扇贝多肽抗紫外线A对无毛小鼠皮肤的氧化损伤

目的:探究扇贝多肽(PCF)抗紫外线UVA对无毛小鼠皮肤氧化损伤的作用.方法:昆明种无毛小鼠,随机分为双蒸水未照射组和模型组(双蒸水照射组、5％PCF组、20％PCF组、10％维生素 C组).酶法测定皮肤匀浆抗氧化酶(GSH-Px、T-AOC、SOD)活性和MDA的含量;免疫组织化学法测定皮肤Bc1-2和NOS蛋白表达;电镜观察皮肤组织超微结构.结果:PCF能明显增加皮肤组织匀浆总抗氧化能力及GSH-Px、SOD活性,降低MDA含量.免疫组化结果表明,PCF能上调Bcl-2蛋白的表达;抑制NOS蛋白的表达.超微结构显示20％PCF组表皮细胞结构正常,成纤维细胞的细胞器结构正常;模型对照组表皮细胞损伤,胞质内可见空泡形成,真皮成纤维细胞内可见囊泡状扩张的滑面内质网,粗面内质网等细胞器减少,PCF组与模型对照组比较各项指标均有改善(P<0.05).结论:扇贝多肽具有抗紫外线UVA对无毛小鼠皮肤氧化损伤的作用.其机制与扇贝多肽上调BCl-2蛋白表达,下调NOS蛋白的表达,提高抗氧化酶含量,抑制脂质过氧化有关.     

扇贝多肽对UVB辐射小鼠胸腺淋巴细胞的保护作用

目的：研究扇贝多肽(polypeptide ftom Chlamys，farreri)对中波紫外线(UVB)辐射损伤小鼠胸腺淋巴细胞的保护作用。方法：UVB辐射小鼠胸腺淋巴细胞，MTT法检测胸腺淋巴细胞的活性；酶生化法检测细胞谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性；流式细胞术检测细胞内活性氧(ROS)的产生量以及细胞凋亡率。结果：PCF能明显减轻UVB辐射对小鼠胸腺淋巴细胞的损伤；提高细胞GSH-Px、SOD和CAT活性；降低细胞中ROS的产生量和细胞凋亡率。结论：PCF对小鼠胸腺淋巴细胞具有明显的辐射保护作用。     

扇贝多肽保护单次UVA氧化损伤HaCaT细胞

目的探讨扇贝多肽对单次长波紫外线辐射HaCaT角质形成细胞氧化损伤的保护作用机制。方法从栉孔扇贝中提取扇贝多肽(PCF,Mr=879)。UVA辐射强度为5J·cm-2。酶生化法测定胞质SOD,GSH-px活性,ROS、MDA水平;透射电镜观察细胞超微结构的改变;原位杂交技术检测细胞内p21mRNA的变化。结果在5J·cm-2UVA辐射下,在给定浓度范围内PCF能剂量依赖性降低胞质ROS、MDA水平;提高SOD,GSH-px活力;电镜下可见PCF可保护细胞超微结构;p21mRNA原位杂交发现PCF可明显抑制其表达。结论扇贝多肽对单次UVA诱导的人HaCaT细胞氧化损伤有保护作用。其机制与清除氧自由基、提高抗氧化酶活性、抑制p21mRNA表达及细胞凋亡有关。     

扇贝多肽保护单次UVA氧化损伤HaUaT细胞

目的探讨扇贝多肽对单次长波紫外线辐射HaCaT角质形成细胞氧化损伤的保护作用机制。方法从栉孔扇贝中提取扇贝多肽(PCF,Mr=879)。UVA辐射强度为5J·cm-2。酶生化法测定胞质SOD,GSH-px活性,ROS、MDA水平;透射电镜观察细胞超微结构的改变;原位杂交技术检测细胞内p21mRNA的变化。结果在5J·cm-2UVA辐射下,在给定浓度范围内PCF能剂量依赖性降低胞质ROS、MDA水平;提高SOD,GSH-px活力;电镜下可见PCF可保护细胞超微结构;p21mRNA原位杂交发现PCF可明显抑制其表达。结论扇贝多肽对单次UVA诱导的人HaCaT细胞氧化损伤有保护作用。其机制与清除氧自由基、提高抗氧化酶活性、抑制p21mRNA表达及细胞凋亡有关。     

海洋生物面膜对中波紫外线氧化损伤的HeLa细胞的保护作用

目的 :探讨中波紫外线 (UVB)照射下天然生物膜对HeLa上皮细胞氧化损伤的保护作用。方法 :建立UVB(辐照强度为 7.15× 10 - 5J/cm2 )对HeLa上皮细胞氧化损伤模型。MTT法测定细胞活性 ;流式细胞仪测定细胞的凋亡和死亡率 ;酶法测定抗氧化酶 (GSH Px、CAT、SOD)活性和MDA含量及总抗氧化能力。结果 :离体条件下生物膜能①显著增加Hela细胞的活性 ;②提高细胞上清液中GSH Px、CAT、SOD的活性 ,降低MDA含量 ,且呈量效关系。结论 :生物膜在体外有抗UVB氧化损伤的作用。其作用机制可能是通过提高抗氧化酶含量、清除自由基等 ,减轻细胞损伤而发挥其保护作用的     

扇贝多肽对紫外线B辐射人真皮成纤维细胞线粒体的保护作用

目的:研究扇贝多肽(PCF)对中波紫外线(UVB)辐射人真皮成纤维细胞线粒体的影响。方法:检测丙二醛(MDA)含量以及细胞内抗氧化酶(SOD、GSH-PX)的活性;流式细胞术测定线粒体膜电位;透射电镜观察细胞超微结构的变化。结果:UVB(1.176×10~(-4)J·cm~(-2))导致真皮成纤维细胞线粒体损伤,PCF(0.25％-1％)剂量依赖性地减轻UVB对线粒体的损伤;而且,PCF也可剂量依赖性地维持线粒体膜电位的相对稳定,PCF能够减少MDA的生成量,提高SOD及GSH-PX的活性,PCF各组与UVB模型组相比差异有显著性(P<0.05,P<0.01)。结论:PCF保护成纤维细胞的线粒体免受UVB的损伤。     

扇贝多肽对紫外线辐照小鼠胸腺淋巴细胞的保护作用

建立体外培养的小鼠胸腺淋巴细胞紫外线（UV）辐射损伤的病理模型，采用MTT法检测细胞活性；酶生化法测定胞浆超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、活性氧（ROS）、丙二醛（MDA）含量以及细胞抗超氧阴离子能力（A-SAC）和总抗氧化能力（T-AOC）；流式细胞仪Rhodamine 123荧光染色测定线粒体膜电位（△ψM）等方法，探扇讨贝多肽（PCF）对小鼠胸腺淋巴细胞辐射损伤的保护作用及机制。结果显示PCF能显著提高小鼠胸腺淋巴细胞的活性；抑制紫外线辐射所致淋巴细胞的损伤，显著提高细胞荣中SOD、GSH-Px的活性，降低ROS，MDA含量，提高A-SAC及T-AOC；稳定线拉体的膜电位；表明扇贝多肽对紫外线辐射损伤的小鼠胸腺淋巴细胞具有保护作用。其作用机制与PCF能清除氧自由基、提高抗氧化酶活性，保护细胞膜组织结构有关。     

扇贝多肽对紫外线B所致人真皮成纤维细胞凋亡和DNA损伤的抑制作用

目的:研究扇贝多肽(PCF)对中波紫外线(UVB)所致体外培养人真皮成纤维细胞的凋亡和DNA损伤的影响。方法:用噻唑兰(MTT)法检测成纤维细胞的存活率;流式细胞术测定细胞的凋亡率和胞浆内游离[Ca~(2+)]_i;彗星电泳法检测单细胞DNA受损的程度。结果:PCF(0.25％-1％)能显著提高UVB(1.176×10~(-4) J·cm~(-2))辐射后人真皮成纤维细胞的增殖活性,降低UVB所致细胞的凋亡率和死亡率,减轻受损的程度,同时可降低胞浆内游离[Ca~(2+)]_i(和UVB模型组相比,P<0.01).结论:PCF对抗UVB所致的光老化是基于其减轻UVB对DNA的损伤和降低UVB所致细胞的凋亡,因此,PCF能够在起始阶段阻止紫外线所致光老化的发展。     

扇贝多肽对UVA损伤HaCaT细胞UCP2表达及线粒体功能的影响

目的探究紫外线A(ultraviolet A,UVA)损伤对HaCaT角质形成细胞线粒体解偶联蛋白2(uncoupling protein2,UCP2)表达的影响以及扇贝多肽(polypeptide from Chlamysfarreri,PCF)的调节作用,并研究PCF对UVA损伤HaCaT细胞线粒体功能的保护作用。方法复制8 J.cm-2 UVA辐射损伤HaCaT角质形成细胞模型,免疫印迹法检测UVA损伤后HaCaT细胞UCP2蛋白表达的变化及PCF的影响、PCF对UVA损伤HaCaT细胞凋亡蛋白酶激活因子(apoptot-ic protease-activating factor 1,Apaf-1)、细胞色素C(Cyto-chrome C,Cyt C)蛋白表达的影响;电子自旋共振(electronspin resonance,ESR)技术检测ROS的释放量;流式细胞术检测线粒体膜电位;紫外分光光度法测定PCF对UVA损伤HaCaT细胞线粒体呼吸链复合酶Ⅰ(NADH-辅酶Q还原酶)活性的影响。结果正常HaCaT细胞UCP2几乎不表达,UVA照射后表达升高,3 h达高峰,6 h开始逐渐下降;1.42～5.69 mmol.L-1剂量范围内的PCF对UCP2的表达有抑制作用;PCF可明显抑制UVA诱导的ROS产生、线粒体膜电位下降、Cyt C的释放及Apaf-1的表达,可有效抑制UVA损伤后HaCaT细胞呼吸链复合酶I活性的下降。结论 PCF对UVA引起的HaCaT细胞线粒体损伤具有保护作用。     

扇贝多肽通过调节c-jun和COX-2抑制UVA诱导的HaCaT细胞凋亡

目的复制UVA诱导的HaCaT细胞凋亡模型,研究UVA对细胞内c-jun和环氧合酶-2(cyclooxygenase-2,COX-2)的影响,从而探究扇贝多肽(Polypeptide from Chlamys far-reri,PCF)抑制UVA引起的HaCaT细胞凋亡的分子机制。方法实验分为5组:正常对照组、UVA模型组、UVA+5.69mmol.L-1PCF组、UVA+2.84mmol.L-1PCF组、UVA+1.42mmol.L-1PCF组。应用实时荧光定量PCR和蛋白印迹法检测细胞内c-jun的表达;RT-PCR结合蛋白质印迹法检测细胞内COX-2的表达;琼脂糖凝胶电泳分析PCF和COX-2特异性抑制剂celecoxib对UVA诱导HaCaT细胞凋亡的影响。结果预先加入PCF和celecoxib均可明显抑制8J.cm-2UVA诱导的HaCaT细胞凋亡;UVA照射HaCaT细胞后COX-2mRNA及蛋白表达水平增加,与对照组相比差异有显著性(P<0.01);1.42~5.69mmol.L-1剂量范围内的PCF可剂量依赖性抑制UVA引起的细胞内COX-2mR-NA及蛋白表达(P<0.05,P<0.01);PCF也抑制了UVA引起的HaCaT细胞内c-jun表达的增加,且呈量效关系(P<0.05,P<0.01)。结论UVA诱导HaCaT细胞发生凋亡时,细胞内COX-2和c-jun的表达明显增加,PCF通过抑制细胞内COX-2和c-jun的表达而发挥其抗凋亡作用。     

肿瘤坏死因子相关凋亡诱导配体及扇贝多肽在UVA诱导HaCaT细胞凋亡中的作用

目的研究UVA对HaCaT细胞肿瘤坏死因子相关凋亡诱导配体(tumour necrosis factor related apoptosis inducing ligand,TRAIL)表达的影响;复制UVA诱导的HaCaT细胞凋亡模型,探究TRAIL在UVA诱导的HaCaT细胞凋亡中的作用及扇贝多肽(Polypeptide from Chlamys farreri,PCF)对UVA诱导的TRAIL凋亡通路的影响。方法实验设计分为5组:对照组、UVA模型组、UVA+5.69mmol·L-1PCF组、UVA+2.84mmol·L-1PCF组、UVA+1.42mmol·L-1PCF组。Real-TimePCR检测TRAIL mRNA表达;蛋白质印迹法检测TRAIL蛋白表达及caspase-8活性;琼脂糖凝胶电泳分析TRAIL中和性抗体对UVA诱导的HaCaT细胞凋亡的影响。结果8J·cm-2UVA照射HaCaT细胞后TRAIL mR-NA及蛋白表达增加,与对照组相比差异有显著性(P<0.01);TRAIL中和性抗体对UVA诱导的HaCaT细胞凋亡有抑制作用;1.42~5.69mmol·L-1剂量范围内的PCF可剂量依赖性抑制UVA引起的HaCaT细胞TRAIL mRNA及蛋白表达(P<0.05,P<0.01);PCF对UVA引起的HaCaT细胞caspase-8的活化有抑制作用,且呈量效关系。结论UVA可增强HaCaT细胞TRAIL表达;TRAIL参与了UVA诱导的HaCaT细胞凋亡;PCF对UVA诱导的HaCaT细胞TRAIL表达有抑制作用,也可减弱UVA诱导的caspase-8活化,以其抗氧化活性抑制TRAIL凋亡通路而发挥抗凋亡作用。     

Inhibitory effect of polypeptide from Chlamys farreri on ultraviolet A-induced oxidative damage on human skin fibroblasts in vitro.

We have previously reported that polypeptide from Chlamys farreri (PCF) inhibits the oxidative damage of ultraviolet A (UVA) on HeLa cells in vitro [Acta Pharm. Sin. 23 (2002) 961]. To further elucidate a possible role for PCF on UVA-damaged normal human cells, we established the oxidative damage models of normal human dermal fibroblasts (NHDF) exposed to UVA to study the protective effect of PCF on human dermal fibroblasts in vitro. In this study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cell viability. The intracellular superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase (CAT), xanthine oxidase (XOD), malondialdehyde (MDA), reactive oxygen species (ROS), total antioxidative capacity (T-AOC), and anti-superoxide anion capacity (A-ASC) were measured. The effect of PCF on UVA-induced apoptosis were investigated by Annexin V-FITC assay. Intracellular calcium was determined with the calcium-sensitive fluorochrome Fluo-3, and mitochondrial transmembrane potential with rhodamine 123. Comet assay was employed to detect the UVA-induced DNA damage. The ultrastructure of cell was observed under transmission electron microscope. The results indicated that PCF could greatly enhance the viability of NHDF and markedly promote SOD, GSH-px, T-AOC, and A-ASC, while the amounts of MDA and ROS, the activity of XOD were decreased. PCF could inhibit UVA-induced apoptosis and DNA damage in NHDF. The concentration of cellular free calcium was decreased and the mitochondrial transmembrane potential was increased by PCF. In ultrastructure of NHDF, PCF could greatly decrease UVA-induced damage, especially membrane. Our results suggest that the supplementation of PCF appears to reduce the UVA-induced normal human dermal fibroblasts damage efficiently. It may be involved in the PCF's abilities of scavenging oxygen free radical, inhibiting lipid peroxidation, increasing antioxidative enzymes, decreasing intracellular calcium and protection of membrane structure in NHDF irradiated by UVA.     

苦马豆素诱导人胃癌细胞SGC-7901凋亡作用机制的实验研究

目的:探讨SW体外诱导人胃癌细胞株SGC7901凋亡的机制。方法:应用MTT法确定SW对体外培养的SGC7901细胞的作用剂量,通过流式细胞术及凋亡相关调控基因p53、cmyc、Bcl2的检测对细胞凋亡及细胞周期的测定,观察SW对胃癌细胞SGC7901增殖周期的影响以及抑制肿瘤细胞增殖的方式,同时利用激光共聚焦显微镜对细胞内Ca2 浓度的监测,研究SW与细胞内Ca2 超载的关系。结果:SW体外抗SGC7901细胞的完全致死剂量为6.2μg·ml-l,高于0.05μg·ml-l时抑制作用明显(P<0.05),其LC50为0.84μg·ml-l;经SW0.5～1.5μg·ml-l处理24h可引起凋亡抑制基因p53、Bcl2的明显下降、凋亡促进基因cmyc的明显升高以及肿瘤细胞内Ca2 超载,最终诱导SGC7901细胞凋亡。实验还证实SW主要作用于肿瘤细胞的S期,使瘤细胞主要积聚在S期。结论:SW通过多种途径诱导细胞凋亡可能是其发挥抗癌作用的重要机制。     

Molecular mechanisms of polypeptide from Chlamys farreri protecting HaCaT cells from apoptosis induced by UVA plus UVB

Aim： To investigate the mechanism of polypeptide from Chlamysfarreri （PCF） protecting HaCaT cells from apoptosis induced by UVA plus UVB in vitro. Methods： An apoptotic model of UV irradiation-induced HaCaT cells was established. The 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide assay, agarose gel electrophoresis, biochemical methods, and Western blotting were employed in the study. Results： PCF inhibited the UV irradiation-induced apoptosis of HaCaT cells. PCF strongly reduced the intracellular reactive oxygen species level, enhanced activities of superoxide dismutase and glutathione per- oxidase and increased the total anti-oxidative capacity in HaCaT cells following UV irradiation. Furthermore, we found that PCF could inhibit the phosphorylation of c-Jun amino-terminal kinase and the activity of caspase-3 in a concentration- dependent manner. Conclusion： PCF protected HaCaT cells from apoptosis induced by UVA plus UVB, mainly through decreasing the intracellular ROS level and increasing the activities of anti-oxidative enzymes to block the ROS-JNK-caspase-3-apoptosis signaling pathway.     

三氧化二砷通过升高细胞内钙离子浓度介导肺癌细胞凋亡

目的　研究三氧化二砷 (As2 O3 )对人肺腺癌细胞株AGZY 83 a的抑制作用 ,探讨As2 O3 诱导人肺腺癌细胞株凋亡的机制。方法　采用MTT法、透射电镜、激光扫描共聚焦仪及流式细胞术等方法观察三氧化二砷对人肺腺癌细胞株AGZY 83 a的影响。结果　①As2 O3 能抑制人肺腺癌细胞株AGZY 83 a的生长 ,呈剂量依赖关系 (P <0 0 1) ,IC50 =1 6 1mg·L-1;②在光学显微镜及电镜下 ,可见As2 O3 作用后的人肺腺癌细胞株AGZY 83 a呈现凋亡中、早期的变化特征 :表面绒毛脱失 ,染色质块状散于核内 ;③流式细胞术显示 :As2 O3 作用后的人肺腺癌细胞株AGZY 83 a细胞凋亡百分率与药物剂量呈依赖关系 ;④LSCM显示 :As2 O3 作用后的人肺腺癌细胞株AGZY 83 a细胞内钙浓度 ([Ca2 + ]i)升高 (P <0 0 1) ,呈剂量时间依赖关系。结论　As2 O3 能明显抑制人肺腺癌细胞株AGZY 83 a细胞的生长 ,并诱导人肺腺癌细胞株AGZY 83 a细胞凋亡 ,细胞内 [Ca2 + ]升高可能是诱导细胞凋亡的机制之一。     

B103 neuroblastoma cells predominantly express endothelin ET(B) receptor; effects of extracellular Ca(2+) influx on endothelin-1-induced mitogenesis

We sought to examine the effects of endothelin-1 on the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and mitogenic response in the neuroblastoma cell line, B103 (B103 cells). The results obtained from an [125I] endothelin-1 binding assay demonstrated that B103 cells express the endothelin receptor. The B(max) and K(d) values for [125I]endothelin-1 binding were 70+/-36 fmol/mg protein and 52+/-13 pM, respectively. Endothelin-1 failed to stimulate cAMP formation, but it did inhibit forskolin-induced cAMP formation. Endothelin-1 also stimulated the accumulation of [3H]inositol phosphates. These results indicate that the endothelin receptor in B103 cells couples with G(i) and G(q) but not with G(s). Monitoring of [Ca(2+)](i) showed that endothelin-1 evoked a transient increase in [Ca(2+)](i); this remained even in the absence of extracellular Ca(2+). However, no sustained, endothelin-1-induced increase in [Ca(2+)](i) due to extracellular Ca(2+) influx was detected. The endothelin B receptor-selective antagonist, 2,6-Dimethylpiperidinecarbonyl-gamma-Methyl-Leu-N(in)-[Methoxycarbonyl]-D-Trp-D-Nle (BQ 788), abolished the endothelin-1-induced increase in [Ca(2+)](i), while the endothelin ET(A) receptor-selective antagonist, cyclo-D-Asp-Pro-D-Val-Leu-D-Trp (BQ 123), failed to inhibit it. These results indicate that B103 cells express endothelin ET(B) receptor or an endothelin ET(B)-like receptor predominantly and have no Ca(2+) channels activated by endothelin-1. Endothelin-1 activated mitogen-activated protein kinase in B103 cells. However, based on the data for 3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl tetrazolium bromide, [3H]thymidine incorporation, and apoptosis screening assays, endothelin-1 induces neither mitogenesis nor apoptosis. These results suggest that endothelin-1 has no role in the mitogenic response in B103 cells, and this is consistent with the notion that an endothelin-1-induced sustained increase in [Ca(2+)](i) plays a role in endothelin-1-induced cell proliferation.     

卡托普利对缺氧诱导的肺动脉平滑肌细胞增殖和胶的合成的抑制作用

AIM: To study the effect of captopril (Cap) on hypoxia-induced proliferation and collagen synthesis in vascular smooth muscle cells (VSMC). METHODS: VSMC were isolated from rabbit pulmonary artery. Cultured VSMC were evaluated by incorporation of [3H]thymidine and [3H]proline, cell number, and intracellular calcium concentration ([Ca2+]i). RESULTS: Pretreatment of pulmonary VSMC with Cap 1 mumol.L-1 blocked hypoxia-induced increase in cell number and incorporation of [3H]proline and [3H]thymidine, which were decreased 25%, 21%, and 36%, respectively, as compared with hypoxic control. It also inhibited the increase of intracellular Ca2+ concentration under hypoxic condition. Addition of nifedipine inhibited hypoxia-stimulated increase in the collagen, DNA synthesis, and [Ca2+]i. Bay-K-8644 increased cell number (35%), DNA (55%), collagen synthesis (36%), and [Ca2+]i (33%) in pulmonary VSMC, that was completely abolished by Cap 1 mumol.L-1. CONCLUSION: Cap inhibited hypoxia-induced proliferation and collagen synthesis in VSMC.     

青蒿琥酯诱导人脐静脉内皮细胞凋亡

目的　探讨青蒿琥酯对人脐静脉内皮细胞(HUVEC)的诱导凋亡作用 ,以及这种诱导作用与内皮细胞增殖及细胞内游离Ca2 + 的关系。方法　采用MTT比色法测定青蒿琥酯对HUVEC增殖的影响 ,Fura 2 /AM荧光标记法测定HUVEC胞内游离Ca2 + 浓度 ([Ca2 + ]i) ,应用吖啶橙 /溴化乙啶荧光双染色、DNA琼脂糖凝胶电泳、流式细胞术检测细胞凋亡。结果　青蒿琥酯可抑制HUVEC增殖 ,明显诱导HUVEC凋亡 ,并呈浓度依赖性和时间依赖性效应关系。在发生凋亡的同时伴有 [Ca2 + ]i 显著升高。结论　青蒿琥酯可诱导HUVEC凋亡 ,其机制可能与[Ca2 + ]i 升高有关