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17В Красную книгу Российской Федерации занесены следующие виды насекомых: Xylocopa violacea, Parnassius apollo, зафиксированные как в Зилаирском, так и в Стерлитамакском районе, Lucanus ceryus, Parnassius mnemosyne, зафиксированы только в Зилаирском районе, Bombus proteus зафиксирован в Стерлитамакском районе. Все насекомые относятся ко 2 категории - сокращающийся в численности вид [2, с. 274-292].
В Красную книгу Республики Башкортостан занесены следующие насекомые: Parnassius apollo - категория 2 - сокращающиеся в численности виды, Xylocopa violacea - категория 1 - находящиеся под угрозой исчезновения, оба экземпляра зафиксированы в обоих районах. Насекомые, зафиксированные в Зилаирском районе - Lucanus ceryus, находящийся во 2 категории - сокращающиеся в численности виды, Parnassius mnemosyne и Papilio machaon, относящиеся к 3 категории - редкие виды, Mantis religiosa, находящийся в 5 категории - восстанавливаемые и восстанавливающиеся виды. Насекомые, зафиксированные в Стерлитамакском районе - Bombus proteus, относящийся ко 2 категории - сокращающиеся в численности виды, Vanessa atalanta и Iphiclides podalirius, относящиеся к 3 категории - редкие насекомые [6, с. 5-10].
Всего на территории двух районов было зафиксировано 156 видов (103 вида в Зилаирском и 94 вида Стерлитамакском районах), из которых 42 вида общие: 2 вида отряда прямокрылые (Orthoptera), 7 видов отряда перепончатокрылые (Hymenoptera), 1 вид двукрылых (Díptera), 16 видов
чешуекрылых (Ьер1^р1ега), 8 видов стрекоз (0^па!а), 3 вида отряда полужесткокрылых (Иеш1р1ега) и 5 видов жесткокрылых (Со1еор1ега) (см. таблицу).
Сравнение видового разнообразия энтомофа-уны Зилаирского и Стерлитамакского районов с помощью коэффициента Жаккара показало, что эти районы по видовому составу энтомофауны сходны на уровне 27%.
Исходя из выше изложенного, результаты исследований могут быть использованы для дальнейшего изучения географического распространения и экологии насекомых, обитающих на территории Республики Башкортостан и разработать более эффективные меры по их охране.
Литература
1. Козлов М.А. Школьный атлас-определитель беспозвоночных. - Просвещение, 1991. - 159 с.
2. Красная книга Российской Федерации. -Эксмо, 2017. - 480 с.
3. Мамаев Б.М. Определитель насекомых европейской части СССР. - Просвещение, 1976. - 304 с.
4. Мигранов М.Г. Булавоусые чешуекрылые Башкирии: (определитель). - БНЦ УрО АН, 1991. -132 с.
5. Насекомые европейской части России: Атлас с обзором биологии. - Фитон XXI, 2013. - 568 с.
6. Редкие и исчезающие виды животных и растений Республики Башкортостан. Вып. III. -РИЦ БашГУ, 2011. - 20 с.
EXPRESSION OF SPECIFIC GLIOMA MARKERS AND PROLIFERATIVE ACTIVITY INDEX IN TUMOR TISSUES OF RATS AT EXPERIMENTAL ASCARIASIS AND TRICHINOSIS
Pobyarzhin V.
Vitebsk Peoples ' Friendship State Medical University, Candidate of Biological Sciences, assistant professor Vitebsk the Republic of Belarus
ABSTRACT
Objective - to study the expression of specific glioma markers and indicators of the proliferative activity index in tumor tissues of rats with experimental ascariasis and trichinosis depending on the dose of infection and the duration of observation.
Materials and methods. Studies were performed on female rats of the Wistar line. The experiment consisted of 5 series, C6 rat glioma in situ was simulated in all animal groups, and on the 14th, 21st, 28th, 35th day of tumor development, the expression of immunohistochemical markers GFAP, S 100, as well as the proliferative activity index Ki 67 were studied. Rats of the first series of the experiment served as control.
The obtained results described a «pure» tumor of rat C6 glioma in situ. The second and third series of the experiment were carried out in order to assess the effect of Ascaris suum at different periods of parasite development at different doses of infection of experimental animals (20 and 40 eggs per 1 gram of body weight, relatively). The fourth and fifth series of experiments were carried out to assess the effect of Trichinella spiralis depending on the timing of the development of the invasion and the dose of infection (10 and 20 larvae per 1 gram of body weight, relatively).
Results. It was found that the invasion of A. suum at a dose of 20 eggs per 1 gram of body weight of the animal does not lead to significant changes in the expression of immunohistochemical markers GFAP, S 100 and the proliferative activity index Ki 67 in tissues of rat C6 glioma in situ compared to «clean» tumor. The invasion of A. suum of 40 eggs per 1 gram of body weight of the animal increases the expression of GFAP, S 100, and also increases the proliferative activity index by the 7th day and the 14th day after infection. The results show a clear dose-dependent effect when infected with roundworms.
Infection of T. spiralis at a dose of 10 or 20 larvae per 1 gram of mass increases GFAP expression in biopsy samples of tumor tissue by the 21st and 28th days, but S 100 expression and Ki 67 proliferative activity index is increased on all terms of observation. No dose-dependent effect was observed upon infection with T. spiralis.
Conclusion. Thus, the invasion of A. suum and T. spiralis promotes the expression of glioma markers and an increase in the proliferative activity index, which can contribute to the intensive growth of the tumor and increase its aggressiveness.
Keywords: rat, glioma, immunohistochemical markers, GFAP, S 100, Ki-67, Ascaris suum, Trichinella spi-
ralis.
Introduction. Recently, there has been an increase in the percentage of incidence of helminth infections worldwide. Among the entire share of helminthi-ases, a small fraction is occupied by geohelminthoses (ascariasis) and biohelminthoses (trichinosis). Geohel-minthes include worms, which for development do not need a change of owners. In turn, parasitic worms, which need several organisms of various species to complete the full development cycle, are classified as biohelminths. Despite such a difference in the development cycles, the negative impact of both is very multi-faceted.
It is shown that in the migration phase of development both Ascaris and Trichinella, there is a sensitization of an organism by products of metabolism, moulting and decay of dead parasites. Due to the secreted antigens arise massive pathological changes, accompanied by an allergic response [1, 2]. The causes of systemic organ lesions also include the formation of immune complexes that trigger a complement dependent reaction. Despite the formation of a powerful host immune response, helminths and their metabolites are able to act as immunosuppressants. As a result, parasites survive and contribute to a decrease in the protective properties of the invasive organism relative to secondary infections, and this, in turn, contributes to the protracted course of diseases, complications, and the formation of carriage [3, 4, 5].
Important links of pathogenetic mechanisms also include mechanical, chemical, and mutagenic effects [3, 6], which, in turn, can lead to the activation of blas-tomogenesis. At the moment, the role of roundworms in blastomogenesis is not studied.
Objective: to study the expression of specific glioma markers and indicators of the proliferative activity index in tumor tissues of rats with experimental ascari-asis and trichinosis depending on the dose of infection and the duration of observation.
Materials and methods. The experiment was performed on 200 female Wistar rats weighing 180-200 g. Before the experiment, experimental animals were quarantined.
All manipulations with animals were carried out in accordance with the recommendations of the Council of Europe Convention on the Protection of Vertebrate Animals for Experimental and Other Scientific Purposes: Strasbourg, Council of Europe, 51 pp; 03/18/1986), Council Directive on the Approximation of Laws, Regulations and Administrative Provisions of the Member States Regarding the Protection of Animal Used for Experimental and Other Scientific Purposes), recommendations of the FELASA Working Group Report (1994-1996), Helsinki Declaration (2008), TCH 125-2008 and guidelines «The Regulation on the use of
laboratory animals in scientific research and the pedagogical process of the educational institution Vitebsk State Medical University», and measures to implement the requirements of biomedical ethics .
The animals were divided into 20 groups of 10 animals in each and 5 series of the experiment were carried out. In accordance with the author's method of reproducing rat glioma, animals of all groups were injected with tumor cells of rat glioma C6 into the inner thigh at a concentration of 10x106 subcutaneously to simulate the tumor in situ. Dexamethasone was injected into the inner surface of the other thigh at a dosage of 0.001 ml (4.0 mg / ml) per 1 g of animal weight. Dexa-methasone injection was performed daily for 7 days after inoculation, and starting from the 8th day - with frequency rate in a day within 14 days.
The first series of the experiment included the first, second, third, fourth groups of female rats, which served to obtain results in a «clean» tumor of rat C6 glioma in situ and served as a control. In animals of the first series of the experiment, on the 14th, 21st, 28th, 35th day of tumor development (after killing under the influence of ether anesthesia), tumor material was collected for histological and immunohistochemical analyses in order to subsequently determine the expression of the main glioma markers: GFAP (glial fibrillary acidic protein), S 100, as well as estimates of the pro-liferative activity of Ki-67 [7].
The second and third series of experiments aimed at assessing the effect of roundworms depending on the timing of the development of invasion on the expression of immunohistochemical markers GFAP, S 100, Ki 67 in tissues of rat C6 glioma in situ.
The fifth, sixth, seventh and eighth groups were included in the second series of the experiment (the dose of animals infection with A. suum eggs, 20 eggs per 1 gramm of body weight), and the ninth, tenth, eleventh and twelfth groups in the third series (the dose of of animals infection with A. suum eggs, 40 eggs per 1 gramm of rat body weight) [8]. Rats were infected orally on the 7th day after the introduction of tumor cells C6. Thus, the 7th day after infection corresponded to the 14th day of tumor development, the 14th day to the 21st day of tumor development, the 21st day after infection to the 28th day of glioma development, the 28th to the 35th days of tumor development.
The fourth (13-16 groups) and fifth (17-20 groups) series of the experiment was performed on 40 female Wistar rats to evaluate the effect of T. spiralis on the expression of immunohistochemical markers in the tissues of experimental rat gliomas (GFAP, S 100, Ki 67) depending on the timing of the development of the invasion and the dose of infection. The animals of the
fourth series were orally infected at a dose of 10 T. spi-ralis larvae per 1 gramm of animal body weight [9] on the 7th day after the introduction of C6 tumor cells, and the fifth series of 20 T. spiralis larvae per 1 gramm of body weight animal also on the 7th day after the introduction of tumor cells C6.
The animals of the fourth and fifth series were removed from the experiment on the 7th day (intestinal stage of Trichinella development, the 14th day of tumor development), on the 14th (migration stage, the 21st day of tumor development) and on the 21st, 28th day (muscle stage, the 28th and 35th day of glioma development, respectively) of the development of trichinella, tumor material was also collected.
The material received from all experimental animals was loaded into buffered formalin for 24 hours, after which it was poured into paraffin [7]. Then prepared tissue samples for survey studying were stained with hematoxylin-eosin and these sections were enclosed in polystyrene [10]
For immunohistochemical studies, serial paraffin sections were made using glasses treated with poly-L-lysine on a Leica RM 2125 RT microtome for subsequent dewaxing and dehydration. The antigens were unmasked by the Wuhan Elabscience Biotechnology Incorporated Company buffer. An immunohistochemi-cal reaction in the finished material to the GFAP receptors (E-AB-10345), S100 (E-AB-32841), Ki-67 (E-AB-22027) was carried out in accordance with the instructions of the manufacturer and the system 2-step plus Poly-HRP Anti-Rabbit / Mouse IgG Detection System imaging (with DAB Solution, Wuhan Elab-science Biotechnology Incorporated Company, China, E-IR-R213).
The evaluation of immunohistochemical staining was carried out using a Leica DM 2500 light microscope in 1000 cells of each section in disjoint fields of view, taking into account the localization of staining in the cell (nucleus, cytoplasm and / or membrane), staining intensity (in the region with maximum expression) and the percentage of stained cells [7]. The tumor was considered negative in the absence of cytoplasmic staining or when staining was less than 10% of the cells; glioma was evaluated at 1 point (1+) when staining the cytoplasm from 10 to 25% of the cells; 2 points (2+) - when staining the cytoplasm from 26 to 50% of the cells; 3 points (3+) - when staining the cytoplasm in more than 50% of cells (GFAP, S 100).
Ki-67 (proliferative activity of the tumor) was evaluated as the percentage of positive nuclei in the tumor cells. The result was considered negative if there was no nuclear expression with antibodies in the tumor tissue or the number of stained nuclei was less than 10%; the result was considered positive when staining more than 10% of tumor cells, evaluated in the region of maximum marker expression; a tumor with high pro-liferative activity included material with Ki-67 expression in more than 40% of cells; low proliferative activity was the characteristic for the expression of Ki-67 in less than 40% of tumor cells [7].
Immune reactivity (IRS) was calculated by summing the scores for the proportion of stained cells and
their staining intensity. The tumor was considered positive with a total score of more than or equal to three [7].
Differences between groups were evaluated using the Mann - Whitney test (Mann - Whitney, U-test) and were considered statistically significant at p <0.05. Data processing was performed using the program Sta-tistica 10.
Results and discussion. When evaluating the results of the first series of the experiment (tumor sampling on the 14th, 21st, 28th, 35th day, control), it was revealed that the tumor of a solid structure is represented mainly by polymorphic tumor cells with figures of mitoses. The intercellular substance is represented by neuropil. Necrosis, vascular proliferation were found in the tumor.
When evaluating tissues with gliomas by the im-munohistochemical method (tumor collection on the 14th, 21st, 28th, 35th day, control, first series), the following was established: GFAP expression by the 14th day was 1+ (12%; 95 % CI: 10.94-13.65%; IRS = 4); by the 21st day - 1+ (17%; 95% CI: 13.70 - 20.61%; IRS = 4); by the 28th day - 1+ (12%; 95% CI: 11.28 - 13.4%; IRS = 4); on the 35th day - 1+ (10%; 95% CI: 10.04 - 11.47%; IRS = 4).
The expression of S 100 in the control samples by the 14th day was at the 1+ level (15%; 95% CI: 12.85 - 17.32%; IRS = 4); by the 21st day - 1+ (17%; 95% CI: 15.32 - 19.15%; IRS = 4); by the 28th day - 1+ (13%; 95% CI: 11.98 - 14.29%; IRS = 4); on the 35th day - 1+ (10%; 95% CI: 10.01 - 10.64%; IRS = 4).
Tumor proliferative activity (Ki-67) in the control group on the 14th day of development was 35% (95% CI: 32.16 - 39.13%); by the 21st day - 36% (95% CI: 33.27 - 38.92%); by the 28th day - 15% (95% CI: 14.22 - 15.57%); on the 35th day -10% (95% CI: 10.04 - 10.41%).
The above immunohistochemical profile and morphological picture corresponded to glioblastoma (glioma).
GFAP expression in biopsy samples of tumor tissue of rats of the second series of the experiment (infection of 20 A. suum eggs per 1 gram of body weight) by the 7th day of invasion development was 1+ (12%; 95% CI: 10.94 -13.65%; IRS = 4); by the 14th day - 1+ (17%; 95% CI: 13.93 - 20.84%; IRS = 4); by the 21st - 1+ (12%; 95% CI: 10.87 -13.24%; IRS = 4); on the 28th day - 1+ (11%; 95% CI: 10.17 - 11.74%; IRS = 4).
Expression of S 100 in the same samples by the 7th day after infection with A. suum was at the 1+ level (14%; 95% CI: 12.71 - 15.48%; IRS = 4); by the 14th day - 1+ (16%; 95% CI: 14.70 - 18.43%; IRS = 4); by the 21st day 21 - 1+ (12%; 95% CI: 11.52 - 13.83%; IRS = 4); on the 28th day - 1+ (10%; 95% CI 10.09 - 10.76%; IRS = 4).
Tumor proliferative activity (Ki-67) was evaluated as follows: on the 7th day of the development of invasion 36% (95% CI: 34.59 - 39.04%); by the 14th day - 35% (95% CI: 32.38 - 37.71%); by the 21st day - 14% (95% CI: 13.74 - 15.05%); on the 28th day - 10% (95% CI: 9.94 - 11.41%).
When comparing the obtained expression data of GFAP, S 100, as well as the proliferative activity of Ki-67, obtained in the second series of the experiment, with the group of the first series ("pure" tumor), no significant differences were revealed at all stages of the development of the parasite.
The results of evaluating the expression of GFAP in the biopsy samples of the tumor tissue of animals of the third series showed (infection of 40 A. suum eggs per 1 gram of body weight), that by the 7th day of the development of invasion it was 2+ (26%; 95% CI 22.97-29.64%; IRS = 6), which is 2.16 times (p = 0.00015) higher than the control indicators; by the 14th day - 1+ (18%; 95% CI 15.41 - 20.74%; IRS = 4); by the 21st day - 1+ (16%; 95% CI 13.74 - 18.18%; IRS = 4); on the 28th day - 1+ (11%; 95% CI: 10.28 - 11.27%; IRS = 4).
S 100 expression in the same samples of the third series by the 7th day after infection with A. suum (infection of 40 A. suum eggs per 1 gram of body weight) was at the 2+ level (38%; 95% CI: 33.40 - 42, 51%; IRS = 6) and exceeded the control by 2.53 times (p = 0.00015); by the 14th day - 2+ (35%; 95% CI: 29.29 - 41.43%; IRS = 4) - 2.05 times higher than the control group (p = 0.0001); by the 21st day - 1+ (15%; 95% CI: 13.09 - 16.56%; IRS = 4); on the 28th day - 1+ (11%; 95% CI: 10.96 - 11.83%; IRS = 4).
The proliferative activity of the tumor (Ki-67) on the 7th day of the development of invasion was 80% (95% CI: 73.56 - 86.07), which is 2.28 times higher than the control (p = 0.00015); by the 14th day - 80% (95% CI: 71.96 - 86.77), which is 2.22 times more than the data of the control group (p = 0.0001); by the 21st day - 14% (95% CI: 13.66 - 14.83%); on the 28th day - 10% (95% CI: 10.08 - 11.63%).
When assessing the expression of GFAP in the tumor tissue of animals of the fourth series of the experiment, by the 7th day of the development of trichinella (the dose of infection of 10 larvae per 1 gram of body weight of the animal) was 1+ (19%; 95% CI: 16.20 - 22.01%; IRS = 4); by the 14th day - 1+ (23%; 95% CI: 14.77 - 31.00%; IRS = 4); by the 21st day - 2+ (37%; 95% CI: 31.31 - 42.68%; IRS = 6), which is higher than the control at 3.08 (p = 0.00015); on the 28th day - 2+ (38%; 95% CI: 31.72 - 44.39%; IRS = 6), which exceeds the data of the first control group by 3.8 times (p = 0.00015).
The expression of S 100 by the 7th day after infection was at the 2+ (45%; 95% CI: 33.12 - 57.71%; IRS = 6) and was 3 times higher than the control (p = 0.00015); by the 14th day - 2+ (45%; 95% CI: 28.14 - 61.93%; IRS = 6), which was 2.64 times higher than the results of the first series (p = 0.02334); by the 21st day - 2+ (40%; 95% CI: 35.35 - 44.68%; IRS = 6) - 3.07 times higher than the control (p = 0.00015); on the 28th day - 2+ (49%; 95% CI: 36.79 - 61.72%; IRS = 6) - exceeded the control by 4.9 times (p = 0.00015).
The proliferative activity of the tumor in the samples of the fourth series of the experiment (Ki-67) on the 7th day of Trichinella development was 71% (95% CI: 56.69 - 86.20%) and exceeded the control by 2.02 times (p = 0.00149); by the 14th day - 61% (95%
CI: 28.14 - 61.93%) and was 1.69 times higher than the control indicators (p = 0.00243); by the 21st day - 40% (95% CI: 29.84 - 46.35%) - exceeded the results of the first series by 3 times (p = 0.00015); on the 28th day - 43% (95% CI: 41.71 - 44.52%), which is 4.3 times more than the control group (p = 0.00015).
A statistical evaluation of the results of immunohistochemical staining of the tumor material of the fifth series of the experiment (the dose of infection of 20 trichinella larvae per 1 gram of body weight of the animal) showed that the expression of GFAP in tumor tissue biopsies by the 7 th day of the development of the parasite was 1+ (18%; 95% CI: 14.45 - 21.98%; IRS = 4); by the 14th day - 1+ (24%; 95% CI: 14.41 - 34.16%; IRS = 4); by the 21st day - 2+ (38%; 95% CI: 31.87 - 43.58%; IRS = 6), which significantly exceeded the control by 3.16 times (p = 0.00015); on the 28th day - 2+ (39%; 95% CI: 32.21 - 46.92%; IRS = 6), which is 3.9 times higher than the data of the control group (p = 0.00015).
The proliferative activity of the tumor (Ki-67) on the 7th day of Trichinella development was 73% (95% CI: 59.35 - 87.34%) and exceeded the control results 2.08 times (p = 0.00076); by the 14th day - 60% (95% CI: 38.87 - 81.88%) - 2.4 times higher than the control (p = 0.000264); by the 21st day - 40% (95% CI: 29.73 - 49.14%) - 2.6 times higher than the control group data (p = 0.005796); on the 28th day - 47% (95% CI: 44.27 - 50.12%), which is 4.7 times more than the control (p = 0.000157).
Thus, for the first time in our experimental rat glioma tumor model C6 in situ, it was shown that invasion of A. suum at a dose of 20 eggs per 1 gram of animal body weight does not lead to significant changes in the expression of immunohistochemical markers GFAP, S 100 as well as in the expression of Ki 67 proliferative activity index in rat glioma tissue C6 in situ in comparison with a «pure» tumor.
It was proved that in comparison with the results obtained in the control group (pure tumor), the invasion of A. suum of 40 eggs per 1 gram of body weight of the animal increases the expression of GFAP in biopsy samples of tumor tissue of rat C6 glioma in situ by the 7th day of the development of the parasite by 2.16 times (p = 0.00015); it raises S 100 expression by the 7th day of invasion development by 2.53 times (p = 0.00015), and by the 14th day after infection - by 2.05 times (p = 0.00015); as well as the proliferative activity index Ki 67 by the 7th day of the development of invasion by 2.28 times (p = 0.00015), by the 14th day after infection by 2.22 times (p = 0.00015).
It was shown that the invasion of T. spiralis at a dose of 10 larvae per 1 gram of mass increases GFAP expression in biopsy specimens of tumor tissue of rat C6 glioma in situ by the 21st and 28th days after infection by 3.08 and 3.8 times, respectively (p = 0.00015); it raises S 100 expression on the 7th day of Trichinella development by 3 times (p = 0.00015), on the 14th day - 2.64 times (p=0.02334), on the 21st day after infection - 3.07 times (p = 0.00015), on the 28th day - by 4.9 times (p = 0.00015); it raises the index of proliferative activity of Ki 67 on the 7th day of the development of invasion by 2.02 times (p = 0.00149),
on the 14th day - by 1.69 times (p = 0.00243), on the 21st day after infection - by 3 times (p = 0.00015), and on the 28th day - by 4.3 times (p = 0.00015) compared with the control.
When analyzing the data obtained in comparison with a «pure» C6 tumor in situ, it was revealed that invasion of T. spiralis at a dose of 20 larvae per 1 g of body weight increases GFAP expression in biopsy samples of tumor tissue of rat C6 glioma in situ by the 21st and 28th days after infection in 3.16 and 3.9 times, respectively (p = 0.00015); it raises S 100 expression by the 7th day of invasion development in 3.4 times (p = 0.00015), by the 14th day - in 2.58 times (p = 0.02334), by the 21st day after infection - in 3.23 times (p = 0.00015), by the 28th day - in 5.4 times (p = 0.00015); the proliferative activity index Ki 67 by the 7th day of the development of invasion in 2.08 times (p = 0.00076), by the 14th in 2.4 times (p = 0.00026), by the 21st day after infection - in 2.6 times (p = 0.00579), and by the 28th day - in 4.7 times (p = 0.00015).
The results obtained allow us to draw the following conclusions:
1. When reproducing ascariasis in the experiment, a clear dose-dependent effect is noted in relation to changes in the expression of both specific glioma markers and the proliferative activity index in C6 tumor tissues in situ.
2. During invasion of T. spiralis in experimental animals, a dose-dependent effect in changing the expression of glioma markers GFAP, S 100 and prolifer-ative activity index in C6 tumor tissues in situ was not detected.
3. The data obtained indicate that invasion by trichinella and roundworms can contribute to the activation of pathogenetic mechanisms leading to more rapid growth of C6 rat glioma in situ, especially during the period of high biological activity of the parasite.
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