靶向大鼠沉默信息调节因子1基因小干扰RNA慢病毒载体对大鼠视网膜神经节细胞中沉默信息调节因子1表达的影响

The effect of shRNA interference lentivirus vector targeting rat Sirtl gene on the expression of Sirtl in retinal ganglion cell

目的观察靶向大鼠沉默信息调节因子1（Sirt1）基因的小干扰（si）RNA慢病毒载体对大鼠视网膜神经节细胞（RGC）中Sirt1表达的影响。方法设计4条靶向大鼠Sirt1基因的siRNA靶点序列，构建靶向大鼠Sirt1基因的siRNA慢病毒载体。采用菌落聚合酶链反应（PCR）和DNA测序鉴定阳性克隆。应用阳性重组质粒与包装质粒共转染293T细胞，进行慢病毒包装及病毒滴度测定。将RGC分为空白对照组、转染阴性对照病毒载体组（NC组）以及分别转染4个带有干扰序列的si-Sirt1-1组、si．Sirt1．2组、si—Sirt1-3组、si-Sirt1．4组。采用荧光定量PCR及蛋白免疫印迹法（Westernblot）分别检测各组RGC中Sirt1mRNA、蛋白相对表达量。结果菌落PCR鉴定和DNA测序结果显示，靶向大鼠Sirt1基因的siRNA慢病毒载体构建成功。在293T细胞中成功包装后，其病毒滴度为8×10^8TU／ml。荧光定量PCR和Westernblot检测结果显示，与NC组比较，si-Sirt1-1组、si-Sirt1-2组、si-Sirt1-3组、si．Sirt1．4组RGC中Sirt1mRNA、蛋白相对表达量均明显下降，差异有统计学意义（F=27．682、1185．206，P=-0．000、0．000）；其中，以si—Sirt1．2组Sirt1mRNA、蛋白相对表达量下降幅度最为明显。结论靶向大鼠Sirt1基因的siRNA慢病毒载体可降低大鼠RGC中sirtlmRNA、蛋白表达。

Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirtl gene on the expression of Sirtl in retinal ganglion cell （RGC）. Methods Four short hairpin （sh） RNA interference sequences targeting rat Sirtl gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction （PCR） and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirtl-1, si-Sirtl-2, si-Sirtl-3, si-Sirtl-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirtl mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8- 10s TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirtl mRNA and protein were significantly decreased in the si-Sirtl-1, si-Sirtl-2, si-Sirtl-3 and si-Sirtl-4 groups （F=27.682, 1 185.206; P=0.000, 0.000）. The si-Sirtl-2 group had the strongest effect in reducing the expression of Sirtl mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirtl gene can effectively down regulate the expression of Sirtl mRNA and protein in RGC cells.