Association Between Surfactant Protein B C/T1580Polymorphism And Susceptibility To Hyperoxic Lung Injury

Part I The preparation of polyclonal antibodies against the surfactant protein B intermediatesObjective To obtain the rabbit polyclonal antibodies against the surfactant protein (SP) B intermediates. Methods The human preproSP-B sequence was analyzed using Lasergene7.0and Macvector11.0.4software, and the epitopes were predicted by the method of secondary structure, hydrophilicity, antigenicity, surface probability and flexibility. Three peptide sequences(Serl45-Leu160, Glnl86-Gln200, Gly284-Ser304) were chosen and the peptides were synthesized commercially. The peptides were conjugated to keyhole limpet hemocyanin(KLH) via cysteine residue, emulsified in Freund’s complete adjuvant and injected subcutaneously into rabbits. Freund’s incomplete adjuvant was used to comprise booster immunizations. Each peptide was used to immunize two rabbits. The reactivity against the immunizing peptide of pre-immune and post-immune sera were screened by indirect enzyme-linked immunosorbentassay(ELISA). The antisera obtained were immunoaffinity purified using specific antigen to become specific antibody. The purity of antibody was tested using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), and the reactivity against the immunizing peptide of antibody was screened by indirect enzyme-linked immunosorbentassay(ELISA). Results The reactivity against the immunizing peptide of pre-immune sera of six rabbits was low, so the rabbits were fit to be immunized. The reactivity against the immunizing peptide of post-immune sera of six rabbits were enough high. The antisera obtained were immunoaffinity purified using specific antigen successfully, so the purity and reactivity of antibodies obtained were qualified. Conclusion Three polyclonal antibodies against SP-B intermediates were prepared successfully, which provide a tool for further study of the processing of SP-B. Part II Construction of pIRES2-EGFP-SP-B-C/T1580eukaryotic expression vectorsObjective To construct two kinds of eukaryotic expression vectors pIRES2-EGFP-SP-B-C/T1580and evaluate their expressions in293T cells. Methods SP-B-C/T1580cDNA were obtained by polymerase chain reaction(PCR) by using pEE14-SP-B-C/T1580as templates. The PCR products and pIRES2-EGFP plasmid were digested by restriction enzymes EcoR I and XhoA549I, and then the digested products were purified and ligated by T4DNA ligase. The ligated products were transfected into Escherichia coli. Kanamycin was used to choose the Escherichia coli transfected by recombinant plasmids. Of course, pIRES2-EGFP-SP-B-C/T1580were confirmed by DNA sequencing. The recombinant expression vectors were transfected into293T cells by Lipofectamine2000. The expression of green fluorescence protein in293T cells was observed by fluorescence microscopy. The mRNAs and proteins of SP-B-C/T1580were tested and identified by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) and western blot. Results Two recombinant plasmids contained the complete cDNA of SP-B with the same sequence as in gene bank. The base of SP-B1580gene of pIRES2-EGFP-SP-B-C1580was C, that of pIRES2-EGFP-SP-B-T1580was T. After being transfected into293T cells, highly efficient expression of SP-B-C/T1580gene were found at mRNA and protein levels. Conclusion The pIRES2-EGFP-SP-B-C/T1580 eukaryotic expression vectors were successfully constructed, which provide a tool for further study of the genetic polymorphism of SP-B1580position. Part III Establishment of monoclone of SP-B-C/T1580A549cell linesObjective To establish the monoclone of SP-B-C1580A549cell line and the monoclone of SP-B-T1580A549cell line. Methods The eukaryotic expression vectors pIRES2-EGFP-SP-B-C/T1580were transfected into A549cells by Lipofectamine2000. Passage cells were seed into fresh growth medium at a proper dilution24hours after transfection. Selective medium (containing400μg/ml G418) were added48hours after transfection. Limiting dilution method and culture dish method were used to choose cell clone. PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism) was used to detect SP-B-C/T1580mRNA, and Western blot was used to detect SP-B-C/T1580protein. Results The A549cells stably transfected by pIRES2-EGFP-SP-B-C/T1580growed normally in selective medium (containing400μ.g/ml G418) for12days, however, the control A549cells died. The monoclone cell lines were chosen and tested, and one monoclone cell line for a plasmid was consistent with the requests, which has the base C or T at SP-B1580position and expressed the correspond protein of SP-B. Conclusion The two A549monoclone cell lines of SP-B-C1580and SP-B-T1580with stable expression of correspond SP-B protein were established successfully. Part IV Relation between surfactant protein B C/T1580polymorphism and hyperoxia-induced A549cells injury Objective To observe the biological characters of SP-B-C/T1580A549cells exposed to hyperoxia for different time, detect the SP-B expression and processing and the SP-A expression of the cells exposed to hyperoxia or normoxia, and to study the mechanism of relation between surfactant protein B C/T1580polymorphism and hyperoxia-induced A549cells injury. Methods SP-B-C/T1580A549cells were randomly assigned to two groups:the normoxia group and hyperoxia group. The cells of normoxia group were exposed to5%CO2and95%air, and the cells of hyperoxia group were exposed to5%CO2and95%oxygen. The cells were used for experiments after exposure for24h,48h,72h and96h. MTT method was used to test cell growth. Trypan blue staining method and lactate dehydrogenase(LDH) method were used to test cell injury and death. Thiobarbituric acid(TBA) method was used to test lipid peroxidation of cells. Flow cytometry was used to test cell apoptosis. The SP-A expression was test by western blot, and the SP-B expression and processing were tested by western blot and laser confocal microscopy. Results The cell injury and cell inhibition were not significantly different between the two kinds of cells exposed to hyperoxia for24h and48h. However, the cell injury, cell inhibition and lipid peroxidation of SP-B-C1580A549cells were significantly serious than those of SP-B-T1580A549cells exposed to hyperoxia for72h and96h (P<0.05). A few dead cells of both kinds of cells exposed to hyperoxia for72h were observed. The dead cells were increased at96h exposure to hyperoxia, and those of SP-B-C1580A549cells were significantly increased than those of SP-B-T1580A549cells (P<0.05). Exposure to hyperoxia for72h mainly induced the necrosis of cells, but exposure to hyperoxia for96h mainly induced the apoptosis of cells. The necrosis and apoptosis of SP-B-C1580A549cells were more serious than those of SP-B-T1580A549cells exposed to hyperoxia for72h and96h. Hyperoxia caused the increase of proSP-B and the intermediates in A549cells. The proSP-B and it’s25kDa intermediate in SP-B-T1580A549cells exposed to hyperoxia for24h,48h,72h and 96h were more than those of SP-B-C1580A549cells significantly (p<0.05). The17kDa intermediate in SP-B-T1580A549cells exposed to hyperoxia for48h,72h and96h were more than those of SP-B-C1580A549cells significantly (p<0.05) Hyperoxia caused the increase of SP-A in A549cells. The SP-A in SP-B-T1580A549cells exposed to hyperoxia for24h,48h,72h and96h were more than those of SP-B-C1580A549cells significantly (p<0.05). Conclusion T allele at SP-B1580position alleviate the hyeroxia-induced A549cells injury, the mechanism of which probably was the enhancement of the SP-B expression and processing and the increase of SP-A expression.