Role Of Serum Amyloid P Component On Hyperoxia-inducedlung Fibrosis And Construction Of The Recombinat Adenovirus Of AD5-SAP

PART ONE: ROLE OF SERUM AMYLOID P COMPONENT ON HYPEROXIA-INDUCEDLUNG FIBROSISObjective: Althrough long—time hyperoxia inhalation maybe cause lung fibrosis, and BPD, however, hyperoxia is very useful in neonatal medicine, which have attached so much attention of people in medical science. At present, we have not understood the pathogenesis of hyperoxic lung injury in neonate completely. SAP correlated with lung fibrosis. Try to explory the dynamic change of SAP level in modle of hyperoxia-induced lung fibrosis of rats, and understand the role of SAP on hyperoxic lung fibrosis. First we established the modle of hyperoxia-induced lung fibrosis with rats, then we observed the the expression level of SAP in the blood of neonatal rats and the dynamic changes of the pathologic alteration in neonatal rats which were exposed to the hyperoxia inhalation.Methods: We enrolled 60 clean Wistar rats in this study ,and the rats were less than 12 hours old. First the rats were divided randomly into 2 groups: hyperoxia group and normal air control group. The rats in normal air control group breathed the room air,while the rats in hyperoxia group were placed into oxygen cabins which filled with >90%02. After the exposure to high concentration oxygen(02>95%), at 72 hours,7days,14days the pathologic alteration in lung were measured, and we measured the expression level of SAP by ELISA.Results: 72 hours in hyperoxia, we found edema,microvessel hyperemia,inflammatory cell infiltration and hemorrhage by using light microscope. After 7 days in hyperoxia, it was still obvious that lung interstitium thickened, and edema,microvessel hyperemia,inflammatory cell infiltration did not disappear. At hyperoxia 14 days, edema,hemorrhage,inflammatory cell infiltration reduced, which with marked fibrosis changes in lung. Campared with the control, the SAP level in rats in hyperoxia group of peripheral blood is higher at hyperoxia 72 hours, but lower at hyperoxia 7 days and 14 days.ConcIusion:Lung fibrosis can be caused by hyperoxia inhalation in new born rats. Long—time hyperoxia inhalation may cause the decline of SAP level in peripheral blood,which aggravates the lung fibrosis. PART TWO: CONSTRUCT THE RECOMBINAT ADENOVIRUS OF AD5-SAPObjective: First, we construct the recombinant adenovims plasmid Ad5-SAP expressing SAP. Second, we transfect the plasmid into the HEK293 which is package cell and generate the recombinant adenovirus.Methods: Firstly, we amplified the target gene SAP containing the linker by using PCR, then we intergrated the target gene SAP into the shuttle vector of pAdTrace-TO4 by restrict ligation. Secondly, we recombined the plasmid in the competent E coli which containing the adenovirus skeleton plasmid Adeasy-1 to get Ad5-SAP plasmid. Finally, transfected the recombinant plasmid Ad5-SAP into the package cell HEK293, which can package and generate the high titre adenovirus.Results: We successfully constructed the adenovirus plasmid Ad5-SAP which can generate the adenovirus expression SAP and the titre was about 2.8x108 pfu/ml.Conclusion: Constructing the adenovirus plasmid by recombining the plasmids in the E coli cells is an effective method. We cloned the SAP gene into recombinant adenovirus, which can offer the basic work for the next investigation.