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。     

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     

丙型肝炎患者外周血单核细胞中丙型肝炎病毒复制的研究

９例临床诊断为丙型肝炎患者，研究其外周血单核细胞中ＨＣＶＲＮＡ的存在及复制。９例患者血清标本抗－ＨＣＶ及ＨＣＶＲＮＡ均为阳性，采用高敏感的逆转录一套式ＰＣＲ法测定其外周血单核细胞中ＨＣＶ正、负链ＲＮＡ，结果９例患者外周血单核细胞中７例ＨＣＶ正链ＲＮＡ阳性，３例ＨＣＶ负链ＲＮＡ阳性，证实部分丙肝患者外周血单核细胞中存在ＨＣＶ的复制，表明肝细胞并非为ＨＣＶ感染与复制的唯一场所。     

Short utterance recognition using a network with minimum training

A feedforward network is used to recognize short, digitized, isolated utterances. A high, multispeaker recognition rate is achieved with a small vocabulary with a single training utterance. This approach makes use of the pattern recognition property of the network architecture to classify different temporal patterns in the multidimensional feature space. The network recognizes the utterances without the need of segmentation, phoneme identification, or time alignment. We train the network with four words spoken by one single speaker. The network is then able to recognize 20 tokens spoken by 5 other speakers. We repeat the above training and testing procedure using a different speaker's utterances for training each time. The overall accuracy is 97.5%. We compare this approach to the traditional dynamic programming (DP) approach, and find that DP with slope constraints of 0 and 1 achieve 98.5% and 85% accuracies respectively. Finally we validate out statistics by training and testing the network of a four-word subset of the Texas Instruments (Tl) isolated word database. The accuracy with this vocabulary exceeds 96%. By doubling the size of the training set, the accuracy is raised to 98%. Using a suitable threshold, we are able to raise the accuracy of one network from 87% to 98.5%. Thresholding applied to all networks would then raise the overall accuracy to well over 99%.

This technique is especially promising because of the low overhead and computational requirements, which make it suitable for a low cost, portable, command recognition type of application.     

Expression of IGF-I and IGF-II mRNA in breast tissue

Summary Expression of IGF-I and IGF-II was studied in human breast cancer tissues by in situ hybridization. IGF-I mRNA was detected only in stromal cells adjacent to normal breast epithelial cells. Stromal cells associated with the tumor cells did not contain IGF-I, nor did malignant or benign breast epithelial cells. In contrast, IGF-II mRNA was found in both the malignant epithelial cells and their adjacent stromal cells. These data imply that stromal cells associated with breast epithelium may switch expression from IGF-I to IGF-II during breast cancer evolution. This appearance of IGF-II expression may identify cancer-associated stromal cells that have a fetal phenotype.     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

甲硫氨酸脑啡肽对抗体和白细胞介素－2的产生和基因转录的影响

甲硫氨酸脑啡肽对抗体的产生和Ｔ细胞免疫功能具有调节作用。为了明确这种作用机制，本实验观察了抗体和白细胞介素－２（ＩＬ－２）的产生和基因表达。甲硫氨酸脑啡肽对３Ｆ３杂交瘤细胞的抗体产生及其轻链和重链基因表达影响不明显。较高浓度的甲硫氨酸脑啡肽（０．１ｎｍｏｌ·Ｌ￣（－１）－１μｍｏｌ·Ｌ（－１））不仅促进ＩＬ－２的产生和ＩＬ－２ｍＲＮＡ的转录，而且能提高ＩＬ－２ｍＲＮＡ的稳定性。