靶向Slug基因腺相关病毒载体的构建及其抑制视网膜母细胞瘤细胞生长的体外研究

Construction of adeno-associated virus vector targeting Slug genes and its inhibitive effect on growth of retinoblastoma cells in vitro

目的构建并制备携带目的基因的rAAV-EGFP-Slug-siRNA病毒载体。方法首先构建pDC316-EGFP-Slug-siRNA质粒,以PCR鉴定正确的pDC316-EGFP-Slug-siRNA克隆为模板扩增EGFP-Slug-siRNA片段,EcoRI和SalI双酶切后回收PCR产物。酶切pSNAV2.0-LacZ-α载体质粒,并与上述产物进行连接。转化感受态大肠杆菌DH5α,得到重组质粒pSNAV2.0-EGFP-Slug-siRNA,酶切和测序对其进行鉴定。建立载体细胞株BHK/Slug-siRNA,大规模制备rAAV2-EGFP-Slug-siRNA并对其纯化、鉴定和滴度测定。将体外培养的视网膜母细胞瘤(retinoblastoma,RB)细胞转染rAAV-EGFP-Slug-siRNA和pSNAV2.0-EGFP空载体。鸡胚绒毛尿囊膜实验检测血管生成,MTT比色法检测细胞存活率,建立鸡胚绒毛尿囊移植瘤。结果携带编码靶向Slug的小发夹状干扰RNA序列的腺相关病毒载体质粒pSNAV2.0-EGFP-Slug-siRNA,经PCR、酶切和测序鉴定,质粒构建正确。将构建成功的载体质粒与重组Ⅰ型单纯疱疹病毒HSV1-rc/UL2共转染包装细胞BHK-2,成功复制和包装出重组腺相关病毒rAAV2-EGFP-Slug-siRNA,经检测目的片段插入成功,滴度为90.21×109puf。Slug-siRNA有效抑制RB细胞内Slug表达后,肿瘤血管密度明显降低,血管生成被抑制,细胞存活率明显降低。实体瘤血管生成和生长受到抑制。结论成功制备出高滴度的携带目的基因的rAAV-EGFP-Slug-siRNA病毒载体。干扰Slug可抑制肿瘤血管生成,抑制RB细胞的生长增殖。

Objective To construct and prepare the recombinate adeno-associated virus（rAAV）-EGFP-Slug-siRNA virus vector containing Slug genes.Methods The plasmid of pDC316-EGFP-Slug-siRNA was constructed,and then this vector was amplified by PCR to obtain the EGFP-Slug-siRNA fragment.The PCR product and pSNAV2.0-LacZ-α vector were double-enzyme digested by EcoR I and Sal I,then recovered the fragments and linked them together.The recombinants were transformed into escherichia coli DH5α and then the reconstructed pSNAV2.0-EGFP-Slug-siRNA plasmid DNA was extracted,and sequentially,the constructs were identified by restriction enzyme analysis and sequencing.The shuttle vector BHK/Slug-siRNA was constructed,the rAAV2-EGFP-Slug-siRNA were large-scale prepared,purified and identified,and detected its titer.Sequentially,the retinoblastoma cells cultured in vitro were infected by rAAV-EGFP-Slug-siRNA and its empty vector pSNAV2.0-EGFP,respectively,and the angiogenesis was detected by the chick embryo chorioallantoic membrane and cell survival rate was detected by MTT colorimetry,meanwhile the transplant tumor of chick embryo chorioallantoic membranes was established.Results The pSNAV2.0-EGFP-Slug-siRNA shuttle plasmid vectors containing the targeted Slug genes were successfully constructed and identified by PCR,double-enzyme digestion and sequencing.The constructed plasmid vectors and the HSV1-rc/UL2 infected the BHK-2 cells,the rAAV2-EGFP-Slug-siRNA virus vector with the titer being as high as 90.21×109 puf were successfully replicated and packaged.After the expression of Slug genes in retinoblastoma cells being effectively inhibited by Slug-siRNA,the blood vessel density and cells survival rate were significantly decreased,and the angiogenesis was inhibited.The angiogenesis and growth of solid tumor were inhibited as well.Conclusions The virus vector rAAV-EGFP-Slug-siRNA with high titer can be successfully constructed,which contains siRNA targeting Slug genes.Silencing the Slug genes can inhibit the tumor angiogenesis,growth and proliferation of retinoblastoma cells.