趋化因子RANTES的研究进展

RANTES是一类重要的趋化因子,其不仅作为一种趋化蛋白参与炎症反应和介导白细胞的游走和浸润,而且可调节细胞的生长和分化,在HIV和动脉粥样硬化(AS)中均起着重要的作用。因而研究RANTES在生物体内的作用,阐明其详细的作用机制可以指导临床应用。     

大肠杆菌在葡萄糖和甘油碳源培养条件下的基因表达谱分析

【目的】探讨葡萄糖和甘油碳源对大肠杆菌(Escherichia coli)基因表达谱的影响。【方法】通过基因表达数据库GEO下载基因芯片数据集GSE2037和高通量测序数据集GSE156143,分别对两个数据集中的葡萄糖和甘油碳源样本进行差异表达基因筛选,对筛选到的差异表达基因进行并集后,进行GO功能富集分析、KEGG通路富集分析以及蛋白质相互作用网络构建。【结果】筛选出701个差异表达基因,差异表达基因主要富集于小分子分解代谢过程、碳水化合物运输、趋化性等生物学过程,细胞器、细菌型鞭毛、甲基接受趋化蛋白复合物等细胞组分,离子跨膜转运蛋白活性、碳水化合物结合、肌动活动等分子功能以及不同环境中的微生物代谢和ABC转运体相关通路。此外,筛选出的14个核心基因在鞭毛合成和趋化中发挥作用。【结论】大肠杆菌在葡萄糖和甘油碳源培养条件下具有不同的基因表达模式,所确定的14种与趋化和鞭毛合成有关的基因有助于进一步揭示大肠杆菌在应对不同营养环境变化时采取的分子调控机制。     

胰升血糖素样肽-1受体激动剂高通量筛选细胞模型研究

为建立GLP-1受体激动剂的高通量筛选方法,将GLP-1受体质粒和报告基因质粒(3×CRE/3×MRE/SRE-LUC/pGL3)按照1∶5的比例共转染到CHO细胞中,建立GLP-1受体激动剂筛选细胞株。利用GLP-1内源激动剂探索和优化每孔细胞接种密度、荧光素酶表达时间、Bright-GloTM试剂用量、DMSO浓度等筛选条件,建立可靠的筛选方法。当细胞数目为4×104个/孔,激动剂孵育时间为8 h,Bright-GloTM试剂4倍稀释,每孔DMSO终质量分数小于1%时,系统Z′-因子为0.75。利用此模型对中药提取物库进行检测,发现有5个样品显示出活性。结果表明,该模型能够用于GLP-1受体激动剂的高通量筛选。     

猪白细胞干扰素的制备及四种纯化方法的比较

本文以猪白细胞—鸭新城疫病毒Ⅱ系(NDVⅡ)为诱生系统探讨了猪白细胞干扰素(PLeIFN)诱生条件,并比较了四种方法纯化PLeIFN的效果。实验表明:(1)每毫升猪白细胞悬液加入40～80HA的NDVⅡ,产生PLeIFN效价>1500 u/ml;(2)猪白细胞经低浓度PLeIFN预处理可增强其产生PLeIFN能力,(3)PLeIFN预处理时间以3小时为宜;(4)猪白细胞干扰素在猪肾细胞上的抗病毒活性高于在鸡胚细胞上的活性;(5)四种方法中以硫氰酸钾—冷乙醇法纯化效果为好,该法去除杂蛋白能力高于其它方法;(6)用未酸化处理的PLeIFN粗制品可提高硫氰酸钾—冷乙醇法的纯化效果。     

滇西地区10种植物抗疟活性研究

目的:研究10种采自滇西地区植物的抗疟活性,为从天然产物中寻找新的抗疟成分或先导化合物奠定基础.方法:将植物样品粉碎,依次用75%乙醇、水回流提取,提取物经冷冻干燥后,采用β-羟高铁血红素形成抑制试验进行筛选,以IC50值评价其活性.结果:在供试的10种植物样品中,梁王茶地下部分、球花报春花、糙毛杜鹃、西域蜡瓣花等4种植物粗提物具有不同程度的β-羟高铁血红素形成抑制活性,相关提取物的IC50值均大于1 388.9μg/mL.结论:梁王茶、球花报春花、糙毛杜鹃、西域蜡瓣花4种植物具有一定的抗疟活性,值得深入研究.     

CCK-8法和MTT法检测人前列腺癌PC3细胞活性的最佳条件比较研究

以人前列腺癌PC3细胞作为研究对象,探讨了CCK-8法和MTT法的最佳实验条件。CCK-8的最佳检测波长为450nm,最佳检测时间为加入CCK-8试剂后4h,适宜细胞数量范围为8×10^2-1×10^5个。在检测抗肿瘤药物阿霉素（ADM）对PC3细胞的增殖影响实验中,发现CCK-8法测得数据的线性相关性优于MTT法,并且数据偏差较MTT法小。实验结果表明CCK-8法较MTT法检测的灵敏度高,准确性好,是一种优于MTT法的检测细胞增殖活性的方法。     

麻黄内生真菌的分离及其生物活性初步研究

从内蒙古赤峰等地的中草药麻黄中分离得到141株内生真菌,体外抗氧化、抗肿瘤等活性测定表明,有11株内生真菌对H2O2具有较好的清除活性(LD50均大于50,LD50为抑制率为50%时的样品稀释倍数),占菌株总数的7.80%;有27株内生真菌对Raji和HepG-2具有肿瘤细胞毒活性(LD50大于50),占菌株总数的19.15%,其中以抑制悬浮Rajii细胞为主.此外,在141株菌中,有74株对一种或二种以上的指示菌显示出抑菌活性,占菌株总数的52.48%.本实验结果表明,麻黄中含有丰富的内生真菌,是抗氧化、抗菌及抗肿瘤等活性物质的重要来源.为进一步从中草药植物中分离筛选新药先导化合物提供了科学依据.     

舟山群岛近海底栖真菌及其抗生活性初筛研究

对分离自舟山群岛海域海底46个沉积物样品中的丝状真菌进行种属鉴定,及其代谢产物抗菌、细胞毒和乙酰胆碱酯酶抑制活性的检测.结果显示:舟山群岛海域海底沉积物中存在着丰富的真菌资源.46个沉积物样品中,真菌含量最高可达到2 250 CFU/g(样品).在进行活性检测的89份真菌代谢产物中,有29.2%的样品对一株或一株以上的指示细菌有不同程度的抑制作用;各有10%左右的样品对5株植物致病真菌显示抑制作用,有49.4%的样品对白色假丝酵母显示抑制作用,但没有样品对黑曲霉有抑制作用;在细胞毒实验中,分别有2个、4个和12个样品在浓度为25μg/mL时,对KB细胞、Raji细胞和Hep G2细胞的抑制率高于50%;有55个样品显示微弱的乙酰胆碱酯酶抑制活性.通过此次研究,我们对舟山群岛海域的底栖真菌资源有了初步了解,将有助于将来更全面深入的研究.     

不同植物激素对绿豆下胚轴愈伤组织影响

以绿豆下胚轴再生愈伤组织效率为评价指标 ,对TDZ、4Pu 30、6 BA ,3种激素的生物学活性进行比较 ,结果表明TDZ、4Pu 30的细胞分裂素活性比 6 BA高 10 0倍 ,而在TDZ和 4Pu 30中 ,以TDZ的活性最强     

应用小鼠胸腺细胞法与脾细胞法测定IL—2活性的比较

分别应用ＣｏｎＡ激活的小鼠胸腺细胞增殖法和脾细胞增殖法对样品内白细胞介素－２（ＩＬ－２）的活性进行生物学测定．细胞增殖状况以３Ｈ－ＴｄＲ掺入强度显示，并对实验条件进行了探讨．结果表明；添加氢化考的松（ＨＣ）的胸腺细胞法适用于检测培养物上清内的ＩＬ－２活性，其实验操作较脾细胞法更为省时省事     

Up—Regulation of CCR5 and CXCR4 Expression on Human Monocytes by Interferon Gamma

Chemokine receptors, mainly CCR5 and CXCR4, have been proved to be the important coreceptors in HIV-1 entry. HIV-1 disease progression is, in general, characterized by an initial predominance of CCR5 using macrophage tropic, non-syncytium-inducing (NSI) isolates, switching later to CXCR4 using T-cell tropic,syncytium-inducing (SI) isolates. How this shift occurs and how the shift can be controlled are still unclear.Since patients with rapid decline of T cell counts have constantly high levels of IFN-7 in the sera and lymphoidnodes, we investigated the influence of this cytokine on the expression of the HIV-1 coreceptors CCR5 and CXCR4 on the cell surfaces of human monocytic cell line U937 and promonocyte NB4. IFN-γ could intensively enhance the expression of both, while a low level of CCR5 expression was detected in two cell lines before stimulation. The results of semiquantitative RT-PCR also confirm the up-regulation. As the newly generated X4-strains have been demonstrated to be insensitive to chemokine in some reports, IFN-7 may play an important role in selecting CXCR4-used strains.     

Regulation of cancer cell migration and bone metastasis by RANKL

Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain. Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile 'soil'. In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs. However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour, other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-kappaB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis, in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.     

CXCR7对HeLa细胞增殖、粘附和侵袭能力的影响

基质细胞衍生因子-1(SDF-1)在肿瘤侵袭、转移过程中起着重要的调控作用.此前认为SDF-1是通过其唯一受体CXCR4来起作用.近年来发现SDF-1还有另一作用受体——CXCR7,SDF-1/CXCR7在部分肿瘤侵袭转移过程中起重要作用,但其在宫颈癌HeLa细胞中的作用目前尚未明确.通过Western blotting检测HeLa细胞中CXCR4和CXCR7的表达,阻断CXCR4或CXCR7后,通过MTT法评价细胞增殖能力,细胞粘附实验评价细胞粘附能力,Transwell实验评价细胞侵袭能力.结果表明,CXCR4和CXCR7在HeLa细胞中表达.阻断CXCR4或CXCR7后,SDF-1诱导的HeLa细胞增殖、侵袭和与内皮细胞的粘附能力均被阻断.结果提示CXCR7在SDF-1诱导HeLa细胞增殖、侵袭和与内皮细胞的粘附过程中起着重要作用,将有望成为治疗宫颈癌转移的新靶点.     

Mesenchymal stem cells within tumour stroma promote breast cancer metastasis

Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells, when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by eliciting reversible changes in the phenotype of cancer cells.     

鲈鱼cxcr4 cDNA克隆和序列分析

趋化因子受体(cxcr4)是一类重要的免疫调节因子受体,对原始生殖细胞的迁移和存活具有十分重要的作用。在对脊椎动物cxcr4进行初步生物信息学分析基础上设计引物,采用RT-PCR和RACE相结合的方法从鲈鱼卵巢克隆了cxcr4a全长和4b部分cDNAs序列,并预测其编码的氨基酸序列。结果表明,由于鱼类基因组经历一次特有的复制,cxcr4在鱼类存在两个复制基因。鲈鱼cxcr4a cDNA全长为1432 bp,编码311个氨基酸;cxcr4b部分cDNA片段长为905 bp,编码285个氨基酸。将鲈鱼cxcr4序列与所有已克隆的硬骨鱼类进行同源性比较,结果显示硬骨鱼类保守性较高,所有已经克隆的cxcr4按照进化地位的不同成簇聚类,表现出了较高的同源性。     

人体内汞分子形态的转化

利用气相色谱－原子吸收联用技术对人体内汞分子形态的转化进行了研究,其方法是收集长期接触汞蒸气的职业工人的尿样和血样,用联用系统进行测定。样品中的甲基汞和二甲汞都可被检出,这说明汞蒸气进入人体后可以发生分子形态的转化。为汞的毒性研究和临床治疗提供了重要依据。     

Host origin of marrow stromal cells following allogeneic bone marrow transplantation

Although it is generally agreed that stromal cells are important in the regulation of haematopoietic cell development, the origin of these phenotypically diverse cells has been a subject for debate for more than 50 years. Data which support the concept of a separate origin for the haematopoietic stem cell and the marrow stroma are derived from cytogenetic or enzyme marker studies of explanted and expanded stromal cells grown under conditions that do not allow haematopoiesis in vitro. Recent evidence in man and in mouse suggesting that the stromal cells capable of transferring the haematopoietic microenvironment in vitro are transplantable seemingly questions this dichotomy, one interpretation being the existence of a common haematopoietic/stromal 'stem cell'. We used in situ hybridization to discriminate donor cells from host in blood and bone marrow samples obtained from patients with functioning sex-mismatched but HLA-identical allografts. Without exception, marrow-derived stromal cells that proliferate in long-term cultures were found to be of host genotype, whereas the macrophage component of the adherent layer in these cultures originated from the donor.     

IDENTIFICATION OF WOOD AND BARK EXTRACTIVES AND THEIR TOXICOLOGICAL EFFECTS ON THE TMP EFFLUENTS

INTRODUCTIONThermomechanical pulp (TMP) mill effluents contain toxicants that may cause acute death of fish in a short term and/or affect long-term growth and reproduction of fish in receiving water. The toxicants in the effluents considerably vary with pulping processes and pulpwood species. The former effect is referred as acute toxicity, and the latter chronic or sublethal toxicity (Spinger, 2000). Unlike chemical pulping, TMP is produced through physically shredding wood chip…     

维氏气单胞菌对草鱼背鳍细胞的毒性及凋亡研究

草鱼(Ctenopharyngodon idellus)在我国是一种重要的淡水经济鱼类,但是由于近些年的集约化高密度养殖导致各种水产疫病暴发,不仅对养殖行业造成巨大的损失,同时对人类生命安全也存在着巨大威胁,维氏气单胞菌(Aeromonas veronii)就是其中一种人畜共患的条件致病菌。本研究旨在探索维氏气单胞菌导致宿主细胞死亡的致死机制,为今后进一步针对维氏气单胞菌开发抗菌渔用功能产品提供数据支持和理论依据。本文首先测定维氏气单胞菌胞外分泌物(Extracellular products,ECPs)的蛋白浓度,然后以不同浓度的ECPs为基准,根据细胞光镜观察结果及细胞活力检测结果确定维氏气单胞菌胞外ECPs的细胞毒性;再利用Hoechst 33342及TdT-mediated dUTP Nick-End Labeling(TUNEL)检测细胞核的变化情况,最终确定草鱼源维氏气单胞菌对宿主细胞的毒性致死机制。从毒性实验中可以看出高浓度的维氏气单胞菌ECPs对草鱼背鳍(Grass carp pectoral fin,GCPF)细胞具有明显的毒性,且从Hoechst 33342结果可以看到凋亡小体的出现,而TUNEL也出现了阳性结果,实验组出现了绿色荧光,而对照组没有出现荧光。说明维氏气单胞菌胞外分泌物对宿主细胞具有明显的毒性,而且会导致宿主细胞发生细胞凋亡。     

Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL

Organ-specific metastasis is governed, in part, by interactions between chemokine receptors on cancer cells and matching chemokines in target organs. For example, malignant breast cancer cells express the chemokine receptor CXCR4 and commonly metastasize to organs that are an abundant source of the CXCR4-specific ligand stromal cell-derived factor-1alpha (ref. 1). It is still uncertain how an evolving tumour cell is reprogrammed to express CXCR4, thus implementing the tendency to metastasize to specific organs. Here we show that the von Hippel-Lindau tumour suppressor protein pVHL negatively regulates CXCR4 expression owing to its capacity to target hypoxia-inducible factor (HIF) for degradation under normoxic conditions. This process is suppressed under hypoxic conditions, resulting in HIF-dependent CXCR4 activation. An analysis of clear cell renal carcinoma that manifests mutation of the VHL gene in most cases revealed an association of strong CXCR4 expression with poor tumour-specific survival. These results suggest a mechanism for CXCR4 activation during tumour cell evolution and imply that VHL inactivation acquired by incipient tumour cells early in tumorigenesis confers not only a selective survival advantage but also the tendency to home to selected organs.