RU2507599C1 - Method for prevention of tumour development in experimental animals with high-frequency spontaneous tumour formation - Google Patents
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Abstract
Description
Изобретение относится к медицине, а именно к способам профилактики развития опухолей у экспериментальных животных с высокой частотой спонтанного опухолеобразования.The invention relates to medicine, namely to methods for preventing the development of tumors in experimental animals with a high frequency of spontaneous tumor formation.
Известен способ профилактики развития опухолей у мышей линии СЗН, предрасположенных к развитию рака молочной железы, при котором добавление в питание животных синтетического антиоксиданта 2-меркаптоэтиламина увеличило среднюю продолжительность жизни на 26%. Возникновение опухолей наблюдалось в более отдаленные сроки, однако частота развития спонтанных опухолей не изменилась [Harman D. Free-radical theory of aging: increasing the functional life span // Ann. N.Y. Acad. Sci.- 1994.-Vol.717. - P. 1-15].A known method of preventing the development of tumors in mice of a line of CHF predisposed to the development of breast cancer, in which the addition of a synthetic antioxidant 2-mercaptoethylamine to the animals, increased the average life expectancy by 26%. The occurrence of tumors was observed at a longer time, but the incidence of spontaneous tumors did not change [Harman D. Free-radical theory of aging: increasing the functional life span // Ann. N.Y. Acad. Sci.- 1994.-Vol. 717. - P. 1-15].
Задачей изобретения является создание способа повышения эффективности профилактики развития опухолей у экспериментальных животных с высокой частотой спонтанного опухолеобразования.The objective of the invention is to provide a method for increasing the effectiveness of prophylaxis of the development of tumors in experimental animals with a high frequency of spontaneous tumor formation.
Поставленная задача решается тем, что в заявляемом способе используется комплексный фитоадаптогенный препарат фитомикс-40 (фм-40) на основе компонентов 40 экстрактов растений, включая женьшень, родиолу розовую, элеутерококк, лимонник, аралию, заманиху, зверобой, календулу, подорожник, калган, бессмертник, сосновые и березовые почки, толокнянку, эвкалипт, можжевельник, солодку, ромашку, чагу, валериану, спорыш, пустырник, душицу, мяту перечную, одуванчик, пижму, толокнянку, тысячелистник, хвощ полевой, чабрец, череду, кориандр, шиповник, калину, рябину черноплодную, боярышник, клюкву, бруснику, чернику, смородину черную [патент РФ №2099410; Шейченко В.И., Бочарова О.А., Шейченко О.П., Бочаров Е.В., Барышников А.Ю., Быков В.А. Аналитические возможности метода ЯМР для определения компонентов препарата фитомикс-40. // Заводская лаборатория. - 2006. - Т. 72. - №8. - с.15-23].The problem is solved in that the claimed method uses a complex phytoadaptogenic preparation phytomix-40 (fm-40) based on components of 40 plant extracts, including ginseng, Rhodiola rosea, Eleutherococcus, Schisandra, aralia, zamanika, St. John's wort, calendula, plantain, galanga, immortelle, pine and birch buds, bearberry, eucalyptus, juniper, licorice, chamomile, chaga, valerian, knotweed, motherwort, oregano, peppermint, dandelion, tansy, bearberry, yarrow, horsetail, thyme, marigold, thyme, marigold, thyme, thyme, , R Bina chokeberry, hawthorn, cranberry, lingonberry, blueberry, blackcurrant [RF patent №2099410; Sheychenko V.I., Bocharova O.A., Sheychenko O.P., Bocharov E.V., Baryshnikov A.Yu., Bykov V.A. The analytical capabilities of the NMR method to determine the components of the phytomix-40 preparation. // Factory laboratory. - 2006. - T. 72. - No. 8. - p. 15-23].
Заявляемый способ осуществляли следующим образом.The inventive method was carried out as follows.
Исследование проводили на 230 мышах-самцах линии СВА с высокой частотой спонтанного образования гепатокарцином. В исследование были включены опытная группа - 70 мышей и 2 контрольные группы (I группа - 90 мышей и II группа - 70 мышей).The study was performed on 230 CBA male mice with a high frequency of spontaneous formation of hepatocarcinomas. The study included an experimental group of 70 mice and 2 control groups (group I - 90 mice and group II - 70 mice).
Мыши опытной группы получали 10% раствор препарата фм-40 с питьевой водой, который применяли в течение первого месяца постнатального развития, включая период завершения дифференцировки ткани печени.The mice of the experimental group received a 10% solution of fm-40 with drinking water, which was used during the first month of postnatal development, including the period of completion of liver tissue differentiation.
Мыши I контрольной группы получали в качестве питья воду, мыши II контрольной группы - 3% раствор этанола в профилактическом режиме.Mice of the control group I received water as a drink, and mice of the control group II received a 3% solution of ethanol in a prophylactic mode.
В каждой группе забивали 10-13 мышей в возрасте 22 месяцев. Остальные мыши находились под наблюдением до их естественной гибели. Определяли объем опухолей (мм3) в печени, который вычисляли по стандартной формуле. Гистологическое исследование печени проводили на парафиновых срезах. Наличие лимфоцитарной инфильтрации и деструкции опухолевых участков в печени мышей опытной группы являлось благоприятным прогностическим фактором течения опухолевого процесса [Оbl D., Loewe R., Yu P., Mihm M. Focus on TILs: Prognostic significance of tumor infiltrating lymphocytes in human melanoma // Cancer Immun. - 2009. - Vol.9. - №3. - p.245-251; Wansom D., Light E., Thomas D., Worden F., Prince M., Urba S., Chepeha D., Kumar В., Cordell K., Eisbruch A., Taylor J., Moyer J., Bradford C, D'Silva N. Infiltrating lymphocytes and human papillomavirus-16-associated oropharyngeal cancer. // Laryngoscope. - 2012. - Vol.122. - №1. - p.121-127]. Для оценки качества жизни определяли вес мышей, наличие алопеции, двигательную активность животных оценивали в тесте «открытого поля» в автоматическом режиме.In each group, 10–13 mice aged 22 months were scored. The remaining mice were monitored until their natural death. The volume of tumors (mm 3 ) in the liver was determined, which was calculated by the standard formula. Histological examination of the liver was performed on paraffin sections. The presence of lymphocytic infiltration and destruction of tumor sites in the liver of mice of the experimental group was a favorable prognostic factor for the course of the tumor process [Оbl D., Loewe R., Yu P., Mihm M. Focus on TILs: Prognostic significance of tumor infiltrating lymphocytes in human melanoma // Cancer Immun. - 2009. - Vol. 9. - Number 3. p.245-251; Wansom D., Light E., Thomas D., Worden F., Prince M., Urba S., Chepeha D., Kumar B., Cordell K., Eisbruch A., Taylor J., Moyer J., Bradford C , D'Silva N. Infiltrating lymphocytes and human papillomavirus-16-associated oropharyngeal cancer. // Laryngoscope. - 2012 .-- Vol. 122. - No. 1. - p.121-127]. To assess the quality of life, we determined the weight of mice, the presence of alopecia, the motor activity of animals was evaluated in the test of the "open field" in automatic mode.
Определяли уровни ИЛ-6 и ИЛ-10 в сыворотке крови, т.к. цитокины ИЛ-6 и ИЛ-10 участвуют в патогенезе кахексии животных, повышая уровень С-реактивного белка и усиливая расщепление мышечных белков, а также ИЛ-6 оказывает негативное влияние на функциональную активность волосяных фолликулов [Krzystek-Korpacka М., Matusiewicz М., Diakowska D., Grabowski К. et al. Acute-phase response proteins are related to cachexia and accelerated angiogenesis in gastroesophageal cancers. // Clin. Chem. Lab. Med. - 2008. -Vol.46. - №3. - p.359-364; Deans D., Tan В., Ross J. et al. Cancer cachexia is associated with the IL10-1082 gene promoter polimorphism in patients with gastroesophageal malignancy. // Am. J. Clin. Nutr. - 2009. - Vol.89. - №4. - p.1164-1172; Biswas S., Pinson D., Bronshteyn I., LeVine S. IL-6 deficiency allows for enhanced therapeutic value after bone marrow transplantation across a minor histocompatibility barrier in the twitcher (globoid cell leukodystrophy) mouse. // J. Neurosci. Res. - 2001. - Vol.65. - №4. - p.298-307]. Содержание цитокинов ИЛ-6 и ИЛ-10 в сыворотке крови определяли с использованием иммуноферментного анализа.The levels of IL-6 and IL-10 in serum were determined, because the cytokines IL-6 and IL-10 are involved in the pathogenesis of animal cachexia, increasing the level of C-reactive protein and enhancing the breakdown of muscle proteins, as well as IL-6 has a negative effect on the functional activity of hair follicles [Krzystek-Korpacka M., Matusiewicz M., Diakowska D., Grabowski K. et al. Acute-phase response proteins are related to cachexia and accelerated angiogenesis in gastroesophageal cancers. // Clin. Chem. Lab. Med. - 2008 .-- Vol. 46. - Number 3. p.359-364; Deans D., Tan B., Ross J. et al. Cancer cachexia is associated with the IL10-1082 gene promoter polimorphism in patients with gastroesophageal malignancy. // Am. J. Clin. Nutr. - 2009 .-- Vol.89. - No. 4. p.1164-1172; Biswas S., Pinson D., Bronshteyn I., LeVine S. IL-6 deficiency allows for enhanced therapeutic value after bone marrow transplantation across a minor histocompatibility barrier in the twitcher (globoid cell leukodystrophy) mouse. // J. Neurosci. Res. - 2001 .-- Vol.65. - No. 4. - p. 298-307]. The content of IL-6 and IL-10 cytokines in blood serum was determined using enzyme-linked immunosorbent assay.
Уровень экспрессии лейкоцитарных интегринов LFA-1 (CD11a) и Мас-1 (CD11b) на лимфоцитах периферической крови определяли с использованием проточной цитофлуометрии.The expression level of leukocyte integrins LFA-1 (CD11a) and Mac-1 (CD11b) on peripheral blood lymphocytes was determined using flow cytometry.
Среднюю продолжительность жизни (СПЖ) и медиану выживаемости определяли по методу Каплан-Мейера. Статистический анализ результатов проводили с использованием однофакторного дисперсионного анализа ONE-WAY ANOVA программы "STATISTICA-6,0», достоверными считали различия при P<0,05.Average life expectancy (LIF) and median survival were determined by the Kaplan-Meier method. Statistical analysis of the results was carried out using ONE-WAY ANOVA univariate analysis of variance of the STATISTICA-6.0 program, the differences were considered significant at P <0.05.
Результаты определения изучаемых параметров в контрольных группах не имели различий, поэтому мыши двух контрольных групп были объединены в одну контрольную группу (n=160).The results of determining the studied parameters in the control groups did not differ, therefore, mice of two control groups were combined into one control group (n = 160).
У мышей опытной группы, получавших препарат фм-40, выявлено снижение частоты возникновения спонтанных опухолей на 30,8% по сравнению с мышами контрольной группы (P<0,001). Количество опухолей на мышь в опытной группе было ниже, чем у мышей контрольной группы (1,0±0,3 и 2,7±0,3 соответственно; P<0,001). Средний объем опухолевой массы на мышь в опытной группе также был ниже, чем у мышей контрольной группы, и составил 268,4±78,0 мм3 и 1043,1±230,0 мм3 соответственно, P<0,05.In mice of the experimental group treated with the drug fm-40, a decrease in the incidence of spontaneous tumors by 30.8% was detected compared with mice in the control group (P <0.001). The number of tumors per mouse in the experimental group was lower than in mice of the control group (1.0 ± 0.3 and 2.7 ± 0.3, respectively; P <0.001). The average volume of tumor mass per mouse in the experimental group was also lower than that in mice of the control group, and amounted to 268.4 ± 78.0 mm 3 and 1043.1 ± 230.0 mm 3, respectively, P <0.05.
Вес мышей опытной группы был выше, чем в контрольной группе, и составил 34,6±0,5 г и 24,4±0,2 г соответственно, P<0,01. Мыши опытной группы имели полноценный шерстный покров в отличие от мышей контрольной группы, у которых в 16,6% случаев наблюдалась алопеция. Эти данные коррелировали со снижением уровня сывороточных цитокинов ИЛ-6 и ИЛ-10 в опытной группе по сравнению с контрольной группой (114,8±12,3 пг/мл и 46,8±5,3 пг/мл) и (139,1±6,6 пг/мл и 60,9±3,9 г/мл) соответственно, P<0,05. Экспрессия лейкоцитарных интегринов LFA-1 и Мас-1 у мышей опытной группы была выше значений в контрольной группе и составила (40,7±1,9% и 11,5±1,1%) и (35,4±1,6% и 7,8±1,0%) соответственно, Р<0,05. Эти данные свидетельствуют об усилении иммунореактивности животных под воздействием препарата фм-40 при его применении в профилактическом режиме. Двигательная активность мышей опытной группы в возрасте 31 месяца соответствовала двигательной активности мышей контрольной группы в возрасте 24 месяцев. СПЖ мышей контрольной группы составила 22 месяца.The weight of the mice of the experimental group was higher than in the control group, and amounted to 34.6 ± 0.5 g and 24.4 ± 0.2 g, respectively, P <0.01. The mice of the experimental group had a full coat, in contrast to the mice of the control group, in which alopecia was observed in 16.6% of cases. These data correlated with a decrease in the level of serum cytokines IL-6 and IL-10 in the experimental group compared with the control group (114.8 ± 12.3 pg / ml and 46.8 ± 5.3 pg / ml) and (139, 1 ± 6.6 pg / ml and 60.9 ± 3.9 g / ml), respectively, P <0.05. The expression of leukocyte integrins LFA-1 and Mac-1 in mice of the experimental group was higher than the values in the control group and amounted to (40.7 ± 1.9% and 11.5 ± 1.1%) and (35.4 ± 1.6 % and 7.8 ± 1.0%), respectively, P <0.05. These data indicate an increase in the immunoreactivity of animals under the influence of the drug fm-40 when used prophylactically. The motor activity of mice of the experimental group at the age of 31 months corresponded to the motor activity of mice of the control group at the age of 24 months. The LSS of the control group mice was 22 months.
СПЖ мышей опытной группы увеличилась на 14,5% (25,2 месяца). Медиана выживаемости мышей опытной группы увеличилась на 23% по сравнению с мышами контрольной группы и составила 25,9 и 21,1 месяцев соответственно.The LSS of mice in the experimental group increased by 14.5% (25.2 months). The median survival of mice of the experimental group increased by 23% compared with mice of the control group and amounted to 25.9 and 21.1 months, respectively.
Изобретение иллюстрируется фиг. 1-2.The invention is illustrated in FIG. 1-2.
На фиг.1 представлен участок трабекулярно-ацинарной гепатокарциномы низкой степени дифференцировки у мыши контрольной группы (гистология печени, окраска гематоксилином и эозином, увеличение х 400, указано стрелками).Figure 1 presents a plot of trabecular acinar hepatocarcinoma of a low degree of differentiation in a mouse of the control group (histology of the liver, staining with hematoxylin and eosin, magnification x 400, indicated by arrows).
На фиг.2 представлен участок лимфоцитарной инфильтрации гепатокарциномы у мыши опытной группы, получавшей препарат фм-40 в профилактическом режиме (окраска гематоксилином и эозином, увеличение ×400, указано стрелками).Figure 2 presents the plot of lymphocytic infiltration of hepatocarcinoma in the mouse of the experimental group treated with the drug fm-40 in a prophylactic mode (stained with hematoxylin and eosin, magnification × 400, indicated by arrows).
Технический результат заявляемого способа заключается в увеличении средней продолжительности жизни и медианы выживаемости, в снижении частоты возникновения спонтанных гепатокарцином и улучшении качества жизни мышей-самцов линии СВА с высокой частотой спонтанного образования гепатокарцином.The technical result of the proposed method is to increase the average life expectancy and median survival, to reduce the incidence of spontaneous hepatocarcinomas and to improve the quality of life of male CBA mice with a high frequency of spontaneous formation of hepatocarcinomas.
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