Method of estimating impact of igg and igm antibodies to phosphatidylserine in placenta of pregnant women at development of apoptosis in syncyotrophoblast of placenta pili in case of exacerbation of herpes-virus infection during pregnancy

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to diagnostic methods and deals with method of diagnosing entry of nuclei of placenta pili syncyotrophoblast into apoptosis by determining in placenta homogenate content of IgG and IgM antibodies to phosphatidylserine in pregnant women, who had herpes-virus infection in the third trimester of pregnancy. Method lies in determination of IgG and IgM antibodies to phosphatidylserine in placenta homogenate by ELISA method. If titre of antibodies to HSV-1 equalled 1:6400, 4.70±0.08 U/ml of IgG and IgM antibodies to phosphatidylserine were detected; if titre was 1:12800, content of IgG and IgM antibodies to phosphatidylserine increased to 5.60±0.06 U/ml (control - 2.50±0.07 U/ml), number of syncyotrophoblast nuclei in state of apoptosis constituted 4.50±0.20% (control - 1.5±0.20%).

EFFECT: method possesses high sensitivity and allows to predict development of apoptosis in nuclei of placenta pili syncyotrophoblast in case if number of IgG and IgM antibodies to phosphatidylserine in placenta grows.

 

The purpose of the study show changes in the content of antibodies to phosphatidylserine classes IgG and IgM in the placenta of pregnant women who are in the third trimester outbreak of herpes viral infection, accompanied by an increase in the number of syncytiotrophoblast nuclei of the villi of the placenta, entering into apoptosis.

The cell can be sent in apoptosis damaging factors (products of lipid peroxidation, TNF α), the applicable factors from the outside. In this case an important role is played by the phospholipid phosphatidylserine the outer surface of the membrane. Transport ATP-ASE carries phosphatidylserin of the outer layer of the plasma membrane in the inner (Vladimirov, Y.A., 1991; knyazikhin IV, 2007; Skulachev V.P., 1999; 2001; Alareon-Segovia D., 1987).

In the existing literature there are no links pointing to change the content of antibodies IgG and IgM to serine in the placenta during exacerbation during pregnancy herpes viral infection, leading to the growth of the nuclei in the syncytiotrophoblast of the villi of the placenta in the state of apoptosis.

The claimed method has the following methods:

Investigated the placental homogenate in 20 pregnant women who are in the third trimester outbreak of herpes viral infection, and 20 pregnant, not ill during the whole period of gestation (control). Research was conducted on the basis of hospital obstetric clinic of the far Eastern scientific center is etiologii and pathology of respiration SB RAMS. All studies were conducted with regard to the requirements of the Helsinki Declaration of the world Association "Ethical principles for medical research with regard to person" with amendments of 2000, and the "Rules of clinical practice in the Russian Federation", approved by Order of Ministry of health of the Russian Federation dated 19.06.2003, No. 266.

Morphological detection of apoptosis was performed on paraffin sections of the placenta in the tag ends of the DNA fragments in situ (ISEL - method) [3]. The slices after dehydration in distilled water for 30 min, incubated in 3% hydrogen peroxide solution and washed buffered 0.15 M saline (PBS) (0.15 M NaCl in 0.1 M phosphate buffer pH 7.5). Then the slices are incubated in a mixture of 0.3 M NaCl and 30 mm sodium citrate (pH 7.0; 80°C, 20 min) and washed in 0.15 M PBS. Then the slices are incubated in a solution of pronase at room temperature for 30 min (Calbiochem; 1 mg/ml in 0.15 M PBS). Washed in 0.15 M PBS and buffer A (50 mm Tris-hydrochloride, 5 mm magnesium chloride, 10 mm β-mercaptoethanol and 0.005% solution of bovine serum albumin (BSA), pH 7.5). Sequentially incubated (18°C, 2 hours) in a mixture of 4 nucleotides (0.01 mm dATF, dCTF, dGTF; "Promega Madison WI", 0.001 mm biotin-II-dUTP, Sigma) and DNA polymerase-1 E coli (20 U/ml; Promega), up to 2-3 cm and a thickness of 1 mm Pieces of tissue were placed in a chemical beakers containing 200 ml of physiological solution, washed prepared in buffer A. Then washed b is from A, followed by 0.5 M PBS (0.5 M NaCl in 0.1 M phosphate buffer pH 7.5). Finally slices incubated with peroxidase conjugate (Vectastam Elite ABC Kit, Vector, Burliname, CA), diluted 1:25 in 0.5 M PBS, which introduced the 1% BSA (bovine serum albumin) and 0.5% tween-20, and within 10 min paint 0,04% solution of DAB (3,3-diaminobenzidine tetrachloride) 0.05 M Tris buffer pH 7.5 and hydrogen peroxide at a final concentration of 0.015% of the 30% solution. Washed with distilled water, dehydrated and embrace balm. The counting of nuclei located in the state of apoptosis, was performed on 100 terminal villi of the placenta in each individual case. In counting to 100 fibers were selected 2000 nuclei.

To obtain a homogenate of fruit part of the placenta (the villous chorion) were cut with a scalpel small plates ranging from blood cells, mixing on a magnetic stirrer for 15 min and dried on filter paper. Then the tissue was ground with pestle in a porcelain mortar and homogenized until smooth. To bad the homogenate was added FRANCS in the amount equal to the original weight of tissue (1 g ml - 1 FR). Suspension was placed in a plastic test tube "Falcon" and were subjected to freezing at -20°C during the day. Then the homogenate was thawed and was ultracentrifugation at 4000 rpm at a temperature of +4°C. Insufficient liquid was poured into a small aliquot and store the ri -20°C prior to conducting the assay.

The content of antibodies to phosphatidylserine classes IgG and IgM were determined using enzyme-linked immunosorbent assay using kits company "Bender MedSystems" (Austria). Verification of herpes simplex virus (HSV-1) and severity of disease was assessed by the dynamics of the titers of IgG antibodies using standard test systems of closed joint stock company "Vector-best" (Novosibirsk). Analysis was performed with a microplate reader (Stat Fax-2100" (USA).

Statistical data processing was performed using t-student test.

Studies have shown that exacerbation of herpes-virus infection in the third trimester of pregnancy was accompanied by an increase in the placental homogenate antibodies IgG and IgM antibodies to phosphatidylserine. When the titer of antibodies to HSV-1 1:6400 noted the increasing antibodies IgG and IgM to PS to 4.70±0,08 IU/ml, Such as high levels of antibodies IgG and IgM to PS was determined when the titer of antibodies to HSV-1 1:12800 - the ceiling of 5.60±0,06 U/ml (control - 2,50±0,07 U/ml).

Exploring sections of the placenta after the reaction ISEL, noted that in 2000 nuclei in the villi of the placenta of pregnant women who have had herpes viral infection in the third trimester when the titer of antibodies to HSV - 1:6400, defined 2,30±0,015% (control - 1,50±0,02%; p<0.001) in the syncytiotrophoblast nuclei in a state of apoptosis, while the titer of 1:12800 number of cores in a state of apoptosis in the syncytiotrophoblast was increased to 4.50±0.20 per cent.

Thus, su the cap herpes viral infection during pregnancy stimulates the production of antibodies IgG and IgM antibodies to phosphatidylserine (PS), which leads to an increase prematurely entering into apoptosis cores placental barrier.

Literature

1. Vladimirov, Y.A. Free radicals in living systems / Waslimited, Oaasypow, Aigai and other // Results of science and technology. Ser. Biophysics. - 1991. - 29 I.

2. Knyazikhin IV Apoptosis in oncourology / Iwhrekan, Vincian - sank. St: "Science". - 2007. - 240 S.

3. Pogorelov V.M. Morphology of apoptosis in normal and malignant haematopoiesis / Vampilov, Geekazine // the Hematology and Transfusiology. - 1995. - t. No. 5. - p.21-24.

4. Skulachev VP Panoptes: the programmed death of an organism / Vpostolati // Biochemistry. - 1999. - 64 so - WIP. - s-1688.

5. Skulachev V.P. Phenomena programmed death. Mitochondria, cells and organs: role of reactive oxygen / Vpostolati // Soros educational journal. 2001. No. 7. p.4-10.

6. Alareon-Segovia D. Antiphospholipid antibodies and the antiphospholipid syndrome in systemic lupus lupus. A perspective analysis of 500 consecutive patients, D. Alareon-Segovia, M. Debze // Medicine, 1987 v.68, p.353-365.

The method for determining the number of syncytiotrophoblast nuclei of the villi of the placenta in the state of apoptosis in parturients undergoing in the third trimester of pregnancy outbreak of herpes viral infections, including the determination 4,70±0,08 U/ml of IgG antibodies, IgM antibodies to phosphatidylserine and titer of antibodies to herpes virus 1:6400, and determines that 2,30±0.015% nuclei sincity the trophoblast villi of the placenta is in a state of apoptosis; when the concentration of the antibodies of IgG and IgM antibodies to phosphatidylserine ceiling of 5.60±0,06% IU/ml (control - 2,50±0,07 U/ml) and the magnitude of the titer of antibodies to herpes virus 1:12800 determines that 4,50±0,20% of syncytiotrophoblast nuclei of the villi of the placenta is in a state of apoptosis.



 

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2 ex, 3 tbl

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