CN105002172A - Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment - Google Patents
Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment Download PDFInfo
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Abstract
The invention discloses a molecular marker-EAPP gene for early diagnosis of the Alzheimer disease. An experimental result shows that the mRNA level of the EAPP gene in blood of the patient suffering from the Alzheimer disease is remarkably lower than that of normal people, so that whether a tested person has the risk of the Alzheimer disease or not or whether the tested person has suffered from the Alzheimer disease or not is judged by determining the expression level of the EAPP gene in the blood of the tested person. The EAPP gene can be used for preparation and application for the patient suffering from the Alzheimer disease or the crowd with the high Alzheimer disease risk so as to be used for treating the Alzheimer disease or preventing the Alzheimer disease. The new diagnosis method is provided for diagnosing the Alzheimer disease clinically and the new candidate medicine is provided for treating the Alzheimer disease.
Description
Technical field
The present invention relates to biological technical field, relate to the purposes of people EAPP gene in the diagnosis, treatment of alzheimer's disease particularly.
Background technology
Alzheimer's disease (Alzheimer ' s disease, AD) also known as senile dementia, be a kind of nerve degenerative diseases of severe human's health.Clinical with Progressive symmetric erythrokeratodermia hypomnesis, cognition dysfunction and dystropy for feature, to occur in encephalatrophy, brain that senile plaque and neurofibrillary tangles, neuron loss are its pathological characters.
Known at present, various types of dementia has 32 kinds at least, and wherein AD just account for 50 ~ 60% more than of its sickness rate.Find according to epidemiology survey, there is the AD patient of more than 2,500 ten thousand in the current whole world.U.S. patient AD in 2008 estimates to reach 5,200,000,1 is just had for AD patient in 65 years old and over-65s (13%) crowd 8,1 people within every 71 seconds, is just had to catch AD, then within every 33 seconds, just there is 1 people to become AD patient to Middle Ages, do not comprise the direct expenses that nurse fees is used for the treatment of AD every year and exceeded 1,480 hundred million dollars.The elderly population sample extracting the different living environment in town and country by the strict statistics methods of sampling in China carries out statistical study, and result shows, the morbidity of China AD is low unlike western countries, belongs to AD severely afflicated area.After AD patient is made a definite diagnosis usually, on average can live 8 ~ 10 years, what have reaches 20 years, therefore be used for the treatment of every year AD and rehabilitation nursing cost and to the mental burden that families of patients brings make AD become 21 century many families have to faced by disaster, the society brought thus, economy, family and social public health problem also become increasingly conspicuous.
Because the cause of disease of AD and pathogenesis are still not clear, do not have effect method to reverse at present and stop disease progression, therefore current mainly through carrying out symptomatic treatment in early days, and the AD patient of clinical diagnosis at present is substantially in middle and advanced stage, existing treatment only can improve symptom, can not organize or the progress of reverse disease.Therefore, early diagnosis and intervention are carried out to it, significantly can reduce the harm of AD, thus alleviate the burden of society and family.
Summary of the invention
In order to make up the deficiencies in the prior art, the object of the present invention is to provide a kind of molecular marker that can be used for alzheimer ''s disease early diagnosis.Compare the diagnostic method of traditional alzheimer's disease, what use gene marker to carry out diagnosis of alzheimer's disease has promptness, specificity and susceptibility, thus make patient just can know disease risks in early days in disease, for risk height, take corresponding prevention and therapy measure.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides a kind of people EAPP gene and the application of expression product in the product preparing diagnosis of alzheimer's disease thereof.
Further, the diagnostic products mentioned above comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection EAPP gene and expression product thereof with the product of diagnosis of alzheimer's disease.
Further, the product of described RT-PCR diagnosis of alzheimer's disease at least comprises the primer of a pair specific amplified EAPP gene; The product of described real-time quantitative PCR diagnosis of alzheimer's disease at least comprises the primer of a pair specific amplified EAPP gene; The product of described immunodetection diagnosis of alzheimer's disease comprises: the antibody be combined with EAPP protein-specific; The product of described in situ hybridization diagnosis of alzheimer's disease comprises: with the probe of the nucleic acid array hybridizing of EAPP gene; The product of described chip diagnosis of alzheimer's disease comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with EAPP protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of EAPP gene.
Preferably, described product comprises chip, test kit.
Present invention also offers the application of people EAPP gene in high-flux sequence platform.Along with the development of high throughput sequencing technologies, will become the structure of the gene expression profile of a people and work very easily.By contrasting the gene expression profile of Disease and normal population, easily analyze exception and the disease-related of which gene.Therefore, in high-flux sequence, know that the abnormal Ahl tribulus sea silent sickness of people EAPP gene is correlated with also belong to the purposes of people EAPP gene, equally within protection scope of the present invention.
Present invention also offers people EAPP gene and the application of expression product in the medicine of preparation treatment alzheimer's disease thereof.
Further, described medicine comprises: the medicine that the carrier of the medicine containing people EAPP gene, carrier EAPP gene or host cell are formed, people EAPP pharmaceutical grade protein or other can promote the medicine of EAPP genetic expression.Medicine of the present invention may be used for disappearance or the deficiency of supplementary endogenic people EAPP albumen, by improving the expression of people EAPP albumen, thus the alzheimer's disease that treatment causes because of people EAPP hypoproteinosis.
The carrier carrying gene of the present invention is various carrier known in the art, as commercially available carrier, comprises plasmid, clay, phage, virus etc.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of conventional prokaryotic host cell comprises intestinal bacteria, Bacillus subtilus etc.Conventional eukaryotic host cell comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
Further, medicine of the present invention also comprises pharmaceutically acceptable carrier, carrier, and this kind of carrier comprises (but being not limited to): thinner, vehicle are if water etc., weighting agent are as starch, sucrose etc.; Tackiness agent is as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone; Wetting agent is as glycerine; Disintegrating agent is as agar, calcium carbonate and sodium bicarbonate; Absorption enhancer quaternary ammonium compound; Tensio-active agent is as cetyl alcohol; Absorption carrier is as kaolin and soap clay; Lubricant is as talcum powder, calcium stearate and magnesium, polyoxyethylene glycol etc.
The mode of drugs delivery tissue of the present invention or cell can be divided into the mode in external or body.Vitro formats comprises by the medicine containing people EAPP gene or containing in the drugs delivery cell of people EAPP protein, then by Transplanted cells or feed back in body.In body, mode comprises directly by the medicine containing people EAPP gene or containing in the infusion of medicine in-vivo tissue of people EAPP protein.
Medicine of the present invention also can with the drug combination of other treatment alzheimer's disease, multi-medicament conbined usage can mention the success ratio for the treatment of greatly.
Present invention also offers a kind of chip of diagnosis of alzheimer's disease, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for EAPP gene for detecting EAPP gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of EAPP albumen of solid phase carrier.
Further, described gene chip can be used for detecting the expression level of the multiple genes (multiple genes that such as, Ahl tribulus sea silent sickness is relevant) comprising people EAPP gene.Described protein chip can be used for detecting the expression level of the multiple protein (multiple protein that such as Ahl tribulus sea silent sickness is relevant) comprising people EAPP albumen.By being detected by the mark of multiple Ahl tribulus sea silent sickness simultaneously, the accuracy rate of diagnosis of Alzheimer disease greatly can be improved.
The invention provides a kind of test kit of diagnosis of alzheimer's disease, described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting EAPP gene transcription level; Described protein immunization detection kit comprises the specific antibody of EAPP albumen.
Further, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection EAPP gene expression dose process.Preference, described reagent comprises primer for EAPP gene and/or probe.The primer and probe that may be used for detecting EAPP gene expression dose is easily designed according to the nucleotide sequence of EAPP gene.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of EAPP gene.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
Further, the specific antibody of described EAPP albumen comprises monoclonal antibody, polyclonal antibody.The specific antibody of described EAPP albumen comprise complete antibody molecule, any fragment of antibody or modification (such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with EAPP albumen.Well known to a person skilled in the art during preparation for the antibody of protein level, and the present invention can use any method to prepare described antibody.
In the context of the present invention, " EAPP gene " comprises the polynucleotide of any function equivalent of people EAPP gene and people EAPP gene.EAPP gene comprises and has more than 70% homology with EAPP gene (NC_000014.9) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of EAPP gene comprises any DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table;
(2) under strict conditions with 1) DNA sequence dna that limits hybridizes and identical function protein DNA sequence of encoding;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described EAPP gene is the DNA sequence dna shown in SEQ ID NO.1.
In the context of the present invention, EAPP gene expression product comprises the partial peptide of people EAPP albumen and people EAPP albumen.The partial peptide of described EAPP albumen contains the relevant functional domain of Ahl tribulus sea silent sickness.
" EAPP albumen " comprises any function equivalent of people EAPP albumen and people EAPP albumen.Described function equivalent comprises people EAPP albumen conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of people EAPP under high or low stringent condition.
Preferably, EAPP albumen is the protein with following amino acid sequences:
(1) protein be made up of the aminoacid sequence shown in SEQ ID NO.2 in sequence table;
(2) aminoacid sequence shown in SEQ ID NO.2 had the protein derivative by the aminoacid sequence shown in SEQ ID NO.2 of identical function with the aminoacid sequence shown in SEQ ID NO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQ ID NO.2, there is at least 80% homology (being also called sequence iden), more preferably, with the aminoacid sequence shown in SEQ ID NO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.
In specific embodiment of the invention scheme, described EAPP albumen is the protein with the aminoacid sequence shown in SEQ ID NO.2.
Usually, it is known that in a protein one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with identity function.Intimate amino acid whose Conservative substitution tables is provided to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is EAPP albumen.Peptide or protein with EAPP protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of EAPP albumen.
EAPP albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQ ID NO.2, as long as the protein through modifying still can retain the biologic activity of EAPP albumen.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosis of alzheimer's disease " had both comprised and had judged whether experimenter has suffered from alzheimer's disease, also comprised and judge whether experimenter exists the risk suffering from alzheimer's disease.
In the context of the present invention, " treatment alzheimer's disease " divides from the change of state of disease, can comprise the healing completely of the alleviation of disease, disease.
Because Beta 4 amyloid (Amyloid-beta is abbreviated as A β usually) is considered to the morbid substance of AD.A β self-polymerization ability is strong, forms the oligomer stronger than A beta monomers toxicity and fiber.Therefore, judge whether function that whether a kind of gene or its expression product have a treatment alzheimer's disease just can have and suppress the effect of A beta peptide aggregation, have the effect of removing A β in brain and have block A β neurovirulent to be used for judgement by detecting gene or its expression product.
In the specific embodiment of the present invention, extraction be that total serum IgE in serum is to carry out the research of EAPP gene differential expression.Those skilled in the art are known, and the tumour cell in tumor in situ tissue can be shed in blood and circulate, and therefore in serum, the expression of EAPP gene just can represent the expression of EAPP gene in tumor in situ tissue.According to achievement in research of the present invention, the expression being detected EAPP gene by the total serum IgE extracted in tumor tissues or body fluid (including but not limited to blood, urine, tissue juice) all can be used for judging whether experimenter suffers from alzheimer's disease.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention EAPP genetic expression Ahl tribulus sea silent sickness is correlated with, by detecting the expression of EAPP in experimenter's blood, can judge whether experimenter suffers from alzheimer's disease or judge whether experimenter exists the risk suffering from alzheimer's disease, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-EAPP gene, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, the early diagnosis of alzheimer's disease can be realized, thus reduce the mortality ratio of alzheimer's disease.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of EAPP gene in alzheimer's disease blood;
Fig. 2 display utilizes QPCR to detect the situation of EAPP gene overexpression;
Fig. 3 shows EAPP genetic expression causes nerve cell death impact on A β;
Fig. 4 shows EAPP genetic expression causes nervous process sex change impact on A β.
Concrete embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring HarborLaboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1 screens the relevant gene marker of Ahl tribulus sea silent sickness
1, the collection of peripheral blood sample
AD patient from Beijing 301 Hospital, totally 60 examples, age 53-84 year, all cases are diagnosed as AD, and its Case definition is with reference to Americanism medical diagnosis on disease statistic handbook third edition revised edition.Contrast crowd totally 50 example, be selected from Beijing Hospital routine physical examination crowd, all examination persons of entering all get rid of the diseases such as blood lipid metabolism, age 60-82 year.All research objects all endorsed the Informed Consent Form to this test item, and provide the detection of peripheral blood for gene.
2, blood Total RNAs extraction
Hundred Tyke blood rnas are used to extract the extraction that test kit carries out blood total serum IgE
(1) get whole blood 250 μ l (or 0.25g) in RNase-Free Filter column, centrifugal 2 minutes of 13000rpm, collect lower liquid, add 0.75ml lysate RLS.
(2) homogenised sample concuss is mixed, hatch under 15-30 DEG C of condition and decompose completely to make ribosome for 5 minutes.
(3) optional step: under the condition of 4 DEG C 12,000rpm centrifugal 10 minutes, carefully gets supernatant and proceeds in a new centrifuge tube without RNA enzyme.
(4) every 1ml RLS adds 0.2ml chloroform.Cover tightly sample hose lid, it is also at room temperature hatched 3 minutes by thermal agitation 15 seconds.
(5) centrifugal 10 minutes in 4 DEG C 12,000rpm, sample can be divided into three layers: lower floor's organic phase, and the colourless aqueous phase in middle layer and upper strata, RNA is present in aqueous phase.The capacity of aqueous phase layer is approximately 60% of added RLS volume, and aqueous phase is transferred in new pipe, carries out next step operation.
(6) add 1 times of volume 70% ethanol, put upside down mixing (now may occur precipitation), the solution obtained proceeds to (adsorption column is enclosed within collection tube) in adsorption column RA together with may precipitating.
Centrifugal 45 seconds of (7) 10,000rpm, discard waste liquid, adsorption column are recovered collection tube again.
(8) add 500 μ l protein liquid removal RE, centrifugal 45 seconds of 12,000rpm, discards waste liquid.
(9) add 700 μ l rinsing liquid RW, centrifugal 60 seconds of 12,000rpm, discards waste liquid.
(10) add 500 μ l rinsing liquid RW, centrifugal 60 seconds of 12,000rpm, discards waste liquid.
(11) put back in sky collection tube by adsorption column RA, centrifugal 2 minutes of 12,000rpm, removes rinsing liquid as far as possible, in order to avoid residual ethanol suppresses downstream reaction in rinsing liquid.
(12) take out adsorption column RA, put into a centrifuge tube without RNA enzyme, add the water of 50-80 μ l without RNA enzyme according to expection RNA output in the middle part of adsorption film, room temperature places 2 minutes, centrifugal 1 minute of 12,000rpm, collects elutriant.
3, QPCR amplification
(1) design of primers
According to the encoding sequence design QPCR amplimer of EAPP gene and GAPDH gene in Genbank, synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
EAPP gene:
Forward primer is 5 '-CTCTGAGGATGAAGTGGAT-3 ' (SEQ ID NO.3);
Reverse primer is 5 '-CGGTAAGACATTCTCTGATG-3 ' (SEQ ID NO.4).
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1 PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulation.Using SYBR Green as fluorescent marker, in the enterprising performing PCR reaction of Light Cycler quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
4, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
5, result
As shown in Figure 1, compared with normal population, the EAPP gene expression amount contained in Alzheimer patients serum significantly reduces result, and difference has statistical significance (P<0.05).
Embodiment 2 EAPP gene expression plasmid builds
1, by human nerve cell strain SH-SY5Y, with DMEM (high sugar) substratum containing 10% calf serum at 37 DEG C, 5%CO
2, relative humidity is cultivate in the incubator of 90%.Within 2-3 days, change liquid 1 time, use 0.25% trypsinase conventional digestion to go down to posterity.
2, the process LAN of EAPP gene
The structure of 2.1 EAPP expression vectors
According to encoding sequence (as shown in the SEQ ID NO.1) design of amplification primers of EAPP gene, primer sequence is as follows: forward primer is 5 '-ATGAACCGGCTTCCGGAT-3 ' (SEQ ID NO.7), and reverse primer is 5 '-TTAGGAATGGCTTGCTAAAAC-3 ' (SEQ ID NO.8).From cDNA library (the clontech company becoming Human fetal spleen, article No.: the encoding sequence of the EAPP gene of amplification total length 638831), above-mentioned cDNA sequence is inserted in the eukaryotic expression vector pcDNA3.1 of restriction enzyme BamHI and XhoI double digestion after restriction enzyme BamHI and XhoI double digestion, connects the recombinant vectors pcDNA3.1-EAPP obtained and is used for subsequent experimental.
2.2 transfection
Cell is divided into two groups, is respectively control group (transfection pcDNA3.1 empty carrier) and EAPP process LAN group (transfection pcDNA3.1-EAPP).Use liposome 2000 to carry out the transfection of carrier, the instruction to specifications of concrete transfection method is carried out.The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-EAPP is 0.5 μ g/ml.
2.3 QPCR detect
Concrete steps are with embodiment 1.
3, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
4, result
As shown in Figure 2, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-EAPP, the mRNA level in-site of EAPP significantly raises, and difference has statistical significance (P<0.05).
Embodiment 3 EAPP genetic expression causes the antagonistic action of nerve cell death to A β
1, cell transfecting: the transfection according to the method for embodiment 2, neurocyte strain R2L1 being carried out to pcDNA3.1-EAPP and pcDNA3.1.
2, after transfection 24h, cultivated by neurocyte strain R2L1 in 96 orifice plates, cell density is 0.5 × 10
4cells/well.Cell is divided into following several groups:
Do not induce group: transfection pcDNA3.1, do not add 20 μMs of A β 42;
Negative control group (pcDNA3.1+A β 42): transfection pcDNA3.1, adds 20 μMs of A β 42 in substratum simultaneously;
EAPP gene overexpression group (pcDNA3.1-EAPP+A β 42): transfection pcDNA3.1-EAPP, adds 20 μMs of A β 42 in substratum simultaneously;
Often group arranges three wells.After hatching 20h at 37 DEG C, then add MTT, hatch 4h.Adding lysate, hatching 12h again for 37 DEG C, 600nm place reads optical density value.Optical density value is higher, shows that cell survivaling number is more.
3, result
Result as shown in Figure 3, compare with not inducing group and (compare for convenience, this optical density value organized is set to 100%), the optical density value of negative control group (pcDNA3.1+A β 42) is 35%, the optical density value of EAPP gene overexpression group (pcDNA3.1-EAPP+A β 42) is 75%, and difference has statistical significance (P<0.05).Above-mentioned experiment shows, EAPP gene overexpression can the neurovirulent effect of antagonism A β 42.
Experimental example 4 EAPP genetic expression causes the antagonistic action of nervous process sex change to A β
1, build the cell strain stablizing high expression level EAPP gene conventionally to carry out.
2, cultivated in 24 orifice plates by the neurocyte strain SH-SY5Y of stable transfection, cell density is 0.5 × 10
3cells/ hole, adds 10 μMs of all-trans retinoic acid in substratum DMEM, cultivates 7 days for 37 DEG C.Afterwards cell is divided into three groups:
Do not induce group: stable transfection pcDNA3.1, do not add 1 μM of A β 42;
Negative control group (pcDNA3.1+A β 42): stable transfection pcDNA3.1, adds 1 μM of A β 42 in substratum simultaneously;
EAPP gene overexpression group (pcDNA3.1-EAPP+A β 42): stable transfection pcDNA3.1-EAPP, adds 1 μM of A β 42 in substratum simultaneously;
Often group arranges three wells.5 days are hatched at 37 DEG C.Remove substratum, then add 4% paraformaldehyde fixed cell.With anti-Beta-tubulin antibody, carry out conventional immunohistochemical dyeing.Take pictures under 20 times of mirrors, measure cell process length.
3, result
Result as shown in Figure 4, compare with not inducing group and (compare for convenience, 100% is set to) by not inducing the cell process mean length of group, the cell process length of negative control group (pcDNA3.1+A β 42) is 30%, the cell process length of EAPP gene overexpression group (pcDNA3.1-EAPP+A β 42) is 78%, and difference has statistical significance (P<0.05).Above-mentioned experimental result shows that EAPP gene overexpression can the nervous process Denaturation that causes of antagonism A β 42.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (10)
1. people EAPP gene and expression product thereof the application in the product preparing diagnosis of alzheimer's disease.
2. application according to claim 1, it is characterized in that, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection EAPP gene and expression product thereof with the product of diagnosis of alzheimer's disease.
3. application according to claim 2, is characterized in that, the product of described RT-PCR diagnosis of alzheimer's disease at least comprises the primer of a pair specific amplified EAPP gene; The product of described real-time quantitative PCR diagnosis of alzheimer's disease at least comprises the primer of a pair specific amplified EAPP gene; The product of described immunodetection diagnosis of alzheimer's disease comprises: the antibody be combined with EAPP protein-specific; The product of described in situ hybridization diagnosis of alzheimer's disease comprises: with the probe of the nucleic acid array hybridizing of EAPP gene; The product of described chip diagnosis of alzheimer's disease comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with EAPP protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of EAPP gene.
4. the application according to any one of claim 1-3, is characterized in that, described product comprises chip, test kit.
5. the application of people EAPP gene in high-flux sequence platform, is characterized in that, can be known that the generation of the abnormal expression Ahl tribulus sea silent sickness of EAPP gene is relevant with development by high-flux sequence.
6. people EAPP gene and expression product thereof the application in the medicine of preparation treatment alzheimer's disease.
7. application according to claim 6, it is characterized in that, described medicine comprises: the medicine that the carrier of the medicine containing people EAPP gene, carrier EAPP gene or host cell are formed, people EAPP pharmaceutical grade protein or other can promote the medicine of EAPP genetic expression.
8. a chip for diagnosis of alzheimer's disease, is characterized in that, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for EAPP gene for detecting EAPP gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of EAPP albumen of solid phase carrier.
9. a test kit for diagnosis of alzheimer's disease, is characterized in that, described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting EAPP gene transcription level; Described protein immunization detection kit comprises the specific antibody of EAPP albumen.
10. test kit according to claim 9, is characterized in that, described reagent comprises primer for EAPP gene and/or probe.
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