rhddADAM15抑制肝癌细胞Bel-7402增殖的作用研究

ADAMl5属于跨膜蛋白ADAM家族中的一员,在乳腺癌、宫颈癌、卵巢癌等多种实体瘤中均发现其表达量提高。ADAMl5在降解细胞外基质、介导细胞的黏附、细胞间信号转导及在肿瘤发展进程中起到重要作用。因其去整合素区域含有RGD序列,ADAMl5可与多种整合素相互作用。在前期工作中,实验组利用大肠杆菌表达系统表达了重组人ADAMl5去整合素区域蛋白,记作rhddADAMl5。该研究将进一步针对rhddADAMl5抑制肝癌细胞Bel-7402增殖的机理进行分析及探讨。SRB法显示,rhddADAMl5可抑制Bel-7402细胞增殖并呈剂量依赖性,IC50为1．14gmol／L;利用DAPI核染发现,rhddADAMl5可显著诱导Bel-7402细胞凋亡;流式细胞仪分析发现,rhddAD—AMl5浓度为6gmol／L时,（87．44±7．25）％的细胞发生凋亡;PI单染分析细胞周期表明,rhddADAMl5作用后,部分Bel-7402细胞周期被阻滞于S期及G2／M期,并呈剂量依赖性,4gmol／LrhddADAMl5处理后G0／Gl期含量下降约14％;Westernblot分析显示,rhddADAMl5可下调Bel-7402细胞周期蛋白CDC26的表达,抑制CDC2-TyrM的去磷酸化,引起G2／M期的阻滞。     

Induction of Fas and Fas-ligand expression in plasmacytoma cells by a cytotoxic factor secreted by murine macrophages

Induction of tumoricidal activity is one of the major functions of activated macrophages. Our previous study demonstrated that P388D1 murine macrophage-like cells secreted a plasmacytoma cytotoxic factor (PCF) that selectively killed certain tumor cell lines including MOPC-315 plasmacytoma in vitro. Our subsequent studies demonstrated that PCF killed MOPC-315 cells by induction of apoptosis. In this report, the involvement of Fas and Fas ligand (FasL) in PCF-induced apoptosis was investigated. Results suggest that expression of Fas mRNA time-dependently increased in PCF-treated cells and reached an optimal level after 36 h of treatment. The augmented effect of PCF on Fas mRNA expression was significantly reduced by the addition of CB7.C2, an anti-PCF monoclonal antibody. The expression of FasL mRNA was also induced by PCF and reached an optimal level at 24 h, but sharply decreased after 36 h of treatment. Caspase-3 is one of the proteolytic enzymes that can be activated by the Fas-FasL interaction. In our studies, the enzymatic activity of caspase-3 was significantly induced by PCF after 6 h of treatment and reached an optimal level at 12 h. The augmented effect of PCF on caspase activity was significantly reduced by the addition of CB7.C2 and the caspase-3-specific inhibitor, DEVD-fmk. Therefore, PCF-treated plasmacytoma cells might undergo apoptosis via interaction between Fas and FasL.     

Characterization and partial amino acid sequence of human plasma glutathione peroxidase

Human plasma glutathione peroxidase was purified to homogeneity and partially sequenced. Overlapping peptide fragments from three endopeptidase digests permitted the determination of one sequence of 32 contiguous amino acids and one sequence of 23 contiguous amino acids. Five additional unique peptide sequences without obvious overlaps were obtained. The sequence of 32 amino acid residues aligns with positions 82-113 of human cytosolic glutathione peroxidase with nine mismatches without gaps or insertions. The sequence of 23 amino acid residues aligns with positions 157-178 with six mismatches and an insertion of one residue. Three additional peptide sequences with no obvious sequence homology to glutathione peroxidase can be aligned based on the sequence of a cDNA clone encoding plasma glutathione peroxidase that was isolated from a human placental library. The plasma enzyme is a homotetramer composed of 21-kDa subunits which cannot reduce phospholipid hydroperoxides. These results indicate that the plasma glutathione peroxidase is distinct from both the classical cytosolic enzyme and the monomeric phospholipid hydroperoxide glutathione peroxidase. Only a negligible amount of glutathione peroxidase activity was detected in bile, indicating that the liver exports plasma glutathione peroxidase exclusively to the circulation.     

PI3KγmRNA 3′非翻译区可能存在基因表达负调控区

为探索人磷脂酰肌醇 3 激酶γ(phosphoinositide 3 kinasePI3Kγ)基因 3′端非翻译区内AU富含区是否在基因表达调控中起作用 ,首先通过生物信息学分析发现在其 3′端非翻译区 (UTR)内存在0 9kb的AU富含区 ,其中包括 4个AU富含元件 ,以及 1个与众多基因非翻译区高度同源的长 130个碱基的区域 .将AU富含区插入报告基因egfp的下游构建pcDNA3 egfp AUR表达载体 .将表达载体转导NIH 3T3,74 0 2及K5 6 2细胞 ,流式细胞检测egfp的表达情况 .PI3Kγ基因 3′非翻译区AU富含区可显著降低egfp的表达 2～ 3倍 (P <0 0 1) .利用放线菌素D阻断RNA转录后 ,Northern印迹分析结果显示egfp AURmRNA较egfpmRNA不稳定 .实验结果提示 ,PI3Kγ基因 3′非翻译区AU富含区内可能存在转录后水平的基因表达负调控区 ,该负调控区可在一定程度上加速mRNA的衰变     

人神经元素3基因重组逆转录病毒表达载体的构建及其包装细胞株的建立

为构建表达人神经元素3基因(neurogenin 3,ngn3)的重组逆转录病毒载体,建立稳定表达ngn3的包装细胞株,本研究以流产人胎儿胰腺组织为材料,通过RT-PCR方法克隆出人ngn3基因,将其连接到pMD18-T载体上并测序,结果表明,测序得到的基因序列与发表的人ngn3基因序列(GenBank Accession No.BC126468)完全一致。将EcoRI和HpaI双酶切后的基因片段构建到pMSCV-neo逆转录病毒载体中,酶切鉴定结果表明,pMSCV-ngn3重组逆转录病毒载体构建成功。脂质体法将pMSCV-ngn3重组载体导入PT67包装细胞,G418筛选后,对得到的细胞株进行RT-PCR和免疫组化检测,结果显示,该细胞株在mRNA水平和蛋白水平均稳定表达Ngn3;收集该细胞株的培养上清液,进行RT-PCR检测及电镜观察,结果表明,该细胞株将导入的重组逆转录病毒载体pMSCV-ngn3包装成了具有感染性的病毒颗粒,并将其释放到了培养上清液中。以上结果表明PT67-ngn3包装细胞株建立成功。该细胞株的成功建立,为下一步将ngn3基因应用于提高人胎儿胰腺祖细胞诱导分化效率方面的研究奠定了基础。     

Biotype status and distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) in Shandong province of China based on mitochondrial DNA markers

Bemisia tabaci has caused significant crop losses in China during the last decade. Recent research has shown that two potentially invasive variants, biotypes B and Q, have been found in several regions of China. Our objective was to determine the biotype status and the distribution of B. tabaci in Shandong province, an important agricultural region of China. Based on mitochondrial DNA markers, both biotypes B and Q were detected, with B being the predominant biotype. The results indicate that the more recently introduced biotype Q has not only been located in China but also has established and spread in some regions.     

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH(2) terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulmonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Polymorphism of bone morphogenetic protein 4 gene and its relationship with litter size of Jining Grey goats

Two pairs of primers (P1 and P2) were designed to detect single nucleotide polymorphisms of exon 2 and intron 2 of bone morphogenetic protein 4 (BMP4) gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Boer, Angora and Inner Mongolia Cashmere goats) by single strand conformation polymorphism. Results showed that no polymorphism was detected for exon 2 (primer P1) of BMP4 gene in four goat breeds. For intron 2 (primer P2), three genotypes (AA, AB and BB) were detected in Jining Grey and Inner Mongolia Cashmere goats, two genotypes (AB and BB) in Angora goats, and only one genotype (AA) in Boer goats. Sequencing revealed one mutation (2203G>A) of BMP4 gene in the genotype BB in comparison to the genotype AA. The differences of litter size between AA, AB and BB genotypes were not significant (P > 0.05) in Jining Grey goats. A pair of primer (P3) was designed to detect polymorphism in the 3' flanking region of BMP4 gene that contained dinucleotide repeated sequence (CA) in the four goat breeds by microsatellite analysis. For primer P3, three genotypes (CC, CD and DD) were detected in four goat breeds. Sequencing revealed one more CA dinucleotide in genotype DD than in genotype CC. The Jining Grey does with genotype CC had 0.55 (P < 0.05) or 0.72 (P < 0.05) kids more than those with genotype CD or DD. These results preliminarily indicated that allele C of BMP4 gene is a potential DNA marker for improving litter size in goats.     

Rapid determination of gatifloxacin in biological samples and pharmaceutical products using europium‐sensitized fluorescence spectrophotometry

A europium‐sensitized fluorescence spectrophotometry method using an anionic surfactant, sodium dodecyl benzene sulphonate (SDBS), was developed for the determination of gatifloxacin (GFLX). The GFLX–Eu³⁺–SDBS system was studied and it was found that SDBS significantly enhanced the fluorescence intensity of the GFLX–Eu³⁺ complex (about 25‐fold). The optimal experimental conditions were determined as follows: excitation and emission wavelengths of 338 and 617 nm, pH 7.5, 3.0 × 10^–6 mol/L europium(III), and 5.0 × 10^–5 mol/L SDBS. The enhanced fluorescence intensity of the system (ΔI_f) showed a good linear relationship with the concentration of GFLX over the range 1.0 × 10^–8–8.0 × 10^–7 mol/L with a correlation coefficient of 0.9990. The detection limit (S:N = 3) was determined as 1.0 × 10^–9 mol/L. This method has been successfully applied for the determination of GFLX in pharmaceuticals and human urine/serum samples. Compared with most other methods reported, the rapid and simple procedure proposed here offered higher sensitivity, wider linear range and good stability. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.