Overview of the different methods used in the primary culture of oral mucosa cells.

The culture of primary cells in vitro has enabled to gain knowledge in the field of cell biology, disease mechanisms and to offer great potential in drug testing. To date, two main techniques of isolating and culturing oral mucosal cells, the direct explant method and the enzymatic method, dominate the literature and practice. In the present study, both techniques are discussed in detail, comparing the advantages and disadvantages of the two approaches in setting up a primary culture of oral mucosal cell. The direct explant technique is well-established and has been commonly used for the past 20-30 years. Although the method of setting up the cultures did not show much variations in the methodology described by authors, the culturing conditions varied according to the aims of the projects.

Expression of PTX3, HAS2 AND TNFAIP6 genes in relation to real-time proliferation of porcine endometrial luminal epithelial cells in primary cultivation model.

Endometrial cells undergo very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume. Then, if fertilization fails, endometrial cells are liable for apoptosis, preparing new cells that are ready for subsequent processes related to the possibility of embryo implantation and the development of pregnancy. PTX3 and TNFAIP6 are absent or reduced in cultured COCs, resulting in a functional change in COC in vitro. In this work, we want to check how PTX3, HAS2 and TNFAIP6 behave in luminal epithelium primary cell culture. Cells obtained during slaughter from porcine specimens were cultured primarily in vitro for 7 days. Their proliferation patterns were then analysed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes in the expression of the genes of interest were analysed on each of the seven days of the porcine luminal primary cell culture. Our study showed the increased level of PTX3, HAS2 and TN¬FAIP6 expression at the same hours of primary culture. Rt-qPCR showed a higher level of expression of the PTX3 gene in the first 72 h, at the end of the lag phase (in the phase of stasis in which the cells adapt to the new environment and often die). In contrast, TNFAIP6 expression increases about 96 hours when the cells are in the full log phase (logarithmic phase growth) and continue this trend in the plateau phase. We did not observe such drastic changes in the HAS2 expression pattern, which leads us to hypothesize that PTX3 and TNFAIP6 are designed to maintain a constant level of HAS2 in the cell throughout its lifetime. The obtained results could become a point of reference for further in vivo and clinical research.

Gene expression regulating lipopolysaccharide and lipid metabolic processes in porcine oral mucosal cells in long-term primary in vitro culture.

Lipids are an alternative energy source for cells and provide structural integrity in cell membrane and their metabolism is regulated with the use of different pathways, such as integrin signalling, oxidative stress, mechanical stress, and pH changes. All of those processes take place in the oral mucosa which is subject to different environmental impacts. In this study, porcine buccal pouch mucosal cells (pBPMCs) were used during long-term primary in vitro culture. The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation. In the results of the following microarray analysis, we analyzed the genes detected, belonging to ontology groups, such as "cellular lipid metabolic process", "response to lipid" and "response to lipopolysaccharides. All of the genes involved in these ontological groups were expressed at higher levels at 7 days of IVC and substantially decreased in expression at days 15 and 30 of primary culture. We observed new genes, which may be recognized as markers in regulation of lipid metabolism in mucosal cells in vitro. The results suggested that the biochemical mechanism-involved lipids were accompanied by increased enzymatic activation and synthesis of crucial growth factors reaching high activity at day 7 of culture, which is also well documented as a stage of tissue regeneration period within oral mucosa. Therefore, this "biochemical fingerprint" may be an additional checkpoint of the integrity, resistance and easy adaptability of oral tissues, which are important conditions of success in tissue engineering and grafting for tissue reconstruction.

Balance between epithelial and stromal marker expression and distribution in primary culture model of porcine endometrium during real-time cell proliferation..

The similarity between humans and pigs, when it comes to tissue morphology, makes Sus scrofa not only a good research model, but also a potential source of cells for tissue engineering. Cell samples obtained from the pig donor, could be influenced in vitro, in order to become a source of tissue material for xenotransplantation, reconstructive and regenerative medicine. Significant amounts of data point to especially major similarities in pig and human reproductive systems. Because of that, particular scientific focus is centered on research concerning porcine COCs, theca and granulosa cells in primary cultures. One of the aspects of the reproductive process, that is still largely undiscovered, is the interaction between preimplantation blastocyst and maternal uterine tissues. In this study, we used molecular analysis techniques, such as RT-qPCR and immunocytochemistry, to analyze the expression and distribution of cytokeratin 18 and panCytokeratins 8, 18 and 19 and vimentin in porcine luminal endometrial epithelial cells, coupled with analysis of their behavior in RTCA. The results have confirmed the presence of epithelial, as well as stromal cell markers in the cells, varying in levels at different stages of culture. They have also given insight into the modes of proliferation and differentiation of studied cells in in vitro culture, as well as providing additional proof for the possible mesenchymal transdifferentiation of epithelial cells.

Enzyme linked receptor protein signaling pathway is one of the ontology groups that are highly up-regulated in porcine oocytes before in vitro maturation.

Before being able to fully participate in the processes associated with its function as a female gamete, the oocyte needs to undergo a range of changes to achieve its mature form. These morphological, biochemical and metabolomic processes are induced by the somatic tissues surrounding the oocyte, through the expression of specific transcription and growth factors. The maturation of the oocyte is highly important for the proceedings that lead to successful fertilization, early embryonic development and implantation. Domestic pigs were used as models for our study, with the cumulus-oocyte complexes obtained from the ovaries that were recovered at slaughter. After shedding of the cumulus, oocytes were assessed with BCB test, with the viable ones chosen to undergo in vitro maturation. With the use of expression microarrays, we analyzed gene expression before and after IVM and detected major changes in both genes that were proven to be associated with oocyte maturation before (FOS, VEGFA, CHRDL1, TGFBR3, FST, INSR, ID1, TXNIP, SMAD4, MAP3K1, EIF2AK3 and KIT) and genes not previously linked with reproduction associated processes (MYO1E, PHIP, KLF10 and SHOC2). All the genes were briefly described, with consideration of possible involvement of the newly discovered elements of the transcriptome in the process of oocyte maturation.

[Preliminary results of testing The WHO Guidelines for Tuberculosis Control in the Republic of Chuvashia].

The authors describe the first results of introduction of the WHO recommendations for tuberculosis control in the Republic of Chuvashia. The total number of studies performed in the general therapeutic network and in tuberculosis facilities has increased by 1.3 and 1.5 times, respectively. The microscopic diagnosis of Mycobacterium tuberculosis in the sputum has improved, increasing the detection rate of tuberculosis from the smear, particularly in the general network, from 2.2% to 24.2%, but at the same time leading to higher morbidity in the republic. There has been improvement of the efficiency of treatment in patients by the generally accepted statistical indicators: abacillation by 19.7%, decay cavity closure by 11.3%, and being taken off the book of bacterial isolates by 15%. On completion of intensive-phase treatment of patients with pulmonary tuberculosis, negative sputum tests have been 84.7% in the tuberculosis facilities and 91.7% in the Federal Services penitentiaries. The interaction has become better between the penitentiary system and the tuberculosis-controlling service.