葡萄糖神经酰胺合成酶siRNA表达载体的构建

Construction of glucosylceramide synthase siRNA expression vectors

目的构建针对葡萄糖神经酰胺合成酶(GCS)mRNA的siRNA表达载体,并观察其对转染细胞中GCS表达的影响。方法根据人GCSmRNA编码序列,设计并合成3对针对GCS的发卡状RNA的寡核苷酸,各64个碱基,退火后插入pSUPEREGFP载体重组构建RNAi质粒,进行双酶切及测序鉴定。重组质粒用脂质体包裹转染入SGC7901/VCR细胞,采用RT-PCR检测GCSmRNA表达水平的变化。结果重组构建的载体经酶切鉴定与测序分析,显示特定位点靶序列与设计序列完全一致。转染SGC7901/VCR细胞后,GCSmRNA表达明显降低。结论GCSsiRNA表达载体构建成功,并可有效抑制人胃癌耐药SGC7901/VCR细胞中GCS的表达,为后续研究及肿瘤的基因治疗奠定了基础。

Aim： To construct glueosyleeramide synthase（GCS） siRNA expression vectors and to observe their effect on GCS mRNA expression in transfeeted cells. Methods：According to the encoding sequence of mRNA of human GCS, three pairs of GCS specific target gene sequence were designed and synthesized. The annealed oligonueleotide fragments were inserted into the pSUPER EGFP vector. After being identified by double digestion with the enzymes and DNA sequencing, the recombinants were transfected into SGC7901/VCR cells using Lipofectamine^TM 2000. The expression of GCS mRNA was detected by RT-PCR. Results： Recombinant plasmids were identified by digestion with EcoR Ⅰ and Hind Ⅲ ,and confirmed by sequencing analysis. The results demonstrated that the insertion sequence was exactly correct. GCS mRNA expression significantly reduced after transfeetion. Conclusion： The expression vector of GCS siRNA has been constructed successfully and can suppress the GCS mRNA expression in gastric carcinoma cells, which lays a basis for further studies of GCS function and its application in the gene treatment of tumors.