CN107058468A - A kind of kit that acute mountain sickness onset risk is predicted by circulating the 3p expressions of microRNA 369 - Google Patents
A kind of kit that acute mountain sickness onset risk is predicted by circulating the 3p expressions of microRNA 369 Download PDFInfo
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- CN107058468A CN107058468A CN201611002734.9A CN201611002734A CN107058468A CN 107058468 A CN107058468 A CN 107058468A CN 201611002734 A CN201611002734 A CN 201611002734A CN 107058468 A CN107058468 A CN 107058468A
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Abstract
The present invention relates to a kind of microRNA marks for detecting acute mountain sickness susceptible person, and its application in the kit for preparing prediction acute mountain sickness onset risk.Mainly the 3p molecules of microRNA 369 in the blood plasma of subject person Plain are detected by methods such as real-time fluorescence quantitative PCRs, and acute mountain sickness onset risk is predicted by the height of its expression, the microRNA is used as molecular marker, with high specificity, sensitivity is high, it is non-invasive, the features such as convenience, it is particularly suitable for carrying out the screening of extensive acute mountain sickness susceptible person in Plain.
Description
Technical field
The present invention relates to detection kit, microRNA-369- is followed particular by one kind is related to by Plain blood plasma
3p expressions predict acute mountain sickness onset risk, so that the neurological susceptibility for evaluating human body acute mountain sickness.
Background technology
Acute mountain sickness (acute mountain sickness, AMS), also referred to as Acute Mild altitude sickness, are to occur
The people of low altitude area is being moved in for a long time, 1-3 days after more than 2500m plateaus are rapidly entered in the case of not shaking down
Produce, including insomnia, headache, dizziness, anorexia, mood uneasiness and vomit etc. a series of symptoms, wherein violent headache is
AMS classical symptom.And AMS, according to the rate of climb and the difference of specific height above sea level, its incidence of disease is up to 50% to 85%
(Bartsch P and Swenson E R (2013), Clinical practice:Acute high-altitude
Illnesses,《N Engl J Med》, 368 (24), 2294-302.), for entering the crowd on plateau, AMS can be serious
Influence its work and viability.More seriously, if AMS is not controlled effectively and treated, it is likely that develop into
For plateau brain edema (high altitudecerebral edema, HACE) (the Boos C J et al. with high fatal rate
(2016), High Altitude and Acute Mountain Sickness and Changes in Circulating
Endothelin-1, Interleukin-6, and Interleukin-17a,《High Altitude Medicine&
Biology》, 17 (1), 25-31.).
AMS has obvious genetic predisposition and private medical service, and the environmental factor and inherent cause of plateau hypobaric hypoxia are equal
AMS generation can be influenceed.For many years, AMS genetic predisposition and private medical service are always what domestic and foreign scholars were paid close attention to
Focus, although it is proposed that being carried out using hypoxemia sensitive gene EGLN1 and HIF-1AN SNP site to AMS Susceptible population
Prediction, but current apparently these labels (Zhang E, Zhang all not fully up to expectations in terms of accuracy and specificity
J, Jin J, Qin J, Li H, Huang L:Variants of the low oxygen sensors EGLN1and HIF-
1AN associated with acute mountain sickness.《(International journal of
molecular sciences》, 2014,15 (12):21777-21787.).The high original area of China is vast (to account for area
1/5) since in recent years, Qinghai-Tibet mean sea level is more than 4000 meters, with domestic and international highland tour's industry and plateau economic construction
Flourish, the increasing Plain generation crowd of occupying enters plateau, and the live and work that AMS generation has given people is caused
Extremely serious influence, so urgently searching out the effective ways being predicted in Plain to AMS Gene susceptibility.
MicroRNA is the endogenous small molecule non-coding RNA that a class is widely present in eucaryote, length is 18~
24 nucleotides.MicroRNA suppresses the expression of target gene by post-transcriptional level, and regulating cell differentiation, propagation, apoptosis etc. are raw
Life activity, plays a significant role in a variety of physiology such as embryonic development, organism metabolism, disease development and pathologic process.Closely
Nian Lai, researcher detects microRNA in a variety of body fluid such as blood, saliva, urine, proposes that circulation microRNA's is general
Read.Also, the height of microRNA expression and its genetic otherness height correlation, it is in recent years, substantial amounts of to grind
Study carefully and show, in advance diagnosis and morbidity prediction of the circulation microRNA to tumour, hypertension, palsy and a series of diseases have very well
Specificity and sensitiveness.It is clinical workable and traumatic small furthermore the humoral specimens such as blood are easily obtained, and circulation
MicroRNA stability is good, and detection facility, therefore, circulation microRNA have the potential as the non-invasive biomarkers of AMS,
Suitable for AMS neurological susceptibilities Mass screening (Ghai V and Wang K (2016), Recent progress toward the
Use of circulating microRNAs clinical biomarkers,《Arch Toxicol》.).
And on the correlation between circulation microRNA and AMS neurological susceptibilities, there is not been reported.
More having no a kind of is used to detect in blood microRNA-369-3p expressions to predict the report of AMS onset risks.
Examination is composed by Plain native Tibetan Plain plasma circulation microRNA chip, after the exposure of its high altitude anoxia
AMS incidences, it is found that Plain plasma circulation microRNA-369-3p has difference between AMS susceptible person and the resistance to receptors of AMS
It is different.Pass through the method inspection of SYBR (i.e. SYBR GREEN dyestuffs, abbreviation SYBR) real-time fluorescence quantitative PCR (polymerase chain reaction) again
Plain plasma circulation microRNA-369-3p expressions are surveyed, it was confirmed that Plain plasma circulation microRNA-369-3p is expressed
Correlation is there is between level and AMS neurological susceptibility.
The content of the invention
It is an object of the invention to find the Plain blood plasma new bio mark related to AMS, and another purpose
It is to provide utilizations of the Plain blood plasma microRNA-369-3p in the kit for preparing Plain prediction AMS onset risks.The examination
Agent box can be used for screening of people from Plain to AMS susceptible person before plateau is entered, and instruct AMS prevention, mitigate AMS threat.
Inventor is analyzed by the blood plasma microRNA express spectras to 13 AMS patients and 9 normal healthy controls,
The blood plasma microRNA-369-3p expressions of 41 AMS patients and 46 normal healthy controls are compared afterwards, studied
MicroRNA-369-3p and AMS correlation, has searched out the susceptible biological heredity marks of sensitive believable AMS.Step is to use
EDTA-Na anticoagulant tubes gather 22 2ml, 3000 × g, 25 degrees Celsius lower point of peripheral bloods for intending rapidly entering plateau crowd from Plain
Extract and saved backup under -80 degrees Celsius of upper plasma after from 10 minutes, pass through microRNA chip of expression spectrum (miRCURYTM
LNA Array (v.18.0)) microRNA expressions in blood plasma are detected, after crowd enters plateau, according to AMS states
Border universal diagnostic standard Lake Louise scoring diagnostic system distinguishes AMS and healthy population (Maggiorini M et al. (1998)
Assessment of acute mountain sickness by different score protocols in the
Swiss Alps Aviat Space Environ Med, 69 (12), 1186-92.), compare AMS and healthy population microRNA
Express spectra, screens the susceptible related microRNA of AMS, it is found that microRNA-369-3p is expressed in AMS susceptible person and healthy population
There is significant difference.
Using qPCR technologies in another independent crowd to microRNA-369-3p in AMS susceptible person (41) and health
Expression and distribution in control group (46) is detected.Blood plasma RNA is extracted using the triumphant outstanding limited public affairs of technology of Germany in whole process
Blood plasma microRNA pillar extraction agents box (the miRNeasy Serurn/Plasma Kit, article No. of department:217184) extract, so
Real-time fluorescence quantitative PCR (Hairpin-itTMmiRNAs RT-PCR Quantitation Kit, article No. are used afterwards:E01008)
Method carry out microRNA-369-3p and outer ginseng cel-miR-39 are expanded;Every sample is being calculated respectively
Normalization expressions of the microRNA-369-3p with respect to cel-miR-39, as a result tests by SPSS 19.0, with P <
0.05 is significance test standard, finds susceptible group of sample of AMS (41) and normal population (46) blood plasma microRNA-369-
3p expression has significant difference (table 1)
The technical problems to be solved by the invention are to find a kind of Plain blood plasma microRNA marks to filter out
AMS susceptible person and Nai receptor.By detecting the content of the microRNA-369-3p in the blood plasma of people Plain, pass through expression quantity height
To distinguish AMS susceptible person and Nai receptor, and then predict the onset risk for entering AMS behind plateau.
Technical proposal that the invention solves the above-mentioned problems is:Plain human plasma microRNA-369-3p content is detected, with
AMS susceptible person and Nai receptor are distinguished, microRNA-369-3p specifying informations are shown in Table 2.
The present invention benefit be:The microRNA filtered out has good prediction efficiency to AMS, can be predicted in Plain
AMS onset risk after up to plateau, instructs AMS prevention and treatment, mitigates AMS threat.
The present invention has also done following improvement in addition to above-mentioned technical proposal.
Present invention additionally comprises blood plasma microRNA (microRNA-369-3p) is preparing the reagent of prediction AMS onset risks
Effect in box.
Brief description of the drawings
MicroRNA-369-3p expressions are (i.e. with cel-miR-39 phases in the blood plasma of Fig. 1 .AMS susceptible persons and Nai receptor
Normalization level than microRNA-369-3p).Wherein AMS be AMS hair patient, non-AMS be normal healthy controls.*:P <
0.05, that is, there are notable significant difference, * *:P < 0.01, that is, have extremely notable significant difference, * * *:P < 0.001, that is, have and extremely show
Write significant difference.
Fig. 2 is AMS susceptible person's blood plasma microRNA-369-3p performance curve (receiver operating
Characteristic curve, abbreviation ROC curve), the ROC curve, the curve that show AMS susceptible person well in the figure
Lower area (area under the curve, AUC), sensitivity (sensitivity), specific (specificity), wherein
AUC reflects prediction efficiency, and (AUC=0.5 is not previously predicted efficiency;The predictive value of the very littles of 0.5 < AUC < 0.7;0.7 < AUC
The fairly accurate predictive values of < 0.9;0.9 < AUC < 1, very accurate predictive value).
Embodiment
Present invention is described below in conjunction with the accompanying drawings, and cited embodiment is merely illustrative the present invention, not
For limiting the scope of the present invention.
The plasma specimen microRNA-369-3p of embodiment 1 expression and the correlation research of AMS onset risks
First, material and specimen collection are described
AMS patient of AMS susceptible person's plasma specimen in Acute Exposed Altitude crowd enters before plateau, adds up to 41.Just
Normal health crowd of the plasma specimen of ordinary person group in Acute Exposed Altitude crowd enters before plateau, adds up to 46.AMS's examines
It is disconnected to be confirmed by international diagnostic criteria-Lake Louise scoring.All groups do not take any before blood is taken
Preventive medicine.Each sample collects 2ml blood with EDTA-Na anticoagulant tubes are total.
2nd, sample treatment and RNA are extracted
After intravenous blood collection in 10min, after being centrifuged 10 minutes under 3000 × g, 25 degrees Celsius, with without RNase and
Abacterial suction nozzle takes upper plasma to be saved backup for -80 DEG C in without RNase and abacterial EP pipes.RNA in blood plasma passes through
Blood plasma microRNA pillar extraction agents box (the miRNeasy Serum/Plasma Kit, goods of German Kai Jie Technology Co., Ltd.
Number:217184) operating procedure is extracted and purified to specifications.
3rd, real-time fluorescence quantitative PCR (SYBR dye methods)
With the microRNA real-time fluorescence quantitative PCR kits of Chinese Shanghai Ji Ma Pharmaceutical Technology Inc.
(Hairpin-itTMmiRNAs RT-PCR Quantitation Kit, article No.:E01008) to microRNA-369-3p and outside
Ginseng cel-miR-39 is expanded;Ct values (cycle threshold) are respectively obtained, pass through formula:Expression quantity=2Ct (cel-
MiR-39)-Ct (microRNA-369-3p) the microRNA-369-3p for calculating every sample respectively expression, specifically
Specification is shown in operating process.Each sample is tested in triplicate.MicroRNA-369-3p and cel-miR-39 primer sequences are shown in Table
3.
4th, statistical analysis technique.
Counted with statistics software SPSS 19.0.Test of normality uses Shapiro-Wilk methods, and conspicuousness is poor
It is different to be examined with Mann-Whitney (Mann-Whitney Test), performance curve (receiver operating
Characteristic curve, abbreviation ROC curve) and line under area (area under the curve, AUC) be used for evaluate
MicroRNA-369-3p prediction efficiency.Think there is significant difference as p < 0.05.
6th, interpretation of result
1.AMS susceptible person and the resistance to receptors of AMS, blood plasma microRNA-369-3p expression quantity are compared, and p < 0.001 have pole
Notable significant difference.
2. blood plasma microRNA-369-3p can be with by ROC curve to the prediction efficiency of AMS susceptible person and the resistance to receptors of AMS
Know, microRNA-369-3p is good to the prediction efficiency of the resistance to receptor of AMS susceptible person and AMS, and AUC is 0.859 (95%
CI, 0.783-0.985).
7th, conclusion
MicroRNA-369-3p has good prediction efficiency to AMS susceptible person and the resistance to receptors of AMS in blood, can predict AMS
Onset risk.
Susceptible group of the AMS of table 1 and AMS tolerance group blood plasma microRNA-369-3p expression
Susceptible group of VS.AMS tolerance group of * * AMS:P < 0.001, data are represented with median (25%-75% quantiles)
The microRNA-369-3p essential informations of table 2
MicroRNA-369-3p the and cel-miR-39 primer information of table 3
Claims (3)
1. a kind of microRNA molecule mark for being used to predict acute mountain sickness susceptible person, it is characterized in that, the microRNA
Molecular marker is microRNA-369-3p.
2. a kind of microRNA molecule mark for being used to predict acute mountain sickness susceptible person as claimed in claim 1, it is special
Levy as the sequence of the microRNA-369-3p is SEQ ID NO:1.
3. claim 1, a kind of microRNA molecule mark for being used to predict acute mountain sickness susceptible person described in 2 any one
Purposes of the thing in the prediction kit of acute mountain sickness susceptible person.
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CN109897897A (en) * | 2019-04-23 | 2019-06-18 | 中国人民解放军陆军军医大学 | Application and kit of the hsa-miR-15b-5p as molecular marker |
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US20110003704A1 (en) * | 2008-02-01 | 2011-01-06 | The General Hospital Corporation | Use of microvesicles in diagnosis and prognosis of medical diseases and conditions |
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ACCESSION NO: LM379230: "TPA:Homo sapiens microRNA hsa-mir-369-3p", 《GENBANK》 * |
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CN109897897A (en) * | 2019-04-23 | 2019-06-18 | 中国人民解放军陆军军医大学 | Application and kit of the hsa-miR-15b-5p as molecular marker |
CN109897897B (en) * | 2019-04-23 | 2022-04-01 | 中国人民解放军陆军军医大学 | Application of hsa-miR-15b-5p as molecular marker and kit |
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