ObjectiveTo evaluate the effect of using Schwann-like cells derived from human umbilical cord blood mesenchymal stem cells (hUCBMSCs) as the seed cells to repair large sciatic nerve defect in rats so as to provide the experimental evidence for clinical application of hUCBMSCs. MethodsFourty-five male Sprague Dawley (SD) rats in SPF grade, weighing 200-250 g, were selected. The hUCBMSCs were harvested and cultured from umbilical cord blood using lymphocyte separating and high molecular weight hydroxyethyl starch, and then was identified. The hUCBMSCs of 3rd generation were induced to Schwann-like cells, and then was identified by chemical derivatization combined with cytokine. The acellular nerve basal membrane conduit was prepared as scaffold material by the sciatic nerve of SD rats through repeated freezing, thawing, and washing. The tissue engineered nerve was prepared after 7 days of culturing Schwann-like cells (1×107 cells/mL) on the acellular nerve basal membrane conduit using the multi-point injection. The 15 mm sciatic nerve defect model was established in 30 male SD rats, which were randomly divided into 3 groups (10 rats each group). Defect was repaired with tissue engineered nerve in group A, with acellular nerve basal membrane conduit in group B, and with autologous sciatic nerve in group C. The nerve repair was evaluated through general observation, sciatic function index (SFI), nerve electrophysiology, weight of gastrocnemius muscle, and Masson staining after operation. ResultsThe hUCBMSCs showed higher expression of surface markers of mesenchymal stem cells, and Schwann-like cells showed positive expression of glia cell specific markers such as S100b, glial fibrillary acidic protein, and P75. At 8 weeks after operation, the acellular nerve basal membrane conduit had no necrosis and liquefaction, with mild adhesion, soft texture, and good continuity at nerve anastomosis site in group A; group B had similar appearance to group A; adhesion of group C was milder than that of groups A and B, with smooth anastomotic stoma and no enlargement, and the color was similar to that of normal nerve. SFI were gradually decreased, group C was significantly greater than groups A and B, group A was significantly greater than group B (P<0.05). The compound action potential could be detected in anastomotic site of 3 groups, group C was significantly greater than groups A and B, and group A was significantly greater than group B in amplitude and conduction velocity (P<0.05). Atrophy was observed in the gastrocnemius of 3 groups; wet weight's recovery rate of the gastrocnemius of group C was significantly greater than that of groups A and B, and group A was significantly greater than group B (P<0.05). Masson staining showed that large nerve fibers regeneration was found in group A, which had dense and neat arrangement with similar fiber diameter. The density and diameter of medullated fibers, thickness of myelinated axon, and axon diameter of group C were significantly greater than those of groups A and B, and group A was significantly greater than group B (P<0.05). ConclusionTissue engineered nerves from hUCBMSCs-derived Schwann-like cells can effectively repair large defects of the sciatic nerve. hUCBMSCs-derived Schwann-like cells can be used as a source of seed cells in nerve tissue engineering.
ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation. MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro. ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group. ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
【摘要】 目的 探讨经脐单孔腹腔镜胆囊切除术的临床可行性及其优缺点。 方法 回顾分析2010年7-9月行经脐单孔腹腔镜胆囊切除术34例患者临床资料。 结果 患者均顺利完成单孔腹腔镜胆囊切除术，手术平均时间为65 min，术后平均住院时间为3 d，术后未发生出血、感染、胆瘘等并发症。 结论 单孔腹腔镜胆囊切除术是安全可行的，术后腹部无明显瘢痕，美容效果明显。【Abstract】 Objective To evaluate the feasibility and value of the trans-umbilical single-port laparoscopic cholecystectomy. Methods The clinical data of 34 patients who underwent trans-umbilical single-port laparoscopic cholecystectomy from July to September 2010 were retrospectively analyzed. Results The operations of 34 patients were successfully performed. The mean operative duration was 65 minutes, and the mean duration in hospital after the operation was 3 days. No infection, postoperative bleeding, and biliary leakage occurred postoperatively. Conclusion Trans-umbilical single-port laparoscopic cholecystectomy is safe and feasible with good cosmetic effect.
摘要:目的:探讨创伤性感染性假性股动脉瘤的诊断和外科治疗的临床经验。方法:回顾性分析２１例创伤性感染性假性股动脉瘤的临床资料,均行瘤体摘除及彻底的清创后,分别采用了血管结扎术和血管修复重建术两种不同的手术治疗方法。结果:血管修复重建术组中３例术后出现血管破裂大出血,要再次手术,１５例行股动脉结扎术,全部保肢成功。结论:瘤体切除加血管移植术是一种理想的方法,但在无条件行血管移植时,股动脉结扎术可做为一种有效的方案,对伴有皮肤缺损者行对侧胸脐皮瓣转移术。Abstract: Objective: 〖WT5”BZ〗To explore the traumatic infected femoral pseudoaneurysm diagnosis and surgical treatment of clinical experience. Methods: Retrospective analysis of 21 cases of traumatic infected femoral pseudoaneurysm of the clinical data were performed and the tumor removed after thorough debridement, respectively vascular ligation and blood vessel repair and reconstruction surgery of two different surgical treatment. Results: The blood vessel repair and reconstruction surgery group, three cases of postoperative bleeding blood vessel ruptures occurred, we must resurgery, 15 routine femoral artery ligation, all of the success of limb salvage. Conclusion: The tumor resection plus vascular graft is an ideal way, but in an unconditional line of vascular grafts, the femoral artery ligation can be used as an effective program for skin defects associated with the contralateral breast underwent umbilical flap transfer of patients.
ObjectiveTo compare the advantages and disadvantages of transumbilical single port (TUSP) and conventional laparoscopic cholecystectomy (LC). MethodsThe clinical data of 45 patients underwent elective LC were analyzed, 20 patients with TUSP LC (TUSP-LC group), 25 patients with conventional LC (conventional LC group). The operation time, Child-Pugh score and painkiller application frequency within three days after operation, the first time of out of bed and hospital stay after operation, intraoperative blood loss, chronic pain within one month after surgery were compared between two groups. ResultsAll cases were operated successfully except one patient in the conventional LC group. The frequency of painkiller application within three days after operation and postoperative hospital stay in the TUSP-LC group were better than those in the conventional LC group (Plt;0.05). There were no significant differences on postoperative chronic pain of surgical area within 1 month and Child-Pugh score between two groups (Pgt;0.05). The operation time and intraoperative blood loss in the conventional LC group were less than those in the TUSP-LC group (Plt;0.05, Plt;0.01). ConclusionTUSP LC has the advantages of small wound, slight pain, and fast recovery.
目的 探讨利用常规腹腔镜器械完成经脐单孔腹腔镜结直肠手术的可能性和技术要点。方法 收集中国医科大学附属盛京医院微创外科于2009年4月至2010年1月期间施行的12例经脐单孔腹腔镜结直肠手术的临床资料。阑尾炎8例，均为女性，平均年龄40岁； 回盲部肿物2例，均为女性，其中1例为回盲部淋巴水瘤（68岁），另1例为回盲部溃疡性结肠炎（47岁）； 乙状结肠息肉1例，女，55岁； 直肠癌1例，男，52岁。 12例均于脐部行2.5~3.0 cm长单切口，利用常规腹腔镜手术器械完成手术。结果 8例阑尾手术，手术时间20~50 min，出血量均少于10 ml； 2例回盲部切除术手术时间分别为60 min和90 min，出血量分别为10 ml和20 ml； 1例乙状结肠切除术用时120 min，术中出血约50 ml，术后4 d拔除引流管； 直肠癌手术时间210 min，术中出血少于200 ml，术后1周拔除引流管并出院。结论 利用常规腹腔镜手术器械完成经脐单孔腹腔镜结直肠手术安全可行。
目的探讨经脐单孔腹腔镜胆囊切除术（LC）的临床应用。方法分析我院2009年1月至2010年5月期间120例因结石性胆囊炎和胆囊息肉行经脐单孔LC患者的临床资料。结果98例患者手术成功，手术时间38～126 min，平均50.3 min。22例单孔手术失败改成两孔完成手术。住院时间2～4 d，平均2.5 d。全组患者无出血及漏胆并发症发生，仅2例（1.7%）脐部戳孔处术后轻度感染，经局部换药治疗2周愈合。89例（90.8%）采用单孔法患者获得1～15个月（平均7.3个月）随访，均无并发症发生。结论单孔LC安全可行，但使用现有腹腔镜设备操作难度较大，器械及技术尚需进一步完善。
Objective To explore the clinical presentation and diagnosis and treatment of prehepatic portal hypertension （PPH） and discuss its surgical strategies. Methods Forty-six cases of PPH treated in the 2nd Artillery General Hospital and Peking Union Medical College Hospital from January 2000 to May 2009 were analyzed retrospectively, including 2 cases of Abernethy abnormality. All patients were evaluated by indirect portal vein angiography, CT angiography and (or) portal duplex system Doppler ultrasonography before treament. Surgical strategies included: 23 cases with meso-caval shunt, 8 cases with splenectomy and spleno-renal vein shunt, 1 case with porta-caval shunt, 2 cases with paraumbilical vein-jugular vein shunt, 3 cases with portal azygous disconnection, 1 cases with splenectomy and portal azygous disconnection, 1 case with sigmoidostomy and closed the fistula of sigmoid six months later, 1 case with resection of part of small intestine due to acute extensive thrombosis of portal vein system, 4 cases with selective superior mesenteric artery and (or) splenic artery thrombolytic infusion therapy, 2 cases remained no-surgical option and underwent conservative treatment. Results Forty-four patients were followed-up from 2 months to 5 years， average of 23.4 months, one patient without surgical treatment was lost. Satisfactory outcomes were obtained in 34 patients with various shunts, which expressed as a release of hypersplenism and gastrointestinal hemorrhage. Two cases were treated with meso-caval shunt because of rehemorrhage in month 13 and 24 and one died in month 8 after disconnection, one died on day 40 after thrombolytic therapy due to putrescence of intestines, one who remained no-surgical option underwent hemorrhage 4 months later, and then went well by conservative treatment. Conclusion The key of treatment of PPH is to reduce the pressure of hepatic portal vein. Surgical managements of shunt and selective superior mesenteric artery and (or) splenic artery thrombolytic infusion therapy are safe and effective, but individual treatment strategy should be performed.
Objective To observe the effects of cryopreservation and resuscitation on the biological characteristics of mesenchymal stem cells (MSCs) derived f rom human umbilical cord blood. Methods MSCs were isolated and cultured f rom human umbilical cord blood in vitro. The cells were passaged , and the third generation of MSCs were cryopreserved in－196 ℃ liquid nitrogen for 4 weeks with cryopreservation medium , which contained 10 % dimethyl sulfoxide (DMSO) and 90 % fetal calf serum ( FCS) . The morphology , proliferation and differentiation of MSCs were investigated and compared with those of MSCs before cryopreservation. Results There was no significant difference of morphology between pre-cryopreserved MSCs and the ones af ter resuscitation. It was observed that all MSCs were spindle-shaped and showed adherence growth characteristic before and af ter cryopreservation. The cell growth curves of MSCs were also similar before and af ter cryopreservation. Even though the curve of resuscitated MSCs descended a little as compared with that of pre-cryopreserved MSCs , there was no significant difference ( Pgt; 0. 05) . After 2-week adipocytic differentiation induction , fat drops could be found in the kytoplasm of MSCs and they were red when stained with oil-red O staining , which suggested that MSCs could be induced and differentiated into adipocytes. Af ter 4-week osteoblastic differentiation induction , MSCs could be induced and differentiated into osteoblasts , and calcium node showed black when stained with Von Kossa staining. There were no significant changes of the differentiating ability of MSCs into adipocyte and osteoblast before and after cryopreservation. Conclusion MSCs derived from human umbilical cord blood maintains their biological characteristics af ter cryopreservation and resuscitation.
Objective To study the influence of three different ways of myogenic induction on Ca2+ regulation of mesenchymal stem cells (MSCs) derived from umbilical cord blood. Methods From January 2007 to April 2010, three different ways of myogenic induction including the adoptions of 5azacytidine, extraction of myocardium, and myocardial differentiation medium were used to induce MSCs derived from the umbilical cord blood of dogs in Xinhua Hospital of Shanghai Jiaotong University. Confocal laser scanning microscope was used to detect cells induced by the three abovementioned methods, cardiomyocytes and Ca2+ combined with Fluo3/AM inside the MSCs. For each group of cells, 2 to 5 visual fields were chosen, and 30 visual fields were recorded for each kind of cells. The mean fluorescence intensity of ten images shot in one minute was used to reflect the concentration of free intracellular Ca2+. Furthermore, the change of the concentration was continuously monitored by optical density(OD) value. Results After induction, the Ca2+ concentration inside the MSCs was significantly higher than that inside the cardiomyocytes (F=59.400, P=0.000). There was a statistical difference among the intracellular Ca2+ concentration induced respectively by 5azacytidine, extraction of myocardium, and myocardial differentiation medium (F=18.988, P=0.000). No significant difference existed between the intracellular Ca2+ concentration induced by 5-azacytidine and extraction of myocardium (OD value: 1 076.88±44.65 vs. 1 040.90±37.48, P=0.186), while the intracellular Ca2+ concentration induced by 5azacytidine was significantly higher than that induced by myocardial differentiation medium (OD value: 1 076.88±44.65 vs. 973.91±46.49, P=0.001), and the intracellular Ca2+ concentration induced by extraction of myocardium was significantly higher than that induced by myocardial differentiation medium (OD value: 1 040.90±37.48 vs. 973.91±46.49, P=0.001). The concentration of intracellular Ca2+ induced by all the three different methods fluctuated spontaneously, which was quite similar with the cardiomyocytes, but the frequency and the scope of the fluctuation were quite different. Ca2+ was released instantly by KCl stimulation in the two groups of MSCs pretreated by 5-aza and extraction of myocardium. Though MSCs pretreated by myocardial differentiation medium had response to KCl stimulation, Ca2+ could not be released in this group. On the contrary, the duration of Ca2+ release was prolonged. Conclusion Ca2+ regulation system of MSCs derived from umbilical cord blood can be influenced by these myogenic inductions. However, the reason and effect of the differences need to be elucidated by further investigation.