Activated prothrombin complex concentrate (FEIBA®) treatment during surgery in patients with inhibitors to FVIII/IX

Non-activated and activated prothrombin complex concentrates (PCC/aPCC) have been used successfully to treat bleeds in haemophilia patients with inhibitors, but most physicians do not consider these products as effective as factor VIII/IX (FVIII/IX) concentrates in non-inhibitor patients. Thus, surgical procedures in inhibitor patients have been performed reluctantly. We have performed 14 minor and five major surgical and invasive diagnostic procedures in eight patients with congenital haemophilia A and inhibitors and in two patients with acquired haemophilia. When a loading dose of 100 U kg-1 of FEIBA was given followed by 200 U kg-1 day-1 in three divided doses every 8 h for 3 days, and then, when the daily dose was tapered to 100-150 U kg-1, no severe or unexpected bleeding complications were observed. However, one adverse event was observed. A 69-year-old man who suffered a myocardial infarction the third postoperative day following sigmoidectomy was managed safely with opiate analgesia, nitrates and diuretics, and the continued use of FEIBA(R).     

人β-防御素3基因在COS-7细胞中的表达

目的：构建人β—防御素3(hBD3)的真核表达载体，建立稳定表达hBD3的细胞株。方法：用EcoR Ⅰ酶切合有hBD3全长基因的pGEM-hBD3重组质粒，获得其编码区全长序列，将其连接入EcoR Ⅰ预处理过的pcDNA3中。转化大肠杆菌，酶切鉴定筛选出插入方向正确的转化子。采用脂质体转染法将重组pcDNA3-hBD3真核表达载体导入COS-7细胞，用G418进行抗性筛选，用RT-PCR和Western印迹检测目的基因hBD3的mRNA和蛋白表达水平。结果与结论：构建的pcDNA3-hBD3真核表达载体转染COS-7细胞后可稳定表达hBD3的mRNA和蛋白，且蛋白主要以分泌形式存在于培养上清中。     

T7 RNA聚合酶体外转录合成siRNA的方法

目的：建立一种应用T7 RNA聚合酶体外转录合成大量siRNA的方法。方法：以萤火虫荧光素酶为靶标，应用T7 RNA聚合酶体外转录合成siRNA，脂质体转染法将其导入HepG2细胞中，通过测定荧光素酶的量，评价siRNA对萤火虫荧光素酶基因表达的抑制作用。结果：合成了以萤火虫荧光素酶为靶标的siRNA，合成的siRNA能特异性地抑制荧光素酶基因的表达，抑制率为80％。结论：建立了一种经济简便的体外合成siRNA的方法。     

凋亡相关蛋白Apr-2的克隆、测序及初步表达

目的: 从HL-60细胞凋亡模型中克隆凋亡相关蛋白Apr-2编码区基因,并对其进行表达,为进一步研究Apr-2的结构功能及多克隆抗体的制备奠定基础.方法: 建立HL-60细胞凋亡模型,提取HL-60凋亡细胞总RNA,以RT-PCR方法获取Apr-2 cDNA编码区全序列,将其与PGEM-T Easy载体连接,转化E.coli DH5α,构建重组克隆载体PGEM-TEasy/apr-2,测序正确后,将目的片段亚克隆入PGEX-4T-2原核表达载体,并转化大肠杆菌, IPTG诱导重组蛋白质表达,分析蛋白质在细菌中的表达分布,进行凝胶自动扫描分析. 结果:序列分析表明,与GenBank中已登录的Apr-2 cDNA编码区序列比较,完全一致.表达的融合蛋白占菌体总蛋白质的40%以上,主要以包涵体的形式存在. 结论:成功的获得了细胞凋亡模型HL-60中Apr-2 cDNA编码区的克隆及其融合蛋白表达产物.     

Expression and effects of human telomerase RNA in testicular tumor

Human telomerase RNA (hTR) plays an important role in determining repeated telomere sequence and the expression of an antisense telomerase RNA that leads to telomere shortening and cell death.1 Using highly sensitive in situ nucleic acid hybridisation, we investigated the expression of hTR in human testicular tumours and located its cellular expression. Our study may help in elucidating the role of hTR in human testicular tumours, finding a highly sensitive diagnostic method and a target for gene therapy of testicular tumours.     

非同位素PCR方法检测脆性X 综合征FMR-1基因CGG重复序列数目

目的 建立一种可靠、简便的非同位素PCR方法检测脆性X综合征的突变基因。方法 采用生物素标记的CGG寡核苷酸探针，检测通过PCR扩增的FMR-1基因中CGG三核苷酸重复序列数目。从而判断所检样品中FMR—1基因是否正常。结果 该法可检测出正常人及携带者的CGG重复拷贝数。结论 该方法可以简便、安全、可靠地检测FMR—1基因中(CGG)n重复拷贝数，从而确定为正常人或携带者，可作为临床上筛查脆性X综合征的首选方法。     

人正、反义转化生长因子beta1基因真核表达载体的构建

目的 :构建人转化生长因子beta 1(TGFβ1)正、反义基因真核表达载体。方法 :从人胎脑的cDNA文库中扩增出TGFβ1共 12 84bp长的DNA片段后 ,与T载体直接进行高效连接 ,并测序证实无突变。接着利用TGFβ1引物两端所设计的酶切位点将目的片段定向亚克隆到真核表达载体pcDNA3.1( )。构建的正反义表达重组体 (pcDNA3.1 TGFβ1和pcDNA3.1 antiTGFβ1)经限制性内切酶消化证实其中有目的片段完整插入。结果 :本实验成功构建了人正、反义TGFβ1基因真核表达载体。结论 :人正、反义TGFβ1基因真核表达载体的构建 ,为今后研究腹膜纤维化的防治奠定了实验基础。     

A clinical and genetic study of 56 Saudi Wilson disease patients: identification of Saudi-specific mutations

Wilson disease (WD) is a hereditary disorder, with recessive transmission and genetic heterogeneity. Several mutations of ATP7B, the gene underlying WD, were reported in many ethnic groups. In this study, mutation screening in ATP7B of 56 Saudi Arabian WD patients was undertaken. The clinical data of all patients were recorded. The entire ATP7B coding sequence, including intron-exon boundaries were screened for mutation by the polymerase chain reaction (PCR)-based mutation detection technique and DNA sequencing. Thirty-nine patients were symptomatic at presentation and 17 subjects were pre-symptomatic siblings of affected patients. Fourteen patients had neurological, 11 patients had mixed (hepatic and neurological), and 14 patients had hepatic presentations. Family history suggestive of WD was present in 72% of cases and 68% had consanguineous parents. Genetic analysis showed disease-causing mutations in three exons (exons 8, 19 and 21) of the ATP7B gene in 28 patients (50%). Mutations in exons 21 (18 cases) and 19 (one case) were unique for Saudis. This large series of Saudi patients with WD has shown wide variability in the genomic substrate of WD. There is no correlation between genotype and clinical presentation.     

人野生型parkin基因真核表达载体的构建及其在PC12细胞中的表达

目的 克隆人野生型parkin基因并构建真核表达载体pCDNA3．1—parkin，将重组质粒转染PC12细胞获得高表达人野生型parkin基因的PC12细胞克隆。方法 从胎脑组织中提取总RNA，用RT—PCR方法获得人野生型parkin基因的全长cDNA，插入pCR2．1—TA克隆载体中进行序列测定，测序正确后将其亚克隆至表达载体pCD—NA3．1，利用脂质体将重组质粒转染PC12细胞，经G418筛选获得抗性细胞克隆，采用RT—PCR和Western Blot方法鉴定人野生型parkin基因在PC12细胞中的过表达。结果 经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒，RT—PCR和Western Blot证明经G418筛选得到的转基因PC12细胞克隆中存在人野生型parkin基因的表达。结论 成功构建了人野生型parkin基因的真核表达载体，获得了稳定表达人野生型parkin基因的PC12细胞克隆，为进一步研究parkin的生物学功能以及parkin在帕金森病发病机制中的作用奠定了良好的基础。