Effects Of The Expression Of SP-B And Apoptosis In Alveolar Epithelial Type? Cells Induced By Heat Resistant Antigen Of Mycobacterium Tuberculosis

Background and Objective: The existing studies about Mycobacterium Tuberculosis(M.tb),mainly through its M.tb component or the secretory components inducing the different immune cells(dendritic cells(DC),T cells and so on)to study its related mechanism of action.But the alveolar epithelial type?cells(AT2),playing an important role in the process of invasion lung alveolar against M.tb,is rarely reported.The widespread use of known in clinical against tuberculosis(TB)-Bacille-Calmette-Guerin(BCG),there is a lack of stability in adult pulmonary tuberculosis protection,so the new targeting drug development has become a new research topic in the treatment of TB.This study select the human lung AT2 cells which are an important barrier to M.tb invasion as the starting point.As human lung adenocarcinoma cell lines(A549)cells have the same phenotype and feature selection with AT2 cells,A549 cells induced by Mycobacterium tuberculosis heat-resistant antigen(Mtb-HAg).To investigation the secretion of pulmonary surfactant-associated protein B(SP-B)in A549 cells and the effect on apoptosis to A549 cells.Furthermore,the effects of Mtb-HAg on the secretion of SP-B and apoptosis in AT2 cells were studied indirectly,which provides a new theoretical basis for studying the mechanism of AT2 cell defense against M.tb invasion in the early stage of lung infection by TB;preliminary explore the apoptosis of AT2 cells induced by Mtb-HAg,for the further study of the pathway to provide new ideas.Methods:(1)Dulbecco's Modified Eagle Media(DMEM),containing 10% fetal bovine serum(FBS)and 1% double-resistance,was used to culture A549 cells in constant temperature incubator for 37 ? and 5% CO2.DMEM culture medium was diluted to different concentrations of Mtb-HAg,and respectively was to stimulate the induction of A549 cells for 24 h and 48 h,and simultaneously set up the control groups.(2)The expression level of culture supernatant of SP-B in the blank control group1,positive control group(Lipopolysaccharide(LPS)for 24 h)and the experimental group1(different concentrations of Mtb-HAg for 24 h,48 h)were detected by enzyme-linked immunosorbent assay(ELISA).(3)The relative expression changing of SFTPB gene in the blank control group1,positive control group(LPS for 24 h)and the experimental group1(different concentrations of Mtb-HAg for 24 h,48 h)were determined by real time fluorescence quantitative PCR(q RT-PCR).(4)The apoptosis rates of the blank control group2,the positive control group(curcumin for 24 h)and the experimental group2(different concentrations of Mtb-HAg induced to culture for 24 h)were assessed by Flow cytometry(FCM).Results:(1)Compared with the blank control group1,the expression of SP-B in the positive control group(LPS group)and the experimental group1(with different concentrations of Mtb-HAg culturing at the same time)were significantly lower than that in the blank control group1,and the difference was statistically significant(P<0.05).As well as the expression of SP-B in the experimental group1(using the same concentration of Mtb-HAg for different time)was not significant(P>0.05).(2)When cultured for the same time,the relative expression of gene SFTPB in the experimental group1(different concentrations of Mtb-HAg induced)and the positive control group(LPS group)were lower than that of the blank control group1,the difference was statistically significant(P<0.05).However,under the same Mtb-HAg concentration,the relative expression of gene SFTPB in the experimental group1 were not significant with time,and the difference was not statistically significant(P>0.05).(3)The apoptosis rate of A549 cells in the positive control group(curcumin group)was higher than that in the blank control group2,the difference was statistically significant(P<0.05).The apoptosis rates of A549 cells in the experimental group2 were significantly higher than that in the blank control group2,and the difference was statistically significant(P<0.05).But the apoptosis rates of A549 cells in the between experimental group2 were not significant(P>0.05).Conclusions:(1)Mtb-HAg could inhibit the expression of SP-B and gene SFTPB in A549 cells,the model of AT2,thus it is beneficial to study the mechanism of M.tb invading AT2 cells in the early stage of lung.(2)Mtb-HAg could induce apoptosis of A549 cells,and provide a new approach for the study of the mechanism of apoptosis in human AT2 cells by Mtb-HAg of M.tb.