CN107557467B - Clinical marker related to cerebral aneurysm and application thereof - Google Patents
Clinical marker related to cerebral aneurysm and application thereof Download PDFInfo
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- CN107557467B CN107557467B CN201710694179.9A CN201710694179A CN107557467B CN 107557467 B CN107557467 B CN 107557467B CN 201710694179 A CN201710694179 A CN 201710694179A CN 107557467 B CN107557467 B CN 107557467B
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Abstract
The invention discloses a clinical marker related to cerebral aneurysm, which comprises any one or more of miR-628-3p, miR-505-5p, miR-3074-3p, miR-497-5p, miR-224-5p, miR-181b-5p and let-7b-5 p. The invention also relates to a kit for detecting the onset of cerebral aneurysm or a kit for diagnosing the onset of cerebral aneurysm, wherein the kit comprises any one or more of the clinical markers and primers corresponding to the clinical markers. The invention also relates to application of the kit in a judgment method for diagnosing the onset of cerebral aneurysm or clinical application in cerebral aneurysm diagnosis. The invention discovers that the exosome miR-628-3p has high accuracy as a clinical marker of the cerebral aneurysm for the first time. If the miRNA markers are combined, the onset of the cerebral aneurysm can be more accurately judged, namely, by using the miRNA clinical markers of the cerebral aneurysm exosome and the method for judging the onset of the cerebral aneurysm, a diagnosis result with higher reliability can be obtained.
Description
Technical Field
The invention relates to the fields of genetic engineering and neurology, in particular to a clinical marker related to cerebral aneurysm and application thereof.
Background
Cerebral aneurysm is a cerebral hemangioma-like protrusion produced by local weak and bulging vessel wall, and subarachnoid hemorrhage (SAH) caused by rupture is one of the most common cerebrovascular diseases, the incidence rate of the cerebral aneurysm is second to cerebral infarction and hypertensive cerebral hemorrhage, the annual incidence rate is about 8-16 cases/10 ten thousand people, and about 80% of subarachnoid hemorrhage is caused by aneurysm. The death rate after the rupture of the aneurysm reaches 40 percent, the disability rate reaches 33 percent, and if the death rate within 2 years is not diagnosed and treated in time reaches 60 percent. It is generally accepted that the incidence of unbroken aneurysms in the adult population is 2% to 7%, and that the incidence increases with age. Intracranial aneurysms, like "sporadic bombs" left in the cranium, can at any time endanger the life of the patient. Therefore, early detection of timely and radical treatment of cerebral aneurysm to avoid rupture and secondary rupture of cerebral aneurysm is a fundamental measure for treating patients with cerebral aneurysm.
Digital Subtraction Angiography (DSA) is still the gold standard for diagnosing intracranial aneurysms today. However, DSA has the disadvantages of being invasive, radioactive, time consuming, relatively expensive, etc. Although CTA diagnoses intracranial aneurysms quickly and with greater accuracy and sensitivity, CTA requires the use of contrast agents and can have renal impairment and allergic reactions, as well as radiation damage. Although 3.0T MRA is highly accurate and sensitive, it can be used as a primary substitute for DSA for the diagnosis and screening of intracranial aneurysms. Due to the long MRA scan time plus post-treatment time, about 15 minutes, there is significant hysteresis in large scale intracranial aneurysm detection diagnosis.
The mechanism of occurrence and development of intracranial aneurysm is still unclear, and the influencing factors are complex. The occurrence and development of intracranial aneurysm are closely related to age, sex, smoking, alcoholism, hypertension, diabetes, hyperlipidemia, infection and the like, and also closely related to heredity and environment. Various factors stimulate each other, and in addition, the cerebral vessels are weak relative to other blood vessels, have weak adventitia, lack elastic fibers, few media smooth muscle cells and undeveloped inner elastic layers, so that the chronic dilatation of the arteries is promoted, and the aneurysm is finally formed. At present, diagnosis, prognosis and evaluation of intracranial aneurysm mainly depend on imaging, hemodynamics, biological model establishment and the like, and the means have obvious hysteresis and cannot carry out early diagnosis on the aneurysm. There is currently a lack of an effective method for accurate, rapid and large-scale detection of unbroken intracranial aneurysms. The research of clinical biomarkers aiming at intracranial aneurysm is helpful for the rapid and large-scale diagnosis and individualized prevention and treatment of cerebral aneurysm, and lays a foundation for revealing pathogenesis of diseases.
Disclosure of Invention
The present invention is intended to overcome the drawbacks of the prior art and to provide a mirna (microrna) marker that can accurately determine the occurrence and risk of cerebral aneurysm, and the clinical markers reported in the literature so far have been associated with the occurrence of cerebral aneurysm, but the degree of association is insufficient, and there is almost no combination of clinical markers for cerebral aneurysm that can further improve the reliability of risk evaluation.
The present invention uses a useful miRNA having a high correlation with the onset of a cerebral aneurysm as a clinical marker, and more preferably uses several useful miRNA related to a cerebral aneurysm in a range that can be clinically measured in reality as a clinical marker, and implements a related application of the miRNA marker for a cerebral aneurysm.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first object of the invention is to provide a clinical marker related to cerebral aneurysm, which comprises any one or more of miR-628-3p, miR-505-5p, miR-3074-3p, miR-497-5p, miR-224-5p, miR-181b-3p and let-7b-5 p.
The second object of the present invention is to provide an application of the above clinical marker related to cerebral aneurysm in the preparation of a kit for detecting the onset of cerebral aneurysm or a kit for diagnosing the onset of cerebral aneurysm.
The third purpose of the invention is to provide a kit for detecting the onset of cerebral aneurysm or a kit for diagnosing the onset of cerebral aneurysm, which comprises any one or more of miR-628-3p, miR-505-5p, miR-3074-3p, miR-497-5p, miR-224-5p, miR-181b-3p and let-7b-5p and primers corresponding to the any one or more of the above.
Further, the primer sequence of the miR-628-3p is shown in SEQ ID NO: 1, and the primer sequence of the miR-505-5p is shown as SEQ ID NO: 2, the primer sequence of the miR-3074-3p is shown as SEQ ID NO: 3, the primer sequence of the miR-497-5p is shown as SEQ ID NO: 4, the primer sequence of the miR-224-5p is shown as SEQ ID NO: 5, the primer sequence of the miR-181b-3p is shown as SEQ ID NO: 6, the primer sequence of the let-7b-5p is shown as SEQ ID NO: shown at 7.
Further, the kit also comprises an internal reference U6 or an internal reference cel-miR-39 and a primer corresponding to the internal reference U6 or the internal reference cel-miR-39.
Further, the primer sequence of the U6 is shown as SEQ ID NO: 8 to SEQ ID NO: 9, and the primer sequence of the cel-miR-39 is shown in SEQ ID NO: shown at 10.
Further, the kit may further comprise commonly used reagents and enzymes.
The nucleotide sequences of the primers are shown in the following table:
a fourth object of the present invention is to provide an application of the above-mentioned kit for detecting onset of cerebral aneurysm or a kit for diagnosing onset of cerebral aneurysm in a judgment method for diagnosing onset of cerebral aneurysm or a clinical application thereof in diagnosis of cerebral aneurysm.
Further, the clinical marker is miR-628-3 p.
Further, when the miRNA marker is used as a clinical marker for determination of occurrence of cerebral aneurysm, the relative expression level 2 of the miRNA marker-ΔΔCtThe values were more than 10-fold compared to normal controls.
Or, further, when the miRNA marker is used as a clinical marker for determining the onset risk of the cerebral aneurysm, the relative expression level of the miRNA marker is 2 along with the increase of the cerebral aneurysm-ΔΔCtThe value also gradually increases.
It is a fifth object of the present invention to provide a method for detecting a clinical marker associated with a cerebral aneurysm, comprising the steps of:
step 1: extracting exosome miRNA from arterial blood or peripheral venous blood;
step 2: detecting the miRNA extracted in the step (1) by using a MiDETECT A TrackTM miRNA qRT-PCR mature chain detection method by adopting the kit;
wherein, the miRNA comprises any one or more of miR-628-3p, miR-505-5p, miR-3074-3p, miR-497-5p, miR-224-5p, miR-181b-3p and let-7b-5 p.
Further, the detection method of the mature chain of the MiDETECT A TrackTM miRNA qRT-PCR comprises miRNA RT reaction and miRNA qPCR reaction, wherein in the miRNA RT reaction, the concentration range of a primer is 200 nM-800 nM, and the RT reaction program is as follows: 60min at 42 ℃ and 10min at 72 ℃; wherein in the miRNA qPCR reaction, the concentration range of forward and reverse primers is 100 nM-500 nM, the PCR reaction degree is 95 ℃ for 10min, and 1 cycle is carried out; 95 ℃ 2sec, 60 ℃ 20sec, 70 ℃ 10sec, 40 cycles.
The sixth purpose of the invention is to provide an application of the miRNA marker related to the cerebral aneurysm in screening of drugs for clinical intervention of the cerebral aneurysm.
The seventh purpose of the invention is to provide an application of the miRNA marker related to the cerebral aneurysm in preparing animals with miRNA marker gene local deletion.
Further, the miRNA marker gene local deletion animal is obtained by knocking out the animal by using selective microRNA clinical marker genes and breeding, wherein the selective microRNA clinical marker genes comprise any one or more of miR-628-3p, miR-505-5p, miR-3074-3p, miR-497-5p, miR-224-5p, miR-181b-3p and let-7b-5 p.
Further, the miRNA marker gene local deletion small animal is used as a test animal for screening clinical intervention drugs or a model animal with diseases with reduced miRNA marker gene expression.
Further, the preparation step of the miRNA marker gene local deletion animal comprises the following steps:
1) firstly, carrying out DNA extraction and sequencing on a wild mouse;
2) designing and constructing a Cas9 vector plasmid and a targeting vector of a miRNA marker gene sequence;
3) transcribing Cas9RNA and sgRNA in vitro;
4) the sgRNA and Cas9RNA are microinjected into fertilized eggs;
5) embryo transfer and establishment of Founder 0(F0) mouse;
6) breeding and identifying experimental mice;
7) and constructing and verifying a gene knockout homozygote mouse model.
Compared with the prior art, the invention has the following beneficial effects:
the clinical marker for cerebral aneurysm of the present invention contains an exosome microRNA clinical marker having sufficiently high correlation with the onset of cerebral aneurysm, and therefore, by using the clinical marker for cerebral aneurysm exosome microRNA of the present invention, i.e., the method for determining the onset of cerebral aneurysm, a determination result with higher reliability can be obtained.
In addition, according to the present invention, by using any one of a kit for detecting the onset of cerebral aneurysm and a kit for diagnosing the onset of cerebral aneurysm, the clinical marker of cerebral aneurysm of the present invention can be accurately and easily detected, and the risk of onset of cerebral aneurysm can be determined with high accuracy and efficiency.
Detailed Description
In the present invention, the term "cerebral aneurysm" means a saccular protrusion of the side wall or bifurcation of cerebral artery, the tip of which does not branch off from the artery. If the length of the protrusion is more than or equal to 2mm, the patient is defined as a cerebral aneurysm, and if the length of the protrusion is less than 2mm, the patient is defined as a pre-neoplastic lesion or an early aneurysm. The arterial cone is defined as a uniform enlargement of the funnel shape at the root of the cerebral artery with vessel branches at the tip. In addition, cerebral aneurysms can be classified as true or false aneurysms. The true cerebral aneurysm is classified as cystic, fusiform or sandwich cerebral aneurysm, which is a cerebral aneurysm originated from the wall of cerebral blood vessels, while the false aneurysm is mostly caused by trauma, and is not a true aneurysm due to the local accumulation of blood due to the rupture of the wall of blood vessels. Therefore, a true cerebral aneurysm is preferred in the present invention.
In the present invention, the "clinical marker of cerebral aneurysm" refers to a gene that is a marker for the onset of cerebral aneurysm. The clinical marker of the cerebral aneurysm comprises 1 or more than 1 exosomes microRNA and the relative expression level of the microRNA is 2-ΔΔCtThe values were more than 10-fold compared to normal controls.
In the present invention, the "risk of developing a cerebral aneurysm" refers to susceptibility of a cerebral aneurysm (the degree of susceptibility to a cerebral aneurysm). That is, in the present invention, the high risk group indicates that the risk of the human individual developing a cerebral aneurysm is estimated to be high, and the low risk group indicates that the risk of the human individual developing hypertension is estimated to be low.
miR-628-3p is microRNA with the highest expression level in smooth muscle cells and endothelial cells of cerebral aneurysm, has obvious difference in serum exosomes of 154 cerebral aneurysm samples and 55 normal control samples, and has the relative expression level of 2-ΔΔCtThe accuracy and the sensitivity of the method are up to more than 85 percent when calculated by more than or equal to 5; however, this standard still has the potential for false negatives and false positives. The inventors and the like for the first timeThe following were found: exosome miR-628-3p is present in serum samples and its relative expression level 2-ΔΔCtThe value is more than or equal to 5 or more than 10 times of the value compared with a normal control, can be used as a clinical marker of cerebral aneurysm to diagnose most of cerebral aneurysm clinically, and is finally confirmed by CTA, MRA or DSA; exosome miR-628-3p is present in serum samples and its relative expression level 2-ΔΔCtValues < 5 or under 10-fold compared to normal controls, mostly without cerebral aneurysms, but the possibility of cerebral aneurysms cannot be completely excluded. Therefore, high expression of exosome miR-628-3p is clinically significant in patients with cerebral aneurysms compared to normal control populations. In addition, no report is provided to indicate that the exosome miR-628-3p gene is particularly related to the cerebral aneurysm, and the fact that the exosome miR-628-3p gene is a clinical marker of the cerebral aneurysm is the content discovered by the inventor and the like for the first time.
In addition, small animals are knocked out by using genes containing the selective miR-628-3p clinical marker and bred, so that animals with local deletion of miR-628-3p clinical marker genes can be made. The small animals thus prepared having a partial deletion of the miR-628-3p gene preferably exhibit a cerebral aneurysm-free state. In addition, the miR-628-3p gene local deletion small animal can be preferably used as a test animal for screening clinical intervention drugs, and can also be used as a model small animal with diseases of miR-628-3p gene expression reduction.
The following further describes embodiments of the present invention with reference to examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1
This example is the extraction and identification of serum exosomes.
Arterial blood or peripheral venous blood of a cerebral aneurysm patient (n 10) or a normal control human (n 10) is extracted by 5ml, and exosome microRNA is extracted by a high-speed centrifugation method or a kit. And then identifying the extracted exosomes by using an electron microscope, Western Blotting and/or a flow cytometry method.
Example 2
The purpose of this example was to discover differential exosome micrornas of cerebral aneurysms.
The method adopts an Illumina HiSeqTM2500 sequencing method or a gene probe method to carry out bioinformatics analysis on the measured data, examine the difference multiple of exosome microRNAs in a case (cerebral aneurysm) group and a control (no cerebral aneurysm) group and carry out case-related analysis, and find 24 differential exosome microRNAs which are related to the occurrence of the aneurysm and have statistical significance.
Example 3
This example performed qPCR validation on 24 differential exosomes micrornas.
qPCR validation was performed on 24 exosome micrornas in 154 aneurysm samples and 55 normal control samples. Then calculating the relative expression level 2 of the microRNA by a formula-ΔΔCtAnd relative expression level 2 with exogenous cel-miR-39-ΔΔCtCompared with the prior art, the exosomes miR-628-3p, miR-505-5p, miR-3074-3p, miR-497-5p, miR-224-5p, miR-181b-3p and let-7b-5p, in particular the exosomes miR-628-3p are very useful as clinical markers of the cerebral aneurysm. When we set the relative expression level of exosomes in serum samples 2-ΔΔCtThe value is more than or equal to 5 or more than 10 times compared with a normal control, the accuracy and the sensitivity of the exosome miR-628-3p for diagnosing the cerebral aneurysm are more than 85 percent, and the exosome miR-628-3p can be used as a clinical marker of the cerebral aneurysm to diagnose most of cerebral aneurysms clinically. Furthermore, by combining these exosome micrornas, the risk of developing a cerebral aneurysm can be determined more accurately than by using a single exosome microRNA alone.
Example 4
The embodiment is a cerebral aneurysm diagnosis kit for detecting serum exosome microRNAs (miR-628-3 p).
Firstly, designing an amplification primer according to a gene sequence of miR-628-3p, and designing an external reference primer according to a gene sequence of an external reference (cel-miR-39) to manufacture the kit. The kit comprises a primer of miR-628-3p, a primer of an external reference (cel-miR-39), a conventional reagent and enzyme required by amplification reaction, and the like.
A serum exosome microRNAs cerebral aneurysm diagnosis kit is prepared by adopting a MiDETECT A TrackTM miRNA qRT-PCR mature chain detection method.
1.Poly(A)Tailing；
1) Preparing a reaction system on ice, preparing the required reaction system according to the following preparation ratio of reagents, wherein the dosage (10 mu L system) of the polyA tailing reaction system (prepared on ice) is as follows:
Total RNA 1μg
5X Poly(A)Polymerase Buffer(blue)2μL
Poly(A)Polymerase(red)1μL
RNase-free water Up to 10μL
note: firstly, total RNA, enriched small molecular RNA, chemically synthesized miRNA standard products and the like can be used as the RNA template, and if the miRNA standard products are used as the RNA template, the specification of the miRNA standard products is referred to; secondly, the system can be amplified according to requirements, and Total RNA of less than 5 mu g is recommended in a 20 mu L reaction system.
2) Uniformly mixing the reaction system, and reacting for 1h at 37 ℃;
3) after the reaction was completed, the reaction mixture was kept on ice for further use or stored at-80 ℃.
2. Carrying out reverse transcription reaction;
1) preparing a reverse transcription reaction system on ice, and preparing a required reaction system according to the preparation proportion of the following reagents; the amount of reagents used in the tailing reverse transcription reaction system (prepared on ice) was as follows:
RTase mix(yellow)4μL
5X RTase Buffer(violet)4μL
miDETECT A TrackTM Uni-RT Primer 2μL
poly (A) Tailing product 10. mu.L
2) Uniformly mixing the reaction system, reacting at 42 ℃ for 1h, and then incubating at 72 ℃ for 10 min;
3) after completion of the reaction, the resulting cDNA was kept on ice or stored at-20 ℃.
qPCR reaction;
1) preparing a qPCR reaction system on ice, and preparing the required reaction system according to the following preparation ratio of reagents, wherein the dosage of the reagents of the qPCR reaction system (prepared on ice) is as follows:
miDETECT A TrackTM miRNA Forward Primer(10μM)0.5μL
miDETECT A TrackTM Uni-Reverse Primer(10μM)0.5μL
2X SYBR Green Mix(brown)10μL
cDNA 2μL
RNase-free water Up to 20μL
note: firstly, 2 mu L (20 mu L system) of undiluted reverse transcription product is used for the highest cDNA, qPCR can be carried out after the cDNA is diluted for miRNA with high expression abundance, the dilution ratio can be 10-1000 times, and the adjustment is carried out according to a specific Ct value; please use the MiDETECT A TrackTM miRNA Forward Primer to perform qPCR amplification. ③ the kit does not contain ROX dye. Using ABI
7000/7700/7900HT/7300/7500/7500Fast Real-Time PCR System and the like require an extra ROX dye as a fluorescence correction instrument and require the user to prepare the required ROX dye. For a specific method of use, reference is made to the description of the apparatus and the description of the ROX dye reagent.
2) The reaction system was gently mixed (avoiding vigorous vortexing) and qPCR reaction was performed using a three-step method.
Reaction conditions are as follows: setting 1) cycle number 1 of 95 ℃ for 10 min; 2) setting the temperature of 95 ℃ for 2s, the temperature of 60 ℃ for 20s, the temperature of 70 ℃ for 10s and the cycle number of 40; 3) and (4) generating a dissolution curve.
Note: the above procedure is applicable to the BioRad CFX96 instrument, and other qPCR instruments that can be set to collect signals at a constant temperature for 10 seconds or less can also use this procedure. Secondly, a part of brand or model qPCR instruments need longer constant temperature time to collect signals when collecting fluorescent signals, the reaction procedure is changed into a two-step method by using the instruments, annealing and extension reactions are combined, and the time is set as the shortest time required by the instruments to collect signals. Melting curve analysis is required to be carried out immediately after circulation is finished on the detection reaction of qPCR (quantitative polymerase chain reaction) carried out by an SYBR Green I dye method, the detection temperature is 70-95 ℃, the heating rate is 0.5 ℃/time, the constant temperature time is 5 sec/time, or the melting curve analysis is carried out by adopting an instrument default program.
Example 5
This example is the establishment of an animal model for knockout of exosome microRNAs (miR-628-3p) gene.
Constructing a miR-628-3p gene knockout mouse model by using CRISPR/Cas9 technology.
The experimental mice are C57BL/6J mice, the age of the mice is 8-12 weeks, and the body mass is 20-25 g.
The establishing steps comprise:
1. firstly, carrying out DNA extraction and sequencing on a wild mouse;
the tail end of a mouse tail with the length of 0.2-0.5 cm is cut and placed in a 1.5ml centrifugal tube, and after the tail end is cut into pieces by sterile scissors, DNA is extracted by adopting a TIANAmp genomics DNA kit gene extraction kit. miR-628 upstream primer 5'-ATTCAGTTTCAAAGAGGTAAT-3' (SEQ ID NO: 11), downstream primer 5'-TTCTGTGTCAACATA ATAG-3' (SEQ ID NO: 12), and the length of the amplification product is 472 bp. The extracted DNA was PCR amplified using an ABI Veriti96PCR instrument with a 20. mu.l reaction system configured with PCR mix. PCR amplification conditions: denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for 35 cycles. And carrying out agarose gel electrophoresis after amplification, and carrying out DNA sequencing on a PCR product with a good electrophoresis result.
2. Designing and constructing Cas9 carrier plasmid and targeting carrier of miR-628 sequence;
after obtaining miR-628-3 p-related sequences with reference to NCBI, Cas9 vector plasmid (Cas9RNA) (U.S. Addgene, ID No.: 68463) and targeting vector single guide RNA (sgRNA) (U.S. Addgene, ID No.: 26527) cleaved by the sequence of interest were designed and constructed. The activity of sgRNA/Cas9 is detected by using a CRISPR/Cas9 activity detection kit, then the sequencing of a targeting vector is carried out, and the endotoxin-free plasmid extraction is carried out by using an endotoxin-free plasmid extraction kit (Omega Bio-Tek in the United states). sgRNA upstream primer: 5'-CATTCTGCACGCTTCAAAAG-3' (SEQ ID NO: 13), and a reverse primer 5'-GTCGCCCCGACGATTTCGCGTACG-3' (SEQ ID NO: 14). miR-628 sites are targeted and positioned by constructing Cas9RNA and corresponding sgRNAs, and target spots are knocked out.
3. Transcribing Cas9RNA and sgRNA in vitro;
T7-sgRNA-Fw primer sequence: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 15), sgRNA-Rv primer sequence: 5'-GCTAGTTATTGCTCAGCGG-3' (SEQ ID NO: 16). A20. mu.l reaction system was prepared using T7-sgRNA-Fw and sgRNA primers and Cas9RNA and sgRNA as templates for PCR amplification. PCR amplification conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s, for 30 cycles.
Microinjecting sgRNA and Cas9RNA into fertilized eggs;
cas9RNA and sgRNA transcribed in vitro are injected into 50-100 fertilized eggs of a mouse in a micro-injection mode, wherein the fertilized eggs are from a C57BL/6J pregnant female mouse. Fertilized egg in-vitro culture is carried out by using a KSOM culture medium under the condition of 37 ℃, 5 embryos are randomly selected after mouse embryos develop for 24 hours, genomic DNA is extracted for PCR amplification, and then the activity detection of sgRNA is carried out.
5. Embryo transfer and establishment of Founder 0(F0) mouse;
and (3) culturing the fertilized eggs subjected to microinjection in vitro for 2-3 d by using a KSOM culture medium at 37 ℃, and transplanting the embryos to the oviducts of surrogate mice after the fertilized eggs develop into embryos. The surrogate mice are female C57BL/6J mice aged 8-12 weeks. 15-25 embryos are transplanted to each generation of pregnant mice. After the breeding of the surrogate mouse, the tail of the bred F0 surrogate mouse is cut, and genome DNA is extracted for PCR amplification and gene sequencing identification.
6. Breeding and identifying experimental mice;
all mice are raised in clean laminar flow animal rooms for breeding, and mouse cages, water, food, padding and the like are sterilized. The experimental animal adopts male mice and female mice to breed offspring in a ratio of 1:1, the pregnancy period of the female mice is about 21d, and the development conditions of the offspring mice are closely observed after the female mice breed. All offspring mice were 4 weeks old with toe clipped for experimental numbering and tail clipped for gene identification.
7. Construction and verification of gene knockout homozygote mouse model
Extracting all progeny mouse genome DNA, carrying out agarose gel electrophoresis after PCR amplification, and then carrying out gene sequencing to identify the mouse genotype. Through identification and analysis of all mouse genotypes, screening a gene knockout mouse capable of being stably passaged, further establishing a miR-628-3p gene knockout mouse homozygous sub-model, and carrying out verification and analysis of miR-628-3p expression detection on the successfully constructed gene knockout model mouse.
The gene knockout model mouse can be used as a test animal for screening clinical intervention drugs, and can also be used as a model small animal with diseases of miR-628-3p gene expression reduction.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> plum Yongdong
<120> clinical marker related to cerebral aneurysm and application thereof
<160> 16
<170> PatentIn version 3.5
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<223> Forward primer, 5' -3' of internal reference U6 '
<400> 8
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aacgcttcac gaatttgcgt 20
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gtcgccccga cgatttcgcg tacg 24
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Claims (4)
1. The application of the reagent for detecting the serum exosome miR-628-3p in preparing a kit for detecting the onset of cerebral aneurysm or a kit for diagnosing the onset of cerebral aneurysm is disclosed.
2. The use according to claim 1, wherein the kit comprises a primer sequence for detecting the serum exosome miR-628-3 p.
3. The use of claim 1, wherein the kit further comprises an internal reference U6 or an internal reference cel-miR-39 and primers corresponding thereto.
4. The use of claim 3, wherein the primer sequence of U6 is as set forth in SEQ ID NO: 8-SEQ ID NO: shown at 9.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710694179.9A CN107557467B (en) | 2017-08-10 | 2017-08-10 | Clinical marker related to cerebral aneurysm and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710694179.9A CN107557467B (en) | 2017-08-10 | 2017-08-10 | Clinical marker related to cerebral aneurysm and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107557467A CN107557467A (en) | 2018-01-09 |
| CN107557467B true CN107557467B (en) | 2021-05-11 |
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| CN112561871B (en) * | 2020-12-08 | 2021-09-03 | 中国医学科学院北京协和医院 | Aortic dissection method and device based on flat scanning CT image |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560553A (en) * | 2009-04-21 | 2009-10-21 | 中南大学 | Usage of miR-381 as encephaloma occurrence molecule mark and testing method thereof |
| CN101842484A (en) * | 2007-09-14 | 2010-09-22 | 俄亥俄州立大学研究基金会 | Mirna expression in human peripheral blood microvesicles and uses thereof |
| CN102031261A (en) * | 2010-10-27 | 2011-04-27 | 南京医科大学 | Serum/plasma miRNA (micro Ribonucleic Acid) marker relevant to gestational diabetes and application thereof |
| CN102899392A (en) * | 2011-07-28 | 2013-01-30 | 上海生物芯片有限公司 | miRNA set detection method and application used in colorectal cancer diagnosis |
| CN103173449A (en) * | 2013-03-07 | 2013-06-26 | 新乡医学院第一附属医院 | Esophagus cancer postoperative early-stage recurrence and prognosis related miRNA marker and its application |
| CN103667282A (en) * | 2012-08-30 | 2014-03-26 | 苏州博泰安生物科技有限公司 | Molecular marker miR-628-3P for prostatic cancer and application of molecular marker miR-628-3P |
| CN103937888A (en) * | 2014-04-14 | 2014-07-23 | 上海交通大学 | Screening method and application of plasma microRNA markers for identifying gastric cancer |
| CN105483230A (en) * | 2015-12-23 | 2016-04-13 | 宁波大学 | MiR-3197 molecular marker for detecting type 2 diabetic retinopathy and amplification primer and application thereof |
-
2017
- 2017-08-10 CN CN201710694179.9A patent/CN107557467B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101842484A (en) * | 2007-09-14 | 2010-09-22 | 俄亥俄州立大学研究基金会 | Mirna expression in human peripheral blood microvesicles and uses thereof |
| CN101560553A (en) * | 2009-04-21 | 2009-10-21 | 中南大学 | Usage of miR-381 as encephaloma occurrence molecule mark and testing method thereof |
| CN102031261A (en) * | 2010-10-27 | 2011-04-27 | 南京医科大学 | Serum/plasma miRNA (micro Ribonucleic Acid) marker relevant to gestational diabetes and application thereof |
| CN102899392A (en) * | 2011-07-28 | 2013-01-30 | 上海生物芯片有限公司 | miRNA set detection method and application used in colorectal cancer diagnosis |
| CN103667282A (en) * | 2012-08-30 | 2014-03-26 | 苏州博泰安生物科技有限公司 | Molecular marker miR-628-3P for prostatic cancer and application of molecular marker miR-628-3P |
| CN103173449A (en) * | 2013-03-07 | 2013-06-26 | 新乡医学院第一附属医院 | Esophagus cancer postoperative early-stage recurrence and prognosis related miRNA marker and its application |
| CN103937888A (en) * | 2014-04-14 | 2014-07-23 | 上海交通大学 | Screening method and application of plasma microRNA markers for identifying gastric cancer |
| CN105483230A (en) * | 2015-12-23 | 2016-04-13 | 宁波大学 | MiR-3197 molecular marker for detecting type 2 diabetic retinopathy and amplification primer and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| miR-628-3p Regulates Osteoblast Differentiation by Targeting RUNX2: Possible Role in Atrophic Non-Union;Hua Chen等;《Int J Mol Med》;20161228;第39卷(第2期);279-286 * |
| miR-628-3p对胃癌细胞增殖与凋亡的影响;陈希媛等;《广东药学院学报》;20150831;第31卷(第4期);531-534 * |
| 颅内动脉瘤壁显著性差异基因miRNA研究;韩亮;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20120915(第09期);E070-84 * |
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