New Gene Markers for Metabolic Processes and Homeostasis in Porcine Buccal Pouch Mucosa during Cells Long Term-Cultivation-A Primary Culture Approach

Abstract

The oral mucosal tissue is a compound structure composed of morphologically and physiologically different cell types. The morphological modification involves genetically determined lifespan, which may be recognized as the balance between cell survival and apoptosis. Although the biochemical processes and pathways in oral mucosa, with special regards to drug transport, delivery, and metabolism, are well known, the cellular physiological homeostasis in this tissue requires further investigation. The porcine buccal pouch mucosal cells (BPMCs) collected from 20 pubertal crossbred Landrace gilts, were used in this study. Immediately after recovery, the oral mucosa was separated micro-surgically, and treated enzymatically. The dispersed cells were transferred into primary in vitro culture systems for a long-term cultivation of 30 days. After each step of in vitro culture (IVC), the cells were collected for isolation of total RNA at 24 h, 7, 15, and 30 days of IVC. While the expression was analyzed for days 7, 15, and 30, the 24th hour was used as a reference for outcome calibration. The gene expression profile was determined using Affymetrix microarray assays and necessary procedures. In results, we observed significant up-regulation of SCARB1, PTGS2, DUSP5, ITGB3, PLK2, CCL2, TGFB1, CCL8, RFC4, LYN, ETS1, REL, LIF, SPP1, and FGER1G genes, belonging to two ontological groups, namely "positive regulation of metabolic process", and "regulation of homeostatic process" at 7 day of IVC as compared to down-regulation at days 15 and 30. These findings suggest that the metabolic processes and homeostatic regulations are much more intense in porcine mucosal cells at day 7 of IVC. Moreover, the increased expression of marker genes, for both of these ontological groups, may suggest the existence of not only "morphological lifespan" during tissue keratinization, but also "physiological checkpoint" dedicated to metabolic processes in oral mucosa. This knowledge may be useful for preclinical experiments with drugs delivery and metabolism in both animals and humans.

Keywords: homeostasis; metabolic process; oral mucosa.

Figure 1

The Volcano plot representation between differently expressed genes between 7, 15, and 30 days of culture. The gene selection criteria were p-value beneath 0.05 and expression higher than two fold.

Figure 2

Heat map representation of differentially expressed genes belonging to the “positive regulation of metabolic process” and “regulation of homeostatic process”—GEOTERM BP database. Arbitrary signal intensity acquired from microarray analysis is represented by colors (green, higher; red, lower expression). Log2 signal intensity values for any single gene were resized to Row Z-Score scale (from −2, the lowest expression, to +2, the highest expression for a single gene). Each analysis was run in two samples coming from different in vitro cultures of oral mucosal cells. Changes in transcript levels were analyzed between three time periods-Day 7(D7), Day 15 (D15) and Day 30 (D30), of primary culture. The fold change was calculated in relation to transcript levels at hour 24 of primary culture.

Figure 3

STRING (Search Tool for the Retrieval of Interacting Genes/Proteins)-generated interaction network among differentially expressed genes belonging to the “positive regulation of metabolic process” and “regulation of homeostatic process” GO BP terms. The intensity of the edges reflects the strength of interaction score.

Figure 4

The representation of the relationship between differently expressed genes that belong to the “positive regulation of metabolic process” and “regulation of homeostatic process” GO terms. The ribbons show, which gene belongs to which categories. The genes were sorted by logFC from most to least changed gene. The gene that was downregulated the most, between day 7 and day 30, is presented on the bottom of the chart, the gene exhibiting the lowest amount of change is presented topmost. The intensity of the color indicates the scale of change in expression.

Figure 5

The results of RT-qPCR (Real Time-quantitative Polymerase Chain Reaction) validation of analyzed genes, presented in the form of bar-graph. All the fold changes were described in relation to the transcript levels in 24 h of primary culture. LogFC was used to present the data, in order to improve the clarity and comparability of up and downregulation results.

Figure 6

Photos of cell cultures prior to the material collection taken under an inverted microscope using relief contrast.