Fifty whitespotted bamboo sharks (Chiloscyllium playgiosum) of both sexes were used to establish a large capacity variable domain of the new antigen receptor (VNAR) library with a total capacity of over 10⁹ colony-forming units (CFU). It was applied to screen VNARs against human serum albumin (HSA) and human transcription factor EB (TFEB), respectively. Meanwhile, VNAR libraries specific to HSA and TFEB with capacities above 10⁸ CFU were obtained following conventional immunization. These two approaches were systematically studied in terms of VNAR yield and composition. By comparing the VNAR sequences obtained from naïve and antigen-immunized libraries, we found that the complementary-determining region 3 (CDR3) of the former differs in composition from that of the latter. It shares a higher degree of homology with the naïve library. Meanwhile, the binding efficiency assessed by ELISA is also different between the naïve and antigen-immunized libraries. The binding of VNARs from the TFEB-immunized library appeared to surpass that observed with the naïve libraries, whereas the performance of VNARs from the HSA-immunized library indicated that both the immunized and naïve libraries for HSA had positive binding responses in polyclonal and monoclonal ELISA. The results are useful to develop novel diagnostic and therapeutic products based on shark VNARs.

Objective:To study the relationship between zoledronic acid (ZOL) and vascular endothelial growth factor (VEGF) conformation so as to reveal the mechanism of bisphosphonates inhibiting angiogenesis.Methods:The binding structures of ZOL and VEGF were preprocessed and the molecular dockings were simulated through AutoDockTools, Discovery studio4 and AutoDockVina. The best binding conformation was accurately screened. The effects of various concentrations of ZOL (group A was 0 μmol/L, groups B, C and D were 25, 50 and 100 μmol/L, respectively) on human umbilical vein endothelial cell (HUVEC) proliferation, angiogenesis and angiogenic molecules were detected by using cell counting kit-8 (CCK-8) in vivo and in vitro angiogenesis, immunofluorescence and Western blotting. Results:There was a ZOL binding site on the target protein VEGF conformation. The affinity was -5.2 kcal/mol. This binding site consisted of the hydrophobic region composed of amino acids Cys26, 51, 57, etc. and the hydrogen bond binding region of the A chain (ASP34, SER50) and B chain (CYS61, 68, LEU66, GLY59). The results of CCK-8 showed that the levels of value A in groups B, C and D were significantly lower than that in group A at each time point from 3 to 6 days ( P<0.05). In vitro vascular experiments demonstrated that the numbers of budding in groups B, C and D [(208±28), (151±21) and (62±9), respectively] were significantly lower than that in group A (276±30) ( P<0.05). In vivo vascular experiments displayed that the ratio of Matrigel gel/plasma fluorescence in group A (0.003 1±0.000 3) was significantly higher than those in group B (0.002 1±0.000 2), group C (0.001 6±0.000 2) and group D (0.000 6±0.000 1) ( P<0.05). The results of Western blotting revealed that the expression of VEGF in groups B, C and D [(0.72±0.11), (0.41±0.07) and (0.24±0.04), respectively] were significantly lower than that in group A (1.01±0.02) ( P<0.05), and the expression levels of hypoxia-inducible factor-1α (HIF-1α) in groups B, C and D [(0.68±0.09), (0.55±0.06) and (0.43±0.08), respectively] were significantly lower than that in group A (0.96±0.04) ( P<0.05). Conclusions:ZOL could inhibit cell proliferation, in vivo and in vitro vascularization and expression of VEGF/HIF-1α. The binding site of ZOL with the conformation of VEGF was located in the hydrophobic region and hydrogen-bonding region of amino acids. Designing an antagonist targeting this site might potentially alleviate the effect of ZOL in inhibiting angiogenesis.

BACKGROUND: Traditional solid bone can not receive satisfied effects in repairing irregular bone defects in oral maxillofacial surgery due to uneven distribution of cells and growth factors. Therefore, it is a research direction to prepare injectable tissue engineering bone.OBJECTIVE: To explore the effects of chitosan/β-tricalcium phosphate/recombinant human bone morphogenetic protein-2(CS/β-TCP/rhBMP-2) composite on mandibular defect repair.DESIGN, TIME AND SETTING: A randomized controlled animal experiment. The experiment was performed atthe animal laboratory, Medical College of Jinnan University from May 2008 to March 2009.MATERIALS: The injectable tissue engineering bone was prepared by using complex of liquid CS and solid β-TCP as scaffold materials, and combined with freeze-dried rhBMP-2.METHODS: Twenty-four New Zealand white rabbits were prepared for double sides mandibular defect models, and randomized into 4 groups: ①CS/p-TCP/rhBMP-2 group: 1 mL CS/β-TCP/rhBMP-2 complex was injected into the defects.②CS/β-TCP group:0.5 mL CS/β-TCP complex was injected into defects. ③Autograft bone group: repairing the defects with sclerotin of the iliac crest.④Blank control group: no implantation. MAIN OUTCOME MEASURES: At weeks 2, 4 and 8 after surgery, the material degradation and new bone formation were evaluated with, haematoxylin-eosin staining, and electron microscope; the bone mineral density was detected by dual energy X-ray absorptiometry (DXA) to determine bone formation rate and quality.RESULTS: ①Gross observation demonstrated that the size and thickness of osteotylus in CS/β-TCP/rhBMP-2 group was equivalent with the autograft group, which were greater than that of the other groups.②Histologicalobservation demonstrated that there were more bone matrixes in the CS/β-TCP/rhBMP-2 group and autograft group than that in the CS/β-TCP group and blank control group at each time points. ③Scanning electron microscope image suggested that at 8 weeks after operation, the bone bed and the materials in CS/β-TCP/rhBMP-2 group were connected with bone, and the gap was diminished. The degradation of the materials was so obvious that the complete structure of materials could not be found. ⑤DXA detection appealed that the bone density of each group was gradually increased with time prolonged. The quantities of bone density in CS/β-TCP/rhBMP-2 group in weeks 2, 4 and 8 were significantly higher than CS/β-TCP group and blankcontrol group (P < 0.05).CONCLUSION: ①CS/β-TCP/rhBMP-2 has good biocompatibility, degradability and the capacity of bone guidance and bone induction. ②CS/β-TCP can be served as a promising carrier for BMP-2, which is a potential degradable biological material for repairing bone defects.