The Effect Of Aggregatibacter Actinomycetemcomitans On Beta-defensins In Human Gingival Epithelial Cells

Objective: To investigate the effect on the expression of host innateimmune effector molecules by different number of Aggregatibacteractinomycetemcomitans infect, we examined the quantity of beta-defensins andinflammatory cytokins in different time after put Aggregatibacteractinomycetemcomitans in human gingival epithelial cells.Methods: Used tissue block cultivation method to culture the gingivaltissue from clinical. Cultivated A. actinomycetemcomitans（ATCC43717） andadjust its number to5×107cells/ml,1×108cells/ml and2.5×108cells/ml, thenjoined them to the third passage cells which concentration was5×105cells/ml.The MOI was100:1,200:1and500:1. A. actinomycetemcomitans and humangingival epithelial cells were co-cultured for0,2,4,8,12,24and48h.Co-cultured supernatants were investigated by enzyme-linked immunosorbent assay. Total RNA that extracted from gingival epithelial cells was detected byreal-time polymerase chain reaction to determine the quanitity of HBD-1mRNA,HBD-2mRNA, HBD-3mRNA. These data were representative of findings fromthree independent experiments and presented as mean SD or median.Statistical significance was determined using ANOVA and LSD-t test orFreidman-M test, Pearson or Spearman rank correlation (P＜0.05).Result:1. HBD-1, HBD-2, HBD-3, IL-1β and TNF-α expressed by HGECchanged with MOI. Supernatant was taken for ELISA test. After A.actinomycetemcomitans added in, HBD-1expression decreased with the MOIincreased at2h（r=－0.999, P=0.024）. HBD-2reduced with the increase of MOIat8h（r=－0.997, P=0.048）. HBD-1mRNA increseased with MOI after2h ofinfection detected by Real-Time PCR（r=0.998, P=0.040）.2. HBD-1, HBD-2, HBD-3, IL-1β and TNF-α expressed by HGEC changedwith time. MOI for100:1, HBD-1and HBD-3with no statistically significantdifference （F=0.753, P＞0.05; F=0.485, P＞0.05）. HBD-2began to increasein2h (P＜0.001), from0to24h express quantity increased with the extension oftime（r=0.904, P=0.013）. IL-1β upregulated with the time extension from8to48h（r=0.931, P＜0.001）. MOI for200:1, HBD-3increased from0to12h（r=0.961, P=0.009）. MOI for500:1, HBD-1increased after2h（r=0.949,P=0.014）.Test by Real-Time PCR, with three different MOI, HBD-2mRNAreached its peak after24h of infection, HBD-3mRNA reached its peak respectively at12,8and24h.Conclution: HBD-2and HBD-3were upregulated more obviously thanHBD-1in human gingival epithelial cells induced by aggregatibacteractinomycetemcomitans.