Department of Chemistry, Centre of Nanotechnology and Tissue Engineering-Photobiology and Photomedicine Research Group, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14040-901, Brazil.
Centre for Cell Therapy and Regional Blood Centre, National Institute of Science and Technology in Stem Cell and Cell Therapy, Medical School, University of São Paulo, Ribeirão Preto, 14051-140, Brazil.
Stem Cell Rev Rep. 2018 Aug;14(4):585-598. doi: 10.1007/s12015-017-9796-3.
The purpose of the present study was to evaluate the neural protein expression pattern of human multipotent mesenchymal stromal cells (hMSCs) treated with forskolin (free-form/FF). The study investigated forskolin's capacity to enhance intracellular levels of cyclic adenosine monophosphate (cAMP) by activating adenylate cyclase and probably by inducing neuron-like cells in vitro. In addition, because nanotechnology is a growing field of tissue engineering, we also assessed the action of a new system called the nanostructured-forskolin (NF) to examine the improvement of drug delivery. Afterwards, the cells were submitted to low-level laser irradiation to evaluate possible photobiostimulatory effects. Investigations using the immunofluorescence by confocal microscopy and Western blot methods revealed the expression of the neuronal marker β-tubulin III. Fluorescence intensity quantification analysis using INCell Analyzer System for β-tubulin III was used to examine significant differences. The results showed that after low-level laser irradiation exposure, there was a tendency to increase the β-tubulin III expression in all groups, as expected in the photobiostimulation process. Notably, this process induced for irradiation was more pronounced in irradiated nanoforskolin cells (INF) compared to non-irradiated free-forskolin control cells (NFFC). However, there was also an increase in β-tubulin III protein expression in the groups: irradiated nanocontrol cells (INC) compared to non-irradiated free-forskolin control cells (NFF) and after treatment with non-irradiated free-forskolin (NFF) and non-irradiated nanoforskolin (NNFC). We concluded that the methods using low-level laser irradiation and/or nanoparticles showed an up-regulation of neural-protein expression in hMSCs that could be used to facilitate cellular therapy protocols in the near future.
本研究旨在评估用福司可林（游离型/FF）处理后的人多能间充质基质细胞（hMSCs）的神经蛋白表达模式。该研究调查了福司可林通过激活腺苷酸环化酶并可能在体外诱导神经元样细胞来增强环磷酸腺苷（cAMP）细胞内水平的能力。此外，由于纳米技术是组织工程的一个新兴领域，我们还评估了一种名为纳米结构福司可林（NF）的新系统的作用，以研究药物输送的改善。之后，将细胞进行低水平激光照射，以评估可能的光生物刺激作用。通过共聚焦显微镜免疫荧光和 Western blot 方法的研究揭示了神经元标志物 β-微管蛋白 III 的表达。使用 INCell Analyzer 系统对 β-微管蛋白 III 的荧光强度定量分析用于检查显著差异。结果表明，在低水平激光照射暴露后，所有组β-微管蛋白 III 的表达都有增加的趋势，这在光生物刺激过程中是预期的。值得注意的是，与未照射游离福司可林对照细胞（NFFC）相比，照射诱导的纳米福司可林细胞（INF）中该过程更为明显。然而，在照射纳米对照细胞（INC）与未照射游离福司可林对照细胞（NFF）和未照射游离福司可林（NFF）与未照射纳米福司可林（NNFC）的组中也观察到 β-微管蛋白 III 蛋白表达增加。我们得出结论，使用低水平激光照射和/或纳米颗粒的方法显示出 hMSCs 中神经蛋白表达的上调，这可能有助于在不久的将来促进细胞治疗方案。
