Antisense Smad2 inhibits high glucose-stimulated production of extracellular matrix in cultured rat glomerular mesangial cells

Transforminggrowthfactor-β1(TGF-β1)is amultifunctionalpolypeptidethatregulatesanum-berofcellularprocesses,includingcellprolifera-tion,differentiation,apoptosis,migration,matrix synthesis,andtheimmuneresponse[1,2].Inchron-icrenaldiseases,TGF-β1isakeymediatorofex-tracellularmatrix(ECM)accumulation[3].Oneof thetargetrenalcellsforTGF-β1isglomerular mesangialcellsthatarecapableofproducingcom-ponentsofECM,suchascollagens,lamininand fibronectin[4,5].Recentstudiesindicatedthatinhi-bitionofT…     

Lines of Murine Oligodendroglial Precursor Cells Immortalized by an Activated neu Tyrosine Kinase Show Distinct Degrees of Interaction with Axons In Vitro and In Vivo

Replication-defective retroviruses expressing the t- neu oncogene, or a hybrid protein with the neu tyrosine kinase linked to the external region of the human epidermal growth factor receptor ( egfr-neu ), were used to establish lines of murine oligodendroglial precursor cells. Differentiation of the t- neu lines into myelin-associated glycoprotein (MAG)-positive oligodendrocytes was induced by dibutyryl cAMP, and the egfr-neu line showed limited differentiation in vitro upon withdrawal of epidermal growth factor. Cerebellar granule cell neurons expressed mitogens for the cell lines. Upon transplantation into demyelinated lesions, t- neu line cells engaged with the demyelinated axons whereas the egfr-neu line cells differentiated further and ensheathed the axons. These cell lines thus interact with neurons in vitro and in vivo and can be used as tools to define the molecules involved in different stages of neuron-glia interaction.     

A Drosophila melanogaster hobo-white+ vector mediates low frequency gene transfer in D. virilis with full interspecific white+ complementation

Transformation of a Drosophila virilis white mutant host strain was attempted using a hobo vector containing the D. melanogaster mini-white⁺ cassette (H[w⁺, hawN]) and an unmodified or heat shock regulated hobo transposase helper. Two transformant lines were recovered with the unmodified helper (HFL1), one containing only the white⁺ marked vector, and a sibling line containing the vector as well as an HFL1 helper integration. An approximate total transformation frequency of 1% is deduced. A high frequency of wing and eye morphology mutants were also observed, suggesting that hobo may have mobilized a related element in D. virilis. The data reaffirms a relatively low transformation vector activity for the hobo transposon in D. virilis; however, nearly full interspecific expression of the white⁺ marker supports its possible function in other species as well.     

一种直接高效克隆PCR产物的载体制备

Ｂｌｕｅｓｃｒｉｐｔ经ＥｃｏＲ Ｖ酶切后，在Ｔａｑ酶作用下加入ｄＴＴＰ使其３’端加上碱基“Ｔ”而成为３’端突出的粘性末端载体。用此载体直接与ＰＣＲ产物连接进行克隆。克隆效率比采用平端载体克隆的效率提高约５０倍。     

GALC transduction leads to morphological improvement of the twitcher oligodendrocytes in vivo

Globoid cell leukodystrophy (GLD, Krabbe disease) is a severe demyelinating disease caused by a genetic defect of beta-galactocerebrosidase (GALC). To date treatment to GLD is limited to hematopoietic stem cell transplantation. Experimental approaches by means of gene therapy in twitcher mouse, an authentic murine model of human GLD, showed significant but only marginal improvements of the disease. To clarify whether the introduction of GALC could provide beneficial effects on the oligodendrocytes in GLD, we transduced twitcher oligodendrocytes by stereotactically injecting recombinant retrovirus encoding GALC-myc-tag fusion gene into the forebrain subventricular zone of neonatal twitcher mouse. In vivo effects of exogenous GALC on twitcher oligodendrocytes were studied histologically by combined immunostaining for the myc-epitope and the oligodendroglial specific marker, pi form of glutathione-S-transferase, at around 40 days of age. We show here that GALC transduction led to dramatic morphological improvement of the twitcher oligodendrocytes comparing with those in untreated twitcher controls. This study provided direct in vivo evidence that GALC transduction could prevent or correct aberrant morphology of oligodendrocytes in GLD which may be closely related to the dysfunction and/or degeneration of oligodendrocytes and the demyelination in this disease.     

EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。     

人类白细胞抗原-G突变体cDNA克隆及在K562细胞上的表达

目的：克隆人类白细胞抗原-G(Human leukocyte antligen-G，HLA-G)突变体cDNA，并使其在HLA-I类阴性的K562细胞上获得稳定表达，为研究配-受体之间的识别机制奠定基础。方法：用RT-PCR方法从人子宫蜕膜组织扩增出HLA-GcDNA，得到全长HLA-GPCR产物后，用桥式PCR方法进行定点点突变，将突变的目的基因亚克隆于逆转录，将突变的目的基因亚克隆于逆转录mG-pLNCX表达载体，采用感染的方法将重组质粒转入K562细胞，最后经G418筛选及有限稀释，利用单克隆抗体W6/32进行FACS及mRNA检测，观察HLA-G突变体在靶细胞表面的表达。结果：HLA-G突变体分子在经mG-pLNCX转染的靶细胞表面获得稳定高表达。结论：成功构建了mG-pLNCX表达载体，并使HLA-G突变体分子在HLA-I类阴性的靶细胞K562细胞上获得稳定表达。     

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     

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。