The Effects of LPS With or Without Forskolin on RT4-D6P2T Cell Morphology

Schwann cells can be used as a model to study different signaling pathways involved during injury and inflammation in the peripheral nervous system. For this study, the immortalized rat RT4-D6P2T Schwannoma cell line from ATCC (#CRL-2768) was used. The cells were treated with different doses of lipopolysaccharide (LPS) with or without 2 uM of forskolin for 3 hours to determine the effects of LPS with or without forskolin on Schwann cell morphology. LPS is a cell wall immunostimulatory component found in Gram-negative bacteria. It activates the nuclear factor kappa B (NF-KB) pathway, which plays a major role in inflammation through the production of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). Forskolin is a root extract from the Indian plant Coleus forskohlii that activates the cAMP pathway, which plays a major role in cell proliferation, axonal regeneration, inflammation, and more. Although it is well-known that both the NF-KB and cAMP pathways are involved in inflammation, not much is known regarding the effects of NF-KB activation, with or without cAMP activation, on Schwann cell morphology. It was hypothesized that cells treated with LPS and forskolin would have significant morphological differences compared to cells treated with LPS only. Before and after a 3-hour incubation with the different treatment combinations, images of the cell cultures were taken at a magnification of 10X using a Zeiss Primovert iLED microscope equipped with Zeiss Axiocam 208 color microscope camera. The provided figure shows representative images of the cells from three independent experiments. Unexpectedly, the cells did not appear to have any obvious morphological changes after a 3-hour incubation with each of the treatments. However, there were large gaps on the surface of the culture flasks where the cells used to be. There is not enough information to determine what exactly caused these gaps. Some possibilities include apoptosis/cell death or cell retraction/migration. Further studies need to be done before conclusions can be made.雪旺细胞（Schwann cells）是研究外周神经系统损伤与炎症过程中相关信号通路的理想模型系统。本研究采用源自美国典型培养物保藏中心（ATCC，货号#CRL-2768）的永生化大鼠RT4-D6P2T雪旺细胞瘤细胞系开展实验。将细胞以不同剂量的脂多糖（LPS）处理，并分别联合或不联合2 μM福斯高林孵育3小时，以探究LPS单独给药与联合给药对雪旺细胞形态的影响。脂多糖为革兰氏阴性菌细胞壁所含的免疫刺激成分，可激活核因子κB（NF-κB）通路；该通路通过促炎细胞因子（如肿瘤坏死因子α（TNF-α））的产生介导炎症反应，发挥核心调控作用。福斯高林是印度植物毛喉鞘蕊花的根提取物，可激活环磷酸腺苷（cAMP）通路，该通路在细胞增殖、轴突再生、炎症调控等多种生理病理过程中均发挥关键作用。尽管学界已明确核因子κB与环磷酸腺苷通路均参与炎症调控，但目前针对单独或联合激活这两条通路对雪旺细胞形态的影响，相关研究仍较为匮乏。本研究提出科学假说：与仅接受LPS处理的细胞相比，经LPS与福斯高林联合处理的细胞将呈现显著的形态学差异。 在采用不同处理组合孵育3小时前后，使用搭载蔡司Axiocam 208彩色显微相机的蔡司Primovert iLED显微镜，以10倍放大倍率对细胞培养物进行成像。本数据集所附图像为三次独立重复实验的代表性细胞成像结果。 出乎意料的是，经各处理组孵育3小时后，细胞未观察到明显的形态学变化。但培养瓶表面出现大片原本存在细胞的区域空缺。目前尚无足够数据明确该空缺的具体成因，潜在可能性包括细胞凋亡/死亡或细胞回缩/迁移。需开展进一步研究后方可得出可靠结论。