At AIG hospitals, we investigate how decreased pancreatic ductal secretions and decreased pH affects the gene expressions in the pancreatic ductal cells, especially focusing on the oncogenes, exploring the possibility of cellular adaptation to acidic microenvironments. The challenge here is the heterogeneity of pancreatic tumors, which complicates the treatment as well as the experimental approaches. So we conducted RNAC analysis of pancreatic ductal cells under reduced pH conditions, and we found that there is increased expression of three oncogenes highlighting the potential links between the acidic environment and oncogene expression in pancreatic ductal cells.
Our study has shed light on the oncogene expression under reduced pH conditions, and this system can advance our research in studying pancreatic ductal adenocarcinoma under the conditions of inflammation. So future scope of this study is to evaluate the role of increased oncogene expressions in the initiations of tumors under inflammatory conditions, such as in chronic pancreatitis. To begin, procure the human normal pancreatic ductal cell line.
Thaw the cells at 37 degrees Celsius in a water bath. Now filter freshly prepared complete medium through a syringe filter. Add six milliliters of complete medium to a new 15 milliliter conical tube.
Then transfer the thawed cell suspension to the tube and mix thoroughly. Centrifuge the suspension at 1, 800 G for six minutes. Discard the supernatant and add one milliliter of culture medium to the pellet before mixing gently.
Next, add four milliliters of complete medium into a 60 millimeters Petri dish. Then pipette the cell suspension dropwise into the flask. Incubate the culture at 37 degrees Celsius under 5%carbon dioxide supplementation.
To change the pH of the culture medium, first, prepare 100 milliliters of 0.5 molar sodium phosphate buffer stock solutions with pH values of 6.0, 6.5, and 7.0. Check the pH with a pH meter and adjust it using one molar hydrochloric acid or one molar sodium hydroxide. Then filter, sterilize the buffers in a laminar airflow hood using a 0.45 micrometer syringe filter.
Next, add 20 milliliters of sterile 0.5 molar sodium phosphate buffer of the respective pH to 80 milliliters of culture medium to prepare media of different pH. Add the media with the altered pH to 85 to 90%confluent human pancreatic ductal cells. Observe morphological changes under a brightfield microscope at one hour intervals for six hours.
To isolate RNA from the culture, first aspirate the culture medium and rinse the cells with one milliliter of PBS. Place the 60 millimeter dish on ice, then one milliliter of PBS to the 60 millimeter dish and scrape the cells with a cell scraper. Now transfer the suspension into a fresh tube.
Centrifuge it at 8, 000 G for 10 minutes and discard the supernatant. Next, add 600 microliters of lysis buffer containing beta-mercaptoethanol to the pellet. Pipette the suspension up and down for five to 10 minutes on ice.
Add an equal volume of 70%ethanol to the lysate and pipette the suspension to mix thoroughly. Transfer the solution to a silica membrane column. Then centrifuge the column at more than 8, 000 G for 30 seconds and discard the flow through.
Next, add 500 microliters of wash buffer to the sample and centrifuge again. Centrifuge the empty tube for one minute at more than 8, 000 G.Finally, add 30 to 50 microliters of nuclease free water to the column. After a five minute incubation at room temperature, centrifuge the column again at speeds higher than 8, 000 G for five minutes.
A significant reduction in cell size and shrinkage of ductile cells in acidic medium was observed compared to cells maintained in a normal medium. The most pronounced changes in cell death occurred after six hours of incubation with a further decrease in cell count and signs of cellular stress at 22 hours. The number of upregulated genes was highest at pH 6.5 with 148 genes followed by pH seven with 109 genes and pH six with 90 genes.
Downregulated genes were fewer with 20 at pH six, 14 at pH 6.5 and 23 at pH seven. Three oncogene, LCK, FGR and ASAP three were upregulated across all tested pH levels, suggesting a role in cell proliferation under acidic conditions.
