Anti-sense RNA Inhibits the Expression of Synaptotagmin Ⅱ in RBL-2H3 and Enhances the Exocytosis of Lysosomes in RBL-2H3

摘要

The expression of synaptotagmin Ⅱ (Syt2) in RBL-2H3 (RBL) and its role during exo-cytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of ca-thepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8% and 10% of control cells) , indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathep-sin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.