摘要
BACKGROUND： Lentiviral vectors, a type of retroviral vector, are able to infect cells at all phases of cell cycle. They are able to express exogenous target genes in vivo over long periods of time with limited immunological reaction. OBJECTIVE： To inhibit neuronal apoptosis by blocking the apoptotic cascade reaction, gene silencing of Caspase 3, and transfection of Caspase 3 short hairpin ribonucleic acid （shRNA） into Neuro 2a cells using a lentiviral vector.DESIGN： TiME-AND SETTING： An observational, genetic engineering cellular biology experiment was performed in Guangzhou Red Cross Hospital and Guangzhou Institute of Traumatic Surgery between March 2007 and June 2008. MATERIALS： PLL3.7, PCMV-VSV-G, and PH＇8.9∧PR plasmids were provided by the CBR Institute for Biomedical Research, Harvard Medical School, USA. Staurosporine was purchased from Sigma, USA.METHODS： Caspase 3 siRNA was synthesized and cloned into the PLL3.7 plasmid. The Caspase 3 shRNA-PLL3.7 Ientivirus was generated in 293T cells using a calcium phosphate transfection kit. After the lentivirus was transfected into Neuro 2a cells, apoptosis was induced in both the transfected and untransfected cells by staurosporine. Cell apoptosis was assessed by flow cvtometrv.MAIN OUTCOME MEASURES： Caspase 3 mRNA expression was measured by RT-PCR and Caspase protein expression was assessed by Western blot. Cellular apoptosis was determined by flow cytometry using Annevin V-PE/Taad-Cy7. RESULTS： The transfection rate of caspase 3 shRNA was 〉 98% using the lentiviral vector, RT-PCR and Western blot results demonstrated that significantly reduced Caspase 3 mRNA and protein expression in the transfected Neuro 2a. The control group exhibited 38.7% Annexin V/7aad-positive cells, which suggested apoptotic anaphase, while only 5.0% cells in the Caspase 3 gene silencing group entered apoptotic anaphase. CONCKUSION： Caspase 3 shRNA inhibited Caspase 3 expression in Neuro 2a ceils and decreased drug-induced apoptosis of Neuro 2a cells.
BACKGROUND： Lentiviral vectors, a type of retroviral vector, are able to infect cells at all phases of cell cycle. They are able to express exogenous target genes in vivo over long periods of time with limited immunological reaction. OBJECTIVE： To inhibit neuronal apoptosis by blocking the apoptotic cascade reaction, gene silencing of Caspase 3, and transfection of Caspase 3 short hairpin ribonucleic acid （shRNA） into Neuro 2a cells using a lentiviral vector.DESIGN： TiME-AND SETTING： An observational, genetic engineering cellular biology experiment was performed in Guangzhou Red Cross Hospital and Guangzhou Institute of Traumatic Surgery between March 2007 and June 2008. MATERIALS： PLL3.7, PCMV-VSV-G, and PH＇8.9∧PR plasmids were provided by the CBR Institute for Biomedical Research, Harvard Medical School, USA. Staurosporine was purchased from Sigma, USA.METHODS： Caspase 3 siRNA was synthesized and cloned into the PLL3.7 plasmid. The Caspase 3 shRNA-PLL3.7 Ientivirus was generated in 293T cells using a calcium phosphate transfection kit. After the lentivirus was transfected into Neuro 2a cells, apoptosis was induced in both the transfected and untransfected cells by staurosporine. Cell apoptosis was assessed by flow cvtometrv.MAIN OUTCOME MEASURES： Caspase 3 mRNA expression was measured by RT-PCR and Caspase protein expression was assessed by Western blot. Cellular apoptosis was determined by flow cytometry using Annevin V-PE/Taad-Cy7. RESULTS： The transfection rate of caspase 3 shRNA was 〉 98% using the lentiviral vector, RT-PCR and Western blot results demonstrated that significantly reduced Caspase 3 mRNA and protein expression in the transfected Neuro 2a. The control group exhibited 38.7% Annexin V/7aad-positive cells, which suggested apoptotic anaphase, while only 5.0% cells in the Caspase 3 gene silencing group entered apoptotic anaphase. CONCKUSION： Caspase 3 shRNA inhibited Caspase 3 expression in Neuro 2a ceils and decreased drug-induced apoptosis of Neuro 2a cells.
基金
Open Foundation of Key Laboratory of Biomedical Engineering,Guangdong Province,No.21030400
Key Project of Guangzhou Medical and Health Care Foundation,No. 2006-ZDi-04
