中文摘要：

目的：构建小鼠热休克蛋白47（47 kDa heat shock protein，HSP47）基因的小干扰RNA（small interference RNA，siRNA）表达载体．及HSP47第一外显子克隆表达载体，观察其在小鼠成纤维细胞（NIH3T3细胞株）中对HSP47基因表达的抑制作用。方法：针对小鼠HSP47的mRNA序列，在368、718位点分别设计两对2Int的，编码HSP47siRNA基因片断的寡核苷酸，退火成双链后将其连接到pSilencer1．0质粒的U6启动子下游，构建pSilencer—HSP47siRNA重组质粒：同时构建HSP47第一外显子重组质粒。经酶切、测序鉴定后，用脂质体介导的基因转染方法，转入NIH3T3细胞．用半定量RT—PCR法检测HSP47表达水平的变化。结果：经酶切、测序鉴定，成功构建了siRNA真核表达载体及靶基因克隆表达载体。经脂质体转染NIH3T3细胞后，RT—PCR显示NIH3T3细胞中HSP47基因的mRNA表达水平明显降低，所构建的HSP47-siRNA基因的真核表达载体，成功地抑制了目的基因的表达。结论：构建的小鼠HSP47基因的RNA干扰真核表达载体pSilencer—HSP47siRNA，在NIH3T3细胞中有效地发挥了对HSP47基因表达的干涉作用．从而提示HSP47特异的siRNA具有抑制皮肤瘢痕形成的潜力。

英文摘要：

Objective： To construct the expression vector of siRNA targeting 47 kDa heat shock protein （HSP47） gene of the mouse, and to observe the inhibitory effect of the vector on HSP47 gene expression in mouse fibroblast cell line NIH3T3. Methods： 21 base-pair oligonucleotide for small interfering RNA expression targeting HSP47 gene at site of 368 and 718 was designed and chemically synthesized. After annealing, double oligonucleotide were inserted into the downstream of U6 promotor of pSilencer1.0 to recombine pSilencer-HSP47siRNA plasmids. The HSP47 Exonl expression vector was also constructed meanwhile. Then pSilencer-HSP47siRNA plasmids and the HSP47 Exon 1 expression vector were transiently transfected into mouse fibroblast cell line NIH3T3 with lipofectamine-mediated transfection. Semi-quantitative RT-PCR was used to detect the expression levels of HSP47 gene. Reults： Recombined pSilencer-HSP47siRNA vector was confirmed by restriction enzyme and sequencing analysis. The results demonstrated that both 64nt and the HSP47 Exonl had been inserted into the expected site. Furthermore, the insert sequence was exactly correct. The recombined pSilencer-HSP47siRNA vector significantly suppressed the expression of HSP47 in NIH3T3. Conclusion： The constructed siRNA expression vector of HSP47 gene psiRNA-HSP47 recombinant plasmid can block the expression of HSP47 gene in mouse fibroblast cell line NIH3T3. It suggests that HSP47 specific siRNA may has the potential of suppressing the formation of scar.