CN105018484A - CRTAP gene and expression product thereof capable of serving as target for diagnosing and treating Alzheimer's disease - Google Patents
CRTAP gene and expression product thereof capable of serving as target for diagnosing and treating Alzheimer's disease Download PDFInfo
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Abstract
The invention discloses a CRTAP gene and an expression product thereof capable of serving as a molecular marker for early diagnosis of Alzheimer's disease, namely judging whether a subject suffers from the Alzheimer's disease by detecting the CRTAP gene expression level in blood of the subject. According to the research result of the invention, a medicament capable of inhibiting CRTAP gene expression or inhibiting functions of a CRTAP gene expression product can be researched, thus realizing prevention and treatment of the Alzheimer's disease in clinic.
Description
Technical field
The present invention relates to biological technical field, relate to the purposes of people CRTAP gene in the diagnosis, treatment of alzheimer's disease particularly.
Background technology
Alzheimer's disease (Alzheimer disease, AD), is senile dementia again, and be that a kind of central nervous system degeneration is sick, insidious onset, the course of disease is chronic progressive external, is the modal type of senile dementia.Main manifestations is the neuropsychic symptoms such as gradual memory obstacle, cognition dysfunction, personality change and aphasis, has a strong impact on social activity, occupation and vital function.The cause of disease and the pathogenesis of AD are not yet illustrated, and characteristic pathological changes into neurofibrillary tangles in the extracellular senile plaque of amyloid beta formation of deposits and the neurocyte of Protein tau Hyperphosphorylationof formation, and neuron loss companion glial cells hyperplasia etc.
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 CRTAP 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 CRTAP 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 CRTAP gene; The product of described real-time quantitative PCR diagnosis of alzheimer's disease at least comprises the primer of a pair specific amplified CRTAP gene; The product of described immunodetection diagnosis of alzheimer's disease comprises: the antibody be combined with CRTAP 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 CRTAP 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 CRTAP protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of CRTAP gene.
Preferably, described product comprises chip, test kit.
Present invention also offers the application of people CRTAP 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 CRTAP gene is correlated with also belong to the purposes of people CRTAP gene, equally within protection scope of the present invention.
Present invention also offers people CRTAP gene and the application of expression product in the medicine of preparation treatment alzheimer's disease thereof.
The main active ingredient of " medicine for the treatment of alzheimer's disease " of the present invention comprises the material of the material suppressing CRTAP genetic expression, the material suppressing CRTAP gene expression product stability and/or suppression CRTAP gene expression product activity.
Further, the medicine for the treatment of alzheimer's disease of the present invention comprises: the double stranded RNA being suppressed CRTAP genetic expression by RNA interfering, or based on the tumor vaccine of CRTAP antigen protein or for suppressing the protein of CRTAP protein-active.
Present invention also offers a kind of pharmaceutical composition being used for the treatment of alzheimer's disease, described pharmaceutical composition comprises CRTAP gene and/or its expression product inhibitor.Described inhibitor comprises the material of the material suppressing CRTAP genetic expression, the material suppressing CRTAP gene expression product stability and/or suppression CRTAP gene expression product activity.
Further, inhibitor of the present invention comprises: the double stranded RNA being suppressed CRTAP genetic expression by RNA interfering, or based on the tumor vaccine of CRTAP antigen protein or for suppressing the protein of CRTAP protein-active.
Present invention also offers above-mentioned CRTAP gene and/or the application of its expression product inhibitor in preparation treatment Alzheimer disease drugs.
In the present invention, described RNA disturbs (RNA interference, RNAi) refer to high conservative during evolution, brought out by double-stranded RNA (double-stranded RNA, dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Use RNAi technology can specific depletion or close the expression of specific gene, this technology be widely used in the field of gene exploring gene function and communicable disease and malignant tumour.RNAi screening based on cell has many advantages in functional gene research, is mainly manifested in most cell types and can uses RNAi method, and the expression of relatively easy downward or reticent any goal gene.
Can efficiently to be rejected in order to ensure CRTAP gene or reticent, the siRNA specific fragment according to the mRNA sequences Design of CRTAP gene.General design principle (the Elbashiret.al 2001 that the design consideration of siRNA has been delivered, Schwarz et.al 2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al2004, Ui-Tei et.al 2004), by online tool complete design, this online tool is:
SiRNASelectionProgram of Whitehead Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/siRNAext/) and BLOCK-iTTM RNAi DesignerofINVITROGEN (winner of the 2004Frost & Sullivan Excellence in Research Award, https: //rnaidesigner.invitrogen.com/sirna/).In order to improve the validity of siRNA segment further, the advantage of comprehensive two Photographing On-line instruments is designed for the siRNA segment of screening.Finally, filter siRNA sequence by sequence analysis (NCBI BLAST), with improve siRNA segment specificity and reduce RNAi interference effect of missing the target.
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.
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 product of diagnosis of alzheimer's disease, described product includes but not limited to chip, test kit.
Wherein, 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 CRTAP gene for detecting CRTAP gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of CRTAP albumen of solid phase carrier; 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 CRTAP 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 CRTAP 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.
Wherein, described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting CRTAP gene transcription level; Described protein immunization detection kit comprises the specific antibody of CRTAP albumen.Further, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection CRTAP gene expression dose process.Preference, described reagent comprises primer for CRTAP gene and/or probe.The primer and probe that may be used for detecting CRTAP gene expression dose is easily designed according to the nucleotide sequence information of CRTAP gene.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of CRTAP 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 CRTAP albumen comprises monoclonal antibody, polyclonal antibody.The specific antibody of described CRTAP 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 CRTAP 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, " CRTAP gene " comprises the polynucleotide of any function equivalent of people CRTAP gene and people CRTAP gene.CRTAP gene comprises and has more than 70% homology with CRTAP gene (NC_000003.12) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of CRTAP 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 CRTAP gene is the DNA sequence dna shown in SEQ ID NO.1.
In the context of the present invention, CRTAP gene expression product comprises the partial peptide of people CRTAP albumen and people CRTAP albumen.The partial peptide of described CRTAP albumen contains the relevant functional domain of Ahl tribulus sea silent sickness.
" CRTAP albumen " comprises any function equivalent of people CRTAP albumen and people CRTAP albumen.Described function equivalent comprises people CRTAP 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 CRTAP under high or low stringent condition.
Preferably, CRTAP 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 CRTAP 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 CRTAP albumen.Peptide or protein with CRTAP protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of CRTAP albumen.
CRTAP 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 CRTAP 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 CRTAP 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 CRTAP gene just can represent the expression of CRTAP gene in tumor in situ tissue.According to achievement in research of the present invention, the expression being detected CRTAP 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 CRTAP genetic expression Ahl tribulus sea silent sickness is correlated with, by detecting the expression of CRTAP 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-CRTAP 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 CRTAP gene in alzheimer's disease blood;
Fig. 2 display utilizes QPCR to detect siRNA to the impact of CRTAP genetic expression;
Fig. 3 shows the impact that CRTAP gene pairs A β causes nerve cell death;
Fig. 4 shows the impact that CRTAP gene pairs A β causes nervous process sex change.
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 CRTAP gene and GAPDH gene in Genbank, synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
CRTAP gene:
Forward primer is 5 '-TCAAGGACTTCAAGGATT-3 ' (SEQ ID NO.3);
Reverse primer is 5 '-ATTCTTCAGGTCGTTCAA-3 ' (SEQ ID NO.4).
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.11);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.12).
(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 CRTAP gene expression amount contained in Alzheimer patients serum significantly increases result, and difference has statistical significance (P<0.05).
Embodiment 2 disturbs the expression of CRTAP gene
1, siRNA design and synthesis
SiRNA sequence for CRTAP:
siRNA1-CRTAP：
Positive-sense strand is 5 '-UUAUUUGCCUUGAAGUAAGCG-3 ' (SEQ ID NO.5);
Antisense strand is 5 '-CUUACUUCAAGGCAAAUAAUC-3 ' (SEQ ID NO.6),
siRNA2-CRTAP：
Positive-sense strand is 5 '-UAUGGAAAGGUAGAAAUCCUU-3 ' (SEQ ID NO.7);
Antisense strand is 5 '-GGAUUUCUACCUUUCCAUAGC-3 ' (SEQ ID NO.8),
siRNA3-CRTAP：
Positive-sense strand is 5 '-UCGUUCAACUUAUAAUAGGCA-3 ' (SEQ ID NO.9);
Antisense strand is 5 '-CCUAUUAUAAGUUGAACGACC-3 ' (SEQ ID NO.10)
Negative control siRNA sequence (siRNA-NC):
Positive-sense strand is 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO.13);
Antisense strand is 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO.14).
By neurocyte strain R2L1 cell by 1 × 10
4/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO
2cell cultures 24h in incubator, without dual anti-, containing in the DMEM substratum of 10%FBS, transfection is according to the specification sheets transfection of lipofectamine 2000 (purchased from Invitrogen company), experiment is divided into normal group, negative control group and experimental group (20nM), wherein, normal group is not transfection group, and the sequence of negative control group siRNA and CRTAP gene is without homology, concentration is 20nM/ hole, simultaneously transfection respectively.
2, QPCR detects the transcriptional level of CRTAP gene
The extraction of 2.1 cell total rnas
Adopt TRIzol Reagent (Invitrogen Cat.No.15596-018) total RNA extraction reagent, by specification supplying method extracts the total serum IgE of QBC939 cell.Concrete grammar is: get cell, rinses 3 times with the PBS that concentration is 0.01M, adds appropriate TRIzol reagent, and room temperature places 5min lysing cell, is filled in 1.5mL Eppendorf pipe after piping and druming evenly with 1mL/ pipe point.Often pipe adds 0.2mL chloroform, concuss 15s, and room temperature places 2-3min, 4 DEG C, the centrifugal 15min of 12000r/min, move to upper water mutually in clean Eppendorf pipe, add 0.5mL Virahol, mix gently, room temperature places 10min, 4 DEG C, the centrifugal 10min of 7500r/min.Abandon supernatant, 75% washing with alcohol RNA precipitation, the centrifugal 5min of 7500r/min, drying at room temperature RNA precipitate, and are dissolved in appropriate DEPC water after 5-10min.Massfraction is the integrity of the agarose gel electrophoresis detection RNA sample of 1.0%, and application Bio-Photometer carries out quantitative assay to the RNA extracted.
2.2 reverse transcription step are with embodiment 1.
2.3 QPCR amplification step 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, employing SPSS13.0 statistical software carries out statistical study, difference between interference CRTAP genetic expression group and control group adopts t to check, and thinks to have statistical significance as P<0.05.
4, result
Result such as Fig. 2 shows, compared with siRNA2-CRTAP, siRNA3-CRTAP, siRNA1-CRTAP can the expression of more effective suppression CRTAP gene, and difference has statistical significance (P<0.05), uses siRNA1-CRTAP to carry out follow-up experiment.
Embodiment 3 CRTAP gene pairs A β causes the antagonistic action of nerve cell death
1, cell transfecting: the transfection according to the method for embodiment 2, neurocyte strain R2L1 being carried out to siRNA1-CRTAP and siRNA-NC.
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 siRNA-NC, do not add 20 μMs of A β 42;
Negative control group (siRNA-NC+A β 42): transfection siRNA-NC, adds 20 μMs of A β 42 in substratum simultaneously;
CRTAP Gene interfere group (siRNA-CRTAP+A β 42): transfection siRNA-CRTAP, 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 (siRNA-NC+A β 42) is 32%, the optical density value of CRTAP Gene interfere group (siRNA-CRTAP+A β 42) is 91%, and difference has statistical significance (P<0.05).Above-mentioned experiment shows, interference CRTAP genetic expression can the neurovirulent effect of antagonism A β 42.
Experimental example 4 CRTAP gene pairs A β causes the antagonistic action of nervous process sex change
1, the cell strain of stable low-expression CRTAP gene is built
Select siRNA1-CRTAP design and synthesis shRNA, be inserted in shRNA expression vector, this work is completed by Shanghai JiMa pharmacy Technology Co., Ltd, obtain the structure of the cell strain of CRTAP gene shRNA expression vector laggard line stabilization low expression CRTAP gene from Shanghai Ji Ma company, construction process is known for those skilled in the art.
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 shRNA-NC plasmid, do not add 1 μM of A β 42;
Negative control group (shRNA-NC plasmid+A β 42): stable transfection shRNA-NC plasmid, adds 1 μM of A β 42 in substratum simultaneously;
CRTAP Gene interfere group (shRNA-CRTAP plasmid+A β 42): stable transfection shRNA-CRTAP plasmid, 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 (shRNA-NC plasmid+A β 42) is 31%, the cell process length of CRTAP Gene interfere group (shRNA-CRTAP plasmid+A β 42) is 89%, and difference has statistical significance (P<0.05).Above-mentioned experimental result shows to disturb CRTAP genetic expression 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 CRTAP 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 CRTAP gene and expression product thereof with the product of diagnosis of alzheimer's disease.
3. the application of people CRTAP 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 CRTAP gene is relevant with development by high-flux sequence.
4. people CRTAP gene and expression product thereof the application in the medicine of preparation treatment alzheimer's disease.
5. a product for diagnosis of alzheimer's disease, is characterized in that, described product can carry out diagnosis of alzheimer's disease by the expression detecting CRTAP gene in bile duct tissue.
6. product according to claim 5, is characterized in that, described product comprises chip or test kit.Wherein, 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 CRTAP gene for detecting CRTAP gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of CRTAP albumen of solid phase carrier; Described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting CRTAP gene transcription level; Described protein immunization detection kit comprises the specific antibody of CRTAP albumen.
7. test kit according to claim 6, is characterized in that, described reagent comprises primer for CRTAP gene and/or probe.
8. be used for the treatment of a pharmaceutical composition for alzheimer's disease, it is characterized in that, described pharmaceutical composition comprises the inhibitor of CRTAP gene and/or its expression product.
9. pharmaceutical composition according to claim 8, is characterized in that, described inhibitor is the siRNA for CRTAP.
10. the application of the inhibitor described in claim 8 or 9 in the medicine of preparation treatment alzheimer's disease.
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Denomination of invention: CRTAP gene and expression product thereof capable of serving as target for diagnosing and treating Alzheimer's disease Effective date of registration: 20181225 Granted publication date: 20171013 Pledgee: Huaxia Bank Beijing branch Wanliu Limited by Share Ltd Pledgor: Beijing Yang Shen biology information technology company limited Registration number: 2018990001252 |
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Date of cancellation: 20191206 Granted publication date: 20171013 Pledgee: Huaxia Bank Beijing branch Wanliu Limited by Share Ltd Pledgor: Beijing Yang Shen biology information technology company limited Registration number: 2018990001252 |
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Effective date of registration: 20191225 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Patentee after: Qingdao Yangshen biomedical Co., Ltd Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Patentee before: Beijing Yang Shen biology information technology company limited |
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