人KLK1和EGFP双顺反子重组腺病毒构建及其在血管平滑肌细胞中的表达

目的：构建携EGFP为报告基因和人组织激肽释放酶1(hKLK1)基因双顺反子的重组腺病毒（Ad-hKLK1-IRES-EGFP）,并观察在自发性高血压大鼠（SHR）血管平滑肌细胞（VSMCs）中的表达变化。方法:通过双酶切质粒pBluescritII KS-hKLK1，将hKLK1基因定向克隆至带有IRES-EGFP的腺病毒穿梭质粒pDC316中，构建成重组穿梭质粒 ，将其与腺病毒骨架质粒BHGloxE1，3Cre共转染293A细胞，经包装并获得重组腺病毒，用PCR、酶切及测序方法对其进行鉴定，测定病毒滴度；用重组腺病毒感染VSMCs，用荧光显微镜下观察到的绿色荧光来测定感染率，用RT－PCR和Western blotting法测定hKLK1基因在VSMCs中的表达。结果:PCR、酶切和测序表明携EGFP的重组穿梭质粒pDC316-hKLK1-IRES-EGFP构建正确，并与骨架质粒在293A细胞中成功包装出重组腺病毒Ad-hKLK1-IRES-EGFP，其滴度为4.5×10¹¹/L。重组腺病毒感染VSMCs后，在荧光显微镜下观察到明亮绿色荧光蛋白表达，感染率高达90％以上，可检测到hKLK1基因的mRNA及蛋白表达，并与感染时间(1 d-7 d) 有显著依赖关系，峰值感染复数为100 MOI。结论:成功构建并包装了携EGFP和hKLK1基因的双顺反子重组腺病毒载体系统Ad-hKLK1-IRES-EGFP；感染VSMCs可检测到基因hKLK1和EGFP的独立共同高表达，该系统为一直观、安全、高效的基因转移系统。     

survivin的siRNA靶向干扰膀胱癌的实验研究

目的实验研究靶向转染survivin的小分子干扰RNA（small interfering RNA，siRNA）对膀胱癌生物学行为的影响。方法设计1对survivin编码基因序列特异的siRNA，用脂质体转染T24膀胱癌细胞株。采用流式细胞术检测转染效率和细胞凋亡；荧光实时定量PCR检测干扰前后survivin的mRNA表达；用survivin的siRNA片段插入空载体后建立重组载体pRNAT-U6、1／Neo-survivin。结果脂质体转染T24细胞后的转染效率为92．3％；survivin编码基因序列特异的siRNA能有效下调survivin基因的表达，呈时间和剂量依赖性，最大效应浓度为100nmol／L，此时survivin表达水平下调了75．91％，并显著地抑制了细胞生长，抑制率达55．29％，差异有统计学意义（P〈0．01）；细胞凋亡率亦增至45．70％，与对照相比差异有统计学意义（P〈0．01）。用限制性内切酶将空载体线性化，T4DNA连接酶将siRNA片段插入空载体中，对该重组表达载体进行鉴定，获得RNA干扰质粒载体pRNAT-U6．1／Neo-survivin。结论siRNA．survivin能显著下调膀胱癌细胞survivin基因表达水平，促进细胞凋亡，抑制肿瘤细胞增殖，可能成为膀胱癌基因治疗的新工具。成功构建的靶向survivin基因表达的RNA干扰质粒载体为进一步应用RNA干扰技术进行survivin基因功能研究奠定了基础。     

Gene therapy of amyotrophic lateral sclerosis

Two-year experiments were performed to evaluate the neurotrophic effect of hypoxia-inducible factors (vascular endothelial growth factor and angiogenin) expressed in recombinant human adenoviruses in amyotrophic lateral sclerosis. Randomized placebo-controlled trial demonstrated safety and good tolerability of the recombinant antiviral drugs. The life span of patients under conditions of hypoxia increased after treatment with the test drug, which was probably related to improved resistance of motoneurons. The presence of virus-neutralizing antibodies decreases the effectiveness of adenoviral vectors, which necessitates differential approach to the selection of patients and continuous monitoring of gene therapy. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 4, pp. 467–470, April, 2008     

逆转录病毒载体介导乙型肝炎病毒反义基因的转录表达

为了探索在真核细胞内转录表达乙型肝炎病毒（ＨＢＶ）反义核酸的方法，用基因重组技术将ＨＢＶ前Ｃ／Ｃ基因（ＰｒｅＣ／Ｃ）和前Ｓ／Ｓ基因（ＰｒｅＳ／Ｓ）片段反向插入逆转录病毒载体质粒，再将重组体分别转染ＰＡ３１７包装细胞，进而获得能够介导ＨＢＶ反义基因向小鼠ＮＩＨ３Ｔ３细胞转移表达的重组逆转录病毒。经分子杂交试验表明，含有ＨＢＶ反义基因的重组逆转录病毒序列已经整合到转染的ＰＡ３１７细胞染色体上；转导的ＮＩＨ３Ｔ３细胞内有ＨＢＶ反义ＲＮＡ转录表达。结论：逆转录病毒载体包装细胞系统能够介导ＨＢＶ反义基因在真核细胞中转录表达，因而有可能利用反义技术和基因转移方法进行抗－ＨＢＶ基因治疗     

Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

基因功能预测问题中的样本不平衡处理

应用机器学习进行分类是基因功能预测的一种重要手段。但是许多预测集中的阳性样本过少，会降低功能预测的效果。针对此问题，本研究对结合支持向量机（SVM）算法的几种常用非平衡数据分类方法进行实验比较，包括投票整合分类器和移动分类面等。在此基础上提出通过加权修正投票的整合策略，以提高预测效果。实验结果显示，结合多数类样本限数取样及整合思想的投票整合法预测效果优于移动分类面法，而在投票整合法基础上的加权修正整合方法在所有方法中获得更好更稳定的结果。     

Analyzing Event-Related Potentials: The Utility of High and Low Pass Filtering in Improving the Relationship Between Various Amplitude Measures and Principal Components Analysis Factor Scores

The present investigation sought to determine whether the relationship between event-related potential (ERP) principal components analysis (PCA) factor scores and analogous waveform amplitude measures could be improved by high- and low-pass filtering the waveforms at a suitable cutoff value. Visual oddball ERPs were submitted to a varimax-rotated PCA performed on the variance/covariance matrix. Principal components corresponding to P300 and Slow Wave were obtained. In keeping with the fact that the variance/covariance PCA analyzes sources of variance around the grand mean waveform, the grand mean waveform was subtracted from each of the original waveforms, and baseline-referenced amplitude measurements were then made of P300 and Slow Wave. P300 was measured both as the maximum positive peak between 275 and 425 ms, and as the average amplitude during that interval. Slow Wave was measured as the average amplitude during the interval 400–700 ms. The P300 measurements were then repeated after high-pass filtering the difference waveforms at 2 Hz. Slow Wave measurements were repeated after low-pass filtering at 2 Hz. The value of 2 Hz was chosen as giving a reasonable cutoff based upon estimates of the wavelengths of the two components derived from inspection of their respective factor loading vectors. The correlation between factor scores and amplitude measurements was .94 for unfiltered Slow Wave and actually declined slightly but significantly to .91 when the waveforms were low-pass filtered. It would appear that Slow Wave factor scores emerging from a PCA can be fairly well approximated by a time-band measurement algorithm, and that this approximation is not improved by low-pass filtering. For both filtered and unfiltered measurements of P300, the amplitude/factor score correlation was significantly higher for the time-band method than for the peak method. Further, high-pass filtering at 2 Hz improved the time-band/factor score correlation significantly from .62 to .75. This improvement is probably because the unfiltered measurements were tapping sources of variance due both to the higher frequency P300 component as well as a simultaneously active, lower frequency Slow Wave component. Theoretical implications of these findings are discussed.     

一种独立成分分析的鲁棒算法及其在脑磁图数据分析中的应用

独立成分分析是一种新的信号处理统计方法。被广泛用于各个领域。在信号分析中面临的难题是：源信号的不同特性（既包括超高斯信号又包括亚高斯信号）；未知的独立源数目；传感器信号受到较大的加性噪声污染。针对以上难题，本文提出了一种独立成分分析的鲁棒算法。该方法先对观测数据作预处理，将包含噪声的高维传感器观测信号降维分解到信号子空间和噪声子空间。利用交叉验证法估计出独立源的数目（解决了独立成分分析本身不能确定源数目的缺陷）；然后利用快速稳定的FastICA算法分离独立成分。通过人工合成的数据和实际的脑磁图数据分析。验证了这种方法的功效。     

小鼠pdx-1基因真核表达载体的构建及其在胚胎干细胞中的表达

目的： 克隆小鼠pdx-1基因，构建其真核表达载体，并在小鼠胚胎干细胞中表达，为糖尿病的细胞移植治疗奠定基础。方法： PCR扩增小鼠胰腺pdx-1基因 cDNA，酶切后和携带绿色荧光蛋白报告基因的真核表达载体pEGFP-N1重组，将pdx-1基因 cDNA片段连接到pEGFP-N1载体的多克隆位点，形成重组载体pEGFP/pdx-1，转化大肠杆菌DH5α菌株，构建成pdx-1基因真核表达载体质粒。扩增DH5α后抽提质粒DNA，Hind Ⅲ 和BamHⅠ酶切，电泳，DNA测序鉴定。鉴定正确的质粒DNA用脂质体包裹后转染小鼠胚胎干细胞MESPU13。结果： 从小鼠胰腺cDNA扩增出876 bp的DNA片段并成功重组到pEGFP-N1载体中。经酶切和DNA测序验证，插入载体的DNA片段为pdx-1基因，插入方向正确。重组质粒经脂质体转染胚胎干细胞MESPU13，24 h 后观察到绿色荧光蛋白报告基因和目的基因的pdx-1表达。结论： 小鼠pdx-1基因的克隆和真核表达载体构建获得成功，为进一步研究其功能奠定了基础。     

氯霉素乙酰转移酶检测(CAT assay)

本文介绍一种快速方便的外源基因在哺乳动物细胞中瞬间表达检测系统,即氯霉索乙酰转移酶检测系统。该项检测技术不仅对基因的调控顺序(Cis-acting elements)如启动子、增强子等检测提供了有效的工具,而且为真核基因在哺乳动物细胞表达体系中,质粒的构建及筛选提供了行之有效的手段。并介绍了该技术的全过程及其操作要点。