CN107190328B - Decidua and villus matched cDNA chip - Google Patents
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Abstract
The invention relates to an ecdysis and villus matched cDNA chip, which is prepared by the following method: the decidua and villus tissues of early-stage abortion women are taken, RNA is extracted and is reversely transcribed into cDNA, and the cDNA is preset in a PCR reaction plate to be manufactured into a cDNA chip. The invention also provides a preparation method and application of the cDNA chip. Its advantages are: each abortive woman takes decidua and villus tissue cDNA to form pairing respectively, and the pairing is concentrated on the same chip, so that the function presentation of a maternal-fetal interface on a physiological structure on an in vitro chip is realized. The kit has the advantages of high flux, strong specificity, high sensitivity, simple and quick operation, high quantitative accuracy, strong result reliability, simple result analysis, complete case clinical information of a sample and the like, and can be used for quantitatively detecting the expression condition of a target gene in different patients and carrying out deep clinical information correlation analysis.
Description
Technical Field
The invention relates to the technical field of biochips, in particular to an ecdysis and villus matched cDNA chip.
Background
Biochip refers to high-throughput microarray of high-density tissues, cells, proteins, nucleic acids, saccharides and other biological components coated on a solid support such as silicon wafer, nylon membrane, etc. The high-flux biochip is used to detect the detected sample by specific molecular hybridization and dyeing technology, so that a great amount of molecular detection information can be accurately and quickly obtained. The method is widely applied to the fields of life science research, clinical medical molecular diagnosis, biological antibody targeted drug screening and verification and the like.
Normal pregnancy is a complex physiological process in which the fetus, while carrying HLA antigens inherited from the paternal lineage, does not elicit maternal rejection of this particular semi-allogenic "natural transplant" to the fetus. A complex molecular dialogue mechanism exists between the pregnant mother and the fetus to maintain the normal development of the fetus placenta. In the early stage of pregnancy, fetal villous ectotrophic cells (EVT) invade decidua tissues and directly contact with maternal Decidua Immune Cells (DIC) and Decidua Stromal Cells (DSC) to form a finely regulated maternal-fetal interface. The maternal-fetal interface is mainly composed of 3 cells: embryonic-derived trophoblasts (trophoblast, Tro), maternal-derived Decidual Stromal Cells (DSC), and Decidual Immune Cells (DIC). As the only trophoblast carrying paternal antigen in the maternal-fetal interface, invasion and migration are key steps in blastocyst implantation, placenta development and establishment of maternal-fetal relationship. Besides being involved in decidua nutrition supply, the decidua stromal cells also secrete a plurality of active molecules to regulate blastocyst implantation and placenta development, and as immune potential cells, participate in antigen presentation and secrete cytokines to play an important role in immune regulation. Decidua immune cells are the basis of maternal-fetal immune tolerance, and locally play a role in immune regulation different from that of the periphery at the maternal-fetal interface by expressing a special activation marker and secreting a large amount of cytokines. The female-fetal interface presents Th2 type immunodominance and regulatory T cell (Treg) expansion phenomena during normal physiological pregnancy, and once this tolerance state is broken, Th1 type immune response predominates, which leads to pregnancy failure or pregnancy complications such as recurrent abortion, preeclampsia, etc.
Intensive research on maternal-fetal interface formation is an important approach and means to reveal normal pregnancy maintenance mechanisms and pathological pregnancy pathogenesis.
Chinese patent document CN103614473A discloses a cDNA chip for detecting the drug resistance level of bactrocera dorsalis against trichlorfon and beta-cypermethrin, a preparation method and application thereof. Chinese patent document CN101235420A discloses a chip of a drought-resistant cDNA expression profile of stigmata arundinacea and application thereof. Chinese patent document CN101245386A discloses a cDNA chip for detecting a variety of tumor suppressor genes. Chinese patent document CN101942515A discloses a cDNA chip for detecting expression information of related genes of tea tree secondary metabolism. At present, no patent and literature reports on the technology and application of preparing tissue cDNA chip by using decidua and villus tissues exist.
Disclosure of Invention
The invention aims to provide an decidua and villus matched cDNA chip aiming at the defects in the prior art.
Another objective of the invention is to provide a method for preparing an ecdysis and villus matched cDNA chip.
Another objective of the invention is to provide an application of the decidua and villus matched cDNA chip.
In order to achieve the purpose, the invention adopts the technical scheme that: an decidua and villus matched cDNA chip, which is prepared by the following method: taking decidua and villus tissue, extracting RNA, reverse transcribing into cDNA, pre-setting in PCR reaction plate and making into cDNA chip.
Further, the PCR reaction plate is 96-well or 384-well.
Further, the decidua and villus tissues are decidua and villus tissues of women who have abortion in early pregnancy.
In order to achieve the second object, the invention adopts the technical scheme that: the preparation method comprises the following steps: the decidua and villus tissues of early-stage abortion women are taken, RNA is extracted and is reversely transcribed into cDNA, and the cDNA is preset in a PCR reaction plate to be manufactured into a cDNA chip.
Further, the method for extracting RNA is according to RNA extraction kit OligotexTMThe process is carried out.
Further, the reverse transcription into cDNA was carried out according to the Stratagene cDNA library construction kit set instructions.
Further, placing the prepared cDNA chip into a wet box for hydration, taking out the cDNA chip, placing the cDNA chip on a platform at the temperature of 90-120 ℃ for drying for 30 seconds to 1 minute, and then irradiating by ultraviolet light for 10-40 minutes for fixation.
Furthermore, the sample solution for preparing the cDNA chip was 2 XSSC buffer, 50% dimethyl sulfoxide, 3 XSSC +1.5mol/L betaine, or 150mmol/L sodium phosphate.
In order to achieve the third object, the invention adopts the technical scheme that: the cDNA chip is applied to the preparation of products for detecting gene expression.
Furthermore, the product is used for pathological pregnancy etiology screening and molecular typing, novel marker screening and verification, individualized treatment patient screening and curative effect and prognosis judgment; the pathological pregnancy is gestational time limit abnormality, ectopic pregnancy, pregnancy-specific diseases, late pregnancy bleeding or other pregnancy diseases, wherein the gestational time limit abnormality comprises abortion, premature delivery and overdue pregnancy, the pregnancy-specific diseases comprise pregnancy hypertension diseases and hyperemesis gravidarum, the late pregnancy bleeding comprises placenta prearranged and placental premolarity, and the other pregnancy diseases comprise amniotic fluid volume abnormality, fetal distress, premature rupture of fetal membranes, multiple pregnancy and maternal-fetal blood type incompatibility.
The invention has the advantages that:
1. each abortive woman takes decidua and villus tissue cDNA to form pairing respectively, and the pairing is concentrated on the same chip, so that the function presentation of a maternal-fetal interface on a physiological structure on an in vitro chip is realized.
2. High flux, one chip can integrate at least 45 patients with decidua and villus matched tissue cDNA, so that a large amount of information can be obtained once, and the efficiency is improved by hundreds of times.
3. The kit has the advantages of strong specificity, high sensitivity, simple and quick operation, high quantitative accuracy, strong result reliability, simple result analysis, complete case clinical information of a sample and the like, and can be used for quantitatively detecting the expression conditions of target genes in different patients and carrying out deep clinical information correlation analysis.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
The present invention takes decidua and villus tissue of abortive women in early pregnancy, extracts RNA, carries out reverse transcription to obtain cDNA, and is preset in a PCR reaction plate (96 holes or 384 holes) to prepare a cDNA chip, which is a cDNA array for detecting the expression of a target gene by a real-time fluorescence quantitative method. It is characterized in that: 1) each abortive woman takes decidua and villus tissue cDNA to form pairing respectively, and the pairing is concentrated on the same chip, so that the function presentation of a maternal-fetal interface on a physiological structure on an in vitro chip is realized; 2) high flux, one chip can integrate at least 45 patients' decidua and villus matched tissue cDNA, so as to obtain a large amount of information once, and improve efficiency by hundreds of times; 3) the kit has the advantages of strong specificity, high sensitivity, simple and quick operation, high quantitative accuracy, strong result reliability, simple result analysis, complete case clinical information of a sample and the like, and can be used for quantitatively detecting the expression conditions of target genes in different patients and carrying out deep clinical information correlation analysis.
EXAMPLE 1 preparation of cDNA chip
1. Extraction of RNA from human decidua tissue
Collecting decidua and villus tissues in the uterine curettage, recording the age, BMI, abortion times, treatment scheme, endocrine, autoantibody, hemagglutination, immunization, chromosome screening or hereditary thrombosis susceptibility screening data parameters of the patient, and establishing a clinical data database. Oligotex using RNA extraction kitTMRNA is directly extracted from decidua and villus tissues of women who have abortion in early pregnancy. The decidua and villus tissues are from the first women and infants health care institute in Shanghai (requiring that the menstrual cycle is regular for 28-32 days, the last menstruation is exact, no abdominal pain and vaginal bleeding history, no contraceptive history and anti-early pregnancy history). The whole material taking process strictly complies with aseptic operation. The method and the steps for extracting RNA should strictly refer to the RNA extraction kit OligotexTMThe process is carried out.
2. construction of cDNA library
This was done according to the Stratagene cDNA library construction Serial kit instructions. Synthesizing a first cDNA chain by using the synthesized mRNA as a template, oligo (dT) as a guide and catalysis of reverse transcriptase RT; second strand synthesis was performed with cDNA synthetase I. Then, the cDNA ends were modified with pfuDNA polymerase. EcoRI linkers were ligated to the ends of the DNA fragments, and the EcoRI ends were phosphorylated using T4 polynucleotide kinase. Allowing it to be ligated to a non-phosphorylated EcoRI site thereafter. Inactivating the activity of the T4 polynucleotide kinase enzyme. The cDNA was digested with Xho I enzyme, cleaved into small fragments, precipitated with ethanol and dissolved in 1 XSTE buffer. The cDNA was subjected to gel filtration chromatography using Sepharose CL-2B gel filtration column, samples were collected step by step, and larger cDNA fragments were pooled. The quality of the obtained cDNA fragments was verified using EB plates.
The cDNA fragments were ligated with the Uni-ZAP XR vector. The ligation products were packaged using the ZAP-cDNA Gigapack III gold cloning kit to form packaged phage particles. The VCS257 bacteria and wild type DNA provided by the kit were used as pre-experiments to test the packaging efficiency of the kit. XL1-blue MRF' host bacteria were then cultured, infected with the packaging product and plated on agar plates.
3. Preparation of cDNA chip
Taking 10 ul of cDNA solution or PCR product, adding 3 ul of 2 × SSC buffer solution, mixing, transferring to 96-well plate, placing the plate on automatic sample spotting machine platform, sequentially using 2000 spots/cm2The cDNA chip was prepared on a glass slide. Placing the prepared cDNA chip in a wet box for hydration for 5-10 minutes, taking out the cDNA chip, placing the cDNA chip on a platform at 90-120 ℃ for drying for 30 seconds to 1 minute, placing the cDNA chip on a UV transmission analyzer with the surface facing downwards, and using the power of 0.5mW/cm2Irradiating the mixture for 10 to 40 minutes by using 320nm ultraviolet light for fixing.
EXAMPLE 2 preparation of cDNA chip (II)
1. Extraction of RNA from human decidua tissue
Collecting decidua and villus tissues in the uterine curettage, recording the age, BMI, abortion times, treatment scheme, endocrine, autoantibody, hemagglutination, immunization, chromosome screening or hereditary thrombosis susceptibility screening data parameters of the patient, and establishing a clinical data database. Oligotex using RNA extraction kitTMRNA is directly extracted from decidua and villus tissues of women who have abortion in early pregnancy. The decidua and villus tissue is obtained from the first women and infants health care institute (requirement: menstrual cycle standard)The menstruation is 28-32 days, the last menstruation is exact, no history of abdominal pain and vaginal bleeding, no history of contraceptive and anti-early pregnancy drugs). The whole material taking process strictly complies with aseptic operation. The method and the steps for extracting RNA should strictly refer to the RNA extraction kit OligotexTMThe process is carried out.
2. construction of cDNA library
This was done according to the Stratagene cDNA library construction Serial kit instructions. Synthesizing a first cDNA chain by using the synthesized mRNA as a template, oligo (dT) as a guide and catalysis of reverse transcriptase RT; second strand synthesis was performed with cDNA synthetase I. Then, the cDNA ends were modified with pfuDNA polymerase. EcoRI linkers were ligated to the ends of the DNA fragments, and the EcoRI ends were phosphorylated using T4 polynucleotide kinase. Allowing it to be ligated to a non-phosphorylated EcoRI site thereafter. Inactivating the activity of the T4 polynucleotide kinase enzyme. The cDNA was digested with Xho I enzyme, cleaved into small fragments, precipitated with ethanol and dissolved in 1 XSTE buffer. The cDNA was subjected to gel filtration chromatography using Sepharose CL-2B gel filtration column, samples were collected step by step, and larger cDNA fragments were pooled. The quality of the obtained cDNA fragments was verified using EB plates.
The cDNA fragments were ligated with the Uni-ZAP XR vector. The ligation products were packaged using the ZAP-cDNA Gigapack III gold cloning kit to form packaged phage particles. The VCS257 bacteria and wild type DNA provided by the kit were used as pre-experiments to test the packaging efficiency of the kit. XL1-blue MRF' host bacteria were then cultured, infected with the packaging product and plated on agar plates.
3. Preparation of cDNA chip
Taking 10 ul of cDNA solution or PCR product, adding 3 ul of 50% dimethyl sulfoxide, mixing, transferring to 96-well plate, placing the plate on automatic sample spotting machine platform at 2000 spots/cm in sequence2The cDNA chip was prepared on a glass slide. Placing the prepared cDNA chip in a wet box for hydration for 5-10 minutes, taking out the cDNA chip, placing the cDNA chip on a platform at 90-120 ℃ for drying for 30 seconds to 1 minute, placing the cDNA chip on a UV transmission analyzer with the surface facing downwards, and using the power of 0.5mW/cm2Irradiating the mixture for 10 to 40 minutes by using 320nm ultraviolet light for fixing.
EXAMPLE 3 preparation of cDNA chip (III)
1. Extraction of RNA from human decidua tissue
Collecting decidua and villus tissues in the uterine curettage, recording the age, BMI, abortion times, treatment scheme, endocrine, autoantibody, hemagglutination, immunization, chromosome screening or hereditary thrombosis susceptibility screening data parameters of the patient, and establishing a clinical data database. Oligotex using RNA extraction kitTMRNA is directly extracted from decidua and villus tissues of women who have abortion in early pregnancy. The decidua and villus tissues are from the first women and infants health care institute in Shanghai (requiring that the menstrual cycle is regular for 28-32 days, the last menstruation is exact, no abdominal pain and vaginal bleeding history, no contraceptive history and anti-early pregnancy history). The whole material taking process strictly complies with aseptic operation. The method and the steps for extracting RNA should strictly refer to the RNA extraction kit OligotexTMThe process is carried out.
2. construction of cDNA library
This was done according to the Stratagene cDNA library construction Serial kit instructions. Synthesizing a first cDNA chain by using the synthesized mRNA as a template, oligo (dT) as a guide and catalysis of reverse transcriptase RT; second strand synthesis was performed with cDNA synthetase I. Then, the cDNA ends were modified with pfuDNA polymerase. EcoRI linkers were ligated to the ends of the DNA fragments, and the EcoRI ends were phosphorylated using T4 polynucleotide kinase. Allowing it to be ligated to a non-phosphorylated EcoRI site thereafter. Inactivating the activity of the T4 polynucleotide kinase enzyme. The cDNA was digested with Xho I enzyme, cleaved into small fragments, precipitated with ethanol and dissolved in 1 XSTE buffer. The cDNA was subjected to gel filtration chromatography using Sepharose CL-2B gel filtration column, samples were collected step by step, and larger cDNA fragments were pooled. The quality of the obtained cDNA fragments was verified using EB plates.
The cDNA fragments were ligated with the Uni-ZAP XR vector. The ligation products were packaged using the ZAP-cDNA Gigapack III gold cloning kit to form packaged phage particles. The VCS257 bacteria and wild type DNA provided by the kit were used as pre-experiments to test the packaging efficiency of the kit. XL1-blue MRF' host bacteria were then cultured, infected with the packaging product and plated on agar plates.
3. Preparation of cDNA chip
Taking 10 mu L of cDNA solution or PCR product, adding 3 × SSC +1.5 mol/L3 mu L, mixing uniformly, transferring to a 96-well plate, placing the plate on an automatic sample spotting machine platform, sequentially using 2000 spots/cm2The cDNA chip was prepared on a glass slide. Placing the prepared cDNA chip in a wet box for hydration for 5-10 minutes, taking out the cDNA chip, placing the cDNA chip on a platform at 90-120 ℃ for drying for 30 seconds to 1 minute, placing the cDNA chip on a UV transmission analyzer with the surface facing downwards, and using the power of 0.5mW/cm2Irradiating the mixture for 10 to 40 minutes by using 320nm ultraviolet light for fixing.
EXAMPLE 4 preparation of cDNA chip (IV)
1. Extraction of RNA from human decidua tissue
Collecting decidua and villus tissues in the uterine curettage, recording the age, BMI, abortion times, treatment scheme, endocrine, autoantibody, hemagglutination, immunization, chromosome screening or hereditary thrombosis susceptibility screening data parameters of the patient, and establishing a clinical data database. Oligotex using RNA extraction kitTMRNA is directly extracted from decidua and villus tissues of women who have abortion in early pregnancy. The decidua and villus tissues are from the first women and infants health care institute in Shanghai (requiring that the menstrual cycle is regular for 28-32 days, the last menstruation is exact, no abdominal pain and vaginal bleeding history, no contraceptive history and anti-early pregnancy history). The whole material taking process strictly complies with aseptic operation. The method and the steps for extracting RNA should strictly refer to the RNA extraction kit OligotexTMThe process is carried out.
2. construction of cDNA library
This was done according to the Stratagene cDNA library construction Serial kit instructions. Synthesizing a first cDNA chain by using the synthesized mRNA as a template, oligo (dT) as a guide and catalysis of reverse transcriptase RT; second strand synthesis was performed with cDNA synthetase I. Then, the cDNA ends were modified with pfuDNA polymerase. EcoRI linkers were ligated to the ends of the DNA fragments, and the EcoRI ends were phosphorylated using T4 polynucleotide kinase. Allowing it to be ligated to a non-phosphorylated EcoRI site thereafter. Inactivating the activity of the T4 polynucleotide kinase enzyme. The cDNA was digested with Xho I enzyme, cleaved into small fragments, precipitated with ethanol and dissolved in 1 XSTE buffer. The cDNA was subjected to gel filtration chromatography using Sepharose CL-2B gel filtration column, samples were collected step by step, and larger cDNA fragments were pooled. The quality of the obtained cDNA fragments was verified using EB plates.
The cDNA fragments were ligated with the Uni-ZAP XR vector. The ligation products were packaged using the ZAP-cDNA Gigapack III gold cloning kit to form packaged phage particles. The VCS257 bacteria and wild type DNA provided by the kit were used as pre-experiments to test the packaging efficiency of the kit. XL1-blue MRF' host bacteria were then cultured, infected with the packaging product and plated on agar plates.
3. Preparation of cDNA chip
Taking 10 ul of cDNA solution or PCR product, adding 3 ul of 150mmol/L sodium phosphate sample solution, mixing, transferring to 96-well plate, placing the plate on automatic sample spotting machine platform at 2000 spots/cm in sequence2The cDNA chip was prepared on a glass slide. Placing the prepared cDNA chip in a wet box for hydration for 5-10 minutes, taking out the cDNA chip, placing the cDNA chip on a platform at 90-120 ℃ for drying for 30 seconds to 1 minute, placing the cDNA chip on a UV transmission analyzer with the surface facing downwards, and using the power of 0.5mW/cm2Irradiating the mixture for 10 to 40 minutes by using 320nm ultraviolet light for fixing.
EXAMPLE 5 evaluation of cDNA chip
The cDNA chips prepared in examples 1 to 4 were hybridized with Cy 5-labeled probe, and the scanned images were analyzed: the shapes of the points in the embodiments 1 to 4 are all relatively regular, wherein the most regular points in the embodiment 1 are; the DNA immobilization efficiencies of examples 1-4 were all high (> 50%), with example 1 being the highest (95%). Example 1 the spotting time was short, the spot shape was regular, and the immobilization efficiency was high.
EXAMPLE 6 use of cDNA chip
The mRNA obtained by matching decidua and villi was labeled, and added to the cDNA chip prepared in example 1 to hybridize, and the target gene was searched by differential expression of decidua and villi tissues of normal pregnant women on the cDNA chip.
1. Marking of samples
Extracting total mRNA in decidua and villus tissues of normal pregnant women and early pregnant women after abortion, performing reverse transcription by using Cy3 and Cy5 for fluorescence labeling, and mixing the labeled fluorescence samples 1:1 for later use.
2. Hybridization and detection
The labeled sample solution was applied to a previously heat-denatured cDNA chip, covered with a cover glass, placed in a wet box for hybridization for 5 hours, then washed in 2 XSSC, 0.1% SDS solution for 5 minutes, 0.1 XSSC, 0.1% SDS solution for 5 minutes, rinsed several times in 0.1 XSSC solution, and air-dried.
Scanning the hybridized and air dried cDNA microarray chip with laser confocal scanner under proper condition to detect its hybridization signal.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (3)
1. An decidua and villus matched cDNA chip is characterized in that the cDNA chip is prepared by decidua and villus tissues taken from patients with recurrent early pregnancy abortion and is prepared by the following method: (1) extracting RNA from decidua and villus tissues of patients with recurrent early pregnancy abortion; (2) constructing a cDNA library: taking RNA to synthesize mRNA, using mRNA as a template, oligo (dT) as a guide, and catalyzing by reverse transcriptase RT to synthesize a first chain of cDNA; synthesizing a second strand by using cDNA synthetase I, modifying the cDNA terminal by pfuDNA polymerase, connecting an EcoRI connector at the terminal, phosphorylating the EcoRI terminal by using T4 polynucleotide kinase to enable the EcoRI terminal to be connected with a non-phosphorylated EcoRI site behind the T4 polynucleotide kinase to inactivate the activity of the T4 polynucleotide kinase, digesting the cDNA by using Xho I enzyme to enable the cDNA to be split into small fragments, precipitating by using ethanol, dissolving the small fragments in 1 XSTE buffer solution, carrying out gel filtration chromatography on the cDNA by using a Sepharose CL-2B gel filtration column, collecting samples step by step, combining the cDNA fragments, connecting the cDNA fragments with a Uni-ZAP XR carrier, and preparing a cDNA solution; (3) constructing a cDNA chip: and adding 2 XSSC buffer solution into the cDNA solution or PCR product, transferring the cDNA solution or PCR product into a 96-well plate or a 384-well plate, placing the plate on an automatic spotting machine platform, and preparing a cDNA chip on a glass slide.
2. The method for preparing cDNA chip according to claim 1, wherein the method comprises the steps of: (1) extracting RNA from decidua and villus tissues of patients with recurrent early pregnancy abortion; (2) constructing a cDNA library: taking RNA to synthesize mRNA, using mRNA as a template, oligo (dT) as a guide, and catalyzing by reverse transcriptase RT to synthesize a first chain of cDNA; synthesizing a second strand by using cDNA synthetase I, modifying the cDNA terminal by pfuDNA polymerase, connecting an EcoRI connector at the terminal, phosphorylating the EcoRI terminal by using T4 polynucleotide kinase to enable the EcoRI terminal to be connected with a non-phosphorylated EcoRI site behind the T4 polynucleotide kinase to inactivate the activity of the T4 polynucleotide kinase, digesting the cDNA by using Xho I enzyme to enable the cDNA to be split into small fragments, precipitating by using ethanol, dissolving the small fragments in 1 XSTE buffer solution, carrying out gel filtration chromatography on the cDNA by using a Sepharose CL-2B gel filtration column, collecting samples step by step, combining the cDNA fragments, connecting the cDNA fragments with a Uni-ZAP XR carrier, and preparing a cDNA solution; (3) constructing a cDNA chip: and adding 2 XSSC buffer solution into the cDNA solution or PCR product, transferring the cDNA solution or PCR product into a 96-well plate or a 384-well plate, placing the plate on an automatic spotting machine platform, and preparing a cDNA chip on a glass slide.
3. The use of the cDNA chip according to claim 1 in the preparation of products for detecting gene expression, wherein said products are used for pathological pregnancy etiology screening and molecular typing, novel marker screening and verification, individualized treatment patient screening and therapeutic effect and prognosis judgment; the pathological pregnancy is gestational time limit abnormality, ectopic pregnancy, pregnancy-specific diseases, late pregnancy bleeding or other pregnancy diseases, wherein the gestational time limit abnormality comprises abortion, premature delivery and overdue pregnancy, the pregnancy-specific diseases comprise pregnancy hypertension diseases and hyperemesis gravidarum, the late pregnancy bleeding comprises placenta prearranged and placental premolarity, and the other pregnancy diseases comprise amniotic fluid volume abnormality, fetal distress, premature rupture of fetal membranes, multiple pregnancy and maternal-fetal blood type incompatibility.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1263161A (en) * | 1999-12-29 | 2000-08-16 | 中国科学院上海冶金研究所 | cDNA microarray chip, preparation method and its application |
CN1940557A (en) * | 2006-09-28 | 2007-04-04 | 广东省人民医院 | Gene chip sampling liquid and its use |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7790463B2 (en) * | 2006-02-02 | 2010-09-07 | Yale University | Methods of determining whether a pregnant woman is at risk of developing preeclampsia |
WO2008030065A1 (en) * | 2006-09-08 | 2008-03-13 | Macrogen Inc. | Microarray chip and method for detection of chromosomal abnormality |
US20080233583A1 (en) * | 2007-02-20 | 2008-09-25 | Regents Of The University Of California | Biomarkers for preeclampsia |
US20140113288A1 (en) * | 2012-10-22 | 2014-04-24 | Academia Sinica | Methods of identifying risk of preeclampsia and pregnancy-related disorders |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1263161A (en) * | 1999-12-29 | 2000-08-16 | 中国科学院上海冶金研究所 | cDNA microarray chip, preparation method and its application |
CN1940557A (en) * | 2006-09-28 | 2007-04-04 | 广东省人民医院 | Gene chip sampling liquid and its use |
Non-Patent Citations (7)
Title |
---|
E-钙粘蛋白在胚胎停育患者绒毛和蜕膜中的差异性表达;李三阳 等;《陕西医学杂志》;20130228;第42卷(第2期);全文 * |
Global analysis of differentially expressed genes in early gestational decidua and chorionic villi using a 9600 human cDNA microarray.;Chen HW. et al.,;《Mol Hum Reprod.》;20020531;第8卷(第5期);摘要 * |
MMP-2在胚胎停育患者绒毛和蜕膜组织中的表达及其临床意义;李智慧 等;《临床合理用药》;20160331;第9卷(第3C期);全文 * |
复发性流产患者子宫蜕膜基因表达谱;张娟娟 等;《中国细胞生物学学报》;20130328;第35卷(第4期);全文 * |
子宫内膜基质细胞分化过程中细胞周期界面的调控机制研究;胡琳莉;《中国博士学位论文全文数据库(电子期刊)》;20091115(第11期);全文 * |
早期自然流产蜕膜组织基因表达谱和蛋白质组研究;余秋波;《中国博士学位论文全文数据库(电子期刊)医药卫生科技辑》;20080215(第2期);全文 * |
自然流产患者绒毛和蜕膜中ZEB1的差异性表达及意义;李三阳 等;《现代生物医学进展》;20140131;第14卷(第2期);全文 * |
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