从鼠黑色素瘤细胞系中分离和鉴定癌干细胞样细胞

目的：从鼠黑色素瘤BL6F10细胞系中分离与鉴定癌干细胞（CSC）样细胞,为今后对CSC的鉴定及靶向治疗奠定基础。方法：用不同免疫磁珠标记的单克隆抗体,从BL6F10细胞系中分离有特征性CD表型的瘤细胞,体外观察不同CD表型瘤细胞在软琼脂培养基上形成克隆的能力;将这些瘤细胞皮下注射到C57BL/6小鼠,比较其致瘤性。结果：从BL6F10细胞系中分离出不同CD表型的特征性瘤细胞;在软琼脂培养基上,CD133^＋、CD44^＋和CD44^＋CD133^＋细胞克隆形成率分别高于CD133^-、CD44^-和CD44^＋CD133^-细胞;CD133^＋、CD44^＋、CD44^＋CD133^＋和CD44^＋CD133^＋CD24＋细胞在小鼠体内的致瘤性分别强于CD133^-、CD44^-、CD44^＋CD133^-和CD44^＋CD133^＋CD24^-细胞。结论：CD44^＋CD133^＋CD24＋表型的BL6F10细胞的某些生物学特性与CSC样细胞相似,具有CSC特征,这些实验结果为进一步鉴定BL6F10细胞系中的CSC提供了重要的实验资料。     

人源CLP的表达、纯化及生化特性研究

目的：为进一步研究CLP（coactosin-likeprotein）与5’-脂氧合酶、肌动蛋白的相互作用机制及功能,开展CLP克隆表达、分离纯化研究,以得到高纯度的CLP,并对其生物化学特性进行分析测定。方法：从人的胎肝cDNA文库中经PCR扩增得到CLP基因,克隆到原核表达载体pGEX-6p-1中并获得高效表达,经过Glutathione Sepharose 4B亲和层析和Su-perdex 75分子筛纯化,得到高纯度的CLP;在此基础上进行SDS-PAGE、动态光散射和分析型超速离心等实验,并进一步分析实验结果。结果：CLP在溶液中主要以单体形式呈现;CLP的摩擦率为1．909,证实该蛋白质具有线性化趋势存在的可能性,且线性化程度较高。结论：实验结果揭示了CLP作为线性化蛋白质的可能性,为进一步搭建CLP和丝状肌动蛋白的作用模型奠定了一定的数据基础。     

脆性组氨酸三联体与复制蛋白A相互作用关键氨基酸残基的鉴定

目的：研究脆性组氨酸三联体（Fhit）对ATR/CHK1通路的影响,在确定Fhit与复制蛋白A（RPA）存在相互作用的基础上鉴定Fhit与RPA相互作用的关键氨基酸残基,为进一步研究Fhit特异的信号通路奠定基础。方法：构建一系列Fhit缺失突变体基因Fhit1～Fhit11及6种Fhit点突变体基因,将这些基因插入含GST基因的原核表达载体中,在大肠杆菌中表达并纯化GST-Fhit1～GST-Fhit11融合蛋白、突变体GST-FhitSIYEEL、GST-FhitIY、GST-FhitEL、GST-FhitF、GST-FhitA,以及GST-FhitD融合蛋白,用GST沉降技术研究Fhit与RPA相互作用的关键氨基酸残基。结果：Fhit蛋白第112～117（SIYEEL）残基可能是Fhit与RPA相互作用的关键区域,而第114（Y）残基可能是Fhit与RPA相互作用的关键氨基酸残基。结论：确定了Fhit与RPA相互作用的关键氨基酸残基,为阐明Fhit在维持基因组完整性方面的机理提供了线索。     

Effects of drought stress and N supply on the growth, biomass partitioning and water-use efficiency of Sophora davidii seedlings

A greenhouse experiment was conducted in order to understand the adaptation responses to different water and N conditions, and further explore if additional N supply could improve the water-use efficiency (WUE) and adaptability of Sophora davidii seedlings under dry conditions. Two-month-old seedlings were subjected to a completely random design with three water (80, 40 and 20% water field capacity (FC)) and three N supply (N0: 0, Nl: 92 and Nh:184 mg N kg⁻¹ soil) regimes. Drought stress dramatically decreased seedlings height, basal diameter, leaf number, leaf area, root length, and biomass production. An increase in below-ground biomass was observed indicating a higher root/shoot ratio (R/S) under drought stress conditions, and drought further decreased relative water content (RWC) and WUE. On the other hand, S. davidii seedlings exhibited strong responses to N supply, but the responses were inconsistent with the various N supply levels. Low N supply (Nl) increased seedlings height, basal diameter, leaf number, leaf area, and biomass production, but decreased root length. In contrast, high N supply (Nh) decreased or had little effect on these growth characteristics. N supply increased leaf percentages, but decreased fine root percentages. In addition, Nl rather than the other two N treatments increased leaf area ratio (LAR), leaf/fine root mass ratio (L/FR), R/S and RWC under severe drought stress (20% FC), even though these parameters could increase with the Nh treatment under well-watered condition (80% FC). Moreover, Nl also increased WUE under three water conditions, but Nh had little effect on WUE under drought stress conditions (40% FC and 20% FC). The results suggested that water and N co-limited the growth of S. davidii seedlings, and the seedlings exhibited great positive responses to Nl in this study. Appropriate or low N supply, therefore, would be recommended to stimulate growth, enhance WUE, alleviate drought stress, and consequently contribute to S. davidii seedling establishment under dry condition, but excess N supply should be avoided.     

Human alpha-synemin interacts directly with vinculin and metavinculin

Synemin is a very large, unique member of the IF (intermediate filament) protein superfamily. Association of synemin with the major IF proteins, desmin and/or vimentin, within muscle cells forms heteropolymeric IFs. We have previously identified interactions of avian synemin with alpha-actinin and vinculin. Avian synemin, however, is expressed as only one form, whereas human synemin is expressed as two major splice variants, namely alpha- and beta-synemins. The larger alpha-synemin contains an additional 312-amino-acid insert (termed SNTIII) located near the end of the long C-terminal tail domain. Whether alpha- and beta-synemins have different cellular functions is unclear. In the present study we show, by in vitro protein-protein interaction assays, that SNTIII interacts directly with both vinculin and metavinculin. Furthermore, SNTIII interacts with vinculin in vivo, and this association is promoted by PtdIns(4,5)P(2). SNTIII also specifically co-localizes with vinculin within focal adhesions when transiently expressed in mammalian cells. In contrast, other regions of synemin show distinct localization patterns in comparison with those of SNTIII, without labelling focal adhesions. Our results indicate that alpha-synemin, but not beta-synemin, interacts with both vinculin and metavinculin, thereby linking the heteropolymeric IFs to adhesion-type junctions, such as the costameres located within human striated muscle cells.     

Comparative proteomics profile of osteoblasts cultured on dissimilar hydroxyapatite biomaterials: an iTRAQ-coupled 2-D LC-MS/MS analysis

Hydroxyapatite (HA) and its derived bioceramic materials have been widely used for skeletal implants and/or bone repair scaffolds. It has been reported that carbon nanotube (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. However, interaction between osteoblasts and these bioceramics, as well as the underlying mechanism of osteoblast proliferation on these bioceramic surfaces remain to be determined. Using iTRAQ-coupled 2-D LC-MS/MS analysis, we report the first comparative proteomics profiling of human osteoblast cells cultured on plane HA and CNT reinforced HA, respectively. Cytoskeletal proteins, metabolic enzymes, signaling, and cell growth proteins previous associated with cell adhesion and proliferation were found to be differentially expressed on these two surfaces. The level of these proteins was generally higher in cells adhered to HA surface, indicating a higher level of cellular proliferation in these cells. The significance of these findings was further assessed by Western blot analysis. The differential protein profile in HA and CNT strengthened HA established in our study should be valuable for future design of biocompatible ceramics.     

人SUMO-3基因原核表达及多克隆抗体制备

构建人SUMO-3基因的原核表达载体pET41a(+)-SUMO-3,表达重组GST-SUMO-3融合蛋白,制备人SUMO-3多克隆抗体。试验结果显示,通过PCR方法从重组质粒pEYFP-SUMO-3中克隆到的SUMO-3 N端93个氨基酸的基因序列与NCBI上提供的序列一致,重组质粒pET41a(+)-SUMO-3构建成功;重组pET41a(+)-SUMO-3在E.coli.BL21 (DE3) pLysS中表达GST-SUMO-3融合蛋白,分子量为44.0 kDa,与预期分子量一致;采用亲和层析纯化融合蛋白GST-SUMO-3并免疫家兔,获得人SUMO-3抗体;Western blot 检测显示该抗体可以特异性识别SUMO-3,ELISA检测结果成阳性,抗体效价约为1: 20000。实验结果为进一步研究人SUMO-3及SUMO第二类家族的功能提供了有用工具。     

Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

Porcine circovirus type 1 （PCV1） contains two major open reading frames encoding the replication-associated proteins and the major structural capsid （Cap） protein. PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals （NLS）. The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCVI Cap versions fused to enhanced green fluorescent protein （EGFP）. The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RR- RR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.     

椎体成形术的生物力学研究现状和进展

椎体成形术是通过向椎体病损部位注入骨粘合剂,达到缓减疼痛、增强椎体稳定性的目的。由于椎体成形术的治疗原理、远期疗效、并发症均与其生物力学密切相关,其生物力学引起广泛的关注。本文通过椎体强化的生物力学研究方法及椎体强化后的生物力学特性,总结影响椎体强化效果的因素及影响程度,并对椎体强化生物力学研究中存在的问题及研究前景进行展望。     

锌离子存在下EGCG对前列腺癌细胞生长的影响

本文研究了锌离子存在下EGCG对前列腺癌细胞PC-3生长的影响．研究发现Zn^2＋可以增强EGCG抗癌活性,Zn^2＋存在下。EGCG处理后前列腺癌细胞PC-3克隆形成率显著下降。以RT—PCR、免疫组化方法研究Zn^2＋、EGCG对67kD层粘连蛋白受体（67kD Laminin Receptor,67LR）表达调控,结果表明Znn可通过上调67LR的表达,为EGCG提供更多作用的靶位点,增强EGCG对前列腺癌细胞PC-3的毒性作用。MMP-9是肿瘤侵袭转移过程中关键的基质金属蛋白酶。MMP-9活性与癌细胞的转移潜能密切相关。本文研究发现Zn^2＋、EGCG处理可通过抑制MMP-9活性,降低前列腺癌细胞PC-3的迁移率．其中80umol／LEGCG＋80umol／L Zn^2＋处理24h后显著抑制了PC-3细胞的迁移率。