CN104313145A - Application of miRNA-30e in preparation of diagnostic reagents as marker molecules - Google Patents
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Abstract
The invention provides application of miRNA-30e in preparation of reagents for diagnosing contrast-induced acute kidney injury as marker molecules. The invention further provides application of miRNA-30e combined with any one or two of miRNA-30a and miRNA-188 in preparation of reagents for diagnosing contrast-induced acute kidney injury as marker molecules. The invention further provides a kit containing miRNA-30e. Experiments prove that miRNA-30e provided by the invention can be used for early diagnosis of CI-AKI (contrast-induced acute kidney injury). When miRNA-30e is used alone for diagnosing CI-AKI, the sensitivity is 64.8% and the specificity is 93.0%; and when miRNA-30e is combined with miRNA-30a and miRNA-188 for diagnosing CI-AKI, the sensitivity is 71.8% and the specificity is 88.7%. The adoption of the marker in early diagnosis of CI-AKI has great application prospects and market prospects.
Description
Technical field
The invention belongs to biomedical sector, particularly relate to a kind of molecular marked compound, miRNA-30e is preparing the purposes in diagnostic reagent as tagged molecule specifically.
Background technology
Other factors except the acute injury of kidney (Contrast Induced Acute Kidney Injury, CI-AKI) that contrast medium is induced refers to, after contrast medium administration, kainogenesis acute kidney injury or renal insufficiency increase the weight of.CI-AKI is the 3rd cause of disease of iatrogenic acute renal failure, accounts for 11% of iatrogenic acute renal failure, is mainly seen in the patient accepting Percutantnoeus coronary intervention diagnosis and treatment operation at present clinically.In recent years along with the increase of China's Incidence of CHD and extensively carrying out of calcification score technology, the sickness rate of CI-AKI, absolute number of falling ill have and raise trend year by year.
Raise although most of CI-AKI patients is transient serum creatinine level, renal failure is really caused to need the ratio of dialysis treatment to be less than 1%, but CI-AKI patient's while in hospital is dead, the incidence of apoplexy and various cardiovascular event all significantly raises, and long-term survival rate is also starkly lower than the patient without kidney injury performance.The control of CI-AKI is significant to improving patient clinical prognosis.
Research shows, early intervention can improve the chance that renal tubal dysfunction improves, and is the key of control CI-AKI.And the prerequisite of early intervention is early diagnosis.The difficult point of current CI-AKI diagnosis is to lack the early diagnosis biomarker that the susceptibility that in similar acute myocardial infarction, troponin is the same is high, specificity is good.The Case definition used clinically take serum creatinine as foundation, and regulation accepts contrast medium 48-72h serum creatinine absolute value increase more than 0.5mg/dL or CI-AKI is made a definite diagnosis in relative value rising more than 25%.But serum creatinine is not the reliability index of the acute change of a reflection renal function, its concentration before renal function loss 50% can not change, raise 48-72 hour after contrast medium exposes usually, seriously delayed, and affect (Thomsen HS by many Non renal factors such as weight in patients, race, age, or sex, body volume, medicine, muscle metabolism and protein absorptions, et al., Br J Radiol 76 (2003) 513-518; Tomlanovich S, et al., Am J Kidney Dis 8 (1986) 332-337; Van Biesen W, et al., Clin J Am Soc Nephrol 1 (2006) 1314-1319).
Some research and inquirement of nearest 10 years novel markings things such as NAGL, IL-18, KIM-1 are as the reliability of CI-AKI early diagnosis index and feasibility (Ferguson MA, et al., Toxicology 245 (2008) 182-193; Devarajan P, et al., Contrib Nephrol 156 (2007) 203-212).In these diagnostic markers, blood NAGL correlative study is maximum, evaluates relatively better.Bachorzewska Gajewska etc. find that PCI postoperative patient blood NGAL obviously raises (Bachorzewska-Gajewska H, et al., Kidney Blood Press Res30 (2007) 408-415) at 4h.Its sensitivity and specific degree are respectively 0.9 and 0.74 (Bachorzewska-Gajewska H, et al.Int J Cardiol 127 (2008) 290-291).But NAGL stability and specificity poor, be subject to the impact of many variable factors of having deposited, as basic kidney disease and urinary tract infections, and the acute kidney injury that Different factor causes can not be differentiated further, serum creatinine therefore can not be replaced as the diagnostic marker of clinical CI-AKI.
At present in the urgent need to searching out, susceptibility is good, high specificity, be easy to measure, stable that do not affect by other variable biotic factors, the early stage biomarker that contrast medium causes acute kidney injury can be evaluated.
MiRNAs is high conservative, endogenous strand non-coding RNA in a class 23-25 Nucleotide size, evolution.Meanwhile, miRNA is that a class has bioactive tiny RNA.MiRNA is by being combined with the pairing of mRNA, and degraded mRNA or disturb it to translate, plays biological regulation effect (Saal S, et al., Curr Opin Nephrol Hypertens18 (2009) 317-323).
Research finds, miRNA expresses has tissue specificity.Yu Liang, Pablo Landgraf etc. study different plant species, different tissues miRNA express spectra respectively.Find that part miRNAs is in specific tissue specific expression or enrichment.Such as, miR-122 is liver specific expression, and miR-133a, miR-499 express (Liang Y, et al., BMC Genomics 166 (2007) 8) at skeletal muscle, Cardiac-specific.
Meanwhile, miRNAs expresses and has disease specific.Research finds that different types of cancer cell miRNA expression characteristic is different.First Calin etc. find miRNA-15, miRNA-16 down-regulated expression (Barbarotto E, et al., Curr Pharm Des 14 (2008) 2040-2050) in lymphocytic leukemia.And Nozomu Yahaihara etc. observe miR-155 high expression level in adenocarcinoma of lung, the low expression of let-7a-2 (Yanaihara N, et al., Cancer Cell 9 (2006) 189-198).The expression demonstrating miRNAs has disease specific.MiRNA expresses also has changes with time, and miRNA express spectra dynamically changes with disease course.Jonathan Godwin etc. carry out contrast to kidney defects Reperfu-sion mouse model different time points kidney miRNA express spectra and find, miRNAs presents different variation tendencies.Such as, miRNA-21 raises rapidly in early days at Reperfu-sion, reaches plateau.And miRNA-146a starts to raise (Godwin JG, et al., Proc Natl Acad Sci U S A 107 (2010) 14339-14344) gradually Reperfu-sion 3-7 talent.
Part research finds that miRNA changes can react in blood plasma in the expression of pathological tissues, organ.Research finds the miR-141 of prostate cancer tissue high expression level in serum of patients with prostate cancer also apparently higher than healthy population, and there is higher Sensitivity and Specificity (Mitchell PS, et al., Proc Natl Acad Sci U S A105 (2008) 10513-10518).In liver lesion induced by drugs model mice, miRNA-122 down-regulated expression in mouse liver that liver is special, but in blood plasma, express obviously rise, and there is dosage/temporal correlation.In blood plasma, miRNA also has good stability (Wang K, et al., Proc Natl Acad Sci U S A 106 (2009) 4402-4407).Although there is the enzyme of a large amount of degradation of rna in blood plasma, substantially there is not the RNA of long section in blood plasma, miRNA can stable existence with some form in blood plasma.Research finds that the miRNAs in blood plasma all keeps when room temperature (24 hours) and multigelation (8 times) stablizing (Chen X, et al., Cell Res 18 (2008) 997-1006).
Just because of tissue, the disease specific of miRNA, determine miRNA with the closely-related characteristic of disease pathology physiological status, as diagnostic marker, there is higher specificity.Simultaneously in blood plasma or serum, miRNA can reflect the change that pathological tissues miRNA expresses.Therefore blood plasma miRNA is more and more paid attention to as a kind of noninvasive, special medical diagnosis on disease marker and is approved.
Have a large amount of research and inquirement status of miRNA in chronic renal disease, comprising (Sun H, et al., Mol Biol Rep 37 (2011) 2951-2958 such as POLYCYSTIC KIDNEY DISEASE, diabetic nephropathy, tumor of kidney; Kato M, et al., Proc Natl Acad Sci U S A 104 (2007) 3432-3437; Zhang Z, et al., FEBS Lett583 (2009) 2009-2014; Hanke M, et al., Urol Oncol 28 (2009) 655-661).But pay close attention to the diagnostic marker of blood plasma miRNA as CI-AKI without correlative study both at home and abroad at present.
Whole blood, serum or blood plasma are the most widely used clinical pathway sample source.Search out the contrast medium inducing acute injury of the kidney plasma markers thing that early diagnosis information is provided, the experiment of a kind of diagnostic can be produced, greatly assisted diagnosis and this disease of process.
Summary of the invention
A kind of miRNA-30e is the object of the present invention is to provide to prepare the purposes in diagnostic reagent as tagged molecule, described this purposes solve in prior art the molecular marked compound stability of the acute injury of kidney diagnosing contrast medium to induce and specificity poor, be subject to the technical problem of many variable factor impacts of having deposited.
The invention provides miRNA-30e as the purposes of tagged molecule in the acute injury of kidney reagent of preparation diagnosis contrast medium induction, the base sequence of described miRNA-30e is as shown in SEQ ID NO:2.
Present invention also offers miRNA-30e as the purposes of tagged molecule in the acute injury of kidney reagent of preparation early diagnosis contrast medium induction, the base sequence of described miRNA-30e is as shown in SEQ ID NO:2.
Further, wherein said early diagnosis refer to contrast medium expose after 4-6 hour.
Present invention also offers miRNA-30e and to combine in miRNA-30a, miRNA-188 any one or two kinds as the purposes of tagged molecule in the acute injury of kidney reagent of preparation diagnosis contrast medium induction, the base sequence of described miRNA-30e is as shown in SEQ ID NO:2, the base sequence of described miRNA-30a is as shown in SEQ ID NO:1, and the base sequence of described miRNA-188 is as shown in SEQ ID NO:3.
Present invention also offers miRNA-30e and to combine in miRNA-30a, miRNA-188 any one or two kinds as the purposes of tagged molecule in the acute injury of kidney reagent of preparation early diagnosis contrast medium induction, the base sequence of described miRNA-30e is as shown in SEQ ID NO:2, the base sequence of described miRNA-30a is as shown in SEQ ID NO:1, and the base sequence of described miRNA-188 is as shown in SEQ ID NO:3.
Present invention also offers a kind of test kit, containing miRNA-30e, the base sequence of described miRNA-30e is as shown in SEQ ID NO:2.
Present invention also offers the acute injury of kidney diagnostic method of contrast medium induction, the method comprises the following steps: a) provide the plasma sample obtained by individuality; B) measure contrast medium and expose miR-30e change in concentration multiple in Plasma Before And After sample, and c) (b) is diagnosed with CI-AKI be associated.
Present invention also offers another preferred embodiment diagnostic method of the acute injury of kidney relating to contrast medium induction, the method comprises the following steps: a) measure contrast medium and expose miR-30e change in concentration multiple in Plasma Before And After sample, the change in concentration multiple that one or more other miRNA b) optionally measuring CI-AKI in plasma sample mark; And c) be used in the change in concentration multiple measured in step (a) and optional step (b), diagnosis CI-AKI.
As used herein, below each of term there is the implication relevant to it in this part.
Term " individuality " is used in reference to the patient accepting calcification score diagnosis and treatment operation in this article, and these patients do not accept unfractionated heparin anticoagulant therapy, or give unfractionated heparin anticoagulant therapy more than 4 hours.
Term " measurement " is used in reference in this article and comprises microRNA chip, Solexa order-checking, Taqman low density array, real-time fluorescence quantitative PCR or the various MicroRNA detection method based on microballoon.
Term " plasma sample " is used in reference to the patients blood plasma obtained for in-vitro evaluation object in this article, and this blood plasma is hung oneself the centrifugal rear acquisition of individual whole blood of EDTA (working concentration is 1.8mg/ml) anti-freezing.
In preferred embodiments, the present invention relates to by various MicroRNA detection method vitro detection blood plasma miR-30a, miR-188 and miR-30e change in concentration, and use the change in concentration diagnosis CI-AKI measured.
The present invention proposes the novelty teabag of a kind of miR-30e.The present invention may be used in early days (after contrast medium exposure 4-6 hour) diagnosis CI-AKI through experimental verification miR-30e.MiR-30e is separately for diagnosing the susceptibility of CI-AKI to be 64.8%, and specificity is 93.0%; Be 71.8% with the susceptibility of miR-30a and miR-188 Combining diagnosis CI-AKI, specificity is 88.7%.Above-mentioned marker early diagnosis CI-AKI is adopted to have great application prospect and market outlook.
The invention described above content, to invention has been detailed description, in order to make the clearer summary of the invention of the present invention of those skilled in the art and technique effect, is described in further details the present invention below in conjunction with the drawings and specific embodiments.
Accompanying drawing explanation
Fig. 1 illustrates the acute injury of kidney marker flow process by the induction of miRNA chip method screening contrast medium.
For detecting by real-time PCR method, Fig. 2 A proves that miR-30e is at rat kidney organizing specific expression.
Fig. 2 B retrieves the people delivered to organize miRNAs express spectra document to show miR-30e at human kidney tissue specific expression.
Fig. 3 real-time PCR method proofing chip result, CI-AKI rat plasma miR-30e up-regulated.
Fig. 4 real-time PCR method proofing chip result, CI-AKI rat plasma miR-30e is 4h rising peaking after contrast medium exposes.
Fig. 5 real-time PCR method proves, CI-AKI rat plasma miR-30e raises special relevant to the injury of the kidney that contrast medium is induced.
Fig. 6 real-time PCR method compares the level of miR-30e in CI-AKI and non-CI-AKI patients blood plasma, proves that CI-AKI patient significantly raises.
Fig. 7 miR-30e evaluates as the ROC of CI-AKI diagnosis marker.
Embodiment
Below in conjunction with specific embodiments and the drawings, the present invention is further illustrated.
The target miRNA that embodiment 1 cDNA microarray people kidney specific is expressed
1. build CI-AKI rat model
The present invention is by the method establishment CI-AKI rat model through the hypotonic contrast medium of tail vein injection on the basis of prohibiting water 3 days, diuresis " pre-treatment ".This is a kind of novel C I-AKI rat model that the present inventor sets up, and concrete scheme is shown in pertinent literature (Sun SQ, et al.Int Journal of Cardiol 172 (2014) e48-50).
2.miRNA chip differences expression pattern analysis
1) miRNA chip preparation
On the basis adopting aforesaid method success modeling, leave and take 8 hours CI-AKI rat plasmas, kidneys after modeling, for chip loading.Adopt Agilent miRNAs 10.0version chip technology, carry out loading with blood plasma and kidney total serum IgE, mark with phosphorylation Cyanine3-pCp, then carry out chip hybridization, washing and data gathering.
2) bioinformatic analysis
Chip fluorescence intensity signals value adopts AgilentG4450AA Feature Extraction Software 9.5 and Agilent Scan Control Software version A7.0 software to carry out collection analysis.Sample signal value is carried out log2 conversion by raw data and is represented after normalization method (normalization).Sample signal Flag value A (Absent) representation signal and background difference are remarkable, P (Present) representation signal and background difference remarkable.Sample data analysis adopts Agilent Gene Spring software 9.0, and by contrasting kidney and the blood plasma chip data of CI-AKI and non-CI-AKI rat, obtain the microRNA of differential expression, result is as shown in table 1.
Table 1.miRNAs expression pattern analysis
3) according to chip data preliminary screening target miRNA
MiRNAs express spectra document (Liang Y, et al.BMC Genomics.2007Jun 12 is organized by retrieval miRbase (release 16) database and the people that delivers; 8:166.), the miRNAs of the special or enrichment of screening CI-AKI rat plasma up-regulated, people mouse homology, kidney is as the early stage candidate diagnosis marker of CI-AKI, and idiographic flow as shown in Figure 1.The target miRNA determined after screening has miR-30a, miR-30e and miR-188 tri-, and can emphasis of the present invention has probed into miR-30e as CI-AKI early diagnosis marker, also have rated the ability that the multiple miRNAs of associating diagnoses CI-AKI simultaneously.
Above result is the result obtained based on miRNA chip technology, below we adopt more accurate real-time PCR method verify.
Embodiment 2 verifies the specific expressed of target miRNA kidney
With the total serum IgE that the Trizol method extraction rat of classics is respectively organized, real-time PCR detects the expression level that target miRNA respectively organizes rat, simultaneously by retrieving the expression level that the people delivered organizes miRNAs express spectra document contrast people respectively to organize.Fig. 2 is the express spectra result of miR-30e in rat (A) and people (B) are respectively organized, and miR-30e is all greater than other organs more than 2 times and 2 times in the renal expression amount of rat and people, therefore thinks that this miRNA kidney specific is higher.
The extracting of embodiment 3 blood plasma microRNA
The method that the present invention adopts two steps centrifugal to blood plasma, eliminate the hemocyte in blood and cell debris, RNA extracting adopts mirVana PARIS Kit (Applied Biosystem, P/N AMl556), and this is a kind of test kit being applicable to RNA in blood plasma or serum.The internal reference that in current blood plasma, RNA does not quantitatively generally acknowledge can be used, the present invention adds the ectogenic internal reference cel-miR-39 (miRNA that nematode is expressed in RNA extractive process, this sequence is not expressed in human body), be used for the RT-PCR detected result of correction target miRNA.
Plasma sample pretreatment process: whole blood collection is in EDTA anticoagulant tube (working concentration is 1.8mg/ml), first through 4 DEG C, the centrifugal 10min of 820g, remove hemocyte, supernatant is again through 4 DEG C, and the centrifugal 10min of 16000g, removes cell debris, finally draw supernatant in the centrifuge tube of 1.5ml RNase/DNase-free ,-80 DEG C of packing are frozen for subsequent use.
The concrete steps of RNA extracting are as follows:
1) get 400 μ L blood plasma, add 2 × Denaturing Solution of equivalent, vortex mixes, and places 5 minutes on ice.
2) add the acid-phenol:chloroform with cumulative volume equivalent, vortex mixes 30-60 second, room temperature, centrifugal 5 minutes of maximum speed.
3) careful supernatant of drawing is in new 1.5ml centrifuge tube, and add 100% ethanol of 1.25 times of volumes, vortex mixes.
4) be placed into by the centrifugal column of cleaning in clean collection tube, draw the liquid of previous step in centrifugal column, 10000g, centrifugal 30 seconds of room temperature, abandoned stream crosses liquid, is reapposed in collection tube by centrifugal column.Repeat this step, until all liquid crosses centrifugal column.
5) draw 700 μ L miRNA Wash Solution 1 in centrifugal column, 10000g, centrifugal 15 seconds of room temperature, abandoned stream crosses liquid, is reapposed in collection tube by centrifugal column.
6) 500 μ L Wash Solution 2/3 cross post twice, centrifugal 1 minute of void column.
7) be placed into by centrifugal column in new collection tube, post center adds the nuclease-free water of 100 μ L, 95 DEG C of preheatings, centrifugal 30 seconds of room temperature maximum speed, and the liquid in collection tube is the Total RNA of extraction, puts-80 DEG C of preservations.
The real-time PCR of embodiment 4 blood plasma microRNA detects
The miRNA detection kit (comprising Reverse Transcriptase kit, specificity miRNA primer and probe, fluorescence quantitative detection kit etc.) of Ambion biotech firm of the U.S. is used to detect the related levels of miR-30e and internal reference cel-miR-39.
Detailed process is as follows:
1. reverse transcription reaction: use Taqman microRNA Reverse Transcription kit (P/N:4366596) and target miRNA specific reverse transcriptase primer to carry out reverse transcription reaction, concrete steps are as follows:
1) reaction system is prepared: prepare according to table 2
The reaction system of table 2.microRNA reverse transcription
2) reverse transcription reaction: carry out reverse transcription reaction upper, reaction parameter is: 16 DEG C, 30 minutes; 42 DEG C, 30 minutes; 85 DEG C, 5 minutes.
2.real-time PCR reacts: use Taqman Universal PCR MasterMix (P/N:4324018) and target miRNA specific probe and primer to carry out quantitative fluorescent PCR reaction.Concrete steps are as follows:
1) reaction system is prepared: the multiple hole of each template-setup of each target miRNA three, reaction system is as shown in table 3.
Table 3.Real time PCR reaction system
2) Real-time PCR reacts: on the LightCycler480 real-time fluorescence quantitative PCR instrument of Roche company, carry out real-time PCR reaction: 95 DEG C of denaturations 10 minutes, and loop parameter is 95 DEG C, 15 seconds; 60 DEG C, 1 minute, cycle index was 40.So reaction in triplicate, get fixing fluorescence threshold, the mean value obtained is the CT value of target miRNA in this sample at every turn.
The analysis of 3.real-time PCR result: the CT value that can record target miRNA and internal reference miRNA in sample blood plasma with aforesaid method.Take cel-miR-39 as internal reference, with 2 of classics in PCR detection
-△ CTtry to achieve the relative content of target miRNA in sample blood plasma (△ CT is the difference of the CT value of target miRNA and internal reference cel-miR-39), then with 2
-△ △ CTtry to achieve the relative changes multiple (△ △ CT is the difference of postoperative △ CT and baseline △ CT) of target miRNA.
Embodiment 5 verifies the change of target miRNA plasma level
Adopt the chip results of real time PCR method to target miRNA of above-mentioned blood plasma miRNA to verify, result, as Fig. 3, shows the consistence of both results.
It is more than the checking to chip results, result shows the consistence of chip results and real-time PCR result, ensure that the reliability of the selection result, for the mark whether miRNA can become the diagnosis of contrast medium inducing acute injury of the kidney, still need further checking.
The change of embodiment 6 rat CI-AKI model blood plasma miRNA
First the present invention verifies on the animal model of CI-AKI whether target miRNA can become the mark of contrast medium inducing acute injury of the kidney diagnosis.
1. first the present invention has carried out collecting to the blood plasma of different time after modeling and has detected the level change of miR-30e in blood plasma, and result is as Fig. 4.MiR-30e after modeling 2 hours, blood plasma level obviously raises, and presents maximum at 4-6 hour, starts after 8 hours to decline, and is down to baseline values after 24 hours.
2. because in CI-AKI modeling process, the process factors such as taboo water, contrast medium are related to, the possibility of the non-specific rising of blood plasma miRNA is caused for getting rid of these process factors, the present invention have detected again the level of target miRNA respectively in the rat plasma of modeling group (pre-treatment+contrast medium), pretreated group and contrast medium group, and result is as Fig. 5.MiR-30e does not significantly raise in pretreated group and contrast medium group rat plasma, and obviously raise in modeling group rat plasma, show that target miRNA causes learning blood plasma level rising because there is acute injury of kidney, instead of because of the non-specific rising that process factor causes.
The clinical CI-AKI patient of embodiment 7 large sample verifies the change of target miRNA
Above-mentioned animal verification portion shows target miRNA as the diagnosis marker of CI-AKI, can reach peak value in 4-6 hour after surgery, below we will verify on human body.
1. patient cases collects
Plasma sample amounts to 392 examples, wherein 71 routine CI-AKI patients blood plasma, 321 routine non-CI-AKI patients blood plasma.Blood sample is from Renji Hospital Attached to Medical College of Shanghai Jiaotong Univ.'s Cardiological, and blood sample is respectively collected once after patient admits He after visualization for 4-6 hour, and the time of collecting is from year June in July, 2013 to 2014.According to patient age (<50,50-70, >70), diabetes (Yes, No), renal function by stages (Stage1,2,3 phases) 71 routine CI-AKI patients carried out 1:1 join
Table 4. clinical characteristic
Right, amount to 142 routine patients and include in.Patient clinical case feature is as shown in table 4.
2) expression and distribution of target miRNA
As a desirable disease diagnosis marker, the background level in blood plasma should be lower, ensure that the susceptibility of Testing index is high, and expression level is relatively homogeneous, ensures the stability of detected result.The present invention have detected the level of target miR-30e in blood plasma by real-timePCR method, and result is as table 5.The CT value about 28-29 of result display miR-30e before visualization, illustrates that the level in the blood plasma of miR-30e in non-CI-AKI patient is lower.Can see that the CT value of miR-30e under different clinical risk factors in blood plasma is close in addition, describe the stably express of miR-30e in blood plasma.
The baseline values of table 5.miR-30e
3) change of miRNA in CI-AKI patients blood plasma
The present invention analyzes the expression of target miRNA blood plasma in CI-AKI and non-CI-AKI group, and as shown in Figure 6, in CI-AKI patient, the expression level of miR-30e is significantly higher than non-CI-AKI group.
4) ROC that miRNA diagnoses as CI-AKI evaluates
Carry out ROC analysis according to the data obtained, result is as Fig. 7.It is 0.805 that miR-30e can distinguish area under CI-AKI and non-CI-AKI, AUC preferably, and diagnosis accuracy is higher.Under the condition of diagnosis dividing value, miR-30e diagnoses the sensitivity of CI-AKI, specific degree is respectively 64.79%, 92.96%, represent that miR-30e has higher sensitivity and specific degree as the early diagnosis marker of CI-AKI, can as a kind of potential mark that can be used for diagnosis CI-AKI newly.
5) scope of miR-30e level diagnosis CI-AKI
According to the data obtained, in blood plasma, the diagnosis threshold value of miR-30e is 2
-△ △ CT>1.428.After patient's visualization, in 4-6 hour blood plasma, the level of miR-30e compares with diagnosis threshold value, is then diagnosed as CI-AKI, is then diagnosed as non-CI-AKI lower than threshold value higher than threshold value.
6) evaluation of blood plasma miRNA Combining diagnosis CI-AKI
For the limitation avoiding single miRNA to diagnose CI-AKI, have detected the change in concentration of blood plasma miR-30a and miR-188 in preferred version of the present invention simultaneously, expression level in result display CI-AKI patient is significantly higher than non-CI-AKI group, under AUC, Line Integral is not 0.802,0.784, and diagnosis accuracy is higher.Obtain according to current data, in blood plasma, the diagnosis threshold value of miR-30a is 2
-△ △ CTthe diagnosis threshold value of >1.405, miR-188 is 2
-△ △ CT>1.343.The present invention proposes a Combining diagnosis model on this basis, namely in patients blood plasma, the concentration of arbitrary miRNA changes and exceedes above-mentioned dividing value and can be diagnosed as CI-AKI, now diagnose the sensitivity of CI-AKI, specific degree is respectively 71.83%, 88.73%, increase when integral diagnostic capability is comparatively used alone miR-30e.
Conclusion: as a novel diagnosis marker, blood plasma miR-30e just can detect the change of expression level in early days at injury of the kidney, can be used in the early diagnosis of CI-AKI, be better than serum creatinine, it is better to combine other miRNA diagnosis effects.
Claims (6)
1.miRNA-30e is as the purposes of tagged molecule in the acute injury of kidney reagent of preparation diagnosis contrast medium induction, and the base sequence of described miRNA-30e is as shown in SEQ ID NO:2.
2.miRNA-30e is as the purposes of tagged molecule in the acute injury of kidney reagent of preparation early diagnosis contrast medium induction, and the base sequence of described miRNA-30e is as shown in SEQ ID NO:2.
3. purposes according to claim 2, wherein said early diagnosis refers to the 4-6 hour after contrast medium exposure.
4.miRNA-30e to combine in miRNA-30a, miRNA-188 any one or two kinds as the purposes of tagged molecule in the acute injury of kidney reagent of preparation diagnosis contrast medium induction, the base sequence of described miRNA-30e is as shown in SEQ ID NO:2, the base sequence of described miRNA-30a is as shown in SEQ ID NO:1, and the base sequence of described miRNA-188 is as shown in SEQ ID NO:3.
5.miRNA-30e to combine in miRNA-30a, miRNA-188 any one or two kinds as the purposes of tagged molecule in the acute injury of kidney reagent of preparation early diagnosis contrast medium induction, the base sequence of described miRNA-30e is as shown in SEQ ID NO:2, the base sequence of described miRNA-30a is as shown in SEQ ID NO:1, and the base sequence of described miRNA-188 is as shown in SEQ ID NO:3.
6. a test kit, is characterized in that: containing miRNA-30e, and the base sequence of described miRNA-30e is as shown in SEQ ID NO:2.
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| WO2010105275A2 (en) * | 2009-03-13 | 2010-09-16 | Cornell University | Method to assess human allograft status from microrna expression levels |
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