【摘要】 目的:研究Synaptotagm inⅡ(简称Syt2)在RBL-2H3(简称RBL)中的表达及其在胞吐中的作用。方法:通过W estern b lot检测Syt2在RBL中的表达情况。用高保真酶扩增Syt2,与pEGFP-N1构建全长反义基因表达载体,电穿孔转染RBL,G418筛选获得稳定转染细胞。通过钙离子载体和抗原刺激,应用W estern b lot检测稳定转染细胞和对照细胞分泌的组织蛋白酶D,分析Syt2对胞吐的影响。结果:在RBL中检测到Syt2表达。构建了pEGFP-N1-Syt2-AS质粒,插入片段测序结果与GenBank登录号NM012665(rat Syt2)序列完全一致。经转染和G418筛选,获得了稳定转染细胞RBL-Syt2-AS,W estern b lot结果显示,两株RBL-Syt2-AS表达的Syt2均明显减少,分别只有对照的8%和10%。经刺激后,RBL-Syt2-AS分泌的组织蛋白酶D较对照明显增加。结论:Syt在RBL中表达,其对RBL溶酶体胞吐起负调控作用。更多还原

【Abstract】 Objective: To investigate the expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3(RBL) and its role during exocytosis of RBL.Methods:The expression of Syt2 in RBL was detected by western blot.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,stable transfectants were selected using G418.To analysis the role of Syt2 during exocytosis of RBL,the release of cathepsin D was assayed by immunoblotting.Results:The expression of Syt2 in RBL was confirmed by immunoblotting.The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely coincidenced with rat Syt2(accession number in GenBank : NM012665).The stable transfectants(RBL-Syt2-AS) were obtained.Anti-RNA down-regulated the expression of Syt2 in RBL-Syt2-AS markedly.Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased.Conclusion:Syt2 expressed in RBL and it played a negative regulation during exocytosis of lysosomes in RBL.更多还原