The Establish Of OA Damage A549 Cells Model And Its Effect On SP-B

The main research content:There is a large number of studies have shown that OA caused by acute lung injury(acute lung injury,ALI)animal models and are very similar with clinical pathology change.However,the mechanism still remains unclear,and there has been no research about SP-B in vitro.In this study,we established oleic acid induced acute lung injury of A549 cells in vitro to explore the effects of different concentrations of OA on SP-B,at the same time,comparing with the LPS model.The main experimental methods:1.Experimental grouping:The cells were cultured for 3 generations.The cells grew well without contamination and with the fusion rate of >80% were selected and divided into 5 groups:(1)OA control group: there is no oleic acid add,and the same amount of DMEM without fetal bovine serum was added to be cultured for 24 hours.;(2)OA experimental groups: there were 4 groups,the final concentrations of 300umol/L,400 umol/L,500 umol/L and 600 umol/L oleic acid solution were added respectively in the cells to be cultured for 24 hours.LPS model group also can be divided into 5 groups:(1)LPS control group: there same with OA;(2)LPS experimental groups: there were 4 groups,the final concentrations of 0.1 ug/ml,1 ug/ml,10 ug/ml and 100 ug/ml LPS solution were added respectively in the cells to be cultured for 24 hours.2.The cell morphological change observed by microscope and the proliferative activity detected by CCK-8:When the cells grew as expected,The inverted microscope was used to observe the morphological change of cells and take photos under 200 magnifications.After that 10 ul CCK-8 solution was added and the mixture was kept in incubator for 2 hours.The microplate reader and GEN5 software were applied.The wavelength was set as 450 nm to detect the OD values.3.SP-B expression detected by ELISA:There were 5 groups with 5 repeated well in each group.After 24 hours,ELISA was used to detect the SP-B levels in the supernatant(ELISA was completed according to the protocol in the kit).4.SP-B secretion detected by Western Blot:The cells were cultured for 3 generations,the supernatant and the cells were collected.Western Blot was used to detect the SP-B levels(Western Blot was completed according to the protocol in the kit).5.Statistical analysis:The SPSS16.0 statistical package was used to analyze the data.Data were presented as mean ± SD and compared using a t test for independent samples.A p value of < 0.05 was considered as statistically significant.The Origin software was used to prepare graphs.The main results:1.The morphological change of cells:The A549 cells in the control group showed polygonal shape with abundant cytoplasm and obvious protuberance,the cells grew adhering to the wall and fused together,there was no karyopyknosis;in the 300umol/L oleic acid group,the cells slightly became round,the volume was slightly decreased,and there were different sizes of granules;in the 400 umol/L oleic acid group,the cells obviously became round and volume was decreased,the protuberances were less and there were many different sizes of granules;in the 500 umol/L oleic acid group,the cells started to be broken and the volume was significantly decreased,the organelles were disintegrated and fragmented;in the 600 umol/L oleic acid group,there cells were mostly broken,there was obvious shirking of nucleus,the organelles were obviously disintegrated and fragmented.LPS model has the same morphological change.2.The proliferative activity of A549 cells detected by CCK-8:In CCK-8 results,the optimal density(OD)values of A549 cells in 300,400,500 and 600μmol/L oleic acid groups were dose-dependently decreased,which were 87.0%,75.7%,64.4% and 56.8% of the value in the control group(P<0.05).T test was used for pairwise comparison,the differences were statistically significant(P<0.05).In CCK-8 results,the optimal density(OD)values of A549 cells in 0.1 ug/ml,1 ug/ml,10 ug/ml and 100 ug/ml LPS solution were dose-dependently decreased,which were 86.7%、75.4%、48.9%和 44.0% of the value in the control group(P<0.05).T test was used for pairwise comparison,the differences were statistically significant(P<0.05).3.The SP-B expression in supernatant detected by ELISA:ELISA results showed that SP-B expressions in 300,400,500 and 600μmol/L oleic acid groups were dose-dependently increased,which were 145.5%,187.4%,223.9% and 235.3% of the expression in the control group(P<0.05),being in accordance with nolinear distribution(R=1.00).T test was used for pairwise comparison,except the 600μmol/L oleic acid group,the differences were statistically significant(P<0.05).ELISA results showed that SP-B expressions in 0.1 ug/ml,1 ug/ml,10 ug/ml and 100 ug/ml LPS solution groups were dose-dependently increased,which were102.7%、124.2%、152.5%和 157.2% of the expression in the control group(P<0.05).4.SP-B detected by Western Blot:Western Blot results showed that SP-B expressions in the cells were decreasd as the oleic acid and Lps concentration increased.43 KD represents pro SP-B,28-32 KD represents incomplete SP-B segments.Conclusion:The injury of oleic acid on A549 cells is dose-dependent.If the concentration is higher,more SP-B is released to extracellular space and less SP-B is secreted.This can provide a reference for the ALI model in vitro.