摘要
目的构建晚期糖化终产物受体(RAGE)反义RNA腺相关病毒载体,并在大鼠肾脏系膜细胞中表达。方法构建腺相关病毒介导的RAGE反义RNA载体,3个质粒共转染293细胞,获得病毒原液,感染大鼠肾脏系膜细胞,流式细胞术、RT-PCR、ELISA检测重组病毒感染的细胞RAGE的表达和分泌细胞外基质的情况。结果经酶切鉴定、序列分析显示RAGE基因片段正确完整反向插入pAAV-MCS。利用293细胞包装获得病毒原液的滴度为8.7×107VP/mL。感染重组病毒的细胞与正常细胞比较RAGE表达被抑制(48.2±6.1)%,分泌Ⅳ型胶原(ColⅣ)水平明显下降(P<0.05)。结论成功构建具有抑制功能的RAGE反义RNA腺相关病毒载体,为进一步研究RAGE的作用机制,以及基因治疗RAGE相关疾病提供一个重要工具。
Objective To construct recombinant receptor for advanced giycation endpreduets （RAGE）anfisense RNA adeno-associated virus vector and express the vector in rat mesangial cell. Methods Total RNA was acquired by TRIZOL method and the gene fragment of rat RAGE was amplified by RT-PCR. The target gene was connected to pAAV-MCS vector, and then identified by restriction endonuclease digestion and DNA sequence analysis. Recombinant AAV-RAGE- was derived by co-transfecting DNAs of rAAV-RAGE- , pAAV-RC, and pAAV- helper into AAV-293 cells using liposome methods. The titer of recombinant AAV-RAGE- was calculated by staining the cells expressed the re- port gene LacZ with X-Gal. After transduction of recombinant AAV-RAGE- in rat mesangial cell, flow cytometry （FCM） and RT-PCR were used to determine the expression of RAGE and ELISA was used to examine the levels of fibronectin （FN） and type IV collagen （ Col Ⅳ ） in cell supematant inducedby AGEs. Results The recombinant AAV-RAGE- wasobtainedwiththetiters of 8.7×10^7 viruspartieles/mL. AfterAG- Es induction, the expressions of RAGE was inhibited by （48.2 ± 6.1 ） % and the amount of Col Ⅳ in the medium was lower than that of vector virus transducted coils （ P 〈 0.05）. Conlusion The success in constructing recombinant AAV/RAGE- promotes the further research in melecular biology of RAGE and the recombinant AAV/RAGE- can be potentially used in gene therapy for RAGE associated diseases.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第2期135-138,143,共5页
Immunological Journal
基金
福建省科技开发计划项目(2003D09)
福建省卫生厅青年科研基金(2004-1-1)资助
关键词
反义RNA
晚期糖化终产物受体
腺相关病毒
Antisense RNA
Receptor for advanced glycation endproducts
Adeno-associated virus vector
