Effect Of Lipopolysaccharide With Different Concentration On Apoptosis Of Trophoblast Cell Lines JEG-3

ObjectiveLPS lies in the adventitial coat of Gram-negative bacteria.It is the important matter of inducing injury and apoptosis of cells.Some studies indicate that LPS can induce excess apoptosis of macrophage and alveolar epithelial cell el at through the different signal conduction pathway.Some studies indicate that some pathological pregnancies such as eclampsism,abortion,fetal growth restriction and postterm pregnancy exist excess apoptosis of cytotrophoblast cell(CTB).Can LPS participate in the onset of pathological pregnancy through inducing over apoptosis of CTB? This experiment will invest effect of LPS with different concentration on apoptosis of CTB and approach the effect of LPS in the onset of pathological pregnancy.Materials and Methods1.MaterialsJEG-3 cell lines were purchased by the apartment of animal of Chinese Academy of Medical Sciences and inoculated by FD medium with 10%calf serum,100u/ml penicillin and 100u/ml streptomycin and cultured in humid incubator with 5%CO₂ at 37℃.The cells of logarithmic phase chose in experiment.2.Methods(1)Control group(CG):no LPS,carry out conventional cell culture and passage.Experimental group(EG):The cells were inoculated for 24 hours and putted in six pore plates and then coverslip handled withâ… -type collagen was putted in six pore plates.When the cell was fulled in coverslip for 80%,different dose LPS was added in culture to make its final concentration to be 25,50,100ng/ml and collected the cells after 24 hours.(2)After dealt with LPS for 24 hours,CTB were stained and observed by fluorescence microscope and electron microscope and analyzed by flow cytometry (FCM).3.Statistic analysisThe ANOVA was used by SPSS 12.0.Apoptosis rate of JEG-3 cell dealt with different concentration of LPS was analyzed.The result had the meaning if p＜0.05.Results1.morphology of apoptotic CTBNucleus of CTB is large and chromosome is well-distributed in CG by Hoechst33258 staining under fluorescence microscope(FRM).The apoptotic body is obviously observed in EG.The ultrastructure of apoptotic cell is observed under transmission electron microscope(TEM).2.Detection of rate of LPS inducing apoptosis of CTBThe result of FCM found that with increase of concentration of LPS,power of LPS inducing apoptosis of CTB increased.In CG the apoptosis rate was (0.81±0.15)%,in EG apoptosis rates were(2.06±0.33)%,(7.44±0.68)%,(13.44±0.90)%(concentration of LPS were 25,50,100ng/ml).ConclusionsLPS can induce apoptosis of plenty of CTB,apoptotic index of CTB is obviously increased with increase of concentration of LPS.They are dose-dependent relation.It is close correlation between apoptosis of plenty of CTB and LPS.