CN109868315A

CN109868315A - For early detection cerebral aneurysm subarachnoid hemorrhage severity and the in-vitro method of prognosis

Info

Publication number: CN109868315A
Application number: CN201910081959.5A
Authority: CN
Inventors: 赖年升; 李真保; 方兴根; 姚阳; 赵心同; 袁金龙; 刘佳强; 吴德刚
Original assignee: Yijishan Hospital of Wannan Medical College
Current assignee: Yijishan Hospital of Wannan Medical College
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2019-06-11
Anticipated expiration: 2039-01-28
Also published as: CN109868315B

Abstract

The embodiment of the invention discloses a kind of for early detection cerebral aneurysm subarachnoid hemorrhage severity and the in-vitro method of prognosis, passes through the microRNAs expression of measurement blood plasma excretion body.The embodiment of the present invention also provides the product of a kind of early detection cerebral aneurysm subarachnoid hemorrhage severity and prognosis, and the product determines cerebral aneurysm subarachnoid hemorrhage severity and prognosis by measuring the microRNAs expression of blood plasma excretion body.The embodiment of the present invention uses excretion body microRNANA sequencing, real-time PCR method and technology for the first time, the potential source biomolecule marker closely related with aSAH occurrence and development is filtered out from blood excretion body, it is related with prognosis mala to be determined that microRNA-193b-3p and microRNA-486-3p expression is increased, and microRNA-369-3p and microRNA-410-3p expression reduction is related with prognosis mala, new method is provided for the monitoring and evaluation prognosis of aSAH, the treatment effect for improving such disease is provided fundamental basis and experimental basis.

Description

For early detection cerebral aneurysm subarachnoid hemorrhage severity and prognosis In-vitro method

Technical field

The present invention relates to technical field of medical detection, and in particular to one kind is under early detection cerebral aneurysm arachnoid The in-vitro method of chamber bleeding severity and in-vitro method, the product of prognosis.

Background technique

Cerebral aneurysm subarachnoid hemorrhage (Aneurysmal subarachnoid haemorrhage, aSAH) is Intracranial subarachnoid hemorrhage caused by cerebral aneurysm bleeding.Clinical onset case fatality rate and disability rate are up to 45%, account for cerebral apoplexy 5%.Majority influences the reason of prognosis predominantly cerebral injury of early stage and vasopasm and resulting Delayed onset brain and lacks Blood.Many researchers have found that early stage cerebral injury is to lead to the deciding factor of aSAH poor prognosis recently, so early for aSAH The research of phase pathophysiological change is particularly important.Early stage cerebral injury may with microcirculation deficiency, the destruction of ionic homeostasis, inflammation and Capilary shrinks related.A kind of discovery of noninvasive biomarker can be possible pre- with early detection patient change of illness state and evaluation Afterwards.

MicroRNAs (microRNANAs) is the non-coding RNA of 17-24 nucleic acid, by with mRNA combination controlling gene Post-transcriptional level.Many research prompt microRNANAs participate in many physiology and pathologic process, while being also used as a variety of diseases Diagnosing and treating means, comprising: stroke, Parkinson's disease, brain trauma and alzheimer's disease.Researcher also prompts MicroRNANAs unconventionality expression in the cerebrospinal fluid of aSAH patient.And microRNANAs is relative to lincRNAs and mRNAs It is more stable, it is not degradable, it is prevalent in tissue, peripheral blood and cerebrospinal fluid.

Cerebral aneurysm subarachnoid hemorrhage (aSAH) is a kind of serious age of onset cerebral apoplexy earlier, after the onset The death rate and disability rate are all very high.It is considered that cerebral vasospasm after subarachnoid hemorrhage and relevant delayed cerebral ischemia It is the main reason for influencing prognosis.But it is died of in SAH initial 48 hours more than the inpatient of half, and reverse encephalic Big vasopasm can not also reduce the death rate and promote neurological functional recovery.What nearest research prompt SAH morbidity early stage occurred Early stage cerebral injury (EBI) may be to influence patient's prognosis key factor, and basic research and clinic show EBI principal element after SAH May be related with cerebral ischemia, but how to diagnose the severity of SAH patient EBI SAH is treated it is most important, especially pair In screening the damage for not causing patient's disturbance of consciousness but so far there are no a kind of reliable, convenient and accurate monitoring method, this is just So that more accurate, sensitive, the noninvasive EBI diagnostic method of research and development is particularly important, and prediction well can be played to prognosis and is made With.But prior art means can only be by the consciousness situation and head CT amount of bleeding that judge patient come subjective judgement.Urgently research and develop A kind of in-vitro method and cerebral aneurysm arachnoid for early detection cerebral aneurysm subarachnoid hemorrhage severity Cavity of resorption bleeding prognosis.

Summary of the invention

Being designed to provide for the embodiment of the present invention is a kind of tight for early detection cerebral aneurysm subarachnoid hemorrhage Weight degree and the in-vitro method of prognosis, can only be by judging the consciousness situation and head CT bleeding of patient to solve the prior art Amount carrys out the subjective judgement state of an illness and causes forecasting inaccuracy true, is unable to the defect of look-ahead.

To achieve the above object, the embodiment of the present invention provides a kind of goes out for early detection cerebral aneurysm cavum subarachnoidale Blood severity and the in-vitro method of prognosis, by the microRNAs expression for measuring blood plasma excretion body.

Preferably, the microRNAs is microRNA-193b-3p, microRNA-486-3p, microRNA-369- One or more of 3p, microRNA-410-3p.

Preferably, microRNA-193b-3p, microRNA-486-3p expression quantity increases and cerebral aneurysm spider web Film lower cavity hemorrhage severity and prognosis mala are related.

Preferably, the microRNA-369-3p and microRNA-410-3p expression reduces and cerebral aneurysm arachnoid Cavity of resorption bleeding severity and prognosis mala are related.

The embodiment of the present invention also provides a kind of early detection cerebral aneurysm subarachnoid hemorrhage severity and prognosis Product, the product by measure blood plasma excretion body microRNAs expression determine cerebral aneurysm cavum subarachnoidale Bleeding severity and prognosis.

Preferably, the product is detection chip or detection kit；

The chip includes being fixed with specificity on solid phase carrier to correspond to microRNA-193b-3p, microRNA- Some or all of at least one of 486-3p, microRNA-369-3p, microRNA-410-3p microRNA few nucleosides Acid sequence；

The kit includes for detecting microRNA-193b-3p, microRNA-486-3p, microRNA-369- The reagent of the expression of at least one of 3p, microRNA-410-3p microRNA.

Preferably, the microRNA-193b-3p primer sequence is as shown in SEQ ID NO.1； microRNA-486-3p Primer sequence is as shown in SEQ ID NO.2；The primer sequence of microRNA-369-3p is as shown in SEQ ID NO.2； The primer sequence of microRNA-410-3p is as shown in SEQ ID NO.4.

Preferably, the detection kit includes reverse transcription agents useful for same, and the reagent includes that total serum IgE is template, reverses Record buffer, triphosphoric acid base deoxynucleotide, RNAase inhibitor, MMLV reverse transcriptase, ploy A tailing enzyme.

Preferably, the detection kit further includes real-time quantitative PCR agents useful for same, and the reagent includes microRNA- It is the positive PCR primer of 193b-3p, microRNA-486-3p, microRNA-369-3p and microRNA-410-3p specificity, outer Join cel-microRNA-39 primer, general reverse primer, real time fluorescent quantitative SYBR dyestuff, without enzyme water.

In the embodiment of the present invention, it includes albumen, lipid, mRNA and non-volume that excretion body, which is the 30 lipid membrane vesicles for arriving 100nm, Code RNA etc. can pass through blood-brain barrier, can also carry out the cell-cell communication of specific long range.Brain cell can discharge outer It secretes body and enters circulation across blood-brain barrier, enter blood circulation similar to endothelial cell and peripheral cells secretion excretion body.So Excretion body can be detected in peripheral blood.

The miRNA that miRNA of the invention is primarily referred to as people can also be write as microRNA.For example, in inventive embodiments, " microRNA-193b-3p " also represents " hsa-miR-193b-3p ".

The embodiment of the present invention has the advantages that

The embodiment of the present invention uses excretion body microRNANA sequencing, real-time PCR method and technology for the first time, from blood The potential source biomolecule marker microRNANA closely related with aSAH occurrence and development, its work of clinical verification are filtered out in liquid excretion body For the specificity, sensitivity and feasibility of aSAH, and inquire into the correlation of itself and patient's aSAH prognosis, it is determined that microRNA- , and microRNA-369-3p and microRNA- related with prognosis mala is increased in 193b-3p and microRNA-486-3p expression 410-3p expression reduction is related with prognosis mala, provides new method for the monitoring and evaluation prognosis of aSAH, improves such disease Treatment effect provide fundamental basis and experimental basis.

Detailed description of the invention

Fig. 1 is the excretion body electron microscope of the embodiment of the present invention；

Fig. 2 is the expression electrophoretogram of the mark memebrane protein of the embodiment of the present invention, is respectively CD63 albumen and Alix egg in figure It is white；

Fig. 3 is the differential expression schematic diagram of the microRNANAs of the embodiment of the present invention；

Fig. 4 is the expression schematic diagram of the candidate microRNANAs of the embodiment of the present invention；

Fig. 5 is the ROC curve figure of 4 kinds of candidate microRNANAs of the embodiment of the present invention；

Fig. 6 is the relation schematic diagram of four kinds of candidate microRNANAs of the embodiment of the present invention and severity of being admitted to hospital；

Fig. 7 is the relation schematic diagram of four kinds of candidates microRNANAs and patient's prognosis of the embodiment of the present invention.

Specific embodiment

Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation Content disclosed by book is understood other advantages and efficacy of the present invention easily.

The embodiment of the present invention is provided through detection cerebral aneurysm subarachnoid hemorrhage marker blood plasma excretion body MicroRNAs and its evaluation disease severity and prognosis external detection method, with excretion body microRNANA sequencing, Real-time PCR method filters out and cerebral aneurysm subarachnoid hemorrhage aSAH occurrence and development from blood excretion body Closely related potential source biomolecule marker microRNAs, its specificity, sensitivity and feasibility as aSAH of clinical verification, Pass through monitoring the severity of disease and prognosis to aSAH.

1 experimental design of embodiment and collection of specimens

1, experimental design

Subject signs informed consent form by oneself or family members, and all samples that research process uses pass through ethics The committee passes through, and the embodiment of the present invention divides three phases: discovery phase, coach group, Qualify Phase.To cerebral aneurysm arachnoid Cavity of resorption bleeding (Aneurysmal subarachnoid haemorrhage, aSAH) and control group blood plasma excretion body carried out for two generations Sequencing, finding differences property microRNANAs.The research for carrying out coach group by the small sample of 10 pairs of blood plasma again is determined to enter and be tested The candidate microRNANAs in card stage.The verifying of large sample is then carried out to candidate microRNANAs.Function outcome is logical Cross improvement Rankin scoring evaluation.Wherein, 0-2 points are prognosis bona, and 3-6 points are prognosis mala.

2, collection of specimens

All blood specimens are all to be acquired with EDTA anticoagulant tube, and blood plasma is centrifuged in 2 hours.Blood specimen first exists 500 turns of centrifugations, 10 minutes removal haemocytes at 4 DEG C, the rear 1.5mlEP for shifting supernatant to no enzyme is managed, and 300 turns of centrifugations 10 at 4 DEG C Minute removal blood platelet and cell fragment.Transfer supernatant deposits in -80 DEG C of refrigerators.

Extraction, Electronic Speculum observation and the WB verifying of 2 excretion body of embodiment

1, excretion body separates

Excretion body passes through exoEasy Maxi Kit (from plasma；Qiagen, Valencia, CA) it is separated.First Sample is filtered by 0.8 μm of screen pipe.By sample and buffer XBP carries out isometric mixing by 1:1 and room temperature is incubated It educates 5 minutes.It is then transferred in the centrifuge tube of filter membrane, 500g is centrifuged 1 minute removal centrifugate.Isolated excretion body application Buffer XWP carries out cleaning 5000g and is centrifuged 5 minutes removal foreign proteins.Finally, carrying out the outer of dissolution collection with buffer XE Secrete body.Utilize the amount of the excretion body of BCA method measurement concentration.Transmission electron microscopy, excretion body are carried out by carbon coating nickel screen lattice Absorb 1 hour, after washed three times with PBS, 5 minutes every time, then fix ten minutes with 2% formaldehyde.Use uranium acetate and citric acid Lead negative staining is cleaned three times with deionized water, and 80kV transmission electron microscope JEOL JEM-1400 observe and taken pictures after drying.

2, protein immunoblotting first carries out race glue to excretion body with 10%SDS-PAGE, then carries out transferring film.At 4 DEG C CD63 albumen and Alix albumen primary antibody are incubated overnight, then show to immune complex using ECL after two anti-bindings and determine Amount.

3, excretion body characteristics confirm that excretion body about between 30-100nm, is marked as shown in Fig. 1, and to it by Electronic Speculum Will memebrane protein carries out protein imprinted check as shown in Figure 2.

3 NGS of embodiment sequencing, data collection and otherness microRNAs analysis

1, NGS is sequenced

Tiny RNA library construction: the extraction of total serum IgE is carried out using Trizol.Using Bioanalyzer 2100 to total serum IgE into Row is quantitative and detects purity, and it is qualified for purity that RIN is greater than 7.0.About 1ug total serum IgE can build library, using Illumina Hiseq2500 is sequenced.

2, data collection

Initial data is acquired by ACGT101-microRNA, and remove dimer, rRNA, tRNA, snRNA, SnoRNA and repetitive sequence.Then retrieved by BLAST by the unique sequences application in 18~26 nucleotide MicroRNABase 21.0 identifies known microRNANAs and new 3p- or 5p-microRNANAs.

3, otherness microRNANAs is analyzed

It is accurately examined using Fisher, X²2*2 inspection, X²N*n is examined, t is examined or the variance analysis based on experimental design Differentiation inspection is carried out to sequencing result.Conspicuousness threshold value is respectively 0.01 and 0.05.

Excretion body microRNA express spectra: sequencing result, which is shown, reads human genomic sequence 9,000,000-1 1,005,000,000 times It takes.There are > 50,000 kinds of different RNA sequences.There is microRNAs in 746 to be read in microRNAs known to 2139 kinds.Analysis ASAH and control group find the difference of their express spectras.According to correction p value<0.05, | log2 (fold change) |>1 and table Up to it is horizontal be not low standard, there are six kinds of microRNAs to be considered expressing variant, six kinds of microRNAs are respectively as follows: hsa- MicroRNA-369-3p, hsa-microRNA-136-3p, hsa-microRNA-410-3p, hsa-microRNA-195-5p, Hsa-microRNA-486-3p, and hsa-microRNA-193b-3p.

The sequence of above-mentioned six kinds of microRNAs is as follows:

4 reverse transcription of embodiment and RT-PCR

1, reverse transcription

Using (the Beijing microRNAcute Plus microRNANA First-Strand cDNA Synthesis Kit Tiangeng biology) synthesize cDNA and tailing.2×microRNANA RT Reaction Buffer(10μl),2μl microRNANA RT Enzyme Mix,4μl total RNA and 4μl RNase-Free ddH₂O, 20 μ l volume altogether, 42 DEG C keep 60 Minute, 95 DEG C are kept for 3 minutes, and it is spare that the cDNA of synthesis is stored in -20 DEG C of refrigerators.

2、Real time PCR

Using microRNAcute Plus microRNANA qPCR Detection kit, (SYBR Green, Tiangeng are raw Object) it is detected in CFX96TouthTM Real-Time PCR machine (Bio-rad Lab, Inc.).According to 20 μ l reaction systems Solution is configured, 2 μ l forward primers:

MicroRNA-193b-3p (SEQ ID NO.1):

5'AACTGGCCCTCAAAGTCCCGCT 3'；

MicroRNA-486-3p (SEQ ID NO.2):

5'gCGGGGCAGCTCAGTACAGGAT 3'；

MicroRNA-369-3p (SEQ ID NO.3):

5'accggccgcggAATAATACATGGTTGATCTTTT 3'；

MicroRNA-410-3p (SEQ ID NO.4):

5'ccgcgggAATATAACACAGATGGCCTGT 3'；

MicroRNA-136-3p (SEQ ID NO.5):

5'ccgcggCATCATCGTCTCAAATGAGTCT 3'；

MicroRNA-195-5p (SEQ ID NO.6):

5'cgccggTAGCAGCACAGAAATATTGGC 3'；

10μl microRNAcute Plus microRNANA Premix；The general reverse primer of 0.4 μ l (SEQ ID NO.7:5 ' CAGGTCCAGTTTTTTTTTTTTTTTCGT3 ')；2 μ lcDNA and 5.6 μ lddH₂O.Then it is maintained according to 95 DEG C 15min, 5 circulations: 25sec maintains 94 DEG C, and 30sec maintains 65 DEG C and 34sec to maintain 72 DEG C, then 45 circulations: 20sec 94 DEG C are maintained, 34sec maintains 60 DEG C.Ct value, which is greater than 30, to be considered utilizing 2 to express in vain^-△CtCarry out result calculating.

Embodiment 5

1, it statisticallys analyze

Data are analyzed using statistics software MedCalc version 15.0.0.Data are averaged ± standard deviation, answer It is examined with Mann-Whitney U or Kruskal-Wallis inspection is compared two or more sets differences.Two groups of related coefficients Using Spearman rank sum test.ASAH feature can be inquired into using diagnostics method ROC curve.First by prognosis in 1 year Using single factor analysis, the results of univariate logistic analysis is subjected to multiplicity using the standard of P < 0.2 afterwards, calculating aSAH prognosis may Correlative factor, and obtain risk factors OR and its 95% credibility interval.P < 0.05 is considered to have statistical significance.

The relationship of 6 miRNAs of embodiment expression and cerebral aneurysm subarachnoid hemorrhage aSAH severity

Express spectra is verified: first with 10 aSAH patient's samples and 10 normal controls, to this six species diversity MicroRNANAs carries out qRT-PCR test, wherein as shown in figure 3, hsa-microRNA-369-3p, hsa-microRNA- 410-3p, hsa-microRNA-193b-3p and hsa-microRNA-486-3p expression have statistical difference, subsequent large sample These four candidate microRNANAs are studied, sample includes 113 aSAH and 20 normal controls.Relative to control Group, wherein as shown in figure 4,4 kinds of microRNANAs expression are all variant, and application ROC curve is studied, as shown in figure 5, hair Now their AUC area is respectively as follows: microRNA-369-3p:0.994；microRNA-410-3p:0.987；microRNA- 193b-3p:0.999；And microRNA-486-3p:0.976.Candidate microRNANAs and patient's early stage coincident with severity degree of condition With the relationship of prognosis: WFNS classification I-III being turned to light-duty aSAH, IV-V is heavy type aSAH.MicroRNA- as the result is shown 193b-3p and microRNA-486-3p expresses increase in heavy aSAH, and microRNA-369-3p and microRNA- 410-3p expresses reduction in heavy aSAH, as shown in Figure 6.4 kinds of microRNANAs of the embodiment of the present invention are expressed and WFNS The degree of correlation of severity is respectively as follows: microRNA-369-3p (ρ=- 0.660；P<0.001；95% CI:-0.753--- 0.541), microRNA-410-3p (ρ=- 0.626；P<0.001；95%CI:-0.727---0.499), microRNA- (ρ=0.774 193b-3p；P<0.001；95%CI:0.688 -- 0.839) and microRNA-486-3p (ρ=0.818；P< 0.001；95%CI:0.746-- 0.871).According to prognosis bona's standard, analysis shows that microRNA-193b-3p and , and microRNA-369-3p and microRNA-410-3p related with aSAH prognosis mala is increased in microRNA-486-3p expression Expression reduces related with aSAH prognosis mala.

7 qRT-PCR of embodiment verifies the relationship of miRNAs expression and aSAH prognosis

In the embodiment of the present invention, the reagent of early detection cerebral aneurysm subarachnoid hemorrhage severity and prognosis Box, the primer including detecting four kinds of miRNAs,

MiR-193b-3p (SEQ ID NO.1): 5 ' AACTGGCCCTCAAAGTCCCGCT 3 '；miR-486-3p(SEQ ID NO.2): 5 ' gCGGGGCAGCTCAGTACAGGAT 3 '；MiR-369-3p (SEQ ID NO.3): 5 ' accggccgcgg AATAATACATGGTTGATCTTTT 3'；MiR-410-3p (SEQ ID NO.4): 5 ' CcgcgggAATATAACACAGATGGCCTGT 3 ') qRT-PCR primer.

Excretion body is extracted from blood plasma and is extracted RNA and is carried out reverse transcription and all reagents of qRT-PCR.Extract RNA's Method is the same as embodiment 1, comprising: from all reagents for extracting RNA in excretion body in blood plasma: excretion body settling agent is adsorbed with film Test tube, RNA stablizing solution, Trizol reagent, metaformaldehyde, isopropanol, without enzyme water, outer ginseng cel-miR-39.Reverse transcription, with It by miR-193b-3p, miR-486-3p, miR-369-3p and miR-410-3p reverse transcription is cDNA, reverse transcription that total serum IgE, which is template, Agents useful for same includes RT Buffer, triphosphoric acid base deoxynucleotide, RNAase inhibitor, MMLV reverse transcriptase, ploy A tailing enzyme.

Real-time quantitative PCR is carried out, agents useful for same includes miR-193b-3p, miR-486-3p, miR-369-3p and miR- 410-3p specificity positive PCR primer, outer ginseng cel-miR-39 primer (SEQ ID NO.8:5 ' CgUCACCGGGUGUAAAUCAGCUUG3 '), general reverse primer (SEQ ID NO.7:5 ' CAGGTCCAGTTTTTTTTTTTTTTTCGT3 '), real time fluorescent quantitative SYBR dyestuff, without enzyme water.

It is analyzed by blood plasma of the qRT-PCR to 113 aSAH patients and 20 normal controls, as shown in figure 4, excretion Body miR-193b-3p, miR-486-3p, miR-369-3p and miR-410-3p differential expression in the two are anticipated with statistics Justice, and by logistics regression analysis, as shown in fig. 7, excretion body miR-193b-3p, miR-486-3p, miR-369-3p It is closely related with patient's prognosis with miR-410-3p variation, according to prognosis bona's standard, analysis shows that microRNA-193b-3p Increase, and microRNA-369-3p and microRNA-410-3p related with prognosis mala with microRNA-486-3p expression Expression reduces related with prognosis mala.

As shown in figure 4, in the embodiment of the present invention, the data statistics of 40 and 20 testing results or atlas analysis.

Embodiment 8miRNA pcr chip verifies microRNA-193b-3p, microRNA-486-3p, microRNA- The differential expression of 369-3p, microRNA-410-3p

In the embodiment of the present invention, the method that miRNA is extracted is such as embodiment 1, microRNAcute Plus MicroRNANA First-Strand cDNA Synthesis Kit (Beijing Tiangeng biology) synthesis cDNA and tailing, obtain Then complete cDNA will be added to after the dilution of cDNA template containing microRNA-193b-3p, microRNA-486-3p, In tetra- species-specific primer of microRNA-369-3p, microRNA-410-3p and the chip of PCR reaction solution, carry out real-time Quantitative PCR reaction.Ct value is calculated, utilizes 2^-△CtCarry out result calculating.

By measuring microRNA-193b-3p, microRNA-486-3p, microRNA-369-3p, microRNA- 410-3p can be with the severity and prognosis of early detection cerebral aneurysm subarachnoid hemorrhage, i.e. microRNA-193b- , and microRNA-369-3p and microRNA-410-3p related with prognosis mala is increased in 3p and microRNA-486-3p expression Expression reduces related with prognosis mala.

As transformable embodiment, microRNA-193b-3p, microRNA-486-3p can choose, Any one or a few in microRNA-369-3p, microRNA-410-3p is made into PCR detection kit or chip progress Detection.

The early detection cerebral aneurysm subarachnoid hemorrhage severity of the embodiment of the present invention and the chip of prognosis or Kit, the seriously ill degree and prognosis to cerebral aneurysm subarachnoid hemorrhage aSAH are related, cerebral aneurysm arachnoid The closely related potential source biomolecule marker of cavity of resorption bleeding aSAH occurrence and development, microRNA:miR-193b-3p, miR-486-3p, MiR-369-3p and miR-410-3p is in its specificity, sensitivity and feasibility as aSAH marker of clinical verification The monitoring and evaluation prognosis of aSAH provides new method.

Although above having used general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.

SEQUENCE LISTING

<110>the first affiliated hospital, Wannan Medical College (mountain Wannan Medical College Ge Ji hospital)

<120>for early detection cerebral aneurysm subarachnoid hemorrhage severity and the in-vitro method of prognosis

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Claims

1. a kind of for early detection cerebral aneurysm subarachnoid hemorrhage severity and the in-vitro method of prognosis, feature It is, by the microRNAs expression for measuring blood plasma excretion body.

2. the method as described in claim 1, which is characterized in that

The microRNAs is microRNA-193b-3p, microRNA-486-3p, microRNA-369-3p, microRNA- One or more of 410-3p.

3. method according to claim 2, which is characterized in that

MicroRNA-193b-3p, microRNA-486-3p expression quantity increases and cerebral aneurysm subarachnoid hemorrhage Severity and prognosis mala are related.

4. method according to claim 2, which is characterized in that

The microRNA-369-3p and microRNA-410-3p expression reduces tight with cerebral aneurysm subarachnoid hemorrhage Severe and prognosis mala are related.

5. a kind of early detection cerebral aneurysm subarachnoid hemorrhage severity and the product of prognosis, which is characterized in that

The product determines cerebral aneurysm subarachnoid hemorrhage by measuring the microRNAs expression of blood plasma excretion body Severity and prognosis.

6. product as claimed in claim 5, which is characterized in that

The product is detection chip or detection kit；

The chip includes being fixed with specificity on solid phase carrier to correspond to microRNA-193b-3p, microRNA-486-3p, Some or all of at least one of microRNA-369-3p, microRNA-410-3p microRNA oligonucleotide sequence；

The kit include for detecting microRNA-193b-3p, microRNA-486-3p, microRNA-369-3p, The reagent of the expression of at least one of microRNA-410-3p microRNA.

7. product as claimed in claim 6, which is characterized in that

The microRNA-193b-3p primer sequence is as shown in SEQ ID NO.1；MicroRNA-486-3p primer sequence is such as Shown in SEQ ID NO.2；The primer sequence of microRNA-369-3p is as shown in SEQ ID NO.2；MicroRNA-410-3p's Primer sequence is as shown in SEQ ID NO.4.

8. product as claimed in claim 6, which is characterized in that

The detection kit includes reverse transcription agents useful for same, and the reagent includes that total serum IgE is template, RT Buffer, three Phosphoric acid base deoxynucleotide, RNAase inhibitor, MMLV reverse transcriptase, ploy A tailing enzyme.

9. product as claimed in claim 8, which is characterized in that

The detection kit further includes real-time quantitative PCR agents useful for same, the reagent include microRNA-193b-3p, MicroRNA-486-3p, microRNA-369-3p and microRNA-410-3p specificity positive PCR primer, outer ginseng cel- MicroRNA-39 primer, general reverse primer, real time fluorescent quantitative SYBR dyestuff, ROX contrast dye, without RNA enzyme distilled water.