CN104849471A - Down's syndrome map model construction method - Google Patents
Down's syndrome map model construction method Download PDFInfo
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Abstract
The invention discloses a Down's syndrome map model construction method. The method comprises taking pregnant woman peripheral blood separated from the human body, carrying out centrifugation, sucking blood plasma, storing the blood plasma at a temperature of -60 to -100 DEG C, extracting blood plasma proteins to obtain a solution to be detected, detecting protein content of the solution to be detected, carrying out immunoblotting detection on the solution to be detected, carrying out statistical analysis on the immunoblotting detection result and building a Down's syndrome map model. The method utilizes differential expression of proteins in mother blood plasma to provide intermediate result information for antenatal screening and diagnosis of Down's syndrome and has high specificity and sensitivity. The differentially expressed proteins are conducive to expounding of pathogenesis and provide basis for early stage diagnosis of Down's syndrome.
Description
Technical field
The present invention relates to the Down syndrome information of a kind of acquisition as intermediate result, particularly relate to a kind of construction method of Down syndrome spectrum model.
Background technology
Down syndrome (Down Syndrome, DS) is the hereditary disease caused by autosomal abnormalities.This patient often has 3 No. 21 chromosomes, and normally only has two.This disease has the higher incidence of disease, and patient's principal character is serious congenital mental retardation dysnoesia, have special face, anthropometic is slow, and unusual facies, and often with 80 various diseases such as congenital heart disease, hypoimmunity.Relative to normal person, the leukemic probability of DS patients also increases greatly, because patient's great majority can not take care of oneself, and substantially can not work, and causes heavy financial burden therefore to family and society.According to statistics, every routine children with Down syndrome can bring the financial burden of about 39.00 ten thousand yuans to family, and the financial burden caused to society is then up to about 45.00 ten thousand yuans.Nearly 600,000 Down syndrome in patients of current China.Abroad, the morbidity quantity of Down syndrome accounts for 91.6% of all chromosome diseases.
Down syndrome is the aneuploid hereditary disease of numerical abnormalities of chromosomes the most common, and the birth intervention is the effective measures of preventing this disease at present.At present, the major way of intervening the birth of DS patient carries out Prenatal Screening and pre-natal diagnosis.But traditional Prenatal Screening loss and false positive higher.Therefore, in order to improve the recall rate of DS fetus, reduce the risk that intrusive mood pre-natal diagnosis brings, researchers are devoted to explore by female blood fetal nucleated cell, female approach such as blood fetus dissociative DNA and female blood fetus dissociative RNA, sets up new non-invasive methods for prenatal diagnosis.
Genome in fetal nucleated cell includes all hereditary information of fetus, the therefore initial research mainly concentrating on complete fetal nucleated cell in pregnant woman's circulating without wound pre-natal diagnosis.Because fetal nucleated red blood surface has specific marker, not easily breed, and the half life period is short, is thus considered to the optimal fetal cell type without wound pre-natal diagnosis.Mainly cell enrichment is comprised to the analysis of female blood fetal nucleated red blood, differentiates and aimed detection (as PCR, FISH etc.).But because in female blood, fetal cell quantity is very few, often less than 1 in every milliliter of female blood, constrain the development of this approach.
The DNA fragmentation of fetus dissociative is present in the circulating of maternal peripheral, because the content of DNA in pregnant woman blood plasma of fetus dissociative is large, probably account for 3% ~ 6% of pregnant woman blood plasma STb gene content, account for large many of the ratio of the total cell of female blood than fetal cell, therefore fetus dissociative DNA is starting to receive much concern without the application in wound pre-natal diagnosis.The research the earliest fetus dissociative DNA in maternal peripheral circulating being used for prenatal detection of Down syndrome finds, when the conceived time is substantially close, pregnant DS fetal DNA in maternal plasma dissociative DNA is approximately pregnant normal fetus pregnant woman 1 to 2 times.But because there is a large amount of source of parents dissociative DNA to exist, fetus dissociative DNA content is lower comparatively speaking, thus need to adopt the fetus dissociative DNA in the method enrichment maternal peripheral circulatings such as sulphite conversion or formaldehyde, but there is certain defect in these enrichment methods, what make the detection of fetus dissociative DNA also exists very large difficulty, and the ratio between its Cost and benefit paid also needs to investigate.
The source of fetus dissociative RNA is identical with fetus dissociative DNA, and is be present in blood plasma with the form of protein-RNA complex, and therefore stability is better.Utilize fetus dissociative RNA to carry out Non-invasive Prenatal Diagnosis, than fetus dissociative DNA, there is advantage, be mainly manifested in: the dissociate absolute content of RNA of fetal DNA in maternal plasma is high, analyzes difficulty and reduces; In pregnant woman blood plasma, mRNA is completely from placenta tissue, the interference of the RNA that dissociates without parent.
At present, female blood fetus dissociative RNA is utilized to carry out the method for Prenatal Screening Down syndrome mainly based on the proportionate relationship of RNA-SNP.See have 2 to derive from father (mother) in 3 No. 21 chromosomes of Down syndrome theoretically, remaining 1 then derives from mother (father).The ratio of nucleotide in normal cell different in the heterozygosis SNP site of No. 21 chromosome related genes is 1: 1, but the ratio in Down syndrome in patients is 2: 1 or 1: 2.The transcriptional efficiency of hypothetical gene is identical, and so in blood plasma, the proportionate relationship of mRNA-SNP is consistent with the ratio of DNA-SNP.Adopt digital pcr and mass spectrum two kinds of technology to measure the mRNA-SNP proportionate relationship of PLAC4 gene in pregnant woman blood plasma, the clinical specificity of 96.5% and the Clinical Sensitivity of 90% can be obtained.But based on female blood fetus dissociative RNA detection method of mass spectrum and digital pcr technology, complex steps, cost are higher, thus until be not all widely used at present.
In sum, based on the defect of traditional Prenatal Screening and pre-natal diagnosis, and the deficiency of existing Non-invasive Prenatal Diagnosis method, make to explore new non-invasive Down syndrome fetus pre-natal diagnosis mark and have very important significance.
Summary of the invention
Based on this, be necessary for the problems referred to above, a kind of construction method of Down syndrome spectrum model is provided.
A construction method for Down syndrome spectrum model, comprises the following steps:
Obtain the maternal blood having departed from human body, centrifugal, draw blood plasma, and the environment described blood plasma being placed in subzero 60 DEG C ~ subzero 100 DEG C is preserved;
Extract the protein of described blood plasma, obtain liquid to be measured;
Measure the protein content of described liquid to be measured;
Described liquid to be measured is carried out immune-blotting method;
Immune-blotting method result described in statistical study, sets up described Down syndrome spectrum model.
Wherein in an embodiment, the protein of the described blood plasma of described extraction comprises the following steps: by described blood plasma with distilled water by volume for 1:1 mixes, mixed liquor is obtained after shaking up, and by described mixed liquor and 5 × SDS-PAGE sample-loading buffer by volume 4:1 mix, boil 10min, after slowly returning to room temperature, be placed in subzero 20 DEG C of environment and preserve.
Wherein in an embodiment, the volume of described maternal blood is 2 ~ 4mL.
Wherein in an embodiment, the volume of described maternal blood is 3mL.
Wherein in an embodiment, described centrifugal temperature is 4 DEG C, and rotating speed is 10000 ~ 150000 revs/min, and centrifugation time is 10 ~ 20 minutes.
Wherein in an embodiment, the environment that described blood plasma is placed in subzero 80 DEG C is preserved.
Wherein in an embodiment, BCA method is adopted to measure the protein content of described liquid to be measured.
Wherein in an embodiment, measure the protein content of described liquid to be measured, comprise the following steps:
The BSA standard solution of preparation variable concentrations;
Liquid to be measured is diluted;
Preparation BCA working fluid;
BSA standard solution and the liquid to be measured that diluted are added in 96 orifice plates respectively, then add BCA working fluid, be 37 DEG C in temperature, under the condition of lucifuge, hatch 30 minutes;
Absorbance when being 560nm with microplate reader mensuration wavelength;
Drawing standard curve, and the concentration calculating protein in liquid to be measured.
Wherein in an embodiment, described immune-blotting method specifically comprises the steps: electrophoresis, transferring film, closes, primary antibodie hatches, two anti-ly hatch and develop the color.
Wherein in an embodiment, described Down syndrome spectrum model comprises Apolipoprotein E, Serum amyloid P-component, Complement factor B, 2-oxoglutaratedehydrogenase-like, mitochondrial, Nucleosome assembly protein 1-like 1.
The construction method of above-mentioned Down syndrome spectrum model, utilize the differential expression of protein in female blood blood plasma, for Prenatal Screening and prenatal detection of Down syndrome are provided as the Down syndrome spectrum model information of intermediate result, there is higher specificity and susceptibility, and the protein of these differential expressions contributes to the pathogenesis illustrating disease, and provide part auxiliary foundation for the early diagnosis of Tang Shi fetus.
Accompanying drawing explanation
Fig. 1 is the construction method schematic diagram of the Down syndrome spectrum model of one embodiment of the invention;
Fig. 2 is the canonical plotting of the protein concentration of one embodiment of the invention;
Fig. 3 is the immune-blotting method figure of the pregnant Down syndrome maternal plasma protein of one embodiment of the invention;
Fig. 4 is the immune-blotting method figure of the pregnant normal fetus maternal plasma protein of one embodiment of the invention;
Fig. 5 is the histogram of protein differential expression in DS group and NC group in the blood plasma of one embodiment of the invention.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, are described in detail the specific embodiment of the present invention below in conjunction with accompanying drawing.Set forth a lot of detail in the following description so that fully understand the present invention.But the present invention can be much different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar improvement when intension of the present invention, therefore the present invention is by the restriction of following public specific embodiment.
The invention provides a kind of construction method of Down syndrome spectrum model, comprising: obtain the maternal blood having departed from human body, centrifugal, draw blood plasma, and the environment described blood plasma being placed in subzero 60 DEG C ~ subzero 100 DEG C is preserved; Extract the protein of described blood plasma, obtain liquid to be measured; Measure the protein content of described liquid to be measured; Described liquid to be measured is carried out immune-blotting method; Immune-blotting method result described in statistical study, sets up described Down syndrome spectrum model.And for example, the protein of the described blood plasma of described extraction comprises the following steps: by described blood plasma with distilled water by volume for 1:1 mixes, mixed liquor is obtained after shaking up, and by described mixed liquor and 5 × SDS-PAGE sample-loading buffer by volume 4:1 mix, boil 10min, after slowly returning to room temperature, be placed in subzero 20 DEG C of environment and preserve.And for example, the volume of described maternal blood is 2 ~ 4mL.And for example, the volume of described maternal blood is 3mL.And for example, described centrifugal temperature is 4 DEG C, and rotating speed is 10000 ~ 150000 revs/min, and centrifugation time is 10 ~ 20 minutes.And for example, the environment described blood plasma being placed in subzero 80 DEG C is preserved.And for example, BCA method is adopted to measure the protein content of described liquid to be measured.And for example, measure the protein content of described liquid to be measured, comprise the following steps: the BSA standard solution of preparation variable concentrations; Liquid to be measured is diluted; Preparation BCA working fluid; BSA standard solution and the liquid to be measured that diluted are added in 96 orifice plates respectively, then add BCA working fluid, be 37 DEG C in temperature, under the condition of lucifuge, hatch 30 minutes.Absorbance when being 560nm with microplate reader mensuration wavelength; Drawing standard curve, and the concentration calculating protein in liquid to be measured.And for example, described immune-blotting method specifically comprises the steps: electrophoresis, transferring film, closes, primary antibodie hatches, two anti-ly hatch and develop the color.And for example, a spectrum model is obtained by above-mentioned steps, i.e. Down syndrome spectrum model, such as, described Down syndrome spectrum model comprises Apolipoprotein E, Serum amyloid P-component, Complement factor B, the at least one of 2-oxoglutarate dehydrogenase-like, mitochondrial and Nucleosome assembly protein1-like 1.And for example, described Down syndrome spectrum model comprises Thymosin beta-10 and ERO1-like protein alpha.
Refer to Fig. 1, one embodiment of the present of invention are, a kind of construction method of Down syndrome spectrum model, it comprises the steps.
S110, obtain and departed from the maternal blood of human body, centrifugal, draw blood plasma, and the environment described blood plasma being placed in subzero 60 DEG C ~ subzero 100 DEG C is preserved.
Such as, particularly, collect pregnant Down syndrome fetus and normal fetus maternal blood 3mL inserts in 5mL anticoagulant heparin pipe, being placed in hydro-extractor, is 4 DEG C in temperature, and rotating speed is under the condition of 12000 revs/min, centrifugal 15 minutes, after layering, upper liquid and blood plasma are placed in the 1.5mL EP pipe through protease inhibitors process, frozen for subsequent use in-80 DEG C.Such as, the pregnant woman that test group can comprise multiple pregnant Down syndrome fetus has departed from the peripheral blood of human body, same, and the pregnant woman that reference group can comprise multiple pregnant normal fetus has departed from the peripheral blood of human body.Preferably, after drawing blood plasma, be placed in 5 ~ 15 minutes in the environment of subzero 60 DEG C ~ subzero 100 DEG C and preserve, and for example, after drawing blood plasma, be placed in 5 ~ 15 minutes in the environment of subzero 80 DEG C and preserve.
S120, extract the protein of described blood plasma, obtain liquid to be measured.
Add the ratio of 100 μ l distilled waters according to every 100 μ l blood plasma, in blood plasma, add distilled water, mixing shakes up and is placed on ice.In the present embodiment, distilled water refers to the water after single flash, again distills the water obtained.
In above-mentioned mixed liquor, add 5 × SDS-PAGE sample-loading buffer, 5 × SDS-PAGE sample-loading buffer volume is 1/5 of cumulative volume, and fully mixing boils 10 minutes, after slowly recovering room temperature, slightly centrifugal, discards precipitation, is placed in-20 DEG C of preservations.
Such as, taken out by described blood plasma, naturally thaw to room temperature, then add distilled water in the ratio of 1:1 from the environment of subzero 60 DEG C ~ subzero 100 DEG C, mixing, obtains mixed liquor, for subsequent use.And for example, described blood plasma is taken out from the environment of subzero 60 DEG C ~ subzero 100 DEG C, naturally thaw to 0 DEG C ~ 4 DEG C, then add the distilled water being chilled to 0 DEG C ~ 4 DEG C in advance in the ratio of 1:1, mix under the environment of 0 DEG C ~ 4 DEG C, obtain mixed liquor, for subsequent use.Such as, described blood plasma is taken out from the environment of subzero 60 DEG C ~ subzero 100 DEG C, naturally thaw to 0 DEG C ~ 4 DEG C in frozen water, then add the distilled water being chilled to 0 DEG C ~ 4 DEG C in advance in the ratio of 1:1, mix in frozen water, obtain mixed liquor, for subsequent use.
And for example, described mixed liquor is added the 5 × SDS-PAGE sample-loading buffer being chilled to 0 DEG C ~ 4 DEG C in advance, then take out from frozen water, at room temperature fully mix, then boil 10 minutes.And for example, by the rate of temperature fall of 3 DEG C ~ 10 DEG C after boiling, slowly room temperature is recovered.And for example, carry out centrifugal 10 ~ 60 seconds with 20 ~ 200rpm, then discard precipitation, be placed in-20 DEG C of preservations.
In the present embodiment, the component of 5 × SDS-PAGE sample-loading buffer (10mL) is: Tris-HCl pH6.8 (60mM); SDS (2%); Bromjophenol blue (0.1%); Glycerine (25%); Beta-mercaptoethanol (14.4mM).
Such as, preparing 5 × SDS-PAGE sample-loading buffer process is: measure 1M Tris-HCl (pH6.8) 0.6mL respectively; The glycerine 5mL of 50%; The SDS solution 2mL of 10%; The bromjophenol blue 1mL of 1%, adds deionized water and is settled to 10mL after mixing.
S130, measure the protein content of described liquid to be measured.
Such as, BCA method is adopted to measure the protein content of described liquid to be measured.
And for example, measure the protein content of described liquid to be measured, comprise the following steps:
The typical curve of S131, configuration variable concentrations: the extension rate of each standard items is as shown in table 1.
Each solution allocation of table 1 typical curve
S132, liquid to be measured to be diluted: each liquid to be measured is diluted 20 times, namely adds 38 μ L H in every 2 μ L liquid to be measured
2o.
S133, configuration BCA working fluid: by A liquid 50 volume, A, B two kinds of liquid are fully mixed, obtain BCA working fluid by B liquid 1 volume.
S134, BSA standard solution and the liquid to be measured that diluted are added (20 μ L/ hole) in 96 orifice plates, then add BCA working fluid (200 μ L/ hole), be 37 DEG C in temperature, under the condition of lucifuge, hatch 30 minutes.
S135, with microplate reader measure wavelength be 560nm time absorbance.
S136, drawing standard curve, according to typical curve and light absorption value, calculate the concentration of protein in liquid to be measured.
With standard items BSA concentration μ g/ μ L for x-axis, OD value is y-axis, draws and obtains typical curve as shown in Figure 1.As shown in Figure 2, R
2=0.9945 (R>0.98), the linear response relationship of detection method is more satisfactory, can be used for the detection of sample protein matter concentration.
S140, described liquid to be measured is carried out immune-blotting method (Western blot).
Such as, described immune-blotting method specifically comprises the steps: electrophoresis, transferring film, closes, primary antibodie hatches, two anti-ly hatch and develop the color.
Wherein, electrophoresis specifically comprises: by described liquid to be measured with 5 × SDS-PAGE sample-loading buffer by volume for 5:1 mixes, be added to after boiling 5 minutes in electrophoresis tank, be under the condition of 80V at voltage, electrophoresis is after 50 minutes, change voltage to 120V, appear at the bottom of polyacrylamide gel to bromophenol blue after, stop electrophoresis.
Such as, transferring film specifically comprises: the polyacrylamide gel after being terminated by electrophoresis takes out and is placed on filter paper, in transfer printing liquid, form gel transfer printing accumulation horizon according to the sequence stack of filter paper, polyacrylamide gel, pvdf membrane, filter paper, be shift 60 ~ 120 minutes under the condition of 100V at voltage by described gel transfer printing accumulation horizon.
In the present embodiment, pvdf membrane is before use through methyl alcohol pre-service 3 ~ 5 seconds, and transfer printing immersion moistens halfhour process.
Such as, close and specifically comprise: take out the pvdf membrane after transferring film, clean 3 times with TBST, each 5 minutes, add 5% skim milk powder solution and close 4 DEG C of conditions and spend the night.
In the present embodiment, the concentration of TBST is 0.01M, and wherein the TBST compound method of 0.01M is: take Tris 1.21g, and NaCl 5.84g is added to 800mL H
2in O, regulate pH to 7.5 with HCl, add 0.05% or 0.1%Tween-20, use H
2o is settled to 1000mL.
Such as, primary antibodie is hatched and is comprised: take out the pvdf membrane after closing, and after cleaning 3 times with TBST, each cleaning five minutes, to add after primary antibodie solution overnight incubation under 4 DEG C of conditions.
Such as, primary antibodie is at least one of Anti-NAP1L1antibody, Anti-Thymosin beta 10antibody, Anti-Apolipoprotein E antibody, Anti-ERO1L antibody, Anti-OGDHL antibody, Anti-Complement factor B antibody or Anti-Serum Amyloid P antibody.And for example, primary antibodie is Anti-ERO1L antibody or Anti-Thymosin beta 10antibody.
Such as, the concentration of TBST is 0.01M.And for example, the 5% skim milk powder solution dilution of primary antibodie solution obtains.And for example, the TBST dilution of primary antibodie solution 0.01M obtains.And for example, primary antibodie solution primary antibodie diluted obtains.And for example, the concentration of primary antibodie is 1 μ g/mL, and certainly, the concentration of primary antibodie also can be selected as the case may be.
Such as, the pvdf membrane of test group and reference group to be added respectively after the Anti-Thymosin beta10antibody of 1 μ g/mL overnight incubation under 4 DEG C of conditions.And for example, the pvdf membrane of test group and reference group to be added respectively after the Anti-ERO1L antibody of 1 μ g/mL overnight incubation under 4 DEG C of conditions.And for example, the pvdf membrane of test group and reference group to be added respectively after the Anti-NAP1L1antibody of 1 μ g/mL overnight incubation under 4 DEG C of conditions.And for example, the pvdf membrane of test group and reference group to be added respectively after the Anti-Apolipoprotein E antibody of 1 μ g/mL overnight incubation under 4 DEG C of conditions.And for example, the pvdf membrane of test group and reference group to be added respectively after the Anti-OGDHL antibody of 1 μ g/mL overnight incubation under 4 DEG C of conditions.And for example, the pvdf membrane of test group and reference group to be added respectively after the Anti-Complementfactor B antibody of 1 μ g/mL overnight incubation under 4 DEG C of conditions.And for example, the pvdf membrane of test group and reference group to be added respectively after the Anti-Serum Amyloid P antibody of 1 μ g/mL overnight incubation under 4 DEG C of conditions.
Such as, two anti-hatching comprise: take out primary antibodie hatch after pvdf membrane, clean 3 times with TBST, each cleaning 5 minutes, adds two anti-solution and hatch 1 hour under 37 DEG C of condition.
Such as, two resist for peroxidase-conjugated goat anti-rabbit IgG.And for example, the concentration of TBST is 0.01M.And for example, the 5% skim milk powder solution dilution of two anti-solution obtains.And for example, the TBST dilution of two anti-solution 0.01M obtains.And for example, in two anti-dilutions, two concentration resisted are 1 μ g/mL, and certainly, two concentration resisted also can be selected as the case may be.
Such as, colour developing specifically comprises:
Resist the pvdf membrane after hatching with TBST cleaning through two, each 2 minutes, repeat twice.
Take out pvdf membrane, the protein powder of pvdf membrane is placed on clean transparent plastic sheet upward, and keeps the moistening of pvdf membrane.
By ECL A liquid: the proportional arrangement ECL working fluid of ECL B liquid=1:1.
ECL working fluid is added to pvdf membrane surface equably, places 5 minutes.
Suck the ECL working fluid of pvdf membrane excess surface, and put it into magazine.
Developing fixing.
Immune-blotting method result described in S150, statistical study, sets up described Down syndrome spectrum model.Such as, immune-blotting method result described in statistical study, set up described Down syndrome spectrum model, it comprises Apolipoprotein E, Serum amyloid P-component, Complement factor B, 2-oxoglutarate dehydrogenase-like, mitochondrial, Nucleosome assembly protein1-like 1; Preferably, it also comprises Thymosin beta-10 (T β 10) and ERO1-like protein alpha (ERO1L); Such as, set up a Down syndrome spectrum model thus, it comprises 2-oxoglutaratedehydrogenase-like (OGDHL), Serum amyloid P-component (SAP), Apolipoprotein E (AopE), Nucleosome assembly protein 1-like 1 (NAP1L1), Thymosin beta-10 (T β 10), Complement factor B and ERO1-like protein alpha (ERO1L).
Such as, adopt the analysis of SPSS 17.0 statistical software, adopt t inspection, P<0.05 thinks that difference has statistical significance.
Below for the concrete pregnant woman of pregnant Down syndrome fetus and the pregnant woman of pregnant normal fetus, provide example further and the present invention is described.
The present invention collects maternal peripheral blood specimen 16 example altogether, derive from heredity room, Shenzhen people's hospital clinic study center and centralab of No.181 Hospital, PLA, comprise maternal peripheral blood specimen 8 example (6 male 2 female of pregnant Down syndrome fetus, pregnant fetus be all diagnosed as standard form Down syndrome fetus through the karyotyping of conventional G banding chromosome) and maternal peripheral blood specimen 8 example (3 male 5 female of pregnant normal fetus, B ultrasonic display fetus is normal type), be respectively DS (Down syndrome) group and NC group (reference group).The time of specimen collection is year January in April, 2013 to 2014.Wherein, the pregnant woman age of DS group is 32.0 ± 7.010, and pregnant week is 17.13 ± 0.836, and fetus number is single tire; The pregnant woman age of control group is 31.5 ± 4.106, and pregnant week is 16.75 ± 0.886, and fetus number is single tire.
Obtain the immune-blotting method result of DS group and reference group peripheral blood blood plasma according to said method, result is as shown in table 2.
Table 2 immune-blotting method result
Refer to Fig. 2 and Fig. 3, in DS group, the band of each protein is all more obvious than reference group, and IOD (integral optical density) value is all higher than control group.Carry out t check analysis to its IOD value, the P value obtained all is less than 0.05, and therefore each protein has statistical significance between DS group and reference group.Wherein, the multiple of 2-oxoglutaratedehydrogenase-like (OGDHL), Serum amyloid P-component (SAP), Apolipoprotein E (AopE), Nucleosome assembly protein 1-like 1 (NAP1L1), Thymosin beta-10 (T β 10), Complement factor B, ERO1-like protein alpha (ERO1L) differential expression in female blood blood plasma is followed successively by 1.527,1.541,1.336,2.188,14.776,2.567,11.100.Wherein, the differential expression of T β 10 in DS group and reference group is the most obvious, and fold differences is up to 14.766 times, and be secondly ERO1L, fold differences is 11.1 times.
From the result of above-described embodiment, the differential expression of application protein builds Down syndrome spectrum model and contributes to providing a kind of possible approaches distinguishing Down syndrome fetus and normal fetus, but consider due to the pathogenetic complicacy of Down syndrome, therefore content disclosed in medical knowledge of the prior art and the present patent application, the diagnostic result of this Down syndrome directly can not be drawn from obtained information itself, this spectrum model is merely able to the reference frame as subsequent analysis diagnosis, this spectrum model how is utilized to need further medical research.But along with carrying out of follow-up study work, adopt the construction method of above-mentioned each example and combination thereof, the spectrum model of the Down syndrome constructed by it, using the effective information as intermediate result, the analytical work for next step provides good Informational support and auxiliary data.
The various embodiments described above of the present invention, analyze with the maternal plasma of Western blot technology to pregnant Down syndrome, find that the protein expression of the protein expression of the maternal plasma of pregnant Down syndrome and the maternal plasma of pregnant normal fetus has significant difference, by the analysis different to protein expression result, not only can successfully distinguish DS group and reference group, and Down syndrome can be built there is spectrum model compared with high specific and susceptibility, and the protein of these differential expressions contributes to the pathogenesis illustrating disease, and provide foundation for the early diagnosis of Tang Shi fetus.
You need to add is that, direct object of the present invention is not obtain diagnostic result or health status, and just to the body fluid departing from human body, i.e. maternal blood, carry out the method processing or detect the information obtained as intermediate result, or process the method for this information, according to current medical knowledge and content disclosed in this invention, the diagnostic result of disease directly can not be drawn from obtained information itself.
Each technical characteristic of the above embodiment can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this instructions is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a construction method for Down syndrome spectrum model, is characterized in that, comprises the following steps:
Obtain the maternal blood having departed from human body, centrifugal, draw blood plasma, and the environment described blood plasma being placed in subzero 60 DEG C ~ subzero 100 DEG C is preserved;
Extract the protein of described blood plasma, obtain liquid to be measured;
Measure the protein content of described liquid to be measured;
Described liquid to be measured is carried out immune-blotting method;
Immune-blotting method result described in statistical study, sets up described Down syndrome spectrum model.
2. construction method according to claim 1, it is characterized in that, the protein of the described blood plasma of described extraction comprises the following steps: by described blood plasma with distilled water by volume for 1:1 mixes, mixed liquor is obtained after shaking up, and by described mixed liquor and 5 × SDS-PAGE sample-loading buffer by volume 4:1 mix, boil 10min, after slowly returning to room temperature, be placed in subzero 20 DEG C of environment and preserve.
3. construction method according to claim 1, is characterized in that, the volume of described maternal blood is 2 ~ 4mL.
4. construction method according to claim 3, is characterized in that, the volume of described maternal blood is 3mL.
5. construction method according to claim 1, is characterized in that, described centrifugal temperature is 4 DEG C, and rotating speed is 10000 ~ 150000 revs/min, and centrifugation time is 10 ~ 20 minutes.
6. construction method according to claim 1, is characterized in that, is preserved by the environment that described blood plasma is placed in subzero 80 DEG C.
7. construction method according to claim 1, is characterized in that, adopts BCA method to measure the protein content of described liquid to be measured.
8. construction method according to claim 7, is characterized in that, measures the protein content of described liquid to be measured, comprises the following steps:
The BSA standard solution of preparation variable concentrations;
Liquid to be measured is diluted;
Preparation BCA working fluid;
BSA standard solution and the liquid to be measured that diluted are added in 96 orifice plates respectively, then add BCA working fluid, be 37 DEG C in temperature, under the condition of lucifuge, hatch 30 minutes;
Absorbance when being 560nm with microplate reader mensuration wavelength;
Drawing standard curve, and the concentration calculating protein in liquid to be measured.
9. construction method according to claim 1, is characterized in that, described immune-blotting method specifically comprises the steps: electrophoresis, transferring film, closes, primary antibodie hatches, two anti-ly hatch and develop the color.
10. construction method according to claim 1, it is characterized in that, described Down syndrome spectrum model comprises Apolipoprotein E, Serum amyloid P-component, Complement factor B, 2-oxoglutarate dehydrogenase-like, mitochondrial, Nucleosome assembly protein1-like 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107167612A (en) * | 2017-06-20 | 2017-09-15 | 深圳市人民医院 | The female blood mark related to Down syndrome and application |
| CN107478845A (en) * | 2017-08-04 | 2017-12-15 | 戴勇 | One group of Down syndrome marker protein and its application |
| CN109251969A (en) * | 2017-07-14 | 2019-01-22 | 戴勇 | Down syndrome parent tire source property circRNA differential expression spectrogram spectrum model and its construction method and building system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2053407A1 (en) * | 2007-10-26 | 2009-04-29 | Foundation for Biomedical Research of the Academy of Athens | The protein serum amyloid P-component (SAP, SAMP) as prognostic and diagnostic marker for the prenatal diagnosis of Trisomy 21 (Down syndrome) |
| US20090305259A1 (en) * | 2006-03-21 | 2009-12-10 | Yale University | Early Diagnosis of Congenital Abnormalities in the Offspring of Diabetic Mothers |
| US20120288873A1 (en) * | 2011-05-09 | 2012-11-15 | University Health Network | Biomarkers for the detection and screening of down syndrome |
-
2015
- 2015-04-22 CN CN201510193659.8A patent/CN104849471A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090305259A1 (en) * | 2006-03-21 | 2009-12-10 | Yale University | Early Diagnosis of Congenital Abnormalities in the Offspring of Diabetic Mothers |
| EP2053407A1 (en) * | 2007-10-26 | 2009-04-29 | Foundation for Biomedical Research of the Academy of Athens | The protein serum amyloid P-component (SAP, SAMP) as prognostic and diagnostic marker for the prenatal diagnosis of Trisomy 21 (Down syndrome) |
| US20120288873A1 (en) * | 2011-05-09 | 2012-11-15 | University Health Network | Biomarkers for the detection and screening of down syndrome |
Non-Patent Citations (7)
| Title |
|---|
| AGGELIKI KOLIALEXI等: "Application of proteomics for the identification of differentially expressed protein markers for Down syndrome in maternal plasma.", 《PRENAT DIAGN》 * |
| GEORGE TH.TSANGARIS等: "Proteomic analysis of amniotic fluid in pregnancies with Down syndrome", 《PROTEOMICS》 * |
| SRINIVASA R.NAGALLA等: "Proteomic analysis of maternal serum in Down syndrome identification of novel protein biomarkers.", 《JOURNAL OF PROTEOME RESEARCH》 * |
| WEIGUO SUI等: "Quantitative proteomic analysis of Down syndrome in the umbilical cord blood using iTRAQ.", 《MOLECULAR MEDICINE REPORTS》 * |
| 刘志国主编: "《生物化学实验》", 30 April 2007 * |
| 汪家政、范明主编: "《蛋白质技术手册》", 31 August 2000 * |
| 蒋滢: "早期诊断唐氏综合征的母体血清蛋白新标志物筛选与验证", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107167612A (en) * | 2017-06-20 | 2017-09-15 | 深圳市人民医院 | The female blood mark related to Down syndrome and application |
| CN113311165A (en) * | 2017-06-20 | 2021-08-27 | 深圳市人民医院 | Maternal blood marker related to Down syndrome and application thereof |
| CN109251969A (en) * | 2017-07-14 | 2019-01-22 | 戴勇 | Down syndrome parent tire source property circRNA differential expression spectrogram spectrum model and its construction method and building system |
| CN107478845A (en) * | 2017-08-04 | 2017-12-15 | 戴勇 | One group of Down syndrome marker protein and its application |
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