Adenoviral vectors expressing siRNAs for discovery and validation of gene function

RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GalphaS, we have measured inhibition of ligand-dependent, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.     

B超诊断老年性白内障及合并症价值

０引言老年性白内障严重影响着老年人的生活质量．手术是根治本病的最佳方法．现将我院１９９８－０１～１９９８－１１经Ｂ超检查并经手术证实的１２９例,共２５８只眼,２２３只老年性白内障的结果及声像图特点报告如下．旨在探讨Ｂ超在诊断该病中的应用价值及对手术的...     

A new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF)

A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high- resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands: a more intense central one and four less intense, two moving faster and two slower than the central band. In normal platelets, each multimer could also be resolved into five bands. The central one had a mobility similar to that in plasma, whereas the four satellite bands had a mobility that differed from that of the corresponding plasma bands. In the patient, platelet von Willebrand factor antigen content and ristocetin cofactor activity were normal, and von Willebrand factor showed the same structure of individual multimers as seen in normal platelets. On the other hand, plasma von Willebrand factor antigen and ristocetin cofactor activity were decreased, and the structure of individual von Willebrand factor multimers was different from that of normal plasma and similar to that seen in normal and patient's platelets. After infusion of 1-deamino-8-D-arginine vasopressin, the largest von Willebrand factor multimers, as well as new satellite bands with a mobility similar to those in normal plasma, appeared in the patient plasma, and the levels of von Willebrand factor antigen and ristocetin cofactor activity became normal. Yet no relevant change in the prolonged bleeding time was observed. This new variant of von Willebrand disease, therefore, is characterized by the presence of a dysfunctional von Willebrand factor molecule that exhibits unique structural abnormalities in plasma but appears to be normal in platelets. The designation of Type IIF is proposed for this type of von Willebrand disease in accordance with the terminology that has been previously used.     

角质形成细胞的培养及其表皮生长因子受体表达

目的:在成功分离人皮肤角质形成细胞的基础上,观察表皮生长因子受体在人皮肤角质形成细胞中的表达情况。方法:实验于2006-3/10在北京大学深圳医院中心实验室进行。采用dispase Ⅱ-trypsin两步消化法获取表皮基底层细胞,用小鼠皮肤成纤维母细胞滋养层和黄素腺嘌呤二核苷酸培养液进行培养。小鼠皮肤成纤维母细胞的预处理:向对数生长期的小鼠皮肤成纤维母细胞培养液中加入丝裂霉素C至终浓度为4mg/L,37℃下培养4h,弃去培养液,用D-Hank’s液洗3次,加入浓度为0.25g/L的胰蛋白酶消化,分离出细胞,离心(200g,5min),用黄素腺嘌呤二核苷酸培养液悬浮细胞,计数,以5.0×104/cm2的密度种于培养皿内,37℃、体积分数0.05的CO2培养箱下培养。角质形成细胞的培养:将分离的角质形成细胞悬浮在黄素腺嘌呤二核苷酸培养液中,以2.0×104/cm2的密度接种在前1天经丝裂霉素C处理的小鼠皮肤成纤维母细胞滋养层上,37℃、体积分数0.05的CO2培养箱下培养。24h换液,以后每3d换1次液。采用免疫细胞化学的方法检测表皮生长因子受体的表达,采用复合逆转录聚合酶链反应检测角质形成细胞中表皮生长因子受体mRNA的表达。结果:采用dispaseⅡ消化法分离了真皮和表皮,获得较多的角质形成细胞,可以避免真皮成纤维细胞的污染。人皮肤角质形成细胞在黄素腺嘌呤二核苷酸培养液中培养5d可见明显的集落,约10d可长满单层。免疫细胞化学显示表皮生长因子受体在细胞表面有明显的表达,复合逆转录聚合酶链反应显示表皮生长因子受体mRNA有明显的表达。结论:用小鼠皮肤成纤维母细胞滋养层和黄素腺嘌呤二核苷酸培养液可以较好地培养原代人皮肤角质形成细胞,表皮生长因子受体在细胞表面有明显的表达,这些结果为与表皮生长因子受体相关的皮肤病(如银屑病)的研究奠定了基础。     

葡萄糖酸镁对离体大鼠心肌缺血再灌注细胞凋亡的抑制效应

目的:观察葡萄糖酸镁对离体大鼠心肌缺血再灌注损伤心肌细胞凋亡的影响。方法:实验于2005-10/2006-01在辽宁医学院药理实验室完成。选用雄性SD大鼠48只,体质量250～300g,按随机排列表法将大鼠分为对照组、缺血再灌注组、葡萄糖酸镁组,每组16只,建立Langendorff离体大鼠心肌缺血再灌注损伤模型。(1)每组各取8只:①对照组:改良的K-H缓冲液持续灌流110min。K-H缓冲液成分(mmol/L)如下:NaCl118.1,NaHCO325.0,KH2PO41.2,MgSO40.6,CaCl22.0,KCl4.7,Glucose11.0。②缺血再灌注组:改良的K-H缓冲液持续灌注至各项指标稳定后,约20min,停灌30min,再灌60min。③葡萄糖酸镁组:灌注方法同缺血再灌注组,但将K-H缓冲液内加葡萄糖酸镁2.4mmol/L。取左室心肌标本测总超氧化物歧化酶活性,丙二醛、Ca2 含量。(2)每组其余8只:①对照组K-H缓冲液持续灌流170min。②缺血再灌注组和葡萄糖酸镁组持续灌注至各项指标稳定后停灌30min,再灌注120min。观察对细胞凋亡的影响。(3)组间计量资料差异比较采用单因素方差分析。结果:SD大鼠共48只均进入结果分析。①缺血再灌注组大鼠心肌组织中丙二醛、Ca2 含量较对照组明显升高(P<0.01),总超氧化物歧化酶活性较对照组明显降低(P<0.01)。②葡萄糖酸镁组与缺血再灌注组比较,总超氧化物歧化酶活性明显升高(P<0.01),丙二醛、Ca2 含量明显降低(P<0.01)。③缺血再灌注组细胞凋亡指数显著高于对照组(P<0.01)。④葡萄糖酸镁组细胞凋亡指数与缺血再灌注组比较显著降低(P<0.01)。结论:葡萄糖酸镁有抑制心肌缺血再灌注损伤中心肌细胞凋亡的作用。其机制可能与葡萄糖酸镁减轻钙超载、清除氧自由基、减少脂质过氧化有关。