CN108707656A - The marker of preeclampsia at the genetic level - Google Patents
The marker of preeclampsia at the genetic level Download PDFInfo
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- CN108707656A CN108707656A CN201810575897.9A CN201810575897A CN108707656A CN 108707656 A CN108707656 A CN 108707656A CN 201810575897 A CN201810575897 A CN 201810575897A CN 108707656 A CN108707656 A CN 108707656A
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- Prior art keywords
- plek
- reagent
- preeclampsia
- gene
- albumen
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2857—Seizure disorders; Epilepsy
Abstract
The invention discloses the diagnosis and treatment target-PLEK genes of preeclampsia a kind of.The present invention may determine that whether subject suffers from preeclampsia or diagnosis subject whether there is the risk with preeclampsia by detecting the content of PLEK genes and its expression product in subject's placenta tissue.In addition, the present invention proves that PLEK genes are used as the drug target for the treatment of preeclampsia by studying the proliferative index of the preeclampsia cell of in vitro culture.
Description
Technical field
The present invention relates to biomedical sectors, more particularly it relates to which PLEK genes are preparing diagnosis and treatment preeclampsia
Product in application.
Background technology
Preeclampsia is one of the principal disease for causing female youngster's disease rate and the death rate to increase in obstetrics, there is apparent something lost
Tendency is passed, pathophysiological change is related to multisystem, the lesion of multiple organ, and the cause of disease and pathogenesis do not illustrate completely yet so far,
It is always the focus of obstetrics' research.And the variation of gene expression is the core mechanism of regulating cell vital movement process, it is abnormal
Metabolic process or pathological change, are either still controlled by Polygene system by single-gene, are inherently gene differential expressions
It is caused, thus can adjust blood pressure, body fluid volume, placenta growth, thrombosis, blood vessel double teeming, function of vascular endothelium, lipid metabolism,
The gene of immune and mitochondrial function etc. all may be tumor susceptibility gene (Wang Xipeng, Lin Qide the gestation hypertensions of preeclampsia
The research China journal of obstetrics and gynecology of syndrome tumor susceptibility gene, 2001, (36) 4：248-251), the abnormal expression of these genes is
Preeclampsia occurs and the basic reason of development, in recent years, with the rapid development of infrastest method, especially human gene
The center of gravity of comprehensive completion of group plan, human genome research is shifted from " structure " to " function ", finds differential expression base
Because having become one of research hotspot of molecular biology (Gabrielson E, Berg K, Anbazhagan R, et
al.Functional genomics,gene arrays,and the future of pathology.Mod Pathol,
2001,(14)12:1294-1299), the gene of different tissues or same tissue different developmental phases differential expression is detached, clones,
Molecule mechanism for understanding disease all has the research emphasis of highly important meaning, the preeclampsia cause of disease and pathogenesis
It is summarized from initial clinical data and cytohistological observation is gradually converted into the tumor susceptibility gene for exploring disease and studies its table
Up to situation.Although oneself is it is found that many preeclampsia related genes at present, as proangiotensin (AGT) gene (Ward K,
Hata A,Jeunemaitre X,et al.A molecular variant of angiotensiongen associated
with preeclampsia.Nature Genet,1993,4:59-61.), Prothrombin activity (Kupferminc MJ,
Eldor A,Steinman N,et al.Increased frequency of genetic thrombophilia in
women with complications of pregnancy.N Engl J Ned,1999,340:9-13), tumor necrosis factor
Sub- a (TNF a) gene (Rinehart BK, Terrone DA, Lagoo-Deenadayalan S, et al.Expression
of the placental cytokines tumor necrosis factor alpha,interleukin 1beta,and
interlukin 10is increased in preeclampsia.Am J Obstet Gynecol,1999,181(4):
915-920.]Deng, but because its generation, development are related to polygenic abnormal expression, therefore still need to do and be studied deeper into ground.
Placenta assumes responsibility for the function of the nearly all organ of immature fetus, and can secrete as gravidic special organ
The substances such as a variety of hormones, enzyme, cell factor, growth factor coordinate parent endocrine, immune and metabolic function, to remaining normal
Gestation plays an important role.Placental ischemia theory is one of preeclampsia etiology, and trophoblast is along spiral shell when normal pregnancy
It revolves parteriole and drives in the wrong direction and infiltrate, gradually replace blood vessel endothelium, so that lumen of vessels is expanded, blood flow increase, this process is referred to as blood vessel weight
Casting, depth is up to myometrial interior 1/3.And trophoblast of preeclampsia wetting capacity declines, and immerses only up to decidua section,
Make uterine spiral parteriole physiology double teeming obstacle, causes Placental ischemia anoxic；Blood vessel endothelium injury theory is generally acknowledged at present eclampsia
The key link of morbidity early period, the vaso-excitor material and cytotoxic factor such as Endothelin of placenta of preeclampsia secretion
(ET), AGT, thromboxane A2, the increases such as TNF a, and the reductions such as vasodilator such as prostacyclin, nitric oxide (NO),
Aggravate the damage of body vascular endothelial cell；And after clinical confirmation fetal placenta is given birth to, the state of an illness of preeclampsia can just be alleviated very
To disappearance, show that placenta is inseparable with the generation of preeclampsia and development.This technology of this experimental applications passes through placenta tissue
The difference of gene expression product (mRNA) understands the expression of gene, can directly be found from gene level and clone preeclampsia
Patient and normal pregnancies placenta tissue difference expression gene.The difference that own major gene can not only be detected, can be also found that and eclampsia
Early period relevant new gene, the research for the science of heredity and tumor susceptibility gene of preeclampsia provides new thinking.
Invention content
One of the objects of the present invention is to provide one kind realizing that preeclampsia examines by detecting PLEK gene expression differences
Disconnected method.
The second object of the present invention is to provide a kind of method for treating preeclampsia by inhibiting PLEK gene expressions.
The third object of the present invention is to provide a kind of method for screening preeclampsia medicine.
The fourth object of the present invention is to provide a kind of drug for treating preeclampsia.
To achieve the goals above, present invention employs following technical solutions：
The present invention provides application of the reagent of detection PLEK in the tool for preparing diagnosis preeclampsia.
Further, the reagent of the detection PLEK includes the reagent for detecting PLEK gene expression amounts.
Further, the reagent of the detection PLEK includes that can quantify the reagent of PLEK gene mRNAs, and/or can determine
Measure the reagent of PLEK albumen.
The reagent of the quantitative PLEK gene mRNAs of the present invention can play its work(based on the known method of nucleic acid molecules is used
Energy：Such as PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO
Method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the reagent.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR methods etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods detect.
The reagent that PLEK gene mRNAs can be quantified can be the specific primer of PLEK genes or transcript, also may be used
To be the specific recognition probe of PLEK genes or transcript, or include primer and probe simultaneously.
The specific primer of PLEK genes or transcript recited above includes the specific amplified used in real-time quantitative PCR
The primer of PLEK genes.In the specific embodiment of the present invention, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2.
Primer can be prepared by chemical synthesis, by using those skilled in the art will know that method refer to known letter
Breath is prepared to be suitably designed by chemical synthesis.
Probe can be prepared by chemical synthesis, by using those skilled in the art will know that method refer to known letter
Breath appropriately designs, and is prepared by chemical synthesis, or can contain desired nucleic acid sequence by being prepared from biomaterial
Gene, and using it is expected designed for amplification nucleic acid sequence primer amplification it prepare.
The reagent of the quantitative PLEK albumen of the present invention can play its function based on the known method of antibody is used：For example,
May include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The reagent of the quantitative PLEK albumen of the present invention includes the antibody or its segment for specifically binding PLEK albumen.It can make
With the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This
The antibody or its segment that the detection product of invention includes can be monoclonals or polyclonal.Antibody fragment refers to reservation antibody
Peptide to the active antibody of the combination of antigen a part of (Partial Fragment) or containing an antibody part.Antibody fragment may include F
(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the areas dimerization V it is (dual anti-
Body) or peptide containing CDR.The reagent of the quantitative PLEK albumen of the present invention may include encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of amino acid sequence, includes the carrier of the nucleic acid, and carries the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.Retain target all or in part for example, preparing
The polypeptide of protein integrates the mammalian cell expression vector for encoding their polynucleotides as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.The antibody of acquisition can finally be implemented by using PLEK albumen for being used as antigen or part thereof
Antigentic specificity purifies to obtain the monoclonal antibody for PLEK albumen.Polyclonal antibody can be prepared as follows：With with it is above
Identical antigen-immunized animal, collects blood sample from by immune animal, serum is isolated from blood, then uses upper
It states antigen and antigentic specificity purifying is implemented to serum.It can be by the antibody that is obtained with enzymatic treatment or by using the antibody of acquisition
Sequence information obtain antibody fragment.
The combination of marker and antibody or its segment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide：Clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2,
B- phycoerythrin labelling kits-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- marker protein marks
Remember kit (Funakoshi Corporation).For correct labeling, can be detected by label using suitable instrument
Antibody or its segment.
The acquisition of the sample for detecting the detection of PLEK gene expression amounts of the present invention is the ordinary skill in the art, preferably
Noninvasive may be selected or the method with minimally-invasive property obtains.
The sample can be (but are not limited to)：Tissue, peripheral blood, marrow, lymph node, abdominal cavity cleaning solution, urine, sweat
Liquid.In specific embodiments of the present invention, tissue of the sample from subject.
The present invention also provides a kind of tool for diagnosing preeclampsia, the tool can detect PLEK gene expressions
Amount.
Further, the tool includes that can quantify the reagent of PLEK gene mRNAs, and/or can quantify PLEK albumen
Reagent.
In general, the reagent is present in container appropriate.Diluent can be used to draw as described in deionized water by each
Object or probe are adjusted to the concentration of at least one requirement, are sub-packed in container.
Further, the reagent that can quantify PLEK gene mRNAs includes the specific amplified used in real-time quantitative PCR
The primer of PLEK genes, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, described for diagnose the tool of preeclampsia to include but not limited to chip, kit, test paper or high pass
Measure microarray dataset；High-flux sequence platform is a kind of special tool(s), with the development of high throughput sequencing technologies, to the base of a people
It very easily works because the structure of express spectra will become.By comparing the gene expression profile of Disease and normal population, hold
The exception for easily analyzing which gene is related to disease.Therefore, the exception and eclampsia of PLEK genes are known in high-flux sequence
The generation correlation of early period also belongs to the new application for having used the PLEK of the present invention, equally within protection scope of the present invention.
It may also include the reagent for extracting nucleic acid in the kit of the present invention, the reagent of PCR be used for, for dyeing or showing
The reagent etc. of color.For example, these reagents include but not limited to：Extract, amplification liquid, hybridization solution, developing solution, washing lotion etc..
In addition, may also include the specification etc. of the method for description detection PLEK gene expressions in the kit.
Kit of the present invention may include being suitable for a variety of different of practical (being such as directed to different detection methods)
Reagent, however it is not limited to cited reagent at present, as long as diagnosing preeclampsia based on the detection of PLEK genes or transcript
Reagent be all contained in the scope of the invention.
The present invention also provides a kind of methods of diagnosis preeclampsia, and described method includes following steps：
(1) sample of subject is obtained；
(2) PLEK gene expression doses in Samples subjects are detected；
(3) the PLEK gene expression doses measured are associated with the disease associated of subject.
(4) compared with normal control, PLEK gene expression doses statistically reduce, and show that subject is judged and suffer from
Preeclampsia or judge that risk of the subject with preeclampsia is high.
The present invention also provides a kind of therapy of preeclampsia, the method includes promote PLEK gene expressions or
Promote the activity of PLEK gene expression products.
The present invention also provides a kind of screening techniques of drug candidate that treating preeclampsia, can be by thin to model
Some period after born of the same parents' addition testing drug measures PLEK genes or the expression of PLEK albumen changes to measure drug candidate
The effect of kind prognosis.More specifically, when the expression of PLEK genes or PLEK albumen is adding or applying testing drug
When increasing afterwards or when restoring normal level, the drug may be selected as the medicine for improving preeclampsia.
The present invention also provides a kind of drug for treating preeclampsia, the drug includes the reagent for promoting PLEK.
The reagent of the promotion PLEK of the present invention is unrestricted, as long as the reagent can promote PLEK or be related to the upstreams PLEK
Or expression or the activity of the substance of downstream pathway, and for treating preeclampsia effective drug.
The present invention also provides the application of PLEK genes or its expression product in the drug for preparing treatment preeclampsia.
Further, the drug include include uncorrected gene expression sequence, the expression vector containing gene order, promote property miRNA,
Transcription regulatory factor or targeting micromolecular compound etc..
The drug of the present invention can be used by any of mode compounding pharmaceutical composition in this field.This combination
Object includes active constituent, in addition one or more pharmaceutically acceptable carriers, diluent, filler, bonding agent and other figurations
Agent, this depends on administering mode and designed dosage form.This field known treatment of branch art personnel is inert inorganic or has
The carrier of machine includes but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, wax, fat, polyhydroxy chemical combination
Object such as polyethylene glycol, water, sucrose, ethyl alcohol, glycerine, such, various preservatives, lubricant, dispersant, corrigent.It protects
It is wet cut to pieces, antioxidant, sweetener, colorant, stabilizer, salt, buffer solution is such can also be added thereto, these substances according to
It needs to be used to help the stability of formula or helps to improve activity or its biological effectiveness or generated in the case of oral
Acceptable mouthfeel or smell, the preparation that can be used in such a composition can be the form of its original chemical itself, or
The form of its pharmaceutically acceptable salt is optionally used, drug of the invention can be administered alone, or with various combination medicine-feedings,
And combining form is administered together with other healing potions.People in the art may be selected in the composition so prepared as needed
Any mode appropriate is administered drug known to member.It is by the present invention of safe and effective amount when using pharmaceutical composition
Medicament administration in people, specific dosage is also contemplated that the factors such as administration route, patient health situation, these are all skilled practitioners skills
Within energy range.
The drug of the present invention can be prepared into various dosage forms as needed.Including but not limited to, percutaneous, mucous membrane, nose, buccal,
Tablet, solution, granule, patch, paste, capsule, aerosol or suppository sublingual or orally use.
The administration method of the drug of the present invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to intravenously, in peritonaeum, intraocular, intra-arterial, intrapulmonary takes orally, in vesicle, intramuscular, and tracheal strips, subcutaneously
, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, it can systematically be administered.It is to be locally administered in some cases.
The dosage of the drug of the present invention is unrestricted, as long as obtaining desired therapeutic effect or preventive effect.This
The medicine of invention or the dosage of prophylactic agent can use therapeutic effect for example to disease or preventive effect as referring to
It marks to determine.
" PLEK genes " (Chromosome 2, NC_000002.12 (68365190..68397453)) sequence of the present invention
It can be to be inquired in ncbi database.
In the context of the present invention, " preeclampsia diagnosis " include judge subject whether suffered from preeclampsia,
Judge that subject whether there is the risk with preeclampsia or judge that placenta in preeclampsia recurs.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with relevant disease or illness
Disease or morbid state, and include：
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) inhibit disease or morbid state, that is, prevent its generation；Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, development is made to stop) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To developing into illness not yet but developing into illness danger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention：
Present invention firstly discovers that and confirming the Close relation that PLEK gene expressions occur with preeclampsia, the sample of verification
Amount is more, as a result accurately.The it is proposed of the correlation provides new approach for the Clinics and Practices of preeclampsia.
Description of the drawings
Fig. 1 shows the difference in placenta from patients with pre-eclampsia and normal placenta tissue using QPCR detection PLEK genes
The statistical chart of different expression；
Fig. 2 is shown using Western blot experiment detection PLEK albumen in placenta from patients with pre-eclampsia and normal tire
The statistical chart of differential expression in disk tissue；
Fig. 3 shows the statistical chart using Western blot experiment detection PLEK gene overexpression situations；
Fig. 4 shows the result figure using CCK-8 methods detection PLEK gene expression cell proliferation capacities.
Specific embodiment
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.
Unconventionality expression gene in the placenta tissue of the screening placenta in preeclampsia of embodiment 1
1, tissue collecting
The placenta tissue totally 5 for collecting the patient of the preeclampsia of obstetrics and gynecology hospital childbirth, selects same time blood pressure normal
The placenta tissue 5 of the late pregnancy pregnant woman for terminal pregnancy of performing the operation because of social factor or anomaly of pelvis is Normal group, two
Group excludes multifetation, communicable disease, chemicals dependence, Maternal Smoking Smoking, fetus congenital malformation and other gestation and merges
Disease and complication, and pregnant week is strictly verified according to pregnant early stage B ultrasound data, pregnant number of days is calculated, all research objects being included in are equal
Informed consent form is signed before making a collection of specimens.The diagnosis of preeclampsia and indexing standard are with reference to the 7th edition national higher medical education
Teaching material gynecotokology (Le Jie chief editors).
Germ-free condition is kept after placenta is given birth in cesarean section, avoids clip placenta tissue at calcification in maternal surface immediately
Size about 0.5cm x 0.5cm x 0.5cm, are cleaned with sterile saline and remove blood as possible repeatedly, until cleaning solution is in clear
Physiological saline is drained after bright, and tissue is shredded as early as possible, is placed in and is put into liquid nitrogen container in sterile cryopreservation tube and freezes.
2, tissue RNA extractions
Using RNA extracts kits (the Trans Zol purchased from Beijing Quanshijin Biotechnology Co., LtdTM Up Plus
RNA Kit) extraction tissue samples RNA.It is as follows：
(1) it after the sample weighing of superfreeze, will be transferred quickly in the mortar with Liquid nitrogen precooler, fully ground with pestle
The sample being ground into powder is transferred in centrifuge tube up to being ground into powder, 1ml is added per 50-100mg samples by mill
Trans Zol TMUp carries out homogenized with Syrup-homogenizing instrument, or with rifle pressure-vaccum mixing repeatedly, is stored at room temperature 5 minutes.
(2) 1ml Trans Zol are often usedTMUp adds 0.2ml chloroforms, acutely oscillation 30 seconds, is incubated at room temperature 3 minutes.
(3) 4 DEG C of 10000 × g are centrifuged 15 minutes.Sample is divided into three layers at this time, colourless aqueous phase (upper layer), and middle layer is pink
Color organic phase (lower layer).RNA draws 500 μ l liquid of colourless aqueous phase layer in colourless aqueous phase.
(4) water phase colourless 500 μ l of transfer absorption is added the absolute ethyl alcohol of 500 μ l, gently runs in new centrifuge tube
Mixing.
(5) solution for obtaining 700 μ l and precipitation are added in centrifugal column together, and 12000 × g room temperatures centrifuge 30 seconds, discard
Efflux.
(6) 500 μ l CB9 are added, room temperature 12000 × g room temperatures centrifuge 30 seconds, discard efflux.
(7) it is primary to repeat step (6).
(8) 500 μ l WB9 (please first checked whether before use and absolute ethyl alcohol is added), the centrifugation of room temperature 12000 × g room temperatures is added
30 seconds, discard efflux
(9) it is primary to repeat step (8).
(10) 12000 × g of room temperature room temperatures centrifuge 2 minutes, thoroughly remove remaining ethyl alcohol, are being stored at room temperature several minutes thoroughly
Dry centrifugal column.
(11) centrifugal column is put into RNase-free Tube (kit has been matched), adds 30 μ l RNase-free Water
In the center of centrifugal column, it is stored at room temperature 1 minute.
(12) 12000 × g of room temperature room temperatures centrifuge 1 minute, eluted rna.
(13) RNA -80 DEG C of refrigerators are placed in preserve.
3, the measurement of the RNA concentration and purity extracted
The concentration and purity of RNA are detected using the ND-1000 instruments of Nano Drop companies of the U.S..
Using UV detector Nano Drop 1000, the Nano Drop icons on computer screen are double-clicked, into system
System menu；According to system prompt after selecting nucleic acid to measure option, 2 μ l distilled waters are added in well；It clicks and determines, with initial
Change system；2 μ l distilled waters are added in well, select RNA, click Blank with measuring system background.It is cleaned with rear lens wiping paper
RNA sample to be measured is clicked and entered after distilled water, clicks Measure.Reading numerical values simultaneously record, and RNA concentration (ng/ μ l) must need at this time
It is noted that A values 260/280, if numerical value between 1.8-2.0, illustrates that RNA sample quality is preferable.By the RNA of each sample
It is frozen after concentration markers are complete in -70 DEG C of refrigerators.
4, the RNA integrity mensuration's extracted
Ago-Gel detects RNA sample operations steps：
1) electrophoresis tank, the cleaning of glue apparatus：Detergent wash clean (general soaked overnight), water are used after rinsing, 3%H2O2
Electrophoresis tank is filled, 10min is placed at room temperature for, is rinsed, is dried spare with 0.1% (V/V) DEPC water.
2) glue：0.5g agarose powders are weighed, addition is placed in the conical flask of DEPC water of 45ml, and heating makes agarose
It is completely dissolved.10 × TAE electrophoretic buffers of 5ml and the Ethidum Eremide of final concentration of 0.5 μ g/ml are added after slightly cooling down.Then
The Casting of gels in glue groove, is plugged comb, is horizontally arranged rear use to be solidified.
3) it is loaded：After sample is mixed with 6 × electrophoretic buffer, it is loaded onto in gel loading wells.
4) electrophoresis：Open electrophoresis apparatus, voltage stabilizing 80V electrophoresis.
5) it (about 30 minutes) after electrophoresis, is observed under ultraviolet lamp, gel imaging system is used in combination to take pictures preservation.
Integrity mensuration' is detected by agarose gel electrophoresis, if 28s rRNA, 18s r RNA, 5s can be clearly apparent
Three bands of rRNA, and the brightness of 28s rRNA should be twice of 18s rRNA.Illustrate that the total serum IgE integrality of extraction is preferable, RNA
Satisfactory quality.
4, high-throughput transcript profile sequencing
(1) RNA-seq reads position
Low-quality read is removed to obtain cleaning read first, then utilize TopHat v1.3.1 will clean segment and
UCSC H.sapiens reference genes groups (hg19) are matched, the index of H.sapiens UCSC hg19 editions built in advance
It is downloaded from TopHat homepages, and as with reference to genome, when being matched with genome using TopHat, allows each read (acquiescence
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signals
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat methods.
(2) transcript abundance is assessed
The read file matched is by Cufflinks v1.0.3 processing, and Cufflinks v1.0.3 are by RNA-seq pieces
Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to being matched in every 1,000,000 sequencing segment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated values is calculated by Bayesian inference method.
The GTF comment files for the reference that Cufflinks is used download (Homo_ from Ensembl databases
sapiens.GRCh37.63.gtf)。
(3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF files of download and by the matched original documents of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF files using original matching files, detects difference table
It reaches.The only q values < 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.
4, result
RNA-seq is the results show that compared with normal pregnancies, and the gene of high expression is in placenta from patients with pre-eclampsia
458, the gene of low expression is 579, and difference all has statistical significance (P<0.05).
2 large sample of embodiment verifies expression of the difference expression gene on transcriptional level
1, tissue collecting
According to the placenta tissue 35 of the standard collection placenta in preeclampsia of embodiment 1, the placenta tissue 40 of normal pregnancies
Example.
2, tissue RNA extractions and identification
Step is the same as embodiment 1.
3, the design and preparation of primer
The primer sequence that qRT-PCR detects the primer sequence of PLEK gene expressions and qRT-PCR expands internal reference GAPDH is equal
It is designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and through the retrieval of UCSC databases, by its sequence information
Typing NCBI software Design primers, and the blast in gene pool confirm correctly.
PLEK gene primers：
Sense primer：5'-ATTGACTTAGGTGCCTTAT-3'(SEQ ID NO.1)；
Downstream primer 5 '-AACAGACTGGTTGGATAC-3 ' (SEQ ID NO.2),
GAPDH gene primers：
Sense primer：5'-GGGAGCCAAAAGGGTCA-3'(SEQ ID NO.3)；
Downstream primer：5'-GAGTCCTTCCACGATACCAA-3'(SEQ ID NO.4).
6, real time fluorescent quantitative cDNA reverse transcriptions detecting step
Reverse transcription is carried out to l μ g total serum IgEs with RT Buffer and synthesizes cDNA.Using 25 μ l reaction systems, each sample
It takes 1 μ g total serum IgEs as template ribonucleic acid, following components is separately added into PCR pipe：DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
7、PCR
(1) Bio-RAD real-time fluorescence quantitative PCR instruments are applied, reaction system is prepared by table 1.
1 PCR reaction systems of table
(2) it is carried out using following qPCR response parameters：95 DEG C of pre-degeneration 10min, then, 95 DEG C of denaturation 15s, 52.6 DEG C are moved back
Fire extends 1min, totally 45 cycle；Then, the acquisition of fluorescence signal and the making of product solubility curve, 3, each sample are carried out
It repeats, takes its average value.Using 2-△△CtRelative quantification method analyzes the expression of PLEK, and Ct is that thermal cycler detects reaction
The intensity value of fluorescence signal in system.Computational methods are：Δ Δ Ct=(Ct target gene-Ct reference genes) placenta in preeclampsia
Placenta tissue experimental group-(Ct target gene-Ct reference genes) normal pregnancies placenta tissue control group, 2-△△CtWhat is indicated is real
Variation multiple of the expression relative to control group of group target gene is tested, analysis of experimental data is completed by Bio-RAD analysis softwares.
5, statistical analysis carries out data analysis using statistics software SPSS19.0, judges eclampsia using paired-samples T-test
Early period placenta tissue with the expression of PLEK in normal pregnancies placenta tissue sample with the presence or absence of the difference on statistical significance.Statistics
It is all two-sided test to examine, and P < 0.05 are that difference is statistically significant.
6, result
Compared with normal pregnancies placenta tissue, PLEK genes in the placenta tissue of 35 patients in 35 Cases with Preeclampsia patients
Expression significantly reduces, and difference has statistical significance.Statistical result is as shown in Figure 1, compared with normal pregnancies placenta tissue, eclampsia
PLEK genes significantly reduce in early period Placenta, and difference has statistical significance (P < 0.05).
2 large sample of embodiment verifies expression of the difference expression gene on protein level
1, the total protein organized in embodiment 2 is extracted
The operation of protein extraction is carried out according to the specification of EpiQuik tissue/cell total protein extraction kits.
2, electrophoresis
Using β-actin as internal reference.50 μ g total proteins are after SDS-PAGE points, electrotransfer to pvdf membrane, with containing 5% defatted milk
1 × TBST room temperature jogs of powder close 1h；It is separately added into PLEK monoclonal antibodies and β-actin monoclonal antibodies, 4 DEG C overnight；1 × TBST washes film 4
It is secondary, secondary antibody is added, is incubated at room temperature 1h；After 1 × TBST washes film 4 times, it is placed in Super Signal chemical illuminating reagents and reacts
2min makes X-ray expose in darkroom, conventional method developing fixing.
3, statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by PLEK albumen
The gray value of band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical softwares are next for statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
4, result
Compared with normal pregnancies placenta tissue, PLEK albumen in the placenta tissue of 35 patients in 35 Cases with Preeclampsia patients
Level significantly reduces, and difference has statistical significance.Statistical result is as shown in Fig. 2, compared with normal pregnancies placenta tissue, eclampsia
PLEK protein levels significantly reduce in early period Placenta, and difference has statistical significance (P < 0.05).
Embodiment 3 promotes PLEK gene expressions
1, the structure of PLEK expression vectors
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.5) of PLEK genes.From at Human fetal spleen
CDNA library (clontech companies, article No.：638831) coded sequence of the PLEK genes of amplification overall length, above-mentioned cDNA sequence in
It is inserted into the eukaryon through restriction enzyme BamHI and XhoI double digestion after restriction enzyme BamHI and XhoI double digestion
In fibrocyte expression vector pcDNA3.1, the recombinant vector pcDNA3.1-PLEK for connecting acquisition is used for subsequent experimental.
2, the culture of placental trophoblasts
JEG-3 cells are placed in 37 DEG C with containing the dual anti-and pools 10% fetal calf serum DMEM high culture medium, 5%CO2Training
Culture in case is supported, every changing 1 culture solution for 24 hours, 48h is passed on 1 time.The cell of logarithmic growth phase carries out subsequent experimental.
3, cell transfecting
Use liposome Lipofectamine2000 for transfection reagent.2 groups of experiment point：Negative control group (transfection
pcDNA3.1)；Experimental group (transfection pcDNA3.1-PLEK).The JEG-3 cells of logarithmic growth phase are inoculated in 6 hole cell culture
Plate.Tissue culture plate coverage rate is about 70%-80% after for 24 hours.Rotaring transfecting mode with reference to Lipofectamine2000 specifications into
Row.
4, the promotion efficiency of Western blot experiments detection pcDNA3.1-PLEK
1) prepared by protein sample
Culture solution is outwelled, and bottle tipped upside down on blotting paper makes blotting paper blot culture solution (or uprightly to place bottle for a moment
Youngster makes residual culture flow to bottom of bottle and then be siphoned away again with pipettor).Every bottle of cell adds the PBS of 4 DEG C of precoolings of 3m1
(0.01M pH7.2-7.3).It keeps flat and gently shakes 1min washing cells, then discard washing lotion.It repeats above operation twice, washes altogether
Cell is three times to wash away culture solution.Culture bottle is placed on ice after PBS is abandoned only.Add 10 μ 1PMSF by lml lysates
(100mM), shakes up and is placed on ice.Every bottle of cell adds 400 lysates of the μ 1 containing PMSF, in cracking 30min on ice, to make cell fill
Division solution culture bottle wants frequent waggle.After having cracked, with clean scraper then cell scraper is used in the side of culture bottle
Rifle moves to cell fragment and lysate in l.5m1 centrifuge tube in -20 DEG C of preservations.
2) electrophoresis
It is carried out according to the step of embodiment 2.
5, result
As shown in figure 3, compared with empty carrier group (transfection pcDNA3.1), experimental group (transfection pcDNA3.1-PLEK) cell
Middle PLEK expressing quantities significantly increase, and difference has statistical significance (P<0.05).
The influence that the expression of 4 PLEK genes of embodiment is proliferated placental trophoblasts
The detection of cell Proliferation uses CCK-8 methods
(1) it is transfected according to the method for embodiment 3；
(2) the JEG-3 cells after transfection for 24 hours, culture medium is abandoned in suction, with 0.25% trypsin digestion and cell, with crystal violet
Dye liquor counts cell, and cell is resuspended with the low blood serum mediums of DMEM/F12 containing 0.5%FBS and adjusts cell concentration as 2x
104/ml；
(3) cell suspension 100 μ l, i.e. 2x 10 is added under aseptic condition per hole into 96 new well culture plates3A cell,
37 DEG C, 5%CO2Stand 20h；
(4) 10 μ l CCK solution are added into each hole respectively, cultivate 2h；
(5) 450nm wavelength, ultraviolet specrophotometer measure each experimental group light absorption value.
As a result：
The results are shown in Figure 4, compared with empty carrier group (transfection pcDNA3.1), experimental group (transfection pcDNA3.1-PLEK)
Cell Proliferation is obviously suppressed, and difference has statistical significance (P<0.05).
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
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>The marker of preeclampsia at the genetic level
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
attgacttag gtgccttat 19
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aacagactgg ttggatac 18
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gggagccaaa agggtca 17
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gagtccttcc acgataccaa 20
<210> 5
<211> 1053
<212> DNA
<213>People source (Homo sapiens)
<400> 5
atggaaccaa agcggatcag agagggctac cttgtgaaga aggggagcgt gttcaatacg 60
tggaaaccca tgtgggttgt attgttagaa gatggaattg aattctataa gaagaaaagt 120
gacaacagcc ccaaaggaat gatcccgctg aaagggagca ctctgactag cccttgtcaa 180
gactttggca aaaggatgtt tgtgtttaag atcactacga ccaaacagca ggaccacttc 240
ttccaggcag ccttcctgga ggagagagat gcctgggttc gggatatcaa gaaggccatt 300
aaatgcattg aaggaggcca gaaatttgcc aggaaatcta ccaggaggtc cattcgactg 360
ccagaaacca ttgacttagg tgccttatat ttgtccatga aagacactga aaaaggaata 420
aaagaactga atctagagaa ggacaagaag atttttaatc actgcttcac aggtaactgc 480
gtcattgatt ggctggtatc caaccagtct gttaggaatc gccaggaagg cctcatgatt 540
gcttcatcgc tgctcaatga ggggtatctg cagcctgctg gagacatgtc caagagtgca 600
gtggatggaa ctgctgaaaa ccctttcctg gacaaccctg atgccttcta ctactttcca 660
gacagtgggt tcttctgtga agagaattcc agtgatgatg atgtgattct gaaagaagaa 720
ttcagagggg tcattatcaa gcagggatgt ttactgaagc aggggcatag aaggaaaaac 780
tggaaagtga ggaagttcat cttgagagaa gaccctgcct acctgcacta ctatgaccct 840
gctggggcag aagatcccct gggagcaatt cacttgagag gctgtgtggt gacttcagtg 900
gagagcaact caaatggcag gaagagtgag gaagagaacc tttttgagat catcacagca 960
gatgaagtgc actatttctt gcaagcagcc acccccaagg agcgcacaga gtggatcaga 1020
gccatccaga tggcctcccg aactgggaag taa 1053
Claims (10)
1. detecting application of the reagent of PLEK in the diagnostic tool for preparing preeclampsia.
2. application according to claim 1, which is characterized in that the reagent of the detection PLEK includes detection PLEK gene tables
Up to the reagent of amount.
3. application according to claim 1 or 2, which is characterized in that the reagent of the detection PLEK includes that can quantify
The reagent of PLEK gene mRNAs, and/or the reagent of PLEK albumen can be quantified.
4. application according to claim 3, which is characterized in that the reagent that can quantify PLEK gene mRNAs includes real
When quantitative PCR in the primer of specific amplified PLEK genes that uses；The reagent that PLEK albumen can be quantified includes specificity
In conjunction with the antibody of PLEK albumen.
5. a kind of tool for preeclampsia diagnosis, which is characterized in that the tool includes that can detect PLEK gene expressions
The tool of amount；Preferably, the tool includes that can quantify the reagent of PLEK gene mRNAs, and/or can quantify PLEK albumen
Reagent.
6. tool according to claim 5, which is characterized in that the reagent that can quantify PLEK gene mRNAs includes real
When quantitative PCR in the primer of specific amplified PLEK genes that uses, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2；The reagent that PLEK albumen can be quantified includes the antibody for specifically binding PLEK albumen.
7. tool according to claim 5 or 6, which is characterized in that the tool includes kit, chip, test paper, high pass
Measure microarray dataset.
8. a kind of drug for treating preeclampsia, which is characterized in that the drug includes the reagent for promoting PLEK.
9. drug according to claim 8, which is characterized in that the reagent includes that can promote PLEK or be related on PLEK
The expression of the substance of trip or downstream pathway or active reagent.
The application of 10.PLEK genes or its expression product in the drug for preparing treatment preeclampsia.
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