AIM
The use of efficient and inexpensive coatings (2D matrices) for the cultivation and differentiation of nerve cells in vitro is important for the development of tissue-engineered constructs intended for the treatment of pathologies of the nervous system. Promising for such constructs are recombinant analogs of the dragline silk fiber of the orb-weaving spiders web, namely, spidroins 1 and 2. The aim of the study was to evaluate the effects of coatings obtained from mixtures of recombinant spidroins (RSs) rS1/9 and rS2/12 with hybrid proteins (HPs) containing the rS1/9 monomer fused with biologically active peptides on the levels of expression of key synapse-specific genes and viability of SH-SY5Y human neuroblastoma cells in directed cholinergic differentiation.
MATERIAL AND METHODS
A two-stage scheme of directed cholinergic differentiation using retinoic acid and brain-derived neurotrophic factor (BDNF) was applied. Cell viability was assessed using the MTT test and crystal violet staining. The expression levels of the studied genes were assessed using real-time PCR.
RESULTS
Directed differentiation of SH-SY5Y cells was accompanied by a significant increase in the expression levels of the genes of synaptophysin, synapsins I, II, and the postsynaptic protein PSD-95. The highest cell viability and increased levels of PSD-95 expression were found during differentiation on a coating consisting of a mixture of RSs rS1/9 and rS2/12 with HPs containing the RGDS peptide (present in extracellular matrix proteins) and the heparin-binding peptide HBP (a fragment of laminin).
CONCLUSION
The coating consisting of a mixture of RSs rS1/9 and rS2/12 and HP based on the RS monomer rS1/9 fused with RGDS (a ligand for integrins) and HBP (a ligand for growth factors and syndecans) showed the highest efficiency in directed neuronal differentiation of SH-SY5Y cells. Coatings consisting of rS2/12 alone or a mixture of rS2/12 with HP(RGDS) demonstrated less efficiency, but the fusion with the GRGGL peptide (which interacts with NCAMs and is a fragment of rS1/9) resulted in an increase in efficiency.