RU2592244C1 - Method for assessing inducing action of cytomegaloviral infection on lipoic acid activity in placental plasmodiotrophoblast in third trimester of gestation - Google Patents
Method for assessing inducing action of cytomegaloviral infection on lipoic acid activity in placental plasmodiotrophoblast in third trimester of gestation Download PDFInfo
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Abstract
Description
1. Цель изобретения - разработать способ оценки влияния цитомегаловирусной инфекции на активность липоевой кислоты в синцитиотрофобласте плаценты на третьем триместре гестации.1. The purpose of the invention is to develop a method for assessing the effect of cytomegalovirus infection on the activity of lipoic acid in the placenta syncytiotrophoblast in the third trimester of gestation.
2. Изобретение относится к медицине, а именно к разработке способа, позволяющего оценить влияние цитомегаловирусной инфекции на активность липоевой кислоты, играющей роль кофермента в окислительно-восстановительном процессе цикла Кребса.2. The invention relates to medicine, namely to the development of a method to assess the effect of cytomegalovirus infection on the activity of lipoic acid, which plays the role of coenzyme in the redox process of the Krebs cycle.
3. Раскрытие изобретения3. Disclosure of invention
Липоевая кислота - это химическое соединение из группы витаминоподобных кислот, которое в небольших количествах синтезируется в организме. Липоевая кислота является коферментом в комплексах (дегидрогеназ), обеспечивающих этапы метаболизма глюкозы [2, 6, 7]. Липоевая кислота как кофактор вовлечена в митохондриальный метаболизм. В аэробных условиях пируват проникает в митохондрии и в ходе комплексной реакции пируватдегидрогеназы конвертируется в ацетил-СоА для участия в цикле Кребса. В этом процессе участвует целый ряд коферментов, в том числе и липоевая кислота [7-9]. Увеличение липоевой кислоты повышает эффективность конвертации пирувата в ацетил-СоА и тем самым увеличивает количество медиаторов анаэробного метаболизма. Липоевая кислота наряду с участием в синтезе ацетил-СоА выполняет в организме еще и роль антиоксиданта[1-5].Lipoic acid is a chemical compound from the group of vitamin-like acids, which is synthesized in small quantities in the body. Lipoic acid is a coenzyme in complexes (dehydrogenases) that provide the stages of glucose metabolism [2, 6, 7]. Lipoic acid as a cofactor is involved in mitochondrial metabolism. Under aerobic conditions, pyruvate penetrates the mitochondria and, during the complex reaction of pyruvate dehydrogenase, is converted to acetyl-CoA to participate in the Krebs cycle. A number of coenzymes are involved in this process, including lipoic acid [7–9]. An increase in lipoic acid increases the efficiency of the conversion of pyruvate to acetyl-CoA and thereby increases the number of anaerobic metabolism mediators. Lipoic acid, along with participation in the synthesis of acetyl-CoA, also plays the role of an antioxidant in the body [1-5].
4. Новая техническая задача4. New technical challenge
Разрабатывается способ, позволяющий путем определения активности липоевой кислоты в синцитиотрофобласте оценить влияние цитомегаловирусной инфекции на характер окислительно-восстановительных процессов в плаценте в период гестации.A method is being developed that allows, by determining the activity of lipoic acid in syncytiotrophoblast, to assess the effect of cytomegalovirus infection on the nature of redox processes in the placenta during gestation.
5. В доступной литературе не имеется сведений, касающихся оценки активности липоевой кислоты в окислительно-восстановительном процессе в синцитиотрофобласте плаценты. В настоящем исследовании учитывается еще и характер участия липоевой кислоты в окислительно-восстановительных процессах в плаценте при обострении цитомегаловирусной инфекции.5. In the available literature there is no information regarding the evaluation of the activity of lipoic acid in the redox process in the placenta syncytiotrophoblast. This study also takes into account the nature of the participation of lipoic acid in redox processes in the placenta during exacerbation of cytomegalovirus infection.
6. Осуществление изобретения6. The implementation of the invention
6.1. Обследовано 20 беременных в возрасте 18-25 лет с хронической цитомегаловирусной инфекцией (ХЦМВИ) в стадии обострения на 25-26 неделях гестации с титром антител к ЦМВ 1:1600 и 10 беременных, не болевших на всем протяжении беременности. Диагноз обострения ХЦМВИ устанавливался при комплексном исследовании периферической крови на наличие IgM к ЦМВ или четырехкратного и более нарастания титра антител IgG в парных сыворотках в динамике через 10 дней, индекса авидности более 65%, а также наличия ДНК ЦМВ. Исследования проводились с учетом требований Хельсинской декларации Всемирной медицинской ассоциации «Этические принципы проведения научных исследований с участием человека» с поправками 2008 г. и правилами клинической практики в РФ, утвержденными приказом Министерства РФ №266 от 19.06.2003 г. Верификация ЦМВ, определение типоспецифических антител, индекс авидности осуществлялись методами ИФА на спектрофотометре «Stat-Fax-2100» (США) с использованием тест-систем ЗАО «Вектор-Бест» (Новосибирск), методами ПНР на аппарате ДТ-96 с использованием наборов НПО «ДНК-технология» (Москва).6.1. We examined 20 pregnant women aged 18-25 with chronic cytomegalovirus infection (HCMVI) in the acute stage at 25-26 weeks of gestation with a titer of antibodies to CMV 1: 1600 and 10 pregnant women who were not ill throughout the pregnancy. The diagnosis of exacerbation of HCMVI was established by a comprehensive study of peripheral blood for the presence of IgM to CMV or a fourfold or more increase in the titer of IgG antibodies in paired sera in dynamics after 10 days, the avidity index of more than 65%, and the presence of CMV DNA. The studies were carried out taking into account the requirements of the Helsinki Declaration of the World Medical Association “Ethical Principles for Conducting Research with a Person”, as amended in 2008 and the rules of clinical practice in the Russian Federation, approved by Order of the Ministry of the Russian Federation No. 266 of 06/19/2003. Verification of CMV, determination of type-specific antibodies , the avidity index was carried out by ELISA on a Stat-Fax-2100 spectrophotometer (USA) using Vector-Best CJSC test systems (Novosibirsk), and on a DT-96 apparatus using commissioning methods using a set NGO "DNA Technology" (Moscow).
6.2. Статистический анализ и обработку данных проводили с использованием пакета прикладных программ «Statistica v. 6.0» (StatSoft Inc., США). Для определения достоверности различий использовали непарный параметрический критерий Стьюдента. Для определения достоверности различий в случае негауссовых распределений - непараметрические критерии Колмогорова-Смирнова и Манна-Уитни. Критический уровень значимости был принят за 5% (0,05). Данные представлены как среднее арифметическое (М) ± стандартная ошибка среднего арифметического (m).6.2. Statistical analysis and data processing was performed using the software package Statistica v. 6.0 "(StatSoft Inc., USA). To determine the significance of differences, unpaired parametric student criterion was used. To determine the significance of differences in the case of non-Gaussian distributions, non-parametric Kolmogorov-Smirnov and Mann-Whitney criteria are used. The critical level of significance was taken as 5% (0.05). Data are presented as arithmetic mean (M) ± standard error of arithmetic mean (m).
6.3. Реакция проводилась на свежезамороженных срезах. Активность липоевой кислоты определяли гистохимическим методом. Субстратом служил 0,1 М раствор липоевой кислоты фирмы ICN (США) на фосфатном 0,1 М буфере рН 7,5-1 мл, нитросиний тетразолий фирмы ICN (США) - 1 мг. Инкубация проводилась при температуре 37°С в течение 30 мин. Срезы промывались в дистиллированной воде, слегка подсушивались и заключались в глицерин-желатин. Полученные препараты изучались под цифровым микроскопом, соединенным с компьютером по программе Scion (США), с целью цитофотометрическим методом по количеству продуктов реакции на липоевую кислоту определять ее активность в синцитиотрофобласте.6.3. The reaction was carried out on freshly frozen sections. The activity of lipoic acid was determined by a histochemical method. The substrate was a 0.1 M solution of lipoic acid from ICN (USA) on a phosphate 0.1 M pH 7.5-1 ml, nitrosine tetrazolium from ICN (USA) - 1 mg. Incubation was carried out at a temperature of 37 ° C for 30 minutes Slices were washed in distilled water, slightly dried and enclosed in glycerin-gelatin. The obtained preparations were studied under a digital microscope connected to a computer using the Scion program (USA) in order to determine its activity in syncytiotrophoblast by the cytophotometric method by the number of reaction products to lipoic acid.
7. Полученные данные7. Received data
В процессе исследования получены следующие результаты. В синцитиотрофобласте ворсинок плаценты рожениц, перенесших на 25-26 неделях гестации обострение цитомегаловирусной инфекции до титра антител к ЦМВ 1:1600, резко подавлялась активность на липоевую дегидрогеназу, выполненную гистохимическим методом: 0,1М раствор липоевой кислоты на фосфатном буфере рН 7,5-1 мл с добавлением нитросинего тетразолия - 1 мг, с инкубацией в течение 30 мин при температуре 37°С. Срезы промывались в дистиллированной воде и заключались в глицерин-желатин. Интенсивность реакции подсчитывали цитофотометрическим способом по программе Scion до 23,50±0,95 усл. ед. (контроль - 68,30±1,50 усл. ед.). Полученные данные свидетельствуют, что при обострении цитомегаловирусной инфекции на третьем триместре гестации в синцитиотрофобласте снижается активность кофакторов, обеспечивающих синтез ацетил-СоА, подавляя тем самым уровень окислительно-восстановительных процессов в фетоплацентарном барьере, что снижает функциональную активность плаценты беременной и создает угрозу развития плода.During the study, the following results were obtained. In syncytiotrophoblast of the placenta villi villi, which underwent exacerbation of the cytomegalovirus infection to an anti-CMV titer of 1: 1600 at 25-26 weeks of gestation, activity on lipoic dehydrogenase performed by histochemical method was sharply inhibited: 0.1 M lipoic acid solution on phosphate buffer pH 7.5- 1 ml with the addition of nitro-blue tetrazolium - 1 mg, with incubation for 30 min at a temperature of 37 ° C. Slices were washed in distilled water and enclosed in glycerin-gelatin. The reaction intensity was calculated by cytophotometric method according to the Scion program to 23.50 ± 0.95 srvc. units (control - 68.30 ± 1.50 conventional units). The data obtained indicate that with exacerbation of cytomegalovirus infection in the third trimester of gestation in syncytiotrophoblast, the activity of cofactors that ensure the synthesis of acetyl-CoA decreases, thereby inhibiting the level of redox processes in the fetoplacental barrier, which reduces the functional activity of the pregnant placenta and poses a threat to fetal development.
На фигуре 1 изображена гистохимическая реакция на активность липоиддегидрогеназы в синцитиотрофобласте ворсинок плаценты рожениц, не болевших на всем протяжении гестации. Гистохимическая реакция по Лойда. Цитофотометрически активность - 68,30±1,50 усл. ед. Увеличение - 10×90.The figure 1 shows the histochemical reaction to the activity of lipoid dehydrogenase in the syncytiotrophoblast of the villi of the placenta of the parturient woman who did not suffer from the entire gestation. Histochemical reaction according to Loyd. Cytophotometric activity - 68.30 ± 1.50 srvc. units The magnification is 10 × 90.
На фигуре 2 изображена гистохимическая реакция на активность липоиддегидрогеназы в синцитиотрофобласте ворсинок плаценты рожениц с обострением ХЦМВИ на 25-26 неделях гестации. Цитофотометрически по сравнению с контролем активность липоиддегидрогеназной реакции цитофотометрически снижена до 23,50±0,95 усл. ед. Увеличение - 10×90.The figure 2 shows a histochemical reaction to the activity of lipoid dehydrogenase in the syncytiotrophoblast of the villi of the placenta of the parturient woman with exacerbation of HCMVI at 25-26 weeks of gestation. Cytophotometrically, compared with the control, the activity of the lipoid dehydrogenase reaction is cytophotometrically reduced to 23.50 ± 0.95 srvc. units The magnification is 10 × 90.
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| UA23634U (en) * | 2006-09-12 | 2007-06-11 | Halyna Bohdanivna Mateiko | Method for prognostication the complications of pregnancy and births in expectant mothers with herpetic and cytomegalovirus infections |
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| RU2164417C2 (en) * | 1998-11-11 | 2001-03-27 | Московский областной научно-исследовательский институт акушерства и гинекологии | Prophylaxis of placental insufficiency under condition of perinatal viral-bacterial infection |
| UA23634U (en) * | 2006-09-12 | 2007-06-11 | Halyna Bohdanivna Mateiko | Method for prognostication the complications of pregnancy and births in expectant mothers with herpetic and cytomegalovirus infections |
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| НОВИЦКИЙ В.В. и др. "Механизмы повреждения эритроцитов при токсическом действии метгемоглобинобразователей"// Бюллетень Сибирской медицины, 2006,No1, с.32-37. MURPHY J.R. Erythrocyte metabolism. I. The equilibration of glucose-C14 between serum and erythrocytes// J Lab Clin Med., 1960 Feb;55:281-5, abstract. * |
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