Localized delivery of brain-derived neurotrophic factor from PLGA microspheres promotes peripheral nerve regeneration in rats.

BACKGROUND: Repair of peripheral nerve defect presents a considerable challenge for reconstructive surgeons. The aim of this study is to develop a brain-derived neurotrophic factor (BDNF) from poly(D,L-lactide-co-glycolide) (PLGA) microspheres for the treatment of the peripheral nerve defect. METHOD: BDNF microspheres were prepared by using an oil-in-water emulsification-solvent evaporation method. The morphology, particle size, encapsulation efficiency, drug loading and sustained release performance of microspheres was observed and calculated. Adipose mesenchymal stem cells (ADSCs) were isolated and expanded. ADSCs were divided into four groups: control, BDNF, blank microsphere and BDNF microsphere groups. Cell count kit-8 (CCK-8) assays were used to assess cell proliferation. Cell migration was determined by Transwell assays. Twenty-eight male Sprague-Dawley rats underwent transection damage model on the right sciatic nerve. The wet weight ratio of the gastrocnemius muscle was calculated by comparing the weight of the gastrocnemius muscle from the operated side to that of the normal side. Neuroelectrophysiological testing was performed to assess nerve function recovery. Nerve regeneration was evaluated by histological analysis and immunohistochemical staining. RESULTS: The microspheres were spherical and had uniform size (46.38 ± 1.00 µm), high encapsulation efficiency and high loading capacity. In vitro release studies showed that BDNF-loaded microspheres had good sustained release characteristics. The duration of BDNF release was extended to more than 50 days. BDNF or BDNF microsphere promote the proliferation and migration of ADSCs than control group (P < 0.05). Compared with control group, BDNF significantly decreased the nerve conduction velocity (NCV) and compound amplitude (AMP) (P < 0.05). The nerve fibers in the BDNF microsphere group were closely arranged and uniformly distributed than control group. CONCLUSION: BDNF/PLGA sustained-release microsphere could promote the migration of ADSCs and promoted neural differentiation of ADSCs. Moreover, BDNF/PLGA sustained-release microsphere ameliorated nerve conduction velocity and prevented neuralgic amyotrophy.

Long noncoding RNA LINC00314 facilitates osteogenic differentiation of adipose-derived stem cells through the hsa-miR-129-5p/GRM5 axis via the Wnt signaling pathway.

BACKGROUND: Many studies have shown that long noncoding RNAs (lncRNAs) are closely related to the stimulation of osteogenic differentiation of adipose-derived stem cells (ADSCs) and the prevention of osteoporosis. Current research aimed to investigate the novel lncRNA and explored the function and molecular mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs. METHODS: LncRNA and miRNA sequencing was performed in normal and osteogenic differentiation-induced ADSCs (osteogenic group). Abnormally expressed lncRNAs and miRNAs were obtained by the R software and the relative expression of LINC00314, miR-129-5p, and GRM5 during osteogenic induction was measured by RT-PCR. ADSCs were then transfected with pcDNA3.1-sh-LINC00314 and agomiR-129-5p. Alizarin red staining (ARS) and alkaline phosphatase (ALP) staining were performed to identify the mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs. RESULTS: LINC00314 was significantly upregulated in the group of osteogenic-induced ADSCs. LINC00314 and GRM5 mimics increased the early and late markers of osteogenic differentiation, which manifest in not only the markedly increased ALP activity but also higher calcium deposition, while miR-129-5p mimic had the opposite effects. LINC00314 directly targeted miR-129-5p through luciferase reporter assay, and miR-129-5p suppressed GRM5 expression. Moreover, the LINC00314/miR-129-5p/GRM5 regulatory axis activated the Wnt/ß-catenin signaling pathway. CONCLUSIONS: LINC00314 confers contributory function in the osteogenic differentiation of ADSCs and thus the LINC00314/miR-129-5p/GRM5 axis may be a novel mechanism for osteogenic-related disease.

Development of a multi-mimotope peptide as a vaccine immunogen for infectious bursal disease virus.

To explore the mimotope vaccine approach against infectious bursal disease virus (IBDV), five IBDV-specific monoclonal antibodies (mAbs) were prepared and their binding peptides were screened against a phage-displayed 12-mer peptide library. After three rounds of biopanning, 12 phages were selected for each mAbs and their specificity to IBDV was verified by sandwich and competitive inhibition ELISAs. Seven phages per mAb were sequenced and their amino acid sequences were deduced. The five representative sequences of mimotopes corresponding mAbs were determined. An artificial gene, designated 5epis (5 epitopes) and consisting of the five mimotopes arranged in tandem (F1-F7-B34-2B1-2G8) with four GGGS spacers, was chemically synthesized and cloned into a prokaryotic expression plasmid pET28b. The protein, designated r5EPIS, was efficiently expressed in Escherichia coli and showed a size of 10kDa in SDS-PAGE. The r5EPIS protein reacted with anti-IBDV mAbs and polyclonal antibodies in Western blot immunoassays. Immunization of SPF chickens with r5EPIS protein (with Freund adjuvant, 50mug per injection on day 0 and 14) evoked high levels of antibody (12,800 by ELISA/1600 by virus neutralizing assay at day 21) and protected 100% of the chickens against a challenge of 200 ELD(50) of IBDV GX8/99 strain, which sharply contrasted with the, respectively, 13.3% and 6.6% survival rate in the adjuvant group and the untreated group. The multi-mimotope protein r5EPIS promises to be a novel subunit vaccine candidate for IBDV.