CN107058472A - A kind of diagnostic kit by four kinds of blood plasma microRNA Combining diagnosis acute mountain sicknesses - Google Patents
A kind of diagnostic kit by four kinds of blood plasma microRNA Combining diagnosis acute mountain sicknesses Download PDFInfo
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Abstract
The present invention relates to a kind of microRNA marks for being used to detect acute mountain sickness and its application in acute mountain sickness detection kit is prepared.By the methods such as quantitative PCR detect measured's blood in microRNA 676 3p, microRNA 181b 5p, microRNA 193b 5p, microRNA 3591 3p content, and compared with normal microRNA levels and carry out diagnosing acute mountain sickness, acute mountain sickness molecular marker microRNA sensitivity is high, high specificity, it is easy to operate, noninvasive injure of safety is easy to a large amount of examinations, effect is used in combination more excellent.
Description
Technical field
It is that a kind of microRNA using in blood plasma is used as biological marker the present invention relates to Medical Molecular Biology technical field
Thing is used for the diagnosis of acute mountain sickness.
Background technology
Acute mountain sickness (acute mountain sickness, AMS), also referred to as Acute Mild altitude sickness, are people couple
The headache that plateau natural environment is not adapted to and produced, anorexia, nausea, the body reaction such as dizziness and fatigue.It is Acute Exposed Altitude
Most common plateau spy hair disease in crowd.Existing research is due to population ages, into plateau elevation height, speed and AMS
The difference of diagnostic method, the AMS incidence of disease is 16-100% (MacInnis M J et al. (2013) A prospective
epidemiological study of acute mountain sickness in Nepalese pilgrims
Ascending to high altitude (4380m),《PLoS One》, 8 (10), e75644., McDevitt M et al.
(2014)Risk determinants of acute mountain sickness in trekkers in the Nepali
Himalaya:A 24-year follow-up,《Wilderness Environ Med》, 25 (2), 152-9., Gaillard S
Et al. (2004) Awareness, prevalence, medication use, and risk factors of acute
mountain sickness in tourists trekking around the Annapumas in Nepal:a 12-
Year follow-up,《High Alt Med Biol》, 5 (4), 410-9.), in randomized control study, AMS middle position hair
Sick rate is 60% (Waeber B et al. (2015) Impact of Study Design on Reported Incidences
of Acute Mountain Sickness:A Systematic Review,《High Alt Med Biol》, 16 (3), 204-
15.) it is, to influence a major obstacle of Acute Exposed Altitude crowd's live and work.If AMS is not controlled timely and effectively
System, may progress to the plateau brain edema with high fatal rate and then threat to life (Basnyat B and Murdoch D R
(2003) High-altitude illness,《Lancet》, 361 (9373), 1967-74.).
Although the diagnosis to AMS has carried out substantial amounts of research, the outer diagnosis Main Basiss to AMS of Current Domestic in recent years
The order of severity for the associated clinical symptoms that patient is shown carries out scoring diagnosis, and existing symptom score diagnostic system has 1980
Environment Symptom Questionnaire score diagnostic system, the People's Republic of China (PRC) GJB GJB proposed for 1991 of year proposition
1098-91, the Canadian international diagnostic criteria in Lake Louise in 1993 and nineteen ninety-five Chinese Medical Association are in GJB base
The Acute Mild plateau disease symptoms indexing formulated on plinth and points-scoring system.Because symptom score has larger subjectivity, therefore
Finding relatively reliable objectivity diagnosis index turns into the another study hotspot that AMS is diagnosed.
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, numerous studies show, follow
In advance diagnosis and morbidity prediction of the ring microRNA to tumour, hypertension, palsy and a series of diseases have good specificity
And sensitiveness.Furthermore the humoral specimens such as blood are easily obtained, clinic can have strong operability and traumatic small, and circulation microRNA is steady
Qualitative good, detection facility, therefore, circulation microRNA have the potential as the non-invasive biomarkers of AMS.
And on the correlation between circulation microRNA and AMS morbidities, there is not been reported.
More having no a kind of is used to detect microRNA-676-3p, microRNA-181b-5p, microRNA- in blood plasma
193b-5p, microRNA-3591-3p expression come diagnose AMS morbidity report.Equally, 4 Combining diagnosis also do not appear in the newspapers
Road.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of microRNA marks for being used to detect AMS, pass through inspection
Survey in measured's serum or blood plasma microRNA-676-3p, microRNA-181b-5p, microRNA-193b-5p and
MicroRNA-3591-3p content, and with microRNA-676-3p, microRNA- in normal human serum or blood plasma
181b-5p, microRNA-193b-5p and microRNA-3591-3p level are respectively compared to diagnose AMS, should
MicroRNA sensitivity and accuracy are high.
The present invention solve above-mentioned technical problem technical scheme be:A kind of diagnosis AMS microRNA marks, the mark
Will thing is and characteristic sequence is as follows:
microRNA-676-3p:CUGUCCUAAGGUUGUUGAGUU
microRNA-181b-5p:AACAUUCAUUGCUGUCGGUGGGU
microRNA-193b-5p:CGGGGUUUUGAGGGCGAGAUGA
microRNA-3591-3p:AAACACCAUUGUCACACUCCAC
The beneficial effects of the invention are as follows:The microRNA marks that examination goes out, are used as accuracy and the spirit for detecting AMS
Sensitivity is high.
Further, on the basis of above-mentioned technical proposal, the present invention can also do following improvement.
Present invention additionally comprises a kind of purposes of diagnosis AMS microRNA marks in AMS diagnostic reagents are prepared.
Brief description of the drawings
Purpose microRNA (microRNA-676-3p, microRNA- in the blood plasma of Fig. 1 .AMS hair patients and normal healthy controls
181b-5p, microRNA-193b-5p, microRNA-3591-3p) expression (i.e. compared with cel-miR-39 purpose
MicroRNA normalization level).
Wherein AMS be AMS hair patient, non-AMS be normal healthy controls.*:P < 0.05, that is, have notable significant difference, * *:
P < 0.01, that is, have extremely notable significant difference, * * *:P < 0.001, that is, have extremely notable significant difference.
Fig. 2 is 4 purpose microRNA of AMS patients blood plasmas individually with the performance curve (receiver of diagnosis
Operating characteristic curve, abbreviation ROC curve), the figure shows that the ROC of AMS hair patients is bent well
Line, TG-AUC (area under the curve, AUC), sensitivity (sensitivity), specificity
(specificity), wherein AUC reflects diagnostic (AUC=0.5, without diagnostics value;The very littles of 0.5 < AUC < 0.7
Diagnostic value;The fairly accurate diagnostic values of 0.7 < AUC < 0.9;0.9 < AUC < 1, very accurate diagnostic value).
Fig. 3 be AMS patients blood plasma's purposes microRNA (microRNA-676-3p, microRNA-181b-5p,
MicroRNA-193b-5p, microRNA-3591-3p) Combining diagnosis performance curve (receiver operating
Characteristic curve, abbreviation ROC curve), the figure shows the AMS hairs ROC curve of patient, below curve well
Product (area under the curve, AUC), sensitivity (sensitivity), specific (specificity).
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 purpose microRNA of embodiment 1 (microRNA-676-3p, microRNA-181b-5p,
MicroRNA-193b-5p, microRNA-3591-3p) expression with AMS fall ill correlation research
First, material and specimen collection are described
AMS patients blood plasmas specimen collection adds up to 37 from the AMS patient in Acute Exposed Altitude crowd.The blood plasma of normal population
Specimen collection adds up to 31 from the normal health crowd in Acute Exposed Altitude crowd.AMS diagnosis is marked by international diagnosis
Score to confirm in quasi- Lake Louise.AMS patient does not take any medicine before blood is taken.Each sample is anti-with EDTA-Na
Solidifying pipe is total to collect 2ml blood.
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 purpose microRNA and outer ginseng
Cel-miR-39 is expanded;Ct values (cycle threshold) are respectively obtained, pass through formula:Expression quantity=2Ct (cel-
MiR-39)-Ct (purpose microRNA) is in the expression for the purpose microRNA for calculating every sample respectively, concrete operations
Journey is shown in specification.Each sample is tested in triplicate.Purpose microRNA and cel-miR-39 primer sequence is 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
Purpose microRNA diagnostic.Think there is significant difference as p < 0.05.
Returned with binary logistic, set up 4 purpose microRNA dependent equation, obtain prediction probability P, and with
Prediction probability P is that test variable carries out ROC curve analysis, then evaluates the diagnostic of purpose microRNA conjunctive uses.Work as p
Think there is significant difference during < 0.05.
5th, interpretation of result
1. mesh microRNA (microRNA-676-3p, microRNA-181b-5p, microRNA-193b-5p,
MicroRNA-3591-3p) the expression in AMS and normal population.
As shown in figure 1, the blood plasma purpose microRNA (microRNA-676- compared with outer ginseng cel-miR-39 level
3p, microRNA-181b-5p, microRNA-193b-5p, microRNA-3591-3p) normalization level in AMS morbidity groups
There is significant difference (P < 0.001) between normal person group.
Purpose microRNA (microRNA-676-3p, microRNA-181b-5p, microRNA-193b- in 2.AMS
5p, microRNA-3591-3p) level can serve as biomarker.
AMS patients blood plasma purpose microRNA (microRNA-676-3p, microRNA-181b-5p, microRNA-
193b-5p, microRNA-3591-3p) separately as biomarker prediction performance pass through ROC curve (Fig. 2).AMS
Patients blood plasma microRNA-676-3p, microRNA-181b-5p, microRNA-193b-5p, microRNA-3591-3p are mono-
Solely show good as the diagnostic of biomarker, its AUC is respectively:0.713 (95%CI, 0.588-0.838),
0.735 (95%CI, 0.614-0.855), 0.650 (95%CI, 0.519-0.780), 0.8047 (95%CI, 0.699-
0.911)。
3.microRNA-676-3p、microRNA-181b-5p、microRNA-193b-5p、microRNA-3591-3p
United diagnostic understands its AUC better than independent utilization, as the diagnostic of biomarker by ROC curve (Fig. 3)
For 0.872 (95%CI, 0.792-0.951).
6th, conclusion
MicroRNA-676-3p, microRNA-181b-5p, microRNA-193b-5p, microRNA-3591- in blood
3p level can come examination and diagnosis AMS as biomarker, and conjunctive use diagnostic is better than exclusive use.
Blood plasma purpose microRNA expression in table 1AMS morbidities group and healthy control group
* * AMS morbidity group VS healthy control groups:P < 0.001, * * AMS morbidity group VS healthy control groups:P < 0.01, number
Median (25%-75% quantiles) is represented according to this.
Table 2microRNA essential informations
The purpose of table 3 and cel-miR-39 primer information
Claims (3)
1. a kind of microRNA molecule mark for diagnosing acute mountain sickness, it is characterized in that, the microRNA molecule mark
Will thing is:microRNA-676-3p、microRNA-181b-5p、microRNA-193b-5p、microRNA-3591-3p.
2. a kind of microRNA molecule mark for diagnosing acute mountain sickness as claimed in claim 1, microRNA-
Its characteristic sequence of 676-3p, microRNA-181b-5p, microRNA-193b-5p and microRNA-3591-3p is respectively:
SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 and SEQ ID NO:4.
3. claim 1, a kind of microRNA molecule mark for diagnosing acute mountain sickness described in 2 any one is in urgency
Purposes in the diagnostic kit of property mountain sickness.
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CN109868318A (en) * | 2019-04-23 | 2019-06-11 | 中国人民解放军陆军军医大学 | Application and kit of the hsa-miR-3654 as acute high altitude reaction susceptible person's molecular marker |
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