Objective To construct the lentiviral vector to co-express enhanced green fluorescent protein (EGFP) gene and human insul in (insulin) gene, and to explore the condition to transfect human umbil ical cord mesenchymal stem cells (hUCMSCs) so as to lay a foundation for tissue engineered adipose reconstruction and transplantation in vivo infuture. Methods The insulin gene was cloned to lentiviral expression vector with EGFP [pLenti6.3-internal ribosome entrysite (IRES)-EGFP] by recombinant DNA technology, the positive clones were screened, and lentiviral packaged systems and target gene plasmid were co-transfected to package virus in 293T cells by lipofectin. The reporter gene expression was observed by fluorescent inverted phase contrast microscope, virus supernatant was collected, purificated and concentrated, and the titer of recombinant viruses was determinated. hUCMSCs from umbilical cord tissue of mature neonates were isolated and cultured by different multiple of infection (MOI, 0, 1, 3, 5, 7, 10, 15, and 20). By recombinant lentiviral infected hUCMSCs with reporter gene green fluorescent protein expression, the best MOI was screened; recombinant lentiviral infected hUCMSCs at the best MOI, then real-time PCR and Western blot methods were appl ied to detect insulin gene and insul in protein expression levels in cells. Results The recombinant lentiviral vector of co-expressing insulin gene and EGFP gene (pLenti6.3-insulin-IRESEGFP) was successfully constructed. Virus could be packaged, purificated and concentrated successfully. The virus titer was 1.3 × 108 TU/mL. The best MOI was 10 and the transfer efficiency was up to 90% in the same time. Real-time PCR results showed that insulin gene expression of transfected group was positive and non-transfected group was negative; Western blot detection confirmed that insul in protein expression of transfected group was positive in cells and supernatant, but that of non-transfected group was both negative. Conclusion Lentiviral vector pLenti6.3-insulin-IRES-EGFP carrying recombinant insulin gene could effectively transfect hUCMSCs and express insul in protein.
目的 回顾性研究老年系统性红斑狼疮（SLE）患者合并感染的危险因素。 方法 选取1995年1月－2009年12月间在四川大学华西医院确诊为SLE，起病年龄为50周岁以上的158例患者，收集性别、临床表现、疾病活动度、实验室检查指标、合并症以及并发症等进行单因素分析或多因素非条件logistic回归分析。 结果 所纳入的158例患者中，合并感染53例（占33.5%），采用单因素分析显示疾病活动性（P=0.001）、低蛋白血症（P=0.030）、糖尿病（P=0.003）、肺间质病变（P=0.000）与老年SLE患者感染发生有关。经logistic回归分析显示，疾病活动性（OR=7.533，P=0.000）、肺间质病变（OR=19.762，P=0.000）、糖尿病（OR=6.862，P=0.025）是老年SLE患者感染发生的危险因素。 结论 积极控制老年SLE的疾病活动度，减少危险因素的发生是控制老年SLE患者并发感染的有效手段。
目的：机械分离、培养小鼠耳蜗螺旋神经元，并进行免疫荧光细胞学鉴定，为后期进一步的实验研究提供实验材料。方法：采用初出生1～5天以内的昆明小鼠进行解剖、机械分离以获得螺旋神经节组织，进行原代培养后，应用神经微丝蛋白（Neurofilament protein,NFP－H）单克隆抗体进行免疫荧光细胞学鉴定。结果：机械分离后获得的螺旋神经节组织中的螺旋神经元，在体外培养条件下可以存活并进行正常分化。典型的螺旋神经元，其细胞形态呈椭圆形,胞体透明光滑、接近生理形态。荧光染色标记后，胞体和神经突起均显色好，Schwann细胞和成纤维细胞未着色。结论：应用机械分离的方法获得小鼠耳蜗螺旋神经节组织并进行培养，耳蜗螺旋神经元在体外可以稳定地存活生长。培养获得的细胞形态和生存状态接近生理状态，满足电生理、免疫细胞化学、药理学等研究。应用特异性的神经微丝蛋白对培养获得的螺旋神经元进行免疫荧光细胞学鉴定，特异性好，荧光显色好。
Objective To investigate the impacts of cytokines （interleukin-4，IL-4；tumor necrosis factor-α，TNF-α） and medications of bronchial asthma （dexamethasone，aminophylline，salbutamol） on the activity of histamine N-methyltransferase（HMT） in tracheal epithelial cells.Methods BEAS-2B bronchial epithelial cells were cultured and treated with different concentration of TNF-α, IL-4, dexamethasone, salbutamol and aminophylline respectively. The activity of HMT in BEAS-2B cells was determined by high performance liquid chromatography.Results The activity of HMT in tracheal epithelial cells was (50±7) pmol?min-1?mg pro-1.TNF-α and IL-4 lowered the activity of HMT significantly at the concentration equal to or higher than 1 ng/mL and 5 ng/mL respectively，and reached the maximum inhibitory effect at the level of 10 ng/mL.Dexamethasone and aminophylline could ameliorate distinctly the inhibitory effect of TNF-α on the activity of HMT, while salbutamol had no significant inhibitory effect.Conclusions TNF-α and IL-4 exert the lowering effect on the activity of HMT，which would be one important cause of airway hyperreactivity.Glucocorticoids and theophyllines are administered to treat asthma partly due to its relieving mechanism of TNF-α negative effects on HMT.
Objective To improve the knowledge of lung injury induced by rituximab. Methods Clinical data of 5 lymphoma patients with lung injury caused by rituximab chemotherapy were analyzed. Results Five patients received chemotherapy including rituximab, and had fever, cough and dyspnea after 3 to 5 chemotherapy cycles. Chest CT showed bilateral diffuse interstitial infiltrates. All 5 cases experienced hypoxemia or respiratory failure. Clinical symptoms were improved 3 to 5 days after the treatment of glucocorticoids, and pulmonary lesions were significantly alleviated 1 to 2 weeks after the treatment. According to the literature, the incidence rate of lung injury caused by rituximab was 0. 03% to 4. 9%, which has increased recently. Conclusions With the comprehensive application of rituximab, lung injury caused by this drug is not rare. The good prognosis depends on early diagnosis and treatment by further recognition of the side effect of rituximab.
Abstract：?Objective?To evaluate clinical outcomes of single utility port complete video-assisted thoracoscopic surgery （VATS） lobectomy for patients with early-stage lung cancer.?Methods?We retrospectively analyzed the clinical data of 162 consecutive patients with early-stage lung cancer who underwent single utility port complete VATS lobectomy from September 2009 to October 2011 in Chinese PLA General Hospital （single utility port group），and compared them with 221 patients with early-stage lung cancer who underwent video-assisted mini-thoracotomy （VAMT） lobectomy in the same period （VAMT group）. The clinical outcomes including operation time, intraoperative blood loss, lymph node dissection number, time to first activity out of bed, chest drainage duration and postoperative complications, were compared between the two groups.?Results?No perioperative death was observed in both groups. There were statistical differences in the intraoperative blood loss （162.8±75.6 ml vs. 231.4±62.8 ml）, time to first activity out of bed （2.2±0.3 d vs. 3.7±0.5 d） , and chest drainage duration （3.5±0.2 d vs. 4.6±0.4 d） between the two groups （P＜0.05）. There was no statistical difference in operation time （133.7±22.0 min vs. 124.9±25.7 min） , lymph node dissection number （11.7±1.9 vs. 12.5±2.7）， and incidence of serious postoperative complications （7.4% vs. 8.1%）between the two groups.?Conclusion?Single utility port complete VATS lobectomy and lymph node dissection are safe and reliable for patients with early-stage lung cancer with less injury and better postoperative recovery compared with VAMT.
Objective To study the influence of different mechanical environments on repair cartilage defect with marrow mesenchymal stem cells as seed cells. Methods The rabbit marrow mesenchymal stem cells were isolated and cultured. The cartilage defects were repaired by autologous tissue engineered cartilage with the marrow mesenchymal stem cells as seed cells. Fifteen rabbits with cartilage defect were divided into 3 groups: dislocation group with cell-free scaffold(controlgroup), dislocation group with cartilaginous construct and normal mechanical environment group with cartilaginous construct. The repaired tissue was harvested and examined 6 weeks postoperatively. Results The repair tissue in normal mechanical environment group with cartilaginous construct showed cartilage-like tissue in superficial layer and subchondral bone tissue in deep layer 6 weeks postoperatively. The defect was filled with bone tissue in dislocation group with cartilaginous construct 6 weeks postoperatively. The surrounding normal cartilage tissue showed vascular invasion from subchondral area and the concomitant thinningof the normal cartilage layer. The cartilaginous construct left in the femoral trochlea groove formed hyaline cartilage-like tissue. The defect was repaired byfibrous tissue in control group. Conclusion The repaired tissue by tissue engineered cartilage with marrow mesenchymal stem cells as seed cells showed the best result in normal mechanical environment group, which indicates that it will be essential for the formation and maintenance of tissue engineered cartilage to keep the normal mechanical stress stimulus.
OBJECTIVE To study the feasibility of constructing tissue engineered cartilage by differentiated rabbit bone marrow mesenchymal stem cells(MSC) cultured in vitro and in vivo. METHODS The MSC were isolated from the nucleated cells fraction of autologous bone marrow by density gradient centrifuge, and then induced into chondrogenic differentiation by adding dexamethasone, transforming growth factor-beta 1 (TGF-beta 1) and ascorbic acid in vitro. After 3 weeks, some cells turned to round shape and secreted metachromatic matrix. The cartilaginoid grafts composed of chondrogenic MSC. Bovine type I collagen and human fibrin were cultured within the chondrogenic medium for 2 weeks in vitro or transplanted subcutaneously adjacent to the knee joint for 3 weeks in vivo. RESULTS The most cells in the grafts were degenerated and disappeared after cultured in vitro. But the residual cells were survival and secreted metachromatic staining proteoglycan with toluidine blue, which was characteristic cartilage matrix. The grafts developed into matured cartilage tissue assessed by histological examination after 3 weeks of transplantation in vivo. CONCLUSION MSC can be used as functional cells to constructing tissue engineered cartilage.
Objective To evaluate the effects of intraocular lenses (IOL) of silicone and polymethylmethacrylate (PMMA) on posterior capsule opacification (PCO). Methods We searched MEDLINE (1966 to 2003), EMBASE (1980 to 2003), Cochrane Central Register of Controlled Trials (Issue 1, 2003) and CBMdisc (1979 to 2003). Only randomized controlled trials (RCTs) were identified. Two reviewers independently assessed the quality of the included trial and extracted data. The following outcomes were assessed: PCO index, percentage of neodymium: YAG (Nd: YAG) laser posterior capsulotomy and visual acuity after cataract surgery. RevMan 4.2 was used for meta-analysis.Results Nine RCTs including 672 eyes were included. In patients with senile cataract, the results of meta-analysis suggested that silicone lens were better than PMMA lens on PCO index (the pooled SMD=－0.92, 95%CI －1.19 to －0.64) and percentage of Nd: YAG (the pooled OR 0.35, 95%CI 0.22 to 0.57) with a statistically significant difference, but there was no significant difference on visual acuity (the pooled OR 1.22, 95%CI 0.43 to 3.50) after cataract surgery between silicone lens and PMMA lens. However, in patients with uveitic cataract, the results of meta-analysis showed that PMMA lens were better than silicone lens in visual acuity (the pooled OR 0.38, 95%CI 0.15 to 0.91) after cataract surgery with a statistically significant difference, but there was no significant difference on PCO index (SMD 0.23, 95%CI －0.59 to 1.05) and percentage of Nd: YAG (the pooled OR 1.82, 95%CI 0.47 to 6.95) between silicone lens and PMMA lens. Conclusions Current evidence indicates that silicone lens are superior to PMMA lens in reducing PCO. Further trials with high quality on methodology are required.
Superficial temporal artery (STA) - middle cerebral artery (MCA) bypass surgery has been widely used to treat patients with moyamoya disease, and its application value in symptomatic internal carotid artery (ICA)/MCA stenosis/occlusion remains controversial. With the development of imaging, micro-devices and surgical techniques, and the deepen understanding of diseases, the effectiveness of STA-MCA bypass surgery in the treatment of symptomatic ICA/MCA stenosis/occlusion is further required. This article reviews the process of development and evolution of this surgical technique, as well as the significance and deficiencies of several randomized controlled trials of ICA/MCA treatment in the past, and looks forward to possible improvements in future research, so as to clarify the way for further randomized controlled study.