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Morphological parameters of the primary tumor in mice C57Bl/6 with transplantanted Lewis lung carcinoma under the effect of allogeneic mesenchimal stem cells
The investigation was carried out in C57Bl/6 male mice weighing 20–22 g aged 2 to 3 months. All researches on animals were carried out according to Guide for the Care and Use of Laboratory Animals.
The strain of metastatic Lewis lung carcinoma (LLC) was kindly given by National Bank of Cell Lines and Tumor Strains of R.E. Kavetsky Institute of experimental pathology, oncology and radiobiology of National Academy of Sciences of Ukraine (IEPOR, NASU). The tumor cell suspension (1х106/0.1ml of Haenk’s solution) which was obtained from LLC primary tumor tissues with routine procedure of trypsinization was inoculated intramusculary into mice. After tumor cell inoculation mice were divided into experimental and control groups not After inoculation of tumor cells was formed three groups of animals. The first group consisted of mice with Lewis lung transplant carcinoma, the second – mice with Lewis lung transplant carcinoma on the 8th day after inoculation of tumor cells was administered intravenously allogeneic MSCs 4th passage in the amount 1,25x104. The third group were combined animal tumor, which was administered saline 0.9 % NaCl (8 animals in each). Mice of experimental group received the course of inoculation of MSCs in concentration 1.25x104 cells were administrated on 8th day after tumor cell inoculation.
MSCs were obtained from male mice bone marrow. Six-week-old С57BL/6 mice were sacrificed by cervical dislocation. Their femurs were carefully cleaned of soft tissues; epiphyses were removed in sterile conditions; and the bone marrow was harvested by inserting a syringe needle (20-gauge) into one end of the bone and flushing with medium DMEM (Sigma, USA). Cell suspension was layered on ficoll-triombrast with index density gradient 1.074 and centrifuged at 300 g. Then select cell fraction which was on the verge ficoll-triombrast, added Dulbeco’s phosphate buffered saline solution, made suspension and centrifugation at 300 g. The cell pellet was resuspended in medium DMEM and cultured in standard condition with the addition of 20 % FBS (fetal bovine serum) and 1 mkl/sm3 antibiotic-antimycotic. Medium was replaced every 3–4 days. Visual assessment carried out by forming a monolayer every 24 hours using an inverted microscope Axiovert 40 (Carl Zeiss). When the cells formed a monolayer, they were shooting from the culture dishes 0.025 % trypsin containing 0.02 % EDTA. The trypsin action was stopped by adding FBS. After centrifugation cells were seeded on the larger diameter cultural dishes and cultivated by the formation of a next passage cells monolayer.
In the 18 th and 24 th day of the experiment was determined pathomorphological changes in muscular tissue of mice with Lewis lung carcinoma perescheplenoyu (LLC) under the influence of allogeneic MSCs. For this purpose produced histological preparations. For this selection of material performed immediately after euthanasia of animals research groups. The selection of tumor tissue was performed with the central, peripheral areas of the tumor and on the verge of muscle tissue unaffected. The selected material is crushed into pieces the size of 3–4 mm3, fixed, embedded in paraffin, sections were produced using sleigh Microtomes MS-2. Slice thickness reaches 5–9 microns. The resulting sections were applied to slides prepared (cleaned, degreased and treated with a mixture of egg protein with glycerol) using distilled water t=37–40 °C for smoothing. Subject glass with slices kept on the table for drying samples within 24–36 hours. Painting cuts performed with hematoxylin and eosin and by Van Ghisone. Painted cuts light optic studied by microscope Olympus, Lomo.
In the 18 th and 24 th day of the experiment determined the morphological changes of the primary tumor. On the 18th day of research on the impact of MSCs in primary tumor tissue were registered 1.3 times higher density of blood vessels per unit area than in groups of animals without the influence of MSCs. In vascular hemostasis recorded the type of sludge-effect of erythrocytes. On the 24th day the samples when exposed to MSCs devitalization foci of tumor tissue were recorded dystrophic tumor cells, and between them – lymphocytes. Registered many areas of necrosis, surrounded by degenerative modified tumor cells. On the 24th day study on the impact of allogeneic MSCs areas recorded higher levels of devitalization and necrosis area of tumor with reduce vessel.
Key words: allogeneic mesenchymal stem cells, Lewis lung carcinoma, morphological indexes, primary tumor mouse.
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