CN106645736A - Screening method for differentially expressed proteins of heterotopic-pregnancy and non-pregnancy decidua tissue - Google Patents
Screening method for differentially expressed proteins of heterotopic-pregnancy and non-pregnancy decidua tissue Download PDFInfo
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- CN106645736A CN106645736A CN201610426995.7A CN201610426995A CN106645736A CN 106645736 A CN106645736 A CN 106645736A CN 201610426995 A CN201610426995 A CN 201610426995A CN 106645736 A CN106645736 A CN 106645736A
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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
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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Abstract
The invention discloses a screening method for differentially expressed proteins of heterotopic-pregnancy and non-pregnancy decidua tissue. The method is characterized by comprising the following steps: carrying out proteomics analysis to discover specific proteins and differential proteins of heterotopic-pregnancy and non-pregnancy decidua tissue; subjecting the specific proteins and differential proteins to separation and purification; and using the purified specific proteins and differential proteins as special biological markers for discrimination of intrauterine and extrauterine pregnancy. After pregnancy, specific proteins can be synthesized in the bodies of both a mother and a fetus. Detection of the specific proteins with different methods is beneficial for diagnosis of pregnancy. Searching for a special marker for pregnancy, especially for distinguishing intrauterine and extrauterine pregnancy, is of critical significance to determination of clinical medical liabilities and medical jurisprudence and is a great breakthrough to the basic theory of medical science.
Description
Technical field
The present invention relates to a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein.
Background technology
Gestation is particularly significant for clinic with the judgement of non-pregnant uterus internal film tissue, and endometrium is from secretion late period
There is no absolute boundary to the transition state between First Trimester, but in practical work, frequently encounter decidua graviditatis and decidua sample
Antidiastole, the clinic of tissue and non-pregnant decidua tissue there is no at present full with dilatation and curettage material requirements Diagnosis of Ectopic Pregnancy problem etc.
The solution of meaning, factor Endometrium is from secretion late period to hormone secretion level and internal film tissue pair in First Trimester conversion process
The difference of hormone response, some non-pregnant factors cause the decidua of inner membrance to become etc., in the presence of without fine hair, in form
On nuance therebetween be difficult to differentiate, to be made whether gestation, be in utero or ectopic pregnancy is very difficult.
For the difference in ultra microstructure between the two and molecular biology, seldom report on document, even if having been reported that its specificity also not
By force, or experiment be difficult to repeat.The interstitial of endometrium is made up of versatility mesenchymal cell, and when the menstrual cycle starts, interstitial is thin
Born of the same parents break up in fusiformis, and endochylema projection is coupled to each other, and core is long, and chromatin enriches.In the latter half of secretory phase, interstitial cell swashs in property
In the presence of element, to both direction differentiation, wherein a semicell becomes fat circular decidua like cell, second half then dwindles into little
Circular endometrial granulocytes.It is the physiology of endometrium experience series of complex, biochemical and morphologic change after embryo nidation
Become, with the development of this change, inner membrance is by secreting the decidua sample structural transformation in late period into a kind of new class epithelium spline structure：Slough off
Film.Then there is more granular cell aggregation around embryo.Endometrium decidualization is fetal cortical neurons and gestation in human reproduction
One of essential condition of maintenance.Decidua be it is a kind of by various kinds of cell into be grouped into heterogeneity tissue, except the battalion to fetus, placenta
Support that effect is outer, it in gestation and during maintaining normal pregnancy, sends out simultaneously or a kind of complicated local immunity organ of function
Wave key effect.Decidua is made up of body of gland, immunocyte and nonimmune cell.The nonimmune cell of decidua is mainly decidual cell
(accounting for 85% or so % of whole decidua TCSs), under normal pregnancy state, the immunity of the immunocyte in decidua
Learn activity to be inhibited, the nonimmune cell of decidua for accounting for the decidua overwhelming majority is possible to have decidua immunocyte immunity tune
The effect of section.After gestation, parent and fetus can synthesize some specific proteins.Using distinct methods to these specificity into
Divide and detected, each contribute to the diagnosis of gestation.
Find the special marking thing that can particularly differentiate the inside and outside gestation in palace for gestation, no matter to facing, malpractice insurance
Judgement and all have great importance in medical jurisprudence, also will be important breakthrough in basic theory.
The content of the invention
It is an object of the invention to provide a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein.
This research adopts the tissue factor expression of simple and easy to do, quick sensitive technology for detection different type cell.
The present invention is achieved through the following technical solutions：
A kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, it is characterised in that：Carry out proteomics
Analysis, finds respective differential protein and differential protein, it is separated, is purified, and in this, as gestation inside and outside discriminating uterus
Special biological marker.
Methods described further includes to carry out test on immune function：Detection tissue culture medium, separating-purifying it is special and poor
The immunization of the basement membrane sample material having found in M-band and decidual cell ultra microstructure, analysis purification and synthesize its effectively into
Point, it is applied to the immunity disease of clinic；Check BMDC in gestation, non-pregnant and cycle endometrial tissue point
Cloth.
Methods described further includes preparation and mass spectral analysis to destination protein spot PMF samples, finally finds and differentiates
The special marking thing of gestation inside and outside uterus；
Methods described further includes to detect specific component immunologic function.
Methods described further includes to check BMDC in gestation, non-pregnant and cycle using flow cytometer etc.
The in utero distribution in membrane tissue.
The preparation of the destination protein includes
1) preparation of protein sample, is completed：Will in utero outer pregnant detached decidua crack respectively, freeze thawing, grinding, centrifugation, survey
Determine protein concentration in supernatant.
2) the protein group of in utero outer pregnant decidua, is individually separated with solid phase gradient two dimensional gel electrophore- sis, photography after colour developing is stayed
Deposit, corresponding software graphical analysis.
3), the preparation and mass spectral analysis of destination protein spot PMF samples, is eventually found special and differentially expressed protein point,
In this, as the special marking thing for differentiating gestation inside and outside uterus；
4), specific component is separated, purified, identification and its immunologic function are detected, completes the screening of differentially expressed protein.
The differential protein and difference of pathology, tissue cultures and separating-purifying are improved and have expanded in whole research process
Protein data.
The present invention compared with prior art, has the advantages that：
The present invention finds the special marking thing that can particularly differentiate the inside and outside gestation in palace for gestation, no matter to clinical, medical treatment
The judgement of responsibility and all have great importance in medical jurisprudence, also will be important breakthrough in basic theory.This research is using easy
The tissue factor expression of easy, quick sensitive technology for detection different type cell.The successful research and development of the present invention, will effectively improve
The technical merit of company, the market competitiveness and research and development ability, for company's Extension of service scope strong support is provided.Project is complete
Cheng Hou, will largely improve the detectability in company laboratory, and annual income is expected to increase by ten million yuan.At the same time, find
For the special marking thing that can particularly differentiate the inside and outside gestation in palace of gestation, no matter to the clinical, judgement to malpractice insurance and
All have great importance in medical jurisprudence, also will be important breakthrough in basic theory, the popularization of project will realize economic benefit
With social benefit bumper harvests.
Description of the drawings
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below will be attached to what is used needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, thus be not construed as it is right
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can be with according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 .1：S-100 albumen is positive in decidua tissue body of gland.
Fig. 1 .2：S-100 albumen cytoplasm and nuclear positive reaction in the decidual cell of part.
Fig. 2：Masson's trichrome stains show the pale red " featheriness wins collagen inclusion " in decidual cell endochylema.
Fig. 3 .1:Decidual cell is larger, circular and oval, and the organelles of endochylema and Qi Nei have projection on cell membrane
And basilar memebrane sample ectoplast is formed（The black arrow in figure lower-left）18,000X.
Fig. 3 .2：Show the connection between decidual cell, see desmosome, gap couples and is tightly linked（Arrow indication）12,000X, endochylema
Inside see endoplasmic reticulum, the mitochondria of more expansion.
Fig. 3 .3：For Fig. 3 .2 partial enlargements, arrow indication is desmosome, and gap couples and is tightly linked, more collagenous fibril（Star
At number）40,000X.
Fig. 3 .4：Show the tonofibril in decidual cell（Arrow）, the big portion in right side is nucleus（）55,000X.
Fig. 4 .1：Conventional H E is cut into slices, and rarely seen sheet is red to be contaminated without structure sphacelus.
Fig. 4 .2：VG chromoscopies, by the remaining necrosis fine hair for being difficult to recognize in conventional section in Fig. 4 .1 examples scarlet is dyed.
Fig. 4 .3：Conventional H E is cut into slices, and main is denaturation decidual cell.
Fig. 4 .4：HPL immunohistochemical detections, intermediate trophoblastic cell positive expression in decidual cell in Fig. 4 .3 examples.
Fig. 5：The screening process figure of histological difference expressing protein.
Specific embodiment
The present invention is described in further detail with reference to specific embodiment, but embodiments of the present invention are not limited to
This.
Embodiment 1：
The present invention by utero, ectopic pregnancy when uterine decidua tissue carry out respectively tissue cultures, light microscopic, Electronic Speculum and immunity inspection
Look into, main contents include：
1st, to two kinds of deciduas separation, cell culture, to nutrient solution；
2nd, distribution of the BMDC in gestation, non-pregnant and cycle endometrial tissue is checked using flow cytometer etc.；
3rd, electron microscopy is applied, is studied to different internal film tissue's ultrastructural characteristics, made decidua granular cell in order and reported with document
The intracellular lymphocyte containing particle in road, the relation of BMDC, illustrate the sertoli cell of the cell surface having been found that
Like cell podocytic process belongs to any cell and its function.
Complete the preparation of protein sample：Will in utero outer pregnant detached decidua crack respectively, freeze thawing, grinding, centrifugation, survey
Determine protein concentration in supernatant, with solid phase gradient two dimensional gel electrophore- sis the protein group of in utero outer pregnant decidua, colour developing are individually separated
Photography afterwards is retained, corresponding software graphical analysis.
The preparation and mass spectral analysis of destination protein spot PMF samples, specific component is separated, purified, identification, finally in uterus
Inside and outside early pregnancy sends out the differentially expressed protein S-100 albumen that pregnancy-associated plasma protein and specificity are found that in decidua tissue.It is described
It is a kind of macromolecular glycoprotein compound, molecular weight is 8000KD, is the tetramer of four identical subunit's compositions.
Embodiment 2
It was found that S-100 albumen is positive in decidua body of gland, but also has negative reflection in inner membrance body of gland, in part decidua
Also there is the reflection (as shown in accompanying drawing 1-4) of the positive in cell；
" featheriness collagen inclusion " in the provable denaturation decidual cell of Masson ' s trichrome stains, and point out once occurred it is pregnant
It is pregnent, because it has false-positive result, accuracy rate is 85% or so.
Electron microscopic examination finds：Decidual cell is larger, circular and oval, and the organelles of endochylema and Qi Nei have development
Good rough surfaced endoplasmic reticulum (RER), mitochondria, phagosome, inclusion body etc..Cell surface has cell of the cellulose sample similar to basilar memebrane
Ectoplast.Between flanking cell in addition to seeing gap connection, being tightly linked, also typical desmosome is formed, and between decidua like cell
Have no this structure.It is found that in the decidual cell of part compared with polydispersion and bundles of tension force silk, the structure of this scaly epithelium
Feature, occurs in decidua tissue.
Downright bad fine hair and nourish thin just as osculant outside the fine hair of decidual cell that VG, hPL dyeing can cannot show HE
Born of the same parents show, such that it is able to prove uterogestation.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, for the skill of this area
For art personnel, the present invention can have various modifications and variations.It is all within the spirit and principles in the present invention, made any repair
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, it is characterised in that：Carry out protein group
Credit is analysed, and finds respective differential protein and differential protein, it is separated, is purified, and pregnant inside and outside uterus in this, as differentiating
Special biological marker.
2. as claimed in claim 1 a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, its feature
It is：Methods described further includes to carry out test on immune function：Detection tissue culture medium, the special and difference egg of separating-purifying
The immunization of the basement membrane sample material having found in bletilla decidual cell ultra microstructure, analysis purifies and synthesizes its active ingredient,
It is applied to the immunity disease of clinic；Check distribution of the BMDC in gestation, non-pregnant and cycle endometrial tissue.
3. as claimed in claim 1 a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, its feature
It is：Methods described further includes preparation and mass spectral analysis to destination protein spot PMF samples, finally finds and differentiates uterus
The special marking thing of inside and outside gestation.
4. as claimed in claim 1 a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, its feature
It is：Methods described further includes to detect specific component immunologic function.
5. as claimed in claim 1 a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, its feature
It is：Methods described further includes to check BMDC in gestation, non-pregnant and cycle uterus using flow cytometer etc.
Distribution in internal film tissue.
6. as claimed in claim 1 a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, its feature
It is：The preparation of the destination protein includes
1) preparation of protein sample, is completed：Will in utero outer pregnant detached decidua crack respectively, freeze thawing, grinding, centrifugation, survey
Determine protein concentration in supernatant；
2) the protein group of in utero outer pregnant decidua, is individually separated with solid phase gradient two dimensional gel electrophore- sis, photography after colour developing is retained, phase
Answer image analysis software；
3), the preparation and mass spectral analysis of destination protein spot PMF samples, is eventually found special and differentially expressed protein point, with this
As the special marking thing for differentiating gestation inside and outside uterus；
4), specific component is separated, purified, identification and its immunologic function are detected, completes the screening of differentially expressed protein；
The differential protein and differential protein of pathology, tissue cultures and separating-purifying are improved and have expanded in whole research process
Data.
7. as claimed in claim 1 a kind of ectopic pregnancy, the screening technique of non-pregnant decidua tissue differentially expressed protein, its feature
It is：The preparation and mass spectral analysis of destination protein spot PMF samples, specific component is separated, purified, identification, in uterus, finally
Outer early pregnancy sends out the differentially expressed protein S-100 albumen that pregnancy-associated plasma protein and specificity are found that in decidua tissue；The S-
100 albumen are a kind of macromolecular glycoprotein compounds, and molecular weight is 8000KD, are the tetramer of four identical subunit's compositions.
8. a kind of ectopic pregnancy, non-pregnant decidua tissue differentially expressed protein, it is characterised in that：The labelled protein in uterus,
Outer early pregnancy is sent out in decidua tissue.
9. ectopic pregnancy as claimed in claim 8, non-pregnant decidua tissue differentially expressed protein, it is characterised in that：The mark
Albumen is a kind of macromolecular glycoprotein compound, and molecular weight is 8000KD, is the tetramer of four identical subunit's compositions.
10. ectopic pregnancy as claimed in claim 8, non-pregnant decidua tissue differentially expressed protein, it is characterised in that：The mark
Application of the albumen in pregnant detection reagent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112034180A (en) * | 2020-08-18 | 2020-12-04 | 四川大学华西第二医院 | Use and product of keratin 1 in ectopic pregnancy |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009004340A3 (en) * | 2007-07-02 | 2009-03-12 | Univ Edinburgh | Identification of ectopic pregnancies |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009004340A3 (en) * | 2007-07-02 | 2009-03-12 | Univ Edinburgh | Identification of ectopic pregnancies |
Non-Patent Citations (9)
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| RENU SHANKAR ET AL: "An emerging role for comprehensive proteome analysis in human pregnancy research", 《REPRODUCTION》 * |
| SARIA AGARWAL ET AL: "Immunoreactivity with S100 protein as an indicator of pregnancy", 《INDIAN J MED RES》 * |
| SINOSICH MJ ET AL: "Circulating and tissue concentrations of pregnancy-associated plasma protein-A (PAPP-A) in tubal ectopic gestation", 《CLINICAL REPRODUCTION AND FERTILITY》 * |
| 余传霖等: "《分子免疫学》", 30 May 2001, 复旦大学出版社 * |
| 余秋波: "早期自然流产蜕膜组织基因表达谱和蛋白质组研究", 《中国优秀博士学位论文全文数据库医药卫生科技辑》 * |
| 尹学敬等: "血清妊娠相关血浆蛋白A在诊断异常妊娠中的临床意义", 《现代生物医学进展》 * |
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| 莫菲特等: "《滋养层生物学与病理学》", 30 May 2008, 浙江大学出版社 * |
| 许良中: "《实用肿瘤病理方法学》", 30 September 1997, 上海医科大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112034180A (en) * | 2020-08-18 | 2020-12-04 | 四川大学华西第二医院 | Use and product of keratin 1 in ectopic pregnancy |
| CN112034180B (en) * | 2020-08-18 | 2021-09-24 | 四川大学华西第二医院 | Application and products of keratin 1 in ectopic pregnancy |
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