SmacsiRNA慢病毒载体的构建及其对Smac基因沉默效果的鉴定

Construction of Smac siRNA lentiviral vector and its silencing effect on Smac gene in human LECs

背景研究证实半胱氨酸天冬氨酸蛋白水解酶激活剂（Smac）蛋白是肿瘤细胞的促凋亡蛋白，我们先前的研究表明，Smac蛋白在白内障患者LECs中表达量明显高于正常人，因此推测沉默LECs中Smac的表达可抑制LECs的过度凋亡，但如何成功将SmacsiRNA转染进入LECs是研究的关键。目的构建人SmacsiRNA慢病毒载体并对人LECs进行感染，研究慢病毒载体构建方法及其在人晶状体上皮细胞HLE—B3中的干扰效率，利用RNA干扰（RNAi）技术建立Smac低表达的HLE—B3株。方法根据我们前期的工作设计并合成针对Smac基因序列的siRNA序列，将合成的双链DNA以T4噬菌体DNA连接酶与GV118慢病毒载体相连接，转化DH5α感受态细胞，采用PCR筛选阳性克隆，经测序鉴定正确后，感染293T细胞，收集上清液并标定病毒滴度。用重组慢病毒感染HLE—B3细胞，荧光显微镜下观察转染后的阳性HLE—B3细胞数并计算病毒感染复数（MOI）。将常规培养的HLE—B3细胞分为阴性对照组、空质粒组和GV118-Smac—siRNA1组，阴性对照组为常规培养的细胞，空质粒组细胞仅转染不含慢病毒载体的siRNA质粒，GV118-Smac·siRNA1组细胞转染GV118-Smac—siRNA1质粒，采用实时荧光定量PCR检测各组细胞中SmacmRNA的相对表达量。结果构建的重组载体GV118-Smac—siRNAl转化DH5c感受态细胞后阳性克隆产物大小为340bp，空载的克隆产物为299bp，与预期结果相符，测序结果显示合成的Smac—siRNA1与预期目的序列一致。构建的GV118-Smac—siRNA1病毒滴度为3．0x10。TU／m1。感染HLE．B3细胞后荧光显微镜下可见MOI为100时，细胞毒性低且感染效率高，约为82％。阴性对照组、空载质粒组和GV118-Smac—siRNA1质粒组细胞中SmacmRNA相对表达量分别为（101．290±8．349）％、（92．330±6．320）％和（32．540±4．221）％，3个组间总体比较差异有统计学意义（F=32．871，P〈0．01），其中GV118-Smac—siRNA1质粒组细胞中SmacmRNA相对表达量较阴性对照组明显下降，差异有统计学意义（P=0．000），而空载质粒组细胞中SmacmRNA相对表达量略低于阴性对照组，但组间差异无统计学意义（P=0．535）。结论本研究中成功构建了siRNA慢病毒载体GV118-Smac—siRNA1，其转染HLE—B3效率高，该siRNA载体能够有效地抑制人LECs中Smac基因的表达。

Background Research comfirmed that second mitochondrial activator of caspase （Smac） is a promoting tumor cell apoptosis protein. Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes. We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs. The way to transfect Smac siRNA into LECs is a key step. Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line （HLE-B3） and establish low-expressed Smac HLE-B3 line. Methods Based on the genebank and our previousstudy,siRNA sequence of Smac was designed and composed. The synthetic double-stranded DNA was linked to the lentiviral vector GVll8 by 3-4 DNA ligase and then transformed DH5a competent cells, The plasmids were transformed into the DH5a competent cells. Recombinant colonies were screened by PCR and sequenced. Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells. Cell culture supernatant was collected for the measurement of viral titer. Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection （MOI） under the fluorescence microscope. Transfection efficiency was examined by calculating the GFP-positive cells. HLE-B3 ceils were divided into negative control group, siRNA plasmid tranfected group and GVll8-Smac-siRNA1 tranfected group. The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR. Results GVllg-Smac- siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp,and viral titer was 3.0xl0s TU/ml. At a MOI of 100,the infecting efficiency of the vector on HLE-B3 cells was about 82% and the cytotoxicity was low. The relative expression levels of Smac mRNA were （ 101. 290± 8. 349）% , （92. 330 ±6. 320 ）% and （32. 540±4. 221 ）% in the negative control group, siRNA plasmid tranfected group and GVllg-Smac-siRNA1 tranfected group, respectively, showing a significant difference among the three groups（ F = 32.871, P〈0.01 ）, and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group （ P= 0. 000）. However, no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group （P= 0. 535）. Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed. Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs.