Specific targeting of pancreatic islet cells in vivo by insulin-promoter-driven adenoviral conjugated reporter genes.
Specific targeting of pancreatic islet cells in vivo by insulin-promoter-driven adenoviral conjugated reporter genes.
复制标题
通过胰岛素启动子驱动的腺病毒缀合报告基因在体内特异性靶向胰岛细胞。
DOI：
10.1007/s00268-005-0688-3
复制
发表时间：
2006
影响因子：
2.6
通讯作者：
Brunicardi,FCharles
中科院分区：
文献类型：
--
作者：
Wang,Xiaoping;Olmsted-Davis,Elizabeth;Davis,Alan;Liu,Shihe;Li,Zhijun;Yang,Jie;Brunicardi,FCharles
BackgroundThe本研究的目的是确定是否大鼠胰岛素启动子（RIP）可以，在小鼠模型中，直接表达的腺病毒介导的报告基因特异性进入胰岛通过systemicdelivery.Methods508个碱基对的RIP DNA序列被构建成腺病毒载体含有lacZ报告基因（Adeno-RIP-lacZ）。构建巨细胞病毒（CMV）启动子以驱动lacZ转录因子基因表达（Adeno-CMV-lacZ）并用作对照。进行体外瞬时转染测定以确定转录因子基因表达水平，并与脂质体介导的质粒转染进行比较。将SCID小鼠饲养并圈养在屏障BL-4动物设施中。在2月龄时，将人胰腺癌细胞PANC-1腹膜内注射到雄性小鼠中。肿瘤细胞接种后2个月，小鼠尾静脉注射107腺病毒颗粒。基因递送后，在不同时间点处死小鼠以确定转基因表达水平。进行完整尸检。形态学改变进行了测定，使用苏木精和伊红（H&E）染色，和分布的报告lacZ基因的免疫组化analysis.ResultsAdenoviral-driven的hepatitis-gene的表达导致了5倍以上的转基因表达相比，传统的质粒转染。在Adeno-RIP-lacZ注射小鼠中，在胰岛中特异性检测到lacZ表达。相反，在Adeno-CMV-lacZ注射小鼠中，在多个器官和组织中观察到lacZ基因表达。Adeno-CMV-lacZ处理组小鼠出现单核细胞浸润和肝细胞炎症。在Adeno-RIP-lacZ处理的小鼠的胰岛细胞中观察到类似的现象。一个显着更高的水平，也发现在体内接种的人胰腺tumors.ConclusionThese结果表明，RIP定向的胰腺癌基因表达的边缘被发现特异性在小鼠胰岛和植入的人胰腺癌细胞。因此，这些数据表明，腺病毒载体和组织特异性启动子的组合可以导致体内增强的和更特异的转基因表达。
BackgroundThe objective of this study was to determine whether rat insulin promoter (RIP) could, in a mouse model, direct expression of an adenovirus-mediated reporter gene specifically into pancreatic islets via systemic delivery.MethodsFive hundred and eight base pairs of the RIP DNA sequence were constructed into an adenoviral vector containing a lacZ reporter gene (Adeno-RIP-lacZ). The cytomegalovirus (CMV) promoter was constructed to drive lacZ reporter-gene expression (Adeno-CMV-lacZ) and used as controls. In vitro transient transfection assays were performed to determine levels of reporter-gene expression and compared with that of liposome-mediated plasmid transfection. SCID mice were bred and housed in the barrier BL-4 animal facility. At 2 months of age, the human pancreatic cancer cell PANC-1 was intraperitoneally injected into male mice. Two months after the tumor cell inoculation, mice were injected with 107adenoviral particles via tail veins. After gene delivery, mice were sacrificed at different time points to determine transgene expression levels. Complete necropsies were performed. Morphological alterations were determined using hematoxylin and eosin (H&E) staining, and distribution of the reporter lacZ gene was determined by immunohistochemistry analyses.ResultsAdenoviral-driven reporter-gene expression resulted in more than 5 times higher transgene expression compared with conventional plasmid transfections. In Adeno-RIP-lacZ-injected mice, lacZ expression was specifically detected in pancreatic islets. By contrast, in Adeno-CMV-lacZ-injected mice, lacZ gene expression was observed in multiple organs and tissues. Mononuclear cell infiltration and liver cell inflammation were found in Adeno-CMV-lacZ-treated mice. Similar phenomena were observed in islet cells of Adeno-RIP-lacZ-treated mice. A significantly higher level of reporter-gene expression was also found at the edge of in-vivo-inoculated human pancreatic tumors.ConclusionThese results demonstrate that RIP-directed reporter-gene expression was found specifically in mouse pancreatic islets and implanted human pancreatic cancer cells. These data thus demonstrate that the combination of an adenoviral vector and a tissue-specific promoter could lead to an enhanced and more specific transgene expression in vivo.