CONSTRUCTION AND IDENTIFICATION OF EUKARYOTIC VECTOR EXPRESSING SIRNA SPECIFIC FOR BACE

Objective To generate eukaryotic expression vector of siRNA specific for β-site APP cleaving enzyme(BACE),and detect the interfering effect to the expression of BACE. Methods To clone the BACE targeting siRNA gene by PCR, the PCR products was inserted into the pUC19/EGFP-U6 plasmid. Then it was sub-cloned into the vector named pLXSN. The resultant plasmid was named pLXSN/EGFP-U6-siBACE, it was packaged into AmphoPack-293 cells by calcium phosphate transfection and collected the virus supernatant. The neuroblastoma cells SK-N-SH was infected with the pLXSN/EGFP-U6-siBACE retroviral vector, immunohistochemistry method was used to detect whether the pLXSN/EGFP-U6-siBACE infection can inhibit the expression of BACE of the neuroblastoma cells. Results The pLXSN/EGFP-U6-siBACE retroviral vector was constructed successfully and the siBACE can inhibit the BACE of the neuroblastma effectively. Conclusion The siRNA can inhibit the expression of the BACE gene, the endogenous production of BACE protein was decreased. It will lay the important foundation for using RNA technology to prevent the Alzheimer's disease.

Morphological characteristics of the lumbosacral spinal cord urogenital center in Sprague Dawley rats

BACKGROUND： Studies have demonstrated that selective innervation of the sacral nerve tract to the bladder plays an important role in bladder functional reconstruction following spinal cord injury. However, there are very few studies reporting detailed morphological characteristics of urogenital center and lumbosacral nerve roots. OBJECTIVE： To analyze the spinal cord segment of the lumbosacral spinal cord urogenital center, and to observe morphological characteristics. DESIGN, TIME AND SETTING： A neuroanatomical study was performed at the Laboratory of Neuroanatomy, Peking University Health Science Center between September 2007 and March 2008. MATERIALS： Horseradish peroxidase-conjugated cholera toxin B subunit （CB-HRP） was purchased from Sigma, USA; surgical microscope was purchased from Zhenjian Zhongtian Optical Instrument, Jiangsu Province, China; BCL-420 biological and functional experimental system was purchased from Taimeng Science and Technology, Sichuan Province, China. METHODS： A total of 36 adult, Sprague Dawley rats were randomly assigned to groups A （n = 10）, B （n = 10）, C （n = 10）, and D （n = 6）. CB-HRP （3%, 10-15 μL） was injected into the bladder detrusor muscle （group A）, external urethral sphincter （group B）, and perineal muscles （group C）, respectively. Rats in group D were not given any treatments. MAIN OUTCOME MEASURES： At 72 hours after CB-HRP injection, CB-HRP-positive neurons were analyzed in lumbosacral segments using 3, 3＇, 5, 5＇-tetramethylbenzidine staining and an Olympus optic microscope, while anatomical structures in the respective spinal nerve tract were observed using a surgical microscope. RESULTS： CB-HRP-positive neurons were distributed in the L6-S1 segments of the spinal cord, and neurons primarily innervating the bladder detrusor muscle were located at the sacral parasympathetic nucleus and the intermediolateral nucleus. In addition, neurons that primarily innervate the external urethral sphincter and perineal muscles were observed in the ventrolateral portion （Onuf＇s nucleus）. The lumbar-sacral nerve roots were composed of varying nerve tracts, Le., they were typically divided into 1-2 sub-bundles, and the sub-bundles were then divided into 2-3 tiny bundles. There were extensive fibro-connections between the rootlets. CONCLUSION： The urogenital center in Sprague Dawley rats was located in the L6 -S1 segments of the spinal cord, and the rootlets were clearly observed. Therefore, this rat experimental model could be utilized for highly selective anterior/posterior rhizotomy.