男性不育症患者中YRRM2基因缺陷的研究

YRRM基因是控制精子生成与成熟的一个重要因素，一旦该基因缺陷，将会造成无精或少精。本研究对340例无精与少精患者的外周血标本进行了PCR基因扩增筛查，结果发现7例为该基因缺陷，占2％。证明YRRM2的缺陷也是造成中国人群男性不育的一个原因，从而可作为指导医生采用适当治疗方法的一个可靠指标。在实验过程中，采用直接加热白细胞提取基因组DNA，并以此作为模板对YRRM2基因进行扩增，既节省时间和经费，又保持良好的扩增效果，使之可用于临床医院作为常规检查。     

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。     

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。     

Down-regulation amyloid β-protein 42 production by interfering with transcript of presenilin 1 gene with siRNA

AIM:ToinvestigatethepathogenesisofAβ42yieldingandnewdrugtargetsaswellasthepossibilityofRNAinterference(RNAi)techniquefortreatmentofAlzheimerdisease(AD).METHODS:HumanADpresenilin1(PS1)cDNAsequencewasobtainedfromNCBIwebsite.ThethreesitesofRNAiactionandonemissensecontrolsitewereselectedinPS1cDNAthroughonlinedesignofAmbioncompany.Toconfirmspecificityofthesesites,weconductedaBLASTsearchoftheIMAGEESTlibrary.Thecorrespondingdouble-strandedDNAwasusedtoconstructpSilencer3.1-H1p…     

脊髓源星形胶质细胞GFAP表达抑制真核表达载体构建及最佳短发夹样RNA分子筛选

目的构建并筛选大鼠胶质原纤维酸性蛋白（GFAP）表达抑制短发夹样RNA（shRNA）真核表达载体。方法针对GFAP基因全编码序列设计并合成三对9bp茎环结构、19bp干扰序列特异性shRNA模板，体外定向克隆构建特异性重组质粒真核表达载体；通过体外大鼠脊髓源星形胶质细胞GFAP表达抑制模型，脂质体介导RNA干扰分子转染，实时荧光定量RT—PCR及Wesem blot技术观察RNA干扰后原代星形胶质细胞GFAP表达抑制效果．筛选最佳GFAP表达干扰抑制真核表达载体。结果序列测定证实GFAP—shRNA重组质粒真核表达载体构建成功，三对shRNA模板在mRNA及蛋白表达水平抑制靶基因表达效率分别为81％、63％、56％。结论高效率的GFAP—shRNA真核表达载体在大鼠原代星形胶质细胞GFAP表达抑制模型中能高效抑制GFAP基因表达，为后续多靶点RNA干扰技术在脊髓损伤胶质瘢痕抑制基因治疗中的应用奠定了前期基础。     

The Effect of 50 Hz External Electrical Interference on Implanted Cardiac Pacemakers

The effects of injected 50 Hz alternating current on the function of cardiac pacemakers has been observed in 18 patients with implanted unipolar VVI units. Current, in the range 0-600 microA was applied via electrodes attached to the patients' upper body and feet and fed from a specially designed current injection unit at the bedside. Most implanted pacemakers reverted to interference mode in the current range 29-250 microA. At current levels just below the reversion current all units developed irregular and inappropriate pacing. This current level was pacemaker dependent and varied in the range 27-246 microA. The total reversion current depended on the location of the injecting electrodes and on the patients' posture. The sensitivity of the units to injected interference was increased by deep inspiration. Temporary pacing catheters fitted to an additional ten patients were used to monitor the interference voltage which would be sensed by an implanted unit. This voltage was similarly dependent on patient posture and on deep respiration. Current injection has proved to be a safe, controllable and reproducible method of testing the sensitivity of implanted pacemakers to 50 Hz external interference.     

S_(180)肿瘤核糖核酸体外对小鼠脾淋巴细胞蛋白质合成的影响

用放射性同位素标记氨基酸参入法研究了肿瘤细胞RNA对小鼠脾淋巴细胞蛋白质合成的影响。结果表明,肿瘤RNA在体外可显著抑制脾淋巴细胞的(~3H)亮氨酸参入,这种抑制效应无肿瘤细胞来源特异性,但有组织来源特异性。其抑制效应的活性组分可能存在于RNase酶解后的Sephadex G-100凝胶过滤第二组分中。     

慢性丙型肝炎患者丙型肝炎病毒核酸与IgM抗体关系的研究

本文检测了１４０例丙型肝炎病毒（ＨＣＶ）抗体（抗－ＨＣＶ）阳性的慢性丙型肝炎（ＣＨＣ）的ＨＣＶ核酸（ＨＣＶＲＮＡ）和ＩｇＭ抗体（抗－ＨＣＶＩｇＭ）两项指标。结果表明，原始诊断为不同临床型的肝炎（ＨＣ）患者，８年随访时，ＨＣＶＲＮＡ和抗－ＨＣＶＩｇＭ阳性率分别为８０．７％和９０．７％（ｕ＝２．３９Ｐ＜０．０５）．在原始诊断不同临床型ＨＣ转慢者中，上述两项指标均未发现统计学上的差别（均为Ｐ＞０．０５）。ＨＣＶＲＮＡ与抗－ＨＣＶＩｇＭ配对比较，符合率为７８．６％。基因分型初步结果表明，河北省固安ＨＣＶ以基因Ⅱ型为主。本研究提示，随访８年的ＣＨＣ患者绝大多数仍有传染性；本文方法检测的抗－ＨＣＶＩｇＭ不能代表早期感染标志，但代表慢性感染活动化或带毒，所以，在不具备检测ＨＣＶＲＮＡ的地方更具有实用价值．     

Astrocyte RNA in relation to neuronal RNA depletion in Alzheimer's disease

Summary A new double-staining procedure, in which the techniques of immunocytochemistry of glial fibrillary acidic protein (GFAP) and quantitative microdensitometry of azure B-RNA were combined, was used to study nucleic acid alterations in fibrous astrocytes in Alzheimer's disease (AD). RNA contents of GFAP-positive cells of the hippocampal endplate (Rose's H₃-H₅ fields) and the dentate gyrus molecular layer were determined in ten autopsy-proven AD patients (ages 51–88) and ten age-matched, non-demented control. In addition, RNA contents of pyramidal neurons of the endplate were examined. While there were no differences in RNA contents of astrocytes of either region between AD patients and controls, neuronal RNA was markedly depleted. These data suggest that astrocytes maintain protein synthetic capabilities in AD and that RNA loss is limited to the neuronal compartment.Supported by Grants 1P01-AG05119 and 1P50-AG05144 from the National Institutes of Health and by a Small Research Project Award from the University of Kentucky Medical Center