Морфометрична характеристика гепатоцитів інтактних щурів та при введенні кріоконсерованої плаценти

Abstract

В роботі представлений аналіз морфо метричної характеристики гепатоцитів інтактних щурів та при введенні кріоконсервованої плаценти. Дослідження проводилось на 50 статевозрілих щурах, масою 180-200 г. Об’єктом дослідження була печінки щурів, де вимірювались кількісні показники гепатоцитів. Експериментальні тварини були поділені на дві групи: І – інтактна група (5), ІІ – тварини, яким біла введена підшкірно одноразово кріоконсервована плацента (45). Виведення тварин відбувалося в наступні терміни: на 1-у, 2-у, 3-ю, 5-у, 7-у, 10-у, 14-у, 21-у та 30-у доби. Парафінові зрізи виготовлялись згідно загальноприйнятим методикам. Аналіз морфометричних досліджень структурних компонентів печінки тварин був проведений згідно з загальноприйнятими статистичними методами за допомогою програми Microsoft Office Excel 2007. Аналіз метричних показників розміру діаметрів і площі гепатоцитів, розміру ядер гепатоцитів та кількості багатоядерних гепатоцитів дозволив зробити висновки, що у щурів інтактної групи структурна організація печінки принципово не відрізняється від людини. Середній показник площі гепатоцитів складає у тварин інтактної групи 1364,226±35,982 мкм², ядерно-цитоплазматичне співвідношення – 0,196±0,006, середня площа ядер дорівнює 249,304±5,861 мкм². Введення кріоконсервованої плаценти впливає на стан гепатоцитів, що проявляється появою клітин з великими ядрами, збільшенням кількості двоядерних гепатоцитів. Зміни метричних показників гепатоцитів проявляються в збільшенні середньої площі клітин і ядер з 1-ої до 10-ої доби. До 30-ої доби спостерігалась тенденція до зменшення параметрів. Значення ядерно-цитоплазматичного індексу збільшувалось на 2-у і відновлювалось на 3-ю добу. Отримані данні можуть бути використані для обґрунтування та розробки ефективних методів терапії печінки за допомогою препаратів кріоконсервованої плаценти.

Introduction. One of the body’s large digestive glands is the liver, which has about 300 billion cells, 80% – hepatocytes. The shape of hepatocytes is polygonal, they contain one or two large nuclei. In the cytoplasm, organelles have a zonal location, there are well-developed granular and smooth endoplasmic reticulum, numerous mitochondria, lysosomes, peroxisomes, elements of the Golgi apparatus, inclusions – glycogen and lipids. The role of the liver is quite different: participation in metabolism, neutralization of toxins, regulation of homeostasis, as well as absorption, transformation and secretion of blood components. Many mammalian tissues, in particular placental tissue, contain a large number of biologically active substances, which reveals their significant therapeutic effect on various body systems, as well as enzyme systems of energy, protein and other types of metabolism. During the using of cryopreserved placenta in various pathological conditions, significant positive clinical results were observed, in particular on the influence of cryopreserved placenta on the course of inflammatory processes in the body. Despite the large amount of experimental and clinical data, many aspects of the mechanisms of action of tissue therapy remain poorly understood, so there is a need for further research in this area. The aim of the research. Compare the quantitative parameters of hepatocytes in intact rats and with the injection of cryopreserved placenta. Object and methods of research. The research was performed on 50 adult rats weighing 180-200 g. Experimental researches were performed according to the European Convection for the protection of vertebrate animals used for experimental and other scientific purposes.The object of the research was the liver of rats, where were measured the quantitative indicators of hepatocytes. Distribution of experimental animals: Group I – intact (5), II – animals, which were injected subcutaneously with disposable cryopreserved placenta (45). The animals were out from the researching at the following times: (1-3, 5, 7, 10, 14, 21 and 30 days). Paraffin sections were made according to generally accepted methods. Analysis of morphometric research of structural components of animal liver was performed according to generally accepted statistical methods using Microsoft Office Excel 2007. The probability of differences in quantitative results was determined using Student’s t-test. The differences were considered statistically significant in the generally accepted in medical and biological examinations error probability p˂0.05. Research results and their discussion. According to the research, the average diameter of hepatocytes of the intact group was 26.412±0.324 μm (large diameter) and 16.355±0.325 μm (small diameter), their area was 1364.225±35 μm, nuclear cytoplasmic index (IG_S) 0.196±0.006. The average values of the nuclei of hepatocytes of this group: large diameter – 9,976±0,094 μm, small diameter – 7,881±0,134 μm, area – 249,304±5,861 μm. The number of mononuclear hepatocytes ranged from 91.43±0.55, multinucleated – 8.57±0.55. The obtained results of qualitative and quantitative indicators of the liver of intact animals are control and necessary for comparison with the results of experimental researches. With the injection of cryopreserved placenta to experimental animals in the early stages of the experiment (1st, 3rd day) in the liver revealed reactive changes in the ultrastructure of hepatocytes. The average hepatocyte diameter was 25,531 μm (large diameter) and 17,564μm (small diameter), their area was 1423,065 μm, nuclear cytoplasmic index (IG_S) 0,217±0,005. The average hepatocyte nuclei were: large diameter – 10,215 μm, small diameter – 8,772 μm, plane – 284,405 μm. The number of mononuclear hepatocytes ranged from 89.17±0.65, multinucleated – 10.83±0.65 p˂0.05. Gradually there was a physiological reaction of hepatocyte structures, where on the 5-7-10th day of the experiment there were moderate changes of qualitative and quantitative nature. On the 7th and 10th day of the experiment, the following increased: large hepatocyte diameter 27.959±0.363 μm (at p˂0.001), small diameter 21.205±0.257 μm (at p˂0.001), hepatocyte area and nuclear cytoplasmic index 1872.067±40.188 μm (at p˂0.001). The diameters of hepatocyte nuclei (large and small) and the area of the nucleus also tended to increase on the 7th and 10th day of the experiment (11.289±0.149 μm at p˂0.001 – large, 9.836±0.140 μm at p˂0.001 – small, area 353.966±9.270 μm at p˂0.001). The number of multinucleated hepatocytes on the 10th day reached a maximum and was 13.09±1.1 (at p˂0,005). In the later stages of the study (14, 21, 30 days), most hepatocytes have no differences in ultrastructural organization from the liver of animals of the intact group. Correlation analysis between the area of hepatocytes and large diameter of hepatocytes showed a direct correlation between them, which comparing with intact (r=0.53 p<0.001) began to increase from the 1st day of the experiment and gradually decreased on the 30th day ( r=0.59 at p<0.001). Correlation analysis between hepatocyte area and small diameter was not performed, as the data were not very informative. Correlation analysis between the area and diameter of the nuclei showed a direct correlation and the greatest results were found on the 7th and 10th day (r=0.94 at p<0.001). Conclusions. It is proved that in rats of the intact group the structural organization of the liver is not fundamentally different from humans. The average area of hepatocytes in animals of the intact group is 1364.226±35.982 μm², the nuclear-cytoplasmic ratio is 0.196±0.006, the average area of nuclei is 249.304±5.861 μm². The injection of cryopreserved placenta affects the state of hepatocytes, which is manifested by the appearance of cells with large nuclei, an increase in the number of dinuclear hepatocytes. Changes in the metric parameters of hepatocytes are manifested in an increase in the average area of cells and nuclei from the 1st to the 10th day by 37% and 42%, respectively (p˂0,001). By the 30th day, there was a tendency to decrease the parameters. The value of the nuclear cytoplasmic index increased by 24.9% on the 2nd day and resumed on the 3rd. Prospects for further research is to develop methods for correcting some pathological conditions of the liver with the help of cryopreserved placenta.