Association Between SP-B-C/T1580Polymorphism And SP-A In Hyperoxic Lung Injury

PartⅠ The Screening of the Specific Surfactant Protein A/BSiRNAObjective Screening out specific small interference RNA (SiRNA) that were able tosilence genes of lung surfactant protein (SP)A or B. Methods A549cells were transfectedwith synthetic SP-A or SP-B sequence-specific SiRNA(SP-A1、SP-A2、SP-A3and SP-B1、SP-B2、SP-B3) by Lipofectamine2000.The expression of mRNA was detected by the realtime PCR to screen out specific SiRNA. Results Comparing with the control group,SP-A2and SP-B1could significantly interfere the production of mRNA, and the inhibitionratio was above70%(97.1%in SP-A2,96.9%in SP-B1). Conclusion Succeeding inselecting out specific SiRNA that were able to inhibit expression of genes of SP-A or SP-B. PartⅡ The Resurrection and Identification of Monoclones ofSP-B-C/T1580A549Cell LinesObjective To resurrection and identification of SP-B-C/T1580A549Cell Lines. MethodsSP-B-C/T1580A549Cell Lines preserved in liquid nitrogen were resurrected and enlargedcultivation in vitro, and genomic DNA and total protein were gained. The target genes weredetected by PCR and restriction enzyme digestion, and the target proteins were detected bywestern-blot. Results A549monoclonal cells after resurrected were survival and adherent.There was no obvious difference in both of the two cell lines. The product of PCR could becut into154bp and the SP-B preproprotein was45kD in SP-B-C1580A549cells, howeverthe product of PCR could not be cut and the SP-B preproprotein was42kD in SP-B-T1580A549Conclusion The preserved cells were needed SP-B-C/T1580A549monoclonal cells. PartⅢ The Transfection of the Specific SP-A/B SiRNA intoSP-B-C/T1580A549Cell Lines and Interference of HyperoxiaObjective To observe the capacity of proliferation and lipid peroxidation ofSP-B-C/T1580A549monoclonal cells silenced genes of SP-A or SP-B in hyperoixa, and toexplore the interaction between SP-B-C/T1580gene polymorphism and SP-A resistingdamage of hyperoxia. Methods SP-B-C/T1580A549monoclonal cells were transfectedwith specific SiRNA by Lipofectamine2000,continuously exposed to hyperoxia (950mL/LO2,50mL/L CO2). After exposure to hyperoxia for48,72and96hours, total protein andculture supernatant were gained. SP-A and SP-B protein were detected by Western-blot, thecapacity of proliferation was detected by methyl thiazolyl tetrazolium(MTT), andthiobarbituric acid (TAB)colorimetric method was used to detect the malondialdehyde(MDA) in culture supernatant. Results Comparing with control group, the SP-A protein wasthe most clearly decreased in both of the two cell lines transfected SiRNA SP-A2afterexposure to hyperoxia for72h(P<0.001),but the SP-B protein was the most clearlydecreased in both of the two cell lines transfected SiRNA SP-B1after exposure tohyperoxia for96h(P<0.001).The inhibition ratio of hyperoixa in cell silenced gene of SP-Awas more than in silenced gene of SP-B. After exposure to hyperoxia for72h silenced geneof SP-A, comparing with control group, the SP-B protein was significantly reduced only inSP-B-C1580A549cell(P<0.001), the inhibition ratio of hyperoixa in SP-B-T1580A549cellwas higher than in SP-B-C1580A549cell(P<0.001), and the MDA in culture supernatant ofSP-B-T1580A549cell was more than SP-B-C1580A549cell(P<0.001).There was statisticalsignificance in the two groups. After exposure to hyperoxia for96h silenced gene of SP-B,comparing with control group, the SP-A protein was significantly reduced only inSP-B-C1580A549cell(P=0.032), the inhibition ratio of hyperoixa in SP-B-T1580A549cell was higher than in SP-B-C1580A549cell(P<0.001), and the MDA in culture supernatant ofSP-B-T1580A549cell was more than SP-B-C1580A549cell(P=0.029).There was alsostatistical significance in the two groups. Conclusion Specific SiRNA could markedlyrestrain the expression of target protein, and the point-in-time was after exposure tohyperoxia for72h in group by silenced gene of SP-A and96h in group by silenced geneof SP-B. The proteins SP-A and SP-B may protect from hyperoxic lung injury by relievinglipid peroxidation, as well as the effect of SP-A was stronger than SP-B. The superrioritu ofSP-B1580allele T in protecting from hyperoxia could be changeover if the SP-A protein orSP-B protein reduced. There was cooperation between SP-B1580allele T and SP-A geneto resisting damage of hyperoxia..