In vivo and in vitro gene transfer and expression in rat intestinal epithelial cells by E1-deleted adenoviral vector.
In vivo and in vitro gene transfer and expression in rat intestinal epithelial cells by E1-deleted adenoviral vector.
复制标题
E1缺失腺病毒载体在大鼠肠上皮细胞中的体内和体外基因转移和表达。
DOI：
10.1089/hum.1997.8.6-755
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Noel,RA
中科院分区：
文献类型：
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作者：
Cheng,DY;Kolls,JK;Lei,D;Noel,RA
由于肠腺中存在大量的增殖组织和干细胞，肠被认为是体细胞基因治疗的一个有吸引力的靶点。以前的研究表明，使用逆转录病毒载体的报告基因，细菌β-半乳糖苷酶（β-Gal），可以转移和表达在小肠上皮细胞。然而，转导效率在大鼠和小鼠肠道中相对较低。在本研究中，我们采用E1缺失的腺病毒载体（其编码β-Gal基因）来研究基因转移到大鼠小肠上皮细胞系和雄性Sprague-Dawley大鼠小肠中的可行性。在体外研究中，采用X-Gal组织化学和荧光报告基因分析法定量测定AdCMVβgal在IEC-6和IEC-18细胞培养物中的表达。结果表明，AdCMVβgal能有效地转入肠上皮细胞系，且转基因表达具有病毒浓度依赖性。AdCMVβgal主要表达于肠道上皮细胞。在十二指肠、空肠、回肠以及较小程度的结肠中观察到转基因的转导效率。此外，在单次或二次施用重组腺病毒后，AdCMVβgal在肠道中的有效表达在3d达到高峰，并在7和14 d下降。用该病毒单次或二次攻击后，血清中未检测到抗腺病毒抗体应答。这些发现表明，E1缺失的腺病毒载体，当通过口腔-十二指肠管给药时，与大肠相比，在小肠的肠上皮中更成功地转移遗传物质。用腺病毒载体进行单次或二次攻击不会引起宿主对该病毒的免疫应答增强。这表明，通过重复施用腺病毒载体的成功基因转导使其成为肠道疾病和代谢缺陷的基因治疗应用的替代候选者。
The intestine is proposed to be an attractive target site for somatic gene therapy due to a large mass of proliferating tissue and stem cells in the crypts. Previous studies using a retroviral vector have shown that a reporter gene, bacterialβ-galactosidase (β-Gal), can be transferred and expressed in the small intestinal epithelial cell. However, transduction efficiency is relatively low in rat and mice intestines. In the present study, we employed an E1-deleted adenoviral vector (which encodes theβ-Gal gene) to investigate the feasibility of gene transfer into rat small intestinal epithelial cell lines and small intestines in male Sprague-Dawley rats. Inin vitrostudies, expression of AdCMVβgal was quantitatively measured in IEC-6 and IEC-18 cell cultures using X-Gal histochemistry and chemiluminescent reporter gene assays. The results indicate that AdCMVβgal can be efficiently transferred into intestinal epithelial cell lines and transgene expression is virus concentration dependent Inin vivostudies, a 5F intestinal feeding tube was used to deliver the vector to the duodenal segment of the rat. Expression of AdCMVβgal was primarily localized to the epithelium of the intestinal tract. Transduction efficiency of the transgene was seen in the duodenum, jejunum, ileum, and, to a lesser extent, the colon. Moreover, following a single or secondary administration of recombinant adenovirus, efficient expression of AdCMVβgal in the intestinal tract peaked at 3 days and decreased by 7 and 14 days. No antiadenoviral antibody response was detected in the serum after a single or secondary challenge with this virus. These findings demonstrate that an E1-deleted adenoviral vector, when administered through an oral-duodenal tube, transfers genetic material more successfully in the intestinal epithelium in the small intestine when compared to the large intestine. A single or secondary challenge with adenoviral vector does not cause enhanced host immune responses to this virus. It suggests that successful gene transduction by the repeat administration of the adenoviral vector makes it an alternative candidate for gene therapy applications in intestinal diseases and metabolic deficiencies.