BACKGROUND:There is a high morbidity after spinal cord injury, and the therapeutic strategy is limited to early surgical intervention, medication and post-treatment exercise that only can improve the motor function slightly. However, there is no effective cure method. OBJECTIVE:To study the effect of partition-type spinal cord catheter combined with bone marrow stromal stem cels on T8 spinal cord transection damage in rats. METHODS:Fifty rats were randomized into five groups (n=10 per group): group I, T8 spinal cord transection (5 mm) was made in rats with no treatment; group II, the partition-type tube was inserted into the injured site after modeling; group III, partition-type tube combined with bone marrow stromal stem cels was implanted into the injured site after modeling; group IV, partition-type tube combined with polyglycolic acid fibers was implanted into the injured site after modeling; group V, partition-type tube combined with bone marrow stromal stem cels and polyglycolic acid fibers was implanted into the injured site after modeling. RESULTS AND CONCLUSION:At 2 and 12 weeks postoperatively, Basso, Beattie and Bresnahan scores were significantly higher in the groups III and IV than the groups I, II, IV (P < 0.05). At 12 weeks postoperatively, the latency of motor evoked potential below the injury plane was significantly decreased in group V compared with groups I, II, III, IV (P < 0.05). Immunohistochemical results displayed that in the groups III and V, regenerated nerve fibers grew positively and arranged orderly among the tubes, and there was no obvious winding phenomenon. Under transmission electron microscopy, a certain number of myelinated nerve fibers were found as bridges among groups. These findings indicate that the partition-type chitosan tube combined with bone marrow stromal stem cels has a good connection with the injured spinal cord a good connection to restore part of electrophysiological properties, accelerate the axon regeneration, recover the motor function, thereby providing a new direction for the treatment of spinal cord injury. Cite this article:Zhao XW, Liu X, Yu DP, Rong H, Yu XS, Yang CS, Liu T, Zhao TB. Partition-type spinal cord catheter combined with bone marrow stromal stem cels in the repair of spinal cord transection injury in rats. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):42-48.

IFN-λ1 is a member of a new family of interferons called type Ⅲ IFNs with similar functions to type ⅠIFNs. Previously we obtained recombinant soluble human rhIFN-λ1 from Pichia pastoris. However, the hyper-glycosylation from P. pastoris brings immunogenicity and low purification recovery rate. To overcome this disadvantage, in this study, we constructed an rhIFN-λ1 mutant (rhIFN-λ1-Nm) devoid of the potential N-glycosylation sites by site-directed mutagenesis. rhIFN-λ1-Nm was successfully expressed and secreted extracellularly in P. pastoris (GS115) using methanol inducible AOX1 promoter with α-mating factor signal sequence. rhIFN-λ1-Nm was purified and characterized. There was no significant difference between rhIFN-λ1-Nm and rhIFN-λ1 in structure and bioactivity. The molecular weight was low after N-glycosylation mutation whereas the glycosylation was much lower. The mutational rhIFN-λ1 (rhIFN-λ1-Nm) could legitimately be developed as substitutes for rhIFN-λ1, and thus it may be developed into a more promising therapeutic reagent in the future.

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.
Animals ; Antiviral Agents ; Humans ; Interferon alpha-2 ; metabolism ; Interferon-alpha ; biosynthesis ; pharmacokinetics ; Polyethylene Glycols ; pharmacokinetics ; Protein Structure, Secondary ; Rats ; Recombinant Proteins ; biosynthesis ; pharmacokinetics ; pharmacology ; Reproducibility of Results ; Transglutaminases ; metabolism