Ox-LDL与动脉粥样硬化的关系及中药抗氧化作用的研究进展

氧化型低密度脂蛋白在动脉粥样硬化的发生发展过程中具有重要的作用,是导致此病发生的独立致病因素.其主要是通过细胞毒性、化学趋化性以及免疫原性等诱导血管内皮细胞损伤,趋化单核细胞与内皮细胞的黏附,巨噬细胞泡沫化,诱发炎症反应,加重动脉粥样硬化.就近年来氧化型低密度脂蛋白与动脉粥样硬化关系的研究以及中药对其进行的干预作用予以综述,以期为寻找有效的防治动脉粥样硬化的中药提供一定的帮助.     

IL-1α体外诱导血管内皮细胞衰老及其机理初步探讨

IL-1α是一种重要的促炎性细胞因子,它具有广泛的生物学效应,作用于机体多个系统,可参与免疫调节,介导炎症反应及致热作用;可促进一些细胞增殖而抑制另一些细胞增殖.研究IL-1α长期刺激血管内皮细胞的效应,发现与衰老相关的β-半乳糖苷酶染色反应阳性;形态学上细胞变得扁平、不规则,细胞质胀大,有些细胞出现双核现象;流式细胞术检测结果表明83.18%细胞停滞在G0-G1期,S期细胞减少;利用彗星电泳检测到被诱导衰老的内皮细胞DNA严重损伤.对IL-1α诱导内皮细胞衰老机理的探讨发现被诱导的衰老细胞内活性氧水平升高,脂质过氧化物增多;而抗氧化酶活力下降且总的抗氧化能力显著下降,这就造成了细胞损伤并诱发导致了细胞的退行性变化,即衰老的发生.此研究为衰老的炎症假说在细胞水平提供了直接的证据,并为抗衰老及其相关疾病的防治提供了理论基础和实验依据.     

抗氧化剂抑制内皮细胞表面的白细胞粘附不依赖其抗氧化活性

氧张力是炎症反应、再灌注损伤和动脉粥样硬化形成的致病因素。这些病变的共同点是内皮细胞表面的白细胞粘附增加。我们用几种抗氧化剂：ＤＣＩ,Ｃｈｒｙｓｉｎ,ＰＤＴＣ和Ｐｒｏｂｕｃｏｌ在流动条件下（切变力在０６～２４ｄｙｎ／ｃｍ２）实验其对内皮细胞表面白细胞粘附的影响,用化学发光法分析这几种抗氧化剂的抗氧化活性。结果显示：ＰＤＴＣ和Ｐｒｏｂｕｃｏｌ对外周血有核细胞氧自由基的产生没有抑制作用,Ｃｈｒｙｓｉｎ可轻度抑制,ＤＣＩ可明显抑制活性氧的产生。相反,ＰＤＴＣ（最大半抑制量８×１０－４ｍｏｌ和Ｃｈｒｙｓｉｎ最大半抑制量５×１０－５ｍｏｌ）可抑制细胞对经ＴＮＦα刺激的人脐带静脉内皮细胞（ＨＵＶＥＣ）的粘附。ＤＣＩ和Ｐｒｏｂｕｃｏｌ对各种切变力范围的细胞粘附无作用。抗氧化剂对内皮细胞表面的白细胞粘附的抑制作用不能单用其抗氧性特性来解释。可能的作用机理是这类物质直接作用转录因子ＮＦ－ｋＢ的结果     

野木瓜三萜皂苷类化合物抗炎活性研究

为了研究野木瓜藤茎中三萜皂苷类化合物的抗炎活性,以LPS诱导RAW264.7细胞建立细胞炎症反应模型,采用Griess法检测一氧化氮(NO)含量;酶联免疫吸附试验(ELISA)检测IL-1β含量;Hochest33258荧光染色观察细胞凋亡形态。结果表明:从野木瓜藤茎的正丁醇提取物中得到两个皂苷类化合物均可显著抑制NO释放;其中化合物1可显著抑制IL-1β产生,且能观察到明显的细胞凋亡现象,两种皂苷化合物均具有一定的抗炎活性.     

野漆树苷对LPS诱导的RAW264.7细胞的抗炎作用及机制探究

目的:探究野漆树苷(Rhoifolin,RHO)对细菌脂多糖(lipopolysaccharide,LPS)诱导的小鼠单核巨噬细胞RAW264.7细胞炎症模型释放炎性因子的影响,并探讨其作用机制.方法:用脂多糖(0.5μg·mL~(-1))刺激体外生长良好的RAW264.7细胞24h建立体外细胞炎症模型,以MTT法测定不同浓度RHO对RAW264.7细胞的毒性作用,用Griess试剂法检测一氧化氮(Nitric Oxide NO)的含量,RT-PCR法检测细胞中肿瘤坏死因子α(Tumor Necrosis Factor,TNF-α),白介素1β(Interleukin-1β,IL~(-1)β),白介素6(Interleukin,IL-6)的含量,采用Western Blot法检测MAPK信号通路中相关蛋白的含量.结果:与空白对照组相比,在LPS诱导下,RAW264.7细胞分泌致炎因子NO,TNF-α,IL~(-1)β,IL-6(P0.01);与模型组相比,25~100μmol·L~(-1)的RHO可明显下调LPS诱导的RAW264.7细胞释放炎症因子NO,TNF-α,IL~(-1)β和IL-6(P0.05,P0.01),并呈现良好的剂量依赖关系;野漆树苷还不同程度的抑制了Erk和JNK激酶的磷酸化.结论:RHO可以抑制LPS所致的RAW264.7细胞炎症反应,减少炎症因子NO分泌,抑制TNF-α,IL~(-1)β和IL-6mRNA的合成,抑制JNK/SAPK及Erk信号通路可能是其抗炎作用机制之一.     

有伤口，不能吃深色食物？

我们首先看看伤口愈合的过程。伤口的愈合,其实是两个并行的过程：第一个是细胞的增生。伤后24—48小时,在炎症反应的基础上,开始有细胞增生,伤缘上皮增厚,一部分基底细胞与真皮脱离,向缺损区移行并发生分裂。同时来自动脉外膜和其他组织的成纤维细胞和来自血管损伤处的内皮细胞也开始大量增生。细胞增生形成新的组织,逐步填补创伤造成的缺损。     

维药野西瓜醇提物的镇痛作用研究

目的:研究野西瓜的镇痛作用,并探讨其作用机制.方法:体内建立福尔马林致小鼠神经性和炎症性双相疼痛、小鼠热板致痛、冰醋酸致小鼠扭体反应动物模型,观察不同实验组效果.体外以RAW264.7细胞为研究对象,探讨野西瓜醇提物对脂多糖(LPS)诱导的RAW264.7细胞释放一氧化氮(NO)的影响.结果:野西瓜醇提物对福尔马林所致小鼠双相疼痛反应呈剂量依赖性的抑制作用,使小鼠局部炎症组织前列腺素E2(PGE2)含量降低,延长小鼠热板痛阈值,减少醋酸致小鼠扭体次数,抑制RAW264.7细胞内NO的产生.结论:野西瓜醇提物有很好的抗炎镇痛效果,其作用机制之一可能是通过降低PGE2含量来发挥作用.     

爵床提取物抗炎镇痛作用的实验研究

目的探讨爵床提取物的抗炎镇痛作用。方法采用致炎剂二甲苯引起小鼠耳肿胀、新鲜蛋清引起大鼠足跖肿胀2种急性炎症模型以及棉球引起大鼠肉芽肿慢性炎症模型,研究爵床提取物灌胃给药对急慢性炎症的作用。采用热板法、醋酸扭体法和甲醛法致痛,研究爵床提取物灌胃给药的镇痛作用。结果与纯化水对照组相比,爵床醇提取物和水提取物高、低剂量组对致炎剂二甲苯所引起的小鼠耳廓肿胀抑制显著(P 0. 01,P 0. 05),对蛋清所引起的大鼠足跖肿胀抑制显著(P 0. 01),对大鼠棉球肉芽肿抑制显著(P 0. 01)。爵床醇提取物和水提取物高、低剂量组给药后对热板所引起的小鼠痛阈值明显提高,与纯化水对照组和自身给药前比较差异明显(P 0. 01,P 0. 05);爵床醇提取物和水提取物高、低剂量组对致痛剂醋酸所引起的小鼠扭体反应次数明显减少,也明显抑制甲醛所引起的早期(0～5 min)和晚期(15～30 min)小鼠足痛反应,与纯化水对照组比较差异显著(P 0. 01)。爵床醇提取物的抗炎镇痛作用比水提取物强(P 0. 01,P 0. 05)。结论爵床提取物灌胃给药具有一定抗炎、镇痛作用,且醇提取物强于水提取物。     

金雀异黄素对小鼠黑色素瘤B16BL6细胞增殖、迁移和黏附的影响

利用四甲基偶氮唑盐微量酶反应比色法、细胞划痕实验和细胞黏附实验等研究了金雀异黄素对小鼠黑色素瘤B16BL6细胞增殖、迁移以及与血管内皮细胞黏附的影响;利用免疫荧光实验观察了金雀异黄素对B16BL6细胞中微管和肌动蛋白分布的影响.结果表明,与对照组相比,金雀异黄素可以抑制B16BL6细胞的增殖、迁移和与血管内皮细胞的黏附,影响B16BL6细胞中肌动蛋白的组装,但不影响细胞中微管的组装.这些结果提示,金雀异黄素抑制B16BL6细胞的增殖、迁移和与血管内皮细胞的黏附可能是通过诱导细胞中肌动蛋白的重排来实现的.     

新鲜冰冻血浆（FFP）通过AMPK/Akt/eNOS通路促肺内皮细胞一氧化氮释放及机制研究

探讨新鲜冰冻血浆(FFP)对人肺正常微血管内皮细胞(HPMECs)一氧化氮(NO)释放的影响及其机制.采用NO/Nitrite/Nitrate分析法检测了体外培养内皮细胞HPMECs经FFP处理后不同时间点细胞上清NO含量.结果显示:与培养基空白对照组相比,FFP显著增加内皮细胞NO生成;采用磷酸化蛋白激酶抗体芯片和免疫印迹方法筛选和鉴定FFP处理后内皮细胞相关蛋白激酶磷酸化,结果为FFP显著增加内皮细胞AMPKα1,Akt1和eNOS蛋白的磷酸化.进一步分别采用Compound C(AMPK抑制剂),LY294002(PI3K/Akt抑制剂)或L-NAME(NOS抑制剂)预处理细胞,阻挡AMPKα1/Akt1/eNOS信号转导通路.结果显示上述三种抑制剂均能抑制FFP诱导eNOS激活和NO生成,表明AMPKα1/Akt1/eNOS信号转导通路介导FFP诱导NO分泌参与血管保护.     

人纤溶酶原Kringle5基因的克隆、表达及产物纯化和性质鉴定

根据人纤溶酶原的cDNA序列,利用PCR技术获得人纤溶酶原kringle5结构域的基因,并将其克隆至表达质粒pET25b( )中。重组载体pET25b（ ）/kringle5转化至大肠杆菌BL21（DE3）中,经IPTG诱导表达,在12kD处可见明显的表达条带,表达产物大部分以无活性的包涵体形式存在。包涵体经过体外变复性,SP-SepharoseFF离子交换柱一步纯化,15%SDS-PAGE鉴定考染一条带,纯度达95%以上。所得到的目标蛋白明显的抑制bFGF诱导的牛毛细血管内皮细胞增生。     

猪血激肽原的分离纯化及其对白鲢鱼鱼糜的凝胶作用

以猪血为原材料,通过硫酸铵盐析和柱层析等方法,分离纯化出2种半胱氨酸蛋白抑制剂激肽原（kininogen）.激肽原Ⅰ的得率为0.7 %,纯化倍数为613.7;激肽原Ⅱ的得率为24.1 %,纯化倍数为295.7.SDS-PAGE结果表明,激肽原Ⅰ和激肽原Ⅱ的分子质量分别为58 ku和116 ku.激肽原Ⅰ和激肽原Ⅱ对白鲢鱼（Hypophthalmichthys molitrix）鱼浆中肌原纤维蛋白降解都有一定的抑制作用,当激肽原Ⅱ添加量为鱼浆质量的0.03 %时,可以使鱼糜凝胶强度提高24 %.因此,激肽原可作为添加剂有效提高鱼糜制品的凝胶强度     

Rapid neutrophil adhesion to activated endothelium mediated by GMP-140.

Granule membrane protein-140 (GMP-140), a membrane glycoprotein of platelet and endothelial cell secretory granules, is rapidly redistributed to the plasma membrane during cellular activation and degranulation. Also known as PADGEM protein, GMP-140 is structurally related to two molecules involved in leukocyte adhesion to vascular endothelium: ELAM-1, a cytokine-inducible endothelial cell receptor for neutrophils, and the MEL-14 lymphocyte homing receptor. These three proteins define a new gene family, termed selectins, each of which contains an N-terminal lectin domain, followed by an epidermal growth factor-like module, a variable number of repeating units related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Here we demonstrate that GMP-140 can mediate leukocyte adhesion, thus establishing a functional similarity with the other selectins. Human neutrophils and promyelocytic HL-60 cells bind specifically to COS cells transfected with GMP-140 complementary DNA and to microtitre wells coated with purified GMP-140. Cell binding does not require active neutrophil metabolism but is dependent on extracellular Ca2+. Within minutes after stimulation with phorbol esters or histamine, human endothelial cells become adhesive for neutrophils; this interaction is inhibited by antibodies to GMP-140. Thus, GMP-140 expressed by activated endothelium might promote rapid neutrophil targeting to sites of acute inflammation.     

医用藻酸盐敷料体外降解的研究

研究藻酸盐医用敷料体外降解的规律及不同降解时间其产物对内皮细胞增殖情况的影响,为藻酸盐医用敷料在临床使用中提供参考。以PBS缓冲液(模拟体液环境)为降解介质,对藻酸盐敷料进行体外降解;用藻酸盐敷料的失重率、分子质量变化表征本体的变化规律;用降解液中降解产物的糖醛酸含量变化表征降解产物的变化规律。将降解液与内皮细胞共培养,用MTT法检测内皮细胞增殖情况。藻酸盐敷料在模拟体液环境条件下,可以被降解,42 d失重率为61.2%,降解过程中其分子质量逐渐减小,最终产物为糖醛酸,其浓度不断增大。不同时间的降解产物均能促进内皮细胞的增殖。藻酸盐医用敷料体外降解比较快,完全降解的最终产物为糖醛酸,其降解液可以促进内皮细胞的增殖、分化,提高细胞新陈代谢能力,增加新细胞的再生速度,促进伤口的愈合。     

Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands

Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression. Whereas the mechanism of hypoxia-driven angiogenesis is well understood, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.     

CCR5膜外二硫键Cys20-Cys269对其构象完整性以及功能行使起关键作用

人类化学趋化因子受体CCR5是HIV-1病毒进入人体细胞的主要辅助受体.实验表明,CCR5分子的N末端区域对化学趋化因子结合以及HIV病毒入侵起关键作用.用同源模建的方法构建CC R5结构模型,并对该模型的胞外结构域进行了1ns高温分子动力学和200ps常温分子动力学模拟.结果表明,CCR5的胞外结构域整体上表现为一个包装紧密的球形构象,二硫键Cys20 - Cys269对这一构象的形成以及N末端区域的空间取向起关键作用,同时,二硫键的存在使N末端1-19区域定位在胞外结构域的顶部,并增加N末端构象柔性,使其有机会伸展到胞外空间去.根据这一结果和现有的实验证据,提出了HIV-CCR5两步结合机制.     

Embryonic MAP2 lacks the cross-linking sidearm sequences and dendritic targeting signal of adult MAP2

The most prominent microtubule-associated protein of the neuronal cytoskeleton is MAP2. In the brain it exists as a pair of high-molecular weight proteins, MAP2a and MAP2b, and a smaller form, MAP2c, which is particularly abundant in the developing brain. High-molecular weight MAP2 is expressed in dendrites, where its messenger RNA is also located, but is not found in axons; it has been shown to be present in fine filaments that crosslink dendritic microtubules. This correlates with the primary structure of high-molecular weight MAP2, which consists of a short carboxy-terminal tubulin-binding domain and a long amino-terminal arm, which forms a filamentous sidearm on reconstituted microtubules. Here we report that the high- and low-molecular weight forms of MAP2 are generated by alternative splicing and share the entire C-terminal tubulin-binding domain as well as a short N-terminal sequence. In contrast to high molecular weight MAP2, embryonic brain MAP2c lacks 1,342 amino acids from the filamentous sidearm domain. Furthermore, the mRNA for low molecular weight MAP2c is not present in dendrites, indicating that the dendritic targeting signal is specific for the high-molecular weight form.     

Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis

Leukocytes enter sites of inflammation by squeezing through the borders between endothelial cells that line postcapillary venules at that site. This rapid process, called transendothelial migration (TEM) or diapedesis, is completed within 90 s after a leukocyte arrests on the endothelial surface. In this time, the leukocyte moves in ameboid fashion across the endothelial borders, which remain tightly apposed to it during transit. It is not known how the endothelial cell changes its borders rapidly and reversibly to accommodate the migrating leukocyte. Here we show that there is a membrane network just below the plasmalemma at the cell borders that is connected at intervals to the junctional surface. PECAM-1, an integral membrane protein with an essential role in TEM, is found in this compartment and constitutively recycles evenly along endothelial cell borders. During TEM, however, recycling PECAM is targeted to segments of the junction across which monocytes are in the act of migration. In addition, blockade of TEM with antibodies against PECAM specifically blocks the recruitment of this membrane to the zones of leukocyte migration, without affecting the constitutive membrane trafficking.     

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.