CN109988829A - It is a kind of detect neural tube malformation molecular marker and its application - Google Patents
It is a kind of detect neural tube malformation molecular marker and its application Download PDFInfo
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Abstract
The invention belongs to biological fields, and in particular to it is a kind of detect neural tube malformation molecular marker and its application.The molecular marker includes specificity marker gene and/or protein；Wherein, the specificity marker gene is at least one of Pax6, Nestin and Bmp4, and the protein is H2AK119, Mdm2 and/or the gene for encoding at least one of H2AK119 and Mdm2 protein.The molecular marker can prepare diagnosis or detect the product of neural tube malformation, can quickly and easily detect the situation of change of molecular marker, can be used for pre-natal diagnosis, treat in time, reduce the disease incidence of newborn's neural tube malformation.
Description
Technical field
The present invention relates to biological fields, and in particular to it is a kind of detect neural tube malformation molecular marker and its application.
Background technique
Neural tube malformation (neural tube defects, NTDs) is a kind of high-incidence birth defect disease, is birth
The highest type of morbidity and mortality in defect, and influence China or even world population quality is most common and most serious goes out
Raw defective disease.NTDs typically occurs in the early stage of pregnancy, be as nerve channel is not closed or dysraphism and caused by head
Portion to backbone position deformity.The deformity different degrees of to slight spina bifida from anencephaly: including anencephaly, exencephalia, microcephalus, major part
Deformity, hydrocephalus, meninges bulging, craniorachischisis and spina bifida.NTDs abnormal rate is up to 0.1-0.5% in newborn, disease
Cause and pathogenesis are still not very clear so far.The development of nerve channel is a complicated multi-step process, is gene-gene,
Gene-environment and gene-nutrition is coefficient as a result, by gene strict control, environmental factor also plays an important role.Nerve channel
It is a most complicated system of internal formation structure, development earliest, terminates the latest.In growth course, if by it is hereditary because
NTDs may can occur for the interference of element or environmental factor, the compensatory capacity more than embryo.This period develops as nerve channel
Critical period, increase to substance in biochemical metabolism in pregnant woman's body or be extremely sensitive less.According to zoopery, clinical sight
Examine with epidemiological study think inherent cause (multiple-factor inheritance) and environmental factor (folic acid vitamin shortage, high fever, alcohol and
Drug-induced teratism etc.) it can all interfere the closure of nerve channel.
In recent years for pathogenetic research of mankind NTDs mainly include gene expression regulation change (promoter and
The mutation of controlling element) and epigenetic regulation analysis, focus on mostly the gene pleiomorphism of gene and specific site with
The relationship of NTDs.However, the association analysis carried out to the mutational site of folic acid passageway related genes is not found and causes NTDs
Consistent risks and assumptions, the same site of same gene is related to NTDs in certain crowds, in other crowd but not
It is related.There are many participations of signal in neurula growth course, as netted connected signal path, if there is a certain link goes out
Problem this may result in organism metabolism imbalance, gene regulation disorder, and then show disease condition.In recent years, researcher one
Directly in the candidate gene for attempting to look for NTDs, data be mainly derived from biochemistry and auxology approach, animal model with
And three aspect of positional candidate gene, since mouse neural tube developmental mechanism and human nerve's pipe growth course are very close, thus
Become people and studies the most important one mode animal of NTDs mechanism.In recent years, 200 have been had more than in mouse model
It has been investigated that related to NTDs, NTDs related gene main code participates in cell signalling, adjusts transcription, inhibits a gene
Tumour growth and the relevant protein of chromatin remodeling.It is sent out currently, research discovery has several signal paths to be undoubtedly NTDs
The important regulation link of exhibition process, relates generally to: Wnt/ β-catenin signal path (participates in closing for regulation embryo's nerve channel
Close), PCP signal path (participate in nerve channel starting closure), (cross activation leads to NTDs to Hh/Shh signal path, leads to nerve channel
Dysraphism, formed low level spina bifida), BMP signal path (regulation mesoderm is formationed, nerve-inducing, nerve channel carry on the back abdomen shape
At), Notch signal path (pass through adjust nerve to occur participate in neural tube closure).These passageway related genes can be used as people
Class NTDs, the especially candidate gene of craniorachischisis research.For example, (Liu Yang .Wnt/PCP-JNK signal path is in NTDs by Liu Yang
Mediation Ningxia Medical University of generation and taurine prevention NTDs, 2014.) the study found that Wnt/PCP-JNK signal egg
Bai Tonglu key protein molecule DVL expression increases activation downstream RhoA and synchronizes and increase, and activates JNK, makes JNK that phosphorylation, ginseng occur
With the generation of neural tube malformation.(the few side of Shangguan, the .PCP such as Wang Li, Wu Lihua access occur the few side of Shangguan in neural tube malformation
In effect and progress [J] China be eugenic and Journal of Heredity, 2010 (12): 140-142.) etc. the study found that PCP signal
Gene is well-conserved between different genera in access, and its convergence extend this cell polarity change procedure in have it is important
Effect, and had confirmed in animal model, the mutation of several core genes causes neural tube closure defect in PCP approach, leads
Cause the generation of serious NTDs；Therefore, variation and effect of the research PCP passageway related genes in NTDs, for disclosing NTDs's
Pathogenesis understands the regulatory pathway that nerve channel is formed, is very significant.
Although the prior art has been found that the signal paths such as Wnt, PCP, Notch, Hh/SHH, BMP and mankind NTDs have
Relationship, but there is no to single specific protein modified functional experiment research, especially histone and related gene and NTDs
Whether related report, there is no the molecular marker of specific mankind NTDs, more with the clinical diagnosis for mankind NTDs
And detection.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that providing a kind of molecular marker of new detection neural tube malformation
And its application.
For this purpose, technical solution of the present invention is as follows:
It is a kind of for detecting the molecular marker of neural tube malformation, the molecular marker includes specificity marker gene
And/or protein；Wherein, the specificity marker gene is at least one of Pax6, Nestin and Bmp4, the protein
For H2AK119, Mdm2 and/or the gene of coding at least one of H2AK119 and Mdm2 protein.
The molecular marker that the present invention also provides above-mentioned for detecting neural tube malformation is in preparation diagnosis or detection nerve channel
Purposes in the product of deformity.
Further, the product includes the base based on the molecular marker design for detecting neural tube malformation
Because of chip, protein chip or detection reagent.
Further, the genetic chip be include for described in augmentation detection for detecting the molecule of neural tube malformation
The primer of marker or with the described probe hybridized for detecting the molecular marker of neural tube malformation.
Further, the protein chip includes and the molecular marker specificity for detecting neural tube malformation
In conjunction with antibody.
Further, the detection reagent includes using Bradford quantitative analysis albumen H2AK119, Mdm2 content
Reagent.
Further, the detection reagent further include using fluorescence quantitative PCR method analysis specificity marker gene Pax6,
The reagent of Nestin and Bmp4 content.
The present invention also provides a kind of kits for detecting neural tube malformation, including above-mentioned for detecting point of neural tube malformation
Genetic chip, protein chip or the detection reagent of sub- marker design.
Further, the kit, including using Bradford quantitative analysis albumen H2AK119, Mdm2 content
Reagent；It preferably, further include that specificity marker gene Pax6, Nestin and Bmp4 content is analyzed using fluorescence quantitative PCR method
Reagent；It preferably, further include extracting H2AK119, Mdm2, reagent.
Technical solution of the present invention has the advantages that
1, the present invention provides a kind of for detecting the molecular marker of neural tube malformation, which includes specificity
Marker gene and/or protein；Wherein, the specificity marker gene is at least one of Pax6, Nestin and Bmp4, institute
Stating protein is H2AK119, Mdm2 and/or the gene for encoding at least one of H2AK119 and Mdm2 protein.Mdm2 can lead to
Cross the Ubiquitin-Proteasome Pathway for promoting H2AK119 on transfer its protein stability, H2AK119, which has, inhibits downstream base
The function of cause, Research of Animal Model for Study discovery, methotrexate (MTX) can cause DNA double chain fracture that the phosphorylation that ATM is mediated occurs, thus
Mdm2 is recruited to the site H2AK119, single ubiquitination occurs, the up-regulation of Mdm2 level promotes the mono- ubiquitination of H2AK119 to inhibit
The phenomenon that specificity marker gene, and human sample research is it has also been found that H2AK119 and Mdm2 water in the human body of neural tube malformation
Flat up-regulation, specificity marker gene Pax6, Nestin and Bmp4 are all lowered；Therefore, pass through the table of detection H2AK119, Mdm2
Reach and the situation of change of specificity marker gene Pax6, Nestin and Bmp4, may be implemented neural tube malformation early diagnosis and
Intervene.
2, it the present invention also provides a kind of kit for detecting neural tube malformation, is made comprising above-mentioned molecular marker kit
Simple process, safe operation, practical, use easy to spread.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is mouse embryo stem cell RNAsequencing differential gene when MTX is 1uM concentration in experimental example 1 of the present invention
Expression；
Fig. 2 is mouse embryo stem cell internal specific marker gene mRNA level in-site after MTX induction in experimental example 1 of the present invention
Expression；
Fig. 3 is the protein expression situation of change result figure of the mouse embryo stem cell of MTX induction in experimental example 1 of the present invention；
Fig. 4 is the result figure of the CHIP qpcr of mouse embryo stem cell H2AK119 in experimental example 1 of the present invention；
Fig. 5 is the result figure of the CHIP qpcr of mouse embryo stem cell Mdm2 in experimental example 1 of the present invention；
Fig. 6 is the albumen situation for the mouse embryo stem cell that MTX is induced after knocking out Mdm2 in experimental example 1 of the present invention；
Fig. 7 is the specificity marker gene for the mouse embryo stem cell that MTX is induced after knocking out Mdm2 in experimental example 1 of the present invention
Situation of change；
Fig. 8 is the result figure of the CHIP qPCR of H2AK119 after knocking out Mdm2 in experimental example 1 of the present invention；
Fig. 9 is the albumen situation of the ATM phosphorylation of mouse embryo stem cell after MTX induction in experimental example 1 of the present invention；
Figure 10 is that the ATM of mouse embryo stem cell after MTX induction in experimental example 1 of the present invention is suppressed the specificity mark of front and back
Will genetic profile；
The situation of change that Figure 11 is H2AK119 and Mdm2 in mouse Nerve tissue after MTX is induced in experimental example 2 of the present invention；
Figure 12 be experimental example 2 of the present invention in neural tube malformation mouse Nerve tissue in specificity marker gene variation feelings
Condition；
Figure 13 is the situation of change of specificity marker gene in human brain tissue in the embodiment of the present invention 1；
The situation of change that Figure 14 is albumen H2AK119 in human brain tissue in the embodiment of the present invention 1；
The situation of change that Figure 15 is Mdm2 in human brain tissue in the embodiment of the present invention 1.
Specific embodiment
There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party
Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its
The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention
Within protection scope.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional reagent product.
Human normal and deformity group sample are taken from Lvliang City of Shanxi Province family members and know and agree to be transported by dry ice to Beijing
Shoudu Inst. of Pediatrics is stored in -80 refrigerators.
Laboratory apparatus: ThermoForma37 DEG C, carbon dioxide incubator (Thermofisher Scientific), TCS
SP8 Laser Scanning Confocal Microscope (Germany Leica), low-temperature and high-speed refrigerated centrifuge (Eppendorf), ultra low temperature freezer
(Thermo Fisher Scientific), horizontal cataphoresis apparatus (Bio-Rad), Vertial electrophorestic tank (Bio-Rad), complete wet transferring film instrument
(Bio-Rad), 7 Flex PCR instrument of QuantStudioTM (Termofisher Scientific), Nanodrop 1000 divides
Light photometer (Thermofisher Scientific), Nanostring Prep Station (Nanostring
Technology),Nanostringn Counter(Nanostring Tech.),Veriti 96 well thermal
cycler(ABI)。
Specificity marker gene (Pax6, Nestin, Bmp4) uses Nanostring method quantitative detection or use
MRNA extraction method, it is quantitative with RT-qPCR method.
RNA extraction method: cell or tissue total serum IgE is extracted using Trizol extraction method, presses Canada's abm Reverse Transcriptase kit
Total serum IgE reverse transcription at cDNA, is then detected the expression of specificity marker gene by operation with qPCR method, used in
Forward Primer and Reverse Primer are obtained using conventional primer-design software design；The reaction system of qPCR
Are as follows:
Overall reaction system is 20 μ L/ reaction
2 × qPCR of EvaGreen MasterMix, 10ul；
Forward Primer (10 μM), 0.6ul；
Reverse Primer (10 μM), 0.6ul；
CDNA template (100ng/ μ L), 2ul；
Deionized water, 6.8ul；
Reaction condition is as follows:
| Step | Temperature | Time | Circulation |
| Enzyme activation | 95℃ | 10min | 1cycle |
| Denaturation | 95℃ | 15s | 35cycle |
| Annealing/Extension | 60℃ | 60s | 35cycle |
| Melting curve | 95℃ | 15s | 1cycle |
| 60℃ | 60s | 1cycle | |
| 95℃ | 15s | 1cycle | |
| 60℃ | 15s | 1cycle |
After reaction, primer specificity is verified according to solubility curve analysis and agarose gel electrophoresis.Each pair of primer
Reaction includes one without Template-negative controls, and 3 repeating holes are arranged in each gene of every part of template, and reaction is completed
Afterwards, the Threshold cycle (CT value) for extrapolating each reaction amplification automatically according to system software, calculates target gene and pipe
The CT average value of family's gene, is compared with 2- △ △ Ct method, calculates the relative expression quantity of target gene, at least 3 secondary pollutants
It learns and repeats, experimental result is indicated with mean ± SD.
The expression of rH2AX and H2AK119 albumen, since rH2AX is the target egg as the fracture of histone DNA double chain
It is white, it is present in nucleus, and H2AK119 albumen is modified as the ubiquitin site of histone H2A, is extracted histone and is obtained
The expression of rH2AX and H2AK119 albumen, for this purpose, providing the extracting method of histone are as follows: use EPIGENTEK reagent
Box EpiQuik TM Total Histone Extraction Kit.Specific steps are as follows: with 1x107Cell concentration or 100mg tissue
For amount.10xpre-lysis buffer in kit is diluted to 1 × pre-lysis buffer with distilled water.Resuspension is beaten
Cell that is broken or being homogenized is placed in and 10 minutes and shaked gently on ice at every three minutes, then 3000rpm, 4 DEG C be centrifuged 5 minutes.
If cell concentration and less be placed in 1.5-2mLEP pipe of sample are centrifuged 1 minute in 10000rpm, 4 DEG C, supernatant is abandoned, then use
Tissue or cell fragment is resuspended in the lysis buffer of three times volume, is incubated for 30 minutes on ice, 12000rpm, 4 DEG C of 5 points of centrifugations
Supernatant is transferred in new pipe by clock.1:500 solution is configured with DTT solution and Balance buffer, it is vertical with 300uL volume
It is added to the supernatant that previous step is transferred in pipe, surveys protein concentration.Extracted histone can be reserved in -80 DEG C.
Mdm2 can also be used using Nanostring method relative quantification detection Mdm2 in the expression of mRNA level in-site
Then RIPA lysate holoprotein extraction method detects Mdm2 to protein quantification with Bradford method.The RIPA lysate is complete
Protein extraction method, specific steps are as follows: with 1 × 107For cell concentration or 100mg tissue mass, dissolution RIPA lysate is added, mixes
It is even, appropriate cell pyrolysis liquid is taken out, first 2 minutes addition PMSF is being used, is making the ultimate density 1mM of PMSF；It will with liquid-transfering gun
Cell culture medium absorption is thrown away, and 3ml pre-cooling PBS cleaning is added twice.To prevent the residuals pair in phenol red and culture medium
Immunoprecipitation, protein purification, the influence of the experiments such as enzyme activity assay.Be added 500ul lysate, with rifle piping and druming it is several under, make to crack
Liquid and cell come into full contact with, and are incubated for 5 minutes on ice.Sufficiently after cracking, 4 DEG C of centrifugations, 14000g 5 minutes, takes supernatant to be placed in new
Guan Zhong, be stored in -80 DEG C it is spare.
It is described with Bradford method to protein quantification, specific steps are as follows: by the BSA standard items albumen of known concentration according to
Concentration gradient 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0mg/mL dilution；It is added in each EP pipe
245 μ L Coomassie Brillant Blue solutions and 5 μ L standard items or sample；Each standard items and sample are surveyed in 595nm wavelength using microplate reader
The absorbance at place；Standard curve is obtained by Excel software according to the absorbance of standard items, finally obtains the albumen of each sample
Concentration.
Protein s DS-PAGE electrophoresis: glass plate wash with distilled water is put it into glass clamp after drying, bottom alignment
Clamping is vertically stuck on clip and prepares encapsulating；With glue: resolving gel concentration is determined according to destination protein molecular weight, according to reagent
The separation gel of recipe configuration 8% or 12%.Glue has to flow down along side glass plate when encapsulating, avoids generating bubble.It is added
After 7mL separation gel, it is slowly added to distillation water seal.It stands 30min to discard distilled water after gelling to be separated is solid, and will with filter paper
Residual moisture blots, and prepared 5% concentration glue is poured into glass plate, is gently inserted into point sample comb, gas can not be arranged at comb teeth bottom
Bubble.Then add some concentration glue again above comb, stand 30min；Loading: after gelling to be concentrated is solid, it is rinsed with water concentration
Glue residue, puts it into electrophoresis tank, and after enough electrophoresis liquids are added, the both sides that two hands pinch comb respectively gently will straight up
Its extract, according to protein concentration pipette samples, general holoprotein loading 20-30 μ g, should not inspiration bubble, sample injector syringe needle is inserted
Sample and 5 μ L albumen loading indicator electrophoresis are slowly added into well: electrophoresis selects constant pressure, is initially 80V, observes loading
When indicator crosses concentration glue, voltage is changed to 110V, when loading indicator will cross separation gel bottom, stops electrophoresis.Turn
Film: NC film being soaked in transferring film buffer and balances 5min, glue is stripped out, and removal concentration glue prepares two according to the size of glue
Sponge, six filter paper and a NC film, filter paper, film are identical with the size of glue three, and place it in transferring film buffer and balance
Glue containing destination protein is transferred on NC film, it is ensured that do not have bubble between film by 10min.According in electrophoresis tank from cathode
It is placed to anode according to sponge+filter paper+gel+NC film+filter paper+sponge " sandwich " sandwich method, then three is moved into transferring film dress
It sets.Install it is wet turn instrument, adjustment electric current is constant current 250mA, and the transferring film time adjusts according to destination protein size.Whole device is put
It sets in ice bath.It after the completion of transferring film, can be dyed with Ponceaux, observe protein band, judge transferring film efficiency, it then will with distilled water
Ponceaux is cleaned.Immune response closing: film is moved to containing in 5% skim milk, is closed 1 hour on room temperature shaker；It is incubated for primary antibody:
Primary antibody is diluted to debita spissitudo (according to antibody specification) with 5% skim milk, by film transfer to the incubation box containing primary antibody
In, 4 DEG C of overnight incubations；3) wash film: room temperature, 1 × TBST wash film, and horizontal shaker is washed 3 times, each 10min；It is incubated for secondary antibody: according to
The Species origin of primary antibody selects corresponding secondary antibody to be diluted with 5% skim milk (by antibody specification), is incubated at room temperature 45-
60min；5) wash film: room temperature, 1 × TBST wash film, and horizontal shaker is washed 3 times, each 10min；6) ECL, which shines, develops the color: by A liquid and B
Liquid mixes in equal volume according to 1:1, and film is immersed in luminescent solution about 2min, is exposed on fully automatic exposure instrument, uses gel
Imaging system analyzes the gray value of band after exposure.
In the present invention, Mdm2 egg is detected using Western blot method when sample is cell and mouse tissue
The variation of white expression quantity, and when sample behaviour tissue by Nanostring technology detect Mdm2 mRNA level come relatively
Quantitative expression quantity of the Mdm2 in people's nerve fiber.
Experimental example 1
Embryonic stem cell is that mouse embryo stem cell Sv/129 freezes from Beijing Xuan Wu hospital in Beijing capital youngster
Research institute cell bank, methotrexate (MTX) (MTX) are purchased from Pfizer (Perth) Pty Limited, and storage liquid concentration is 500mg/
20mL。
The mouse embryo stem cell is cultivated in DMEM high glucose medium in 37 DEG C, carbon dioxide incubator, culture
Based formulas (mouse embryo stem cell (mESC) Sv/129 maintain Dulbecco improvement Eagle culture medium (DMEM, Gibco,
USA in), contain 0.1mM beta -mercaptoethanol (Invitrogen, Carlsbad, USA) in culture medium, 0.1mMnon-
The U.S. essential amino acid (NEAA) (Invitrogen, Carlsbad)), 0.1mM glutamic acid (Invitrogen,
Carlsbad, USA), 15% fetal calf serum (Gibco, USA) and 1000U/ml mouse leukemia inhibiting factor (Millipore,
Billerica, USA), 0.2% gelatin (Invitrogen, Carlsbad, USA)).Cell is placed in humidified incubator, 37
DEG C, 5%CO2Under the conditions of cultivate, passage in every 2-3 days is primary.After culture, it is divided into experimental group and control group, in experimental group culture medium
It is added MTX (methotrexate (MTX)), so that MTX concentration is 1 μm in culture medium；Control group adds same amount of normal saline；It cultivates 12 small
When, the situation of change of the albumen and specificity marker gene of test experience group and control group embryonic stem cell.
(1) the RNA sequencing of the embryonic stem cell of MTX induction
Experimental group and control group embryonic stem cell total serum IgE are extracted using Trizol reagent (Invitrogen).Pass through
Bioanalyzer 2200 (Aligent) checks RNA mass and saves RNA at -80 DEG C.Detect RNA mass RNA
The RNA of RIN > 8.0 integrity number can be used for later period RNAsequencing.Same RNA integrity
The RNA of RIN > 8.0 number is purified suitable for miRNA.MiRNA is purified using miRNeasy Mini Kit (Qiagen), and
Purification result is verified by gel electrophoresis.
CDNA library building
It according to the manufacturer's instructions, the use of VAHTSTM Total RNA-seq (H/M/R) is each combined RNA sample
Product construction cDNA library.In general, the program comprises the steps of: rRNA exhausts and uses bivalent cation segment at 94 DEG C
150-200bp is turned to, continues 8 minutes.By the RNA segment reverse transcription of cutting at the first chain cDNA, the second chain cDNA synthesis, end
Repair segment, A tail and the connection of index of reference adapter.Target stripe is obtained by VAHTSTM DNA Clean Beads.Pass through
PCR purifying and enriched product are to generate final cDNA library and be quantified by Agilent2200.By the cDNA library of label with
Equal proportion merges, and in NovelBioCorp.Laboratory, the single swimming of the IlluminaHiSeqXten of Shanghai
150bp paired end sequencing is used in road.
Illustrate to prepare according to manufacturer using Ion Total RNA-Seq Kit v2.0 (Life Technologies)
Library complementary DNA (cDNA) for single-ended sequencing.Size selection is carried out to carry out to cDNA library by PAGE gel electrophoresis
MiRNA sequencing.Then cDNA library is handled according to commercially available scheme and is used for proton sequencing procedure.Sample is diluted and mixed, will be mixed
Object is closed to handle on 2 instrument of OneTouch (Life Technologies) and in 2 ES work station (Life of OneTouch
Technologies it is enriched on) to prepare template positive ions PITM ion SphereTM particle (Life according to Ion PITM
Technologies) 200 Kit v2.0 of template OT2 (Life Technologies).After enrichment, the template sun of mixing is loaded
Property ion PITM ion ball particle samples particle to 1 P1v2 proton chip on according to coming from
The 200 Kit v2.0 (Life of Ion PI Sequencing of NovelBioCorp.Laboratory, Shanghai
Technologies it) carries out being sequenced and being sequenced on proton sequenator.
Experimental group and right is indicated as shown in Figure 1 for the RNA sequencing of embryonic stem cell under different condition, Fig. 1 (a)
According to the fold differences between two groups of group, and it is 1642 that Fig. 1 (b), which shows that experimental group compares the discrepant gene of up-regulation with control group,
A, and what is lowered is to have 672, being close to 45 degree of dark parts, to be that difference is insufficient take 2 times after logarithm of gene.
(2) after MTX induction after mouse embryo stem cell specificity marker gene situation of change
Experimental result as shown in fig. 2, it can be seen that MTX induction after mouse embryo stem cell internal specific marker gene
Pax6, Nest in and Bmp4 are lowered relative to normal fetus stem cell, expression.
(3) the protein expression situation of change of the mouse embryo stem cell of MTX induction
With the variation of western method test experience group and control group embryonic stem cell protein content, H2AK119 egg is detected
It is white to detect Mdm2 albumen using GAPDH as internal reference using H2A as internal reference, fig. 3, it is shown that after MTX induction, H2AK119
It is raised with Mdm2 protein expression.
As shown in figure 4, being H2AK119CHIP qpcr, it can be seen that the combination of specificity marker gene and H2AK119 are bright
It is aobvious to reinforce.IgG in Fig. 4 is negative control.
As shown in figure 5, being Mdm2 CHIP qpcr, it can be seen that the combination of specificity marker gene and Mdm2 obviously add
By force.
(4) Mdm2, the protein expression situation of the embryonic stem cell of MTX induction are knocked out
The variation of H2AK119 after knocking out Mdm2 is detected with western blot, as shown in Figure 6, it can be seen that before MTX induction
Afterwards, using H2A as internal reference, albumen H2AK119 variation is unobvious, illustrates the up-regulation of albumen H2AK119 dependent on Mdm2.
The situation of change of specificity marker gene is detected with qPCR method, as shown in Figure 7, it can be seen that knock out Mdm2 base
Cause, MTX induction front and back, the variation of specificity marker gene are unobvious.
H2AK119CHIPqPCR, as shown in Figure 8, it can be seen that the mouse embryo stem cell of MTX induction knocks out mdm2 base
Cause, specificity marker gene variation are unobvious.
In above-mentioned attached drawing 6-8, si-Mdm2 indicates to knock out Mdm2 gene；MTX+ indicates experimental group, and CON- indicates control
Group.NC is negative control, will not be knocked in the reaction therefore can compare the knockout efficiency of Mdm2.
(5) detection of the ATM phosphorylation of the embryonic stem cell of MTX induction
As shown in figs. 9-10, double-strand break occurs for DNA in the state of low folic acid, and Mdm2 is enrolled into DNA damaged site
And have occurred ATM kinases induction phosphorylation, ATM kinases activity together with the Mdm2 for being enrolled into damaged site induction of
H2AK119 ubiquitination occurs.Fig. 9 indicates that DNA double chain fracture is to rely in ATM kinase mediated phosphorylation and not ATR kinases.
Figure 10 indicates that after ATM is suppressed, specificity marker gene is not lowered, and therefore, specificity marker gene is also by ATM phosphorus
The generation of acidification and it is repressed.
To sum up, the mouse embryo stem cell of MTX induction will lead to the expression up-regulation of H2AK119 and Mdm2, specificity marker
Gene Nestin, Pax6 and Bmp4 are lowered.MTX induction front and back, knocks out Mdm2 gene, and albumen H2AK119 expression changes not
Obviously；Specificity marker gene Nestin, Pax6 and Bmp4 variation are unobvious, change with albumen H2AK119 combination degree unknown
It is aobvious.It can be concluded that MTX inducing mouse embryonic stem cell, recruits the site Mdm2 to H2AK119, single ubiquitination occurs, to inhibit
Specificity marker gene expression.
Experimental example 2
1, experimental animal and material
SPF grades of adult C57BL/6J mouse, half male and half female, 7~8 week old, 17~19g of weight.Purchased from Beijing gold shepherd reality
Test animal-breeding responsible company.Methotrexate (MTX) (MTX) is purchased from Pfizer (Perth) Pty Limited, and storage liquid concentration is
500mg/20mL。
2, prepared by mouse model
The mouse adaptable fed bought is random to be grouped after 2 days.Female mice and the male mouse of same a batch after grouping press the ratio of 2:1
Example mates overnight, and morning discovery vaginal plug is decided to be pregnant 0.5 day.Experimental group gives various dose in 7.5 days in pregnancy
MTX intraperitoneal injection；Control group gives isometric physiological saline.When being pregnant 13.5 days, pregnant mouse is carried out to pluck eyeball blood sampling.Blood sampling
The disconnected neck of mouse afterwards is put to death, and abdomen is placed on operating table upwards, 75% alcohol disinfecting.It cuts open the belly along ventrimeson and takes out gravid uterus,
Uterus bunchiness is removed in the culture dish for being placed in and filling PBS, embryo is carefully taken out under stereomicroscope, and examines every nest embryo
Tire observes visible orthodontic condition under stereomicroscope, and takes pictures to embryo.Take part normal fetus and lopsided embryo solid
Due to volumetric concentration be 10% neutral formalin in, for doing histotomy.Lopsided embryo is taken, gene and albumen in Mice Body is surveyed and contains
The variation of amount.
3, experimental method and experimental result
(1) situation of change of Mice Body internal specific marker gene after MTX is induced
Experimental result is as shown in figure 12, it can be seen that neural tube malformation mouse specificity marker gene Pax6, Nestin and
Bmp4 is all lowered relative to normal mouse.
(2) MTX induction neural tube malformation mouse Nerve tissue in protein content variation
Experimental result is as shown in figure 11, it can be seen that Mdm2, H2AK119 and rH2AX are raised in Mice brain tissues, rH2AX
Up-regulation illustrate that DNA double chain fracture occurs in mouse Nerve tissue, con indicates that normal mouse, NTD indicate that MTX induction produces in figure
Raw neural tube malformation mouse；Brain indicates that brain tissue, Spine indicate myeloid tissue.
To sum up, it is known that, neural tube malformation mouse is mediated since ATM has occurred in the DNA double chain fracture of MTX induction
Phosphorylation recruit Mdm2 to DNA damaged site the mono- ubiquitination of H2AK119 have occurred to inhibit the expression of differentiation gene and cause
It is abnormal；I.e. Mdm2, H2AK119 level of neural tube malformation mouse raise, under specificity marker gene Pax6, Nestin and Bmp4
It adjusts.
Embodiment 1
Present embodiments provide a kind of method for detecting neural tube malformation, comprising the following steps:
1) H2AK119, Mdm2 of sample to be tested are extracted；
2) quantitative analysis H2AK119, Mdm2, the content of specificity marker gene Pax6, Nestin, Bmp4；3) with compare
Sample compares, if H2AK119, Mdm2 level raise, and specificity marker gene Pax6, Nestin, Bmp4 are all lowered, then refreshing
Occur through pipe deformity.
Wherein sample to be tested is the brain tissue of neural tube malformation patient, and check sample is the brain tissue of normal human.It is above-mentioned
Tissue is derived from Lvliang City of Shanxi Province family members and knows and agree to be transported by dry ice to Beijing Shoudu Inst. of Pediatrics to be stored in -80 ice
Case.
The extracting method of H2AK119 are as follows: use EPIGENTEK kit EpiQuik TM Total Histone
Extraction Kit.Using the horizontal situation of change of Nanostring method quantitative detection Mdm2.Specificity marker gene
(Cdx2, Gata4, Bmp4) is using mRNA extraction method, using Nanostring method quantitative detection.
It as a result as shown in figure 13, is the situation of change of specificity marker gene in human brain tissue, it can be seen that NTDs patient
Internal specificity marker gene is significantly lower than normal human.
It as shown in figure 14, is the situation of change of albumen H2AK119 in human brain tissue, it can be seen that NTDs patient's body
H2AK119 is apparently higher than normal human, and the NTDs1-NTDs8 in figure indicates the brain tissue sample 1-8 of neural tube malformation patient, figure
In Normal 1-Normal8 indicate normal human brain tissue sample 1-8.
It as shown in figure 15, is Mdm2 situation of change in human brain tissue, it can be seen that NTDs patient's body Mdm2 is apparently higher than
Normal human.
Embodiment 2
Present embodiments provide a kind of kit for detecting neural tube malformation
It is molecular marker, packet with specificity marker gene Pax6, Nestin and Bmp4, protein H2AK119 and Mdm2
Include the reagent for extracting H2AK119, Mdm2, the quantitative analysis reagent and quantitative fluorescent PCR reaction reagent of H2AK119, Mdm2.
Embodiment 3
Present embodiments provide a kind of application method of kit for detecting neural tube malformation
(1) H2AK119, Mdm2 of sample to be tested are extracted；
(2) detect H2AK119, Mdm2 in and specificity marker gene Pax6, Nestin and Bmp4 content；
(3) it is compared with check sample, if H2AK119, Mdm2 level raise, and specificity marker gene Pax6, Nestin
It is all lowered with Bmp4, then neural tube malformation occurs.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (9)
1. a kind of for detecting the molecular marker of neural tube malformation, which is characterized in that the molecular marker includes specificity
Marker gene and/or protein；Wherein, the specificity marker gene is at least one of Pax6, Nestin and Bmp4, institute
Stating protein is H2AK119, Mdm2 and/or the gene for encoding at least one of H2AK119 and Mdm2 protein.
2. the molecular marker described in claim 1 for detecting neural tube malformation is in preparation diagnosis or detection neural tube malformation
Product in purposes.
3. purposes according to claim 2, which is characterized in that the product includes based on described for detecting nerve channel
Genetic chip, protein chip or the detection reagent of the molecular marker design of deformity.
4. purposes according to claim 3, which is characterized in that the genetic chip is to include for described in augmentation detection
For detecting the primer of the molecular marker of neural tube malformation or with described for detecting the molecular marker of neural tube malformation
The probe of hybridization.
5. purposes according to claim 3, which is characterized in that the protein chip includes with described for detecting nerve
The antibody of the molecular marker specific binding of pipe deformity.
6. purposes according to claim 3, which is characterized in that the detection reagent includes using Bradford standard measure point
Analyse the reagent of albumen H2AK119, Mdm2 content.
7. the purposes according to claim 3 or 6, which is characterized in that the detection reagent further includes using quantitative fluorescent PCR
The reagent of method analysis specificity marker gene Pax6, Nestin and Bmp4 content.
8. a kind of kit for detecting neural tube malformation, which is characterized in that including being used to detect mind based on described in claim 1
Genetic chip, protein chip or the detection reagent of molecular marker design through pipe deformity.
9. kit according to claim 8, which is characterized in that including using Bradford quantitative analysis albumen
The reagent of H2AK119, Mdm2 content；Preferably, further include using fluorescence quantitative PCR method analysis specificity marker gene Pax6,
The reagent of Nestin and Bmp4 content；It preferably, further include the reagent for extracting H2AK119, Mdm2, RNA.
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| CN116004790A (en) * | 2022-09-30 | 2023-04-25 | 首都儿科研究所 | Use of a biomarker detection product in the preparation of a product for aiding in the diagnosis, diagnosis or prognosis of a neural tube abnormality |
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| CN116004790A (en) * | 2022-09-30 | 2023-04-25 | 首都儿科研究所 | Use of a biomarker detection product in the preparation of a product for aiding in the diagnosis, diagnosis or prognosis of a neural tube abnormality |
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