增强型绿色荧光蛋白标记技术示踪组织工程化骨形成

目的:观察以脂质体介导法转染的增强型绿色荧光蛋白质粒作为骨组织工程种子细胞示踪剂的可行性。方法:实验于2005-07/2006-11在南昌大学第二附属医院分子医学实验室完成。选取12个月龄中国青山羊2只作为骨髓基质干细胞供体。另取BACB/un裸鼠9只作为骨髓基质干细胞-珊瑚复合物受体。①将已转化pEGFP-N2大肠杆菌109菌种,进行质粒提取并经AvaⅡ酶切鉴定。②脂质体lipofectamine2000介导转染羊骨髓基质干细胞。分别于转染24h、6个月后计算荧光的转染效率。标记与未标记细胞经成骨诱导分化后,检测碱性磷酸酶活性和四环素钙结节染色,以未标记的同期细胞作对照。③将标记细胞接种于珊瑚支架,形成骨髓基质干细胞-珊瑚复合物,分别于体外培养4,7,14d后进行电镜扫描观察。④将体外培养7d的骨髓基质干细胞-珊瑚复合物移植于裸鼠皮下,8周后取出,荧光显微镜下进行示踪观察,苏木精-伊红染色观察组织结构。以未标记的骨髓基质干细胞-珊瑚复合物、单纯珊瑚作对照。结果:共纳入中国青山羊2只,BACB/un裸鼠9只,作为受体的9只裸鼠进入结果分析。①质粒经酶切后电泳,可见3条带,分别为445bp,1938bp,2354bp,确定所提质粒为pEGFP-N2。②转染24h后绿色荧光表达率35%;经G418筛选,转染6个月后绿色荧光表达率75%;与对照组相比,标记细胞碱性磷酸酶活性差异无统计学意义(P>0.05),均具有钙结节形成能力。③标记细胞与材料贴附生长良好,并分泌胶原纤维及钙盐结晶样基质。④组织学示新生骨样组织围绕材料孔隙生成;新生组织细胞内有绿色荧光表达,同时对照组未观察到绿色荧光。结论:脂质体介导法转染的pEGFP-N2标记骨髓基质干细胞,可用于裸鼠体内示踪,是一种理想的组织工程化骨组织的示踪方法。     

西山医院脊髓损伤功能量表内容介绍

目的:介绍一套新的脊髓损伤功能评价量表。方法:综合目前使用的各种常用脊髓功能量表,结合实际工作经验及脊髓损伤患者神经功能改善特点,制定了西山医院脊髓损伤功能量表。结果:西山医院脊髓损伤功能量表共分9大项16小项,采用4分制,正常为3分,最差为0分。总分0～48分,0～16分为重度残障,17～32分为中度残障;33～47分为轻度残障,48分正常。量表包括上肢运动功能(饮食、梳洗、书写)、下肢运动功能(站立、行走)、躯干运动功能(坐、翻身)、全身运动功能(床椅转移、穿脱衣服、洗澡)、括约肌功能(膀胱功能、直肠功能)、肌张力、泌汗、皮肤营养、疼痛、性功能。结论:西山医院脊髓损伤功能量表是一套专门应用于脊髓损伤患者功能评价量表。该量表能全面、精确评定患者功能,简便实用、费时短。应用这份功能量表,既能客观反映患者的功能变化,随访时又不增加患者负担,同时也能减轻医务工作者的负担。     

胚胎嗅鞘细胞再移植治疗晚期脊髓损伤1例

目的:对晚期脊髓损伤患者开展嗅鞘细胞再移植治疗,探讨其对神经功能的继续改善效果。方法:①患者男性,27岁,哈萨克斯坦籍,2001年9月20日遭霰弹枪击急诊手术后,双下肢不能运动及感觉消失,尿便失禁,右下肢阵发性钝痛。诊断为陈旧性完全性脊髓损伤(T12),美国脊髓损伤协会标准脊髓功能分级为A级。2002年9月30日第1次行脊髓胚胎嗅鞘细胞移植术,2007年2月5日行第2次脊髓胚胎嗅鞘细胞移植术。治疗方案患者知情同意。②取4个月流产胚胎(孕妇及其家属均知情同意)的嗅球,消化成单个嗅鞘细胞后培养2周,细胞浓度约2×1010L-1。于患者相应脊髓损伤上端(平T11椎体)做1个锁孔,直径约3cm,在脊髓损伤部位与正常脊髓交界处沿中线于无血管区,分两点用4.5号细针共注入100μL(第1次手术)、50μL(第2次手术)嗅鞘细胞悬液,细胞数均为1×106个。第2次移植术前进行供、受体细胞HLA配型,术后口服FK506胶囊2mg,2次/d,共42d。结果:①第1次术后3个月,排尿能控制6h,双足运动功能改善,性功能改善,阴茎勃起功能改善,疼痛减轻。②第2次术后10d,双下肢双足皮肤泌汗功能改善,右下肢疼痛减轻,腹部皮肤感觉稍有好转,针刺觉左侧由术前T10皮节消失好转至T10减退。较第2次移植前椎旁躯体感觉诱发电位有所改善,双侧椎旁电位从T10下降到T12。结论:胚胎嗅鞘细胞二次移植能继续改善晚期脊髓损伤患者的神经功能。但移植的细胞数量、容积、注射点位置等需深入探讨。     

Single‐port laparoscopic cholecystectomy: A comparative study in 106 initial cases

Introduction: Laparoscopic cholecystectomy has been the standard of care for gallbladder diseases since the late 1980s. Many surgeons have rapidly adopted single‐port laparoscopic cholecystectomy for gallbladder pathologies. The aim of the present study was to analyze the clinical outcome in initial single‐port laparoscopic cholecystectomy. Methods: Data from 106 consecutive single‐port laparoscopic cholecystectomies between May 2008 and April 2009 were analyzed retrospectively. We divided the patients into two groups – an early group (group I, n=56) and a late group (group II, n=50) – to compare clinical outcomes. During each procedure, only one longitudinal transumbilical incision, 1.5 to 2.0 cm in length, was made to access the abdominal cavity. A multichannel port system was assembled with existing devices. Standard laparoscopic instruments were used to perform each cholecystectomy. Results: Patient demographics did not differ between the two groups. Of the eight cases that were converted to conventional laparoscopic surgery, seven were part of group I (P=0.063). Mean operation time for single‐port laparoscopic cholecystectomy was significantly shorter in group II (58.2 versus 71.6 min, P=0.004). There were two operative complications in group I, which were successfully managed with laparoscopic surgery. There was no statistical difference in occurrence of operative complication and hospital stay between the two groups. Conclusion: Single‐port laparoscopic cholecystectomy can be safely performed for various gallbladder lesions in selected cases, and the operation time improved with accumulation of cases.     

Immunohistochemical expression of matrix metalloproteinases in the granulomatous rosacea compared with the non‐granulomatous rosacea

Background There is a granulomatous variant which is recognized in the rosacea spectrum. However, the pathogenesis of granuloma formation in rosacea has not been clearly demonstrated. Matrix metalloproteinases (MMPs) are required for recruitment of inflammatory cells and for tissue remodelling, making way for the development of well‐organized granuloma. Objective The aim of this study was to investigate the expression of transforming growth factor (TGF)‐β, TGF‐β type II receptor (TβRII), Tumour necrosis factor (TNF)‐α, MMP‐1, 2 and 9 in the granulomatous rosacea (GR) compared with the non‐granulomatous rosacea (NGR) and test the hypothesis that the changes of these profiles in GR would be related with chronic ultraviolet radiation (UVR)‐exposure. Methods Facial skin samples were obtained from 20 patients with GR and NGR (control group). The sections were stained using haematoxylin and eosin, Verhoeff’s elastic stain, and antibodies to TGF‐β, TβRII, TNF‐α, MMP‐1, ‐2 and ‐9. Results The amount of elastotic material was significantly increased in the dermis of GR lesions. Expression of TGF‐β was significantly decreased in the epidermis of GR lesions compared with NGR lesions. In addition, the expression of MMP‐9 was significantly increased in the dermis of GR lesions compared with NGR lesions, especially at the centre of the granuloma on a semi‐quantitative analysis. MMP‐2 expression was also increased in GR lesions, although the difference between the two groups was not statistically significant. Conclusions The results of this study suggest that the increased expression of MMPs in the dermis may participate in granuloma formation of GR in association with UVR.     

Roles of attention, memory, and motor preparation in modulating human brain activity in a spatial working memory task

Neuronal activity of the human brain was studied with magnetoencephalography (MEG) in a spatial working memory task similar to those commonly used with nonhuman primates. The subject was required to remember target positions for 3 s and make a same-different judgement with a finger lift comparing the position of the probed target with the probe or to execute a memory-guided saccade to the probed target. In this type of task single-unit studies have shown attention- and memory-related activities independent of movement type during the retention interval in a large number of cortical areas of the primates, including the parietal and prefrontal areas. Consistent with these results, there were strong stimulus-driven transient and sustained responses and modulations of oscillatory activity during the retention period. Although we did not determine the source locations, coarse estimates of the currents responsible for the MEG signals showed activity over a wide area of the cortex, most prominently over the Rolandic, parietal and occipital areas, but also over the frontal area. Some of the activities in these cortical areas reflect processes that may be identified with attention and memory, while others were related to preparation of the overt movements.      

升高空气压力减轻烫伤大鼠组织水肿及加速愈合

目的:应用升高空气压力来减轻大鼠深Ⅱ度烫伤后创面组织水肿,观察其效果及对愈合的影响。方法:实验于2004-12/2006-06在扬州大学医学院外科实验室完成。①实验分组:Wistar雄性大鼠36只,随机分为两组:实验组18只,对照组18只。②实验方法:大鼠以硫化钠脱毛剂脱去背部体毛,24h后采用苯巴比妥腹腔注射麻醉,水浴法(80℃,时间6s)造成背部4cm×4cm深Ⅱ度烫伤(病理切片证实),腹腔立即注射林格氏液5mL复苏。实验组动物伤后在高于大气压2.45kPa的压力箱内饲养,对照组箱内压力与大气压同。③实验评估:喂养48h后麻醉下处死大鼠,取伤部组织皮肤测定组织含水量;在伤后1,3,6,12h检测创面组织液渗出量;观察创面愈合率及愈合时间。结果:36只大鼠均进入结果分析。伤后48h实验组组织含水量低于对照组(P<0.05)。实验组在伤后1,3,6,12h创面组织液渗出量低于对照组(P<0.05)。实验组创面愈合时间短于对照组(P<0.05)。结论:升高空气压力可以降低创面的液体渗出,减轻组织水肿,缩短创面愈合时间。     

小干扰RNA抑制neuro-2a细胞内源β位淀粉样前体蛋白裂解酶1基因的表达

目的:分析小干扰RNA(small interference RNA,siRNA)抑制neuro-2a细胞内源β位淀粉样前体蛋白裂解酶1基因(Beta-site APP cleaving enzyme protein,BACE1)的表达情况。方法:实验于2004-12/2006-06在中山大学中山医学院和上海交通大学农业与生物学院完成。①用脂质体将EGFP基因表达载体pEGFP-C1 Vector和体外转录合成的针对EGFP基因的小干扰RNA(siEGFP)分别或同时转染neuro-2a细胞,在倒置荧光显微镜下计算EGFP在neuro-2a细胞中的表达率。②将体外转录合成的siBACE1-1,siBACE1-2,siBACE1-3转染neuro-2a细胞,干扰24,48,72h后分别用Real time RT-PCR定量分析siBACE1对内源BACE1基因表达的抑制率和干扰的时效性。结果:①外源EGFP基因转染neuro-2a细胞后,43%neuro-2a细胞高表达EGFP蛋白。通过转染siEGFP则可有效抑制EGFP基因表达。②3个干扰位点的siBACE1对BACE1基因表达有不同的抑制效率,siBACE1-3使BACE1m RNA表达水平下降60%,siBACE1-1为13%,siBACE1-2对BACE1 mRNA无明显的抑制作用。③siBACE1抑制内源BACE1基因的表达与干扰时间相关,siBACE1干扰24h、48h后BACE1 mRNA的表达与正常组无明显差异(P>0.05),但干扰72h后,siBACE1-3和siBACE1-1均使BACE1 mRNA表达量下降。结论:体外转录合成的siBACE1能有效抑制neuro-2a细胞内源BACE1基因表达,其抑制率与BACE1基因的干扰位点和干扰时间相关。     

Current management and future perspectives of metastatic renal cell carcinoma

Over the last number of years, the treatment of metastatic renal cell cancer has evolved tremendously with the advent of targeted therapy. Previously, immunotherapies, such as interferon alpha and interleukin‐2, were the only treatment options available for this chemoresistant malignancy. Currently, seven additional agents, including sunitinib, sorafenib, axitinib, pazopanib, bevacizumab, everolimus and temsirolimus, have been approved for use in metastatic renal cell cancer, with several more in development. The efficacy of these agents depends primarily on inhibition of the vascular endothelial growth factor and mammalian target of rapamycin pathways, and have drastically improved the outcomes of patients diagnosed with metastatic renal cell cancer. This article reviews the major treatment advances that have occurred for metastatic renal cell cancer with the advent of targeted treatments, summarizes the evidence to support their use and addresses clinical issues that have arisen with them. To help guide clinicians in their decision‐making with these emerging therapeutic choices, the evidence for sequencing and combining these agents, and the need for biomarkers will be addressed. The role of surgical management options, such as cytoreductive nephrectomy and metastectomy, in the era of targeted treatment is also reviewed. Several novel treatments are also on the horizon, which might serve as future avenues for treatment advancement in metastatic renal cell cancer.     

血小板激活因子对大鼠脑血管通透性的影响及药物的保护作用

颈内动脉注射血小板激活因子(PAF),再给伊文思蓝,可见脑实质染色程度加深,而颈内动脉只注射伊文思蓝,脑实质未见染色。而我们合成的新药SZ-1可剂量依赖性地抑制PAF诱导的脑实质伊文思蓝染色程度的加深。在体外培养的脑微血管平滑肌细胞上,PAF能显著刺激~(14)-花生四烯酸的释放,而SZ-1能剂量依赖性地抑制这种释放,提示PAF在脑内产生的损害除与其他因素相关外,还与其刺激花生四烯酸释放有密切关系,SZ-1对PAF引起的脑部损害有保护作用。