ObjectiveTo investigate the effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells (ADSCs).MethodsADSCs were isolated from 5-week-old Sprague Dawley rats (weight, 100-150 g) and cultivated. The cells at passages 3-5 were inoculated to prepare cell climbing slices, subsequently was exposed to direct-current electrical stimulations (ES) at electric field strengths of 1, 2, 3, 4, 5, and 6 V/cm on a homemade electric field bioreactor (groups A1, A2, A3, A4, A5, and A6); at electric field strength of 6 V/cm, at 50% duty cycle, and at frequency of 1 and 2 Hz (groups B1 and B2) of square wave ES; at electric field strength of 6 V/cm, at pulse width of 2 ms, and at frequency of 1 and 2 Hz (groups C1 and C2) of biphasic pulse wave ES; and no ES was given as a control (group D). The changes of cellular morphology affected by applied ES were evaluated by time-lapse micropho-tography via inverted microscope. The cell alignment was evaluated via average orientation factor (OF). The cytoske-leton of electric field treated ADSCs was characterized by rhodamine-phalloidin staining. The cell survival rates were assessed via cell live/dead staining and intracellular calcium activities were detected by calcium ion fluorescent staining.ResultsThe response of ADSCs to ES was related to the direct-current electric field intensity. The higher the direct-current electric field intensity was, the more cells aligned perpendicular to the direction of electric field. At each time point, there was no obvious cell alignment in groups B1, B2 and C1, C2. The average OF of groups A5 and A6 were significantly higher than that of group D (P<0.05), but no significant difference was found between other groups and group D (P>0.05). The cytoskeleton staining showed that the cells of groups A5 and A6 exhibited a compact fascicular structure of cytoskeleton, and tended to be perpendicular to the direction of the electric field vector. The cellular survival rate of groups A4, A5, and A6 were significantly lower than that of group D (P<0.05), but no significant difference was found between other groups and group D (P>0.05). Calcium fluorescence staining showed that the fluorescence intensity of calcium ions in groups A4, A5, and A6 was slightly higher than that in group D, and no significant difference was found between other groups and group D.ConclusionThe direct-current electric field stimulations with physiological electric field strength (5 V/cm and 6 V/cm) can induce the alignment of ADSCs, but no cell alignment is found under conditions of less than 5 V/cm direct-current electric field, square wave, and biphasic pulse wave stimulation. The cellular viability is negatively correlated with the electric field intensity.
ObjectiveTo study the growth of adipose-derived stem cells (ADSCs) planted in three-dimensional (3D) materials, a 3D cultured ADSCs system based on microbial transglutaminase (mTG) enzyme crosslinked gelatin hydrogel was constructed. MethodsADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated in situ (2D environment) and cultured in the 3D materials (3D environment). The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment. ResultsThe result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant (P < 0.05). But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly. ConclusionADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.
Objective To explore risk factors and treatment strategies of liver resection surface infection following laparoscopic hepatolithiectomy for patient with complicated intrahepatic bile duct stones. Methods The clinical data of 45 patients with complicated intrahepatic bile duct stone underwent laparoscopic hepatectomy from January 2014 to April 2017 in this hospital were analyzed. The liver resection surface infection rate, pathogenic bacteria distribution, factors of operation, antibiotic use time, volume of drainage, and drainage tube placement time were analyzed. Results A total of 13 cases of liver resection surface infection occurred following the laparoscopic hepatolithiectomy in the 45 cases, the infection rate was 28.89%. Totally 24 strains of pathogens were isolated from the infected patients, including 9 strains of gram-positive bacteria and 15 strains of gram-negative bacteria. The mainly postoperative complications included 16 cases of the biliary leakage, 5 cases of the effusion and empyema, the average drainage volume was about 200 mL after the surgery. The double pipes were placed in the 10 patients in the operation. The drainage tubes were placed in the 23 patients under the ultrasound or CT intervention after the surgery, the average time of drainage tube placement was 8 d. The results of univariate analysis showed that the past biliary surgery history, combined with liver cirrhosis, double pipe drainage, operation time, and postoperative biliary leakage were associated with the liver section surface infection following the laparoscopic hepatolithiectomy (P<0.050). The results of multivariate analysis identified that the past biliary surgery history and postoperative biliary leakage were the risk factors (P<0.050), while the double pipe drainage was the protective factor (P<0.050) for liver resection surface infection following the laparoscopic hepatolithiectomy. Conclusions Prophylactic treatment such as perfect preoperative management and careful intraoperation should be taken for risk factors of liver section surface infection following laparoscopic hepatolithiectomy. Actively effective treatment strategies should be given if postoperative liver section surface infection existence.
目的 分析肝脏良性肿瘤的临床诊断、治疗及治疗效果。方法 对川北医学院附属医院2008年2月至2011年2月期间收治并经术后病理学检查证实为肝脏良性肿瘤的156例患者的临床资料进行回顾性分析。结果 156例患者中肝血管瘤119例，肝细胞腺瘤13例，肝内胆管囊腺瘤3例，肝局灶结节性增生9例，肝淋巴管瘤4例，肝血管平滑肌脂肪瘤3例，肝炎性假瘤5例。3例肝局灶结节性增生患者行保守治疗，随访1年，其肿瘤大小和肝功能均无明显变化； 余153例患者行手术治疗，全部切除了病灶，病灶切除率为100％，术后随访6～48个月，平均24个月，均无死亡及复发。结论 肝脏良性肿瘤的症状不典型，缺乏特异性表现，易误诊，应完善超声、CT及MRI检查，并结合病史进行综合分析。对于具有临床症状、肿瘤体积较大（肿瘤直径＞5cm）的肝脏良性肿瘤患者，应积极行手术治疗，术后密切随访。
Bone marrow-derived mesenchymal stem cells (BMSCs) for repairing damaged heart tissue are a new kind of important treatment options because of their potential to differentiate into cardiomyocytes. We in this experiment investigated the effect of different electrical stimulation time on the expression of myocardial specificity gene and protein in rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs of second or third generation were randomly divided into three groups, i.e. electrical stimulation (ES) group, 5-Azacytidine (5-Aza) group and the control group. The rBMSCs in the ES groups with complete medium were exposed to 2 V, 2 Hz, 5 ms electrical stimulation for 0.5 h, 2 h, 4 h, and 6 h respectively every day for 10 days. Those in the 5-Aza group were induced by 5-Aza (10 μmol/L) for 24 h, and then cultured with complete medium for 10 days. Those in the control group were only cultured with complete medium, without any treatment, for 10 days. The rBMSCs' morphological feature in each group was observed with inverted phase microscope. The mRNA expression of myocyte-specific enhancer factor 2C (MEF-2C) and connexin 43 (Cx43) were examined with Real-Time quantitative PCR and the protein expression of MEF-2C, Cx43 were detected with Western Blot method. The results showed that the mRNA expression level of the MEF-2C, Cx43 and the protein expression level of MEF-2C, Cx43 were significantly higher in the ES group and 5-Aza group than those in the relative control group (P < 0.05). It suggests that electrical stimulation could play a part of role in the induction of the rBMSCs to differentiate into the cariomyocyte-like cells in vitro and the effectiveness of the electrical stimulation with 2 h/d had the best in our experiement. But the mechanism how electrical stimulation promotes the differentiation of rBMSC into cardiomyocyte is still unclear.
ObjectiveTo investigate how to shorten the learning curve of the laparoscopic pancreaticoduodenectomy (LPD). MethodsClinical data of 5 patients who underwent the LPD in our hospital from May 2015 to November 2015 were retrospectively analyzed. ResultsThe mean age of 58.8 years old. There were four patients who were diagnosed with periampullary tumor, one patient was distal bile duct carcinoma. The median operative time was 588 min, the average blood loss was 290 mL, the time of feeding was 5 days, the mean hospital stay was 25 days. One case died of cardiovascular event on postoperative day 1. One patient had postoperative bleeding after LPD, who recovered smoothly after reoperation for hemostasis laparoscopiclly. Conciusions LPD needs basic learning curve. The key of this procedure are appropriate treatment of pancreatic head and digestive tract reconstruction. Rich operative experience of surgeon in pancreaticoduodenectomy, optimization of the operation process, skilled in laparoscopic procedures, appropriate cases, appropriate perioperative management, and steady surgical team are also important factor for the success of LPD and shorten learning curve.
ObjectiveTo summarize the key technical points, applicability, feasibility, and safety of laparoscopic spleen-preserving distal pancreatectomy (LSPDP).MethodA retrospective analysis was performed for the clinical data of 22 patients who were admitted to the Affiliated Hospital of North Sichuan Medical College from September 2016 to November 2019, all patients planned to receive LSPDP.ResultsTwenty of the 22 patients successfully completed LSPDP, and 2 patients converted to laparotomy. One patient was transferred to laparotomy to suture the damaged splenic artery. The spleen was observed to have no ischemia and the spleen preservation operation was continued. One patient was converted to laparotomy due to the difficulty of dissecting the tail of the pancreas which caused by severe abdominal adhesion. The operation time of LSPDP patients was (191±86) minutes (170–480 min), intraoperative blood loss was (365±50) mL (200–1 000 mL), and postoperative hospital stay was (9.9±2.6) days (7–16 d). Six patients of pancreatic fistula occurred after operation, including 3 cases of biochemical fistula, which were cured and discharged after symptomatic treatment, 3 cases of grade B pancreatic fistula, who all improved after anti-inflammatory, acid suppression, enzyme suppression, and double catheter drainage. Twenty patients were interviewed after the operation, and the follow-up time was 3–24 months (median of 15 months). During the follow-up period, no patient had recurrence or metastasis.ConclusionsUnder the conditions of strict screening of suitable cases, adequate preoperative imaging evaluation, intraoperative fine manipulation, and the application of appropriate operating instruments and cutting closure devices, LSPDP is safe and feasible to treat benign tumors of the pancreatic body and tail and some borderline tumors. During the operation, attention should be paid to the reasonable treatment and protection of splenic arteries and veins.