Wei Li1, Su Liu, Jianli Ren, Haibo Xiong, Xingyun Yan, Zhigang Wang. 1. Institute of Ultrasound Imaging, Department of Ophthalmology, Second Affiliated Hospital of Chongqing Medical University, No.76, LIJiang Road, YuZhong District, ChongQing City, 400010, China.
Abstract
RATIONALE AND OBJECTIVES: To investigate the expression levels of green fluorescence protein (GFP) into retinal ganglion cells (RGCs) in vitro by ultrasound-mediated microbubble destruction (UMMD) and assess the effect of bcl-xl gene on N-methyl-D-aspartate (NMDA)-induced apoptosis in the cultured RGCs by UMMD. MATERIALS AND METHODS: pEGFP-N1 was transfected to RGCs in vitro by UMMD and liposome was used as the control. The transfection effect was detected using microscope and flow cytometry qualitatively and quantitatively. Monotetrazolium was adopted to measure the cell vitality. NMDA was used to induce apoptosis in the cultured RGCs, and the bcl-xl gene was transfected into RGCs by UMMD before NMDA-induced apoptosis. The expression of bcl-xl protein in RGCs was assessed by immunohistochemistry assay. The amorphous character of RGCs was revealed by acridine orange and ethidium bromide staining. DNA fragment was detected by agarose gel electrophoresis. RESULTS: Ultrasound combined with microbubbles enhanced gene transfection to the cultured cells in some condition. The average transfection rate of pEGFP-N1 with UMMD was 25%. Both ultrasound and microbubble had no effect on cell viability. The expression of bcl-xl protein in transfected and non-transfected RGCs was significantly different. Less apoptotic bodies and no representative DNA fragment were detected in the treatment group. CONCLUSIONS: Microbubble destruction can enhance the reporter gene transfection and expression and have a good target. Transfection of bcl-xl gene has an anti-apoptosis effect on the cultured RGCs induced by NMDA with UMMD.
RATIONALE AND OBJECTIVES: To investigate the expression levels of green fluorescence protein (GFP) into retinal ganglion cells (RGCs) in vitro by ultrasound-mediated microbubble destruction (UMMD) and assess the effect of bcl-xl gene on N-methyl-D-aspartate (NMDA)-induced apoptosis in the cultured RGCs by UMMD. MATERIALS AND METHODS: pEGFP-N1 was transfected to RGCs in vitro by UMMD and liposome was used as the control. The transfection effect was detected using microscope and flow cytometry qualitatively and quantitatively. Monotetrazolium was adopted to measure the cell vitality. NMDA was used to induce apoptosis in the cultured RGCs, and the bcl-xl gene was transfected into RGCs by UMMD before NMDA-induced apoptosis. The expression of bcl-xl protein in RGCs was assessed by immunohistochemistry assay. The amorphous character of RGCs was revealed by acridine orange and ethidium bromide staining. DNA fragment was detected by agarose gel electrophoresis. RESULTS: Ultrasound combined with microbubbles enhanced gene transfection to the cultured cells in some condition. The average transfection rate of pEGFP-N1 with UMMD was 25%. Both ultrasound and microbubble had no effect on cell viability. The expression of bcl-xl protein in transfected and non-transfected RGCs was significantly different. Less apoptotic bodies and no representative DNA fragment were detected in the treatment group. CONCLUSIONS: Microbubble destruction can enhance the reporter gene transfection and expression and have a good target. Transfection of bcl-xl gene has an anti-apoptosis effect on the cultured RGCs induced by NMDA with UMMD.