Construction Of Recombinant Adenovirus Vector Of Human Keratinocyte Growth Factor And Effect Of The Vector To Oxidative Stress Injury On A549Cell In Hypoxic Conditions

Objective: To construct and identify a recombinant adenovirus vector expressingkeratinocyte growth factor（KGF）gene and study the effect of the vector to oxidativestress injury on A549cells in hypoxic conditions. Methods：KGF gene was amplifiedfrom the plasmid pIRES2-EGFP-KGF by polymerase chain reaction（PCR）and theninserted into the plasmid pShuttle-CMV to construct pShuttle-KGF．After beingconfirmed by restriction enzyme digestion and DNA sequencing，the DNA encodingKGF in the new structure was inserted into the vector of recombinant plasmidadenovirus and confirmed by restriction enzyme digestion．Then the humanembryonic kidney cell line293was transfected with correctly identifiedpAdxsi-KGF，and the recombinant adenovirus were packed,producted,purified andthe virus titer was detected using50%tissue culture infective dose（TCID50）assay.We transfected man lung adenocarcinoma A549cancer cells with restructuringadenovirus,then detected the expression of the green fluorescent protein byfluorescent microscope and expression of KGF protein and gene. A549cells wererandomly divided into prevention groups, prevention control groups, treatment groupsand treatment control groups. The prevention groups transfected the Ad-KGF intoA549cells under normoxic conditions，36h later，cultured under hypoxic condition（1%O₂、5%CO₂、94%N₂）,we collected the culture supernatant and cells at12h,24hand36h. The treatment groups were cultured under hypoxic condition（1%O2、5%CO2、94%N2）,24h later, we transfected the Ad-KGF into A549cells, collected theculture supernatants and cells at24h,36h and48h. The control groups were cultured inaccordance with the preventive groups and treatment groups, given the same amountof PBS, we collected the culture supernatant and cell at the same time points. At last,we detected the levels of NOS, LDH ROS content in the cell culture supernatan andthe expression of p53and Bax gene.Results：The recombinant adenovirus Ad-KGFwas constructed successfully and amplified with titer of1.6×1010pfu/mLï¼›the greenfluorescence proteins can be observed from fluorescent microscope in A549cells,and the KGF protein and gene can be stably expressed; When the A549cells weretransfected by Ad-KGF,the levels in cell culture supernatant of ROS and LDH waslower, the levels of NOS was higher in the treatment groups and prevention groupsthan control groups;the the expression of p53and Bax gene was lower in thetreatment groups and prevention groups than control groups.Conclusion：Therecombinant adenovirus Ad-KGF was constructed successfully，which was useful inpreventive and therapeutic of oxidative stress to A549cells in hypoxic conditions.