Association Between Surfactant Protein B Intron 4 Polymorphism And Predisposition To Bronchopulmonary Dysplasia

Part Ⅰ:Establishment of monoclone of SP-B intron4 A549 cell line and monoclone of SP-B intron4del(3-8)m A549 cell lineObjective To establish the monoclone of SP-B intron 4 A549 cell line and the monoclone of SP-B intron4del(3-8)m A549 cell line.Methods the overexpression lentiviral vector contain the wild type SP-B intron4 target gene or the SP-B intron4 deletion 3 ~ 8motifs(del(3-8)m)target gene was transfected into A549 cells via lentiviral transfection.72 h after transfection,used fluorescence microscopy to observe the expression of GFP in A549,then began to use the selective medium containing 2μg/ml Puromycin for selective culture.Limiting dilution method was used for selecting monoclonal A549 cells.PCR was used to detect SP-B intron4 and SP-B intron4del(3-8)m gene.Western blot was used to detect the expression of SP-B.Results When MOI=40,The transfection efficiency of lentiviral vector in A549 cells were more than 80%.Cells with the Ability of resistant puromycin can be observed after selective culture for 3days.The monoclone of A549 cell stably transfected by The LV-SP-B-intron4 or the LV-SP-B-intron4del(3-8)m after selecting monoclone and the amplification of 3 generations.Tested the monoclone cell lines by PCR and western blot and obtained 4 monoclone of SP-B intron4 A549 cell lines and 3 monoclone of SP-B intron4del(3-8)m A549 cell lines,which all can expressed the intermediate SP-B-3Flag fusion protein(29KD).Conclusion The two A549 monoclone cell lines of SP-B intron4 or the SP-B intron4del(3-8)m with stable expression of corresponding SP-B were established successfully.Part Ⅱ Relation between the SP-B intron4 variations and the SP-B gene expression as well as hyperoxia-induced A549 cells injuryObjective To observe the cell morphology and biological characters of SP-B intron4 A549 cells and SP-B intron4del(3-8)m A549 cells exposed for different time to hyperoxia,detect the expression of SP-B m RNA and protein of the two kinds of cells exposed to hyperoxia.To study the association between the SP-B intron4 variations and the SP-B expression as well as hyperoxia-induced A549 cells injury.Methods The SP-B intron4 A549 cells and SP-B intron4del(3-8)m A549 cells obtained from the first part were randomly exposed to 90% oxygen for0 h,24h,48 h,72h.At each time step,MTT method was used to test cell growth,lactate dehydrogenase(LDH)method were used to test cell injury and death,the expression level of SP-B m RNA were detected by real-time PCR,the expression level of pro-SP-B and it’s intermediate were detected by western blot.Results When exposed to 90% oxygen for 0h or 24 h,both the two kind of cells grew normally.When exposed for 48 h,both kind of cells suffered a few death.When exposed for 72 h,the dead cells number were increased significantly and those of SP-B intron4del(3-8)m A549 cells were significantly increased than those of SP-B intron4A549 cells.When exposed to 90% oxygen for 0h or 24 h,the cell proliferation inhibition and cell injury were not significantly different between the two kinds of cells.However,the cell proliferation inhibition and cell injury of SP-B intron4del(3-8)m A549 cells were significantly serious than those of SP-B intron4 A549 cells when exposed to hyperoxia for 48 h or 72h(P<0.05).Hyperoxia caused the increase of SP-B m RNA and protein expression levels.When exposed to hyperoxia for 24 h,48h,72 h,the expression levels of SP-B m RNA of intron4del(3-8)m A549 cells were significantly lower than those of SP-B intron4 A549 cells(P<0.01).When exposed to hyperoxia for0 h,24h,48 h,72h,the expression levels of pro-SP-B(42KD)and intermediate SP-B-3Flag fusion protein(29KD)of intron4del(3-8)m A549 cells were significantly lower than those of SP-B intron4 A549 cells(P<0.01).Conclusion SP-B intron4 deletion(3-8motifs)variation decreased the normal expression of SP-B in A549 cell.SP-B intron4 deletion(3-8motifs)variation decreased the expression of SP-B m RNA and protein of A549 cells exposed to hyperoxia.SP-B intron4 deletion(3-8motifs)variation leaded A549 cells more sensitive to hyperoxia-induced injury.This may be the mechanism of the SP-B intron4 variations increase the risk of BPD.