The Construction Of P66Shc Gene Interfering Lentivirus Vectors And Its Affections On Alveolar Epithelial Cells Apoptosis Induced By Hyperoxia

Objective: To construct p66Shc gene interfering lentivirus vectors，stablytransfect it into human alveolar type II epithelial A549cells, inhibite theexpression of p66Shc.To explore its affections on Alveolar Epithelial Cellsapoptosis induced by hyperoxia.Methods:Design3p66ShcRNA interference targets to compound doublestranded DNA,connecting with digested lentivirus vector by twoenzymes,transformed into competent cells,after identified the positive clonewas correct,then extract and amplify the plasmid,packaging lentivirus vectorwith the addition of3auxiliary packaging plasmid.Determinate of the titer andinfect A549cells,selecting the most obvious silencing effect of p66Shc-Shc1group in subsequent experiments, stablely culture for a month.And the sametime,culture untreated A549cells.The human alveolar epithelial A549cellswere divided into control group,hyperoxia group,empty vector+hyperoxiagroup, lentiviral Vector+hyperoxia group.The control group was contained with10%fetal bovine serum and1%streptomycin1640medium for regularcultivation.The hyperoxia group,empty vector+hyperoxia group,lentiviralVector+hyperoxia group were contained the equal nutrient solution whichabove-mentioned,renewing the solution,then exposed to O2(950ml/L) for10minutes,finally cultivated in a closed environment.Cells were collected,andthen,the change of morphology were observed under inverted microscope.The cellular apoptosis was detected by flow cytometry (FC).The expression ofXIAP,Caspase-9in the cytoplasm were detected by immunohistochemistry.The detection of ROS in the mitochondria was examined by confocalmicroscopy with the vital dye MitoSOX Red.The mitochondria membranepotential△Ψ m was examined by confocal microscopy with the method of JC-1.Results:(1)Construction of lentivirus vector: Digest recombinant plasmid andthe pLenR-GPH empty vector by restriction enzyme and do agarose gelelectrophoresis,the construction of LV-shRNA vecor was successful.(2)Packingof lentivirus:Transfer the recombinant plasmidp66Shc-pLenR-GPH,pLenR-GPH empty vector and packaging plasimdpRsv-REV,pMDlg-pRRE,pMD2G into293T cells.The green fluorescence wereexpressed in293T cells under fluorescence microscope aftertransfection.(3)Construction of stably silencing p66Shc gene A549cell line:Letthe infected A549cells do fluorescence quantitative PCR and Western blot, theexpression of p66ShcRNA and p66Shc protein decreased, the p66Shc-shclgroup decreased more significantly, which indicates that we have successfullyestablished a stably silencing p66Shc gene A549, and selected thep66Shc-shc1group to enter the following experiment.(4)The morphologicalchanges of the cells were observed by inverted phase contrast microscope:Incontrol group, A549cells growth in good condition,grew quickly and sticked toeach other tightly,morphology of cells showed cobblestone,as a typical flatpolygonwere,a small number of suspended cells.Compared with control group,the changes in morphology of A549were most obvious in the hyperoxiagroup and the empty vector+hyperoxia group,the number of suspended cellsincreased,form of the cells changed from typical multy-angle oblate to ellipseor round and loose connections.the number of cells decreased,cell gap visiblemore cell debris.However,the changes in morphology of A549were obviouslyimproved in Lentiviral Vector+hyperoxia group.(5) flow cytometry showedthe following:Compared with the control group,the apoptosis rate of A549cellswas significantly increased in the hyperoxia group and the empty vector+hyperoxia group; But compared with the hyperoxia group,the apoptosis rate ofA549cells was obviously decreased in the Lentiviral Vector+hyperoxia group(P<0.05),but not reached the control group(P<0.5).(6) Immunohistochemicalresults showed the following:Compared with the control group,the expressionof protein Caspase-9were significantly increased and the expression of proteinXIAP were significantly decreased in the hyperoxia group the empty vector+hyperoxia group(P<0.5); But compared with the hyperoxia group, theexpression of protein Caspase-9were significantly decreased and theexpression of protein XIAP were significantly increasedin in the LentiviralVector+hyperoxia group (P<0.5), but not reached the control group.(7)Confocal laser scanning microscopic showed the following:Compared withcontrol group,the mitochondrial membrane potential were decreasedsignificantly(P<0.5),but compared with the hyperoxia group,the production ofmitochondrial were increased(P<0.5),but not reached the control group(P<0.5).Conclusion:Hyperoxia induced intracellular p66Shc high expression,phosphorylation,turn intomitochondria,mitochondrialROS generation increased,and the membrane potentialdecreased.Targeting shRNA lentiviral vector which expressing p66Shc,weretransfected into human alveolar type II epithelial A549cells by RNAinterference,which can cause p66Shc gene silencing,reduced mitochondrialROS generation,reduce membrane potential decrease,reduce the apoptosis ofA549cells,reduce alveolar epithelial cell injury.While the lentiviral emptyvector group had no such changes.