CN103205430A - Related serum microribonucleic acid marker for human severe preeclampsia and application of marker - Google Patents
Related serum microribonucleic acid marker for human severe preeclampsia and application of marker Download PDFInfo
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Abstract
The invention belongs to the fields of genetic engineering and clinical medicine, and discloses a related serum microribonucleic acid marker for human severe preeclampsia and application of the marker. The marker is selected from more of hsa-miR-193a-5p, hsa-miR-424 and hsa-miR-10a. The marker has the specificity and sensitivity on a sufferer with severe preeclampsia, and can be used for preparing a diagnosis or monitoring kit for severe preeclampsia.
Description
Invention field
The invention belongs to genetically engineered and clinical medicine domain, relate to human serious preeclampsia relevant blood serum minuteness ribonucleic acid (microRNA, miRNA) mark and application thereof take place.
Background technology
China's gestation hypertension disease incidence is about 9.4%, is reported as 7%～12% abroad, is pregnant and lying-in women and the major reason of enclosing living youngster's death.The gestation hypertension disease comprises gestation hypertension, preeclampsia, eclampsia, chronic hypertension superimposed eclampsia early stage and chronic hypertension.Preeclampsia, (preeclampsia was the distinctive disease that relates to multisystem of the Gestation period PE), and sickness rate is 1%～5%, is main clinical characters with 20 week of gestation back generation hypertension, proteinuria, oedema etc.Be divided into slight and severe two classes according to the state of an illness weight preeclampsia.Refer to blood pressure 〉=140/90mmHg occur after pregnant 20 weeks slight preeclampsia, and urine protein (+) or urine protein 〉=300mg/24h can be with symptoms such as epigastric discomfort, headaches.Preeclampsia the patient following arbitrary unfavorable condition to occur diagnosable for serious preeclampsia: 1. blood pressure continues to raise: systolic pressure 〉=160mmHg and (or) diastolic pressure 〉=110mmHg; 2. proteinuria 〉=2.0g/24 hour or proteinuria 〉=(++) at random; Just raise before unless 3. serum creatinine 〉=1.2mg/dL is known; 4. thrombocyte＜100,000/ML(＜100 * 10
9/ L); 5. capillary blood vessel characteristic of disease haemolysis-LDH raises; 6. serum transaminase level rising-ALT or AS; , 7. unrelenting headache or other brains or visual disorder; 8. continue epigastrium pain.The person of being in a bad way can cause eclampsia, even merges the many organ injuries of whole body such as liver and kidney dysfunction, hematencephalon, heart failure, disseminated intravascular coagulation etc., the female youngster's of serious threat safety.
The factor of sending out well of preeclampsia comprise unipara, pregnant woman age less than 18 years old or greater than internal diseases such as 40 years old, polycyesis, merging chronic hypertension, chronic nephritis, antiphospholipid syndrome, diabetes, malnutrition and preeclampsia family history etc.Morbidity preeclampsia more early, female youngster's debatable time is more long, complication is more many, prognosis is also more poor.Therefore morbidity is divided into hair style preeclampsia morning and delayed preeclampsia sooner or later again to perinatology according to this disease.Recently discover that early hair style and delayed have certain difference at genetic background, morbidity and risk of recurrence, the change of pathologic, physiologic index, clinical characters, cardiovascular risk at a specified future date preeclampsia.Early hair style mainly shows as placental function disorder and blood vessel endothelium dysfunction preeclampsia; And delayed often shows as the abnormal response of parent preeclampsia.Do you how to determine the boundary of hair style and delayed early? according to national standards such as the U.S. and Canada, generally be decided to be early hair style preeclampsia the preeclampsias that gestation was taken place before 34 weeks; Be called delayed preeclampsia the preeclampsias of 〉=34 week morbidities.But there are some researches show again in recent years gestation before 32 weeks the maternal mortality rate that causes preeclampsias of morbidity be 20 times that take place preeclampsia in mature back, so both at home and abroad some scholar also the boundary of 32 weeks of gestation as morning hair style and delayed preeclampsia.
Preeclampsia, pathogenesis was not illustrated as yet fully, thought mostly that at present placenta is diseases related, and the back state of an illness of giving birth to of placenta is alleviated or cured.Have genetic predisposition, may relate to the relevant gene of blood pressure regulation, thrombosis, blood vessel inner skin cell function, blood vessel double teeming, metabolism immunity etc.Under the polygene background, factor interaction such as life behavior, environment, may influence female tire immunological tolerance, female tire interface nurse cell dysfunction, invasiveness descends and makes the shallow implantation of placenta or helicine artery double teeming deficiency, placenta hypoxic-ischemic, local cells immune response enhancing, placenta part oxidative stress release oxyradical occurs and cause blood vessel endothelium to activate and damage, synthetic or the secretion vasoconstrictive factor relaxing factor dysequilibrium of blood vessel endothelium causes arteriolospasm, placenta and whole body internal organs blood perfusion amount to descend.Blood vessel endothelium injury can make thrombin increase again in addition, and anti-fibrinolytic strengthens, fibrinolytic decline hyperamization liquid hypercoagulative state or microthrombusis, even chronic intravascular coagulation (DIC) takes place, cause many organ injuries.Depressed placental function, placenta hypoxic-ischemic can cause FGR, fetal distress in uterus, iatrogenic premature labor, fetal death in utero even neonatal death.
Treat to monitor female tire state, symptomatic treatment (as spasmolysis, calmness, step-down, short fetal growth etc.) preeclampsia, termination of pregnancy is main treatment measures to reduce female youngster's complication in case of necessity, termination of pregnancy is still unique cure method so far.After clinical diagnosis time preeclampsia is pregnant 20 weeks, but a series of pathophysiological changes just may take place at morbidity body in period the last period in this disease, and female youngster produced in various degree damage, or even the badness come-off that can't retrieve.Therefore we need alleviate female youngster's badness come-off subclinical or more early even before pregnant avoid or reduce the generation of this disease, the course of disease that slows down with regard to the intervening measure of taking to be correlated with.We press for this sick special mark of searching and treatment target spot for this reason, thereby start prevention and intervening measure as early as possible, and generation, the female youngster's prognosis of improvement that reduces this disease is extremely important.
MiRNA (microRNA, be miRNA) be the research focus that has just risen in recent years, it is the single stranded RNA molecule that a class is about 19-23 Nucleotide, multidigit is in the genome non-coding region, high conservative in the evolution can be regulated genetic expression at post-transcriptional level, and with many normal physiological activity of animal, closely related as biont growth, tissue differentiation, apoptosis and energy metabolism etc., also exist closely with the generation of numerous disease and development simultaneously and contact.Since participating in lin-4 that regulation and control nematode sequential grows and being found with let-7, miRNA became the research focus of regulation and control mRNA stability and protein translation gradually, was selected in the annual ten big technological breakthroughs of Science magazine respectively twice at 2002 and 2003.Now forecast miRNA can regulate and control thousands of Human genomes at least, accounts for more than 30% of all genes.Along with going deep into of research, increasing miRNA is found.At present, the relation of miRNA and tumour has become the emphasis of research, has found expression and the lymphocytic leukemia, lung cancer, mammary cancer, colorectal carcinoma height correlation of some miRNA by negative regulator gene.Yet the mutual relationship of serum miRNA and serious preeclampsia etc. is not appeared in the newspapers as yet.
Up-to-date achievement in research finds to exist in the serum hundreds of miRNA, stable in properties, content enrich, are easy to detection by quantitative, and there is significant disease specific, confirmed that in lung cancer, colorectal carcinoma the express spectra of serum miRNA can be used as the potential source biomolecule mark of early diagnosis.This discovery is exciting, and serum miRNA might replace the biomarker that traditional differential protein is representative as the microRNA of the non-coding and regulating of a class, has opened up the frontier of biomarker.This research causes the extensive concern of international media rapidly, Reuter, United Press, " American of science ", U.S.'s " technology review " etc. have all carried out special report to this achievement in research, " Nature " magazine also its website homepage-showed this latest Progress in the latest Progress ‖ special column.Yet miRNA is also paid close attention to accordingly in serious preeclampsia early diagnosis Application in Monitoring in the serum, if can find that the stable special serum miRNA relevant with the serious preeclampsia morbidity is as biomarker, and research and develop diagnosis, the monitoring reagent box of corresponding disease, not only be in the first place in the world in this field, can create the economic benefit that attracts people's attention, also will be once strong promotion to the control of China's gestational hypertension.
Summary of the invention
The purpose of this invention is to provide with human serious preeclampsia relevant serum microRNA mark takes place.
Another object of the present invention provides the primer of above-mentioned serum microRNA mark.
A further object of the invention provides the application of above-mentioned serum microRNA mark or its primer.
The present invention has a purpose to provide to contain the test kit that is used for human serious preeclampsia diagnosis or monitoring of above-mentioned serum microRNA mark or its primer again.
The objective of the invention is to realize by following technical measures:
The serum microRNA mark relevant with human serious preeclampsia is selected from multiple among hsa-miR-193a-5p, hsa-miR-424 and the hsa-miR-10a.
Described serum microRNA mark is made of hsa-miR-193a-5p, hsa-miR-424 and hsa-miR-10a.Described serum microRNA mark, wherein the sequence of hsa-miR-193a-5p is ugggucuuugcgggcgagauga(SEQ ID No.1), the sequence of hsa-miR-424 is cagcagcaauucauguuuugaa(SEQ ID No.2), the sequence of hsa-miR-10a is uacccuguagauccgaauuugug(SEQ ID No.3).
The primer of described serum microRNA mark, wherein sequence is that the upstream primer of the mark of SEQ ID No.1 is SEQ ID No.4, downstream primer is SEQ ID No.5; Sequence is that the upstream primer of the mark of SEQ ID No.2 is SEQ ID No.6, and downstream primer is SEQ ID No.7; Sequence is that the upstream primer of the mark of SEQ ID No.3 is SEQ ID No.8, and downstream primer is SEQ ID No.9.
The application in the diagnosis of preparation serious preeclampsia or monitoring reagent of described serum microRNA mark or its primer.Described reagent is the reagent that can measure these serum microRNA mark expression amount in serum.
A kind of human serious preeclampsia diagnosis or monitoring reagent box, this test kit contain the primer of above-mentioned serum microRNA mark (among hsa-miR-193a-5p, hsa-miR-424 and the hsa-miR-10a multiple).
Described diagnosis or monitoring reagent box, this test kit contain how right in the above-mentioned primer (SEQ ID No.4 and SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9).
Described diagnosis or monitoring reagent box, this test kit contain following 3 couples of primer SEQ ID No.4 and SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9.
Other reagent in the test kit except primer can adopt the common agents of corresponding detection technique in the prior art.
The present invention is described in detail as follows:
The inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP), crowd's Back ground Information and clinical data that systematic collection is complete, and adopted RT-PCR method, TaqMan miRNA Array, Real-time PCR(TaqMan probe and dye method) one or more of method detect.
Yan Jiu experimental technique mainly comprises following components specifically:
One, research object is selected and the grouping foundation
The A group: normal healthy controls group (n=100, the screening of 20 people's chips, the checking of 40 people's first phases, 40 people's independence crowds checking), there are not other general major diseases.
The B group: serious preeclampsia group (n=100, the screening of 20 people's chips, the checking of 40 people's first phases, 40 people's independence crowds checking), there are not other general major diseases.
Two, blood serum is separated and pre-treatment
(1) fresh heparin anti-coagulating 5ml gets the per 100 μ l branch of supernatant and is filled in the clean 1.5ml EP pipe in the centrifugal 5min of whizzer 3000rpm
(2) add 900 μ l TRIzol in the EP pipe, fully behind the mixing, the centrifugal 15min of 12000rpm gets in the clean 1.5ml EP of supernatant to a pipe immediately.
(3) add the dehydrated alcohol of 1.5 times of supernatant water phase volumes in the EP pipe, fully be transferred to centrifugal post behind the mixing, centrifugal 15 seconds of 10000rpm abandons lower floor's waste liquid.
(4) add 700 μ l RWT damping fluids at centrifugal post, centrifugal 15 seconds of 10000rpm abandons lower floor's waste liquid.
(5) add 500 μ l RPE damping fluids at centrifugal post, centrifugal 15 seconds of 10000rpm abandons subnatant.
Come again.
(6) with the pipe of a new 2ml of centrifugal post adding, centrifugal 1 minute of 10000rpm is used for removing the RPE damping fluid.
(7) centrifugal post is contained in the centrifuge tube of a new 1.5ml, and adds the water that 50 μ lDEPC handle, centrifugal 1 minute at pillar.
(8)-70 the sample after ℃ preservation is handled.
The centrifugal post that uses in the present invention's experiment and matched reagent (RWT damping fluid, RPE damping fluid) are all from Qiagen miRNeasy Mini Kit(article No. 217004) this test kit, down together.
Three, the Real-time PCR method is measured serum miRNAs expression amount
1. the learn from else's experience serum of pre-treatment obtains the cDNA sample by the RNA reverse transcription reaction.
The reverse transcription of preparation shown in according to the form below system:
2. PCR pipe is put upside down repeatedly and done behind the mixing 6 times briefly centrifugally, placed on ice 5 minutes.
3. the PCR pipe is put into the PCR instrument and carry out reverse transcription, reaction conditions is as shown in the table:
Reverse transcription product is stored in 4 ℃ of refrigerators to be used for next step pre-amplification.
4. the preparation of the cDNA according to the form below reaction system after the reverse transcription is increased in advance:
The reaction conditions of pre-amplification is as shown in the table:
After being down to 4 ℃, pre-expansion being increased production thing be stored in 4 ℃ of refrigerators to be used for next step Real-time PCR reaction.
5. pre-expansion is increased production thing brief centrifugal after, add 0.1 * TE (pH8.0), 75 μ l, put upside down do again behind the mixing briefly centrifugal.Pre-expansion volume increase thing can be directly used in following Real-time PCR.
Pre-expansion volume increase thing carries out preparing shown in the Real-time PCR according to the form below reaction system:
6. detect and healthier control group, serious preeclampsia group serum sample in the miRNAs expression amount
Difference.
Detected normal healthy controls and the serious preeclampsia patients serum miRNAs that there are differences expression comprises hsa-miR-193a-5p(SEQ ID No.1), hsa-miR-424(SEQ ID No.2), hsa-miR-10a(SEQ ID No.3).The copy number of these miRNAs in the serious preeclampsia patient all significantly is lower than the normal healthy controls group, and these miRNAs express in serum and have stability.
Four, Real-time PCR method checking serum miRNAs expression amount
1. design the primer of 3 target miRNAs: use Stem-loop PCR method design primer.
2. add fluorescence dye and carry out Real-time PCR reaction.The difference of miRNAs expression amount in detection and healthier pregnant woman, the serious preeclampsia patients serum sample (case and each 100 people of contrast).
3. select independent crowd (contrast and case each 40 people) to carry out Real-time PCR detection, unanimity has 3 miRNAs a result, is specially: hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a.
Therefore, finally confirm as the healthy pregnant women and the serious preeclampsia patients serum miRNAs that there are differences expression and comprise hsa-miR-193a-5p(SEQ ID No.1), hsa-miR-424(SEQ ID No.2), hsa-miR-10a(SEQ ID No.3), concrete primer sees Table 1.Their copy numbers in the serious preeclampsia patients serum all significantly are lower than the normal healthy controls group, and these miRNAs express in serum and have stability.Adopt the combination of SEQ ID No.1, SEQ ID No.2, these 3 formations of SEQ ID No.3 control group, serious preeclampsia group differentiation can be opened.Though these 3 also can be separated two groups of crowds separately, if only use 1, may have coincidence, and if use 3 simultaneously, even 1 difference is little, but other 2 differential expressions are all very big, just can carry it into the high group of scoring.
Five, diagnostic reagent box preparation method
According to above-mentioned a series of experimental results, the inventor has also prepared the diagnostic kit that a kind of energy is used for the serious preeclampsia dynamic monitoring, and described diagnostic kit comprises primer and the instrument of measuring stable existence in experimenter's serum and detectable ripe hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a.Diagnostic kit comprises a collection of serum miRNAs primer, can also comprise reagent such as Taq enzyme, triphosphoric acid base deoxynucleotide.
Beneficial effect of the present invention:
The inventor is by separating and compare the miRNAs among normal control and the serious preeclampsia patients serum, found to exist in the serum and can be used for assessing the specificity of whether suffering from the serious preeclampsia patient and susceptibility (the ROC curve prompting of embodiment 5 has sensitivity preferably, embodiment 6 verifies its actual effect, be that the serious preeclampsia patient is all detected identification by correct) the miRNA combination, thereby the serum miRNA mark that has proposed the serious preeclampsia patient makes up, and this serum miRNA mark or the application of its primer in the diagnosis of preparation serious preeclampsia or monitoring reagent, develop the serious preeclampsia diagnosis that can be convenient to clinical application, the monitoring reagent box.
The present invention adopts serum miRNA to be as the superiority of the mark of serious preeclampsia evaluation:
(1) serum miRNA is a kind of new bio mark, be different from the traditional biological mark, not only stable, Wicresoft, be easy to detect, and quantitatively accurately, susceptibility and the specificity of serious preeclampsia diagnosis will be improved greatly, the successful exploitation of such microRNA biomarker is to based on the overturning of the traditional biological mark of albumen, and will start brand-new situation for the control that gestation merges syndromes, for the development of other diseases biomarker is offered reference.
(2) serum miRNA mark provided by the invention can be used as the serious preeclampsia diagnosis marker, can avoid invasive diagnosis, and can carry out auxiliary diagnosis in early days, thereby for the further testing in depth testing of clinician provides foundation, for quick and precisely grasping the disease of patient state and the degree that is in a bad way, in time taking the scheme of preventing and treating of more personalized to provide support, delay and stop progression of disease.
(3) the present invention adopts the sample that meets serious preeclampsia and normal healthy controls crowd to verify, proves that there is significant difference in these several marker expression amounts and has stability, has specificity so that this mark to be described, can be used as mark and uses.
(4) the present invention adopts tight, multistage checking and appraisement system, initial stage is screened multiple serum miRNAs by preliminary experiment, methods such as application Real-time PCR are carried out secondary checking and independent crowd checking, adopt the layering points-scoring system to marking of diagnostic result, and organize among the independent crowd at another serum miRNA mark and diagnostic kit are carried out blind method evaluation, guaranteed the reliability of this serum miRNA biomarker and diagnostic kit.
Description of drawings
Fig. 1 with hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a as a token of thing normal healthy controls and serious preeclampsia group are distinguished.
The horizontal fluctuation analysis of the individual serum miRNAs continuous expression of Fig. 2.
ROC curve between Fig. 3 normal control group and the serious preeclampsia group.
Embodiment
The invention will be further elaborated by the following examples.
Embodiment 1 research object is selected and the grouping foundation
The inventor divides between year October in July, 2009 to 2011 and collects satisfactory serious preeclampsia patient and normal healthy controls pregnant woman blood sample from affiliated hospital of Nanjing Medical University three times, by the arrangement to the sample data, therefrom selected satisfactory 100 routine normal healthy controls (mean age: 27.63 ± 3.25 days), 100 routine serious preeclampsia patients (mean age: 28.56 ± 4.15 days) to detect the experimental subjects that miRNA expresses as Real-time PCR.Concrete sample classification criteria is as follows:
A group: normal healthy controls group (n=100, the screening of 20 people's chips, the checking of 40 people's first phases, 40 people's independence crowds checking):
1. there are not other general major diseases.
B group: serious preeclampsia group (n=100, the screening of 20 people's chips, the checking of 40 people's first phases, 40 people's independence crowds checking):
1. be serious preeclampsia through clinical diagnosis;
2. there are not other general major diseases.
Embodiment 2TaqMan miRNA array screening
Preparation cDNA sample: a) get 100 μ l serum; B) add 900 μ l Trizol, the vibration mixing, 4 ℃, centrifugal 15 minutes of 12000rpm abandons lower floor's waste liquid; C) the dehydrated alcohol concussion mixing of 1.5 times of volumes of adding supernatant goes to centrifugal post, and centrifugal 15 seconds of 12000rpm abandons lower floor's waste liquid; D) add 700 μ l RWT damping fluids at centrifugal post, centrifugal 15 seconds of 10000rpm abandons lower floor's waste liquid.E) add 500 μ l RPE damping fluids at centrifugal post, centrifugal 15 seconds of 10000rpm abandons subnatant.F) repeat e.G) with the pipe of a new 2ml of centrifugal post adding, centrifugal 1 minute of 10000rpm is used for removing the RPE damping fluid.H) add 50 μ lDEPC at pillar and handle the centrifugal collection of water 12000rpm RNA.I) obtain cDNA by the RNA reverse transcription reaction then.The reaction system of reverse transcription comprises 4 μ l5 * AMV damping fluid, 2 μ l10mM dNTP mixed solutions (Takara company), 0.5 μ l RNA enzyme inhibitors (Takara company), 1 μ l AMV(Takara company) and the reverse primer of the single miRNA correspondence of 1.5 μ l.Reactions steps is 16 ℃ hatched 15 minutes, and 42 ℃ were reacted 1 hour, and hatched 5 minutes for 85 ℃.(if then adopting corresponding miRNA reverse primer to be undertaken by above-mentioned steps at different miRNA)
CDNA according to the form below reaction system preparation after the reverse transcription is increased in advance:
The reaction conditions of pre-amplification is as shown in the table:
With pre-expansion increase production thing brief centrifugal after, add 0.1 * TE (pH8.0), 75 μ l, put upside down do again behind the mixing briefly centrifugal.Pre-expansion volume increase thing can be directly used in following real-time fluorescence quantitative PCR (qPCR).
Pre-expansion volume increase thing carries out preparing shown in the qPCR according to the form below reaction system:
Detect the difference of miRNAs express spectra in also healthier contrast, the serious preeclampsia serum sample, filter out the above miRNAs of 4 times of differences.Through bioinformatic analysis and experimentation on animals result, selected wherein 3 become candidate's step card of advancing of going forward side by side, be specially: hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a.
Embodiment 3Real-time PCR method is measured serum miRNA expression amount
Design primer (table 1) carries out the quantitative Real-time PCR detection of each miRNAs respectively to 80 routine normal healthy controls, 80 routine serious preeclampsia patients' serum.
(1) preparation cDNA sample: a) get 100 μ l serum; B) add 900 μ l Trizol, the vibration mixing, 4 ℃, centrifugal 15 minutes of 12000rpm abandons lower floor's waste liquid; C) the dehydrated alcohol concussion mixing of 1.5 times of volumes of adding supernatant goes to centrifugal post, and centrifugal 15 seconds of 12000rpm abandons lower floor's waste liquid; D) add 700 μ l RWT damping fluids at centrifugal post, centrifugal 15 seconds of 10000rpm abandons lower floor's waste liquid.E) add 500 μ l RPE damping fluids at centrifugal post, centrifugal 15 seconds of 10000rpm abandons subnatant.F) repeat e.G) with the pipe of a new 2ml of centrifugal post adding, centrifugal 1 minute of 10000rpm is used for removing the RPE damping fluid.H) add 50 μ l DEPC at pillar and handle the centrifugal collection of water 12000rpm RNA.i) obtain cDNA by the RNA reverse transcription reaction then.The reaction system of reverse transcription comprises 4 μ l5 * AMV damping fluid, 2 μ l10mM dNTP mixed solutions (Takara company), 0.5 μ l RNA enzyme inhibitors (Takara company), 1 μ l AMV(Takara company) and the reverse primer of the single miRNA correspondence of 1.5 μ l.Reactions steps is 16 ℃ hatched 15 minutes, and 42 ℃ were reacted 1 hour, and hatched 5 minutes for 85 ℃;
(2) Real-time PCR: dye method: get 1 μ l cDNA, with the cDNA doubling dilution, add 0.3 μ l Taq enzyme (Takara company), 1 μ l20 * EVA GREEN, 0.25 the forward primer of the above-mentioned single miRNA correspondence of μ l10 μ M, 0.25 the general reverse primer of μ l10 μ M (URP), 1.2 μ l25mM MgCl
2, 1.6 μ l2.5mM dNTP mixed solutions (Takara company), 2 μ l10 * PCR damping fluid, 12.4 μ l pure water, 20 μ l systems are carried out quantitative fluorescent PCR.10 μ l TaqMan universal PCR master Mix, 6.6 μ l H
2O, 20 μ l systems are carried out q-PCR.What instrument used all is ABI Prism7900 quantitative real time PCR Instrument, and the reaction conditions of PCR all is: carried out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carried out 40 circulations in 60 ℃, 1 minute.Detect the variation of miRNA expression amount in also healthier contrast, the serious preeclampsia infant serum sample, each organizes the expression amount ratio of sample serum miRNA can use equation 2
– △ GExpression, wherein △ G=C
Tgroup1– C
Tgroup2For guaranteeing the comparability between each time experiment, we are provided with U6 on every plate, adjust the calculation expression amount with its expression amount as confidential reference items.
Draw from interpretation of result, hsa-miR-193a-5p, hsa-miR-424, these three miRNA of hsa-miR-10a all have marked difference (Fig. 3) between each group.Non-parametric tendency check has also shown identical difference.
The primer sequence of table 1 miRNAs
The stability analysis of embodiment 4 individual serum miRNA expression amounts
Adopt the method for embodiment 3 that the stability of 8 pregnant woman's serum miRNA level is estimated.Gather continuous 3 serum of research object (be 2 days pitch time, no disease in interval) with embodiment 1 same acquisition method.The result shows that hsa-miR-193a-5p, hsa-miR-424, these three miRNA expression levels of hsa-miR-10a are stablized (Fig. 2) in the serum.These have all pointed out the expression amount of individual serum miRNA comparatively stable, possess the characteristic as the diagnose/monitor mark.
Embodiment 5miRNA combination is to the judgement of serious preeclampsia
According to above-mentioned Real-time PCR method, the inventor passes through the analysis to the miRNAs expression level of case and control group serum sample, five fractiles with normal healthy controls group miRNAs expression amount are threshold value, hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a are marked, further try to achieve PTS, draw susceptibility and the specificity that the ROC curve is assessed prediction with this, so assess these three miRNAs low express or high expression level to the evaluation capacity of serious preeclampsia.The ROC analytical results shows, hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a are with 81.00% AUC(ROC area under curve) normal control group and serious preeclampsia component are opened (Fig. 3).
On the basis of above-mentioned a series of results of study, the inventor has proved that employing hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a can separate serious preeclampsia patient and normal healthy controls well.
The blind method checking of embodiment 6miRNA layering scoring and independent crowd
When the expression level to hsa-miR-193a-5p, hsa-miR-424, these three marks of hsa-miR-10a carries out the layering scoring (adding up after the five fractile layerings), can assess whether suffering from serious preeclampsia, it is more high to be expressed as integration, and the risk of confirming as serious preeclampsia is more high.
Three miRNAs of table 3, five fractile scores and summation
Annotate: each miRNA is divided into five grades 0,1,2,3,4 by five fractiles of expression amount, minimum 0 minute, the highest 4 minutes, article 3, miRNA comprehensively can be divided into 0～12 fen, (expression amount is low and the most scores of serious preeclampsia group are very low, integration is by backwards calculation), and the most scores of normal control group are very high.For instance, be 12 minutes (the highest score value group) if any a sample scoring, then it can not be the serious preeclampsia case, and should be normal control.
(namely judge its grouping according to concrete score, what secure satisfactory grades is included into control group, and what make low score is included into the serious preeclampsia group at the point value of evaluation that draws; Comparatively speaking, 〉=9 calculate high score, ≤ 3 calculate low the branch), the independent crowd who gathers of another group is carried out blind method head to be examined, namely adopt double blind trial, the miRNA that independent crowd (the 40 routine pregnant woman that another hospital gathers) is carried out simultaneously routine diagnostic analysis and serum sample detects, the result shows by 3 miRNA detection scorings, serious preeclampsia and contrast pregnant woman can be distinguished well that (40 examples select to have in the sample 7 example scorings lower (≤3 minutes) at random, wherein reach 4 examples that have of 0 minute (best result), this 4 example is serious preeclampsia after diagnosing, all the other scorings higher (〉=9 minutes) be non-serious preeclampsia), point out these several miRNA to can be used as the mark of assessment serious preeclampsia.
Embodiment 7 is used for the making of the miRNA diagnostic kit of serious preeclampsia diagnosis and monitoring
The manufacture craft of this miRNA test kit and operating process are mainly based on RT-PCR, Real-time round pcr.
At first determine to have among normal people and the serious preeclampsia patients serum miRNA that copies more than by method and the Real-time PCR method of order-checking.By technology screenings such as a quantitative PCR class serum miRNA relevant with serious preeclampsia, whether suffers from the index of serious preeclampsia and diagnosis as prediction then.Filter out the quantity control of corresponding serum miRNA at last at several, this is optimized the simplifying of making on the basis of preliminary experiment.This test kit comprises a collection of serum miRNA primer, and wherein the primer of miRNA comprises forward and reverse primer (seeing Table 1) of hsa-miR-193a-5p, hsa-miR-424, hsa-miR-10a, U6.Relevant round pcr reagent commonly used can also be arranged, as Taq enzyme, PCR damping fluid, MgCl
2, reagent such as triphosphoric acid base deoxynucleotide mixed solution, dyestuff, these reagent also can adopt corresponding commercially available prod.The value of this test kit is only to need once to extract out a small amount of (1～2ml) blood, can detect the variation tendency of serum miRNA mark, predict serious preeclampsia possibility occurrence or diagnosis serious preeclampsia disease by this variation tendency again, and be easy to carry out dynamic monitoring and observe result for the treatment of.
Concrete test kit is composed as follows:
Primer also can be two couple in following three pairs of primers or three pairs: SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.6 and SE Q ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, each 10 μ M0.25 μ l.
Can also contain 0.3 μ l Taq enzyme in the test kit, 1 μ l20 * EVA GREEN, 1.2 μ l25mM MgCl
2, 1.6 μ l2.5mM dNTP mixed solutions, 2 μ l10 * PCR damping fluid, 12.4 μ l pure water.
Forward and reverse primer a pair of (table 1) that can also contain confidential reference items U6 in the test kit.
Perhaps except forward primer, also contain the general reverse primer of 1 μ l10 μ M in the test kit, 10 μ l TaqMan universal PC R mixed solutions, 6.6 μ l H
2O.
The reagent of component in the test kit except primer can adopt the corresponding reagent that is used for the miRNA content detection in the prior art.
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Claims (9)
1. the relevant blood plasma microRNA mark of human serious preeclampsia is selected from multiple among hsa-miR-193a-5p, hsa-miR-424 and the hsa-miR-10a.
2. blood plasma microRNA mark according to claim 1 is characterized in that being made of hsa-miR-193a-5p, hsa-miR-424 and hsa-miR-10a.
3. blood plasma microRNA mark according to claim 1 and 2, the sequence that it is characterized in that hsa-miR-193a-5p is SEQ ID No.1, and the sequence of hsa-miR-424 is SEQ ID No.2, and the sequence of hsa-miR-10a is SEQ ID No.3.
4. the primer of the described blood plasma microRNA of claim 3 mark is characterized in that sequence is that the upstream primer of the mark of SEQ ID No.1 is SEQ ID No.4, and downstream primer is SEQ ID No.5; Sequence is that the upstream primer of the mark of SEQ ID No.2 is SEQ ID No.6, and downstream primer is SEQ ID No.7; Sequence is that the upstream primer of the mark of SEQ ID No.3 is SEQ ID No.8, and downstream primer is SEQ ID No.9.
5. claim 1 or the 2 described blood plasma microRNA marks application in the human serious preeclampsia diagnosis of preparation or monitoring reagent.
6. the application of the described primer of claim 4 in the human serious preeclampsia diagnosis of preparation or monitoring reagent.
7. a human serious preeclampsia is diagnosed or the monitoring reagent box, it is characterized in that this test kit contains the primer of claim 1 or 2 described blood plasma microRNA marks.
8. diagnosis according to claim 7 or monitoring reagent box is characterized in that this test kit contains how right in the described primer of claim 4.
9. diagnosis according to claim 8 or monitoring reagent box is characterized in that this test kit contains primer SEQ ID No.4 and SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9.
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| WO2017101172A1 (en) * | 2015-12-15 | 2017-06-22 | 中国人民解放军第二军医大学 | Serum mirna marker for opll diagnosis and application thereof |
| US11124832B2 (en) | 2015-12-15 | 2021-09-21 | The Second Military Medical University | Serum miRNA marker for OPLL diagnosis and application thereof |
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