人GM－CSF基因在昆虫细胞中表达的研究

本工作构建的昆虫表达重组体ｐＡＣ６１０－ＧＭＴ，是在ＡｃＭＮＰＶ的Ｐｏｌｙｈｅｄｒｉｎ启动子控制下，表达去除了信号肽编码顺序的人ＧＭ－ＣＳＦ基因（ｃＤＮＡ）的转染载体。它与野生ＡｃＭＮＰＶ病毒ＤＮＡ共转染Ｓｆ２１细胞，经过筛选得到纯化的可表达人ＧＭ－ＣＳＦ的重组病毒株ｖＡｃＧＭＴ。其感染细胞总ＲＮＡ的Ｎｏｒｔｈｅｒｎ分析结果表明，重组病毒在ｍＲＮＡ水平有人ＧＭ－ＣＳＰ特异性表达，其表达水平在感染后４８ｈ时达高峰，７２ｈ未见明显下降。感染细胞裂解物的Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析和活性测定也证实其蛋白水平的表达，并有人ＧＭ－ＣＳＦ的生物学活性。     

Cryptococcus neoformans CAP10 Gene Regulates the Immune Response in Mice

The capsule associated protein 10 gene (CAP10) is indispensable to the formation of the polysaccharide capsule, and is closely associated with Cryptococcus (C.) neoformans virulence. In this study, we designed the shRNA expression plasmid to interfere with the synthesis of CAP10 gene. We infected mice with yeast cells in the respiratory tract, and monitored the development of infections in lung tissues. Results showed that the cap10-shRNA group may alleviate pathological lesions in pulmonary C. neoformans infection, and a lower degree of inflammatory cells was observed in the cap10-shRNA group. Moreover, the fungal burden was significantly lower in the cap10-shRNA group, indicating that the clearance towards C. neoformans was somehow affected. Down-regulation of CAP10 was beneficial to the balance of Th1/Th2 and Th17/Treg ratios. Collectively, our results showed that the expression of CAP10 was associated with an antifungal immune response in mice, suggesting that CAP10 regulates the inflammatory response. Therefore, we expect that the CAP10 gene will become a new molecular therapeutic target in cryptococcosis treatment.     

Human Monocytic U937 Cells Kill Salmonella In Vitro by NO-Independent Mechanisms

Nitric oxide (NO) has a central role in host defense against intracellular microbes. HLA-B27 has been shown to directly modulate host-microbe interaction in vitro, leading to the impaired elimination of Salmonella in human monocytic U937 cells. Here, we studied whether impaired elimination of Salmonella would result from differences in NO production between HLA-B27- and HLA-A2-transfected U937 cells. Both human monocytic transfectants produced NO equally well and killed Salmonella via NO-independent mechanisms.     

人载脂蛋白A—1基因在中国仓鼠卵巢细胞的表达

本文报道了用人载脂蛋白A-1(apo A-1)的cDNA探针从人基因库中筛选到apo A-1全基因:将2.2kb的apo A-1全基因连接到1.9kb鼠MT-1基因启动子下游,组装成人apo A-1基因表达质粒pLY1。质粒pLY1与质粒pSV2-neo共转化中国仓鼠卵巢(CHO)细胞,经G418筛选,单细胞克隆培养,经酶联免疫测定,结果表明,人apo A-1可在CHO细胞中得到表达。     

Normal Neurogenesis but Abnormal Gene Expression in Human Fragile X Cortical Progenitor Cells

Human stem and progenitor cells offer an innovative way to study early events in development. An exciting new opportunity for these cells is their application to study the underlying developmental consequences of genetic diseases. Because many diseases, ranging from leukemias to developmental disorders, are caused by single-gene defects, stem and progenitor cells that carry disease-causing genetic mutations are invaluable in understanding and treating disease. We have characterized human neural progenitor (hNPCs) cells that carry a single-gene defect that leads to the neurodevelopmental disorder Fragile X syndrome (FX). A loss-of-function mutation in the FMR1 gene leads to subtle changes in neural development and subsequent mental impairment characteristic of FX. hNPCs were isolated from fetal cortex carrying the FMR1 mutation to determine whether aberrations occur in their proliferation and differentiation. As expected, FX hNPCs have reduced expression of the FMR1 gene product Fragile X mental retardation protein (FMRP), and this decrease is maintained in culture and following differentiation. In contrast to a previously published report, the proliferation of FX hNPCs and their differentiation into neurons is not different from unaffected controls. Although the early development of FX hNPCs is essentially normal, microarray analysis reveals novel changes in the expression of signal transduction genes in FX hNPCs. Therefore, hNPCs have intrinsic characteristics that can be investigated to further our understanding and potential treatment of developmental disorders such as FX.     

Interleukin-10 Receptor Signaling in Innate Immune Cells Regulates Mucosal Immune Tolerance and Anti-Inflammatory Macrophage Function

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Expression of Mn-Superoxide Dismutase Gene in Nontumorigenic and Tumorigenic Human Mammary Epithelial Cells

Manganese superoxide dismutase (Mn-SOD), localizedat the mitochondrial matrix, has the ability to protect cellsagainst oxidative damage. It has been reported that low levels ofMn-SOD gene expression cause the development of certain kind oftumors. On the other hand, overexpression of Mn-SOD gene may playan important role in the development of cancer. In our study, wefind that Mn-SOD activity was higher in nonaggressive (MCF-7) andaggressive (BT-549 and 11-9-14) breast cancer cell lines comparedto that of nontumorigenic (MCF-12A and MCF-12F) mammaryepithelial cell lines. We also observed an increased expressionof Mn-SOD gene in cancerous cell lines. The elevated level of SODactivity in nonaggressive and aggressive breast epithelial celllines was associated with some changes in nucleotide sequence.     

Gene Expression Pattern of Human Chorion-Derived Mesenchymal Stem Cells during Adipogenic Differentiation

Purpose: The aim of this study was to identify the adipocyte-specific gene expression patterns in chorion-derived mesenchymal stem cells during adipogenic differentiation. Materials and Methods: Chorionic cells were isolated from the third trimester chorions from human placenta at birth and identified morphologically and by fluorescence-activated cell sorting analysis. After inducing adipogenic differentiation for 28 days, cells at days 3, 10, 21 and 28 were analyzed by Oil red O staining and RNA extraction in order to assess the expression levels of adipocyte marker genes, including CCAAT-enhancer binding protein α (C/EBPα), peroxisome proliferator- activated receptor γ (PPARγ), fatty acid binding protein 4 (FABP4) and Glycerol-3-phosphate dehydrogenase (GPD2). Cells not induced for differentiation were compared with the induced cells as a control group. Results: Chorion-derived cells showed the same pattern as fibroblasts, and expressed CD73, CD105, and CD166 antigens, but not CD45, CD34, and HLA-DR antigens. On day 3 after differentiation, cells began to stain positively upon Oil red O staining, and continuously increased in lipid granules for 4 weeks. The expression level of C/EBPα increased 4.6 fold on day 3 after induction, and continued to increase for 4 weeks. PPARγ was expressed at a maximum of 2.9 fold on day 21. FABP4 and GPD2 were significantly expressed at 4.7- and 3.0-fold, respectively, on day 21, compared to controls, and further increased thereafter. Conclusion: Human chorion-derived mesenchymal stem cells exhibited the sequential expression pattern of adipocyte marker genes during differentiation, corresponding to adipogenesis.     

肝脏内源性microRNA调控乙型肝炎病毒基因的表达与复制

目的探讨肝脏内源性microRNA对乙型肝炎病毒(HBV)复制与基因表达的影响。方法通过miRNA靶点分析软件寻找与HBV序列之间相关联的肝脏内源性microRNA,体外化学合成相应的microRNA分子,将合成寡核苷酸及对照与1.3倍HBV全基因组真核表达质粒pUC18-HBV1.3采用Lipofectamine2000共转染HepG2细胞,转染48h后收集细胞培养上清;通过ELISA检测HBsAg、HBeAg的表达水平;Western印迹检测HBcAg的表达水平;Trizol抽提转染细胞RNA,逆转录后用荧光定量PCR检测HBVmRNA的水平;提取细胞基因组DNA,Southern印迹检测HBV的复制中间体。经以上检测从HBV蛋白表达、转录和复制水平评价相应的microRNA作用效应。结果生物信息学方法提示miR-16和miR-122存在与HBV基因组作用的可能结合位点。经试验初步证实miR-16可下调HBV蛋白的表达及HBVDNA水平;miR-122可下调HBsAg、HBeAg的表达,上调HBVmRNA的水平。结论肝脏内源性microRNA可以调节HBV的复制与基因表达。     

SARS冠状病毒M基因膜内区的克隆、表达与鉴定

目的对SAILS冠状病毒M蛋白膜内部分（Mc蛋白）的基因（Mc区）进行克隆、表达及鉴定。方法采用PCR技术从SAILS-CoV GD322株M基因全长片段上扩增得到Mc区，克隆至pGEM-T载体。利用引物上的EcoR I／Xho I酶切位点切下Mc区插入至pET-32a（＋）表达载体，转化原核表达系统BL21，表达His-融合蛋白，利用SDS-PAGE和Western-blot对表达产物进行鉴定。结果扩增的Mc区长度为360bp，重组子经PCR，双酶切和测序鉴定，获得pET-32a（＋）／Mc原核表达菌株并可进行高效表达，表达产物经SDS-PAGE和Western-blot验证为相对分子质量约43000的融合蛋白，与预期理论值相符。结论实现了Mc蛋白的高效表达，为进一步研究SAILS冠状病毒Mc蛋白的生物学作用奠定了基础。     

人脂联素球状结构域基因在昆虫杆状病毒表达系统中的表达

目的利用杆状病毒表达系统表达人脂联素球状结构域（gAd）基因。方法以人基因组为模板,PCR法扩增人gad基因,将gAd基因与供体质粒pFastBacHTB连接,转化含有穿梭载体Bacmid的大肠杆菌DH10Bac。筛选转座成功的重组穿梭载体Bacmid—gAd,通过脂质体介导,转染昆虫细胞Sf9,经SDS-PAGE、免疫印迹法检测表达产物。结果重组杆状病毒感染的Sf9细胞形态变化明显,SDS-PAGE电泳结果显示,BacmidgAd组和对照组相比．在相对分子质量15000～25000之间多出一清晰的蛋白条带．免疫印迹法证实该条带能与相应的抗His标签抗体结合。结论人gAd基因成功在真核细胞中表达,为后续的实验研究奠定了基础。     

流体切应力对人内皮细胞迁移和整合素基因表达的影响

目的研究流体切应力空间梯度和均匀切应力对内皮细胞（ECs）迁移和整合素基因表达的影响，为阐明应力诱导血管重建的分子机制提供一些实验依据。方法将脐静脉内皮细胞置于平行平板流动腔系统，分别施加11dyn／cm^2的均匀切应力（矩形组）和5～14dyn／cm^2的切应力梯度（梯度组）为受力组，以静态条件下培养的ECs为对照组（静止组）。Transwell方法检测切应力对ECs迁移能力的影响；半定量RT-PCR测定ECs整合素α3β1、α5β1mRNA3、6和12h的表达情况。结果①同静止组相比，切应力梯度组ECs3h的迁移（88±4 vs 23±3，P〈0．01，n=3）能力显著提高；而均匀切应力组ECs3h迁移（21±3VS23±3）同静止组相比，差异无统计学意义（P〉0．05，n=3）；②ECs受切应力梯度作用3h即可诱导ECs整合素α3β1、α5β1mRNA表达（P〈0．01，P〈0．05）；均匀切应力于6h激活ECs整合素理，亚型表达（P〈0．01），α3 、β1亚型表达延迟到12h激活（P〈0．01）。结论切应力通过上调整合素基因表达促进了ECs迁移，提示整合素信号通路参与了切应力条件下ECs迁移过程的信号转导。