【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.
ObjectiveTo summarize the diagnosis and treatment of thoracoomphalopagus conjoined twins according to successful separation of two cases of conjoined twins. MethodsA retrospectives study was performed on two pairs of thoracoomphalopagus twins. One case was omphalopagus with complex congenital heart disease (transposition of the great arteries and single cardiac ventricle), and the other was sternoxiphopagus with single pericardium and complex congenital heart disease (single cardiac ventricle and cardiac atrium). All the two cases of conjoined twins shared the common livers. The separation surgery was performed at one month after birth. Results All the two cases of conjoined twins were successfully separated. These conjoined twins were connected by a liver bridge, and shared a pericardium or had a contact pericardium, respectively. The coloboma of peritoneum, diaphragm， and mediastinum were repaired by Gore-Tex patch. Each patient, to varying degrees, had a wound infection after surgery, and healed by taking out the Gore-Tex, debridement or drainage. The first two infants were discharged on 37 d after operation, but one died of pneumonia after 6 months. The two other infants were discharged on 40 d and 93 d after operation, respectively. ConclusionsDetailed information about the twins’ general and conjoined condition, especially on cardiovascular system and the joined liver, shall be understood by the imagination examinations before the operation. Adequate preoperative preparation, elaborate liver separation and rational reconstruction for thoracic and abdominal wall are the key to successful operation.
Kidney transplantation is an ideal treatment for patients with end-stage renal disease. Circulating alloantibodies against donor human leukocyte antigens and blood group antigens can impair allografts, shorten allograft survival, and limit access to kidney transplantation. Furthermore, the presence of donor specific antibodies is associated with increased incidence of antibody-mediated rejection and decreased graft survival following transplantation. Plasmapheresis, an extracorporeal therapy directed at removing plasma proteins that has been found to minimize the effects of perioperative sensitization in kidney transplantation. Plasmapheresis enables transplantation across the barrier of ABO blood group incompatibility. In addition, it is also an important approach for the treatment of antibody-mediated rejection. Therefore, studying the application of plasmapheresis in perioperative period of kidney transplantation is expected to increase the chance of transplantation and improve the outcomes following transplantation. This article introduces the application of plasmapheresis in the perioperative period of kidney transplantation.
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 provide theoretical and technological support for further study of liver metabolism and disease by comparing the advantages and disadvantages of various artificial liver models (biological). MethodsLiteratures were searched and compared to summarize the requirement for liver donor, isolation, and culture of hepatocyte. ResultsIn the separation method of hepatocyte, mechanical separation method had no requirement for liver donor, and was easy to acquire hepatocyte, while the acquired hepatocyte would be destructed severely, and the survival rate was low. On the other hand, the restriction of the digestion of the hepatocytes to the liver cell samples was unlimited, while the key of the enzyme digestion method was to regulate the balance between enzyme concentration and digestion time, which was limited to function researches of hepatocyte, and research about the responds of hepatocyte against outside, and other few researches. Perfusion digestion method had been widely applied for animal test. The Ca2+, collagenase and perfusion rate, pH value, buffer, and intubation method all play vital roles. During the cultivation, we needed to choose different methods according to several experiments, and add different additives in the appropriate medium. Different biological reactors had different advantages, disadvantages, and applicable conditions. ConclusionsThe donor selection is based on various experimental purposes to harvest hepatocytes from different sources. Whether on the separation process or on the cultivation process, according to the specific circumstances, such as the concentration, perfusion time, and the choice of different kinds of culture medium, we can choose different kinds of bioreactors, but all kinds of methods are still remained with multiple insufficiencies, which require more researchers to improve.
Blind source separation technique based on independent component analysis (ICA) can separate blood volume pulse (BVP) from the facial video and then realize the telemetry of heart rate, blood oxygen saturation, respiratory rate and other vital signs parameters. However, the superiority of ICA in BVP extraction has not been demonstrated in the existing researches. Some researchers suggested using traditional G-channel method for BVP extraction (G-BVP) instead of ICA method (ICA-BVP). This study investigated the applicability of ICA-BVP comparatively. To solve the inherent permutation problem of ICA, a spectral kurtosis-based method was proposed for BVP identification. The experimental results based on the facial video datasets from 9 subjects shows that ICA-BVP method has apparent advantages in motion artifacts attenuation and ambient light changes elimination. The kurtosis-based method achieved a good performance in BVP identification and dynamic heart rate (HR) estimation. In practical application, the proposed ICA-BVP method could present a better stability and accuracy in vital signs parameters extraction.
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。
【Abstract】ObjectiveTo develop a method of adult porcine pancreatic islet isolation.MethodsThe tails of adult porcine pancreas were perfused through the pancreatic duct with 0.1% cold collagenase(type Ⅺ) and incubated at 38.5 ℃.The digested tissue was dispersed in 4 ℃ Hanks balanced salt solution(HBSS).The tissue suspensions were filtered through a 600 μm mesh.The residual tissue was resuspended in cold HBSS,and put in the Ricordi’s chamber and shaken for 5 minutes,then filtered again.The isolated islets were divided into three groups: control group(n=14),Pefabloc(trypsin inhibitor,n=8) group and FOY(trypsin inhibitor,n=5) group.The collagenase solution of the Pefabloc and FOY group was supplemented with 1.0 mmol/L Pefabloc and FOY respectively. ResultsThe islet yields of the Pefabloc group and FOY group 〔(11 848±3 530) islet/g pancreas and (14 496±3 693) islet/g pancreas〕 were significantly higher than that of the control group 〔(8 505±3 349) islet/g pancreas〕,P＜0.05.The activity of pancreatic protein enzyme in digestive fluid after digestion in control group was higher than the activity of pancreatic duct before injection and Pefabloc group(P＜0.01),which the control group, pancreatic duct before injection and Pefabloc group were (114.7±50.0) BAEEU,(4.0±1.8) BAEEU and (5.5±2.7) BAEEU,respectively.The pancreatic duct before injection and Pefabloc group showed no significant difference in statistics. In control group,when the harvest of islet was more than 8 000/g,the activity of pancreatic protoin enzyme was less than that with the harvest of islet below 8 000/g 〔(78.3±26.7) BAEEU vs (137.5±48.4) BAEEU,P＜0.05〕.Islet after purification in control group,Pefabloc group and FOY group showed good insulin secretion ability for different concentration of glucose.ConclusionA higher porcine pancreatic islet yield can be obtained by this method of pancreatic islet isolation and prophylactic administration of trypsin inhibitors consistently produce excellent islet yields.
ObjectiveTo understand the latest progress of enrichment technology of circulating tumor cells (CTCs), and summarize the principle, advantages and disadvantages of various enrichment technologies and their applications in primary liver cancer (PLC). MethodThe literature relevant to the enrichment methods of CTCs in the PLC was reviewed and summarized. ResultsThe clinical significances of CTCs in the early diagnosis and staging, hierarchical diagnosis and treatment, and efficacy monitoring of patients with PLC had been recognized. There were many separation and enrichment technologies for CTC, which were mainly based on the differences of physical and biochemical characteristics, as well as the combination of enrichment methods with various principles. Each enrichment method had corresponding advantages and disadvantages, and few enrichment methods for CTC was applied to PLC. ConclusionsAlthough many problems need to be solved in enrichment method of CTCs at present, it is believed that the existing problems will be solved one by one with continuous improvement of technology. And CTC detection is expected to apply in clinical, so as to provide more efficient diagnosis and treatment methods for patients with PLC.