摘要
BACKGROUND： Adenovirus has been used to develop neuroglobin （Ngb） vectors. Although transfection efficiency is high, induced gene mutation, cytotoxicity, inflammation, and low exogenous gene content have limited its application. OBJECTIVE： To observe the effects of recombinant Ngb plasmid in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： Genetically engineered, randomized, controlled, animal experiment was performed at the Laboratory of Chongqing Medical University from May 2006 and January 2007. MATERIALS： 2, 3, 5-triphenyltetrazolium chloride was purchased from Shanghai Sangon Biological Engineering Technology and Services. Rabbit anti-rat Bcl-2 polyclonal antibody, rabbit anti-rat β-actin monoclonal antibody, and FITC-labeled goat anti-rabbit IgG were purchased from Sigma, USA. TUNEL apoptosis kit was purchased from Roche, Germany. METHODS： A total of 54 male, adult, Wistar rats were randomly assigned to 3 groups （n=18）： normal saline, plasmid control, and recombinant Ngb （pCDNA3.1 （＋）/Ngb）. Normal saline, plasmid pCDNA3.1 （＋）, and recombinant plasmid pCDNA3.1 （＋）/Ngb were separately injected into two sites in the rat cerebral cortex, and models of focal ischemia were established by occlusion of the right middle cerebral artery after 24 hours. MAIN OUTCOME MEASURES： Local ischemic damage was detected by 2, 3, 5- triphenyltetrazolium chloride staining, apoptosis in the penumbra was confirmed using the TUNEL method, and Bcl-2 protein expression in the penumbra was determined by indirect immunofluorescent staining and Western blot analysis. RESULTS： Compared with the normal saline and plasmid control groups, cerebral infarction size and the number of apoptotic cells in the pCDNA3.1 （＋）/Ngb group were significantly reduced （P 〈 0.01）. The percentage of Bcl-2-positive cells in the penumbra of the pCDNA3.1 （＋）/Ngb group was significantly increased （P 〈 0.01）. The relative expression level of Bcl-2 protein was increased by 40%-50%. CONCLUSION： Recombinant plasmid pCDNA3.1/Ngb provides neuroprotection by upregulating Bcl-2 expression and inhibiting cell apoptosis in the penumbra.
BACKGROUND： Adenovirus has been used to develop neuroglobin （Ngb） vectors. Although transfection efficiency is high, induced gene mutation, cytotoxicity, inflammation, and low exogenous gene content have limited its application. OBJECTIVE： To observe the effects of recombinant Ngb plasmid in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： Genetically engineered, randomized, controlled, animal experiment was performed at the Laboratory of Chongqing Medical University from May 2006 and January 2007. MATERIALS： 2, 3, 5-triphenyltetrazolium chloride was purchased from Shanghai Sangon Biological Engineering Technology and Services. Rabbit anti-rat Bcl-2 polyclonal antibody, rabbit anti-rat β-actin monoclonal antibody, and FITC-labeled goat anti-rabbit IgG were purchased from Sigma, USA. TUNEL apoptosis kit was purchased from Roche, Germany. METHODS： A total of 54 male, adult, Wistar rats were randomly assigned to 3 groups （n=18）： normal saline, plasmid control, and recombinant Ngb （pCDNA3.1 （＋）/Ngb）. Normal saline, plasmid pCDNA3.1 （＋）, and recombinant plasmid pCDNA3.1 （＋）/Ngb were separately injected into two sites in the rat cerebral cortex, and models of focal ischemia were established by occlusion of the right middle cerebral artery after 24 hours. MAIN OUTCOME MEASURES： Local ischemic damage was detected by 2, 3, 5- triphenyltetrazolium chloride staining, apoptosis in the penumbra was confirmed using the TUNEL method, and Bcl-2 protein expression in the penumbra was determined by indirect immunofluorescent staining and Western blot analysis. RESULTS： Compared with the normal saline and plasmid control groups, cerebral infarction size and the number of apoptotic cells in the pCDNA3.1 （＋）/Ngb group were significantly reduced （P 〈 0.01）. The percentage of Bcl-2-positive cells in the penumbra of the pCDNA3.1 （＋）/Ngb group was significantly increased （P 〈 0.01）. The relative expression level of Bcl-2 protein was increased by 40%-50%. CONCLUSION： Recombinant plasmid pCDNA3.1/Ngb provides neuroprotection by upregulating Bcl-2 expression and inhibiting cell apoptosis in the penumbra.
基金
a Grant from Chongqing Health Bureau, No. 06-2-177
