糖尿病肛瘘lncRNA芯片分析及CNC图搭建＊

目的 研究糖尿病肛瘘创面特异表达LncRNA与mRNA基因功能之间调控网络。方法 用基因芯片技术对糖尿病肛瘘创面和普通肛瘘创面组织中差异性表达的LncRNA及mRNA进行基因表达谱检测，筛选的标准为2倍差异及P < 0.05,再进行差异表达分析，然后对差异表达的mRNAs进行KEGG通路分析及Pathway Map展示，挑选出显著mRNA指标，将这些显著mRNA指标进行q-PCR验证，得到有意义的阳性指标，再将阳性指标与差异LncRNA交集得出LncRNA-mRNA共表达网络，并基于LncRNA-mRNA共表达网络挑选出不同LncRNA进行功能验证。结果 将芯片标准化后分析差异表达的长链非编码RNA和mRNA，发现上调差异有502个，下调差异有1204个；mRNA分析发现上调差异621个，下调差异505个，KEGG通路分析发现上调和下调的通路均有10条；通过分别对上调、下调最明显的通路进行Pathway Map展示，挑选出显著mRNA指标8个：BMP2、IFNB1、IL6、IL18、PIK3CB、SMAD7、SMAD9、β-actin，分别将其进行q-PCR验证，得到有意义的阳性指标个5个：BMP2、IL6、IL18、PIK3CB、SMAD7，将5个阳性指标和差异LncRNA交集得出LncRNA-mRNA共表达网络，并基于LncRNA-mRNA共表达网络挑选出不同LncRNA进行功能验证。结论 挑选出的20个LncRNA（NR_125383、T323486、ENST00000582334、TCONS_00018312、ENST00000418393、TCONS_00017190、TCONS_00019532、ENST00000601559、ENST00000566575、NR_109882、NR_026913、T301537、NR_109774、ENST00000415536、ENST00000610000、ENST00000412485、ENST00000573220、T175957、ENST00000580756、TCONS_00014747）所调控的mRNA居于LncRNA-mRNA共表达网络，其在各个领域当中均有不同程度的报道，为后续的功能和机制研究提供了方向和重点，为加速慢性难愈合和创面愈合的研究提供了新的思路，为开发促愈药物提供基础研究借鉴。     

Faster bone union progression and less sclerosis at the osteotomy margin after medial opening-wedge high tibial osteotomy using highly porous β-tricalcium phosphate granules versus allogeneic bone chips: A matched case–control study

BackgroundThis study compared bone union progression using highly porous (80% porosity) β-tricalcium phosphate (β-TCP) granules or allogeneic bone chips in the gap created by medial opening-wedge high tibial osteotomy (MOWHTO).MethodsThe study population consisted of 54 patients who received MOWHTO with locking plate fixation: 27 patients using highly porous β-TCP granules, and 27 age- and sex-matched patients using allogeneic bone chips. Bone union progression was evaluated 1, 3, 6, and 12 months postoperatively. The presence of radiographic sclerosis at the osteotomy margin was also assessed.ResultsAmong all patients, the highest degree of bone union observed 12 months postoperatively was grade 4. As postoperative time passed, bone union progression of highly porous β-TCP granules increased linearly and was statistically significant compared with that of cancellous allogeneic bone chips (P = 0.014). The presence of radiographic sclerosis at the osteotomy margin was significantly less common in the β-TCP group than in the allograft group (P = 0.003) and was the strongest predictor of delayed progress of bone union (odds ratio = 6.16, P = 0.006).ConclusionsPatients who underwent MOWHTO using highly porous β-TCP granules had faster new bone remodeling, less radiographic sclerosis at the osteotomy margin, and no inferior clinical outcome compared with allogeneic bone chips, as determined at the 1-year follow up. The presence of radiographic sclerosis at the osteotomy margin in patients undergoing MOWHTO using allogeneic bone or synthetic bone substitute may indicate delayed progress of bone union.     

口腔疣状癌差异基因表达分析

目的:研究口腔疣状癌患者癌组织与正常口腔黏膜上皮组织基因的差异表达。方法:应用 cDNA 芯片技术对4例口腔疣状癌患肯的疣状癌组织及其正常口腔黏膜上皮组织的 mRNA 进行检测。结果:在3900条基因中共发现差异表达基因371条,差异表达基因占9.5%,其中表达增强200条(显著增强56条),表达降低171条(显著降低43条)。结论:口腔疣状癌的发生涉及多基因改变,cDNA 芯片技术能高效研究多个基因的表达水平。     

中间普氏菌内毒素刺激前后下调人牙周膜细胞表达基因的研究

目的 :研究中间普氏菌内毒素刺激前后牙周膜细胞下调表达基因。方法 :应用点样数 5 12点的基因芯片分析中间普氏菌内毒素刺激前后牙周膜细胞下调表达基因。结果 :研究发现中间普氏菌内毒素刺激牙周膜细胞后 8条下调基因 ,包括转录调控基因、凋亡相关基因和受体基因。结论 :中间普氏菌内毒素刺激牙周膜细胞后 ,可引起部分基因表达下调 ,从而影响牙周膜细胞正常的生理功能     

Self-assembled lipid and membrane protein polyhedral nanoparticles

We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation of MPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics-based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation.The functions of many membrane proteins are intimately coupled to the generation, utilization, and/or sensing of transmembrane gradients (1). Despite advances in the structure determination of membrane proteins (2), the high-resolution structural analysis of membrane proteins in a biological membrane is uncommon and in the presence of a functionally relevant gradient remains an as-yet unrealized experimental challenge. This stems from the fact that the primary 2D- and 3D ordered specimens used in structural studies of membrane proteins by X-ray crystallography and electron microscopy lack closed membrane surfaces, thus making it impossible to establish physiologically relevant transmembrane gradients.As an alternative, we have been developing methodologies for the self-assembly of lipids and membrane proteins into closed polyhedral structures that can potentially support transmembrane gradients for structural and functional studies. The possibility of generating polyhedral arrangements of membrane proteins in proteoliposomes was motivated by the existence of polyhedral capsids of membrane-enveloped viruses (3, 4), the ability of surfactant mixtures to self-assemble into polyhedral structures (5, 6), and the formation of proteoliposomes from native membranes containing bacteriorhodopsin (7, 8) and light-harvesting complex II (LHCII) (9). Significantly, the high-resolution structure of LHCII was determined from crystals of icosahedral proteoliposomes composed of protein subunits in chloroplast lipids (10). Whereas detergent solubilized membrane proteins and lipid mixtures can self-assemble to form 2D-ordered crystalline sheets or helical tubes favorable for structure determination by electron microscopy (11–14), simple polyhedral ordered assemblies have only been described to form from select native membranes (7–9). To expand the repertoire of membrane protein structural methods, we have prepared membrane protein polyhedral nanoparticles (MPPNs) of the bacterial mechanosensitive channel of small conductance (MscS) (15, 16) from detergent solubilized protein and phospholipids, and demonstrated that they are amenable to structural analysis using electron microscopy.Conditions for generating MPPNs were anticipated to resemble those for other types of 2D-ordered bilayer arrangements of membrane proteins, particularly 2D crystals, in that membrane protein is mixed with a particular phospholipid at a defined ratio, followed by dialysis to remove the solubilizing detergent (17). The main distinction is that because MPPNs are polyhedral, conditions are sought that will stabilize highly curved surfaces of polyhedra rather than the planar (flat) specimens desired for 2D crystals. We used the Escherichia coli MscS as a model system. MscS is an intrinsically stretch-activated channel identified by Booth and coworkers (15) that confers resistance to osmotic downshock in E. coli. MscS forms a heptameric channel with 21 transmembrane helices (3 from each subunit) and a large cytoplasmic domain with overall dimensions of ∼8 × ∼12 nm parallel and perpendicular to the membrane plane; structures have been reported in both nonconducting (16, 18) and open-state conformations (19). Different phospholipids were added to purified the E. coli MscS solubilized in the detergent Fos-Choline 14 and the system was allowed to reach equilibrium by dialysis at different temperatures. To gain insight into the biophysical parameters that govern MPPN formation, we investigated the role of lipid head group, alkyl chain length, pH, and protein construct. Table S1 shows the observed influence of these various factors on our ability to form uniform MPPNs (as opposed to disordered aggregates or polydisperse proteoliposomes). The optimal conditions for MPPN formation used 1,2-dimyristoyl-sn-glycero-3-phosphocholine [added to ∼1:0.1 (wt/wt) protein:phospholipid] at pH 7 with the His-tagged MscS that is anticipated to be positively charged under these conditions. The biophysical properties of the protein are important as the best results were achieved using a His-tagged construct and the presence of a FLAG tag at the C terminus of MscS interfered with MPPN formation, even though the tag is ∼10 nm from the membrane-spanning region of MscS.To monitor MPPN formation, dynamic light scattering (DLS) was used. Under optimal conditions, we observed (Fig. 1A) the complete transition of solubilized MscS particles with a narrow distribution centered around a mean radii of 4.5 nm to MPPNs with a narrow distribution centered around a mean radii of 20 nm. We further characterized these particles using negative-stain electron microscopy. Fig. 1B is a field view negative-stain electron micrograph of a solution of detergent-solubilized MscS and lipid before initiation of the self-assembly process. Fig. 1C is a field view negative-stain electron micrograph of the same sample after the self-assembly process. We observed the incorporation of MscS into highly uniform polyhedra with mean radii of ∼20 nm (90%) and ∼17 nm (10%) by negative-stain electron microscopy. To gain more insight into the biophysical properties of these particles, we performed protein and phosphorus analysis on multiple samples to determine the lipid:protein ratio (Fig. S1). The observed lipid:protein ratio of the MscS MPPNs was 11 ± 1 (mole lipid:mole protein subunit) and consistent with a single layer of lipids forming a bilayer surrounding each protein. This ratio is comparable to the observed lipid to protein ratio found in 2D crystals of membrane proteins such as bacteriorhodopsin (lipid:protein ratio of 10; refs. 20 and 21) and aquaporin (lipid:protein ratio of 9; ref. 22).Open in a separate windowFig. 1.Preparation of MscS MPPNs. (A) DLS analysis of particles before dialysis and after completion of dialysis when MPPNs are formed. The observed radius of MscS alone was 4.5 nm and the particle radius at the end of dialysis was observed to be 20 nm. In both cases 99% of the scattering mass was observed in the distributions centered at 4.5 nm and 20 nm, respectively. (B) Negative-stain electron microscopy analysis of MscS before dialysis. Individual MscS proteins can be observed as small doughnut-shaped particles. (C) Negative-stain electron microscopy analysis of MPPNs following dialysis of the sample in B. MPPNs can be clearly observed and appear as uniform assemblies of individual MscS molecules. (Scale bars, 100 nm.)To further elucidate the structural nature of these particles and to unambiguously determine the symmetry, we performed electron cryotomography with image reconstruction using IMOD (23) combined with Particle Estimation for Electron Tomography (PEET) program (ref. 24 and SI Materials and Methods). In principle, electron tomography provides a complete 3D map of the particles and would allow us to unambiguously determine the MPPN symmetry. However, the alignment process was highly biased by the missing wedge phenomenon (24) due to poor signal:noise and resulted in an incomplete map (Fig. 2A). To overcome this alignment bias, we assigned random initial orientation values to all particles and constrained possible angular shifts to less than 30° to achieve a more uniform distribution of orientations (Fig. S2). This strategy resulted in a much improved density map (Fig. 2B) that revealed individual molecules with a size and shape that are in good agreement to the known molecular structure of MscS (Fig. 2C and Fig. S3). Building on the analysis of Haselwandter and Phillips (25), a systematic analysis was conducted (Table S2) of the symmetry relationships between MscSs in MPPNs that identified the arrangement corresponding to the snub cuboctahedron (dextro), an Archimedean solid. The snub cuboctahedron has cubic (octahedral) symmetry which, as recognized by Crick and Watson (26), provides an efficient way to pack identical particles in a closed, convex shell. In this particular arrangement, 24 MscS molecules are related by the 432-point group symmetry axes that pass through the faces, but not the vertices, of the snub cuboctahedron. Because the MscS molecules are positioned on the vertices of this chiral polyhedron, they occupy general positions that permit the ordered packing of the heptamers of a biomacromolecule (or indeed any type of particle). This is an important observation as it means that the individual MscS molecules with sevenfold symmetry are capable of packing into a symmetric assembly that is amenable to averaging. Whereas 24 objects can be arranged with identical environments in a snub cuboctahedron, certain integer multiples of this number can also be accommodated using the principles of quasiequivalence (27, 28) to form larger closed shells.Open in a separate windowFig. 2.Cryotomography of MscS MPPNs. (A) The PEET isosurface derived from 162 individual particles selected from eight single-tilt tomograms. The strong bias due to the missing wedge is observed along the lower part of the surface, but individual MscS heptamers are still discernible in the image. (B) The corresponding PEET isosurface, following introduction of randomized starting Euler angles to minimize missing-wedge bias. (C) The use of randomized starting Euler angles results in a much-improved map with apparent octahedral (432) symmetry that could be fit with 24 molecules of the MscS crystal structure. Isosurface renderings of the volume averages were generated using Chimera (31).Using the symmetry derived by electron cryotomography, we proceeded to collect high-resolution single-particle electron cryomicroscopy data. Samples prepared identically for cryotomography were imaged under low-dose conditions and a total of 4,564 particles were processed using the Electron Micrograph Analysis 2 (EMAN2) software package (SI Materials and Methods) (29). The final map had a resolution of 9 Å by Fourier shell correlation (Fig. S4) and allowed us to model the inner and outer helices of the transmembrane pore (Fig. 3). The arrangement of the helices more closely resembles the nonconducting conformation (16, 18) than the open-state structure (19), although some differences in the positioning of the outer helices relative to the nonconducting structure are indicated in sections 2 and 3 of Fig. 3. These results demonstrate that membrane proteins are capable of assembling into MPPNs that are amenable to high-resolution structure analysis by single-particle electron cryomicroscopy. Higher resolution data will be required, however, to detail the precise conformational differences between MscS in the phospholipid environment of MPPNs compared with those in the detergent-solubilized state used in the X-ray crystal structure analyses.Open in a separate windowFig. 3.Single-particle image analysis reconstructed from 4,564 particles processed with EMAN2 and subsequently the density surrounding a single MscS heptamer was extracted and sevenfold averaged as described in SI Materials and Methods. (Left) A cross-section through the electron density revealing the translocation pathway and cytoplasmic vestibule, and showing the overall fit of the closed structure of MscS (red ribbons) fit to the map (cyan). (Right) Stereoviews of cross-sections in the density map normal to the sevenfold axis at sections 1, 2, and 3. The closed-structure coordinates (red ribbons) of MscS were fit to the map using rigid body refinement in Chimera (31) showing the position of the transmembrane helices.In these promising initial studies we used traditional dialysis methods to screen conditions for MPPN formation. These methods are time consuming and require substantial quantities of a sample. To more efficiently screen conditions for MPPN formation with a variety of membrane proteins, we designed and fabricated a free interface diffusion microfluidic device (30) (Fig. 4A and Fig. S5) This device greatly simplifies the screening process and minimizes the amount of sample required for determining suitable conditions for MPPN formation. Using this device, we were able to produce MPPNs from MscS but more importantly from several other proteins that had previously failed to produce MPPNs using traditional dialysis. Fig. 4 B and C shows the results of using this device for the mechanosensitive channel of large conductance (MscL) and the connexon Cx26, respectively, where polyhedra were only observed in the presence of the target protein. Intriguingly, several different particles sizes could be observed for both MscL and Cx26 and we hypothesize that the variable-sized polyhedra may correspond to different packing arrangements similar to triangulation numbers observed in viral polyhedral assemblies. This microfluidic device will provide rapid screening of conditions for the formation of MPPNs and it is hoped will expedite membrane protein structural analysis in native lipid environments.Open in a separate windowFig. 4.Preparation of MPPNs using a microfluidics-based free interface diffusion system. (A) Schematic illustration of the device used for lipid–protein nanoparticle formation. From left to right, molecules in the center flow diffuse into the outer flow by the concentration gradient, with small molecules (larger diffusion coefficient) moving more quickly than larger molecules. Specifically, monomer detergents are removed through interfacial diffusion, whereas larger membrane proteins remain in the center flow, forming nanoparticles. Both the ratio of input:buffer and the flow rate influence particle formation. (B). Negative-stain electron microscopy images of MPPNs of MscL and (C) Cx26 formed using the microfluidic device from A. (Scale bar, 100 nm.) Insets show 2.5× magnification of a select region of interest.The self-assembly of membrane proteins into polyhedral nanoparticles demonstrates a potentially powerful method for studying the structure and function of membrane proteins in a lipid environment. MPPNs represent a novel form of lipid–protein assemblies which lie between single particles and large crystalline sheets or tubes. We have demonstrated that conditions favorable for MPPN formation can be identified and have elucidated the structure, symmetry, and potential application to membrane protein structure analysis. In addition we have designed and fabricated microfluidic devices for high-throughput screening of conditions for MPPN formation. MPPNs may allow a variety of perturbations to be achieved such as pH, voltage, osmotic, concentration gradients, etc. that cannot be achieved with other membrane protein assemblies and will potentially allow us to activate various types of gated channels and receptors so that active conformational states can be structurally investigated. The potential of such materials for targeted drug delivery with precisely controlled release mechanisms offers an intriguing avenue for future biomedical applications.     

Chipping fracture resistance of dental CAD/CAM restorative materials: Part I – Procedures and results

Objective

The edge chipping test was used to measure the fracture resistance of CAD/CAM dental restoration ceramics and resin composites.

Methods

An edge chipping machine was used to evaluate six materials including one feldspathic porcelain, two glass ceramics, a filled resin-composite, a yttria-stabilized zirconia, and a new ceramic-resin composite material. Force versus edge distance data were collected over a broad range of forces and distances. Data were analyzed by several approaches and several chipping resistance parameters were evaluated. The effects of using different indenter types were explored.

Results

The force versus distance trends were usually nonlinear with good fits to a power law equation with exponents usually ranging from 1.2 to 1.9. The order of chipping resistance (from least to greatest) was: feldspathic porcelain and a leucite glass ceramic (which were similar), followed by the lithium disilicate glass ceramic and the two resin composites (which were similar), and finally the zirconia which had the greatest resistance to chipping. Chipping with a Vickers indenter required 28–45% more force than with the sharp conical 120° indenter. The two indenters rank materials approximately the same way. The power law exponents were very similar for the two indenters for a particular material, but the exponents varied with material. The Rockwell C indenter gives different power law trends and rankings.

Significance

Despite the variations in the trends and indenters, simple comparisons between materials can be made by chipping with sharp conical 120° or Vickers indenters at 0.50 mm. Broad distance ranges are recommended for trend evaluation.     

大鼠面神经损伤修复晚期面神经核的基因表达谱

目的:筛选外周损伤修复晚期面神经核区的差异表达基因。方法:建立大鼠面神经断伤吻合模型,90 d后准确解剖定位获取健患两侧面神经核组织的总RNA,再以其逆转录的c DNA为模板转录生成生物素标记的c RNA,纯化与片段化后形成探针并与大鼠基因表达谱芯片杂交,扫描仪检测信号后用生物学分析软件筛选差异表达基因并作初步功能分析。结果:基因芯片检测表明,患侧面神经核区检出1 660个基因,健侧检出1 683个基因,与健侧相比筛选出差异基因300个,其中上调157个(差异倍数≥2),下调143个(差异倍数≤-2)。GO分析电子功能注释发现,差异基因功能涉及代谢反应、细胞黏附、细胞因子、信号传递等。KEGG信号通路分析筛选出最可能相关的信号通路32个(P0.05),包括白细胞跨膜转运、黏着斑激酶途径等。结论:基因表达谱分析表明多基因、多信号通路参与外周损伤修复晚期面神经核内的塑形活动。     

多通道微流控芯片的设计、仿真及细胞迁移应用研究

细胞迁移是指细胞朝着特定的化学浓度梯度发生定向迁移运动,其在胚胎发育、伤口愈合、肿瘤转移中发挥着至关重要的作用。当前研究手段大多通量低,难以综合考虑不同浓度梯度条件对细胞迁移行为的影响。针对上述问题,本文首先设计了一款四通道微流控芯片,其特征如下:借助层流和扩散机制在细胞迁移主通道中建立和维持浓度梯度;可在单一显微镜视野下同时观测四组细胞迁移现象;集成了宽度为20μm的细胞隔离带,可校准细胞初始位置,保证实验结果的准确性。随后,借助Comsol Multiphysics有限元分析软件完成了微流控芯片的仿真分析,证明了芯片上设计S型微通道和水平压力平衡通道有助于在细胞迁移主通道中形成稳定的浓度梯度。最后,采用不同浓度(0、0.2、0.5、1.0μmol·L^-1)与糖尿病及其并发症密切相关的晚期糖基化终末产物(AGEs)孵育中性粒细胞,研究了其在100 nmol·L^-1趋化因子fMLP浓度梯度环境中的迁移行为。结果表明,AGEs抑制了中性粒细胞的迁移能力,证明了四通道微流控芯片的可靠性和实用性。     

卵巢癌基因表达的cDNA微阵列研究

目的应用基因表达谱芯片研究人卵巢癌组织和正常卵巢组织中差异表达的基因,筛选出与卵巢癌发生发展相关的基因,并对这些基因的功能进行初步分析.方法采用cDNA芯片技术从有关癌基因和抑癌基因、细胞周期和细胞凋亡相关、DNA合成、转录和修复以及细胞信号传递等肿瘤相关的2304个基因中,分析研究卵巢癌基因表达谱,即寻找出不同于正常卵巢组织而只在卵巢癌实体瘤中上调或下调的特殊基因.结果与正常卵巢组织比较,卵巢浆液性囊腺癌组织表达有明显差异的基因92个,卵巢粘液性囊腺癌差异表达基因110个,卵巢浆液性囊腺癌和粘液性囊腺癌均呈差异表达的基因65个,卵巢浆液性囊腺癌和粘液性囊腺癌中不同的差异表达基因共有173个.结论与卵巢癌有关的基因涉及多种细胞生物学过程,说明卵巢癌的发生是一个多步骤、多基因调控的过程.基因芯片技术实现了基因分析的快速、高通量、微型化和自动化,它为同时分析几百个基因状况提供了一个有效的方法,所获得的差异基因表达谱为深入研究特异相关基因提供了参考.