The experimental research of R-phycoerythrin subunits on cancer treatment: A new photosensitizer in PDT

The mouse tumor cell 5180 and human liver carcinoma cell SMC 7721 cells were first treated with R-PE and its subunits (alpha, beta, gamma subunits), then irradiated with Argon laser (496 nm, 28.8 J/cm(2)). Survival rate was measured by MTT method. In order to compare the phototoxicity in normal cells, the mouse marrow cells were treated with photofrin II and beta-subunit, irradiated with 45 J/cm(2) of light; survival rate was also measured by MTT method. The result showed that R-PE subunits had better PDT effect on s180 cells than R-PE and lower phototoxicity in marrow cells than photofrin II Flow cytometric analysis showed that PDT results in a growth inhibition and a G(0)-G(1) cell cycle arrest in SMC 7721 cells. The tumor cells inhibited by PDT in vivo were morphologically observed by TEM, the tumor cell death was daze to the occlusion of tumor blood vessels and inducement of cell programmed death in nuclei. Therefore, with the advantage in special fluorescence activity, loth molecular weight, good light absorbent character and weak phototoxicity, R-PE subunit is art attractive option for improving the selectivity of PDT.

N-acetylchitooligosaccharide is a potent angiogenic inhibitor both in vivo and in vitro

N-Acetylchitooligosaccharide (N-acetyl-COs) was prepared by N-acetylation of chitooligosaccharide (COs). In vitro study using human umbilical vein endothelial cells (HUVECs) revealed that both N-acetyl-COs and COs inhibited the proliferation of HUVECs by inducing apoptosis. Treatment of HUVECs by N-acetyl-COs resulted in a significant reduction of density of the migration cells and repressed tubulogenesis process. The antiangiogenic effects of the oligosaccharides were further evaluated using in vivo zebrafish angiogenesis model, and the results showed that both oligosaccharides inhibited the growth of subintestinal vessels (SIV) of zebrafish embryos in a dose-dependent manner, as observed by endogenous alkaline phosphatase (EAP) staining assay. In contrast, no cytotoxicity was found when treating the NIH3T3 and several other cancer cells with the oligosaccharides. Our results also confirmed the antiangiogenic activity of N-acetyl-COs was significantly stronger than the parent oligosaccharide, COs. (c) 2007 Published by Elsevier Inc.

A novel polypeptide from shark cartilage with potent anti-angiogenic activity

Using guanidine-HCl extraction, acetone precipitation, ultra-filtration and chromatography, a novel polypeptide with potent anti-angiogenic activity was purified from cartilage of the shark, Prionace glauca. N-terminal amino acid sequence analysis and SDS-PAGE revealed that the substance is a novel polypeptide with MW 15500 (PG155). The anti-angiogenic effects of PG155 were evaluated using zebrafish embryos model in vivo. Treatment of the embryos with 20 mu g/ml PG155 resulted in a significant reduction in the growth of subintestinal vessels (SIVs). A higher dose resulted in almost complete inhibition of SIV growth, as observed by endogenous alkaline phosphatase (EAP) staining assay. An in vitro transwell experiment revealed that the polypeptide inhibited vascular endothelial growth factor (VEGF) induced migration and tubulogenesis of human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs in 20 mu g/ml PG155 significantly decreased the density of migrated cells. Almost complete inhibition of cell migration was found when HUVECs were treated with 40-80 mu g/ml PG155. PG155 (20 mu g/ml) markedly inhibited the tube formation of HUVECs and a dose-dependent effect was also found when treatment of HUVECs with PG155 at the concentration from 20 to 160 mu g/ml.

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

鲨鱼软骨抗血管生成多肽的分离纯化及其生物活性

以血管生成为靶点的抗肿瘤策略是抗肿瘤领域的研究热点，目前已经发现许多天然和化学合成的抗血管生成药物。鲨鱼软骨作为抗新生血管生成因子的重要来源的研究已有20多年的历史，很多研究显示鲨鱼软骨提取物有抗血管生成活性。但鲨鱼软骨活性多肽的完整分子结构一直未见报道；鲨鱼软骨活性多肽干扰血管生成通路的信号途径尚不明确。 本文应用盐酸胍抽提、丙酮分级沉淀、超滤、凝胶层析等分离技术，从青鲨（Prionace glauca）软骨中分离纯化并鉴定了一种新的具有抗新生血管生成活性的多肽。经SDS-PAGE和N-末端氨基酸序列分析显示，该多肽分子量为15500 Da，采用蛋白数据库分析表明该多肽是一种新发现的鲨鱼软骨多肽（Polypeptide from Prionace glauca，PG155）。 体外实验显示，PG155抑制内皮细胞生长因子（vascular endothelial growth factor，VEGF）介导的人脐静脉内皮细胞（human umbilical vein endothelial cell ，HUVEC）迁移和管腔形成，并呈剂量依赖关系。200 μg/ml PG155对牛主动脉内皮细胞（Bovine Aortic Endothelial Cells，BAECs）和HUVECs及以下癌细胞，包括人肝癌细胞（human hepatoma Bel-7402 cells，Bel-7402）、 口腔上皮癌细胞（human oral epidermoid carcinoma KB cells ，KB）、人结肠癌细胞（human colon cancer HCT-18 cells，HCT-18）和人乳腺癌细胞（human breast MCF7 cancer cells ，MCF7）的增殖均无抑制作用，说明PG155无细胞毒作用。20 μg/ml PG155显著抑制HUVEC的迁移和管腔形成；40-80 μg/ml PG155 对VEGF 介导的HUVEC的迁移和管腔形成几乎完全抑制。 体内实验显示，PG155显著抑制斑马鱼胚胎模型新生血管生成，并呈剂量依赖关系。形态学观察表明PG155显著抑制斑马鱼胚胎肠下静脉（subintestinal vessels��� SIVs）的生长，随着浓度的升高SIVs的生长可受到完全抑制。碱性磷酸酶染色分析显示，在一定浓度范围内，PG155随着浓度的升高对斑马鱼胚胎整体血管生成抑制作用依次增强。160 μg/ml PG155会引起斑马鱼胚胎心脏功能障碍。 由海洋生物中发现新的肿瘤新生血管生成抑制剂国内外的报道较少，我们的工作表明鲨鱼软骨可作为血管生成抑制剂的重要来源，鲨鱼软骨活性多肽PG155由于具有极低的细胞毒作用，并能抑制VEGF介导的血管生成过程，有希望成为一类新型抗肿瘤药物。

玻璃海鞘多肽PCI的分离纯化及其抗血管生成活性研究

血管生成是肿瘤生长、发展的必经之路，并且与实体瘤的发生、转移有着密切的关系，抑制肿瘤新生血管生成具有特异性高、疗效好、不易产生耐药性以及毒副作用低等特点，因此抑制肿瘤血管形成可望成为治疗癌症的一个突破点，以抗血管生成为主的肿瘤生物治疗研究已成为近十年的研究热点。玻璃海鞘（Coina intestinalis）属于内性目、玻璃海鞘科，因其进化上的独特地位常作为研究神经发育、免疫系统进化的材料。在其它种属的海鞘中分离发现了多种具有抗肿瘤活性的多肽，但关于玻璃海鞘抗血管生成活性多肽的分离纯化和活性研究未见报道。本文利用多种分离纯化手段，采用活性追踪的方法首次从玻璃海鞘中分离得到具有抗新生血管生成作用的多肽，并对其抗血管生成活性做了初步研究。 本研究利用冷丙酮分级沉淀，超滤截留分子量小于5kDa的蛋白，再经SephadexG25、Superdex75柱层析，µRPC C2/C18反相柱层析等分离手段，采用活性追踪的方法，由玻璃海鞘（Coina intestinalis）中分离纯化出抗血管生成多肽PCI，据保留时间计算其分子量为1.8 kDa。MTT检测表明其对人脐静脉血管内皮细胞（HUVEC）具有强烈的抑制作用，IC50为7.5 μg/ml，并对多种肿瘤细胞有直接的抑制作用。斑马鱼胚胎体内实验进一步表明PCI在40 μg/ml的浓度下作用12h，斑马鱼胚胎新生血管生成受到显著抑制，肠下静脉血管长度为正常组的30%，斑马鱼胚胎血管生成率为正常组的45%。

深海ROV铠缆绞车及主动升沉补偿装置设计研究

深海ROV是一种可以在深海进行观察和作业的水下机器人。其研究和开发，对海洋环境的调查，海洋资源的钻探以及军事应用方面都具有重要的意义。深海ROV收放在深海区域收放作业时，作业环境复杂，作业深度大，如果铠缆绞车采用单卷筒结构会存在驱动装置功率过大，卷筒的转动惯量过大，载体出水的瞬间铠缆外层压入内层等问题。另外，海面的海况条件比较恶劣，支持母船受风、浪、涌、流影响而产生较大幅度的升沉运动对有缆水下机器人正常作业和收放操作有较大的影响，ROV中继器的升沉运动甚至可以造成系缆的损害而使机器人本体丢失。为了提高ROV在恶劣海况下的安全性能，保护中继器与载体之间的系缆不受损坏，需要配备升沉补偿装置。所以深海ROV铠缆绞车及其升沉补偿装置的研究与设计，对实现深海ROV的收放及保障系统安全及各项关键技术的进一步研究具有重要的意义。 本文在863计划“基于母船升沉预测的深海装备主动升沉补偿测控单元的研究开发”资助下，结合7000m ROV收放操作和升沉补偿的要求，开展深海ROV铠缆绞车及其主动升沉补偿装置的设计研究。针对深海ROV收放作业时，ROV铠缆绞车需要具有ROV快速收放能力及升沉补偿能力，设计适合于深海ROV收放作业的铠缆绞车。本文重点研究适合于深海ROV铠缆绞车的双绞车结构形式；研究双绞车的受力分析，驱动装置的选型；设计深海ROV铠缆绞车的储存绞车和牵引绞车；设计用于深海ROV铠缆绞车的主动升沉补偿装置。本文的主要内容如下： （1）研究深海ROV铠缆绞车的结构形式。分析了如果深海ROV铠缆绞车采用单卷筒结构存在转动惯量过大，驱动装置功率过大，在出水的瞬间铠缆外层缆压入内层等问题，提出了双绞车结构。分析了双绞车的组成，布置及其作用原理，各部件的组成及作用原理。 （2）研究深海ROV铠缆绞车的受力及所需要的功率。利用微元分析，求解出深海ROV牵引绞车每个绞盘提供的摩擦力，从而求出各绞车驱动装置需提供的最大驱动力矩和功率，作为驱动装置的选型依据。 （3）设计深海ROV铠缆绞车。通过类比以前的绞车系统，理论计算，初步选定储存绞车及牵引绞车各个部件参数。采用SolidWorks进行三维建模后，采用ANSYS进行有限元分析，通过计算机仿真技术克服理论计算的缺陷，对各个部件进行强度校核及结构优化。 （4）设计主动升沉补偿装置。根据升沉补偿装置的技术指标，通过理论计算确定升沉补偿装置各个部分结构形式和参数，并进行了三维建模、通过有限元分析对各个部件进行强度校核，最后分析了超长缆释放时自身的张力对绞车定位的影响。 通过以上研究，设计出符合7000m ROV收放作业要求的深海ROV铠缆绞车及其升沉补偿装置，为7000m ROV的实现和收放技术的研究奠定了良好的基础。

自治式水下机器人回收系统的研究与设计

本文介绍了我们承担研制的国家“８６３”计划１０００ｍ及６０００ｍ无缆水下机器人的回收系统．回收系统在４级海况下不用专用母船能够成功地回收水下机器人，依据母船、海况、水下机器人及其他具体情况，介绍了两种不同的回收方案和回收器，经海上试验证明是有效和可行的。

冠状动脉旁路移植术后抑郁焦虑状态随访和分析

To explore the presence of depression, anxiety and cardiac events after coronary artery bypass grafting (CABG) prospectively and comparably. To analysis influential factors of depression and anxiety ( peri-operation and 1 year after operation ), and the effection on cardiac events in the year after operation. We followed the patients who underwent scheduled consecutive CABG. We interviewed patients to assess depression, anxiety symptoms using self-rating depression scale( SDS) and self-rating anxiety scale（SAS）before operation , before discharge and 1 year after operation. And cardiac events were also identified. All patients were divided into the group with depression and/or anxiety symptoms or the other group without depression and/or anxiety symptoms 3 times according to depression and anxiety symptoms before operation , before discharge and 1 year after operation. 69 patients completed the follow-up.24 patients (34.8%) had depression and/or anxiety symptoms before CABG, 31 patients (44.9%) had depression and/or anxiety symptoms before discharge, and 10 patients (14.5%) had depression and/or anxiety symptoms 1 year after operation. 6 patients(8.7%) had cardiac events, and 5 patients were re-admitted. Arhythmia before and after operation, the quantity of blood vessel grafted, length of stay , depression and anxiety symptoms before operation, cardiac events may lead to depression and anxiety symptoms 1 year after operation. The use of cardiopulmonary bypass grafting (CPB), Arhythmia after operation, the prolonged length of stay are influential factors of cardiac events. Though the incidence of depression and/or anxiety symptoms 1 year after operation is lower than before operation and before discharge, it is high still. Arhythmia before and after operation, the more blood vessels grafted, the prolonged length of stay , depression and anxiety symptoms before operation and cardiac events could be risk factors of depression and anxiety symptoms 1 year after operation. Much attention must be paid to this phenomenon. Heavy patient's condition, complicated surgery are risk factors of cardiac events, and bad emotion before operation and before discharge are independent to cardiac events. Key words： perspective research; depression；anxiety; coronary artery bypass grafting; follow up

LED (660 nm) and laser (670 nm) use on skin flap viability: angiogenesis and mast cells on transition line

Skin flap procedures are commonly used in plastic surgery. Failures can follow, leading to the necrosis of the flap. Therefore, many studies use LLLT to improve flap viability. Currently, the LED has been introduced as an alternative to LLLT. the objective of this study was to evaluate the effect of LLLT and LED on the viability of random skin flaps in rats. Forty-eight rats were divided into four groups, and a random skin flap (10 x 4 cm) was performed in all animals. Group 1 was the sham group; group 2 was submitted to LLLT 660 nm, 0.14 J; group 3 with LED 630 nm, 2.49 J, and group 4 with LLLT 660 nm, with 2.49 J. Irradiation was applied after surgery and repeated on the four subsequent days. On the 7th postoperative day, the percentage of flap necrosis was calculated and skin samples were collected from the viable area and from the transition line of the flap to evaluate blood vessels and mast cells. the percentage of necrosis was significantly lower in groups 3 and 4 compared to groups 1 and 2. Concerning blood vessels and mast cell numbers, only the animals in group 3 showed significant increase compared to group 1 in the skin sample of the transition line. LED and LLLT with the same total energies were effective in increasing viability of random skin flaps. LED was more effective in increasing the number of mast cells and blood vessels in the transition line of random skin flaps.

C-Kit(+) Cells Isolated from Developing Kidneys Are a Novel Population of Stem Cells with Regenerative Potential

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. STEM Cells 2013;31:1644-1656

Linear structures in mammographic images: detection and classification

R. Zwiggelaar, S.M. Astley, C.J. Taylor and C.R.M. Boggis, 'Linear structures in mammographic images: detection and classification', IEEE Transaction on Medical Imaging 23 (9), 1077-1086 (2004)

Experimental and numerical simulation of dough kneading in filled geometries

Binding, David; Couch, M.A.; Sujatha, K.S.; Webster, M.F., (2003) 'Experimental and numerical simulation of dough kneading in filled geometries', Journal of Food Engineering 58 pp.111-123 RAE2008

Kontakty społeczności Europy środkowej i strefy egejskiej w drugim tysiącleciu przed Chr. Próba analizy archeologiczno-chronometrycznej

Wydział Historyczny: Instytut Prahistorii

HEATING IN VASCULAR TISSUE AND FLOW-THROUGH TISSUE PHANTOMS INDUCED BY FOCUSED ULTRASOUND

High intensity focused ultrasound (HIFU) can be used to control bleeding, both from individual blood vessels as well as from gross damage to the capillary bed. This process, called acoustic hemostasis, is being studied in the hope that such a method would ultimately provide a lifesaving treatment during the so-called "golden hour", a brief grace period after a severe trauma in which prompt therapy can save the life of an injured person. Thermal effects play a major role in occlusion of small vessels and also appear to contribute to the sealing of punctures in major blood vessels. However, aggressive ultrasound-induced tissue heating can also impact healthy tissue and can lead to deleterious mechanical bioeffects. Moreover, the presence of vascularity can limit one’s ability to elevate the temperature of blood vessel walls owing to convective heat transport. In an effort to better understand the heating process in tissues with vascular structure we have developed a numerical simulation that couples models for ultrasound propagation, acoustic streaming, ultrasound heating and blood cooling in Newtonian viscous media. The 3-D simulation allows for the study of complicated biological structures and insonation geometries. We have also undertaken a series of in vitro experiments, in non-uniform flow-through tissue phantoms, designed to provide a ground truth verification of the model predictions. The calculated and measured results were compared over a range of values for insonation pressure, insonation time, and flow rate; we show good agreement between predictions and measurements. We then conducted a series of simulations that address two limiting problems of interest: hemostasis in small and large vessels. We employed realistic human tissue properties and considered more complex geometries. Results show that the heating pattern in and around a blood vessel is different for different vessel sizes, flow rates and for varying beam orientations relative to the flow axis. Complete occlusion and wall- puncture sealing are both possible depending on the exposure conditions. These results concur with prior clinical observations and may prove useful for planning of a more effective procedure in HIFU treatments.