【摘要】 目的 通过比较两种原代人脐静脉内皮细胞的分离培养方法并对细胞特异性抗原进行鉴定,探索提高原代内皮细胞体外培养存活率及纯化率的方法。 方法 采用一次性无菌注射器向人脐静脉灌注消化液,消化液的浓度和消化时间分别025%(质量体积比)胰蛋白酶,10 min和01%(质量体积比)胶原酶Ⅱ,15 min。通过在倒置显微镜下观察细胞的形态特点和用免疫荧光染色的方法对细胞进行鉴定,比较两种消化方法的优劣。 结果 01%胶原酶Ⅱ,15 min的消化方法较025%胰蛋白酶,10 min对原代人脐静脉内皮细胞有更好的分离效果,活细胞数量多且细胞纯度较高。免疫荧光染色结果表明细胞内有Ⅷ因子相关抗原表达。结论 胶原酶Ⅱ可以有效分离脐静脉内皮细胞,最佳消化条件是01%胶原酶Ⅱ,37℃,15 min。【Abstract】 Objective To explore the optimal method for primary culture of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were prepared from human umbilical cords by 01% collagenase Ⅱ digestion for 15 minutes and 025 trypsinase digestion for 10 minutes,respectively. HUVECs were observed under inverted microscope and identified by immunofluorescence.The two methods of digestion were compared. Results More HUVECs were harvested through the method of 01% collagenase Ⅱ for 15 minutes,which expressed Ⅷ related antigen. Conclusion The method of 0.1% collagenase Ⅱ digestion for 15 minutes is a better choice to isolate HUVECs.
Objective To evaluate the clinical experience and skills of laparoscopic totally extraperitoneal hernia repair with non-stapling of mesh and without using a balloon dissection in order to spread and popularize this kind of operation. Methods Under general anesthesia, 32 patients (42 sides) with inguinal hernias were repaired by laparoscopic totally extraperitoneal hernia repair with non-stapling of mesh and without using a dissection balloon from August 2005 to December 2007. Results All of operations were successfully performed. The operative time was 60-120 minutes, the blood loss was 20-60 ml, and the length of postoperative hospitalization was 3 days. All cases were followed up for 8 to 18 months and found no recurrence. Conclusion Laparoscopic totally extraperitoneal hernia repair with non-stapling of mesh and without using a balloon dissection is feasible, reliable and effective, offering a low recurrence rate, while its price is higher than tension-free herniorrhaphy by traditional method.
Objective To investigate the radiographic and clinical outcomes of dorsal intercarpal ligament capsulodesis (DILC) procedure for chronic static scapholunate dissociation. Methods Between January 2008 and January 2011, 12 patients with chronic static scapholunate dissociation were treated with DILC. Of 12 cases, 10 were male and 2 were female with an average age of 42 years (range, 20-66 years). All injuries were caused by falling. The interval from injury to surgery was 3-19 months (mean, 8 months). Physical examination at admission showed wrist tenderness and limited range of motion (ROM). Radiological examination showed that scapholunate gap was greater than 3 mm on posteroanterior view, and scapholunate angle more than 60° on the lateral view. Before operation, the grip strength was (25.4±8.2) kg; the wrist ROM was (56.7±11.5)° in flexion and (52.0±15.2)° in extension; visual analogue scale (VAS) score was 6.3±1.4; and disabilities of arm, shoulder & hand (DASH) score was 39.5±7.4. According to Garcia-Elias staging criteria, all cases were rated as stage 4, indicating that the scapholunate interosseous ligament was completely injured and reduction could easily be obtained. Eight patients had wrist instability. Results Primary healing of incision was achieved, no complication was found. All patients were followed up 13-34 months (mean, 15.9 months). During surgery, all deformities were corrected completely, but 7 patients (58%) recurred at 1 month after Kirschner wire removal. Compared with preoperative ones, the scapholunate gap, scapholunate angle, radiolunate angle, lunocapitate angle, and wrist height ratio at 1 month after Kirschner wire removal and last follow-up showed no significant difference (P > 0.05); the wrist flexion and extension ROM were significantly decreaed to (46.8±7.2)° and (42.0±9.0)° at last follow-up (P < 0.05); the grip strength was significantly increased to (32.7±9.6) kg at last follow-up (P < 0.05); VAS score and DASH score were improved to 1.7±1.0 and 8.1±8.7 (P < 0.05). Conclusion Carpal collapse will recur in short time after DILC. DILC is not the best way to treat chronic static scapholunate dissociation.
ObjectiveTo evaluate the effects of sevoflurane and propofol on preoperative implicit and explicit memories in general anaesthesia patients of elective surgery. MethodsThe surgical inpatients in Sichuan Provincial People's Hospital were enrolled from December 2013 to May 2014, and were randomly divided into three groups (S, P, M). In Group S, anesthesia was induced and maintained with sevoflurane. In Group P, anesthesia was induced and maintained with propofol. Midazolam was not utilized throughout the whole anaesthesia for the above groups. Patients in Group S and Group P were given a list of test materials to remember and listen before the anesthesia. Within 12 to 36 hours after operation, memory was assessed, based on the Buchner's model applied on the process dissociation procedure (PDP) using a phrases task. The Group M was given the same test materials, and received test with the PDP in 12 to 36 hours before surgery. Value A and value R were used to represent the implicit memory score and the explicit memory score, respectively. ResultsA total of 150 patients were included, and 50 cases were included in each group. During testing, 2 cases were excluded, 3 cases were loss to follow-up, so finally 49 cases were included in the Group S, 47 cases in the Group P and 49 cases in the Group M. The results showed that there were significant differences in the implicit memory score (A) and the explicit memory score (R) among the three groups (all P values <0.05). The explicit memory score (R) of the Group M was higher than those of the Group P and Group S (all P values <0.05), the implicit memory score (A) in the Group M was higher than those of the Group S and Group P (all P values <0.05), and the implicit memory score (A) in the Group S was higher than that of the Group P (P<0.05). ConclusionPropofol and sevoflurane can decrease the score of explicit memory after anesthesia within 12 to 36 hours, and there are no significant differences in explicit memory between the two drugs. Both propofol and sevoflurane can decrease the score of implicit memory, but the influence of sevoflurane on the implicit memory is less than propofol within 12 to 36 hours.
【Abstract】 Objective To summary the effects of staged pelvic closure using external fixator combined withreconstruction plate for old Tile B1 pelvic fracture. Methods From August 2000 to August 2006, 14 patients (9 males and 5 females, age ranging from 21 to 65 years with old Tile B1 pelvic fracture with pubic symphysis separation were treated. The injuries were caused by the traffic accident in 10 patients, high crash in 3, and crush in 1. The duration ranged from 4 weeks to 3 months. The 14 patients were compl icated with other injuries in some degrees. The X-ray and CT showed pubic symphysis separation. In 13 patients, pubic symphysis separation distance was more than 2.5 cm, who also had fracture or dislocation in the posterior structure of pelvis. The X-ray films showed the mean pubic symphysis separation distance was (6.67 ± 2.11) cm preoperatively. The 14 patients underwent pelvic external fixation at first and staged pelvic closure gradually for the pubic symphysis separation. After 2 to 3 weeks, when the pubic symphysis separation distance was less than 1 cm, the patients underwent open reduction and internal fixation with reconstruction plate. Pubic symphysis separation distance was measuredin the preoperative and postoperative pelvic anterioposterior X-ray films. The condition of the posterior structure of pelvis was observed in CT films. The functions of patients were assessed according to Majeed grading system. Results All incisions healed by first intention. The 14 patients were followed up for 6 months to 2 years (15 months on average). The X-ray films showed the mean pubic symphysis separation distance of post-operation was (0.85 ± 0.23) cm, showing statistically significant difference when compared with that of pre-operation (P lt; 0.05). The CT films showed fracture and dislocation of the posterior structure of pelvis had bony heal ing after 6 to 12 months. According to the Majeed grading system, the results were excellent in 5 cases, good in 4 cases, fair in 4 cases and poor in 1 case; the excellent and good rate was 64.29%. Conclusion Staged pelvic closure using external fixation combined with reconstruction plate for old pelvic fracture with pubic symphysis separation can reduce the pubic symphysis separation distance significantly. Satisfactory effects can be expected in treating the patients with TileB1 fracture.
To set up an economic and effective method for islet isolation from rat, and thereby prove a laboratory protocol of animal model for cl inical islet transplantation. Methods Twenty-five adult male SD rats weighing 230-380 g were used as organ donor. In each of 5 repeated experiments, pancreatic islets of 5 animals were isolated by intraductal infusion of compound sodium chloride injection (CSCI), and subsequently, digested with low concentration (0.5 mg/mL)of collagenase V solution. Islet purification was performed by using a discontinuous density gradient centrifugation thatwas prepared with 27.0%, 23.0%, 20.5% and 11.0% of Ficoll 400. Islet yield and purity were determined by dithizon (DTZ)stain, and propidium iodide (PI)/fluorescein diacetate (FDA) double stain was used to check viabil ity of islets. The endocrine secretory function was assessed by insul in secretion in either low (2.8 mmol/L) or high (25.0 mmol/L) glucose incubation after 3 days of culture in RPMI1640 media. Results Average islet digestion time of 5 experiments was (13.8 ± 1.6) min. Before purification, average isolated number was (5 626 ± 422) islets, and the number was significantly reduced to (2 914 ± 485) islets after purification (P lt; 0.01). The average recovery rate was 51.6% ± 6.0%, and the average yield was (583 ± 97) islets/pancreas. The average purity and viabil ity of islets were 90.2% ± 3.4% and 81.6% ± 7.0%, respectively. After 3 days of culture, insul in secretion of the islets was (116.1 ± 17.4) EU/L in high glucose incubation, which was significantly higher than that of low glucose environment [(39.7 ± 7.5) EU/L, P lt; 0.01)]. The average insul in stimulation index was 3.0 ± 0.4. Conclusion The islet isolation with the CSCI solution and digestion with low concentration of collagenase V decrease experimental cost and also have a beneficial effect on islet recovery and their function.
To separate the overlapped protein spots in two-dimensional gel electrophoresis (2-DE) images, we proposed an auto-separating algorithm based on valley characteristics. Firstly, the marker-controlled watershed algorithm was used to detect the initial outlines of the object regions. Secondly, medial axis transform and hierarchical branch pruning method were applied to the main skeletons of the object regions, and each main skeleton was fitted into line segments to describe the overlap directions. Then, the 3-dimensional model of the object region was scanned on the normal planes of the line segments to find the valley locations. And finally, a validation model was adopted to construct separation lines. The experiments on 2 real scanned 2-DE images showed that the true overlap separate (TOSs) were 78.95% and 85.71%, respectively. The results indicated that the proposed algorithm was better than the existing algorithms and could be used in engineering practice.
目的：建立并探讨一种体外分离、培养和扩增骨髓间质干细胞的优化方法。方法：采用密度梯度离心法和全骨髓贴壁法分离培养大鼠骨髓干细胞，应用倒置显微镜和免疫组化法观察和鉴定骨髓间质干细胞。结果：大鼠骨髓干细胞呈均一的梭形成纤维细胞样，形成集落样生长，显微结构表现出干细胞特征；免疫组化显示CD14、CD45阴性，CD44、CD90阳性。与密度梯度离心法比较，贴壁法获得的骨髓干细胞活性高，增殖力强，克隆形成早，传代时间短。结论：全骨髓贴壁法和密度梯度离心法均可获得高纯度贴壁生长的骨髓干细胞，其中全骨髓贴壁法方法简单易行，骨髓干细胞增殖快，活性好，传代力持久，是一种有实用价值的骨髓干细胞培养方法。