ABSTRACT
<p><b>OBJECTIVE</b>To investigate whether short interfering RNAs(siRNAs) of beta-site APP cleaving enzyme (BACE) can inhibit the expression of BACE in mammalian cells.</p><p><b>METHODS</b>The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR, respectively. The PCR products were inserted into plasmid pLXSN. The interfering vector pLXSN/EGFP-U6-siBACE was transferred into SK-N-SH cells to express BACE. The inhibition effect of BACE siRNA on BACE expression was investigated by fluoroscopy and immunohistochemistry method.</p><p><b>RESULT</b>The interfering vector pLXSN/EGFP-U6-siBACE was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cells specifically and effectively, and the production of A beta was reduced.</p><p><b>CONCLUSION</b>BACE siRNA can inhibit the expression of BACE gene of mammalian cells.</p>
Subject(s)
Animals , Humans , Mice , Amyloid Precursor Protein Secretases , Genetics , Metabolism , Green Fluorescent Proteins , Genetics , Metabolism , Immunohistochemistry , Microscopy, Fluorescence , NIH 3T3 Cells , Neuroblastoma , Genetics , Metabolism , Pathology , Plasmids , Genetics , RNA Interference , RNA, Small Interfering , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Tumor Cells, CulturedABSTRACT
OBJECTIVE@#To investigate the tumor-suppression effect of PA combined with GM-CSF, TNF-alpha and IL-4 on cord blood mononuclear cells (CBMC).@*METHODS@#The mononuclear cells were isolated from human umbilical cord blood and cultured with polyacttin A (PA), GM-CSF + TNF-alpha + IL-4 (GTI), and GTI + PA (GTIP) respectively. Six days later, surface antigen expression of the cultured cells, including CD1a and CD83, which were the specialized markers of dendritic cell (DC), were analyzed by immunohistochemistry technique. The CBMC were cultured with GTI for 24 h to enhance DC, then were added apoptotic/necrotic Hela/HepG2 tumor cells, and finally PA was co-cultured. The antitumor cytotoxicity of CBMC was measured by MTT assay.@*RESULTS@#After the culture, CD1a and CD83 positive cell rates of the PA group inreased significantly, reaching (19.63 +/- 3.61)%, (9.28 +/- 4.31) % respectively, much higher than that of the control, but lower than that of the GTI group. The killing rate to the tumor cells of CBMC cultured with GTIP increased remarkably, much higher than the control, GTI and PA groups. After tumor antigens were added to the CBMC of GTIP group (GTIP + Tc), the killing rate increased.@*CONCLUSION@#PA not only promotes the proliferation and maturation of cord blood derived DC, but also improves the tumor-suppression effect of CBMC cultured with GTI.