CN116694747A - Application of Ng protein as target in diagnosis and treatment of vascular cognitive dysfunction - Google Patents
Application of Ng protein as target in diagnosis and treatment of vascular cognitive dysfunction Download PDFInfo
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Classifications
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
Abstract
The invention discloses an application of Ng protein as a target in vascular cognitive disorder diagnosis and treatment, which has a good marking effect on diagnosis of vascular cognitive disorder of human, shows considerable treatment capability in animal experiments, and has good clinical treatment application prospect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of Ng protein serving as a target in diagnosis and treatment of vascular cognitive dysfunction.
Background
Vascular cognitive disorders are clinical strokes or subclinical vascular brain injuries caused by cerebrovascular lesions and their risk factors, and involve at least one clinical syndrome of impaired cognitive domains, covering mild cognitive impairment to dementia, as well as cognitive impairment to varying degrees due to co-mingled pathologies such as alzheimer's disease. In the prior art, there are a few technical solutions for diagnosing and treating vascular cognitive disorders, such as: CN106461645a provides biomarkers, methods and systems for detecting one or more related proteins in a patient sample to assess traumatic brain injury of varying severity and therapeutic efficacy and blood brain barrier or blood cerebrospinal fluid integrity and to assess neurodegenerative disorders (including vascular cognitive disorders); CN111956658A is used for detecting the expression quantity of p35 which is negatively regulated by using a primer of a micro RNA marker aiming at miRNA 148 cluster in a cognitive disorder disease model to be used as a marker of a novel cognitive disorder related disease for auxiliary diagnosis and treatment of the cognitive disorder related disease; CN111269977A detects the expression level of the miRNA 200 cluster in human bodies through an AD model and clinical serum samples, and discovers that the expression level of the miRNA 200 cluster in the Alzheimer disease process is obviously reduced, so that the miRNA 200 cluster can be used as a novel Alzheimer disease marker for auxiliary diagnosis and treatment of Alzheimer disease; the CN109456964A proves that the nucleotide sequence group can improve the spatial learning and memory capacity of an APP/PS1 transgenic mouse of a Alzheimer's disease classical animal model and can regulate key proteins in the beta amyloid cascade reaction process through multiple targets, so that the nucleotide sequence group can be applied to the treatment of cognitive impairment induced by cerebral amyloid cascade reaction and various neuropsychiatric system diseases caused by the cognitive impairment. Based on the research, developing new VD diagnosis and treatment targets still has practical clinical significance.
Disclosure of Invention
The invention aims to provide the application of Ng protein serving as a target in diagnosis and treatment of vascular cognitive dysfunction.
The technical scheme of the invention is as follows:
the application of Ng protein in peripheral blood as a detection target in preparing a diagnosis kit for vascular cognitive impairment.
In a preferred embodiment of the invention, the kit has reagents for detecting abnormal down-regulation of Ng protein expression in peripheral blood.
Further preferred, the kit has a reagent for detecting abnormal up-regulation of miR-29b expression in peripheral blood.
A diagnostic kit for vascular cognitive impairment has reagents for detecting abnormal down-regulation of Ng protein expression in peripheral blood.
In a preferred embodiment of the invention, there are agents that detect aberrant upregulation of miR-29b expression in peripheral blood.
Use of Ng protein in hippocampus as a therapeutic target for the preparation of a therapeutic composition for vascular cognitive disorders.
In a preferred embodiment of the invention, the kit has an agent that antagonizes abnormal down-regulation of Ng protein expression in hippocampus.
Further preferred, the kit antagonizes an agent that abnormally upregulates miR-29b expression in the hippocampus.
A therapeutic composition for vascular cognitive disorders having an agent that antagonizes abnormal down-regulation of Ng protein expression in a hippocampus.
In a preferred embodiment of the invention, there are agents that antagonize aberrant upregulation of miR-29b expression in the hippocampus.
The beneficial effects of the invention are as follows: the invention has good marking effect on diagnosis of vascular cognitive disorder of human, and has considerable treatment capability in animal experiments, and has good clinical treatment application prospect.
Drawings
FIG. 1 is a graph showing the experimental results of example 1 of the present invention.
FIG. 2 is a graph showing the experimental results of example 2 of the present invention.
FIG. 3 is a second experimental result chart of example 2 of the present invention.
FIG. 4 is a third experimental result chart of example 2 of the present invention.
FIG. 5 is a graph showing the experimental results of example 2 of the present invention.
FIG. 6 is a fifth experimental result chart of example 2 of the present invention.
FIG. 7 is a diagram showing the experimental results of example 2 of the present invention.
FIG. 8 is a graph of the experimental results of example 2 of the present invention.
FIG. 9 is a graph eight of the experimental results of example 2 of the present invention.
Detailed Description
The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.
The amino acid sequence of the Ng protein referred to in the following examples is
MDCCTENACSKPDDDILDIPLDDPGANAAAAKIQASFRGHMARKKIKSGERGRKGPGPGGPGGAGVARGGAGGGPSGD (SEQ ID NO. 01) with nucleotide sequence of
agaccggacccgagagcagagctgctgtttcggcgcgggtcggctggcggccgactgccccagagcccccacccggcaccacacagaccccacccccgccctgcgccagccttcgtccccgcagaggaccccccgacaccagcatggactgctgcaccgagaacgcctgctccaagccggacgacgacattctagacatcccgctggacgatcccggcgccaacgcggccgccgccaaaatccaggcgagttttcggggccacatggcgcggaagaagataaagagcggagagcgcggccggaagggcccgggccctggggggcctggcggagctggggtggcccggggaggcgcgggcggcggccccagcggagactaggccagaagaactgagcattttcaaagttcccgaggagagatggatgccgcgtccccttcgcagcgacgagacttccctgccgtgtttgtgaccccctcctgcccagcaacctgccagctacaggagccccctgcgtcccagagactccctcacccaggcaggctccgtcgcggagtcgctgagtccgtgcccttttagttagttctgcagtctagtatggtccccatttgcccttccactccaccccaccctaaaccatgcgctcccaatcttccttcttttgcttctcgcccacctcttcccgcacccagcatgcagctctgcctccgcagcctcagtgcgctttcctgcgcgcactgcggagggcgccctaagcgtcacccaagcacactcacttaaagaaaaaacgagttctttcgttctgtgcgcagctaaaaggggcgccctacatctccgtgccactcccgccccagcctagccccaagactttggatccggggcgagatgaagggaagagggttgttttggtttcggacgacccttgctctgaccggaagagaagtccctatcccacacctgcctgtcacgttccctcccctttccccagcgcactgttgagggcagcctctccagctctcttgtttatgcaaacgccgagcgcctgggaggctcggtaggaggagtcttccacggccccgccccgcccctgtcggtcccgccctcccccccgccgggctcctggggctgtggccgaaaggtttctgatctccgtgtgtgcatgtgactgtgctgggttggaatgtgaacaataaagaggaatgtccaagtgttca(SEQ ID NO.02)
Example 1 clinical trial
1. Study object and method
1. A subject
Selecting 13 post-stroke cognitive impairment (PSCI) patients and 13 post-stroke non-cognitive impairment (PSNCI) patients matched with PSCI patients in age, sex and educational years from 7 months in 2014 to 12 months in 2016 at a affiliated rehabilitation hospital of the university of Fujian traditional Chinese medicine; selecting PSCI and PSNCI patients with matched ages, sexes and education years from the community population of Fuzhou as a normal control group (n=13); 13 post-stroke cognitive impairment (PSCI) patients and 26 post-stroke non-cognitive impairment (PSNCI) patients matched in age, sex, educational age to PSCI patients were collected from mountain market in Guangdong province.
2. Inclusion criteria
The inclusion criteria for this example are as follows: (1) The cerebral apoplexy diagnosis of the embodiment refers to the fourth national cerebral vascular disease academic conference on various cerebral vascular disease diagnosis criteria, and is verified by computer tomography or magnetic resonance imaging; (2) PSCI patients with a simple mental state examination scale (MMSE) score of less than or equal to 24 points; (3) PSNCI patients with MMSE score >24 points; (4) Clinical diagnosis of cerebral apoplexy occurring for the first time within 12 months in the past; (5) ages 45-75 years, beginners and higher; (6) consciousness and stable vital signs of the patient. PSCI patients were diagnosed using interview methods, neuropsychological suites and clinical dementia assessment methods according to the American manual for diagnosis and statistics of mental disorders (4 th edition). Cognitive impairment following a stroke that does not meet post-stroke dementia criteria is classified as no dementia.
3. Exclusion criteria
The exclusion criteria for this example are as follows: (1) Conscious disturbance, severe vision, hearing and aphasia, and other inability to perform health assessment; (2) Memory, vascular cognitive dysfunction, alzheimer's disease, hypothyroidism, and executive dysfunction due to brain trauma; (3) Complications including tumors and severe heart, liver, kidney, hematopoietic and endocrine system disorders; (4) There is a history of mental disorders such as personality disorders and schizophrenia; (5) There are cognitive disorders caused by depression, the Becky depression scale (> 10 points).
4. Neuropsychological testing
MMSE is used to assess general cognitive function. The fraction of MMSE is between 0 and 30. The subjects were divided into three groups. Control group (MMSE score >24 points), PSNCI group (MMSE score >24 points) and PSCI group (MMSE score. Ltoreq.24 points).
5. Peripheral blood mRNA and miRNA chip detection
Collecting peripheral blood of a subject, centrifuging to obtain serum, extracting serum RNA by TRIzol LS (Invitrogen life technologies) method, identifying concentration and purity, and performing analysis by Affymetrix gene expression profile chip (Prime View) TM Human Gene Expression Array) and Agilent miRAN expression profiling chip (Human miRNA Microarray, release 21.0,8x60 k) to detect subject serum mRAN and miRNA expression.
6. Real-time quantitative PCR detection
Serum samples of each group were collected and assayed for Ng mRNA expression. Reverse transcription 2 μl of serum miRNA was converted to cDNA using miRNA cDNA synthesis kit (Thermo Fisher Scientific) following standard procedures. First strand cDNA was pre-amplified for 16 cycles and diluted 1:8 using ABI Quant Studio 12K Flex OpenThe real-time PCR instrument performs qPCR detection. A control was set up in 96-well plates for each batch of reverse transcribed RNA samples to normalize the qPCR assay during data analysis.
2. Results
As shown in fig. 1, in this example, for 12 PSCI patients (clinical nuclear magnetic resonance is diagnosed with ischemic stroke, MMSE score is less than 24 minutes, 1 patient sample is excluded due to RNA quality) and 13 PSCI patients (clinical nuclear magnetic resonance is diagnosed with ischemic stroke, MMSE score is greater than 24 minutes), the gender and age of the two groups of people are matched, education degrees are matched, through peripheral blood mRNA and miRNA expression profile detection, 105 (P < 0.05) of 1.5-fold differential mRNA is found, including Ng, NUMAl, SZRD, MEF2C, RAB and the like, 25 (P < 0.05) of 1.5-fold differential miRNA is found, including miR-29b, miR-29c, miR-192 and the like, miRNA-mRNA pearson correlation analysis is performed on the two, and the miRNA target gene mRNA is predicted by combining TargetScan, miRwalk, miRanda database, and network mapping is performed by using Cytoscape software, so as to find Ng proteins related to the miR-29b and the synaptic plasticity. Further, through the PCR verification of peripheral blood of 13 vascular cognitive impairment patients and 24 vascular non-cognitive impairment patients (2 patient samples are excluded due to RNA quality), the peripheral blood Ng protein expression of the vascular cognitive impairment patients is down-regulated, and the result is basically consistent with the mRNA chip result.
Example 2 animal experiments
1. Materials and methods
1. Experimental animal
This example was based on C57BL/6 mice and Ng conditional knockout mice with C57BL/6 as background. C57BL/6 male mice used in the experiments were purchased from Shanghai Laike laboratory animal liability Co., ltd; ng flox /wt Heterozygote mice, and CaMKII-Cre instrument mice were purchased from Nanjing university-Nanjing biomedical research institute. In the embodiment, a CRISPR-Cas9 system is adopted to construct an Ng conditional knockout mouse strain, gRNA aiming at Ng genes of the mice is firstly constructed, a Donor vector carrying a target site homology arm loxP site is prepared at the same time, then the two mice are injected together to obtain an F0 generation mouse, and a positive F0 generation mouse and a background mouse are backcrossed to obtain a positive F1 generation Ng flox/wt Heterozygote mice are mated with each other to obtain offspring mice, i.e. Ng flox/flox Homozygous mice; subsequent passes through Ng flox/flox The homozygous mice are bred with CaMKII-Cre mice, after the floxed exon is excised by Cre/loxP, the frame shift mutation is caused, the translation is stopped, the purpose of gene inactivation is achieved, and the gene strain of Ng in the conditional knockout of excitatory neurons of the nervous system is obtained. All animals were in the university of chinese medicine laboratory animal center of fowls [ license number: SYXK (Min) 2019-0007]Offspring are bred and propagated, the ambient temperature is kept at 22-26 ℃, and the illumination condition simulates a day and night system (12 h illumination+12 h night), so that free diet and drinking water can be realized. All animal experiments were approved by the ethical committee of animal experiments at the university of Fujian, and followed the national experimentsAnimal health guidelines.
2. Preparation of chronic cerebral ischemia model
In this example, a bilateral common carotid artery stenosis operation was performed, a chronic cerebral ischemia model was prepared, and vascular cognitive impairment was induced. The mice were anesthetized with 2% isoflurane, then supine fixed on a surgical plate, sterilized and shaved Mao Beipi on the neck with 75% alcohol, the right common carotid artery and vagus nerve were fully exposed by incising the skin, two 4-0 medical sutures were placed under the distal and proximal portions of the right common carotid artery, and the common carotid artery was gently held up by the sutures and clamped with a mini-spring coil of 0.18mm diameter to narrow the common carotid artery. After 30min, the left common carotid artery was stenosed by the same procedure. During surgery, the body temperature is maintained using a heating pad and the respiratory rate is monitored using an electrocardiogram. Cleaning wound after operation, suturing skin, preventing infection with penicillin, controlling pain, reducing death rate, placing mice back into cage, and feeding with soft padding. The sham group separated only the bilateral common carotid artery and the vagus nerve, without pinching the bilateral common carotid artery.
3. Stereotactic injection
The mice were anesthetized with 2% isoflurane, and the prone position was fixed on a brain stereotactic device, and the head level was adjusted by adjusting the left and right ear stems and the dental stems. The mice were disinfected with 75% alcohol and shaved Mao Beipi, periosteal tissue removed, bregma exposed and used as origin, and a small hole (AP: -1.9mm, ML:.+ -. 1.2mm, DV: -1.7 mm) was drilled over the bilateral hippocampus using a cranial drill. Then, 300nl of the mixed virus was injected at a rate of 30nl/min using a microinjector, and after the injection was completed, the injection needle was left in place for 10min to reduce virus needle-extending spread. The control group was injected with empty adeno-associated virus.
4. T-shaped labyrinth
The T maze was used to examine the spatial working memory of mice. The T maze consists of two target arms (50 cm long, 10cm wide, 20cm high) and one starting arm perpendicular to it (60 cm long, 10cm wide, 20cm high). The test includes 2 parts: first force test and free selection test. First forced test: the mice were placed in the starter arm and the experimenter randomly closed one side of the target arm forcing the mice into the non-closed target arm. After the mice returned to the starter arm, the starter arm gate 15s was closed. Free selection test: after 15s all gates are opened, the mouse is allowed to explore any target arm at random, after entering one target arm, the gate of the other target arm is closed, and the gate is closed for 15s when the mouse returns to the initial arm. The procedure was repeated 14 times and the direction and number of times the mice entered the target arm each time were recorded. Correct autonomous alternation rate = number of autonomous alternations/total number of selections (14 times) ×100%.
5. New object experiment
New object experiments were used to detect the identifying memory of mice, and the new object device comprised a white uncovered cube (40 cm) and 3 objects, where "A" and "a" are identical and "B" is the new object. The experiment was divided into three phases: adaptation period, learning period and test period. During the adaptation phase, mice were left free to explore for 50min in an open box, which was rubbed with 75% sterile alcohol to remove residual odors prior to the next experiment. The second d is the learning period, two identical objects "a" and "a" are placed in two corners, the mouse is and allowed to explore the area and two objects again for 10min, after 1h the mouse is put again for memory extraction test, object "a" remains unchanged, the position of object "a" is replaced by object "B", the mouse is placed in the box facing away from the object, and free exploration is performed for 5min. The camera recorded the motion trajectories of the mice and the exploration time of the "a", "B" objects, noted TA and TB, respectively. Identification index=tb/(ta+tb) ×100%.
6. Water maze
Morris water maze was used to examine spatial learning memory of mice. The Morris water maze is a circular pool with the diameter of 120cm and the depth of 50cm, the inner wall of the pool is white, the water depth in the pool is 30cm, and the water temperature is kept at about 18-23 ℃. The water maze is divided into four quadrants, the escape platform is arranged in the third quadrant, the escape platform is a transparent circular platform with the diameter of 6cm, is higher than the pool bottom by 28cm and is positioned under the water surface by 1.5cm. The experiment was divided into a learning period (pilot experiment) and a testing period (space exploration experiment). Positioning navigation experiment: the mice were first trained for 4d, placed in water with the mice facing the pool wall, and the time for the mice to find a hidden plateau within 90s was recorded. If the mice find the platform and stay above it for more than 3s, the time taken, i.e. escape latency, will be recorded. If mice cannot find the platform within 90s, the escape latency is 90s and need to be guided to learn, each mouse receives 4 experiments per d. Space exploration experiment: and 5d, removing the escape platform, releasing the opposite side quadrant of the quadrant where the mouse escape platform is positioned, and recording the times of the mouse crossing the corresponding position of the original platform within 90 s.
7. Golgi staining
Brain pieces were stained using FD rapid golgi staining kit to observe the number of dendritic spines. The mice are sacrificed after anesthesia, fresh brain tissues are trimmed into small blocks with the thickness of 5-10mm and put into the mixed solution A and B, and the small blocks are preserved for 14 days at room temperature and in a dark place; after 14d, transferring the soaked brain tissue into the solution C, and preserving for 72h at room temperature in a dark place; then, the fresh brain tissue is embedded on a base by OCT, a frozen microtome is used for preparing the brain tissue into brain slices with the thickness of 100 microns, and the slices are transferred to an anti-drop glass slide dripped with a solution C and dried in the dark for 24-48 hours for the subsequent dyeing experiment. Washing the prepared slice with double distilled water twice for 4min each time; then put into solution D, solution E and double distilled water working solution to soak for 10min (preparation ratio: 1:1:2); washing twice in double distilled water, dehydrating in gradient ethanol of 50%, 75% and 95%, respectively, and soaking for 4min in each gradient; the sections were dehydrated in 4 runs of 100% ethanol for 4min each, and finally transparent treated with xylene, and neutral resin was used for the encapsulation. And (5) observing and shooting under a mirror after airing at room temperature, and carrying out data analysis.
8. Electrophysiology
Mice were sacrificed immediately after anesthesia and their cranium isolated, rapidly placed into artificial cerebrospinal fluid (ACSF) prepared in advance and continuously aerated with 95% o 2 +5％CO 2 The mixture was then prepared into brain tissue sections with a thickness of 400 μm along the coronal plane and rapidly transferred to an incubation tank with ACSF continuously aerated with the mixture. After incubation in a water bath at 30℃for 30min, field potential recordings were performed. Head partFirstly preparing a recording electrode by using a glass electrode drawing instrument, transferring a brain slice into the central position of a recording groove, fixing the brain slice at a proper position by using a cover net, determining the position of a hippocampal brain region by using a microscope, placing a stimulating electrode in a hippocampal CA3 region, and placing the recording electrode in a hippocampal CA1 region. Then, the stimulating current of 1-7 mu A and 20s interval is given to the stimulating electrode, meanwhile, the excitatory postsynaptic potential of the hippocampal region is recorded, after confirming that the potential can steadily increase after receiving different stimulating intensities, 30% of the maximum peak electric stimulating intensity is selected for stimulating, the recorded basic field potential is recorded as basic potential (baseline), the recording duration is 20min, one stimulus is given every 20s, after the baseline recording is finished, the stimulating electrode is given 2 times of 100Hz,30s interval high-frequency stimulus is given, LTP is induced, and 60min is recorded.
9. Double luciferase reporter assay
In order to determine the direct interaction between miRNA and the target gene, a double-luciferase reporter gene detection system is adopted for verification, psiCHECK-2 is selected as a working carrier, the carrier contains Luc marker gene expression, and two enzyme cutting sites of XhoI and NotI are constructed. Then designing full-length overlapping primers including NRGN 3' UTR wild type and mutant based on PCR Accurate Synthesis (PAS) method, and transferring into psi-CHECK2 vector to obtain recombinant vector. Immediately prior to transfection, cells were seeded at 2×104 cells/well on 24-well plates in DMEM-high sugar medium containing 10% fbs; transfection when d, cell confluence was about 50-60%, 300. Mu.L OPTI-MEM medium was added and placed in 5% CO 2 Placing the mixture in an incubator at 37 ℃; then adding lipofectamine2000 diluted by OPTI-MEM culture medium and miRNA1 μl or miRNA inhibitor and 0.5ug plasmid into each well, standing at room temperature for 5min; finally, adding the transfection complex liquid, shaking a 24-pore plate, slightly and uniformly mixing, and after incubating for 5 hours in a 37 ℃ incubator, replacing the culture medium containing the transfection complex with a fresh complete culture medium; after 48h of transfection, the old medium was aspirated, washed twice with PBS, 100. Mu.L of PLB (Passive Lysis Buffer) was added to each well of cells, gently shaken for 15min at room temperature, and cell lysates were collected; after 20. Mu.L of cell lysate is added to the luminescent plate, the background value is read for 2s; then adding 100 mu L of LAR II working solution, quickly and uniformly mixing, and reading for 2s, namely an internal reference value;subsequently add 100. Mu.L Stop&And (3) after the Reagent is quickly and uniformly mixed, placing the Reagent into a luminescence detector, and reading the value for 2s, namely, reporting the luminescence value of the gene.
10. Real-time fluorescent quantitative PCR
Mice were sacrificed immediately after anesthesia and hippocampus was isolated, RNAs in hippocampal brain tissue were extracted by Triozl method, 1000. Mu.L of RNA isolater was added to brain tissue, and the mixture was thoroughly ground by a grinder, followed by 200. Mu.L of chloroform, vigorously mixed, and then allowed to stand still at 4℃for 15 mm, after separation was apparent, by centrifugation at 12000g for 15min at 4℃using a centrifuge, followed by aspiration of a suitable amount of supernatant, addition of an equal volume of isopropanol, after thoroughly mixing, allowed to stand still at 4℃for 10min, centrifugation at 12000g for 20min at 4℃for discarding supernatant, addition of 75% ethanol, centrifugation at 12000g for 20min at 4℃for suspension by flick tube bottom precipitation, washing the precipitate again for 5min, repeated washing for 2 times, and after drying, re-suspension by adding 10. Mu.LDEPC water, followed by detection of RNA concentration by spectrophotometry. Reverse transcription Using Mir-X miRNA First-Strand Synthesis Kit reverse transcription kit manufactured by Takara, the extracted RNAs were reverse transcribed into cDNA, the reaction system was 10. Mu.L, and the conditions of on-machine were: the reaction mixture was run at 37℃for 1 hour and at 85℃for 5 minutes. The obtained cDNA was diluted 10-fold after the end for quantitative experiments. Fluorescent quantitative reaction was carried out using TB Green Premix Ex TaqTM II kit manufactured by Takara Co., ltd., reaction system was 25. Mu.L, and on-machine pre-denaturation reaction: cycling for 1 time at 95 ℃ for 30 s; the PCR reaction was performed at 95℃for 3s and at 60℃for 30s, and the cycle was 40 times.
2. Experimental results
1. Ng conditional knockdown impairs mouse synaptic plasticity
As shown in fig. 2, the golgi results show a decrease in hippocampal dendritic spine density in Ng conditional knockout mice compared to littermate negative mice; while as shown in fig. 3, electrophysiological results showed that Ng conditional knockout mice had impaired hippocampal excitatory postsynaptic potential compared to littermate negative mice. This suggests that conditional knockdown of Ng on excitatory neurons compromises synaptic plasticity in mice structurally and functionally.
2. Ng conditional knockout induces cognitive impairment in mice
As shown in fig. 4, the behavioural results showed that Ng conditional knockout mice had a reduced correct rate of T maze and a reduced rate of recognition of new objects compared to littermate negative mice.
3. Ng conditional knockdown upregulates expression levels of mouse miR-29b-1
The results of the double luciferase report experiments indicate that after the plasmid cloned with Ng 3' UTR is transfected, the fluorescence activity of miR-29b-1 group is reduced compared with that of a blank group and an NC group; after miR-29b-1inhibitor is transfected, the fluorescence activity of the recombinant strain is obviously increased; further transfection of the plasmid cloned with mut RNGN 3' UTR revealed no significant difference in fluorescence activity in the miR-29b-1 group compared to the blank and NC groups (FIG. 9). The above results suggest that miR-29b-1 can directly regulate its expression level by binding to the 3' UTR of the Ng gene. As shown in FIG. 5, further qPCR detection revealed that the expression level of hippocampus miR-29b-1 was increased in Ng conditional knockout mice.
4. Ng overexpression improves cognitive dysfunction in CCH model mice
As shown in fig. 6, the behavioral results show that model mice subjected to bilateral common carotid artery stenosis surgery exhibited a reduced correct alternation rate in the T maze compared to the control group, while local overexpression of Ng in the hippocampus was sufficient to increase the correct alternation rate of model mice in the T maze; similarly, as shown in fig. 7 and 8, the water maze results showed that the CCH model mice exhibited an extended escape latency in the learning period and a reduced number of crossing the escape platform in the test period, as compared with the control group. And the overexpression of the Hippocampus Ng obviously shortens the escape latency period and improves the times of crossing the escape platform.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, i.e., the invention is not to be limited to the details of the invention.
Claims (10)
1. The application of Ng protein in peripheral blood as a detection target in preparing a diagnosis kit for vascular cognitive impairment.
2. The use according to claim 1, wherein: the kit is provided with a reagent for detecting abnormal down-regulation of Ng protein expression in peripheral blood.
3. The use according to claim 2, wherein: the kit is provided with a reagent for detecting abnormal up-regulation of miR-29b expression in peripheral blood.
4. A diagnostic kit for vascular cognitive impairment, characterized in that: has the reagent for detecting abnormal down-regulation of Ng protein expression in peripheral blood.
5. The kit of claim 4, wherein: has the reagent for detecting the abnormal up-regulation of miR-29b expression.
6. Use of Ng protein in hippocampus as a therapeutic target for the preparation of a therapeutic composition for vascular cognitive disorders.
7. The use according to claim 6, wherein: has the effect of antagonizing abnormal down-regulation of Ng protein expression in sea horse.
8. The use according to claim 7, wherein: has the function of antagonizing the abnormal up-regulation of miR-29b expression in the sea horse.
9. A therapeutic composition for vascular cognitive disorders, characterized by: has the effect of antagonizing abnormal down-regulation of Ng protein expression in sea horse.
10. The therapeutic composition of claim 9, wherein: has the function of antagonizing the abnormal up-regulation of miR-29b expression in the sea horse.
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