Hashamdar Somayeh, Parvin Parviz, Ramezani Fatemeh, Ahmadinouri Fatemeh, Jafargholi Amir, Refahizadeh Mitra, Akbarpour Mahzad, Aghaei Mohammadreza, Heidari Omid
Physics Department, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran, Iran.
Physiology Research Center, Iran University of Medical Sciences, Tehran, Iran.
Biomed Opt Express. 2024 Jul 12;15(8):4655-4674. doi: 10.1364/BOE.530876. eCollection 2024 Aug 1.
The lack of regeneration of injured neurons in the central and peripheral neural system leads to the failure of damaged tissue repair in patients. While there is no definitive cure for most neurodegenerative diseases, new therapeutic methods that cause the proliferation and differentiation of neurons are of interest. Challenges such as the inability of neuronal cells to proliferate after injury, the lack of a stimulus for initial stimulation, and the presence of the microenvironment around CNS neurons contain several inhibitory factors that prevent neuron regeneration, thus, creating a structure similar to the extracellular matrix helps the cell proliferation in current treatment. A rapid method of neuron-like cell differentiation of PC12 cells is introduced here based on a novel synthetic scaffold. Initially, poly allyldiglycol carbonate (CR-39) substrate is textured under a high dose of ArF UV excimer laser (1000 shot, 300 mJ/pulse equivalent to 300 J/cm at 193 nm) to create superficial periodic parallel microchannels with the micrometer spacing and sub-micron width. Ultraviolet treated CR-39 (UT CR-39) provides a suitable scaffold to speed up the transformation/differentiation of PC12 cells. The latter is pheochromocytoma of the rat adrenal medulla as an embryonic origin from the neural crest usually exposed to the nerve growth factor (NGF). In fact, PC12 cells are seeded on the microchannels and simultaneously are stimulated by coherent red photons at 660 nm within the therapeutic window. The UT CR-39 scaffold undergoes extra improvement of ∼ 30% after 12 minutes of laser activation regarding the photo-biomodulation (PBM) mechanism. The cell activation due to the coherent photons also gives rise to enhanced proliferation/differentiation. Here, PC12 cells are efficiently differentiated into neurons according to immunocytochemistry (ICC) and Western Blot verification tests based on MAP2 and synapsin-1 protein expression. In general, UT CR-39 acts as a superior bed to elevate the population of neuron-like cells up to threefold against those of untreated (control)ones. We conclude that the surface cross-linking due to UV exposure and subsequent induced hydrophilicity notably contribute to the neuron-like cell differentiation of PC12 without adding NGF.
中枢和外周神经系统中受损神经元缺乏再生能力，导致患者受损组织修复失败。虽然大多数神经退行性疾病尚无确切的治愈方法，但能促使神经元增殖和分化的新治疗方法备受关注。诸如神经元细胞在损伤后无法增殖、缺乏初始刺激的诱因以及中枢神经系统神经元周围微环境中存在多种抑制神经元再生的抑制因子等挑战，因此，在当前治疗中创建类似于细胞外基质的结构有助于细胞增殖。本文基于一种新型合成支架，介绍了一种快速诱导PC12细胞向神经元样细胞分化的方法。首先，在高剂量的ArF紫外准分子激光（1000次脉冲，300 mJ/脉冲，相当于193 nm波长下的300 J/cm²）照射下，对聚二烯丙基碳酸酯（CR-39）基底进行纹理化处理，以创建具有微米级间距和亚微米级宽度的表面周期性平行微通道。经紫外线处理的CR-39（UT CR-39）提供了一个合适的支架，可加速PC12细胞的转化/分化。PC12细胞是大鼠肾上腺髓质嗜铬细胞瘤，起源于神经嵴的胚胎组织，通常会接触神经生长因子（NGF）。实际上，将PC12细胞接种在微通道上，并同时在治疗窗口内用660 nm的相干红光光子进行刺激。根据光生物调节（PBM）机制，UT CR-39支架在激光激活12分钟后会有额外约30%的改善。相干光子引起的细胞激活也会促进增殖/分化。在此，根据基于微管相关蛋白2（MAP2）和突触素-1蛋白表达的免疫细胞化学（ICC）和蛋白质免疫印迹验证试验，PC12细胞有效地分化为神经元。总体而言，与未处理（对照）的细胞相比，UT CR-39作为一个优质的基底，可使神经元样细胞的数量增加两倍。我们得出结论，紫外线照射导致的表面交联以及随后诱导的亲水性显著促进了PC12细胞在不添加NGF情况下向神经元样细胞的分化。
