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.

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.

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 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.

Objective To compare the diagnostic value of MRE and DWI in staging hepatic fibrosis in pa-tients with CHB. Methods In this retrospective analysis ,we investigated 93 patients with CBH and live fibrosis. Ninety-three patients were grouped according to their pathological grading of fibrosis ,from S0 to S4. Sixteen healthy patients were enrolled as the control group. Spearman correlation analysis was used to analyze the correla-tion between the stiffness and ADC value and their staging fibrosis. ROC analysis was conducted to compare the per-formance of the stiffness and ADC value in staging hepatic fibrosis. Results Both liver stiffness value(r=0.962, P<0.01)and ADC value(r=-0.823,P<0.01)were highly correlated with the stage of liver fibrosis. The area un-der ROC(AUC)for the detection of≥S1≥S2/≥S3/S4 stage fibrosis with the stiffness and ADC value were 0.963/0.868、0.995/0.947、0.998/0.948、0.996/0.889 respectively ,with statistically significant differences (P < 0.05 , resectively). Conclusions MRE and DWI have higher value ,and MRE is more accurate than DWI for staging hepatic fibrosis in patients with CHB.

Objective:To evaluate the effects of different exercises on executive function, moter skills and core symptoms in children with attention deficit hyperactivity disorder (ADHD) using network Meta-analysis, and to provide evidence-based basis for the treatment of children with ADHD.Methods:The randomized controlled trails(RCTs)of exercises on children with ADHD were searched in Web of Science, PubMed, Cochrane Library, Embase, PsycINFO, CNKI, Wanfang Data, VIP and CBM databases.The retrieval time limit was from the establishment of each database to May 2023.The Cochrane quality review manual was used to evaluate the quality of the included literatures.Stata 17.0 software was used to perform network Meta-analysis.Results:Finally, twenty-four studies were enrolled including five types of exercise intervention, such as aerobic exercise, multicomponent exercise, exergaming, high intensity interval training (HIIT) and traditional low-to-moderate intensity exercise therapy (TRAD).The results of network Meta-analysis showed that: in terms of improving executive function(working memory, inhibitory control, and cognitive flexibility), motor skills and core symptoms of children with ADHD, the optimal exercise intervention was aerobic exercises (surface under the cumulative ranking were 78.9%, 73.0%, 88.7%, 77.9% and 69.9% respectively).Conclusion:Exercises can improve executive function, improve motor skills, and alleviate ADHD core symptoms.Aerobic exercise has a better effect on improving executive function, motor skills and core symptoms of ADHD children.

Objective:To explore the application status, research hotspots and frontiers of blended teaching mode in undergraduate nursing education at home and abroad, so as to provide reference for related research.Methods:In Chinese, CNKI, Wanfang Database and VIP, and in English, Web of Science database were used as search sources. CiteSpace was used to conduct a quantitative analysis of the retrieved literatures published from 2008 to 2022.Results:A total of 43 literatures in Chinese and 83 in English were retrieved, and the number of publications showed an overall increasing trend. The research hotspots in China focused on education reform, online learning and curriculum system evaluation, and the research frontiers were network teaching, moral education and humanistic care. Foreign research focused on the choice of education model and emphasizes interaction, and the research frontier were to carry out innovative blended teaching and pay attention to the learning experience and learning environment construction of nursing students.Conclusions:In the future, blended teaching should pay attention to carrying out innovative research and dynamic evaluation and feedback of students' satisfaction. In particular, it is necessary to strengthen the cultivation of blended learning competence among nursing teachers, aiming to promote the knowledge and skills of nursing students, and continuously optimize and innovate the application of blended learning in the field of undergraduate nursing education.

Objective:To study the effects of modified citrus pectin (MCP) on the viability and gene expressions of synovial fibroblasts (SF) as well as SF treated by galectin-3 (Gal-3).Methods:Rabbit SF was isolated and cultured in vitro. Then SF was treated with different concentrations of MCP (0, 250, 500, and 750 mg/L). In addition, SF was further treated with the same different concentrations of MCP after treatment with 10 μg/ml Gal-3 for 24 h. The viability of SF was detected by CCK-8 on the first, third, and fifth day after treatment. The mRNA expression of transforming growth factor-β1 (TGF-β1), type I collagen (COL1A2), and Gal-3 in SF was detected by real-time quantitative PCR. The synthesis of type I collagen in SF was investigated by immunofluorescence staining. Results:MCP, especially at a concentration of 500 mg/L can inhibit the proliferation of SF significantly (all P < 0.05) on the first, third, and fifth day after treatment. Compared with the control group, MCP at different concentrations induced different gene expression profiles. In particular, MCP at high concentrations can upregulate the expression of TGF-β1, COL1A2 and Gal-3 in SF. However, MCP shows no significant effect on the synthesis of type I collagen in SF. MCP can down-regulate the expression of TGF-β1, COL1A2, and significantly reduce the synthesis of type I collagen in SF after Gal-3 treatment. Particularly, the effect of MCP at a concentration of 500 mg/L on inhibiting the expression of TGF-β1, COL1A2, and Gal-3 in SF is significant. Conclusions:MCP can inhibit the excessive proliferation of SF and regulate gene expression in SF.