Generierung intestinaler Zelllinien: In vitro Studien zur mechanischen Aktivierung beim postoperativen Ileus

Abstract

Introduction: The mechanical trauma is an iatrogenic consequence of abdominal surgery that results in a massive inflammation of the entire bowel wall, subsequently leading to POI [1]. Former studies of our group demonstrate that a mechanical trauma in vitro results in a opposite proinflammatory activation of primary cultures of resident muscularis macrophages and smooth muscle cells (SMC) [2]. A major disadvantage of these primary cell cultures is the absence or limitation of proliferative capacity in macrophage or SMC cultures, respectively and the differentiation of SMC to myofibroblasts. Therefore, the aim of this study is to generate conditionally immortalized cell lines of both, macrophages and SMC. Methods: Two different expression vectors, encoding the large-T antigen of the SV40 virus or the v-myc oncogene of the ARK Virus were delivered by electroporation into primary macrophages and SMC. The integration of a 72bp enhancer sequence of the SV40 virus should enhance plasmids targeting to the nucleus. The conditional gene expression of the oncogenes is regulated by a tetracycline dependent promoter. Transfected cells were enriched by geneticin selection and analyzed after several passages in culture for the expression of cell specific differentiation markers. Results: The transfection of primary SMC is much more efficient compared to macrophages. Large-T transgenic SMC (LT-SMC) also showed after several months in culture the expression of α-actin whereas normal SMC cultures lost this specific marker during propagation. Furthermore, LT-SMC retained the fusiform shape that is also lost by normal SMC. In macrophage cultures, the nucleofection does not result in the cellular immortalization. Conclusion: Our results demonstrate for the first time that intestinal SMC can be immortalized by large-T antigen expression. Thereby, the LT-SMC retained the phenotype of normal intestinal SMC for several months. Resident muscularis macrophages transfection by electroporation is insufficient. The macrophage approach will be repeated by microinjection of the v-myc plasmid into the nucleus of these cells. The generation of both cell lines is essential for the investigation of cell- and organ-specific intestinal dysfunctions, subsequently identifying molecular targets to prevent postoperative ileus.