Objective:To explore a simple way to improve seeding efficiency and uniformity in constructing 3-D dermal equivalent,in order to effectively construct tissue engineered skin.Methods: Human dermal fibroblasts were added onto collagen sponge scaffolds and cultured in the following 3 ways:(1)oscillating seeding and oscillating culture(OO),(2)oscillating seeding and static culture(OS),and(3)static seeding and static culture (SS).Samples were obtained and subjected to light microscopy,scanning electron microscopy(SEM) and quantitative cell number assay at planed times.Results: Oscillating seeding achieved more uniform spatial cell distribution within scaffolds as compared to static seeding.Moreover,fibroblast seeding efficiency was significantly higher in oscillating conditions(\%) than in static conditions(\%,P

Objective:To provide reference for the intensive study on polyethylene glycol modified chitosan. Methods:The relat-ed literatures at home and abroad were looked up and retrieved from 2004 to 2014, and the related contents were summarized. Results:The methods for the preparation of polyethylene glycol modified chitosan were various. Polyethylene glycol modified chitosan could be used as the carrier for nano-drug and gene therapy, the base of temperature-sensitive hydrogel, polymeric prodrug and the repair mate-rial of tissue engineering. Conclusion:Polyethylene glycol modified chitosan can be wildely used in biomedical field. However, the re-lated research is only in vitro. The in vivo studies should be further systematic and intensive.

Objective To investigate the effects of collagen concentration and pre-freezing temperature on structure and properties of the scaffold. Method A series of porous collagen scaffolds were fabricated with different collagen concentration and pre-freezing temperature by freezing-drying. The effective pore sizes and other properties of the porous scaffolds were evaluated and compared with each other. Chondrocytes of rabbit were separated and cultured on these scaffolds to evaluate their biocompatibility. Result The collagen scaffolds had interconnected pore ranging from 50 to 200 ?m in pore size. With increasing the collagen concentration density and tensile strength of the scaffolds increased, while pore size and degradation rate of the scaffolds decreased, as well as become less homogeneous. Reducing pre-freezing temperature resulted in smaller poresize and slower degradation rate of scaffolds. MTT analyses demonstrated that all the scaffolds availed to cell attachment and proliferation, while increasing collagen concentration and decreasing pre-freezing temperature evidently restrained chondrocytes attachment and proliferation. Conclusion The collagen concentration and pre-freezing temperature have crucial influence on the structure and properties of collagen scaffolds. The suitable collagen scaffolds were obtained by adjusting the collagen concentration and pre-freezing temperature. The bigger of the pore size was. The faster cell proliferation was achieved.

AIM:To observe the efficacy and safety of ropivacaine supplemented with midazolam for sacral block in pediatric operation.METHODS:40 cases of patients aged 1-6 years old,who were going to be operated in hypogastrium,perineum and lower limb,were randomly and double-blindly divided into four groups with ten cases each:control group(bupivacaine 2.5 mg/kg),low dosage group(ropivacaine 2.5 mg/kg),middle dosage group(ropivacaine 3.5 mg/kg)and high dosage group(ropivacaine 5.0 mg/kg).Sacral block was performed after induction of inhalation anaesthesia with sevoflurane.Sedation was induced by midazolam(0.2 mg/kg)administered through mainline 5 min before the surgical procedure.RESULTS:In a certain dosage range,ropivacaine supplemented with midazolam anesthesia for sacral block showed a slight influence on diastolic blood pressure,mean arterial pressure,heart rate and pulse oxygen saturation.Those parameters remained in the physiological normal range,though they dropped slightly during the operative period.Compared with bupivacaine group,the postoperative analgesic period was similar in the high and middle ropivacaine groups,while it was shorter in low ropivacaine group.There was no significant adverse effect in all groups except for operative stretch reflex and postoperative vomiting in individual patients.CONCLUSION:Ropivacaine supplemented with midazolam anesthesia for sacral block has a slight influence on hemodynamics,prolongs the postoperative analgesic period,and shows less adverse effect.

Objectives: To study the e ect of human P27KIP1 gene AVV virus combining with Chinese herb Pien Tze Huang on human osteosarcoma mice model. Methods: 40 standardized human osteosarcoma mice model were divided into 5 groups, treated with PBS, Paav-MCS, human P27KIP1 gene AVV virus, Chinese herb Pien Tze Huang or human P27KIP1 gene AVV virus combining with Chinese herb Pien Tze Huang in each di erent group. Results: ① Mice model of transplant human osteosarcoma were established successfully. ②Osteosarcoma were inhibited successfully by human P27KIP1 gene AVV virus, Chinese herb Pien Tze Huang or human P27KIP1 gene AVV virus combining with Chinese herb Pien Tze Huang. ③Mice model of transplant human osteosarcoma were active during observation. Conclusions: ①These results suggested that human P27KIP1 gene AVV virus, herb Pien Tze Huang and human P27KIP1 gene AVV virus combined with herb Pien Tze Huang were e ective on human osteosarcoma. ②Among them, the most e ective treatment was human P27KIP1 gene AVV virus combining with herb Pien Tze Huang. ③Chinese herb Pien Tze Huang was possible useful for life improvement of osteosarcoma patient.

BACKGROUND:Liposomes as a new drug delivery system are characterized by few adverse reactions, no immunogenicity and biodegradation. Furthermore, methotrexate-loaded liposomes can significantly reduce drug toxicity and improve anti-tumor effect. OBJECTIVE:To prepare long-circulating methotrexate-loaded liposomes and to evaluate its antitumor activity in MG-63 in vitro. METHODS:The methotrexate-loaded liposomes were prepared using the film dispersion method, and the long-circulating ones were prepared using the post-insertion method. The initial concentrations of methotrexate were 9.1, 1.82, 0.364 g/L. The ultracentrifugation method and spin column method with Sephadex G-10 or G-50 as packing were employed to separate free drugs from the methotrexate-loaded liposomes. Their recovery, size, morphology, encapsulation efficiency and drug-to-lipid ratio were evaluated. The cytotoxity of the long-circulating methotrexate-loaded liposomes purified with ultracentrifugation method and spin column G-50 method under three dose levels (0, 1, 5, 25 mg/L) were determined by MTS method. RESULTS AND CONCLUSION:According to the recovery rates of three methods, the spin column G-50 method was considered as optimal for the long-circulating methotrexate-loaded liposomes. The long-circulating liposomes were spherical or el ipsoidal under transmission electron microscope, about 200 nm in size. At the certain initial concentration of methotrexate, the encapsulation efficiency and drug-to-lipid ratio of the liposomes purified using the spin column G-50 method were remarkably higher than those purified using the other two methods. At the same mass concentration, the cytotoxity of the liposomes purified with ultracentrifugation or spin column G-50 was significantly higher than that of free methotrexate, and furthermore, the cytotoxity of the liposomes purified with spin column G-50 was higher than that of the liposomes purified with ultracentrifugation method. To conclude, the long-circulating methotrexate-loaded liposomes show a higher antitumor activity than free methotrexate in MG-63 cel s in vitro, providing the basis for further investigation of its antitumor effect on human osteosarcoma in vivo.

Objective To investigate the influence of pullulan molecular weight and spacer length on the properties of modified pullulan carriers including morphologies,sizes and in vitro release behaviours of drug-loading carriers.Methods Using cholesterol as hydrophobic ligand,succinic anhydride and 1,6-hexyldiisocyanate as spacers,hydrophobic modified pullulans with different molecular weights were prepared.Self-assembled nanoparticles were then formed in the aqueous solution,and drug-loaded nanoparticles were prepared by dialysis method.The influence of pullulan molecular weight and spacer length on the loading-content,morphologies and in vitro release behaviours of drug-loading nanoparticles were then investigated in detail.Results Self-assembled nanoparticles could be formed by the cholesterol-modified pullulan,and doxorubicin and mitoxantrone could be loaded into cholesterol-modified pullulan to form nanoparticles.Pullulan molecular weight and spacer length show influences on sizes,morphologies and stabilities of pullulan nanoparticles and drug-loaded nanocarriers.Conclusions Before drug loading,nanoparticles with larger moleculare weight and shorter spacer length are more stable in solution,while after drug loading,the influences of these two factors on the nanoparticles are drug-type depended.

This study is to investigate the effect of curcumin on the induction of glutathione Stransferases (GST) and NADP(H):quinone oxidoreductase (NQO) and explore their possible molecular mechanism. The activity of GST, NQO and cellular reduced glutathione (GSH) content were measured by spectrophotometrical methods. Cellular changes in the distribution of NF-E2 related factor 2 (Nrf2) were detected by Western blotting analysis. Nrf2-AREs (antioxidant-responsive elements) binding activity was examined by electrophoretic mobility shift assay (EMSA). Treatment of HT-29 human colon adenocarcinoma cells with curcumin dramatically induced the activity of GST and NQO at the range of 10-30 μmol·L-1. Curcumin exposure caused a significant increase in cellular GSH content rapidly as early as 3 h. Moreover, curcumin triggered the accumulation of Nrf2 in nucleus, and increased Nrf2 content in ARE complexes. These results demonstrated that induction of GST and NQO activity by curcumin may be mediated by translocation of transcription factor Nrf2 from cytoplasm to nuclear and increased binding activity of Nrf2-ARE complexes.

Microspheres made of poly-lactic-co-glycolic acid (PLGA) have been frequently proposed as drug delivery systems.A very significant challenge in the development of controlled PLGA releasing systems is the instability of drugs especially therapeutic peptides and proteins.Additional approaches,particularly the use of additives,are needed to optimize PLGA delivery of drugs.This article reviews the effects of additives,especially the effects of stabilizing protein during the preparation of PLGA microsphere and the sustained drug releasing processes.

ObjectiveThe aim was to construct the recombinant plasmid of pET-28a-G-protein pathway suppressor 2 (GPS2) GPS2,express GPS2 protein in E.coli,and obtain specific polyclonal antiserum of GPS2.MethodsGPS2 gene was obtained and the amplified fragment was then cloned into E.coli expression vector pET-28a to construct recombinant plasmid.The recombinant plasmid was transformed into E.coli expression strain BL21(DE3).IPTG induces the expression protein GPS2 protein,and the induction conditions were optimized.The induced product was purified by Ni2+ affinity chromatography,and the purified product was dialyzed with buffer for refolding.The purified protein can be used as antigen,injected to immunize male New Zealand white rabbit to get polyclonal antiserum.The titer and specificity of the rabbit antiserum were detected by ELISA and Western Blotting.ResultsThe E.coli expression vector pET-28a-GPS2 was constructed successfully and the recombinant protein was efficiently expressed and purified.The purified protein was used to immunize male New Zealand white rabbit to get polyclonal antiserum and the ELISA and Western Blot results showed that the high titer of specific polyclonal antiserum.ConclusionGSP2 could be highly expressed in E.coli.Antiserum of GPS2 protein can be obtained by the purified recombinant to analyze its function.