shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达

目的 构建基于miR-155结构的shRNAmir慢病毒表达载体,在鼻咽癌细胞中沉默COX-2基因的表达,探索全新的COX-2抑制方法.方法 转染小干扰RNA(siRNA),筛选出沉默COX-2表达的RNA干扰序列,以该序列替换miR-155前体序列中的双链部分作为anti-COX-2 shRNAmir转录模板,构建pLVTHM/shRNAmir慢病毒质粒表达载体,利用293FT细胞包装pLVTHM/anti-COX-2 shRNAmir慢病毒.感染鼻咽癌C666-1细胞后,通过流式分选,建立anti-COX-2 shRNAmir稳定表达,COX-2基因沉默的亚系.结果 设计合成的anti-COX-2a siRNA能够使COX-2 mRNA表达下降90%以上,根据其序列构建的plVTHM/sbRNLAmir质粒载体测序无误,pLVTHM/anti-COX-2 shRNAmir慢病毒感染C666-1细胞后,分选出亚系C666-1经反复传代仍可保持COX-2基因沉默的表型.结论 pLVTHM/shRNAmir慢病毒载体能够转录出基于miR-155结构的shRNAmir,沉默鼻咽癌细胞COX-2的表达,为探索更加高效安全的COX-2抑制剂奠定基础,也为鼻咽癌研究和抗肿瘤基因治疗提供了新手段.     

Coordinated β-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for β-thalassemia

A primary challenge in lentiviral gene therapy of β-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVβ-shα2, that allows coordinated expression of the therapeutic β^A-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in β-thalassemia. We demonstrate that LVβ-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and β^A-T87Q-globin gene expression identical to the parent vector. Compared with the first β^A-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVβ-shα2 shows 1.7-fold greater potency to improve α/β ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of β-thalassemia and provide an improved benefit/risk ratio regardless of the β-thalassemia genotype.     

光诱导RNAi系统的构建及优化

利用光诱导基因表达系统,构建光诱导RNAi系统,并对系统转染比例、转录因子的表达量和shRNAmir表达载体的核心启动子进行优化。通过构建不同启动子的光敏转录因子GAVPO表达载体和不同核心启动子的shRNAmir表达载体,分析它们对报告基因的干扰效果的影响。结果表明,GAVPO的表达量对报告基因沉默效果和shRNAmir的背景表达水平至关重要。低表达量的GAVPO能够实现对报告基因的微调控,报告基因的表达量下降到约60%;而高表达量的GAVPO能够高效抑制报告基因表达,报告基因的表达量下降到34%。     

shRNAmir慢病毒载体沉默环氧合酶-2对鼻咽癌细胞放疗敏感性的影响

摘要：目的探讨COX-2基因沉默对鼻咽癌C666-1细胞放射敏感性的影响。方法以稳定沉默COX-2基因表达的鼻咽癌
Anti-COX-2 C666-1细胞和对照细胞Anti-GL-2 C666-1为研究对象,采用克隆形成实验及曲线拟合计算不同剂量辐射后
各放射生物学参数和放射增敏比;采用流式细胞仪检测辐射后细胞周期的变化;采用体内荷瘤裸鼠动物模型检测放疗后
皮下移植瘤的生长曲线,并计算抑瘤率。结果Anti-COX-2 C666-1细胞SF2、D0、Dq值较对照组均明显偏低,α/β显著增
高,放射增敏比为1.4014;COX-2 沉默后降低了辐射诱导的G2/M 期阻滞,并增加辐射后荷瘤裸鼠的抑瘤率。结论
COX-2基因稳定沉默后可改变鼻咽癌C666-1的放射生物学参数,并降低辐射引起的G2/M期阻滞,增大抑瘤率,能够起到
放疗增敏的作用。
     

Coordinated β-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for β-thalassemia

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