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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies
  • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• • 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
  • G01N33/6893—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 diseases not provided for elsewhere
• • 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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2503/00—Use of cells in diagnostics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/36—Gynecology or obstetrics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/38—Pediatrics
  • G01N2800/385—Congenital anomalies
  • G01N2800/387—Down syndrome; Trisomy 18; Trisomy 13

Definitions

• the invention relates to cultures of trisomy 21 cytotrophoblasts, and to their uses.
• Trisomy 21, responsible for Down syndrome more commonly known as mongolism, is the most common autosomal trisomy since it is observed on nearly 1 in 800 newborns during childbirth [FERGUSON-SMITH et al., Prenat. Diagn. , 4, 5-44, 1984)]. It is the major genetic cause of mental retardation [CUNNINGHAM et al., Williams Obstetrics, ed. 19, 919-938, (1993)].
• the prenatal diagnosis of trisomy 21 is essentially based on the establishment of the fetal karyotype, an examination practiced most often on fetal cells of the amniotic fluid collected by amniocentesis, more rarely on the trophoblastic cells taken by biopsy of the cho ⁇ al villi.
• VAN-LITH Prenat. Diagn., 12, 495-504, (1992)].
• this screening method is called "triple test”.
• the present invention aims to provide new means of studying cellular and molecular dysfunctions occurring during placental development in trisomy 21, as well as new markers of trisomy 21.
• the inventors have put in culture cytotrophoblastic cells from normal placentas and placentas with fetal trisomy 21.
• the cytotrophoblastic cell is the key cell of the human placenta. It is differentiated m vi tro and m if you at the placental level as a syncytiotrophoblast.
• the syncytiotrophoblast is the endocrine tissue of the human placenta. It produces steroid and polypeptide hormones at very high rates (several grams per day).
• the cytotrophoblasts isolated and purified from the normal placenta aggregate then merge to form the syncytiotrophoblast.
• This highly polarized cell secretes m vi tro into the culture medium the hormones which it normally pours into the maternal circulation.
• the inventors have isolated, purified and cultured cytotrophoblasts from trisomy 21 placentas to all terms of pregnancy, and have compared their development, as well as their ability to express and secrete different peptide hormones, with that of normal cytotrophoblasts.
• transformed cytotrophoblasts overexpressing copper and zinc-dependent superoxide dismutase (Cu / Zn SOD), exhibited the same abnormalities in syncytiotrophoblast formation, and decreased production of hCG ⁇ , the hCG ⁇ , hPL, and PGH.
• Cu / Zn SOD gene is carried by the long arm of chromosome 21, and this enzyme is one of those that are overexpressed in subjects with Down's syndrome.
• the subject of the present invention is a culture of cytotrophoblasts, characterized in that said cytotrophoblasts are trisomal for human chromosome 21, or for a region thereof comprising at least the gene coding for Cu / Zn superoxide dismutase, and in that '' they express after 3 days of culture at least one peptide hormone chosen from human placental lactogen (hPL), the human cho ⁇ onic gonadotroph
• hCG human cho ⁇ onic gonadotrophm ⁇
• PGH human placental growth hormone
• normal cytotrophoblasts is understood here to mean cytotrophoblasts having a normal karyotype, and which are capable of forming a syncytiotrophoblast when cultivated in vitro.
• Cytotrophoblast cultures according to the invention can be obtained from trisomy 21 placentas, by enzymatic digestion in several stages, followed by purification on a Ficoll gradient.
• the cells thus isolated can then be cultured in a conventional manner, according to the techniques usually used for cultures of normal cytotrophoblasts.
• Cytotrophoblast cultures according to the invention can be used as an in vitro model to study the anomalies of differentiation of the syncytiotrophoblast in trisomy 21, for example cell fusion anomalies.
• the cultures of cytotrophoblasts in accordance with the invention can be used to identify and characterize biological molecules, in particular proteins, released into the blood maternal by placentas of fetuses with Down's syndrome, and quantitatively and / or qualitatively different from the biological molecules released into maternal blood by placentas of normal fetuses, and thus being able to constitute markers of Down's syndrome.
• cultures of cytotrophoblasts in accordance with the invention can be used for obtaining differential expression libraries of nucleic acids between normal cytotrophoblasts and trisomal cytotrophoblasts for chromosome 21, and / or between trisomal cytotrophoblasts for chromosome 21 at different stages of culture.
• the present invention thus encompasses differential expression libraries of nucleic acids obtained using at least one culture of cytotrophoblasts according to the invention.
• Such libraries can consist, for example: of a population of total or partial cDNA sequences of genes expressing themselves in the cytotrophoblast cultures according to the invention, and not expressing themselves in the cytotrophoblast cultures normal to same stage of syncytiotrophoblast formation; a population of total or partial cDNA sequences of genes not expressing themselves in the cultures of cytotrophoblasts in accordance with the invention, and expressing themselves in cultures of normal cytotrophoblasts at the same stage of formation of the syncytiotrophoblast; of a population of total or partial sequences of cDNAs of genes whose expression is greater in the cultures of cytotrophoblasts according to the invention than in the cultures of normal cytotrophoblasts at the same stage of formation of the syncytiotrophoblast; a population of total or partial sequences of cDNAs of
• Differential expression libraries of nucleic acids according to the invention can be obtained by methods known in themselves to those skilled in the art.
• methods such as DDRT-PCR [LIANG and PARDEE, Science, 257, 967-971, (1992)], subtractive hybridization [JIANG and FISHER, Mol. Cell. Different., 1, 285-299, (1993)], RAP-PCR [MCCLELLAND and WELSH, PCR Methods & Applications, 4, S66-81, (1994)], SAGE analysis [VELCULESCU et al., Science , 270, 484-487, 1995], and their different variants. They can then be used in particular to identify, characterize and clone genes involved in abnormalities in the differentiation of the syncytiotrophoblast, and / or to study the regulatory mechanisms of these genes.
• Cytotrophoblast cultures in accordance with the invention can also be used to study the expression and / or differential secretion of proteins during the various stages of syncytiotrophoblast formation, by comparing their expression and / or secretion profiles. proteins with those of normal cytotrophoblast cultures, or with those of other cytotrophoblast cultures according to the invention, at another stage of development or cultivated under different conditions.
• the comparison can be carried out on total proteins, or on protein subpopulations, for example on intracellular proteins or on those secreted in the culture medium, on glycosylated proteins or on those which are not, etc. .
• the glycosylation or the absence of glycosylation of a protein can for example be detected by isoelectric focusing; according to their glycosylation (in particular their sialic acid content), the different glycoforms will migrate until they arrive in a pH zone corresponding to their isoelectric point; this isoelectric focusing profile can be compared with that obtained after treatment with an enzyme (for example neurammidase or mannosidase) which hydrolyzes the glycans.
• Glycosylated proteins can also be detected by affinity electrophoresis on a gel containing a lectme; the glycoforms are separated according to their affinity for lectme.
• Proteins separated on gel can for example be detected by conventional staining or by immunodetection after transfer to nitrocellulose, and their quantity can be evaluated by densitometry. The proteins for which differential expression is observed can then be purified from the gels to complete their identification and characterization.
• the proteins in the culture medium can also be analyzed by chromatography. For example, to detect and / or quantify glycosylated proteins, it is possible to perform affinity chromatography on lectme.
• the comparison of the expression of mRNAs and of the secretion of proteins in cultures of cytotrophoblasts in accordance with the invention and in cultures of normal cytotrophoblasts enabled the inventors to identify proteins which may constitute serum markers for Down's syndrome.
• the inventors have in fact found that the synthesis of certain peptide hormones, which is very important in the cultures of normal cytotrophoblasts, at the time of the formation of the syncytiotrophoblast, was very weak in the cultures of cytotrophoblasts in accordance with the invention. These serum hormones should therefore be present in smaller quantities in the serum of women with a Down's syndrome fetus than in that of women with a normal fetus.
• hCG which is one of the peptide hormones whose expression is reduced in Cytotrophoblast cultures according to the invention is, on the contrary, known to be present in a greater quantity in the serum of women carrying a trisomic fetus 21 than in that of women carrying a normal fetus.
• the proteins which are produced by the cytotrophoblasts in non-glycosylated form will, if they are produced in less quantity, also present in smaller quantity in the serum of women carrying a fetus with Down's syndrome than in that of women carrying a normal fetus, whereas on the contrary the proteins produced in glycosylated form, like hCG, will be glycosylated abnormally in the cytotrophoblasts trisomiques 21, and even if they are produced in smaller quantity, will be found in more quantity important in maternal blood.
• the inventors carried out the assay, in sera of women carrying a trisomic fetus 21 and in sera of women carrying a normal fetus, 1 hCG and another glycosylated hormone, l growth hormone, as well as that of the following hormones: human placental lactogen (hPL) and leptm, which are not produced by cytotrophoblasts in glycosylated form.
• hPL human placental lactogen
• leptm which are not produced by cytotrophoblasts in glycosylated form.
• the subject of the present invention is a method for detecting a risk of trisomy 21 in a fetus, characterized in that it comprises the measurement, in a sample of a biological fluid obtained from the mother, of at least one protein chosen from:
• non-glycosylated proteins secreted by a culture of cytotrophoblasts according to the invention in a smaller amount than by a culture of normal cytotrophoblasts; - the non-glycosylated proteins secreted by a culture of cytotrophoblasts in accordance with the invention in greater quantity than by a culture of normal cytotrophoblasts; glycosylated proteins, secreted by a culture of cytotrophoblasts according to the invention, with the exception of hCG.
• Proteins "secreted by a culture of cytotrophoblasts according to the invention in a smaller amount than by a culture of normal cytotrophoblasts” also include proteins which are not secreted by cytotrophoblasts according to the invention but are secreted by normal cytotrophoblasts .
• proteins "secreted by a culture of cytotrophoblasts according to the invention in greater quantity than by a culture of normal cytotrophoblasts” also include proteins which are secreted by cytotrophoblasts according to the invention but are not by normal cytotrophoblasts.
• biological liquid is meant in particular serum and urine.
• the assay of said non-glycosylated protein will preferably be carried out from a serum sample. Proteins that can be used can be easily identified by those skilled in the art, by comparing the protein secretion profiles of the 2 cultures, and / or by detecting glycosylation or the absence of glycosylation, as indicated above.
• a separate dose of one or more of said proteins different from the average dose encountered in women carrying a normal fetus indicates a risk of Down's syndrome in the fetus.
• a dose of said protein greater than the average dose encountered in women with a normal fetus indicates a risk of Down's syndrome in the fetus.
• cytotrophoblastic cell expressed secreted proteins such as: IGF 2 , Thymosme beta 10, Calgranulme B, "Macrophage colony stimulatmg factor”, “C. reactive protem precursor ", EGF,” FMLP-related receptor 1 ", small cytok e mductible A 5, IGF BPi,” Hepatocyte growth factor like protem “, PDGF, and some of the IL-1 mterleukmes IL-18. They also found that cytotrophoblastic cells expressed the precursor of the amyloid peptide (APP). They also observed that this expression was much greater in the cultures of cytotrophoblasts according to the invention than in the cultures of normal cytotrophoblasts.
• APP amyloid peptide
• the precursor of the amyloid peptide is a transmembrane protein (SWISS-PROT access number:
• P05067) the extracellular domain of which is released in soluble form after proteolytic cleavage by an ⁇ -secretase activity (release of a soluble protein comprising the amino acids 18-699 of APP) or by a ⁇ -secretase activity (release of a soluble protein comprising amino acids 18-671 of APP), (for review, see OCTAVE et al., Medicine / Sciences, 11, 1251-1259, 1995).
• the extracellular domain of the APP protein, or fragments thereof therefore appears usable, in accordance with the invention, as maternal serum markers of trisomy 21.
• the inventors have also identified hPL and leptm, among the non-glycosylated proteins secreted by normal and non-secreted cytotrophoblast cultures, or secreted in less quantity, by the cytotrophoblast cultures in accordance with the invention. They also identified 1 mterleukme 13, among the glycosylated proteins secreted by the cultures of cytotrophoblasts in accordance with the invention. This protein is not secreted by normal cytotrophoblasts.
• the method according to the invention comprises the assay, in a biological sample obtained from the mother, of at least one protein chosen from glycosylated proteins secreted by a culture of cytotrophoblasts in accordance with the invention, and at least one protein chosen from non-glycosylated proteins secreted by a culture of cytotrophoblasts according to the invention.
• said glycosylated protein can be for example hCG, placental growth hormone, the extracellular domain of APP protein or any other glycosylated protein secreted by cultures of cytotrophoblasts in accordance with l 'invention.
• Glycosylated proteins which can be used can be easily identified by a person skilled in the art, from the protein secretion profile of a cytotrophoblast culture according to the invention, by detecting glycosylation or the absence of glycosylation, as indicated above.
• the dosage of the selected proteins is carried out according to the usual methods, known in themselves. same to those skilled in the art.
• immunoassay methods can be used, using antibodies directed against the protein concerned.
• suitable antibodies are available commercially, or can readily be obtained by conventional techniques, from purified preparations of these proteins.
• suitable antibodies can be obtained, for example, from proteins eluted from two-dimensional gels. These antibodies can then be used to purify larger quantities of the protein concerned from cytotrophoblast cultures.
• the detection method according to the invention can be implemented in addition to another method of detecting trisomy 21, for example in addition to an assay method using other serum markers, or a method of detection by ultrasound.
• hPL which constitutes a very discriminating marker from the 19 th week of gestation.
• EXAMPLE 1 OBTAINING TRISOMY CYTOTROPHOBLAST CULTURES 21 Collection of placental tissues:
• Placental tissue samples were taken during abortion for abnormalities severe fetal, between 12 and 35 weeks of gestation, either in cases of trisomy 21 detected by karyotype, or as a control, in cases of fetal malformation without karyotype anomalies.
• the samples were taken in accordance with the provisions of French law for the consent of the patients concerned.
• HBSS balanced solution of HANK
• Cytotrophoblast cells were isolated by trypsin-DNAse digestion, using the protocol described by KLIMAN et al. [Endocrmology, 118, 1567-1582, (1986)], modified as follows:
• the digestion is carried out using an enzymatic preparation prepared extemporaneously, and comprising 0.5% (w / v) of trypsm powder (DIFCO), 5 IU / ml of DNAsel (SIGMA, 375000 U, 2600 Kunitz u / mg solid), 25 mM HEPES, 4.2 mM MgS0 4 and 1% (w / v) penicillin / streptomycin, in HBSS.
• DIFCO trypsm powder
• SIGMA 5 IU / ml of DNAsel
• SIGMA DNAsel
• 25 mM HEPES 4.2 mM MgS0 4
• 1% (w / v) penicillin / streptomycin in HBSS.
• the minced villi are covered with this preparation; after 30 mm. digestion at 37 ° C, the liquid is discarded and replaced by a fresh enzyme preparation. A further 25 min incubation is carried out, at the end of which the liquid is discarded and again replaced by a fresh enzymatic preparation. 5 digests are then carried out successively, each time using a fresh enzymatic preparation: 1 digestion of 20 min., 1 digestion of 15 mm. and 3 digests of 10 mm. The liquids from the last 5 digests are stored and collected, then fractionated on a discontinuous Percoll gradient of 10 to 70%.
• a homogeneous population of mononuclear cells is thus obtained. After centrifugation, the cell pellet is taken up in 4 ml of DMEM medium. These 4 ml are deposited on the gradient comprising more than 90% of viable cytotrophoblastic cells [ALSAT et al. , J. Clin. Encodocrmol. Metab., 73, 288-294, (1991)].
• the cells are cultured in dishes 60 mm in diameter (3 ⁇ 10 6 cells per dish, in 3 ml of DMEM medium, supplemented with 25 mM of HEPES, 2 mM of glutam, 20% of fetal calf serum inactivated by heat, and in the presence of antibiotics (100 IU per ml of penicillin and 100 ⁇ g per ml of streptomycin).
• the cultures are maintained at 37 ° C. in a humid atmosphere: 95% air
• Modified Eagle Medium in the presence of 25mM HEPES, 2mM glutam, 20% heat-inactivated fetal calf serum, and in an atmosphere consisting of 95% air and 5% C0 2 .
• the cytotrophoblasts isolated from the control placentas aggregate, then after 24 to 48 hours, fuse and differentiate into syncytiotrophoblasts.
• Cytotrophoblasts isolated from trisomy 21 placentas remain aggregated in culture dishes. After 3 to 4 days of culture, only very few syncytiotrophoblasts are observed.
• An anti-desmoplaqum or anti-E-cadherm monoclonal antibody, diluted to l / 400 th (SIGMA) is then applied to the treated cells, and the antigen-antibody complex is revealed by anti-mouse goat immunoglobulins, fluorescently labeled. (SIGMA).
• desmoplaqume and E-cadherme are absent from normal syncytiotrophoblasts, but present at mtercellular contacts in cultures of trisomy 21 cytotrophoblasts.
• EXAMPLE 2 CHARACTERIZATION OF THE EXPRESSION AND SECRETION OF PROTEINS IN CULTURES OF TRISOMY CYTOTROPHOBLASTS Study of transcription
• Total RNA is extracted from cytotrophoblast cultures after 24, 48 or 72 hours using _t QIAGEN, following the manufacturer's protocol.
• Human placental lactogen (hPL), human choonic gonadotrophm ⁇ (hCG ⁇ ), human chorionic gonadotrophm ⁇ (hCG ⁇ ), human placental growth hormone (PGH), and leptm mRNAs are quantified as described below, from total RNA in solution, previously transcribed inversely into cDNA.
• the cDNA is amplified by RT-PCR in real time using the TaqMan ⁇ method (PE Applied Biosystems). This method is based on the use of the 5 '3' exonuclear activity of the Taq polymerase in order to digest a labeled probe hybridized to a target sequence, during the extension phase of a PCR amplification.
• the labeling of the probe is designed so that its digestion generates a fluorescent signal.
• the parameter C ⁇ (cycle threshold) is defined as the number of cycles for which the fluorescence generated by the digestion of the probe exceeds a threshold fixed beforehand; this number is lower the higher the initial quantity of the target molecule.
• the quantity of each target sequence in samples of unknown composition is thus determined by measuring C ⁇ and using a calibration curve to determine the initial quantity of the target sequence.
• the primers used for reverse transcription, as well as the amplification primers and the detection probes are chosen from the nucleic sequences of hPL), of hCG ⁇ , of hCG ⁇ , of PGH and of leptum. available on the databases, using OLIGO 4.0 (NATURAL BIOSCIENCES) and Primer express (PERKIN-ELMER BIOSYSTEMS) software. Their specificity, and the absence of polymorphism was verified by research using BLASTN
• the standard curve was established using series of 10 in 10 dilutions of cDNA obtained by reverse transcription from 1 ⁇ g of total RNA extracted from placenta collected during the 1 st trimester of pregnancy.
• an internal control was carried out by quantification of transcripts of the PPIA gene (coding for human peptidyl prolyl isomerase A), and each sample was normalized on the basis of its PPIA content.
• the quantity of target RNA to be quantified and that of the control PPIA RNA was determined from the standard curve. Then, the amount of target RNA was divided by the amount of PPIA RNA to obtain the normalized value.
• the concentration of hCG in the culture media was determined by an ELISA test (VITAS system, BIOMERIEUX) following the protocol indicated by the manufacturer.
• the detection sensitivity of this test is 2 mU / ml.
• the concentration of secreted hPL was determined using the IRMA AMERLEX hPL kit, (AMERSHAM) in concentrated medium 4 times. The sensitivity of the test is 0.5 ⁇ g / ml). HPL has also been detected from cell lysates of cytotrophoblasts. The solubilized proteins (5 ⁇ g) were revealed after electrophoresis and transfer to nitrocellulose using a polyclonal rabbit antibody directed against 1 hPL and diluted to 250 erae (DACO, FRANCE). The band corresponding to 1 hPL is revealed by chemiolummescence (PIERCE SUPERSIGNAL, INTERKIM, FRANCE) after incubation with an anti-lapm antibody coupled to peroxidase.
• chemiolummescence PIERCE SUPERSIGNAL, INTERKIM, FRANCE
• the leptm was measured in concentrated medium 4 times using the RIA kit “Sensitive Human Leptm RIA Kit” (LINCO, ST LOUIS, USA).
• the sensitivity of the assay is 0.5 ng / ml.
• FIGS. 1A and 1B show the expression of the mRNAs of 1 hCG ⁇ and 1 hCG ⁇ during the differentiation of cytotrophoblasts isolated from control placentas (N) and trisomy 21 placentas (T21) and cultured for 24 h, 48 h or 72 h.
• Figure 1C illustrates the secretion of hCG by control cells (N) or T21 cells after 24 h, 48 h or 72 h of culture. In normal placental cell cultures, the level of transcripts of the hCG ⁇ subunit increases by more than 10 3 times between cytotrophoblast and differentiated syncytiotrophoblast m vi tro. Likewise, the secretion of ⁇ hCG gradually increases in the culture medium. In cultures of T21 placenta cells, the level of transcripts and hCG secretion are decreased.
• Figures 2A to 2C illustrate the expression of
• FIG. 2A illustrates the level of transcription, expressed as a normalized amount of mRNA.
• Figure 2B illustrates the secretion of hPL in the culture medium, expressed in ⁇ g / ml.
• Figure 2C shows the detection of intracellular hPL after immunoelectrophoretic transfer. In T21 cells, we do not detect after
• Figure 3 illustrates the expression of leptm (Figure 3A) and PGH (Figure 3C) mRNAs, as well as leptm secretion (Figure 3B) during differentiation of cytotrophoblasts isolated from control placentas (N) or of T21 placentas grown for 3 days.
• hPL is not detectable in trisomy 21 cells, whereas it is present in normal cells.
• the secretion of PGH in the culture medium has not been measured since it is known that this secretion is inhibited by glucose, which is present in this medium.
• the transcription of the hCG ⁇ , hCG ⁇ , PGH, hPL, and leptm genes was measured during placental ontogenesis, using the protocol described above, in samples of normal placentas and T21. the results obtained indicate that throughout pregnancy, the transcription of these genes is much lower in the placentas of trisomy 21 than in normal placentas.
• hPL The assay is carried out on serum.
• the test portion is adapted according to the stage of pregnancy (at the start of pregnancy 200 ⁇ l, at the end of pregnancy 20 ⁇ l).
• the Assay is carried out using the AMERLEX-hPL-IRMA IRMA Kit (Ortho Clmical Diagnostics, United Kingdom).
• Each serum is dosed in duplicate with a precision on the mter-series measurement ranging from 5.9% to 10% coefficient of variation.
• the concentration range is
• the detection limit of the test is
• the assay is carried out on serum.
• the test portion is 100 ⁇ l.
• the assay is performed using the RIA Human Leptm RIA KIT assay (Linco, USA).
• Each serum is dosed in duplicate with a precision on the mter-series measurement ranging from 3.3% to 8.9% of coefficients of variation.
• the concentration range is from 0.5 to 100 ng / ml.
• the detection limit is 0.1 ng / ml
• the assay is carried out on serum.
• the test portion is 100 ⁇ l.
• the assay is carried out using the hPGH IRMA kit (BC1017) BIOCODE.
• Each serum is dosed in duplicate with a precision on the mter-series measurement ranging from 5.5% to 7.9% of coefficients of variation.
• the concentration range is from 1 to 180 ng / ml.
• the detection limit is 0.2 ng / ml.

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