To constructe,package and identificate the lentiviral vector with overexpression gene Grp78 / 重庆医学

Objective To constructe ,package and identificate the lentiviral vector with overexpression gene Grp78 .Methods We used lentiviral vector and genetic engineering technology to obtain the aim gene fragment and to constructe recombinant plas‐mid .we prepared competent cells and transform the cells .Through positive clone sequencing ,lentivirus was packaged and virus titer was tested .Results Positive cloning sequence comparison results show that the test was passed .Melt curve did not appear mixed peak ,also did not appear abnormal peak broadening .It means that does not appear pollution ,primer dimers and nonspecific amplifi‐cation in the experiments .Conclusion The construction ,packaging and identification of lentiviral vector with over expression gene Grp78 are sucessful .

Is activation of AMP-activated protein kinase involved in ropivacaine-induced SH-SY5Y cell apoptosis? / 中华麻醉学杂志

Objective To determine if activation of AMP-activated protein kinase (AMPK) is involved in ropivacaine-induced reactive oxygen species (ROS) production and apoptosis in human neuroblastoma cell line SHSY5Y.Methods SH-SY5Y cell line was purchased from cell center of Shanghai life Science Research Institute,Chinese Academy of Sciences and cultured in DMEM/F12 liquid culture medium containing 15 % bovine calf serum at 37 ℃ in incubator filled with 5% CO2 . Plasmids pGPU6/GFP/Neo-shRNA AMPKα2 and pEGFP-N1-AMPKα2were transfected into the SH-SY5Y cell line. The expression of AMPKα2 was determined by Western blot analysis.The SH-SY5Y cells transfected with recombinant plasmids were exposed to 3 mol/L ropivacaine. Intracellular ROS was detected by flow cytometry. Cell viability was quantitatively determined by MTT colorimetry assay. Apoptosis was assessed by flow cytometry and Hoechst33258 staining. Results The plasmid pEGFP-N1-AMPKα2 upregulated while pGPU6/GFP/Neo-shRNA AMPKα2 down-regulated the expression of AMPKα2 ( P < 0.01). Down-regulation of AMPKα2 expression attenuated while up-regulation of. AMPKα2 expression promoted intracellular ROS production and cell apoptosis induced by ropivacaine ( P < 0.01) . Conclusion AMPK probably mediates ROS production and cell apoptosis induced by ropivacaine.