摘要
背景:人类白细胞抗原(human leukocyte antigen,HLA)是人的主要组织相容性复合物,在移植排斥反应中起关键作用。降低HLA的表达,可以延长移植物的存活时间。目的:设计HLA-A2小干扰RNA,检测其对HLA-A2表达沉默的作用。方法:设计4种HLA-A2 siRNA靶点序列构建其慢病毒表达体系,体外感染HLA-A过表达的293T细胞,观察4种靶点的敲减效率并筛选出有效靶点序列。体外培养人胚肺成纤维细胞,并用携带目的干扰序列的慢病毒感染人胚肺成纤维细胞,荧光显微镜下观察感染效率,应用Western blot检测在人胚肺成纤维细胞中对HLA-A2表达沉默的作用。结果与结论:根据Genbank中HLA-A2的mRNA序列,设计合成了3个小干扰RNA,在体外能有效的沉默HLA-A2在293细胞中的表达,将基因序列亚克隆入干扰载体后感染人胚肺成纤维细胞,也能有效的沉默HLA-A2的表达,效率达80%左右。
BACKGROUND： Human leukocyte antigen （HLA）, the major histocompatibility complex of human, plays an important function in the transplant rejection. Decreasing the expression of HLA will prolong the survival time of transplants. OBJECTIVE： To design small interfering RNA （siRNA） of HLA-A2 and to detect the effect of siRNA-HLA-A2 on the expression of HLA-A2. METHODS： Four kinds of siRNA-HLA-A2 domains were designed, and recombinant lentivirus expression vector were formed. The 293T cells, highly expressing HLA-A2, were infected in vitro. Then the knockout efficacy of four domains was detected to select the highly efficient siRNA-HLA-A2 target sequences. The human embryo lung fibreblasts were cultured in vitro and infected with the lentivirus carrying the target sequence. The infecting efficiency of LV-siRNA-HLA-A2 was observed under the fluorescence microscope and the silence function of this siRNA in human embryo lung fibroblasts was detected by western blot analysis. RESULTS AND CONCLUSION： According to the mRNA sequence of HLA-A2 in Genbank, three siRNAs were designed and synthesized./n vitro, the over expression of HLA-A2 in 293K cells was successfully silenced. The HLA-A2 expression in human embryo lung fibroblasts was also efficiently silenced after the human embryo lung fibroblasts were infected by the highly efficient siRNA of HLA-A2. The efficacy was up to 80%.
出处
《中国组织工程研究》
CAS
CSCD
2014年第29期4605-4610,共6页
Chinese Journal of Tissue Engineering Research
基金
辽宁省自然科学基金资助项目(2013022066
2013022048)
辽宁省教育厅项目(L2013335)
辽宁医学院青年基金项目(Y2011Z022)~~
