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.

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 correlation between dose and effect of thioacetamide (TAA) on rat model of intestinal endotoxemia. Methods The models of intestinal endotoxemia were induced by three different doses of TAA by gavage administration of TAA 200, 400, 600mg/kg respectively once per day for two days.The doses were given at same time point every day. Each group included 10 rats. The rats in the control group were administrated with 2 mL 0.9% NaCl saline gavage. The death of the rats was observed at 24 hours and 48 hours after administration. The blood samples of the living rats were drawn from abdominal aorta for determining the plasma endotoxin levels, serum alanine aminotransferase(ALT)and aspartated transaminase (AST) levels. The histopathological changes of liver were examined. Single factor analysis of variance was performed and comparision between groups was done using t test. Results No rat in the control group died. Two rats of 200 mg/kg TAA group, five rats of 400 mg/kg TAA group and eight rats of 600 mg/kg TAA group died during the experiment. The mean serum ALT levels of TAA model groups [(305.09±116.78)U/L,(901.67±274.31)U/L,(1454.84±473.49)U/L] were all significantly higher than that of the control group(47.81±22.61)U/L(t=14.583, 25.896 and 20.596, respectively; all P＜0.05). The mean serum AST levels of TAA model groups [(465.88±139.96)U/L, (884.37±250.90)U/L,(1889.23±159.67)U/L] were all significantly higher than that of the control group (69.33±22.04)U/L(t=12.988,18. 455 and 13.542, respectively; all P＜0.05). The mean plasma endotoxin levels of TAA model groups [(0.436±0.110)EU/mL, (0.550±0.095) EU/mL, (0.620±0.057)EU/mL] were all significantly higher than that of the control group (0.103±0.056)EU/mL(t=7.335, 5.260 and 8.191, respectively; all P＜0.05). The histological results of TAA model groups showed hepatic cell degeneration and necrosis in different degrees. Conclusions TAA with 200-600mg/kg is proper to establish the rat model of intestinal endotoxemia. The death rate of rats in the 200mg/kg TAA group is lower than those of other model groups, which suggests that 200mg/kg TAA may be the best dosage for establishing rat model for further studies.

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.

Objective To construct a extracellular matrix-like collagen mimetic peptide-PEG hybrid hydrogel and to study the usage of this hydrogel in 3D culture of rabbit bone marrow mesenchymal stem cells (rBMSCs).Methods The hybrid hydrogel was synthesised by conjugating the cysteine at the end of the collagen mimetic peptide with the maleimine-modified multi-arm PEG.The circular dichroism spectra were used to characterize the triple helix structure and thermal stability of the collagen mimetic peptides.The rheology test and scanning electron microscopy were used to study the gelation process,mechanical strength and internal structure of the hydrogel.The rBMSCs were embedded in the hybrid hydrogel for 3D culture.The cell compatibility of the hydrogel and its effect on differentiation of the cells was studied.Results Collagen mimetic peptides could promote spontaneous formation of triple helix structure in the natural collagen,and the thermal transition temperature was 49.4 ℃.The formation process of the collagen mimetic peptides-PEG hybrid hydrogel was rapid,in which the porous network-like fibrous structure was formed.After the encapsulation of rBMSCs within the hydrogel for 24 h,most of the cells remained viable.Gene expression analysis showed that the hybrid hydrogel could affect the differentiation of rBMSCs.Conclusions The collagen mimetic peptide-PEG hybrid hydrogel possesses the characteristics of mild preparation condition,good mechanical strength and good cell compatibility,and is favorable to chondrocyte differentiation of rBMSCs.

Objective To improve the level of diagnosis and differentiation of renal benign mass with renal cell carcinoma(RCC),so as to lower the misdiagnosis rate.Methods This study included 9 cases of benign renal mass,whose age ranged from 30 to 76 years with a mean of 54 years and included 52 patients with RCC.Three subtypes of RCC were noted,including clear cell in 37 cases,papillary RCC in 10 cases and chromophobe RCC in 5 cases.Plain scan and three phase CT(corticomedullary,nephrographic and excretory phases)were done in all patients.The CT features of RCC and benign mass were compared.Results All the cases were underwent radical nephrectomy as RCC,while they were postoperatively diagnosed as benign renal mass.There were 4 cases of angiomyolipoma (AML)with minimal fat,two cases of oncocytoma,one case of leiomyoma,one case of inflammatory pseudotumor,and one case of cyst with hematoma and organization.Fifty-two cases of RCC showed homogenous or inhomogeous,equal,slightly lower,slightly higher or mixed density on unenhanced scan,inhomogenous obvious enhancement after administration of contrast media.And the most obviously enhanced portion of renal carcinomas were isodense or slightly hyperdense relative to adjacent renal cortex in corticomedullary phase.Conclusion CT is an important radiologic approach to diagnose and differentially diagnose malignant or benign kidney mass.For those patients with benign mass that is not a typical case on radiology,the preoperative needle biopsy or intraoperative frozen section pathological diagnosis is the key to avoid misdiagnose and mistake resection of the kidney,and choose the proper treatment approach to avoid unnecessary kidney radical resection.

OBJECTIVE To explore the sterilizing effect of low-temperature vacuum formaldehyde.METHODS The test group used the own-produced 140 L low-temperature vacuum formaldehyde sterilizer for sterilization;and the control group used "Xinhua" hydrogen peroxide plasma sterilizer.Sterilization effect of the two groups was monitored by biological indicator.RESULTS After 50 sterilization procedures run in test group,the biological indicators the bacterial were all killed,the qualification rate of sterilization was 100%.But after 30 sterilization procedures run in control group,only 8 procedures were qualified,the qualification rate of sterilization was 26%.The sterilizing effect of the two groups was significantly different(P

Objective To study the application of extrusion method in inducing liposome membrane fusion or membrane component mixing,and to investigate the effect of different extrusion conditions on liposome membrane fusion rate.Methods N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) labeled 1,2-dioleoyl lecithin (DOPC) liposomes and non-fluorescently labeled DOPC monolayer liposomes were mixed and extruded.The fluorescence changes before and after the extrusion of the mixed liposomes were observed using laser scanning confocal microscope,and the membrane fusion rate of the mixed liposomes was calculated by fluorescence resonance energy transfer method.Besides,the effects of extrusion times,extrusion pressure and temperature on the fusion rate of liposome membrane were studied.Results The results of laser scanning confocal microscopy showed that the distribution density and intensity of the green fluorescence of N-NBD-PE increased significantly after the extrusion of fluorescently labeled and non-fluorescent labeled DOPC liposomes,which confirmed membrane fusion.After 75 times of extrusion treatments,the liposome membrane fusion rate can reach 26％.The number of extrusions,extrusion pressure and temperature had a significant effect on the fusion rate of the liposome membrane.The higher the number of the extrusions,the smaller the extrusion pressure and the higher the efficiency of the liposome membrane fusion were at physiological temperature.Conclusions Extrusion method can induce liposome membrane fusion and membrane component mixing,and the prepared liposome has a narrower particle size distribution,which is expected to be a new method to induce the bilayer membrane fusion of liposome or lipid vesicle.

With an incidence of 1/800 - 1/600, Down syndrome (DS) is the most common chromosomal disorder in humans. Whilst most DS patients has trisomy 21, a small proportion may carry translocations or mosaicisms involving chromosome 21. The main characteristics of DS include mental retardation, peculiar facies, growth retardation, congenital heart disease, duodenal stenosis, Alzheimer's disease, leukemia, and immunodeficiency, which may be due to increased dosage of critical genes. Recent studies also showed that epigenetic changes may also occur in DS. For research on patients with DS or other trisomies have been restricted by ethical considerations, and commonly used mouse models cannot fully replicate the characteristic features of DS, pluripotent stem cells induced from fetal samples or biopsy tissues from DS patients may generate models with the same genetic content, which may provide idea materials for studying the pathogenesis of DS and customized cell and/or gene therapies.

Objective:To study the inhibitory effect of ezetimibe in an experimental model of human hepatoma cell line (HepaRG) infected with hepatitis B virus (HBV) positive human serum in vitro.Methods:Mature HepaRG cells were divided into a treatment group (received drugs) and a control group (did not receive drugs). In the ezetimibe prevention experiment, the cells in the treatment group was treated with drugs 2 h before infection and 24 h during infection. In the ezetimibe treatment experiment, the cells in the treatment group were treated with drugs for 6 ~ 10 days continuously after 24 hours of HBV infection. The expression of HBV DNA and intracellular cccDNA in the supernatant was detected by fluorescence quantitative PCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) content in the cell supernatant were detected by chemiluminescence. Analysis of variance was used to compare the differences between multiple groups. Pairwise comparisons among groups were followed by t- test with normal distribution. P < 0.05 was considered as statistically significant. Results:Ezetimibe prevention experiment showed that compared with control group, the treatment group was added with 20, 60, and 100 μmol/L ezetimibe before and during infection, and the HBV DNA content in the supernatant 2 days before was significantly reduced ( P < 0.05) in the treatment group. Compared with the control group, the HBsAg expression level 2 days before was significantly reduced ( P < 0.05) with the addition of 60 μmol/L ezetimibe in the treatment group. Compared with the control group, the expression level of intracellular cccDNA was significantly reduced ( P < 0.05) after 10 days with the addition of 100μmol/L ezetimibe in the treatment group. Ezetimibe treatment experiment showed that cccDNA content in the cells were significantly lowered with the immediate addition of 60μmol/L ezetimibe 24 hours after infection for 10 days when compared to control group ( P < 0.05). Conclusion:Ezetimibe, as a cytosolic inhibitor, has a certain inhibitory effect on hepatitis B virus infection in both prevention and treatment experiment.