靶向髓样细胞表达的激发受体1基因短发夹RNA真核质粒表达载体的构建及筛选

Construction and screening of eukaryotic plasmid expression vectors encoding the short hairpin RNA targeting triggering receptor expressed on myeloid cells 1 gene

目的 构建编码髓样细胞表达的激发受体1（TREM-1）基因短发夹RNA（shRNA）真核质粒表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体.方法 针对TREM-1的靶位点设计含shRNA的靶序列,分别构建pGCsi-TREM-1 shRNA1和pGCsi-TREM-1 shRNA2的质粒表达载体和pGCsi-NegshRNA阴性质粒表达载体,通过酶切及测序进行鉴定.鉴定后以脂质体包裹转染肺泡上皮A549细胞,分为未转染对照组（A组）、转染空质粒载体pGCsi对照组（B组）、转染阴性shRNA质粒表达载体pGCsi-NegshRNA对照组（C组）、转染TREM-1 shRNA1质粒表达载体组（D组）和转染TREM-1shRNA2质粒表达载体组（E组）,48 h后观察转染细胞绿色荧光蛋白表达效果.转染24、48、72 h后,以A组为对照采用MTT法测定B、C、D、E组细胞的存活率.转染48 h后,通过荧光定量PCR检测各组TREM-1 mRNA的表达水平并与转染前相比较.结果 经酶切和测序鉴定证实,目的 TREM-1基因shRNA1和shRNA2片段已被克隆到pGCsi载体中.荧光显微镜下,B、C、D、E组A549细胞有明显的绿色荧光蛋白表达.各时间点B、C、D、E组细胞存活率均达90%以上.A、B、C 3组转染前后TREM-1mRNA的表达水平差异无统计学意义,D、E组转染的重组质粒均能有效抑制A549细胞的TREM-1mRNA的表达（[（1.945±0.252）×105比（1.010±0.194）×105,（1.933±0.216）×105比（1.202±0.171）×105,均P＜0.05],抑制率分别为48.07%和37.82%.结论 成功构建了靶向TREM-1基因的shRNA质粒表达载体,可有效抑制细胞中TREM-1目的 基因的表达,为后续脓毒症的基因治疗提供了依据.

Objective To construct eukaryotic plasmid expression vectors encoding the short hairpin RNA（shRNA）targeting triggering receptor expressed on myeloid cells 1（TREM-1）mRNA,and to screen the shRNA plasmid expression vectors with most obvious effects for silencing TREM-1 gene.Methods According to the target site of TREM-1 gene,shRNA target sequence was designed.Two plasmid expression vectors of pGCsi-TREM-1 shRNA1 and pGCsi-TREM-1 shRNA2,and negative control plasmid expression vectors of pGCsi-NegshRNA were constructed respectively.After being identified by restriction endonuclease digestion and nucleotide sequencing, the recombinant plasmids with TransFectin lipid were transfected into alveolar epithelial cells A549.A549 cells were allocated into the un-transfected group（group A）, pGCsi transfected group （group B）,pGCsi-NegshRNA transfected group （group C） , TREM-1 shRNA1 transfected group （group D） and TREM-1 shRNA2 transfected group（group E）. The effects of green fluorescent protein expression in transfected cells were studied at 48 h later.Cell viability was assessed by MTT at 24, 48 and 72 h after transfection with group A as reference. The expression of TREM-1 mRNA was detected by quantitative real-time PCR and compared with the level before transfection. Results Restriction endonuclease digestion and nucleotide sequencing showed that TREM-1 shRNA1 and shRNA2 oligonucleotide fragments were correctly inserted into pGCsi vector.Under fluorescence microscopy, A549 cells in groups B, C, D and E clearly showed expression of green fluorescent protein. At all the time spots, over 90% of cells in these groups were viable. There was no change of TREM-1 mRNA expression in groups A, B and C from pre-transfection. The recombinant plasmids showed effective inhibition of TREM-1 mRNA expression in 48.07% and 37.82% of A549 cells in groups D and E respectively[（1.945±0.252）×105 vs （1.010±0.194）×l05, （1.933±0.216）×l05 vs （1.202±0.171）×105, all P〈0.05）. Conclusion The expression vector of TREM-1 shRNA is successfully constructed and can inhibit TREM-1 mRNA expression in A549 cells, which provides evidences for subsequent studies on gene therapy for sepsis.