靶向leptin基因的siRNA对大鼠肝星状细胞生物学特性的影响

目的 构建具有特异性瘦素（leptin）基因小于扰RNA（small interfering RNA，siRNA）功能的表达质粒，研究靶向leptin基因的siRNA对大鼠肝星状细胞（hepatic stellate cell，HSC）中leptin表达及HSC生物学特性的影响。方法 以leptin为目的基因，以产生siRNA质粒载体psiRNA-hHlneo为表达模板，细胞内转录合成4对特异的siRNA和1对对照siRNA。构建能在真核细胞中表达的重组质粒leptinsi RNA，用酶切方法和测序法对重组体进行鉴定。应用脂质体介导重组质粒转染HSC。RT-PCR、Western blot和免疫细胞化学法检测HSC的leptin表达情况，用MTT法检测细胞增殖，用流式细胞仪分析细胞凋亡。结果 酶切和序列测定表明，成功构建了leptinsiRNA表达质粒；外源重组质粒siRNA3和siRNA4能够抑制leptinm RNA和蛋白表达（均P〈0．01）；HSC增殖活性显著降低（P〈0．05），凋亡增加（P〈0．01）。与未转染组相比，consiRNA、siRNA1和siRNA2转染组没有显著改变（均P〉0．05）。结论 构建的leptinsi RNA表达载体可减少leptin的表达，抑制HSC增殖并诱导其凋亡。为肝纤维化基因治疗提供了新的靶点。

Effect of siRNA Targeting Leptin Gene on Biological Behaviors of Rat Hepatic Stellate Cells

Objective To construct the specific expressing plasmids of siRNA targeting leptin and investigate the effect of siRNA targeting leptin gene on the leptin expression and the biological behaviors of hepatic stellate cells(HSCs).Methods The 5 siRNAs targeting leptin gene were synthesized intracelluarly by expressing templates of plasmid vector psiRNA-hH1neo.The recombinant leptin siRNA plasmids expressed in eukaryocytes were constructed and identified by using enzyme digestion and sequencing.The recombinant plasmids were transfected into HSCs by Lipofectamine.The expression of leptin was detected by using RT-PCR,Western blot and immunocytochemical staining respectively.The cell proliferation was assayed by MTT method and the cell apoptosis by flow cytometry.Results Enzyme digestion and sequencing revealed the successful construction of leptin siRNA expression plasmids.Extrogenous recombinant plasmids siRNA3 and siRNA4 could inhibit the expression of leptin mRNA and protein(P<0.01),significantly decrease the proliferating activity of HSC(P<0.05),but increase the apoptosis(P<0.01).As compared with no-transfection group,no obvious changes were found in consiRNA group,siRNA1 group and siRNA2 group(P>0.05).Conclusion The constructed leptin siRNA expressing plasmids could reduce the expression of leptin,inhibit the proliferation of HSC and induce apoptosis.A novel target was provided for gene therapy of liver fibrosis.