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 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 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.
With the development of tissue engineering, a variety of forms of silk fibroin (SF) scaffolds has been applied to research of constructing variety of organization based on cells, which has become scientific focus in recent years. In this paper we introduced the source and structure of SF and the fabrication method of the scaffold, and also address the SF application progress in several relevant fields of tissue engineering, such as bone, cartilage, skin, blood vessel and nerves. Finally, we discuss the future leading prospect of the SF in order to provide reference for subsequent research.
This study aims to construct the plasmid of human Runx1 and observe its possible effects on Runx1 gene expression in human pulmonary adenocarcinoma cells (A549). The shRNA sequence targeting human Runx1 was designed and synthesized, then inserted into pSuper plasmid by DNA recombination technology. The recombinant plasmid was confirmed by bacterial colonies PCR, enzyme digestion analysis and DNA sequencing. A549 cells were transfected by Runx1 shRNA plasmid. The inhibition efficiency of pSuper-Runx1-shRNA plasmid on Runx1 at mRNA level and protein level were measured with real-time PCR and Western blot. The results of real-time PCR and Western blot indicated that the mRNA and protein levels of Runx1 in A549 cells were inhibited by the pSuper-Runx1-shRNA expression plasmid, and the inhibition rate were 33% and 50%, respectively.
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。
Objective To construct gene-modified hepatic stem cells (WB-F344 cells), which have rat IL-13 gene and can secrete the recombinant rat IL-13 cytokine in the cells. Methods Firstly, the rat IL-13 sequences were synthesized. Then the sequences were amplificated in bacterium coli after recombinated with pWPXL-MOD plasmid. After PCR and sequence identification, the positive clones were packaged into lentivirus. After detecting the virus titer, the WB-F344 cells with constructed lentivirus vector with rat IL-13 gene were cultured, then the valid targets (expression level of the IL-13) were detected by real time-PCR and Western blot in cultured WB-F344 cells on 5 days. Results The valid DNA of rat IL-13 was recombinated and packaged in lentivirus vector. The recombinant gene sequence was correct by checking with gene sequence test. Then the recombinant was introducted into the WB-F344 cells cultures. The best multiplicity of infection (MOI) value for effective transfection was 5. IL-13 had been detected on day 5 after transfection by checking with real-time PCR and Western blot. Conclusion The recombinant rat IL-13 gene with lentivirus vector is constructed and gene-modified WB-F344 cells are cultured successfully, which can be used in next animal experiment.
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.
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 yeast eukaryotic expression vector carrying human endostatin (ES) cDNA. Methods The functional fragment of endostatin gene in human hepatic tissue was amplified by using RT-PCR technology, and cloned into yeast pPIC9 expression vector. The positive clone was sequenced by using automatized sequencer. Results The endostatin cDNA was successfully cloned. The positive ES clone gene in pPIC9 expression vector was sieved, and its coding sequence was identified to be as same as the previously reported sequence. Conclusion The successful construction of ES gene in pPIC9 expression vector using molecular biological method maybe helpful for the high expression of ES protein, which may lay the foundation for the treatment of malignant tumor through anti-angiogenesis appoach.
