In vitro biocompatibility study of a water-rinsed biomimetic silk porous scaffold with olfactory ensheathing cells.

Olfactory ensheathing cells (OECs) are one of the most promising candidates for cell-based therapy in repairing spinal cord injury (SCI). However, the conventional method of transplanting OECs, orthotopically or intrathecally, leads to a low rate of cell replacement and limited utilization due to the impediments of chemical and physical barriers after SCI. In this study, we fabricated a three-dimensional (3-D) silk fibroin (SF) scaffold with a novel water-rinsing process. Compared to traditional methanol-treated scaffolds (MTS) and ethanol-treated scaffolds (ETS), the present water-rinsed scaffold (WRS) had a biomimetic nanofibrous structure and softer mechanical properties and was shown to be more biocompatible with OECs. The in vitro study showed that the 3-D SF scaffolds supported OEC adhesion and spreading and that the OECs had a normal cell phenotype. A quantitative study using the CCK8 assay revealed that OEC proliferation on the WRS was higher than that on the MTS and ETS. Moreover, a significantly higher level of GDNF was detected when the OECs were cultured on the WRS. Collectively, these results suggested that the 3-D biomimetic SF porous scaffolds not only provide suitable microenvironments for governing OEC behaviour but also serve as a promising transplantation strategy for OECs to repair SCI.

Immunogenicity of DNA vaccines encoding regulatory/accessory proteins derived from three different prevalent strains in China / 中华微生物学和免疫学杂志

0bjective To determine the immunogenicities of DNA vaccines expressing tat-rev-integrase(c-half)-vif-neffusion genes(TRIVN) derived from prevalent B', B'/C and AE recombinant subtypes of HIV-1 in China. Methods Two DNA vaccines were constructed by inserting the codon optimized tat-revintegrase(c-half)-vif-nef fusion genes derived from B' and B'/C subtype of HIV-1 into mammalian expression vector pSVI. 0. DNA vaccine containing tat-rev-integrase (c-half)-vif-nef fusion gene derived from HIV-1AE2f has been constructed previously. In vitro expression efficiencies of three DNA vaccines were determined by Western blot and their immunogenicities were compared by immunizing female BALB/c mice. IFN-γ ELISPOT assay was used to read out the specific T cell immunity. Results The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blot assay showed three constructed DNA vaccines could be expressed at a comparable level in vitro. After vaccination, AE-TRIVN mounted T cell immune responses at (948.0 ± 330.0) SFCs/106 splenocytes, followed by the mixed DNA vaccine[ (500.0 ± 155.0) SFCs/106 splenocytes ], RL-TRIVN r[ ( 195. 1 ± 44.0) SFCs/106 splenocytes ]and CN-TRIVN [ (89.5 ± 17.0) SFCs/106 splenocytes]. Interestingly, we observed that single DNA vaccination induced specific T cell responses predominantly targeting Integrase (C-half) and Vif, whereas the mixed DNA could significantly improve T cell responses against Nef. Conclusion AE-TRIVN was the most immunogenic among the three DNA vaccines and the mixed DNA vaccination could change the immunogenic hierarchy of T cell epitopes across the fusion genes vaccine.