慢病毒介导的RNAi对大鼠SOCS3基因表达的抑制作用

Inhibition effect of lentiviral vector-mediated RNAi on expression of SOCS3 gene in rat hypothalamic cells

目的研究慢病毒表达载体介导的RNA干扰(RNAi)对大鼠下丘脑细胞中细胞因子信号转导抑制因子3(SOCS3)的抑制作用。方法根据大鼠SOCS3基因(NM053565),用Ambion在线软件选择3个靶序列,设计并合成包含各正反义靶序列的互补单链寡核苷酸,与经BamHⅠ和XhoⅠ酶切后的慢病毒载体质粒pRNA-lenti-GFP连接产生pRNA-Lenti-SOCS3-GFP慢病毒重组质粒,与慢病毒包装混合物共转染293T细胞,包装产生慢病毒,收集病毒上清,采取逐孔稀释滴度法测定病毒滴度,然后转染大鼠下丘脑细胞,通过荧光显微镜观察细胞转染情况,利用荧光实时定量PCR方法检测RNAi组(siRNA1,siRNA2,siRNA3)、空白细胞组和阴性序列组(siRNA-Negtive)中SOCS3的表达情况。结果将目的序列成功连接到载体上,并经测序分析证实载体构建成功。系列稀释法检测慢病毒悬液的滴度为1.0×1010 TU/L。荧光实时定量PCR检测显示慢病毒感染大鼠下丘脑细胞后,与空白细胞组相比,3个RNAi组都有不同程度的抑制SOCS3表达,其中siRNA1组的抑制效果最好,可使SOCS3 mRNA表达下调达80%。结论构建的pRNA-Lenti-SOCS3-GFP慢病毒载体可有效地抑制大鼠SOCS3的表达,为以SOCS3基因为靶点的相关疾病的基因治疗研究奠定基础。

Objective To construct a lentiviral-mediated RNAi vector targeting rat suppressors of cytokine signaling 3 （SOCS3） gene and assess its gene silencing effect in rat hypothalamic cells. Method Three target sequences were selected by on-line designer software on Ambion according to rat SOCS3 mRNA sequence. The complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After annealing, these double strands DNA were cloned to pRNA-lenti-GFP which contained U6 promoter and green fluorescent protein （GFP）. The resulting lentiviral vector containing SOCS3 shRNA was named pRNA-Lenti-SOCS3-GFP. Then pRNA-Lenti-SOCS3-GFP and Lentivector Packaging plasmid mix were co-transfeeted into 293T to package lentivims particles. The virus titer of culture supematant was determined by serial dilution assay after it was harvested. Real-time PCR were used to evaluate the level of SOCS3 expression after the rat hypothalamic cells were transfected with the constructed vectors. Result The SOCS3 mRNA sequence was successfully cloned into pRNA-lenti-GFP, which was proved by PCR and DNA sequencing. The particle titer was ×10^10 TU/L, which was determined by serial dilution assay. Compared with control group, the SOCS3 mRNA expression were obviously suppressed in all three experimental groups （ siRNA1, siRNA2 and siRNA3 ） especially in siRNA1 group, and 80% of the expression was reduced. Conclusion The lentiviral-mediated RNAi vector can inhibit the expression of SOCS3 gene in rat hypothalamic cells, which provides a potential tool for the therapeutic research of SOCS3-related diseases.