Investigation On The Immune-like Cells And Morphology Of Slug During The Dictyostelium Discoideum Development

Dictyostelium discoideum is a lower eukaryotic protozoan, a social amoeba, living in the soil under natural and feeding on Klebsiella in laboratory. Dictyostelium discoideum bred by bipartition as nutrient-rich and the single cell assembled to multicellular aggregate when the food ran out. The entire development process will take about 24 hours and broadly be divided into four stages, aggregation stage, mound stage, slug body stage and fruiting stage. Slug phase can be divided into "tip", "slug" and "topping". Slug cells are divided into two categories, pre-stalk cells and pre-spore cells. Pre-stalk cells are subdivided into PstA cells, PstB cells, PstO cells and PstAB cells. Pre-stalk cells will develop into stalk cells and pre-spore cells will develop into spore cells in the fruiting body stage. Spore cells can germinate and start a new life cycle when the conditions are favorable, while the stalk cells can not germinate under any conditions, so stalk cells are apoptotic cells.It can be seen clearly that Dictyostelium discoideum exist the phenomenon of cells differentiation and apoptosis which is same as higher organisms.Slug period is the initial phase of cell differentiation and cell apoptosis. Due to the environmental impact and the differences of size, the individual development of slug is not fully synchronized. Because the whole slug phase only has two hours, slug period is regarded as an important period but specific morphological development process of slug has not been reported. Because the various types of cells move at different speeds, morphology of slug has undergone tremendous changes. We dyed Dictyostelium discoideum with two kinds of fluorescent dyes, acridine orange and Hoechst 33342. We observed Dictyostelium discoideum cell differentiation through the fluorescence microscope and attempted to understand Dictyostelium discoideum cell development, in particular, the development of cell differentiation and apoptosis occurs during specific development process.The results showed that near 4/5 cell has always been dyed blue in the whole development process, while 1/5 cell located at the front of slug body. With developmental process, the results show that different degree apoptosis of the pre-stalk cells during development were referred to the colors, blue-green, green, orange, and finally colorless. In the later of slug stage, a channel-like structure were observed and dyed green. There is light blue-green net structure in the channel when it developed into fruiting body. The channel could eventually develop into stalk of fruiting body. However, the blue cells are no signs of recession and eventually develop into spores.The cell nuclear of spores within the fruiting body also had been dyed blue. Accordingly, the spore cells germinate to form a new life. These data indicate that prestalk begin apoptosis from the early of slug, but cells never die immediately and their apoptosis are a gradual process.The function of cells nuclear doesn't disappear completely, while losing its function with the process of cell apoptosis. Slug can divide into four periods at least, nucleus was dyed blue and green in the first place, and then it was dyed green, orange, and finally colorless. Determining the development process through the cell nuclears shading different with recession, and can form base for the future study of Dictyostelium discoideum.In the experiment, a special kind of cells is found in the slug stage and these cells have the ability of swallowing toxic substances.It is found that Hoechst33342 dye the nucleus of living cells when its concentration is lower than 10μg/mL and Dictyostelium discoideum are damaged which may be caused as Dictyostelium discoideum cells die or some emergency response in the experiment when the Hoechst33342 concentration is higher than 10μg/mL by Irene Tatischeff. Because the function of these cells is similar to the immune function of higher organisms and has detoxification and immune-like activity, we called these cells immune-like cells. We use fluorescent dye EB as toxic which Dictyostelium discoideum encountered in the grow environment, by fluorescence microscopy and Confocalmicroscope to position and by flow cytometry to determine which cells are immune-like cells and how much is the ratio of cells. We found by study the immune-like cells not only can engulf bacteria, but can block the poison when these poison circulate in the slug body. The poison will expel from slug eventually as the slug mobile and be found in the abandoned sheath. Showed feeding mechanism of the ancient cells before the animal diverse has defense function. It is found that EB is not positioned in the nucleus, so it doesn't combine DNA but is wrapped in the cell.It was found that immune-like cells mainly existed in pre-stalk cells and they are a special class of pre-stalk cells by flow cytometry. However, it still need be further confirmed from the genes examined in depth.