Method of estimating inducing action of granzyme b on apoptosis of placenta syncyotrophoblast nuclei caused by exacerbation of herpes-virus infection during pregnancy

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to diagnostic methods. Homogenate of placenta of pregnant woman who had herpes-virus infection is analysed by ELISA method for activity of granzyme B, titre of antibodies in pregnant woman's peripheral blood, and simultaneously number of syncyotrophoblast nuclei in apoptosis is calculated. If antibody titre is 1:3200 and granzyme B content is 283.14±18.00 pg/ml, percentage of nuclei in apoptosis state is 1.5±0.09%; if antibody titre is 1:6400 and granzyme B content in placenta homogenate is 712.84±23.00 pg/ml content of nuclei in apoptosis state is 2.0±0.12%; if titre of antibodies to herpes virus is 12800 and content of granzyme B in placenta homogenate is 1007.53±31.50 pg/ml (control - 251.10±31,00 pg/ml) percentage of nuclei in state of apoptosis increases to 3.5±0.08% (control - 1.0±0.009%).

EFFECT: method ensures high sensitivity of detecting nuclei in state of apoptosis in syncyotrophoblast at the background of increased content of granzyme B in placenta homogenate.

 

The invention relates to medicine, namely to diagnostic methods.

The purpose of the study show a violation of the syncytiotrophoblast of the villi of the placenta by increasing the number of cores entering into apoptosis due to the high content in the homogenate of the placenta granzyme, during exacerbation of herpes viral infection during pregnancy.

Grasim In is released into the cytosol of target cells when touching the NK-lymphocytes. Penetrating Grasim In the cytosol is a proteolytic enzyme that turns procaspase-3 to active caspase-3, under the influence of which is initiated apoptosis cores(1, 2, 3, 4, 5, 6, 7).

However, in the available literature we did not find information reflecting the effects of herpes virus infections on the increase in the content of granzyme In the placenta parturients undergoing during pregnancy exacerbation of herpes viral infections that affect the increase in the percentage of nuclei in a state of apoptosis in the syncytiotrophoblast.

The claimed method has the following methods.

1. Investigated peripheral blood from 15 pregnant women who are in the third trimester outbreak of herpes viral infection, and in 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 of physiology and 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.

2. Activity granzyme In the homogenate of the placenta of healthy pregnant women and pregnant women who are in the third trimester outbreak of herpes viral infection was determined by an enzyme immunoassay (ELISA) using reagents "Bender MedSystems" (Austria) on spectrophotometer "Stat Fax-2100 (USA).

3. The titer of antibodies to the herpes virus was determined by an enzyme immunoassay (ELISA) on spectrophotometer "Stat Fax-2100 (USA).

Morphological classification of apoptosis was performed on paraffin sections of the placenta on the label, breaking the ends of the DNA fragments in situ end-labeling (ISEL - method) [8]. The slices after dehydration in distilled water for 30 min, incubated in 3% hydrogen peroxide solution and washed 0.15 M buffered 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 trishit chlorid, 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; "Puomega Madison WI", 0.001 mm biotin-II-dUTP, Sigma) and DNA polymerase-1 E coli (20 u/ml; Promega), prepared in buffer A. Then washed with buffer 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-diamino-baseinterface) in 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 conclude in balsam.

The method has a high sensitivity even for kernels that are just starting the programmed death. Using this method, you can see the condensation of chromatin, closely adjacent to the nuclear membrane.

Studies have shown that with the rise of the antibody titer in peripheral blood of pregnant women in the third trimester increases the content of granzyme In the homogenate of the placenta postpartum who are in the third trimester exacerbation of herpes viral infections.

When the antibody titer of 1:3200 content granzyme In homogene the e placenta was 283,14±18,00 PG/ml; when the titer of antibodies to the herpes virus its content was 712,84±23,00 PG/ml, while the titer of antibodies to herpes virus 1:12800 content granzyme In the placenta homogenate increased to 1007,53±31,50 PG/ml (control 251,10±31,00 PG/ml).

In addition, we counted the number of syncytiotrophoblast nuclei, located in the state of apoptosis. 2000 nuclei in the villi of the placenta when the content of granzyme In 283,14±18,00 PG/ml, there were 1,5±0,09% of the nuclei in a state of apoptosis. When 712,84±23,00 PG/ml granzyme In the percentage of nuclei in a state of apoptosis was 2.0±0,12%, and at 1007,53±31,50 PG/ml the number of cores in the syncytiotrophoblast was increased to 3.50±0,08% (control - 1,0±0,009%).

Thus, the outbreak of herpes-virus infection in pregnant women in the third trimester increases the amount of granzyme In the placenta, which initiates the formation of apoptotic nuclei syncytiotrophoblast and the larger the number, the more in the homogenate of the placenta is determined by granzyme Century

Literature

1. Kozlov V.K., Molchanov PU, Zharinov GC Immunotherapy with recombinant cytokines in the treatment of cancer patients // Advances in clinical immunology and Allergology. vol. the drafting. Averella. M.: Publishing house of the regional branch of the Russian Academy of natural Sciences, 2002. s-279.

2. Avdonin A.L., Medvedev N Extracellular heat shock protein 70 and its function // Cytology. 2009, C. No. 2. s-137.

3. Sintsov A.V., Kovalenko, H., Khanin M.A.// Apoptosis, induced grannymom Century Bioorg. chemistry 2008, 34(6), C-733.

4. Agol NR. Genetically programmed cell death // Saratov educational journal 1996, No. 6, p.20-24.

5. Kawaguchi Y., Kono K., Mizukami Y., Mimura, K., Fujii H // Mechanisms of escape from trastuzumal-mediated ADCC in esophageal sguamous cell carcinoma: relation to susceptibility to perforin-granzyme. Anticancer Res. 2009; 29(6), 2137-2146.

6. E.R. Podack, Hengartner H., Lihtenheld M.G. A central role of perform in cytolisis // Annu. Res. Immunol. - 1991. - Vol.9, p.129-157.

7. Fuwthrop D.J., A.R. Boobis, D.S. Davies Mechanisms of cell death // Arch. Toxicol. - 1991. - Vol.65. - p.437-444.

8. Pogorelov V.M. Morphology of apoptosis in normal and malignant haematopoiesis / Vampilov, Geekazine // Haematology and Transfusiology - 1995. - So 43 No. 5. - p.21-24.

The method for determining the cores in a state of apoptosis in the syncytiotrophoblast in the background of the high content of granzyme In the homogenate of the placenta detected by enzyme-linked immunosorbent assay (ELISA) - induced exacerbation of herpes viral infection during pregnancy: antibody titer of 1:3200, the content of granzyme In -283,14±18,00 PG/ml, the percentage of nuclei in a state of apoptosis - 1,5±0,09%; when the antibody titer of 1:6400 and content in the homogenate of the placenta granzyme In 712,84±23,00 PG/ml content of nuclei in a state of apoptosis - 2,0±0,12%; when the titer of antibodies to the herpes virus 1:12800 and content in the homogenate of the placenta granzyme In 1007,53±31,50 PG/ml (control - 251,10±31,00 PG/ml) the percentage of nuclei in a state of apoptosis increased to 3.5±0,08% (control - 1,0±0009%).



 

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2 ex, 3 tbl

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