The use of efficient and inexpensive substrates (2D matrices) for cultivation and differentiation of nerve cells in vitro is important for the creation of tissue engineering constructs intended for the treatment of nervous system pathologies. Recombinant analogues of the orb-weaver spider dragline-silk proteins spidroins 1 and 2 appear promising in addressing this task. The aim of the study was to evaluate the effect of cell substrates derived from mixtures of recombinant spidroins (RS) rS1/9 and rS2/12 with hybrid proteins (HP) containing rS1/9 monomer fused with biologically active peptides on gene expression levels of key synapse-specific proteins and viability of the human neuroblastoma SH-SY5Y cell line during directed cholinergic differentiation. A two-stage scheme of directed cholinergic differentiation of SH-SY5Y cells using retinoic acid and brain-derived neurotrophic factor (BDNF) was implemented. Cell viability was assessed via MTT assay and crystal violet staining. The mRNA levels of the studied genes were assessed by real-time PCR. Directed differentiation of the SH-SY5Y cells was marked by a significant increase in the gene expression levels of synaptophysin, synapsins I and II, and the postsynaptic protein PSD-95. The highest cell viability and increased PSD-95 expression levels were observed during differentiation on a matrix consisting of RS rS1/9 and rS2/12 mixed with the RGDS peptide (present in extracellular matrix proteins) and heparin-binding peptide (HBP, laminin fragment) containing HPs. The highest efficiency during the differentiation of the SH-SY5Y cells was demonstrated by a matrix consisting of the mixture of RS rS1/9 and rS2/12 and a HP made up by RS rS1/9 monomer fused with RGDS (the ligand of integrins) and HBP (the ligand of growth factors and syndecans). Matrices consisting of RS rS2/12 alone or the mixture of rS2/12 with HP(RGDS) showed lower efficiency, although the use of the GRGGL peptide (which interacts with the neural cell adhesion molecules and is a component of RS rS1/9) led to an increase in efficiency.

使用高效且廉价的基质（2D 基质）在体外培养和分化神经细胞对于创建用于治疗神经系统病理的组织工程结构非常重要。圆织蜘蛛拖丝蛋白 spidroins 1 和 2 的重组类似物似乎有望解决这一任务。本研究的目的是评估源自重组蛛丝蛋白 (RS) rS1/9 和 rS2/12 的混合物以及含有与生物活性肽融合的 rS1/9 单体的杂合蛋白 (HP) 的细胞底物对基因表达水平的影响人神经母细胞瘤 SH-SY5Y 细胞系在定向胆碱能分化过程中的关键突触特异性蛋白和活力。使用视黄酸和脑源性神经营养因子（BDNF）实施 SH-SY5Y 细胞定向胆碱能分化的两阶段方案。通过MTT测定和结晶紫染色评估细胞活力。通过实时 PCR 评估所研究基因的 mRNA 水平。SH-SY5Y 细胞定向分化的特点是突触素、突触素 I 和 II 以及突触后蛋白 PSD-95 的基因表达水平显着增加。在由 RS rS1/9 和 rS2/12 与 RGDS 肽（存在于细胞外基质蛋白中）和肝素结合肽（HBP、层粘连蛋白片段）混合组成的基质上分化期间，观察到最高的细胞活力和增加的 PSD-95 表达水平）含有HP。由 RS rS1/9 和 rS2/12 混合物组成的基质以及由 RS rS1/9 单体与 RGDS（整合素配体）融合组成的 HP 证明了 SH-SY5Y 细胞分化过程中的最高效率和HBP（生长因子和多配体的配体）。尽管使用了 GRGGL 肽（与神经细胞粘附分子相互作用，并且是 RS rS1/9 的组成部分），但仅由 RS rS2/12 或 rS2/12 与 HP(RGDS) 的混合物组成的基质显示出较低的效率导致效率的提高。