WO2015012541A2 - Kit de diagnostic précoce de la maladie d'alzheimer - Google Patents

Kit de diagnostic précoce de la maladie d'alzheimer Download PDF

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WO2015012541A2
WO2015012541A2 PCT/KR2014/006582 KR2014006582W WO2015012541A2 WO 2015012541 A2 WO2015012541 A2 WO 2015012541A2 KR 2014006582 W KR2014006582 W KR 2014006582W WO 2015012541 A2 WO2015012541 A2 WO 2015012541A2
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mir
alzheimer
fluorescence
antigen
specific
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WO2015012541A3 (fr
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김명옥
김상태
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경상대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a diagnostic kit, and more particularly to an early diagnostic kit of Alzheimer's dementia.
  • Alzheimer's dementia As an aging society enters, various diseases of the elderly are emerging as a big social problem, causing a huge burden on the medical expenses and the national economy.
  • Alzheimer's dementia (hereinafter referred to as 'AD') is accompanied by a lame progression of motor and cognitive decline, accompanied by a variety of psychological and behavioral disorders, beginning with mild symptoms, and finally progressing to spontaneous personal life. This causes serious economic burdens such as medical expenses on patients, family members and society, and the loss of the economic population in the country is very serious.
  • Alzheimer's disease is a degenerative brain disease that emerges as a serious social problem, with 10% of people aged 65-74, 19% of people aged 75-84, and 47% of people over 85 years old, with an increasing incidence each year.
  • the pathogenesis of Alzheimer's disease known to date is the amyloid hypothesis, in which amyloid beta and plaques that accumulate in dementia patients induce neuronal cell death and inhibit brain nerve transmission. It is hypothesized to cause cognitive impairment and cause illness. It has been reported that histopathological findings of dementia have been linked to the key pathogenesis of the disease. In other words, the pathologic findings of neurofibrillary tangles (hereinafter referred to as 'NFT') and amyloid plaques through post-mortem biopsy are the only confirmation methods.
  • 'NFT' neurofibrillary tangles
  • amyloid plaques through post-mortem biopsy are the only confirmation methods.
  • amyloid necrosis is formed by the accumulation of proteins and dead cells outside the cell, and its main component is a peptide called amyloid-beta (hereinafter referred to as 'A ⁇ ') (Hardy, J. et al. , Nat. Neurosci . 1: 355-358, 1998).
  • 'A ⁇ ' amyloid-beta
  • the progressive loss of cognitive function a key feature of Alzheimer's disease, appears to be caused by abnormally accumulated A ⁇ , which is a ⁇ -secretase (BSCE) from amyloid precursor protein (hereinafter referred to as APP).
  • BSCE ⁇ -secretase
  • APP amyloid precursor protein
  • proteolysis which are degraded by ⁇ -secretase (GSCE).
  • Efforts have been actively made to develop diagnostic indicators of Alzheimer's disease from blood, which is a relatively non-invasive sample, including WO2002 / 088706 and an epigenetic marker for diagnostic kits of Alzheimer's with glutamine synthase.
  • WO2010 / 144634, etc. regarding a method for diagnosing Alzheimer's disease by checking the degree of phosphorus DNA methylation are present.
  • an object of the present invention is to provide an early diagnosis kit of Alzheimer's dementia and a method for providing information for the early diagnosis of dementia.
  • these problems are exemplary, and the scope of the present invention is not limited thereby.
  • an early diagnosis kit for Alzheimer's dementia comprising a probe molecule comprising an oligonucleotide capable of detecting Alzheimer's specific microRNA is provided.
  • the probe molecule may have the following structure:
  • ABC-B'-Z (wherein A is a phosphor, B is a 5'-terminal oligonucleotide of 3 to 10 nt, and C is an oligonucleotide that is complementary to the Alzheimer's specific microRNA) Region, wherein B ′ is a 3′-terminal oligonucleotide capable of complementary binding to B, Z is a quencher capable of canceling the fluorescence of A, and B, C and B ′ are The Alzheimer's specific microRNA-free system forms a stem-loop, in which case fluorescence by A is quenched by Z).
  • the probe molecule may additionally include biotin, and the biotin may be located between B ′ and Z, but the position thereof is not particularly limited.
  • the kit may comprise two probe molecules each having the structure of I and II as follows:
  • A is a phosphor
  • B is a 3 'to 10 nt 5'-terminal oligonucleotide
  • C is a probe oligonucleotide that complements the Alzheimer's specific microRNA
  • D is partially 3 to 12 nt of the 3'-terminal oligonucleotide that complementarily binds the B and acts as a switch to thermocouple the Z from the A when the target microRNA binds to the probe oligonucleotide.
  • I is a linker region part which partially complements the B and forms a stem together with the D
  • Z is a quencher capable of canceling the fluorescence of A, wherein B and C , D and L form a stem-loop in the absence of the Alzheimer's specific microRNA, in which case fluorescence by A is quenched by Z).
  • the stem-loop structure is released.
  • the phosphor and the quencher are spaced apart, thereby causing fluorescence by the phosphor.
  • the kit may further include a reaction vessel smeared with streptavidin and the reaction vessel may be a 24-well, 48-well, 96-well, 192-well, or 384-well microplate.
  • the phosphor is poly-lysineflourescein isothiocynate (FITC), rhodamine-B-isothiocyanate (RITC), rhoda Rhodamine and its derivatives as 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B, sulfonyl chloride, rhodamine B, rhodamine 123, rhodamine X Isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives (Texas Red) of sulforhodamine 101, N, N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA) , Tetramethyl rhodamine, tetramethyl rhodamine isothiocy-anate
  • the quantum dot may have a structure of a central body, a central part in which the central body is enclosed, and a polymer coating layer coating the central part.
  • the specific kind of the quantum dot is not particularly limited, and may be used without limitation as long as it has biocompatibility so that it can be used for biological imaging. In the present invention, those having a particle diameter of 5 nm or less may be used.
  • the constituent constituent of the quantum dots is mainly heavy metals such as CdSe (cadmium-cesium), CdTe (cadmium-telluride), or CdS (cadmium sulfide), and thus the use of quantum dots may be harmful to the human body.
  • the toxicity can be reduced by reducing the residence time in the body through rapid in vitro discharge of the quantum dots (Choi et al., Nat. Biotechnol. 25 (10 ): 1165-1170, 2007).
  • oligonucleotides to which a phosphor is bound and oligonucleotides to which a quencher is bound are neurons, tissues or plasma.
  • This is a diagram schematically illustrating a state in which a region that can be dissociated when bound to a specific target molecule in a sample such as bound is combined.
  • the probe molecule or the fluorescent nanoparticle may include a linker region 12 connected to the biotin 11 and a quencher 13 connected to the linker region 12.
  • a probe region 21 including a region having hybridization or affinity with an Alzheimer's specific target molecule miRNA or an Alzheimer's specific antigen inflammatory mediator or dementia-inducing protein, an oligo linked to both ends of the probe region 21.
  • oligonucleotide 22 and 3'-end oligonucleotide 23 and 5'-end oligonucleotide 22 or 3'-end oligonucleotide 23 which can hybridize with each other as nucleotides.
  • the first molecule 10 and the second molecule 20 may form the probe complex 1 of the present invention.
  • the probe region 21 may include an antibody 21 ', as shown in B of FIG. 1.
  • the quencher 13 and the phosphor 24 may be changed in position, and are not divided into the first molecule 10 and the second molecule 20,
  • the phosphor 13 and the phosphor 24 may be connected to one molecule to form a single probe molecule having a stem-loop structure.
  • the quencher 13 has a 5′-terminal oligonucleotide 22 or 3′-terminal oligonucleotide 23 by the linker region 12 at a position where the phosphor 24 is in close proximity. Since it is complementary with, the phosphor 24 is quenched by the quencher 13. That is, since the linker region 12 coupled to the quencher 13 is hybridized with the second molecule 20 in the absence of the target molecule, the linker region 12 is present in an energy erasing state and is in a fluorescent quenching state, thereby preventing fluorescence.
  • dementia-specific miRNA or dementia-specific protein antigens are present in the sample, these target molecules react with target sequences or antibodies in the probe region of the probe complex 1 to dissociate the quencher 13.
  • the phosphor 24 of the probe complex 1 is excited and exhibits fluorescence. That is, when the target molecule is not present in the signal-off state (signal-off state), but when the target molecule is in contact with the probe molecule or probe complex to provide a molecular fluorescence detection system (signal-on) Can be.
  • the presence or absence of specific target molecules in cells or tissues can be confirmed by simple in vitro experiments, and after transfection into cells or tissues targeted by nucleic acid sensors, The presence or absence of the target molecule to be detected can be confirmed by turning on and off the fluorescent signal.
  • the linker region is used in FIG. 2 to easily bond the phosphor to the left end of the molecular image sensor, the linker region is not necessarily required in the configuration of the molecular beacon according to the present invention. Even if the linker region of the probe molecule or the fluorescent nanoparticle does not exist, it may be modified to have a fluorescent material at one end portion (5'- or 3'-) of the region capable of hybridizing with the target molecule. In addition, it can be modified to attach a biotinylated functional group to the linker region of the fluorescent sensor, but is not necessary.
  • the quencher is 4- (dimethylamino) azobenzene-4-carboxylic acid (DABCYL), 4- (dimethylamino) azobenzene sulfonic acid (DABSYL), BHQ-1, BHQ-2, BHQ-3 And BHQ (Biosearch Technologies, Inc) or Eclipse (Amersham Bioscience).
  • DABCYL 4- (dimethylamino) azobenzene-4-carboxylic acid
  • DABSYL 4- (dimethylamino) azobenzene sulfonic acid
  • BHQ-1, BHQ-2, BHQ-3 And BHQ Biosearch Technologies, Inc
  • Eclipse Analoguesham Bioscience
  • a fluorescent nanoparticle for detecting a target molecule consisting of two molecules each having a structure of I and II as follows:
  • A is a phosphor
  • B is a 5'-terminal single-stranded nucleotide of 3 to 10 nt, respectively
  • Y is a probe molecule that specifically binds to the target molecule
  • E is a loop.
  • B ' is partially complementary to the B and thermodynamically when the target antigen specifically binds to the antibody Y.
  • Z is a 3-12 nt 3'-terminal oligonucleotide that acts as a switch to dissociate from A
  • Z is a quencher
  • L is partially complementary to B and stems with B ' a linker region capable of forming a stem, wherein B, Y, E, B 'and L form the stem-loop of the antigen-free system, in which case fluorescence by A is Quenched).
  • a fluorescent nanoparticle for antigen detection having the structure:
  • A is a phosphor
  • B is a 5'-terminal oligonucleotide of 3 to 10 nt
  • Y is an antibody or functional fragment thereof that specifically binds to the antigen
  • E is a loop
  • B ' is a 3'-terminal oligonucleotide capable of complementarily binding to B
  • Z is capable of canceling the fluorescence of A.
  • a quencher wherein B, Y, E and B 'form the stem-loop in the absence of the antigen, in which case fluorescence by A is quenched by Z).
  • the target molecules for detecting fluorescent nanoparticles may additionally include biotin, and the biotin may be a fluorescent nanoparticle for detecting target molecules, which is connected between the B 'and Z or the terminal of the L.
  • the phosphor is poly L-lysine-flourescein isothiocynate (FITC), rhodamine-B-isothiocyanate , RITC), rhodamine and its derivatives as 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B, sulfonyl chloride, rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N, N, N ', N'-tetramethyl-6-carboxy Rhodamine (TAMRA), Tetramethyl Rhodamine, Tetramethyl Rhodamine Isothiocyanate (FITC), rhodamine-B-iso
  • the quencher is 4- (dimethylamino) azobenzene-4-carboxylic acid (DABCYL), 4- (dimethylamino) azobenzene sulfonic acid (DABSYL), BHQ-1, BHQ-2 , BHQ-3 (Biosearch Technologies, Inc) or Eclipse (Amersham Bioscience).
  • kit for producing fluorescent nanoparticles for antigen detection comprising molecules each having the structures a and b:
  • Y which is an antibody or fragment thereof, that specifically binds to the antigen is linked through the carboxyl group (-COOH) of a and the amine group (NH 2 ⁇ ) of b to form fluorescent nanoparticles for antigen detection.
  • the A is
  • B is a 5-10 terminal single stranded nucleotide of 3 to 10 nt
  • E is a loop forming oligonucleotide forming a loop
  • B ' is partially complementary to B and binds to Y
  • the target antigen specifically binds thermodynamically 3 to 12 nt 3'-terminal oligonucleotide that acts as a switch to dissociate the Z from the A
  • Z is a cow capable of canceling the fluorescence of the A
  • the antibody is a quencher and an antibody or fragment Y thereof that specifically binds the antigen is linked between B and E
  • L is provided as a separate molecule having a structure of ZL as a linker region that is partially complementary to B and forms a stem together with B ').
  • -loop in this case A Fluorescence is quenched by Z).
  • Alzheimer's specific including an Alzheimer's specific antigen-detecting fluorescent nanoparticle having a structure of ABYE-B'-Z or two molecules each having a structure of III and IV as follows Fluorescent nanoparticle complexes for antigen detection are provided:
  • Probe Complex For detecting Alzheimer's specific microRNA having a structure of A 1 -B 1 -CB 1 '-Z 1 or for detecting Alzheimer's specific microRNA comprising two molecules each having a structure of III and IV as follows Probe Complex:
  • a and A 1 are each phosphor, B and B 1 are each 3 to 10 nt of 5′-terminal oligonucleotide, and Y is an antibody that specifically binds to the Alzheimer's specific antigen or A fragment thereof, wherein C is a probe oligonucleotide that complements the Alzheimer's specific microRNA while forming a loop, and B 1 ′ is partially complementary to B 1 and partially binds to the probe oligonucleotide 3 to 12 nt 3'-terminal oligonucleotide that acts as a switch to thermally dissociate Z from the A when the target microRNA binds, wherein L and L 1 are partially in conjunction with B and B 1 , respectively A linker region portion capable of complementary bonding, wherein Z and Z 1 are quenchers capable of canceling the fluorescence of A and A 1 , respectively, B, C, B 'and L form a system-loop in the absence of the Alzheimer's specific microRNA, in which
  • the kit may further include a reaction vessel smeared with streptavidin and the reaction vessel may be a 24-well, 48-well, 96-well, 192-well, or 384-well microplate.
  • a method for detecting Alzheimer specific target molecules for early diagnosis of Alzheimer's dementia comprising the following steps:
  • ABC-B'-Z (wherein A is a phosphor, B is a 5'-terminal oligonucleotide of 3 to 10 nt, and C is a probe oligonucleotide complementary to the Alzheimer's specific microRNA) , B 'is a 3'-terminal oligonucleotide capable of complementarily binding to B, Z is a quencher capable of canceling the fluorescence of A, B, C and B' is the Alzheimer's Form a stem-loop in the absence of specific microRNA, in which case fluorescence by A is quenched by Z); And
  • a method for detecting Alzheimer specific target molecules for early diagnosis of Alzheimer's dementia comprising the following steps:
  • ABYE-B'-Z (wherein A is a phosphor, B is 3-10 nt of 5'-terminal oligonucleotide, Y is an antibody or fragment thereof that specifically binds to the antigen, E is a loop forming oligonucleotide forming a loop, B 'is a 3'-terminal oligonucleotide capable of complementary binding to B, and Z is a quencher capable of canceling the fluorescence of A.
  • B, Y, E and B ' form the stem-loop in the absence of the antigen, in which case fluorescence by A is quenched by Z);
  • a method for detecting Alzheimer specific target molecules for early diagnosis of Alzheimer's dementia comprising the following steps:
  • Fluorescent nanoparticles for detecting Alzheimer's specific antigen having a structure of ABYE-B'-Z And reacting the plasma obtained from the subject in a reaction vessel coated with a probe molecule for detecting Alzheimer's specific microRNA having a structure of A 1 -B 1 -CB 1 '-Z 1 , respectively:
  • a and A 1 are each phosphor
  • B and B 1 are each 3-10 nt of 5′-terminal single stranded nucleotide
  • Y is an antibody or functional fragment thereof that specifically binds to the antigen.
  • C is a probe oligonucleotide complementary to the Alzheimer's specific microRNA
  • B 'and B 1 ' are 3'-terminal oligonucleotides capable of complementary binding to B and B 1 , respectively
  • Z And Z 1 is a quencher capable of canceling the fluorescence of A and A 1 , respectively, wherein B 1 , C and B 1 ′ are stem-loops absent the Alzheimer's specific microRNA.
  • fluorescence by A 1 is quenched by Z 1
  • B, Y, C and B ′ form the stem-loop of the antigen-free system, in which case fluorescence by A is Quenched by Z)
  • an early diagnosis kit for Alzheimer's dementia comprising a probe molecule comprising an oligonucleotide capable of detecting the above Alzheimer's specific microRNA is provided.
  • the kit may further include a reaction vessel smeared with streptavidin and the reaction vessel may be a 24-well, 48-well, 96-well, 192-well, or 384-well microplate.
  • the AD-specific antigen is A ⁇ 1-42 monomer (amyloid-beta 1-42 monomer), A ⁇ 1-42 oligomers (amyloid-beta 1-42 oligomer), UCHL1, NLRP3, Tau (Tau), STAT3, SORL1, cluster It may be clusterin, ApoE3, ApoE4, nogo-A or visfatin.
  • the method may further include analyzing the presence and progress of Alzheimer's dementia in the subject from the presence or absence of the fluorescence and the fluorescence pattern.
  • the Alzheimer's specific microRNA may be miR-200a, miR-181a, miR-124b, miR-146b, miR-34a, miR-106b, miR-153 or miR-155.
  • the probe molecule may additionally include biotin, and the probe molecule may be coated on the reaction vessel by a bond between the biotin and streptavidin smeared on the reaction vessel.
  • the sample may be blood, plasma or serum obtained from a subject.
  • the probe molecules and the fluorescent nanoparticles may each additionally comprise biotin, and the probe molecules are coated in the reaction vessel by a bond between the biotin and streptavidin plated in the reaction vessel. Can be.
  • the sample may be blood, plasma or serum.
  • an information providing method for the early diagnosis of Alzheimer's dementia comprising the following steps:
  • Alzheimer's specifics selected from the group consisting of miR-200a, miR-181a, miR-124b, miR-146b, miR-34a, miR-106b, miR-153 and miR-155 from a sample obtained from a subject Red micro RNA; And A ⁇ 1-42 monomer (amyloid-beta 1-42 monomer), A ⁇ 1-42 oligomers (amyloid-beta 1-42 oligomer), UCHL1, NLRP3, Tau (Tau), STAT3, SORL1, cluster lean (clusterin), Quantifying one or more Alzheimer's specific antigens selected from the group consisting of ApoE3, ApoE4, nogo-A and visfatin; And
  • the inventors selected miR-200a, miR-181a, miR-124b, miR-146b, miR-34a, miR-106b, miR-153 and miR-155, which are dementia-induced inflammation specific miRNAs.
  • Inflammatory factors A ⁇ 1-42 monomer (amyloid-beta monomer), A ⁇ 1-42 oligomer, UCHL1, NLRP3, Tau, STAT3, SORL1, clusterin, ApoE3, ApoE4, nogo-A and visfatin were selected.
  • the antibody in order to determine whether the selected markers can be utilized in the serological diagnosis of the degree of dementia in the actual dementia animal, the antibody is linked to the antibody specifically binding to the A ⁇ 1-42 antigen, a representative dementia-related antigen
  • a ⁇ 1-42 antigen a representative dementia-related antigen
  • it was normal group non-Tg mouse
  • early inflammatory group ApoE knockdown mouse
  • mild cognitive impairment group APP / PS knockdown mouse
  • dementia stage group APP / PS / ApoE knockdown
  • the level of A ⁇ 1-42 in the plasma of the initial inflammation, mild cognitive impairment and dementia stage group than in the control group, A ⁇ 1-42 was confirmed that the antigen is gradually increased as shown in FIG. X.
  • the quantitative value for A ⁇ 1-42 was about 0.2 ⁇ 0.05 ⁇ 100 RFI (arbitrary fluorescence intensity) in the control group, 0.5 ⁇ 0.4 ⁇ 100 RFI (arbitrary fluorescence intensity) in the ApoE knockout mouse, and APP / PS knockout mouse.
  • a ⁇ 1-42 antigen The level of A ⁇ 1-42 antigen was 40% in ApoE knockout mice corresponding to the early inflammatory group expressing NLRP3, and 175% (p ⁇ 0.05) in mild cognitive impaired APP / PS knockout mice in which Tau begins.
  • the present inventors can use the molecular imaging probe molecule or probe complex according to an embodiment of the present invention prepared as described above can be used as a diagnostic kit for determining the presence of dementia and the progress of dementia using plasma obtained from a real human.
  • a molecular image sensor complex and molecular image nanoparticles capable of detecting each of the eight miRNAs and 12 antigens described above, and then reacted with plasma obtained from four patients with different degrees of dementia.
  • the fluorescent image was detected (see FIG. 10). As a result, as shown in Figure 10, it was confirmed that the markers can be very useful for diagnosing the degree of dementia of the patient.
  • the early diagnosis of Alzheimer's dementia by using a relatively low invasive sample, such as plasma of the subject, so that the early diagnosis Alzheimer's dementia treatment and further disease Because it can suppress the progression of, it can realize the saving of social costs due to the development of dementia.
  • a relatively low invasive sample such as plasma of the subject
  • FIG. 1 is a schematic diagram showing the structure of a probe complex according to an embodiment of the present invention:
  • A probe complex for detecting miRNA
  • Figure 2 is a schematic diagram showing the reaction process in a plastic container of a fluorescent sensor that can detect the initial dementia specific miRNA and antigen using the probe complex according to an embodiment of the present invention.
  • Figure 3 is a graph showing the change in the expression pattern of miRNA in HT-22 cells with or without A ⁇ 1-42 oligomer.
  • FIG. 4 is a graph showing changes in expression patterns of various inflammation-related antigens in plasma of normal mice (non-Tg) and dementia-induced model mice (APP / PS / ApoE knockout mice).
  • Figure 5 is a micrograph showing the generation of plaques of hippocampal tissue in normal mice and dementia-induced transgenic mice.
  • FIG. 6 is a photograph confirmed by confocal electron microscopy showing green fluorescence and red fluorescence in comparison with the control miRNA expression when the dementia-induced oligomeric amyloid peptide is treated in hippocampal neurons.
  • FIG. 7 is a photograph showing green fluorescence by confocal electron microscopy when detecting amyloid peptide in cells after treatment of dementia-induced oligomeric amyloid peptide.
  • Figure 8 is a photograph of the immunocytochemical analysis showing the expression of A ⁇ 1-42 , STAT3, Tau, NLRP3 during dementia progression in animal brain tissue.
  • Figure 9 is a photograph of the immunocytochemical analysis showing the expression patterns of A ⁇ 1-42 and nogo-A expressed in the brain tissue of dementia-induced transgenic mice.
  • FIG. 10 is a fluorescence image of expression patterns of various Alzheimer's specific microRNAs and antigens by a molecular image-based dementia early diagnosis kit according to an embodiment of the present invention analyzed for biomarkers present in plasma of dementia patients. .
  • the present inventors treated 40 miRNAs with or without A ⁇ in HT-22 cell line, a hippocampal cell line.
  • the expression level of was analyzed using a microarray in which antisense oligonucleotides specific for the miRNA were integrated (FIG. 3 and Table 1).
  • miR-106b, miR-146b, miR-181a, miR-200a, miR200c, miR29 and miR-124b showed higher expression in the inflamed group compared to the control group (HT-22 cells not treated with A ⁇ ).
  • the present inventors in order to determine what are the inflammatory response factors generated in response to the inflammatory response due to the accumulation of A ⁇ when induced by dementia, non-transformed mice and dementia animal model APP / PS / ApoE knockout mice Immunohistochemistry was performed on the standard A ⁇ and negative controls with the following 20 antigens and positive controls in plasma of (Fig. 3 and Table 2). As a result, A ⁇ 1-42 monomer and oligomer showed very high levels in dementia-inducing mice compared to normal mice.
  • Example 3 Preparation of a Fluorescent Image Sensor Conjugated to a Molecular Image of Fluorescent Nanoparticles, Specific miRNAs, and Antibodies to Detect Inflammation-Dementia Inducers
  • the inventors of the present invention provide a molecular image sensor capable of detecting microRNAs and inflammation mediators that are significantly different in expression from normal mice and dementia-induced model mice. Made as follows:
  • miR-200a, miR-181a, miR-124b, miR-146b, miR-34a, miR-106b, miR-153 and miR-155 specifically expressed in stellate, glial and hippocampal neurons as a were selected, the relevant factors in inflammation and dementia A ⁇ 1-42 monomer (amyloid-beta monomer), A ⁇ 1-42 oligomers (amyloid-beta oligomer), UCHL1, NLRP3, Tau (Tau), STAT3, SORL1, cluster Clinerin, ApoE3, ApoE4, nogo-A and visfatin were selected.
  • nucleic acid sequences comprising regions that can hybridize with miR-153a, 155, and 106b were designed and synthesized.
  • a 5'-terminal oligonucleotide (TCGCTGT) capable of forming a stem is connected, and a quantum dot (QD 565 ) showing red fluorescence is attached to the 5'-end of the 5'-terminal oligonucleotide, and the probe oligo
  • TCGCTGT 5'-terminal oligonucleotide
  • QD 565 quantum dot showing red fluorescence
  • the molecular image sensor nucleic acid molecule to which the 3'-terminal oligonucleotide, which is an arbitrary sequence (GTCGCTTT) which performs a switch function to allow fluorescence to be emitted, was customized (Bioneer, Korea).
  • GTCGCTTT 3'-terminal oligonucleotide
  • CAGCG nucleic acid sequence capable of complementary binding to the molecular image sensor nucleic acid molecule as a linker region
  • the biotin is bonded to the 5'-end of the linker region and the QD 565 at the 3'-end of the linker region.
  • a quencher capable of quenching the fluorescence of BHQ2 (blackhole quencher 2) connected to the switch nucleic acid molecule was also customized (Bioneer, Korea).
  • the biotin serves to allow the molecular image sensor nucleic acid molecule and the switch nucleic acid molecule forming a stem-loop complex to be immobilized on a reaction vessel or microarray coated with streptavidin. .
  • the structure of a molecular imaging sensor nucleotide (hereinafter, abbreviated as 'MISN') and a switch nucleic acid molecule capable of turning on and off a signal of the molecular reversed sensor nucleic acid molecule is shown in Table 3 below. Only sequences for parts).
  • the structure of the antibody-bound molecular sensing nanoparticles formed of a complex of a molecular imaging sensor nucleotide-antibody complex molecule (hereinafter referred to as 'MISNAC') and a switch nucleic acid molecule is as follows (see below).
  • SEQ ID NO shows the entire nucleic acid sequence of the right fragment on the right, based on the antibody):
  • a ⁇ 1-42 monomer (amyloid-beta monomer), A ⁇ 1-42 oligomer (amyloid-beta oligomer), UCHL1, NLRP3, Tau, STAT3, SORL1, clusterin (clusterin) ), ApoE3, ApoE4, nogo-A, and bispatin-specific antibodies (SantaCruz, USA), respectively, were used, and the left fragment of the MISN containing 2 ⁇ g, QD 565 , respectively, was used.
  • the 3'-terminus of the left fragment is modified so that the carboxyl group (-COOH) comes instead of the hydroxy group (-OH), and the 5'-terminus of the right fragment is also an amino group (NH 2- ) instead of the hydroxy group (-OH).
  • 500 pmol of the switch nucleic acid molecule including the quencher (BHQ2) and 500 pmol of MISNAC including the QD 565 prepared above were mixed in 1 ml of hybridization buffer (1x TE buffer, 100 mM NaCl, pH 7.8). The reaction was carried out at a temperature of 93 ° C. for 5 minutes and slowly cooled at a temperature of 4 ° C. to prepare antibody-bound molecular sensing nanoparticles.
  • hippocampal neurons were cultured based on ATCC's cell culture criteria.
  • miRNA targets were identified in hippocampal neuronal cell lines, and even target miRNAs expressed inside the cells were observed. In contrast, no target was observed in the normal group.
  • confocal microscopy of specific expression of mature miR-155 (mature type), miR-153a (mature type) and miR-106b (mature type), miR-181c, miR-9, miR-200a I could check through.
  • HT-22 cells which are hippocampal neurons, overnight in a 35 mm culture dish
  • HT-22 cells were treated with 5 ⁇ M of oligomer A ⁇ 1-42 for 2 hours.
  • Culture was carried out based on the cell culture criteria of the company.
  • HT-22 cells were fixed using 4% formaldehyde solution (Sigma, St. Louis, MO, USA), and then washed three times with PBS for 10 minutes.
  • oligomers A ⁇ 1-42 showed green fluorescence (FIG. 7) and fluorescence signals of miR-155, miR-181c, miR-9, and miR-200a (FIG. 7).
  • the mature miRNA overexpressed in the cell was coupled to the miRNA molecular image sensor, and the quencher was dissociated from the molecular beacon, thereby increasing the red or green fluorescence signal.
  • the miRNA molecular image was dissociated from the quencher.
  • the molecular image sensor nucleic acid complex prepared in Example 3-1, the anti-A ⁇ 1-42 antibody, the anti-Tau antibody, the anti-STAT3 antibody, and the anti-NLRP3 antibody prepared by dissolving the fixed tissue fragment in PBS.
  • the bound fluorescent nanoparticles were each added to 2 pmol and reacted at 4 ° C. for 12 hours. As a result, as shown in FIG.
  • Plasma of four human dementia patients was isolated, blocked with BSA and dispensed 10 ⁇ l onto a 96-well reaction vessel having a surface labeled with gastric streptavidin, and reacted by treating the molecular beacon prepared above. , Immediately observed or quantified with a LAS-4000 confocal to fluorescence spectrum photometer (Table 4 and FIG. 10). As a result, as shown in FIG.
  • a ⁇ 1-42 oligomer (amyloid-beta monomer), A ⁇ 1-42 oligomer, NLRP3, UCHL1, STAT3, Tau, vvisfatin, IL-13, SORL1, clusterin, ApoE4 , ApoE3, and nogo-A were also divided into antigens whose expression patterns differed from the common antigens according to patients (Table 4).
  • the markers in the measurement are As an important biomarker for early diagnosis of dementia, all four patients were positive for A ⁇ 1-42 monomer, Tau was not detected in 3 patients, and 4 patients were A ⁇ 1-42 negative. Reaction and nogo-A were positive. In 3 patients, Tau expression was unresponsive, indicating early dementia, and 1, 2, and 4 patients had already entered dementia. . The results were identical to the actual clinical diagnosis of the patients.
  • miRNA the levels of miR-124b, miR-34a, and miR-153 were significantly lower in the third patient than in the other three patients. Therefore, the level of these miRNAs was determined to confirm the progress of dementia. It was found that it can be used as a molecular label. Furthermore, since miR-106b had the lowest levels in patients 3 and high in patients 1, 2, and 4, it was also an early diagnosis marker to confirm the progress of dementia. It can be seen that it can be used as an argument. Based on the above results, low levels of miR-106b can be used for early diagnosis of dementia, and high levels of miR-153 can be diagnosed later in dementia. Cases 4 and 4 were considered to be severe dementia and the results were consistent with the actual clinical findings.
  • the molecular imaging sensor of the present invention is very useful as a diagnostic kit useful for diagnosing dementia, and it is possible to use multiple markers rather than using only one marker. It can be seen that the accuracy of the diagnosis can be further improved by combining.

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Abstract

La présente invention concerne un kit de diagnostic précoce de la maladie d'Alzheimer permettant de diagnostiquer la maladie d'Alzheimer de manière précoce, ce kit comprenant une molécule sonde contenant un oligonucléotide pouvant détecter au moins un microARN spécifique de la maladie d'Alzheimer, choisi dans le groupe constitué par miR-200a, miR-181a, miR-124b, miR-146b, miR-34a, miR-106b, miR-153 et miR-155, en vue du diagnostic précoce de la maladie d'Alzheimer.
PCT/KR2014/006582 2013-07-26 2014-07-21 Kit de diagnostic précoce de la maladie d'alzheimer WO2015012541A2 (fr)

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CN105567737A (zh) * 2015-12-30 2016-05-11 广州凯拓生物科技开发有限公司 miRNA-34a过表达重组载体的构建及应用
WO2019238807A1 (fr) * 2018-06-15 2019-12-19 Universitat Autonoma De Barcelona Miarn circulants utilisés en tant que biomarqueurs pour le diagnostic d'une déficience cognitive légère et de la maladie d'alzheimer
CN111269977A (zh) * 2020-02-24 2020-06-12 中国医学科学院医药生物技术研究所 miRNA 200簇作为诊断和/或治疗阿尔茨海默病标志物的应用
CN111521592A (zh) * 2020-05-11 2020-08-11 深圳大学 一种信号放大的荧光检测体系、荧光生物传感器及其用途
CN114874198A (zh) * 2022-06-14 2022-08-09 黄淮学院 一种基于罗丹明-铜配合物的Aβ荧光探针及其应用

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KR101734645B1 (ko) * 2015-05-18 2017-05-11 (주)에이엔티랩스 초기 알츠하이머 병 또는 경도 인지 장애 진단 방법
KR102075558B1 (ko) * 2018-03-07 2020-02-10 광운대학교 산학협력단 스크리닝 목적의 올리고머/모노머 기반 알츠하이머 진단칩과 이를 이용한 분석 방법
KR102321753B1 (ko) * 2021-08-13 2021-11-04 주식회사 알츠코리아 알츠하이머성 치매 조기 진단용 신규 마이크로 rna 바이오마커
KR20230153559A (ko) 2022-04-28 2023-11-07 광운대학교 산학협력단 인터디지털커패시터 기반의 멀티플렉싱 조기 진단을 위한 키트

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
CN105567737A (zh) * 2015-12-30 2016-05-11 广州凯拓生物科技开发有限公司 miRNA-34a过表达重组载体的构建及应用
WO2019238807A1 (fr) * 2018-06-15 2019-12-19 Universitat Autonoma De Barcelona Miarn circulants utilisés en tant que biomarqueurs pour le diagnostic d'une déficience cognitive légère et de la maladie d'alzheimer
CN111269977A (zh) * 2020-02-24 2020-06-12 中国医学科学院医药生物技术研究所 miRNA 200簇作为诊断和/或治疗阿尔茨海默病标志物的应用
CN111521592A (zh) * 2020-05-11 2020-08-11 深圳大学 一种信号放大的荧光检测体系、荧光生物传感器及其用途
CN114874198A (zh) * 2022-06-14 2022-08-09 黄淮学院 一种基于罗丹明-铜配合物的Aβ荧光探针及其应用
CN114874198B (zh) * 2022-06-14 2023-08-25 黄淮学院 一种基于罗丹明-铜配合物的Aβ荧光探针及其应用

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