The Study Of Invasive Characteristics And Mechanism Of Human Source S. Flexneri On Chicken Intestinal Epithelial Cells

To establish an in vitro model for studying the pathogenicity and invasioncharacteristic of Human Shigella in chicken, isolation and culture of primary chicken intestinalepithelial cells were studied. Primary intestinal epithelial cells from chicken embryos wereisolated by the digestion with0.1g/mL neutral proteaseⅠand300U/mL collagenaseⅪ.Theoptimal condition for separating chicken embryo intestinal epithelial cells was screened, and theisolated cells were identified by transmission electron microscope. Then primary chickenintestinal epithelial cells were digested by0.125%trypsin and0.01%EDTA, and passaged.Primary chicken intestinal epithelial cells in better condition can be obtained by the digestion withneutral proteaseⅠand collagenase Ⅺ, and attached well. These cultured cells were round orpolygonal cells, and grew into pavestone-like monolayer. The intestinal epithelial cells attachedwithin12h, proliferated significantly within24⁴8h, and poorly after72h. The cultured cells wereidentified as intestinal epithelial cells by transmission electron microscope. The passaged cellsgrew well. The primary intestinal epithelial cells with high purity could be obtained from chickenembryo by the established method, and can be passaged for3generations.In order to further determine pathogenicity of Human S.flexneri in chicken, invasion andinvasive characteristics of human S. flexneri ZD02strain on primary chicken intestinal epithelialcells were studied, and an in vitro model was established. Virulence of the isolated human S.flexneri ZD02strain was validated by invasion of Hela cell using Gentamicin resistance assay andimmunohistochemistry. Invasion and invasive characteristics of ZD02strain on primary chickenintestinal epithelial cells were researched by immunohistochemistry. Human S. flexneri ZD02strain could invade into Hela cells and the invasion ratios were1.43%,2.43%,2.56%,2.72%at30,60,90,120minutes after infection respectively. The result of immunohistochemistry showed thatZD02strain could invade into primary chicken intestinal epithelial cells and the invasion ratioswere1.7%,6.2%,8.0%,11.2%at1,2,3,4hours after infection respectively. Primary chickenintestinal epithelial cells treated with EGTA which disrupts intercellular junctions, significantlyenhanced invasion ratio of ZD02strain. Human S. flexneri ZD02strain can invade into primarychicken intestinal epithelial cells from the basolateral pole of cells, which further validated the possibility of mutual infection caused by S. flexneri between human and avian. Primary chickenintestinal epithelial cells can be used as an in vitro model to study the pathogenic mechanism ofShigella.To study the role of cell signal transduction and cytoskeleton in invasion of Human S.flexneriinto primary chicken intestinal epithelial cell, primary chicken intestinal epithelial cell wastreated with Genistein, sodium orthovanadate, staurosporine, Nifedipine and Cytochalasin Brespectively, which are the corresponding inhibitor of protein tyrosine kinase, protein tyrosinephosphorylase, protein kinase C, Ca2+channel signal transduction and microfilamentpolymerization. The influences of above inhibitors on invasion of ZD02strain into primarychicken intestinal epithelial cells were detected by immunohistochemistry. F-actin distribution ofprimary chicken intestinal epithelial cells infected with Human S.flexneri ZD02strain wasdetected by indirect immunofluorescence double labeling.Genistein and Nifedipine significantlyreduced internalization of ZD02strain into primary chicken intestinal epithelial cells. In contrast,staurosporine and sodium orthovanadate have no effect on internalization of ZD02strain.Cytochalasin B could prevent internalization of ZD02strain. F-actin aggregated when ZD02strain invaded into primary chicken intestinal epithelial cells. The process of Human S.flexneriinvading into primary chicken intestinal epithelial cell depends on protein tyrosine kinase, Ca²⁺channel activation and actin rearrangement.The above results show that Human S. flexneri can invade into primary chicken intestinalepithelial cells from the basolateral pole of cells, which further validated the possibility of mutualinfection caused by S. flexneri between human and avian, and the process of Human S.flexneriinvading into primary chicken intestinal epithelial cell depends on protein tyrosine kinase, Ca²⁺channel activation and actin rearrangement.