Objective To construct AWP1 (associated with protein kinase C related kinase 1) recombinant adenovirus as the tool of transferring the gene and investigate its expression and localization in human vascular endothelial cell ECV304. Methods Cloned AWP1 cDNA was inserted into the multiply clone sites (MCS) of plasmid pcDNA3 for adding flag tag, and the flag-AWP1 gene was subcloned into shuttle vector pAdTrack-CMV. After identified with restrictional enzymes, plasmid pAdTrack-flag-AWP1 was linearized by digestion with restriction endonuclease PmeⅠ, and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination. After linearized by PacⅠ, the homologous recombinant adenovirus plasmid transfected into 293 cells with Lipofectamine to pack recombinant adenovirus. After PCR assay of recombinant adenovirus granules, recombinant adenoviruses infected 293 cells repeatedly for obtaining the high-level adenoviruses solution. And then, the recombinant adenoviruses infected human ECV304 cells for observing the expression and localization of AWP1 under laser scanning confocal microscope (LSCM). Results PCR assay showed that recombinant adenovirus Ad-flag-AWP1 was obtained successfully; and ECV304 cells were infected high-efficiently by the homologous recombinant virus. Then, it was observed that flag-AWP1 protein expressed in ECV304 cells and distributed in the leading edges of the cell membrane. Conclusion The vectors of flag-AWP1 recombinant adenovirus are constructed, and the localization of AWP1 protein in ECV304 cells might show that AWP1 may be a potential role on the cell signal transduction.
Objective To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue. Methods The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay. Results SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates. Conclusion The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.
Objective To summarize the recent progress of construction methods of engineered cell sheet and to forecast the possible prospect. Methods The recent original articles about investigation and appl ication of engineered cell sheet were reviewed. Several common methods were selected and expounded. Results The construction methods of engineered cell sheet mainly include temperature-responsive culture dish, salmon atelocollagen, magnetic force, surface roughness, and polyelectrolytes, which may overcome the l imits of traditional tissue engineering methods. Conclusion The construction methods of engineered cell sheet are feasible and have a bright future in the cl inical appl ication.
Objective To construct and screen neurite outgrowth inhibitory 66-samll interfering RNA (nogo66-siRNA) eukaryotic expression vectors of effective interference, so as to lay a foundation for further reconstruction of related viral vector. Methods The nogo66-siRNA fragments were designed and cloned into pGenesil-1.1, 4 plasmids of pGenesil-nogo66-siRNA-1, pGenesil-nogo66-siRNA-2, pGenesil-nogo66-siRNA-hk, and pGenesil-nogo66-siRNA-kb were obtained, sequenced and identified, then were transfected into C6 cell l ine. The transfection efficiency was measured by fluorescence microscope. RT-PCR and Western blot were used to detect the expression of nogo gene and select the plasmid of effective interference. Results DNA sequencing results showed interference sequences were correct. The bands of 800 bp and 4.3 kb were detected when pGenesil-nogo66-siRNAs were digested by Kpn I /Xho I. The expression of green fluorescent protein could be detected under fluorescence microscope, and the transfection efficiency was about 73%. RT-PCR and Western blot results showed that compared to non-transfected cells, the transfection of pGenesil-nogo66-siRNA-1 made the expression of nogo gene decl ine 22% and the expression of nogo protein decl ine 73%; the transfection of pGenesil-nogo66-siRNA-2 made the expression of nogo gene decl ine 28% and the expression of nogo protein decl ine 78%; the differences were significant (P lt; 0.05); and the transfection of pGenesil-nogo66-siRNA-hk and pGenesil-nogo66- siRNA-kb did not make the expressions of nogo gene and nogo protein decrease significantly (P gt; 0.05). Conclusion Nogo66-siRNA eukaryotic expression vector is successfully constructed, it lays an experimental foundation for repair of spinal cord injury.
【Abstract】 Objective To reconstruct three different kinds of tympanic membrane in vitro by tissue engineeringtechnique, and to examine their histological structures and mechanical properties. Methods The skin and dura of pig(weight 30 kg) were processed with high satuated sal ine and enzymes to make extracellular matrix. Meanwhile, fibroblasts(1×106 /mL,0.2 mL) were seeded on the surface of these two scaffolds and collagen. The composite tissues were cultured in vitro for 1 week and examined in histological structure and mechanical properties. Results Fibroblasts cultured were spindle–shaped and could grow and attach to these scaffolds with a arrangement of sarciniform and parallel. The reconstructed tissue of ECM and collagen appeared to integrate well and had better bio-compatibil ity. The mean thickness of the collagen, the skin and the dura (all covered with fibroblasts) were 9.4, 10.0 and 10.4 μm respectively. The tension of the collagen was (1.417±0.030) N/mm2, of the acellular dermal matrix was (24.500±2.040) N/mm2(being close to the tension of normal tympanic membrane, 26.700 N/ mm2),of the acellular dura was (53.300±2.600) N/mm2. Conclusion The results suggest that the tension and the thinkness of acellular dermal matrix is similar to the normal tympanic membrane of guinea pig, it is an ideal material for tympanoplasty.
Objective To find a feasible method that can reconstruct the composite tissue-engineered skin fast in vitro and can provide enoughskin as soon as possible for covering the surface of the large-area burn. Methods The foreskin was taken during the posthectomy. The epidermal cells and fibroblasts were isolated, identified and cultured. The cytokeratin 19 (K19) flow cytometry and the fluorescein isothiocyanate (FITC)immunofluorescence for K19 and the FITCimmunofluorescence for PAN-cytokeratin of the epidermal cells and the FITCimmunofluorescence for vimentin of the fibroblasts were performed to identify the epidermal cells and the fibroblasts. Then, the epidermal cells were seeded onto the papillary surface of an acellular dermal matrix (ADM) and were submerged into the condition culture medium added with 25 ng/ml of the keratinocyte growth factor (KGF). However, in the control group, no KGFwas added. After 24 hours, the ADM was moved up to the airfluid surface, and the culture was continued. After 6 days, the fibroblasts were seeded onto the other surface of the ADM. After a 24 hour culture, the ADM was harvested and fixed in formalin, and the hematoxylin-eosin staining was conducted. Then, the structure of the reconstructed skin was observed under the microscope and the cell count in the epidermal layer was also conducted. Results All the cultured and expanded epidermal cells stained by the immunofluorescence demonstrated a positive reaction for PANFITC, and a partially positive for K19-FITC, and 17% of the cells demonstrated a positive reaction for K19 identified by the flow cytometry. The fibroblasts could be expanded by more than 100 times after a 7day culture in vitro, and they could demonstrate a positive reaction for vimentin-FITC. After a 7day culture, a composite tissue-engineered skin could be attained. The hematoxylineosin staining of the reconstructed skin showed that there was one continuous layer of the epidermis on the papillary surface of the ADM and there were fibroblasts in the superficial layer of the other one, but the epidermal layer did not stick tightly to the ADM. The cell count demonstrated that KGF promoted the epidermal cells to proliferate better（Plt;0.01）and to form more significantly continuous layers of the epidermis in the experimental group than in the control group（Plt;0.01）. Conclusion Through the seed-cell separation by the digestion of collagenase and trypsin combined with the use of the KGF-added condition ulture medium, a composite tissueengineered skin can be reconstructed within 7 days.
Objective To search the most suitable concentration of calcium in the medium for the basement membrane reconstruction in tissue engineering skin in vitro. Methods Composite chitosan tissue engineering skin was prepared according to previous studies. Four groups were included according to the concentrationof calcium (1.00, 1.45, 1.65 and 1.95 mmol/L respectively). After 7 days and 15 days of culture, the histological manifestation of basement membrane in tissue engineering skin was observed by hematoxylin amp; eosin staining and PAS staining, and collagen Ⅳ of basement membrane was detected immunohistochemicallyatthe dermalepidermal junction. Results This tissue engineering skin shared some histological features of normal skin, including a welldifferentiated stratifiedepidermis and a dense dermis. The epithelium in the group of 1.95 mmol/L calcium differentiated better than those in other groups. PAS staining showing a regularly red dying strap domain at the dermal-epidermal junction. Collagen Ⅳ was positively stained immunohistochemically at the dermalepidermal junction inthe tissue engineering skin. Conclusion The above results suggest that the medium with 1.95 mmol/L calcium should be suitable for the growth of composite chitosan tissue engineering skin and the reconstruction of basement membrane.
Objective To study and summarize the clinical experience and significance of the skeleton reconstruction of human hand allografts. Methods From January 2001 to October 2003, human hand allografts were appliedto treat 4 cases of traumatic hand defect(6 hands) at different levels. During operation, the ulna and radius were reduced anatomically and fixed firmly with 3.5 mm AO-plates and screws according to AO internal fixation principle. The X-ray films were taken periodically andthe function recovery of hand allografts was observed and estimated. Results The 4 cases were followed up for 4-36 months postoperatively. The clinical healing of fracture in 4 cases(6 hands) was achieved after 9 weeks,and by means of comprehensive assessment including the joint function, muscle strength, sensation, appearance, sequela and the ability of work, the satisfactory effects were gained eventually. Conclusion It is significant forhuman hand allografts to reconstruct skeleton firmly.
Objective We aimed to evaluate the current status of the construction and practice of the stroke center in West China Hospital of Sichuan University and develope a future strategy to promote the standardized developement of inpatients care and evidence practice. Methods The current status of the Stroke Center in West China Hospital of Sichuan University was assessed. The procedure of diagnosis and treatment was inspected in detail, including triage, thrombolytic therapy, antithrombotic medication and anticoagulant, primary and secondary prevention, filter of risk factors, rehabilitation and education for patients. After that, new plans were made on the basis of the assessment and experiences acquired from practices in the Stroke Center in West China Hospital. Results The primary Stroke Center had been constructed in West China Hospital. The practice in West China Hospital showed that the Stroke Center significantly reduced the mortality and shortened the length of hospital stay of the patients with stroke. Conclusion It is proved that construction and implementation of the Stroke Center in West China Hospital are feasible.
目的 探讨成都市传染病医院护理应急体系的构建方法、效果。 方法 成立护理应急管理小组；组建护理应急梯队；储备应急物资和设备；加强护理应急人员知识技能培训和实战演练；严格防护措施与消毒隔离流程。 结果 出色地完成了多次突发传染病的救治工作，培养了一支具有丰富应急救治经验的专业护理人员队伍。 结论 建立完善的护理应急体系可有效提高突发事件的应急保障能力。