中文摘要：

目的：构建HLA—G短干扰RNA表达质粒及探讨HLA—G的功能。方法：根据siRNA设计的原则，结合pSilencer2．1-U6-neo质粒的特点，针对HLA—G基因设计并合成两对寡聚DNA片段，退火后将其克隆入pSilencer2．1-U6-neo，将重组真核表达质粒pSi—lencer2．1-U6-neo—HLA—G采用脂质体法转染JEG-3绒癌细胞株。应用RT—PCR、Western—blot等方法检测转染的JEG-3细胞中HLA—G基因的表达水平；MTT法检测转染后JEG-3细胞增殖的变化。结果：RT—PCR和Western—blot结果均表明瞬时转染重组pSilencer2．1-U6-neo—HLA—G质粒明显抑制了JEG-3细胞中HLA—G基因的表达。实验组JEG-3细胞生长较对照组明显受到抑制。结论：pSilencer2．1-U6-neo—HLA—G质粒构建成功，可下调JEG-3细胞中HLG的表达，抑制绒癌细胞生长。

英文摘要：

Objective：To determine the feasibility of RNAi application in investigation of HLA-G functions, and to construct HLA-G-targeting siRNA （small interfering ）-expressing plasmid. Methods： Hairpin siRNA templates were designed based on HLA-G gene sequence and was cloned into pSilencer2.1-U6-neo vector. The resultant plasmid pSilencer2.1-U6-neo-HLA-G was transfected into JEG-3 cells with lipofectamine 2000. The non-transfected cells,the cells transfected by pSilencer2. 1-U6-neo and the non-specific siRNA transfected cells were used as controls. The inhibitory effects of HLA-G mRNA and protein expression were detected by semi-quantitative RT-PCR and Western-blot, respectively. The cellular survival of JEG-3 was determined by MTF. Results：The plasmid pSilencer2.1-U6-neo-HLA-G efficiently down-regulated the expression of HLA-G in JEG-3 cells. The growth of cells transfected was remarkably inhibited. Conclusion：The successful construction of the siRNA expressing plasmid pSileneer2. 1-U6-neo-HLA-G inhibits the expression of HLA-G gene and the proliferation of JEG-3.