FGFBP2 genes；It is described to diagnose pa with chip
The product of the gloomy disease of gold includes：Protein-chip and genetic chip；Wherein, protein-chip includes and FGFBP2 protein-specific knots
The antibody of conjunction, genetic chip include the probe with the nucleic acid array hybridizing of FGFBP2 genes.
Preferably, the product includes chip, kit.
The present invention also provides a kind of products of diagnosis Parkinson's disease, include the reagent of detection FGFBP2 levels.The production
Product include but not limited to chip, kit.
Further, the reagent includes：
The probe of specific recognition FGFBP2 genes；Or
The primer of specific amplification FGFBP2 genes；Or
Specifically bind the antibody or ligand of the albumen of FGFBP2 codings.
As a preferred embodiment, the primer sequence of specific amplification FGFBP2 genes such as NO.1~2 SEQ ID
It is shown.
In the present invention, term " probe " can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives.It is described
There is no limit as long as can complete specific hybrid, be specifically bound with purpose nucleotide sequence, any length is all for the length of probe
It can be with.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe can grow to
60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to hybridization efficiency, letter
Number specificity has different influences, and the length of the probe is typically at least 14 base-pairs, and longest is usually no more than 30 alkali
Base pair, it is best with 15-25 base-pair with the length of purpose nucleotide sequence complementation.The probe self-complementary sequences are preferably few
In 4 base-pairs, in order to avoid influence hybridization efficiency.
" antibody " includes but not limited to monoclonal antibody, polyclonal antibody.The specific antibody packet of the FGFBP2 albumen
Complete antibody molecule, any segment of antibody or modification are included (for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..Only
Want the segment that can retain the binding ability with FGFBP2 albumen.The preparation of antibody for protein level is ability
Well known to field technique personnel, and the present invention may use any method to prepare the antibody.
The present invention provides applications of the FGFBP2 in the drug of screening treatment Parkinson.
The step of screening the drug for the treatment of Parkinson using FGFBP2 is as follows：
In experimental group, untested compound is added into cultivating system, and measures the expression of FGFBP2；In control group
In, untested compound is added without into same cultivating system, and measure the expression of FGFBP2；Wherein, if experimental group
The expression of middle FGFBP2 is less than control group, then illustrates that the substance to be screened is the drug of FGFBP2.
In the present invention, the method further includes：It is further tested to the drug that previous step obtains and inhibits pa gold
Gloomy effect illustrates that the compound is to prevent or treat pa gold if test compound has significant inhibition to Parkinson
Gloomy drug.
The cultivating system includes but is not limited to cell system, subcellular system, solution system, organizational framework, organ
System or animal system (such as animal model, the preferably animal model of non-human mammal, such as mouse, rabbit, sheep, monkey) etc..
When using the compound of screening technique separation through the invention as medicament administration in people or other mammals,
Including but not limited to mouse, rat, cavy, rabbit, cat, dog, sheep, pig, ox, monkey, baboon, chimpanzee when, the compound of separation
It can directly apply, or various dosage forms can be configured to using known process for preparing medicine.For example, as needed, it is described
Drug can be used as sugar coated tablet, capsule, elixir and microcapsules to be administered orally；Or it is acceptable with water or any other drug
Liquid dosage at sterile solution or suspension, the non-oral application in the form of injection.For example, can be by compound with general
Unit dosage forms (unit dose) and pharmaceutically acceptable carrier or medium needed for the medicament administration mode of receiving are blended in one
Rise, the carrier or medium include but not limited to sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant,
Stabilizer, flavoring agent, excipient (excipient), medium (vehicle), preservative, adhesive etc..According to these preparations
The content of middle active ingredient can obtain suitable dosage within the specified range.
The present invention provides the inhibitor of FGFBP2 genes and/or its expression product in preparing treatment anti-parkinson drug
Application, wherein the inhibitor include inhibit FGFBP2 gene expressions substance, influence FGFBP2 gene expression products stablize
Property substance, and/or inhibit the active substance of FGFBP2 gene expression products.
As selectable embodiment, the inhibitor includes：Inhibit the double of FGFBP2 gene expressions by RNA interfering
Chain ribonucleic acid, or the tumor vaccine based on FGFBP2 antigen proteins or the protein for inhibiting FGFBP2 protein actives.
As a preferred embodiment of the present invention, the inhibitor is the siRNA for FGFBP2.
In the present invention, the RNA interference (RNA interference, RNAi) refers to endogenous or exogenous double-strand
Selective degradation occurs for the intracellular mRNA that RNA (double-stranded RNA, dsRNA) is mediated, so as to cause target gene
Expression silencing generates the phenomenon that corresponding function phenotype lacks.RNAi technology is a kind of typical negative regulation mechanism, uses the skill
Art can have been widely used for exploring gene function, gene therapy with the expression of specific depletion or closing specific gene, the technology
And new drug development field.RNAi based on cell is screened has many advantages, main table in terms of functional gene research
Present most cell types can use RNAi methods, and the opposite expression for being easier to downward or silence target gene.
In order to ensure FGFBP2 genes can be rejected efficiently or silence, devised according to the mRNA sequence of FGFBP2 genes
SiRNA specific fragments.SiRNA design according to delivered general design principle (Elbashir et.al 2001,
Schwarz et.al 2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004,
Ui-Tei et.al 2004), by online tool complete design, which is：siRNASelectionProgram of
Whitehead Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bi℃/
) and BL DEG C of K-iTTM RNAi Designer ofINVITROGEN (winner of the 2004Frost＆ siRNAext/
Sullivan Excellence in Research Award, https://rnaidesigner.invitrogen.com/
sirna/).In order to be designed for the advantages of further increasing the validity of siRNA segments, integrate two Photographing On-line tools
The multipair siRNA segments of screening, filter siRNA sequence, to improve siRNA segments by sequence analysis (NCBI BLAST)
Specificity and reduce RNAi interference undershooting-effect, finally, by the best siRNA of experiment screening interference effect be applied to this
In invention.
The present invention provides a kind of pharmaceutical composition for treating Parkinson, described pharmaceutical composition includes being directed to FGFBP2
SiRNA and pharmaceutically acceptable carrier.
Further, the sequence of siRNA is as shown in NO.7~8 SEQ ID.
Pharmaceutically acceptable carrier includes but is not limited in the present invention：Diluent, excipient such as water etc., filler
Such as starch, sucrose；Adhesive such as cellulose derivative, alginates, gelatin and polyvinylpyrrolidone；Wetting agent such as glycerine；
Disintegrant such as agar, calcium carbonate and sodium bicarbonate；Sorbefacient quaternary ammonium compound；Surfactant such as hexadecanol；Absorption
Carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and magnesium, polyethylene glycol etc..
The drug of the present invention can also can significantly be carried with the drug combination of other treatment Parkinson, a variety of Drug combinations
To the success rate for the treatment of.
In the context of the present invention, " FGFBP2 genes " includes people FGFBP2 genes and times with people's FGFBP2 genes
What equivalent polynucleotides of function.A kind of sequence tool of the gene of representative FGFBP2 in specific embodiments of the present invention
Have and the identical DNA sequence dna of FGFBP2 genes (NM_031950.3) in international public GenBank GeneBank at present.
In the context of the present invention, " diagnosis Parkinson's disease " had both included judging whether subject has suffered from Parkinson
Disease also includes the risk that judges subject and whether there is with Parkinson's disease.
In the context of the present invention, " treatment Parkinson's disease " divides from the state change of disease, may include disease
Alleviate, the complete healing of disease；The effect played from drug is different, may include improving DOPA amine effect, promoting dopamine
Synthesis and release, the degradation for blocking dopamine.
Statistical analysis
In a specific embodiment of the present invention, experiment all completed according to being at least repeated 3 times, result data be all with
The mode of mean+SD indicates, using SPSS18.0 statistical softwares come for statistical analysis, difference between the two
It is different to be examined using t, it is believed that work as P<There is statistical significance when 0.05.
The advantages of the present invention：
Present invention firstly discovers that FGFBP2 gene expressions are related to Parkinson's disease, by detecting in subject blood
The expression of FGFBP2, it can be determined that whether subject, which suffers from Parkinson's disease or judge that subject whether there is, suffers from Parkinson
The risk of disease, to instruct clinician to provide prevention scheme or therapeutic scheme to subject.
Present invention finds the proliferation that the expression of FGFBP2 genes can influence Parkinson's cell, which can answer
For the clinical personalized treatment for realizing disturbances in patients with Parkinson disease.
Description of the drawings
Fig. 1 shows the expression in Parkinsonian's blood using QPCR detection FGFBP2 genes；
Fig. 2 shows the influence to FGFBP2 gene expressions using QPCR detections siRNA；
Fig. 3 shows the influence to Parkinsonian cell growth using MTT detection FGFBP2 gene expressions.
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.Test method without specific conditions 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 normal condition proposed by manufacturer.
Embodiment 1 is screened and the relevant gene marker of Parkinson's disease
1, sample collection
10 normal human bloods and Parkinsonian's blood sample are respectively collected, writes sample names, number, sampling day exactly
The acquirement of situations such as phase, sample process process, above-mentioned all samples pass through the agreement of Ethics Committee.
2, the preparation of RNA sample
RNA sample is extracted using the blood rna extracts kit of Invitrogen, concrete operations refer to specification.
3, 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 detects RNA integralities, and 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.
4, rRNA is removed
The rRNA in total serum IgE is removed using Ribo-Zero kits.
5, construction cDNA library
The structure of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit
By specification carries out.
6, upper machine sequencing
CDNA library is sequenced using Illumina X-Ten microarray datasets, concrete operations by specification carries out.
7, 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.
8, result
RNA-seq is the results show that expression quantity of the FGFBP2 genes in Parkinsonian's blood is significantly higher than normal person
Level in blood.
The differential expression of 2 QPCR sequence verification FGFBP2 genes of embodiment
1, FGFBP2 genes are selected to carry out large sample QPCR verifications according to the testing result of high-flux sequence.According to embodiment
Sample collection mode in 1 selects disturbances in patients with Parkinson disease blood and each 90 of normal human blood.
2, RNA extraction steps are the same as embodiment 1.
3, reverse transcription：
(1) it takes 2 μ g of total serum IgE to carry out reverse transcription, Oligo (dT) 2 μ l is added, mix well.70 DEG C of water-baths are stood after five minutes
That is ice bath 2-3min.
(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.
4, QPCR is expanded
(1) design of primers
QPCR amplimers are designed according to the coded sequence of FGFBP2 genes and GAPDH genes in Genbank, are given birth to by Shanghai
Work biotechnology Services Co., Ltd synthesizes.Specific primer sequence is as follows：
FGFBP2 genes：
Forward primer is 5 '-ATGAGGAAGCAAAGAAGA-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-GGAAGAAGCTGATGAGAA-3 ' (SEQ ID NO.2).
GAPDH genes：
Forward primer is 5 '-AACTCTGGTAAAGTGGATATTG-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
(2) PCR reaction systems are prepared according to table 1：
Wherein, SYBR Green PCRs system is purchased from Invitrogen companies.
1 PCR reaction systems of table
(3) PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 cycles.Using SYBR Green as
Fluorescent marker carries out PCR reactions on Light Cycler fluorescence quantitative PCR instruments, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT methods carry out relative quantification.
5, statistical method
Experiment is tested using 3 repetitions, and result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical softwares are next for 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.
6, result
The results are shown in Figure 1, and compared with normal human blood, expression of the FGFBP2 genes in Parkinsonian's blood is aobvious
Up-regulation is write, difference has statistical significance (P<0.05), consistent with RNA-sep results.
The silence of 3 FGFBP2 genes of embodiment
1, cell culture
Dopamine neuronal cell SH-SY5Y, with the DMEM culture solutions containing 10% fetal calf serum, 1% penicillin/streptomycin
In (pH7.2~7.4), in 37 DEG C, 5%CO2, relative humidity be 90% incubator in cultivate.It changed the liquid once, waits for every 2 days
It is passed on when cell growth to 90% contact, 0.25%-EDTA trypsin digestions are added after being cleaned with PBS makes cell from bottle
It is separated on wall, terminates pancreatin digestion reaction with the DMEM culture solutions containing fetal calf serum, 1000g centrifuges 2min, abandons supernatant, uses
The culture solution newly configured is resuspended, with 1:3~1:4 ratios pass on, and cell enters exponential phase replacement culture solution after 24 hours, and
Different interventions is given according to requirement of experiment.
2, siRNA is designed
For the siRNA sequence of FGFBP2：
siRNA1：
Positive-sense strand is 5 '-UUAGAAACUCUCUUCUUCCAG-3 ' (SEQ ID NO.5),
Antisense strand is 5 '-GGAAGAAGAGAGUUUCUAAUC-3 ' (SEQ ID NO.6)；
siRNA2：
Positive-sense strand is 5 '-AAGUAGUUGUGUAUGCUUGUC-3 ' (SEQ ID NO.7),
Antisense strand is 5 '-CAAGCAUACACAACUACUUAU-3 ' (SEQ ID NO.8)；
siRNA3：
Positive-sense strand is 5 '-UACAGAUAAUAAGUAGUUGUG-3 ' (SEQ ID NO.9),
Antisense strand is 5 '-CAACUACUUAUUAUCUGUAGA-3 ' (SEQ ID NO.10).
3, recombined adhenovirus
According to the difference of adenovirus mediated siRNA, cell is divided into five groups, SH-SY5Y groups：Any virus is not transfected to carry
The SH-SY5Y cells of body, as blank control；Ad groups：The empty adenoviral plasmid groups of cells of infection, siRNA1 groups：It is adenovirus mediated
1 infection cell group of interference sequence：SiRNA2 groups：2 infection cell group of adenovirus mediated interference sequence；SiRNA3 groups：Adenovirus is situated between
Lead 3 infection cell group of interference 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；Supernatant is sucked out to discard, is washed twice with serum free medium 1ml,
It is primary to rock culture plate at interval of 20min for each group adenovirus for being 50 with the diluted MOI of 1ml serum free mediums, to increase sense
Effect is contaminated, after infecting 48h, adds a concentration of 1000 μm of ol/L MPP+Complete medium, after being incubated for 24 hours.Cell is collected to be used for
Extract RNA；
3, QPCR detects the transcriptional level of FGFBP2 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.Specific method is：
Culture solution is sucked, is cleaned one time with PBS, appropriate TRIzol reagents are added, 1mL, room temperature are added per hole in six orifice plates
5min lytic cells are placed, are dispensed into 1.5mL Eppendorf pipes with 1mL/ pipes after piping and druming uniformly.By 400 μ l chloroforms/ml
Chloroform is added in Trizol, is fluctuated 30-50 times with hand, is placed at room temperature for 5min.4 DEG C, 12000g centrifugation 15min, by upper water
It mutually moves in clean Eppendorf pipes, 0.4mL isopropanols is added, gently mixing, be placed at room temperature for 10min, 4 DEG C, 7500g centrifugations
10min.Abandon supernatant, 75% ethyl alcohol washs RNA precipitate, and 7500g centrifuges 5min, drying at room temperature RNA precipitate, is dissolved in after 5-10min
Appropriate DEPC water.The agarose gel electrophoresis that mass fraction is 1.0% detects the integrality of RNA samples, using Bio-
Photometer quantitative determines the RNA of extraction.
3.2 reverse transcription steps are the same as embodiment 2.
3.3QPCR amplification steps are the same as embodiment 2.
4, statistical method
Experiment is tested using 3 repetitions, and result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical softwares are next for statistical analysis, and the difference between FGFBP2 gene expression panels and control group is interfered to use t
It examines, it is believed that work as P<There is statistical significance when 0.05.
5, result
As a result such as Fig. 2 is shown, compared to the control group, the expression for the inhibition FGFBP2 genes that experimental group can be different degrees of, and
SiRNA1 group inhibitions are the most apparent, therefore siRNA1 is selected to carry out subsequent experiment.
The influence of 4 FGFBP2 gene pairs nerve cells of embodiment
The influence of FGFBP2 gene pairs SH-SY5Y Parkinson's disease cell model ability of cell proliferation is detected using MTT experiment.
1, cell culture and adenovirus infection step are the same as embodiment 3.
2, MTT is detected
SH-SY5Y cell densities are adjusted to 5 × 104/ mL, with 100 μ l cell inoculations of every hole in 96 well culture plates, according to
Embodiment 3 handles cell, is detected after treatment every 12h applications MTT, until 72h.MTT regression analysis methods detect cell activity：
Solution in hole is discarded, 100 μ l culture mediums are added, then sucked after MTT liquid 10 the μ l, 37 DEG C of culture 4h of addition 5mg/mL per hole
DMSO100 μ l are added per hole for culture medium, and 10min is shaken in room, make the fully dissolving of hyacinthine precipitation, with microplate reader in 490nm wavelength
Survey absorbance value (OD values).Using OD values as the parameter of reflection SH-SY5Y cell activity.This experiment is repeated 3 times, every time in experiment
The same terms set 5 parallel holes.
3, statistical method
Experiment is tested using 3 repetitions, carrys out for statistical analysis, difference between the two using SPSS18.0 statistical softwares
It is examined using t, it is believed that work as P<There is statistical significance when 0.05.
4, result
Result shown in Fig. 3 is shown：The vitro growth rates of siRNA1 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 FGFBP2 is overexpressed the growth for being unfavorable for SH-SY5Y cells, pass through
Inhibit the expression of FGFBP2 genes that can promote the growth of nerve cell.
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 also be fallen into the protection domain of the claims in the present invention.
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Claims (10)
1. detecting application of the reagent of FGFBP2 levels in the product for preparing diagnosis Parkinson's disease.
2. application according to claim 1, which is characterized in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization or the expression of chip detection FGFBP2 genes and its expression product are to diagnose the production of Parkinson's disease
Product.
3. a kind of product of diagnosis Parkinson's disease, which is characterized in that the product includes the reagent for detecting FGFBP2 levels.
4. product according to claim 3, which is characterized in that the reagent includes：
The probe of specific recognition FGFBP2 genes；Or
The primer of specific amplification FGFBP2 genes；Or
Specifically bind the antibody or ligand of the albumen of FGFBP2 codings.
5. product according to claim 4, which is characterized in that the primer sequence such as SEQ of specific amplification FGFBP2 genes
Shown in NO.1~2 ID.
Applications of the 6.FGFBP2 in the drug of screening treatment Parkinson.
The inhibitor of 7.FGFBP2 genes and/or its expression product is preparing the application in treating anti-parkinson drug.
8. application according to claim 7, which is characterized in that the inhibitor is the siRNA for FGFBP2.
9. a kind of pharmaceutical composition for treating Parkinson, which is characterized in that described pharmaceutical composition includes being directed to FGFBP2
SiRNA and pharmaceutically acceptable carrier.
10. pharmaceutical composition according to claim 9, which is characterized in that the sequence of the siRNA such as institutes of SEQ ID NO.7~8
Show.
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| CN201810292179.0A CN108611413B (en) | 2018-03-30 | 2018-03-30 | Parkinson related biomarker and application thereof |
| PCT/CN2018/123794 WO2019129029A1 (en) | 2017-12-29 | 2018-12-26 | Gene as marker for diagnosing and treating parkinson's disease and use thereof |
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| CN111187827A (en) * | 2020-01-20 | 2020-05-22 | 厦门大学 | Parkinson disease biomarker and application thereof |
| CN111549117A (en) * | 2020-05-21 | 2020-08-18 | 天津医科大学总医院 | Biomarker and application thereof in Parkinson |
| WO2021104092A1 (en) * | 2019-11-28 | 2021-06-03 | 中国科学院深圳先进技术研究院 | NEURODEGENERATIVE DISEASE MARKER β-SPECTRIN AND APPLICATION THEREOF |
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