BACKGROUND:Periosteal osteoblasts possess strong reproductive activity,as well as osteoblastic differentiation potential,which is an ideal seed cell if can shorten the culture time.OBJECTIVE:Modified enzymatic digestion was used to culture rabbits'osteoblasts.and to study the adherence and proliferation of osteoblasts on the sudace of sandblasting titanium.MEITHODS:Periostea were harvested from the theanteromedial surface of the proximal tibia of male,Japanese white rabbits,and cultured as follow:①Routine method:Digested with 0.25%trypsinase at 37 ℃ for 30 minutes,followed by digestion with 0.1%type I collagenase at 37 ℃ for 30 minutes,vibration.removed trypsinase and dried.After 2 hours,DMEM containing 15% fetal bovine serums were added.②Modified method:30 minutes culture of type I collagenase was prolonged to 1 hour.The osteoblasts were identified by alkaline phosphatase staining and calcium node staining.The adherence and proliferation of osteoblasts cultured on sandblasting surface were measured by scanning electron microscopy and MTT.RESULTS AND CONCLUSION:Five days after culture.the periosteal steoblasts crawled out from tissues,gathered as monolayer with tdangle or polygon at after 25 days of modified culture.After 1 month of culture,superposition growth of calcium nodus appeared.The cultured cells possessed the morphological characteristic and biological behavior of osteoblasts.which were positive to alkaline phosphorase and calcium node staining.The time of cells cultured with routine method covered flask delayed 12 days than modified method.The osteoblasts were inseted into sandblasting titatium with pseudopodium.However,the adherence and proliferation of osteoblasts cultured on sandblasting surface had no obviously difference between two culture methods.The results suggested that modified enzymatic digestion can sho～en the culture time without effect on adherence and proliferation of osteoblasts.

Objective The aim of this study was to prepare anti-snail polyclonal antibody and make it widely useful in snail detection. Methods The DNA fragment encoding human full length 264 amino acid of snail was obtained by PCR from cDNA library of human umbilical vein epithelial cells and was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST).The GST-snail fusion protein was expressed by E.coli BL21 after IPTG induction and purified from total proteins of BL21 transformed by the recombinant plasmid pGEX-4T-1/snail.The New Zealand rabbit was immunized with the purified fusion protein to prepare polyclonal antiserum.The antiserum was identified by western blotting and immunofluorescent staining. Results The prokaryotic expression plasmid pGEX-4T-1/ snail was successfully constructed,and the fusion protein GST-snail was expressed efficiently.The polyclonal antibody raised in the rabbit could react specifically with snail in human cells.Conclusion The snail antiserum was of good purity with high titer and specificity which could satisfy the requirement for studying immuno-analysis on snail protein.

OBJECTIVE:To study the chemical constituents of the ethyl acetate extraction portion of Polygonum bistorta,pro-vide scientific basis for the basic research of its effective substances. METHODS:Taking the ethyl acetate extraction portion of P. bistorta 95％ ethanol extract,positive phase and reversed phase column chromatography were used for repeated isolation and purifi-cation,and the structures of the purified compounds were identified on the basis of physicochemical properties or spectral data. RE-SULTS:11 monomer compounds were isolated and identified as β-sitosterol(1),dihydromyricetin(2),quercetin(3),kaempferol (4),epicatechin(5),chlorogenic acid(6),rutin(7),gallic acid(8),1,2,3,4-tetrahydro-8-hydroxy-4-isopropyl-1-methylnaph-thalene-6-carboxylic acid (9),protocatechuic acid (10) and ellagic acid (11). CONCLUSIONS:The literature is confirmed that compound 2 is isolated from P. bistorta for the first time and compound 9 is isolated from Polygonum for the first time. The related research results have provided experimental references for further clarifying the effective substances of P. bistorta.

BACKGROUND: Autologous bone, bone substitute materials and guided bone regeneration (GBR) technique can repair jaw defects, but the absorption speed of bone substitute materials and GBR membrane are faster than the formation speed of new bone, therefore, it affects the volume and shape of new bone.OBJECTIVE: To investigate the role of personalized prefabricated titanium template, autologous bone and nano-hydroxyapatite on restoration of maxillary defect in rabbit.METHODS: A total of 18 rabbits were randomly divided into two groups, and maxillary alveolar defect with 10 mm length and 5 mm high was created. The template was implanted in both two groups, and fastened with titanium screws. Autologous and nano-hydroxyapatite were placed into the defect in experimental group; neither autologous bone nor bone substitute materials were implanted into the defect in control group. New bone formation, X-ray findings, and histological changes with HE stain were carded out 4, 8 and 12 weeks postoperatively.RESULTS AND CONCLUSION: The quality of new bone in experimental group was batter than that in control group 4 weeks postoperatively, but the quality of new bone was almost the same 8 and 12 weeks postoperatively. By paired t-test, there was significant difference in new bone density between the experimental group and the control group 4 .weeks after operation (P<0.01), but there was no significant difference in new bone density between the experimental group and the control group 8 and 12 weeks after operation (P > 0.05). Autologous bone and nano-hydroxyapatite can restore the defect of maxillary alveola.Personalized prefabricated titanium template can play an important role of screen membrane and external scaffold in new bone formation, and remain shape of new bone.

Jervine, a novel steroidal alkaloid from Veratrum nigrum L., exhibits both antitumor effect and potential toxicity. The aim of study was to characterize the pharmacokinetic behaviors and enterohepatic circu-lation of jervine in rats. A rapid and simple ultra-high performance liquid chromatography-tandem mass spectrometric method was developed and validated for quantification of jervine and alpinetin (internal standard) in rat plasma. After extraction from rat plasma by a simple protein-precipitation method, the analyte was separated on a C18 column (2.1 mm × 50 mm, 1.7μm) using water with 0.1％formic acid and acetonitrile as the mobile phase delivered at a flow rate of 0.4 mL/min. Jervine and alpinetin were determined in the positive mode with multiple reaction monitoring (MRM) of the ion transitions at m/z 426.3→108.8 and m/z 271.0→166.9, respectively. Molecular docking method was used to investigate the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The method was well validated within acceptance limits including specificity, matrix effect, recovery, precision, accuracy, and stability, and was successfully applied to the pharmacokinetic study of jervine after oral and intravenous administration to rats. Jervine presented a small volume of distribution, fast absorption, high oral bioavailability, and enterohepatic circulation. The enterohepatic circulation was first observed in veratrum alkaloids, and was further investigated by molecular docking studies, which was related to the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The pharmacokinetic properties and enterohepatic circulation of jervine in rats provided a significant basis for the drug-drug interaction and toxicity study in the future.

Objective To investigate the biocompatibility of Ti-6Al-4V scaffolds fabricated by electron beam melting(EBM).Methods Bone marrow mesenchymal stem cells(BMSC) co-cultured with Ti-6A1-4V specimens fabricated with EBM was prepared as experimental group and the regular cells culture was employed as control.The biocompatibility was detected using CCK-8 and cytoskeleton staining.The osteogenic differentiation ability was assessed using mineralization nodule formation.A 24 mm defect was created on the right mandibular body in 12 beagles.The mandibular defects were repaired with Ti-6A1-4V scaffolds mesh fabricated by EBM.General observation,CT and histology examination was carried out to evaluated the biocompatibility of Ti-6A1-4V scaffolds in vivo.Results CCK-8 result showed the A values of the two groups had no significant difference(P ＞ 0.05).There was no significant difference between the two groups (P＞0.05).Cytoskeletal staining showed that cells were fully stretched out and grew well on T-i6A1-4V specimen.The actin fibers were arranged in parallel and stained uniformly with fluorescent.After osteogenic culture,the quantity of the nodule formation of the experimental group and control group were 5.7±0.7 and 5.1 ± 0.6,respectively(P＞0.05).All animals had tolerated the surgery and healed well.CT examination showed that Ti-6A1-4V scaffolds mesh had good retention with surrounding bone and the continuity of mandible was restored.Histological examination showed that no inflammation reaction or toxity was caused in the soft tissue surrounding the scaffolds and in the liver and kidney after implantation.Ti-6A1-4V scaffolds had good retention with surrounding bone.Conclusions Ti-6Al-4V fabricated with electron beam melting has good biocompatibility.

Objective To evaluate the surface characteristics and cytocompatibility of Ti-6Al-4V alloy fabricated using select laser melting (SLM) and electron beam melting (EBM) technique.Methods Ti-6Al-4V alloy specimens were fabricated with SLM and EBM.A wrought form of Ti-6Al-4V alloy was used as a control.Its properties were evaluated using component analysis,contact angle test,surface roughness,surface topography,cell ultrastructure,cell attachment and proliferation observation,metal ion precipitation examination.Results The roughness of SLM and EBM specimens was suitable for cell attachment but not the best.The character of SLM and EBM specimens was hydrophobic (＞65°).The surface topography of EBM and SLM specimens were similar,but were not the best type for cell attachment.The components of Ti-alloy oxide film were detected in all the specimens.The content of Ti,A1,V ions of EBM,SLM and wrought specimens were very low and did not affect the cell attachment and proliferation.The ultrastructure of cell was normal,and the cytomembrane was intact.The number of cells was similar to each other among the three kinds of specimens and increased obviously with the culture time.Conclusions The results of the study suggested that EBM and SLM Ti-6A1-4V specimens possessed good surface characteristics.However,the surface modification are needed further.

Objective:To identify the clinical significance of CDK5 in colon cancer tissues.Methods:Two hundred colon cancer tissues were tested for CDK5 expression by immunohistochemistry on tissue microarrays. The correlation between CDK5 expression and clinicopathological features, prognosis and peripheral inflammation-related cells was analyzed.Results:CDK5 was low expressed in 100 cases (50.0%), and high in another 100 cases (50.0%). Longer time to tumor progression ( P=0.026) and overall survival ( P=0.035) were observed in patients with high CDK5 expression. By multivariate analysis , the expression of CDK5 was an independent risk factor for poor prognosis ( HR=0.45,95% CI: 0.21-0.99, P=0.049). The expression of CDK5 was not related to the counts of white blood cells and neutrophils ( P>0.05). Prognosis of patients with a positive lymph node ratio less than 0.15 was significantly better than that of patients with a higher lymph node ratio ( P<0.001). Conclusions:Patients with low CDK5 expression have poor prognosis, and CDK5 expression is not related to the counts of peripheral white blood cells and neutrophils.