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AIM To clone the cDNA fragment of human TRAIL(TNF-related apoptosis inducing ligand)into a tetracycline-regulated gene expression system,the RevTet-Onsystem,transduce expression vectors into a gastriccarcinoma cell line-NCl-N87 and examine the effects ofcontrolled expression of TRAIL in vitro on the gastriccarcinoma cells.METHODS The full-length cDNA of TRAIL was insertedinto a vector under the control of the tetracycline-responsive element(TRE)to obtain the plasmid pRevTRE-TRAIL,which was transfected into a packaging cell linePT67.In addition,vector pRev-Tet-On and pRevTRE werealso transfected into PT67 separately.After hygromycinand G418 selection,the viral titer was determined.Themedium containing retroviral vectors was collected andused to transduce a gastric carcinoma cell line NCI-N87.The resulting cell line NCI-N87-Tet-On-TRE-TRAIL and acontrol cell line,NCI-N87-Tet-On-TRE,were established.TRAIL expression in the cell line was induced byincubating cells with doxycycline(Dox),which is atetracycline analogue.The killing effect on gastriccarcinoma cells was analyzed after induction.RESULTS The recombinant plasmid pRev-TRE-TRAIL wasconstructed.After hygromycin or G418 selection,theproducer cell lines PT67-TRE,PT67-TRE-TRAIL and PT67-Tet-On were obtained,with titers of about 10~8CFU·L~(-1).By transducing NCI-N87 cells with retroviral vectors fromthese cell lines,stable cell lines NCI-N87-Tet-On-TRE-TRAIL(NN3T)and control cell line NCI-N87-Tet-On-TRE(NN2T)were established.The growth curves of theselected cell lines were the same with the wild type NCI-N87.When Dox was added,cell death was obvious in thetest groups(29%-77%),whereas no difference wasobserved in control and wild type cell lines.With theaddition of a medium from the test group,human leukemiacell line Jurkat was activated till death(83%),indicatingthe secretion of active TRAIL proteins from the test cellsto the medium.CONCLUSION With the use of the RevTet-On system,a regulated expression system for TRAIL was constructed.Using this system,the selected killing effect of TRAIL ongastric carcinoma cell line NCI-N87 could be observed.
AIM To clone the cDNA fragment of human TRAIL(TNF-related apoptosis inducing ligand)into a tetracycline- regulated gene expression system,the RevTet-Onsystem,transduce expression vectors into a gastriccarcinoma cell line-NCl-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastriccarcinoma cells.METHODS The full-length cDNA of TRAIL was insertedinto a vector under the control of the tetracycline-responsive element(TRE) to obtain the plasmid pRevTRE-TRAIL,which was transfected into a packaging cell linePT67.In Addition,vector pRev-Tet-On and pRevTRE werealso transfected into PT67 separately.After hygromycinand G418 selection,the viral titer was determined.Themedium containing retroviral vectors was collected andused to transduce a gastric carcinoma cell line NCI-N87.The resulting cell line NCI -N87-Tet-On-TRE-TRAIL and acontrol cell line,NCI-N87-Tet-On-TRE,we established.TRAIL expression in the cell line was induced by incubating cells with doxycycline(Dox),whi Ch is atetracycline analogue.The killing effect on gastriccarcinoma cells was analyzed after induction.RESULTS The recombinant plasmid pRev-TRE-TRAIL wasconstructed.After hygromycin or G418 selection,theproducer cell lines PT67-TRE,PT67-TRE-TRAIL and PT67-Tet- On were obtained,with titers of about 10~8CFU·L~(-1).By transducing NCI-N87 cells with retroviral vectors fromthese cell lines,stable cell lines NCI-N87-Tet-On-TRE-TRAIL(NN3T)and Control cell line NCI-N87-Tet-On-TRE(NN2T)were established.The growth curves of the selected cell lines were the same with the wild type NCI-N87.When Dox was added,cell death was obvious in thetest groups(29 %-77%), whereas no difference wasobserved in control and wild type cell lines.With the addition of a medium from the test group,human leukemiacell line Jurkat was activated till death(83%),indicatingthe secretion of active TRAIL proteins from the test Cellsto the medium.CONCLUSION With the use of the RevTet-On system,a regulated expression system for TRAIL was constructed.Using this system,the selected killing effect of TRAIL on gastric cancer cell line NCI-N87 could be viewed.