Objective:To construct the sphingosine kinase 1(SPHK1)overexpression lentiviral vector,and to establish the SKOV3 lentiviral stable transfection cell line.Methods:According to the SPHK1 data information provided by the National Center for Biotechnology Information(NCBI)database,the primers were designed and synthesized,the target gene was amplified,and connected to the GV492 plasmid treated with Bam HⅠ and AgeⅠ restriction enzymes to construct the SPHK1 overexpression lentiviral vector;the positive clones were selected for PCR and sequencing identification;the lentiviral plasmid and the lentiviral packaging auxiliary plasmid were co-transfected into the HEK-293T cells for packaging and titer determination;according to the measured optimal multiplicity of infection(MOI)of 10,the corresponding lentiviral amounts in various groups were transfected into the SKOV3 cells,and the SKOV3 cells were divided into blank group(without treatment),GV492 control group(GV492 control lentivirus infected SKOV3 cells),and GV492-SPHK1 overexpression group(GV492-SPHK1 overexpression lentivirus infected SKOV3 cells,ov-SPHK1 group);the optimal concentration of 2 mg·L-1 puromycin was used to screen the stably transfected SKOV3 cell line;after 48 h,the medium was changed and replaced with 1 mg·L-1 puromycin for screening for 14 d;the morphology and fluorescence expression of the cells were observed under fluorescence microscope;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of SPHK1 mRNA in the SKOV3 cells in various groups;Western blotting method was used to detect the expression level of SPHK1 protein in the SKOV3 cells in various groups.Results:The PCR sequencing results showed that the gene sequence of the SPHK1 overexpression lentiviral vector was completely consistent with the target sequence,and the SPHK1 overexpression lentiviral vector was successfully constructed;the titer determination results showed that the lentiviral titers in GV492 control group and ov-SPHK1 group were 5×1011 and 8×1011 TU·L?1,respectively;the SKOV3 cells in GV492 control group and ov-SPHK1 group were in good state and showed strong fluorescence expression,suggesting that the SKOV3 stable transfection cell line overexpressing SPHK1 was successfully established;the RT-qPCR results showed that compared with blank group and GV492 control group,the expression level of SPHK1 mRNA in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01);the Western blotting results showed that compared with blank group and GV492 control group,the expression level of SPHK1 protein in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01).Conclusion:The SPHK1 overexpression lentiviral vector is successfully constructed,and the SKOV3 stable transfection cell line is established.

Objective To analyze the clinical application of the Donabedian-based structure-process-result three-dimensional quality evaluation model for the prevention of stress injury in bone tumor surgery.Methods A total of 284 patients with bone tumor who were admitted to The Second Affiliated Hospital of Naval Medical University from January 2022 to December 2023 were enrolled and assigned to two groups according to the random number table.The control group received routine nursing.In the observation group,the Donabedian-based structure-process-result three-dimensional quality evaluation model was applied to prevent the stress injury in bone tumor surgery.The indexes of the two groups were compared and analyzed.Results Before intervention,there was no significant difference in scores between the two groups(P>0.05).After intervention,the scores of stress injury behavior,skill and knowledge in the observation group were higher than those in the control group(P<0.05).The incidence of stress injury in the observation group was lower than that in the control group(P<0.05).There was significant difference in nursing satisfaction between the two groups(P<0.05).Conclusion Donabedian-based structure-process-result three-dimensional quality evaluation model can not only enhance the self-care ability of the patients undergoing bone tumor surgery,but also prevent stress injury,thus improving patient satisfaction.

Objective To culture and expand primary rat aortic vascular stem cells in vitro and evaluate their characteristics as mesenchymal stem cells.Methods The thoracic and abdominal aortas isolated from 2-to 3-week-old Sprague-Dawley rats were cut into vascular segments 2.0 mm in length and cultured in culture flasks till adhesion and solidification of the outer membranes.The primary cells were further cultured to 80%-90%confluence before passaging.The morphology and growth characteristics of the cells were observed under a microscope,and the expressions of surface marker CD molecules on the cells was analyzed using flow cytometry.Adipogenic and osteogenic differentiation assays were performed to assess the capacity of the cells for multilineage differentiation.Results After 3 days of culture,a small number of spindle,star-shaped or polygonal cells migrated out from the peripheral of the vascular segments.At 5-6 days,island-like cell clusters occurred and the cells began to proliferate rapidly.The cell clusters expanded radially and showed signs of cell cloning.At 7-8 days,the cells fused into sheets and displayed a vortex-like distribution.The cells in the third passage presented with a uniform morphology,showing a typical fibroblast-like arrangement.Flow cytometry showed that the cells expressed predominantly CD44(80.3%),CD73(62.2%)and CD90(46.8%)with low expressions of CD34(1.1%),CD45(0.2%)and CD11b/c(0.2%).Adipogenic and osteogenic differentiation experiments demonstrated that the cells were capable of lipogenic and osteogenic differentiation in vitro.Conclusion Rat aortic vascular stem cells with mesenchymal stem cell characteristics can be successfully isolated and cultured by adherent culture of the segmented outer membrane.

Objective To culture and expand primary rat aortic vascular stem cells in vitro and evaluate their characteristics as mesenchymal stem cells.Methods The thoracic and abdominal aortas isolated from 2-to 3-week-old Sprague-Dawley rats were cut into vascular segments 2.0 mm in length and cultured in culture flasks till adhesion and solidification of the outer membranes.The primary cells were further cultured to 80%-90%confluence before passaging.The morphology and growth characteristics of the cells were observed under a microscope,and the expressions of surface marker CD molecules on the cells was analyzed using flow cytometry.Adipogenic and osteogenic differentiation assays were performed to assess the capacity of the cells for multilineage differentiation.Results After 3 days of culture,a small number of spindle,star-shaped or polygonal cells migrated out from the peripheral of the vascular segments.At 5-6 days,island-like cell clusters occurred and the cells began to proliferate rapidly.The cell clusters expanded radially and showed signs of cell cloning.At 7-8 days,the cells fused into sheets and displayed a vortex-like distribution.The cells in the third passage presented with a uniform morphology,showing a typical fibroblast-like arrangement.Flow cytometry showed that the cells expressed predominantly CD44(80.3%),CD73(62.2%)and CD90(46.8%)with low expressions of CD34(1.1%),CD45(0.2%)and CD11b/c(0.2%).Adipogenic and osteogenic differentiation experiments demonstrated that the cells were capable of lipogenic and osteogenic differentiation in vitro.Conclusion Rat aortic vascular stem cells with mesenchymal stem cell characteristics can be successfully isolated and cultured by adherent culture of the segmented outer membrane.

Objective To investigate the feasibility and safety of transanal total mesorectal excision (TaTME) in re-operation for anastomosis recurrence rectal cancer. Methods Five patients with anastomosis recurrence rectal cancer underwent TaTME at Ruijin Hospital between April 2020 and December 2021 were retrospectively enrolled in this study. The peri-operative situation, pathological examination, and short-term follow-up results were analyzed. Results All cases were operated laparoscopic TaTME successfully. The operative time was (206.00±19.49) min without intraoperative complications. One case encountered incorrect dissection plane. Anastomotic leakage occurred in one case and anastomotic stenosis developed in another case. The specimens quality of mesorectum deemed complete in all cases without both positive circumferential resection margin and positive distal resection margin. There was (15.20±2.39) months of median follow-up and one case found defecation disorder. Tumor recurrence, metastasis and tumor-related death were not found. Conclusions For patients with anastomosis recurrence rectal cancer, laparoscopic TaTME procedure is novel type and would be safe and effective surgical approach with satisfactory short-term follow-up.

Objective    To investigate the feasibility and safety of hybrid coronary revascularization (HCR) in patients with multivessel coronary artery disease (MVCAD). Methods    A total of 50 patients with MVCAD who underwent HCR technique in our heart center from May 2016 to April 2019 were included in this study (a HCR group), including 38 patients who underwent two-stage HCR and 12 patients one-stop HCR. There were 39 males and 11 females, with an average age of 62.4 (46-82) years. Another 482 patients who underwent conventional median incision under off-pump coronary artery bypass grafting (OPCAB) at the same period were selected as control (an OPCAB group), including 392 males and 90 females, with an average age of 64.2 (48-84) years. The safety and feasibility of HCR were evaluated and compared with conventional OPCAB technique. Results    There was no perioperative death in both groups. Compared with the OPCAB, HCR was associated with shorter operation time, less chest tube drainage, lower requirement of blood transfusion, shorter mechanical ventilation time and shorter postoperative intensive care unit (ICU) stay (P<0.05). There was no statistical difference in the incidence of major adverse cardiac or cerebrovascular events during the follow-up of 6 to 36 months between the two groups. Conclusion    HCR provides favorable short and mid-term outcomes for selected patients with MVCAD compared with conventional OPCAB.

Objective:To evaluate the results of butterfly cartilage myringoplasty for anterior quadrant tympanic perforation under endoscope.Methods:Thirty-eight patients with anterior quadrant tympanic perforations who were subjected to endoscopic butterfly cartilage myringoplasty from April 2016 to October 2018 were included in this study, including 16 males and 22 females, with an average age of (34.5±14.2) years. The patients were reviewed retrospectively, and the pre-and post-operative pure tone audiometry (PTA) thresholds, pre-and post-operative air-bone gaps (ABG), post-operative graft success rates and complications were evaluated. SPSS 23.0 was used to analyze data.Results:Mean post-operative follow-up duration was (9.4±3.1) months (range 6-18 months). The graft survival rate was 94.7% (36/38) . The preoperative and postoperative mean PTA was (30.9±8.9) dB HL and (21.4±7.7) dB HL respectively. Preoperative and postoperative mean ABG was (18.4±6.3) dB and (10.8±6.0) dB respectively. There was significant difference between pre-and postoperative PTA and ABG ( t=5.353 and 4.162, P<0.05 for both). A postoperative ABG reduction of (8.3±1.5) dB was reached. Two (4.7%) patients had postoperative myringitis, two (4.7%) had recurrent perforation, and one (2.4%) had lateral healing of transplanted tympanic membrane in the postoperative follow-ups. No intratympanic cholesteatoma was observed. Conclusions:Endoscopic butterfly inlay myringoplasty is a reliable, minimally invasive alternative method to repair anterior tympanic membrane perforations, with high closure rate and low risk of complications.

Objective To identify the predictors of postoperative acute kidney injury in patients undergoing surgery for Stanford type A acute aortic dissection. Methods A total of 220 patients who underwent surgery for type A acute aortic dissection in Qingdao Municipal Hospital from September 2010 to September 2017 were divided into two groups including a group A and a group B based on whether acute kidney injury occurred or not after surgery. There were 40 patients with 29 males and 11 females with the mean age of 54.6±9.2 years in the group A, 180 patients with 133 males and 47 females with the mean age of 48.5±7.9 years in the group B. Univariate and multivariate analyses (logistic regression) were used to identify the predictive risk factors. Results Overall in-hospital mortality was 5.5%. In univariate analysis, there were statistically significant differences with respect to the age, preoperative creatinine, preoperative white blood cell, the European system for cardiac operative risk evaluation (EuroSCORE), total cardiopulmonary bypass (CPB) time, deep hypothermic circulatory arrest (DHCA) time, arch replacement, red blood cell transfusion intraoperative and in 24 hours postoperatively, postoperative mechanical ventilation time, ICU stay duration, hospital stay duration and in hospital mortality. Multivariate logistic analysis showed that preoperative creatinine, preoperative white blood cell, CPB time, and red blood cell transfusion intraoperative and in 24 hours postoperatively were the independent predictors for postoperative acute kidney injury. Conclusion The incidence of acute kidney injury is high after surgery for acute Stanford type A aortic dissection. It can be predicted based on above factors, for patients with these risk factors, more perioperative care strategies are needed in order to induce the incidence of acute kidney injury.

Otorhinolaryngology is a high-risk department of a hospital, where there are many emergency critical diseases, common multiple diseases and major malignant diseases. Therefore, it is easy to cause many medical ethical problems. This paper analyzed the clinical status and characteristics of various otolaryngology diseases systematically, and expounded the related medical ethical issues in the diagnosis and treatment of otorhinolaryngology, including doctor-patient trust, safety and informed consent. Finally, the paper put forward a number of measures to do well the psychological evaluation and nursing care of patients, improve the professional skills of medical and nursing care, formulate the treatment plan of diseases, enhance the supervision and management of the network, and promote the social support of patients. The aim was to alleviate the "doctor-patient conflict" and create a harmonious medical environment.

Objective To understand the needs to reform curriculum for the graduate students in health management, and to provide evidence for the reform of the curriculum system. Methods From Jan-uary 2015 to May from China's colleges and universities carrying out health management professional post-graduate education, graduate students and supervisors from 3 universities were selected, and self-designed questionnaires were used to investigate their views on training objectives, abilities and curriculum settings, as well as the needs and suggestions for curriculum reform. The questionnaire survey data were collected and analyzed by SPSS 17.0. Results 120 questionnaires were issued and 100 were recovered in the study, with a recovery rate of 83.3％. 84.0％ of the graduate students did not manage relevant work experience before entering the school. The graduate students thought that we should focus on the research and teaching ability training related to the management of health services, and then the cultivation of management ability. There were 60 questionnaires for instructors, and 51 were recovered, with a recovery rate of 85.0％. With regard to the demand for ability training , teachers and graduate students tended to agree on the goal and focus of the training. 100.0％instructors believed that learning was an important course, only 11.2％ of the students thought that the course of study was not important, but to the current status of health manage-ment professional postgraduate courses, 79.3％ instructors believed that the current curriculum was reasonable, and only 53.5％of the students thought it reasonable. Conclusion The emphasis should be given to optimizing the course setting, adding practical and innovative courses, and strengthening the training of scientific research, etc., which is the only way to improve the quality of health management postgraduate education and meet the needs of health development.