CN108624678A

CN108624678A - A kind of biomarker for preeclampsia diagnosis and treatment

Info

Publication number: CN108624678A
Application number: CN201810669608.1A
Authority: CN
Inventors: 孙耀兰; 杨承刚; 张冬梅
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2018-06-26
Filing date: 2018-06-26
Publication date: 2018-10-09
Anticipated expiration: 2038-06-26
Also published as: CN108624678B

Abstract

The invention discloses the biomarker for preeclampsia diagnosis and treatment, the biomarker KIAA1949 in preeclampsia pregnant women placental tissue and blood lower by expression, and KIAA1949 is prompted to can be used as the early diagnosis that detection target is applied to preeclampsia；Proliferation and the invasion of trophocyte can be promoted by being overexpressed KIAA1949 genes, can be applied to the treatment of preeclampsia as molecular target accordingly.

Description

A kind of biomarker for preeclampsia diagnosis and treatment

Technical field

The invention belongs to biomedicine fields, are related to a kind of biomarker for preeclampsia diagnosis and treatment, specifically relate to And biomarker be the KIAA1949 significantly lowered in preeclampsia.

Background technology

Preeclampsia (pre-eclampsia, PE), eclampsia (eclampsia) and gestation hypertension are that the gestational period is peculiar Disease, belongs to the scope of hypertensive disorder in pregnancy (hypertensive disorders in pregnancy), and incidence exists China is about 10% or so, wherein especially with preeclampsia multiple (Redman CW, Sargent IL.Latest advances In understanding pre-eclampsia [J] .Science, 2005,308:1592-1594).PE is mainly shown as pregnant The hypertension that occurs after being pregnent 20 weeks, albuminuria, can cause the failure of whole body Analysis of multi-organic functional damages and function, can lead to height Up to 15% maternal mortality rate, it is the main reason for leading to pregnant and lying-in women and perinatal infants dead rate, is always what pathological obstetrics were studied Emphasis, so far unknown (Duley L.The global impact of pre-eclampsia and of pathogenesis eclampsia.Semin Perinatol.2009；33:130-137.).Clinical research is found, after PE patient's gestation, Its symptom often terminates therewith, and only small part delay becomes chronic hypertension.Research at present is it is believed that embryo as a result, Play important role in Attack of Preeclampsia, further research thinks that embryonic period, embryonic phase trophoblast invasion is lowly led Cause Uterine Spiral double teeming obstacle and placentation it is bad be preeclampsia starting pathological change (Rigourd M T Chelbi S,Uaiman D.Preeclampsia.Med Sci.2008,24(12):1017 1 1019.).

The high risk factor that preeclampsia occurs removes by low age or Elderly primipara, nutritional deficiency, multifetation and once had original Outside the such environmental effects such as Essential hypertension, ephritis, diabetes medical history, also by multiple genes or multiple polymorphic positions on science of heredity The influence for the effect that intersects is put, there is apparent genetic predisposition (Serrano N C, Casas JP, Diaz LA, et al.Endothelial NO synthase genotype and risk of preeclampsia:a multicenter case-control study[J].Hypertension,2004,44(5):702-707.).Since 19th century, before eclampsia The familial of phase fall ill it is verified that, also illustrate inherent cause take part in preeclampsia generation (Paula J.Williams, Fiona Broughton Pipkin.The genetics of pre-eclampsia and other hypertensive disorders of pregnancy.Best Pract Res Clin Obstet Gynaecol 2011 August,25(4- 4):405-417.).With the development of the Human Genome Project, the positioning and identification of Attack of Preeclampsia related gene have become Research hotspot.Illustrate pathogenesis of the preeclampsia on molecule and cellular level, and clinically for the prevention of preeclampsia, It timely and effectively predicts and treats and experimental basis is provided, to ensureing that baby's health, reduction illness rate and maternal mortality rate have weight Want meaning.

Invention content

In order to make up for the deficiencies of the prior art, one of the objects of the present invention is to provide a kind of biological markers of preeclampsia Object, the diagnosing and treating of sensitive and specific realization preeclampsia.

The second object of the present invention is to provide biomarker in the biomarker of screening treatment preeclampsia Using.

To achieve the goals above, the present invention adopts the following technical scheme that：

The present invention provides application of the detection reagent of KIAA1949 in the product for preparing diagnosis preeclampsia.

Further, the product includes：Chip, preparation, kit or nucleic acid film item.

The present invention provides a kind of products of diagnosis preeclampsia, and the product includes the detection reagent of KIAA1949.

Further, the reagent includes：

The probe of specific recognition KIAA1949 genes；Or

The primer of specific amplification KIAA1949 genes；Or

Specifically bind the antibody or ligand of KIAA1949 albumen.

Product of the present invention can be used for detecting multiple genes including KIAA1949 genes and encoded egg The expression of (for example, with preeclampsia relevant multiple genes and encoded albumen) in vain.

The present invention provides applications of the KIAA1949 in the pharmaceutical composition for preparing treatment preeclampsia.

Further, described pharmaceutical composition includes the accelerating agent of KIAA1949 functional expressions, wherein the accelerating agent packet Include improve KIAA1949 genes or its expression product stability, up-regulation KIAA1949 genes or its expression product expression, Increase KIAA1949 genes or the substance of its expression product effective acting time.

Further, the accelerating agent is the carrier containing KIAA1949.

The present invention also provides a kind of pharmaceutical composition, the drug includes the accelerating agent of KIAA1949 functional expressions, And pharmaceutically acceptable carrier.

Wherein, the accelerating agent includes improving KIAA1949 genes or its expression product stability, up-regulation KIAA1949 bases The expression of cause or its expression product increases KIAA1949 genes or the substance of its expression product effective acting time.

Further, the accelerating agent is the carrier containing KIAA1949.

The present invention provides applications of the KIAA1949 in the drug of screening treatment preeclampsia.

Further, the step of drug of screening treatment preeclampsia includes：

The system for the albumen expressed or containing KIAA1949 genes or its coding is handled with substance to be screened；With

Detect the expression of the albumen of KIAA1949 genes or its coding or activity in the system.

Wherein, if substance to be screened can promote expression or the activity of KIAA1949, show that the substance to be screened is to control Treat the drug of preeclampsia.

In the present invention, the system includes but is not limited to：Cell system, subcellular system, solution system, organizer System, organ systems or animal system.

In the present invention, the step further includes：To the drug candidate of acquisition carry out further cell experiment and/or Animal experiment, with further selection can treat the drug of preeclampsia from drug candidate.

The advantages of the present invention：

Present invention firstly discovers that with preeclampsia occurrence and development relevant biomarker KIAA1949, by detection by The level of examination person KIAA1949, to realize the early diagnosis of preeclampsia, when otherness low expression is presented in KIAA1949, explanation Patient may suffer from preeclampsia.

The present invention provides the molecular target KIAA1949 for the treatment of preeclampsia to be changed by targeting molecular marker The level of marker has sensibility and specificity to treat disease.

The present invention provides a kind of new means to treat the research and development of preeclampsia drug.

Description of the drawings

Fig. 1 is the expression in placenta in preeclampsia using QPCR detection KIAA1949 genes；Wherein, A is placenta Expression in tissue, figure B are the expressions in blood；

Fig. 2 is the expression figure of KIAA1949 genes；

Fig. 3 is the expression figure of KIAA1949 albumen；

Fig. 4 shows that mtt assay detects the active influence diagram of KIAA1949 cell proliferations；

Fig. 5 shows the influence diagram using the cells Transwell detection KIAA1949 gene pairs cell invasions.

Specific embodiment

The present invention after extensive and in-depth study, passes through high throughput sequencing technologies and bioinformatic analysis, detection Gene expression dose in epilepsy patient's early period and normal pregnancies blood, find wherein with notable difference gene, inquire into its with Relationship between the generation of preeclampsia, early detection and targeted therapy to be preeclampsia find better approach and side Method.By screening present invention firstly discovers that KIAA1949 is to the occurrence and development of preeclampsia related, and demonstrates KIAA1949 The low expression in preeclampsia.KIAA1949 can be used as the independentpredictor of preeclampsia, can also be with other gene marks Will object use in conjunction.

Term " biomarker " is its expression in tissue or cell and normal or healthy cell in the present invention Or the expression of tissue compares any gene or albumen to change.

KIAA1949 genes are located at No. 62 areas 1 of the short arm of a chromosome and take, and the KIAA1949 in the present invention includes wild type, dashes forward Modification or its segment.In a specific embodiment of the present invention, KIAA1949 has such as current international public nucleic acid database Sequence shown in KIAA1949 genes (NM_001134870.1) in GeneBank.

Biomarker as described herein includes gene and albumen.Such biomarker includes containing encoding human mark Complete or partial sequence the DNA of the complementary series of the nucleic acid sequence of object or this sequence.Biomarker nucleic acid further include containing Complete or partial sequence the RNA of any nucleic acid sequence of interest.Biomarker protein is by the DNA biology marks of the present invention The albumen of will object coding or corresponding to the present invention DNA biomarkers.Biomarker protein includes any biological marker The complete or partial amino-acid series of object albumen or polypeptide.The segment and variant of biomarker genes and albumen are also included within this In the range of invention.

The present invention can utilize any method known in the art to measure gene expression.Those skilled in the art should manage Solution, the means for measuring gene expression are not the importances of the present invention.It can be detected on transcribing or translating (i.e. albumen) level The expression of biomarker.

The gene and albumen of the present invention can be examined using multiple nucleic acids technology known to persons of ordinary skill in the art It surveys, these technologies include but not limited to：Nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technologies, protein immunization detection technology.

The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but not limited to chain terminator (Sanger) sequencing and dye Expect terminator sequencing.Those skilled in the art it will be recognized that due to RNA in cell less stable and in an experiment It is more vulnerable to nuclease attack, therefore usually by RNA reverse transcriptions at DNA before sequencing.

The present invention simultaneously can expand nucleic acid (for example, ncRNA) before detection or with detection.Nucleic acid amplification technologies Exemplary, non-limitative example include but not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT- PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleic acid sequence It expands (NASBA).Those skilled in the art will be it will be recognized that certain amplification techniques (for example, PCR) needs will before amplification RNA reverse transcriptions are at DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

The PCR of commonly referred to as PCR uses denaturation, annealing and the primer extend of primer pair and opposite strand Multiple cycles, exponentially increase target nucleic acid sequence copy number；The amplification of the transcriptive intermediate of TMA is (in substantial constant Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more A RNA copies autocatalytically generate other copy；The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid The two groups of complementary DNA oligonucleotides handed over；Other amplification methods include for example：The expansion based on nucleic acid sequence of commonly referred to as NASBA Increase；Use the amplification of rna replicon enzyme (commonly referred to as Q β replicase) amplification probe molecule itself；Amplification method based on transcription； And the sequence amplification of self―sustaining.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Protein immunization technology includes but is not limited to sandwich immunoassay, such as sandwich ELISA, wherein using identification biology Two kinds of antibody of different epitopes carry out the detection of the biomarker on marker；Radiommunoassay (RIA), directly, indirectly Or comparison enzyme linked immunosorbent assay (ELISA) (ELISA), enzyme immunoassay (EIA) (EIA), fluorescence immunoassay (FIA), immunoblotting, Immuno-precipitation and immunoassays based on any particle are (as used gold particle, Argent grain or latex particle, magnetic-particle or amount Sub- point).Can for example immunization be implemented in the form of microtiter plate or item.

It can be used in the detection of the biomarker of the present invention any directly (as used sensor chip) or indirect Method.

Chip, preparation, nucleic acid film item, kit

The present invention provides the product of the expression of KIAA1949 genes in detection, the product includes but is not limited to Chip, preparation, nucleic acid film item or kit.Its chips includes：Solid phase carrier；And it is orderly fixed on the solid phase carrier Oligonucleotide probe, the oligonucleotide probe specifically correspond to KIAA1949 shown in some or all of sequence.

The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but not limited to have silicon carrier, Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..

Term " probe " refers to the molecule that can be combined with the particular sequence of another molecule or subsequence or other parts.Unless another It points out, term " probe " is often referred to match by complementary base and another polynucleotides (often referred to as " target polynucleotide ") In conjunction with polynucleotide probes.Lack sufficient sequence complementarity according to the preciseness of hybridization conditions, probe energy and with the probe Target polynucleotide combines.Probe can make direct or indirect label, and range includes primer.Crossing system, including, but it is unlimited In：Solution phase, solid phase, mixed phase or in situ hybridization measuring method.

Exemplary probe in the present invention includes PCR primer and gene specific DNA oligonucleotide probe, such as fixed In micro probe array, the quantitative nucleic acid enzyme protection on microarray substrate examine probe, the probe that is connect with molecular barcode and The probe being fixed on pearl.

These probes have the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA, Can also be RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or whole nucleotides Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerine nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

In the present invention, term " antibody " includes but not limited to monoclonal antibody, polyclonal antibody.The KIAA1949 eggs White specific antibody include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with KIAA1949 albumen.For protein level The preparation of antibody be well known to those skilled in the art, and the present invention may use any method to prepare the antibody.

In the present invention, nucleic acid film item includes substrate and the oligonucleotide probe that is fixed in the substrate；The substrate Can be any substrate suitable for immobilized oligonucleotide probe, for example, nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass, Silica gel chip, micro magnetic bead etc..

The present invention provides a kind of kit, the kit can be used for detecting the expression of KIAA1949.The kit It include the specific primer pair for expanding KIAA1949；Standard DNA template；PCR reaction solution.

In a preferred embodiment, the specific primer is to including sense primer and downstream primer, and sequence is such as Shown in NO.3~4 SEQ ID.

Embodiment more preferably, the kit are fluorescent quantificationally PCR detecting kit, and the primer is suitable for The detection of SYBR Green, TaqMan probe, molecular beacon, double cross probe, combined probe.

In a further preferred embodiment, the PCR reaction solution in the kit is fluorescence quantitative PCR reaction solution, And further include fluorescent dye.

In a further preferred embodiment, the fluorescence quantitative PCR reaction solution includes dNTP, Mg²⁺, Taq enzyme and Buffer buffer solutions, the fluorescent dye are SYBR Green II, and Taq enzyme is thermal starting enzyme.

Accelerating agent and pharmaceutical composition

The pharmaceutical composition of the present invention includes the inhibitor of KIAA1949 functional expressions and pharmaceutically acceptable load Body.

In the present invention, about " functional expression " of KIAA1949, it is intended that the transcription of functional gene product and/or turn over It translates." functional expression " can lack of proper care at least three levels.First, on DNA level, such as by the missing of gene or It destroys, or there is no transcription (synthesis for all preventing relevant gene product in both cases).The missing of transcription can be with Such as cause by the variation (such as DNA methylation) of epigenetic or by afunction mutation." work(as used herein Energy missing " or " LOF " mutation are for assigning protein enhancing or new active function gain mutation, to hinder Only, the mutation of the function of gene outcome is reduced or eliminated.Functional deficiency can be caused by extensive mutation type, including but unlimited It is mutated in the deletion of whole gene or Gene Partial, splice site, dashed forward with frameshift mutation, nonsense caused by deleting by small insertion Become, instead of essential amino acid missense mutation and prevent product correct cellular localization mutation.This definition also includes The mutation of the promoter or regulatory region of KIAA1949 genes, if these mutation disturbances function of gene.Null mutation is The LOF mutation of gene outcome function are destroyed completely.Null mutation in one allele will usually be such that expression reduces 50% but may to the function of gene outcome have seriously affect.It is worth noting that, functional expression can also be because of function Gain mutation and lack of proper care：By assigning protein new activity, the normal function imbalance of protein, the functional activity egg of expression It is white to reduce.Vice versa, and functional expression can for example increase by gene duplication or by lacking DNA methylation.Function Property expression can also lack of proper care because of function gain mutation：By assigning protein new activity, the normal function of protein The functional activity albumen of imbalance, expression is reduced.Vice versa, and functional expression for example by gene duplication or can pass through shortage DNA methylation and increase.

Second, on rna level, such as by lacking effective translation, such as because the unstable of mRNA (such as passes through UTR variants), the premessenger RNA in translation of transcript can be caused to be degraded.Or effectively transcribed by lacking, such as because it is prominent Become induction of new splice variant.

As a kind of selectable embodiment of the present invention, the accelerating agent includes improving KIAA1949 genes or its table Up to product stability, the expression of up-regulation KIAA1949 genes or its expression product, increase KIAA1949 genes or its expression The substance of product effective acting time.

In general, these accelerating agents can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, Wherein pH ordinarily is about 5-8, and preferably pH is about 6-8, although pH value can be with the property and disease to be treated for being formulated substance Disease and be varied from.Prepared pharmaceutical composition can be administered by conventional route, including (but being not limited to)： It is intramuscular, peritonaeum is interior, intravenous, subcutaneous, intradermal or local administration.

As a kind of preferred embodiment of the present invention, the accelerating agent of the KIAA1949 is a kind of expression load of KIAA1949 Body.The expression vector usually also contains promoter, replication orgin and/or marker gene etc..

Method well-known to those having ordinary skill in the art can be used to build the required expression vector of the present invention.These methods include Recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..The expression vector preferably includes one or more Selected marker, to provide the phenotypic character of the host cell for selecting conversion, such as kalamycin, gentamicin, tide Mycin, amicillin resistance.

In the present invention, be various carriers known in the art, such as commercially available carrier including plasmid, clay, bacteriophage, Virus etc..Importing of the expression vector into host cell can use electroporation, calcium phosphate method, liposome method, the Portugals DEAE poly- The known methods such as sugared method, microinjection, viral infection, the combination of liposome transfection and cell-membrane permeable peptide.

In the present invention, " host cell " born of the same parents can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as Yeast cells；Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, the bacterium of streptomyces Cell；Fungal cell's such as yeast；Plant cell；The insect cell of drosophila S2 or Sf9；The animal of CHO, COS or 293 cells is thin Born of the same parents etc..

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The present invention also provides a kind of pharmaceutical compositions, it contains the accelerating agent of a effective amount of KIAA1949, with And pharmaceutically acceptable carrier.The composition can be used for treating placenta in preeclampsia.Any KIAA1949's above-mentioned Accelerating agent is used equally for the preparation of composition.The pharmaceutically acceptable carrier and/or auxiliary material, including but not limited to dilute Agent, adhesive, surfactant, Humectant, absorption carrier, lubricant, filler, disintegrant.

Wherein, diluent such as lactose, sodium chloride, glucose, urea, starch, water etc.；Adhesive such as starch, pregelatinated form sediment Powder, dextrin, maltodextrin, sucrose, Arabic gum, gelatin, methylcellulose, carboxymethyl cellulose, ethyl cellulose, poly- second Enol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginate, xanthans, hydroxypropyl cellulose and hydroxypropyl methyl Cellulose etc.；Surfactant such as polyoxyethylene sorbitan aliphatic ester, lauryl sodium sulfate, stearic acid list glycerine Ester, hexadecanol etc.；Humectant such as glycerine, starch etc.；Absorption carrier such as starch, lactose, bentonite, silica gel, kaolin and soap Clay etc.；Lubricant such as zinc stearate, glycerin monostearate, polyethylene glycol, talcum powder, calcium stearate and magnesium, polyethylene glycol, Boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyl 40 stearate, single bay sucrose acid ester, laruyl alcohol sulfuric acid Sodium, magnesium laurylsulfate, Stepanol MG etc.；Filler such as mannitol (granular or powdery), xylitol, sorbierite, wheat Bud sugar, erythrose, microcrystalline cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp Polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate etc.；Disintegrant such as cross-linked ethylene pyrrolidones, sodium carboxymethyl starch, low Replace hydroxypropyl methyl, croscarmellose sodium, soybean polyoses etc..

Pharmaceutical composition in the present invention can also include stabilizer, fungicide, buffer, isotonic agent, chelating agent, pH controls The additives such as preparation and surfactant.

Term " treatment " refers to for the purpose of healing, improvement, stabilization or prevention disease, pathological state or illness in the present invention The medical supervision that patient is carried out.The term includes active treatment, i.e., specially for the purpose of improving disease, pathological state or illness Treatment, and further include etiological treatment, i.e. the treatment for the purpose of removing relevant disease, pathological state or the cause of disease of illness. In addition, the term further includes palliative treatment, that is, it is designed for alleviating symptom rather than cures controlling for disease, pathological state or illness It treats；Prophylactic treatment, i.e., utmostly to reduce or partially or completely inhibit the development of relevant disease, pathological state or illness For the purpose for the treatment of；And supportive treatment, that is, it is used to supplement another kind to improve relevant disease, pathological state or illness as mesh Specific therapy treatment.

Statistical analysis

In a specific embodiment of the present invention, experiment all completed according to being at least repeated 3 times, result data be all with The mode of mean+SD indicates, using SPSS18.0 statistical softwares come for statistical analysis, difference between the two It is different to be examined using t, it is believed that work as P<There is statistical significance when 0.05.

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 is screened and preeclampsia relevant gene marker

1, sample collection

1) collection of serum specimen

The blood of each placenta in preeclampsia collected 45 normal pregnancies and do not receive treatment, EDTA anticoagulant tubes are stood 10min centrifuges serum, and -20 DEG C save backup.

2) collection of placenta sample

Each placenta tissue for collecting 45 Cases with Preeclampsia and normal pregnancies, physiological saline are rinsed 2 times, are dispensed after removing moisture removal In in cryopreservation tube, -80 DEG C save backup.

Two groups exclude multifetation, communicable disease, chemicals dependence, Maternal Smoking Smoking, fetus congenital malformation and its His pregnancy complication and complication, all research objects being included in sign informed consent form before making a collection of specimens.It is above-mentioned all Agreement of the acquirement of sample by the committee of organizational ethics.Every group takes the detection and analysis that 5 samples carry out gene expression profile, The screening of difference expression gene is carried out, and confirmatory experiment is carried out in each group all 45 samples.

2, the preparation of RNA sample

Utilize the total serum IgE in the tissue RNA extracts kits extraction placenta tissue of QIAGEN, specific steps reference explanation Book.

3, the quality analysis of RNA sample

By the RNA of said extracted into row agarose gel electrophoresis, using Nanodrop2000 to the concentration of carried RNA and pure Degree is detected, and agarose gel electrophoresis detects RNA integralities, and Agilent2100 measures RIN values.Single requirement for construction data base RNA is total 5 μ g are measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.

4, rRNA is removed

The rRNA in total serum IgE is removed using Ribo-Zero kits.

5, construction cDNA library

The structure of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit By specification carries out.

6, upper machine sequencing

CDNA library is sequenced using Illumina X-Ten microarray datasets, concrete operations by specification carries out.

7, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, RNA-seq read positioning is carried out using TopHat v1.3.1, leads to It crosses Cufflinks v1.0.3 and RNA-seq segment numbers is standardized to the relative abundance for calculating transcript, utilize Cuffdiff detects differential expression, works as p value<When 0.05, it is believed that gene significant difference is expressed.

8, result

RNA-seq is the results show that compared with the placenta tissue of normal pregnancies, and KIAA1949 genes are in preeclampsia organizes Expression quantity significantly lower.

The differential expression of 2 QPCR sequence verification KIAA1949 genes of embodiment

1, large sample QPCR verifications are carried out to KIAA1949 gene differential expressions.

2, RNA is extracted

Utilize the total serum IgE in the tissue RNA extracts kits extraction placenta tissue of QIAGEN, blood rna extracts kit Extract the RNA in serum, specific steps reference explanation book.

3, reverse transcription：

1) dNTP mixture1 μ l, Oligo dT primer 1 μ l, 2 μ g of total serum IgE is added and adds Rnase Free ddH₂O makes total volume to 10 μ l, is denaturalized in PCR instrument, annealing reaction, 65 DEG C, 5min, and reaction is completed to be placed on 4 DEG C.

2) 20 μ l reaction systems are built, 5 × Primer Script Buffer 4 μ l, RNase are continuously added 0.5 μ l, Prime Script RTase of Inhibitor, 0.5 μ l, RNase Free ddH₂5.0 μ l of O, are pressed in PCR instrument Row condition carries out reverse transcription reaction：42 DEG C of 15~30min, 95 DEG C of 5min are placed on ice after reaction is completed.

3) 42 DEG C of heating 15min in water-bath, 95 DEG C of heating 3min, -20 DEG C store for future use.

4, QPCR detects the expression of KIAA1949

1) design of primers

QPCR amplimers are designed according to the coded sequence of KIAA1949 genes and GAPDH genes in Genebank, by winning Mai De biotech firms synthesize.Specific primer sequence is as follows：

The primer sequence of house-keeping gene GAPDH is：

Forward primer：5’-CTCTGGTAAAGTGGATATTGT-3’(SEQ ID NO.1)

Reverse primer：5’-GGTGGAATCATATTGGAACA-3’(SEQ ID NO.2)

KIAA1949 genes：

Forward primer is 5 '-CAGGAACAGAGTTTGGTA-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-TCCGAGTAGTCTTGTCTT-3 ' (SEQ ID NO.4).

2) PCR reaction systems：Forward primer and each 10 μ l of 1 μ l, SYBR Green PCR master mix of reverse primer, CDNA 1 μ l, ddH₂O 7μl。

3) PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 15s) × 40 cycles, 65 DEG C~ 95 DEG C, 0.5 DEG C/5s of temperature ramp-up rate.PCR reactions are carried out on Bio-Rad iQ5 fluorescence quantitative PCR instruments, by melting song Line analysis and electrophoresis determine that purpose band, Δ Δ CT methods carry out relative quantification.

5, statistical method

Using GAPDH as internal reference, calculate preeclampsia tissue with normal placenta tissue, women with pre-eclampsia liquid with normally it is pregnant The experimental result of KIAA1949 fluorescence quantitative RT-RCRs in woman's blood, using SPSS18.0 statistical softwares come for statistical analysis, Difference between the two is examined using t, with P<0.05 has significant difference.

6, result

The results are shown in Figure 1, compared with normal pregnancies, in preeclampsia pregnant woman KIAA1949 gene expressions significantly lower, Difference has statistical significance (P<0.05), the positive rate in placenta tissue=number of cases is expressed in downward/always detect number of cases × 100%=43/45 × 100%=95.56%, the positive rate in blood are 42/45 × 100%=93.33%, prompt inspection The expression for surveying KIAA1949 in blood or placenta tissue can be used for the auxiliary diagnosis of preeclampsia.

The overexpression of 3 KIAA1949 genes of embodiment

1, cell culture

People's villi trophocyte strain (HTR-8/SVneo) is existed with the RPIM-1640 culture mediums containing 10% fetal calf serum 37 DEG C, 5%CO₂Incubator in cultivate.It changes within 2-3 days liquid 1 time, is passed using the 0.25% trypsase conventional digestion containing EDTA Generation.

2, it transfects

1) precellular processing is transfected

By the trophocyte HTR-8/SVneo in logarithmic phase with 1 × 10⁵Density plant respectively in six orifice plates, in 37 DEG C 5%CO₂Incubator in cultivate.

2) structure of gene overexpression carrier

According to the special PCR amplification primer of the sequent synthesis of KIAA1949 in GeneBank, primer sequence is as follows：

Forward primer：5’-CCGGTTTAAACGCCACCATGGCCACCATCCCAG-3’(SEQ ID NO.5)

Reverse primer：5’-CGGGCGGCCGCCCGCCGGCAGGACTCATCCA-3’(SEQ ID NO.6)

Two restriction enzyme sites of PmeI and NotI are added respectively in 5 ' end primers and 3 ' end primers.Suffered from preeclampsia The cDNA that person extracts and reverse transcription obtains is as amplification template, and above-mentioned cDNA sequence is through the bis- enzymes of restriction enzyme PmeI and NotI It is inserted into after cutting in the eukaryotic expression vector pcDNA3.1 through PmeI and NotI double digestions, connects the recombinant vector of acquisition PcDNA3.1-1 is used for subsequent experimental.

3) it transfects

Nerve cell is divided into 3 groups, respectively control group (HTR-8/SVneo), blank control group (transfection pcDNA3.1- NC), experimental group (transfection pcDNA3.1-1).The transfection of carrier is carried out using liposome 3000, specific transfection method is according to explanation The instruction of book carries out.The transfection concentrations of pcDNA3.1 empty carriers and pcDNA3.1-1 are 0.5 μ g/ml.

3, QPCR detects the transcriptional level of KIAA1949 genes

The extraction of 3.1 cell total rnas

The extraction of cell total rna is carried out using the cell RNA extracts kit of QIAGEN, specific steps, which refer to kit, to be said Bright book

3.2 reverse transcription steps are the same as embodiment 2.

3.3 QPCR amplification steps are the same as embodiment 2.

4, statistical method

Experiment is all completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical softwares come difference for statistical analysis, being overexpressed between KIAA1949 gene expression panels and control group It is different to be examined using t, it is believed that work as P<There is statistical significance when 0.05.

5, result

As a result such as Fig. 2 is shown, HTR-8/SVneo and transfection zero load pcDNA3.1-NC, experimental group can be shown compared to the control group Write the expression for increasing KIAA1949 genes.

The protein expression of KIAA1949 in embodiment 4 ELISA detection HTR-8/SVneo cells

Using double-antibody sandwich enzyme-labeled immunity (Enzyme-Linked Immunosorbent Assay, ELISA) analytic approach Measure KIAA1949 protein levels in HTR-8/SVneo cell conditioned mediums.It is thin to collect three groups of HTR-8/SVneo respectively by 48h after transfection The supernatant of born of the same parents quantitatively detects the concentration of KIAA1949 in tumour cell supernatant according to ELISA kit operating process.

1, configuration concentration is that the standard items of 70000pg/ml then carry out 2 times of doubling dilutions after 10 times of dilutions, share 7 it is dilute Degree of releasing.

2, it is loaded：Blank well, gauge orifice, sample to be tested hole are set respectively.Blank well adds 50 μ l of sample diluting liquid, remaining hole difference Add the standard items and each 50 μ l of sample to be tested of various concentration gradient.Mixing is gently shaken, ELISA Plate is plus lid, 37 DEG C of reaction 2h.

3, liquid is discarded, is dried.Add 200 μ l VEGF-C conjugates per hole.37 DEG C, after 120min, liquid in hole is discarded, It dries, PBS board-washings 3 times.

4, sequentially add 200 μ l of substrate solution per hole, 37 DEG C are protected from light colour developing 30min.

5, sequentially add 50 μ l of stop bath per hole, terminate reaction.

6, the optical density (OD values) in each hole is sequentially measured in 450nm wavelength with enzyme-linked instrument.All standard items and sample to be tested OD values be both needed to subtract the OD values in zero hole to obtain corrected value.

7, the actual concentrations of sample are calculated.

8, result is illustrated in fig. 3 shown below, and is overexpressed the KIAA1949 genes of HTR-8/SVneo cells, the albumen of KIAA1949 Content also accordingly increases, and difference has statistical significance.

5 mtt assay of embodiment detects HTR-8/SVneo cell-proliferation activities

1, after cell transfecting for 24 hours, 0.25% trypsin digestion, culture medium count after being resuspended, diluting cells suspension, by every Hole adjusts concentration control 10⁴/ml；

2, it presses cell inoculation per 150 μ l of hole to 96 orifice plates, sets 5 parallel holes again；

3, when transfecting 1~6 day, each hole culture medium is discarded, 100 μ l (0.5mg/ml) of MTT culture solutions are added and continue Cultivate 5h.MTT culture solutions are discarded, 150 μ l DMSO are added per hole and dissolve MTT reduzate first a ceremonial jade-ladle, used in libations, shakes 10min on shaking table, makes knot Brilliant object fully dissolves, and enzyme linked immunological instrument detects absorbance value at 490nm；

4, daily cell absorbance value, obtained numerical value graphical representation are counted.

5, result

The results are shown in Figure 4, and compared with the control, the cell Proliferation of transfection pcDNA3.1-1 groups dramatically increases, and prompts to change The expression of KIAA1949 can change the proliferative capacity of trophocyte.

7 Transwell cells in vitro Matrigels of embodiment

48h collects different groups of other HTR-8/SVneo cells after cell transfecting, is resuspended in culture solution, keeps cell dense eventually Degree is 10⁶/ ml draws 100 μ l cell suspensions and is added in the cells Transwell.It is observed using the cells Transwell method Influence of the KIAA1949 gene overexpressions to HTR-8/SVneo cellular invasiveness.

1, Matrigel is put into 4 DEG C of thawings, prepares ice chest (ice bath environment).After Matrigel is diluted with RPIM-1640 It uses, final concentration of 1mg/ml.

2, the cells Transwell for taking out precooling are put into 24 orifice plates, are uniformly added on each cells Transwell film 1,37 DEG C of 50 μ of Matrigel glue diluted, cell incubator, which places 3~4h, keeps gelling poly-.

3, the cell for collecting exponential phase, is resuspended in culture solution, final concentration of 10⁶/ ml gently adds 100 μ, 1 cells Suspension enters cell.

4, the culture medium that 600 μ 1 contain 20% serum, 37 DEG C, 5%CO are added in 24 orifice plates₂36h is incubated in incubator.

5, cotton swab lightly cleans Matrigel glue and cell in the holes Transwell, and the thin of cell bottom end is fixed with formaldehyde Born of the same parents are placed at room temperature for 25min, are dried after taking out cell.

6,0.4% violet staining 10min, three times, microscopically observation after drying is random to select for physiological saline quick wash It selects eight different visuals field to take a picture and count, statistical result is simultaneously analyzed.

7, result

The results are shown in Figure 5, and HTR-8/SVneo, pcDNA3.1-NC, pcDNA3.1-1 are cultivated in the cells transwell Afterwards, the cell number in room face increases under experimental group pcDNA3.1-1 cell membranes, illustrates that the expression for increasing KIAA1949 can increase Add the wetting capacity of trophocyte, KIAA1949 is prompted to can be used for the treatment of preeclampsia.

The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection domain of the claims in the present invention.

Sequence table

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Claims

Application of the detection reagent of 1.KIAA1949 in the product for preparing diagnosis preeclampsia.
2. application according to claim 1, which is characterized in that the product includes：Chip, preparation, kit or nucleic acid Film item.
3. a kind of product of diagnosis preeclampsia, which is characterized in that the product includes the detection reagent of KIAA1949.
4. product according to claim 3, which is characterized in that the reagent includes：

The probe of specific recognition KIAA1949 genes；Or

The primer of specific amplification KIAA1949 genes；Or

Specifically bind the antibody or ligand of KIAA1949 albumen.
5. product according to claim 4, which is characterized in that the primer sequence of the specific amplification KIAA1949 genes As shown in SEQ ID NO.3 and SEQ ID NO.4.
Applications of the 6.KIAA1949 in the pharmaceutical composition for preparing treatment preeclampsia.
7. application according to claim 6, which is characterized in that described pharmaceutical composition includes KIAA1949 functional expressions Accelerating agent.
8. application according to claim 7, which is characterized in that the accelerating agent is the carrier containing KIAA1949.
9. a kind of pharmaceutical composition, which is characterized in that including the accelerating agent described in claim 7 or 8, and/or can pharmaceutically connect The carrier received.
10.KIAA1949 the application in the pharmaceutical composition of screening treatment preeclampsia.