关键词: Egr1；分泌型肿瘤坏死因子相关的凋亡诱导配体；基因重组；辐射；细胞凋亡

Abstract:

o construct human secreted TRAIL(hsTRAIL) recombinant vetor pEgr1-hsTRAIL mediated by Egr1,and to explore the inhibitory effect on the growth  of  lung adenocarcinoma A549 cells.Methods   The  hsTRAIL vetor mediated by Egr1 was constructed by gene recombination techinique,the A549 cells were transfected with the plasmid after identification by PCR,restrictive enzyme digestion and sequencing,and irradiated by 6 Gy X-rays.There were control group,pEgr1-hsTRAIL group,6 Gy  X-rays group and pEgr1-hsTRAIL + 6 Gy  X-rays group in the experiment.The expression of hsTRAIL　in A549 cells was detected by ELISA method,the cell proliferation was detected by MTT assay,the cycle changes of cell cycle were detected by flow cytometry and the  apoptosis was measured by TUNEL method.Results The hsTRAIL recombinant vector pEgr1-hsTRAIL mediated by Egr1 was constructed successfully.The cells were irradiated by 6 Gy X-rays after transfected with plasmid.The  hsTRAIL protein expressions in control,6 Gy and pEgr1-hsTRAIL groups didn’t change significantly with the time prolongation, but the expression in pEgr1-hsTRAIL+6 Gy group was increased significantly with the time prolongation (P<0.05 or P<0.01),and reached to peak value at 8 h.There was no significant difference of A549 cell proliferation ability between control group and pEgr1-hsTRAIL group,but the proliferation abilities in 6 Gy and pEgr1-hsTRAIL+6 Gy groups were decreased significantly compared with control group,especially in pEgr1-hsTRAIL+6 Gy group (P<0.05 or P<0.01).Compared with control group,the percentages of A549 cells  at different phases in  pEgr1-hsTRAIL group didn’t change significantly,but the percentages of A549 cells at  G0/G1 phase  in 6 Gy and pEgr1-hsTRAIL+ 6 Gy groups were  increased significantly  (P<0.05),the percentages of A549 cells at G2/M phase were decreased significantly  (P<0.05),the percentages of A549 cells at S phase didn’t change significantly.The percentages of A549 cells at different phases in 6 Gy group were basically consistent with those in pEgr1-hsTRAIL+6 Gy group.Compared with control group,the  apoptotic percentage of A549 cells in  pEgr1-hsTRAIL group had no significant change,but they were  increased significantly in 6 Gy and pEgr1-hsTRAIL+6 Gy groups compared with control group (P<0.01),especially in pEgr1-hsTRAIL+6 Gy group.Conclusion The hsTRAIL recombinant vector pEgr1-hsTRAIL mediated by Egr1 is successfully constructed,the vector can enhance the  inhibitory effects and  effect of inducing apoptosis of radiation on the A549 cells,but it affects cell distribution little.

Key words: Egr1;secreted tumor necrosis factor related apoptosis inducing ligand;gene recombination;radiation;apoptosis