大鼠谷氨酰胺合成酶RNA干扰表达载体的构建及其影响星形胶质细胞黏附的初探  

Construction and identification of rat glutamine synthetase RNA interference expression vector

作　　者：刘春兴 张滨[2] 邹健[2] 

机构地区：[1]华东疗养院检验科,江苏无锡214065 [2]南京医科大学附属无锡人民医院中心实验室,江苏无锡214023

出　　处：《南京医科大学学报（自然科学版）》2010年第10期1397-1401,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：南京医科大学科技基金重点项目(09NJ-MU254)

摘　　要：目的:构建并鉴定大鼠谷氨酰胺合成酶(glutamine synthetase,GS)RNA干扰表达载体,并检测GS对星形胶质细胞黏附的影响。方法:利用软件根据GS mRNA序列设计特异性siRNA序列,与GS真核表达载体GS-EGFP以LipofectamineTM2000试剂联合转染Hela-G细胞,以GFP为指标鉴定其有效性;体外合成该siRNA插入序列,将其定向克隆至真核表达载体pRNATH1.l/Neo中并转染至大鼠星形胶质细胞中,以免疫细胞化学方法鉴定GS的表达;通过免疫化学法对actin特异性染色分析GSRNA干扰后对星形胶质细胞黏附的影响。结果:GS siRNA能有效抑制GS-EGFP在Hela-G细胞中的表达;DNA测序证实合成并被克隆入真核表达载体pRNAT HI.1/Neo的siRNA插入序列完全正确,GS siRNA表达载体可特异性抑制星形胶质细胞中GS的表达;GS RNA干扰导致细胞黏附力显著降低。结论:成功构建大鼠GS siRNA真核表达载体,初步表明GS影响星形胶质细胞的黏附,为后期研究GS在中枢神经系统损伤后反应性星形胶质细胞中的功能奠定了基础。Objective：To construct and indentify rat glutamine synthetase （GS） RNA interference expression vector and detect GS on cell adhesion of astrocytes.Methods：The rat GS specific siRNA sequence was designed by online software,and GS eukaryotic expression vector,GS-EGFP were co-transfected into Hela-G cells by LipofectamineTM 2000 regent.GFP expression level was used to identify the interference efficiency of GS siRNA.The effective siRNA sequence was inserted into eukaryotic expression vector pRNAT H1.l /Neo,and the vector was transfected into astrocytes.The expression of GS in astrocytes was detected by immunocytochemistry.After trasfected with GS siRNA vector,the effect of GS on cell adhersion was detected by actin specific immunocytochemistry.Results：GS siRNA inhibited the expression of GS-EGFP in Hela-G cells.The inserted sequence of GS siRNA was confirmed by DNA sequencing.The GS siRNA expression vector can specifically inhibit the expression of GS in astrocytes.GS RNA interference leads to a significant decrease of cell adhesion.Conclusion：The construction of rat GS siRNA eukaryotic expression vector was successful.The initial data showed GS level in astrocytes impacted on cell adhesion.The results of this study lay the foundation for further investigating the role of GS in reactive astrocytes after central nervous system injury.

关 键 词：反应性星形胶质细胞 谷氨酰胺合成酶 真核表达载体 RNA干扰 细胞黏附 SIRNA表达载体 大鼠 免疫细胞化学方法 

分 类 号：Q25[生物学—细胞生物学]