参芎葡萄糖注射液对H2O2诱导的HUVEC细胞氧化损伤的保护作用

目的:探讨参芎葡萄糖注射液(Shenxiong glucose injection,SGI)对H_2O_2诱导的人脐静脉上皮细胞(HUVEC)细胞氧化模型损伤的保护作用及其机制。方法:体外培养HUVEC人脐静脉内皮细胞,用H_2O_2(130 mmol·L~(-1))处理0.5 h,建立HUVEC细胞H_2O_2氧化损伤模型。SGI组HUVEC细胞用(6%,8%,10%)SGI预处理6 h后,再用H_2O_2处理0.5 h。用MTS法检测细胞存活率;用ELISA法检测乳酸脱氢酶(LDH)漏出量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活力;用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白质免疫印迹(Western blot)技术检测凋亡相关基因及蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的mRNA和蛋白表达情况。结果:在130 mmol·L~(-1)H_2O_2作用细胞0.5 h的情况下,细胞存活率降低至50%左右,降低的程度合适,且实验结果重复性好,因此后续实验用该条件建立氧化损伤模型。与H_2O_2组比较,SGI预处理6 h能显著升高细胞存活率(P0.05,P0.01),减少LDH的外漏和MDA的生成(P0.05,P0.01),显著增加SOD,GSH-Px和CAT的活性(P0.05,P0.01)。RT-PCR和Western blot结果表明,SGI能显著上调Bcl-2的表达(P0.05,P0.01),下调Caspase-3,Bax的表达(P0.05,P0.01)。结论:参芎葡萄糖注射液能保护HUVEC细胞对抗H_2O_2诱导的氧化损伤,具有一定的剂量依赖关系,其作用机制可能与抑制细胞凋亡有关。     

牙龈卟啉单胞菌对脐静脉血管内皮细胞表达IL-8和MCP-1 mRNA的影响

目的：在转录水平上观察牙龈卟啉单胞菌对脐静脉血管内皮细胞（human umbilical vein endothelial cells,HUVECs）表达白细胞介素-8（IL-8）和单核细胞趋化蛋白-1（MCP-1）的影响。方法：厌氧培养Pg，原代培养HUVECs，RNA抽提，逆转录-聚合酶链式反应（RT-PCR）和mRNA比色定量法检测IL-8和MCP-1基因表达。结果：HUVECs基础表达IL-8和MCP-1mRNA,Pg以剂量依赖的方式增强IL-8和MCP-1mRNA的表达，Pg感染后1hIL-8mRNA开始增高，3h达到高峰，并持续到5h。而MCP-1的mRNA从Pg感染后1h开始增高，3h达到高峰，5h出现下降趋势。结论：Pg在转录水平上增强UHUVECs表达IL-8和MCP-1，这种上调作用可能是牙周病早期基因水平调控炎症细胞募集的重要因素，可能是牙周疾病的炎症反应和免疫反应中主要调控机制之一。     

扁塑藤素对LPS诱导的HUVEC细胞功能损伤和焦亡的作用

目的研究扁塑藤素对脂多糖(LPS)诱导人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及可能机制。方法 建立LPS诱导HUVEC损伤模型,HUVEC细胞分为对照组、LPS组以及低、中、高剂量扁塑藤素组(0.1、0.2、0.4 μmol/L扁塑藤素)。CCK-8法测定细胞活力;试剂盒法检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量;ELISA法检测白细胞介素-1β(IL-1β)、IL-18蛋白水平;蛋白免疫印迹法、实时荧光定量PCR法检测焦亡相关分子NLRP3、Caspase-1、GSDMD蛋白和mRNA表达量。 结果与对照组比较,LPS组细胞活力和SOD含量显著下降(P＜0.05),LDH和MDA含量、NLRP3、Caspase-1、GSDMD的蛋白和mRNA表达量均显著升高(P＜0.05)。扁塑藤素呈剂量依赖性提高细胞活力和SOD含量,抑制LDH、MDA,降低NLRP3、Caspase-1、GSDMD的蛋白和mRNA表达水平(P＜0.05)。 结论扁塑藤素呈剂量依赖性抑制细胞焦亡和减轻氧化应激,从而改善LPS诱导的HUVEC功能损伤。     

17β-雌二醇对氧化型低密度脂蛋白由钙介导细胞骨架损伤的抑制作用

目的　通过观察 1 7β 雌二醇 (1 7β E2 )与氧化低密度脂蛋白 (OX LDL)作用于血管内皮细胞后细胞内微丝肌动蛋白的变化以及同细胞内钙离子变化间的关系 ,以探讨雌激素的血管保护作用机制。方法　采用ECV 30 4脐静脉内皮细胞株体外培养 ,分为空白对照组 ,OX LDL(2 0 0 μg/ml)组 ,4× 1 0 - 7mol/LE2 作用组 ,E2 保护组 ,采用激光扫描共聚焦显微镜分别观察OX LDL作用后 1 2h时的胞内钙离子的变化及OX LDL作用 2 4h时的细胞骨架微丝的改变。结果　OX LDL作用 1 2h后细胞内钙离子浓度明显升高 ,而细胞骨架微丝也在 2 4h后发生明显的破坏。雌激素预处理后可明显抑制OX LDL作用导致后细胞内钙离子浓度的升高 ,并且明显地防止细胞骨架微丝损伤的发生。结论　 1 7β E2 可能通过抑制OX LDL导致的细胞内钙离子升高从而防止细胞骨架微丝损伤的发生 ,这可能是雌激素产生血管保护作用的机制之一。     

Biological activity of bevacizumab,a humanized anti-VEGF antibody <Emphasis Type="Italic">in vitro</Emphasis>

Bevacizumab (Avastin, Genentech) is a humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), a critical angiogenic factor involved in both physiological and pathological conditions. It has been recently approved by the US FDA as a first-line therapy for widespread metastatic colorectal cancer. This report is a detailed biological characterization of bevacizumab in a variety of in vitro models. It is shown that bevacizumab potently neutralizes VEGF and blocks its signal transduction through both the VEGFR-1 and VEGFR-2 receptors, as demonstrated by the inhibition of VEGF-induced cell proliferation, survival, permeability, nitric oxide production, as well as migration and tissue factor production. Although bevacizumab retains the ability to bind to human Fc receptors and complement protein C1q, it does not demonstrate cell or complement-mediated cytotoxicity in either VEGF producing or targeting cells. Thus the mechanism of anti-tumor activity of bevacizumab is most likely due to its anti-angiogenesis effect through binding and neutralization of secreted VEGF.     

杜鹃素对H_2O_2诱导的内皮细胞凋亡的保护作用研究

目的研究杜鹃素对H2O2诱导的人脐静脉内皮细胞(HUVEC)凋亡的保护作用。方法采用M TT法检测细胞活力,相差显微镜和荧光显微镜观察细胞形态变化。结果采用5、10、20μg/m L浓度的杜鹃素处理细胞后,内皮细胞活性呈浓度依赖性增加,细胞存活率提高;与H2O2组相比,细胞皱缩明显减少。杜鹃素20μg/m L可抑制H2O2诱导的人脐静脉内皮细胞凋亡。结论杜鹃素具有抑制H2O2诱导的人脐静脉内皮细胞凋亡的作用。     

狭鳕鱼皮胶原蛋白肽对过氧化氢诱导的人脐静脉内皮细胞氧化损伤的作用研究

目的：通过建立H2O2 导致人脐静脉内皮细胞损伤的模型来研究狭鳕鱼皮胶原蛋白肽作用。方法：用人脐静脉内皮细胞作为体外研究对象,采用不同的浓度（40M、200M、400M）胶原蛋白和维生素E预处理12h,然后在加入50uM H2O2继续培养12h造成损伤模型。模型成功后用CCK-8检测吸光度,根据试剂盒操作检测细胞内及培养上清液谷胱甘肽过氧化物酶（GXH-PX）,一氧化氮（NO）、过氧化氢酶（CAT）,丙二醛（MDA）等指标的含量和活性变化。结果：经胶原蛋白预保护后细胞增值率升高,GXH-PX、CAT等活性增强,NO含量升高,丙二醛含量降低。     

Cellular senescence determines endothelial cell damage induced by uremia

Renal dysfunction is closely associated with endothelial damage leading to cardiovascular disease. However, the extent to which endothelial damage induced by uremia is modulated by aging is poorly known. Aging can render endothelial cells more susceptible to apoptosis through an oxidative stress-dependent pathway. We examined whether senescence-associated to oxidative stress determines the injury induced by the uremia in endothelial cells.     

MK2 plays an important role for the increased vascular permeability that follows thermal injury

We previously reported Rho kinase is involved in vessel hyper-permeability caused by burns. Here we further explore the Rho kinase downstream signaling, it is found that its specific inhibitor Y27632 significantly diminishes the activation of JNK and p38 MAPKs but not ERK that induced by serum from burned rats (burn-serum). JNK activation was found involved in the expression of HUVEC adhesion molecules following thermal injury, although not in the process of stress fiber formation. Inhibition of various MAPKs by specific inhibitors showed that SB203580 (inhibitor of p38), but neither SP600125 (inhibitor of JNK) nor PD98059 (inhibitor of ERK), abolish activation of the p38 downstream kinase MK2. Demonstration of stress fibers by fluorescent-labeled phalloidin showed that inhibition of MK2, either by its specific inhibitor or by dominant negative adeno-viral-carried constructs, significantly reduced burn-serum-induced HUVEC stress-fiber formation, while inhibition of another downstream p38 MAPK kinase, PRAK, had no such effects. Transfection of dominant negative adeno-viral MK2 (Ad-MK2(A)) significantly inhibited thermal injury-induced blood vessel hyper-permeability in rats and, moreover, prolonged the survival of burned rats beyond 72 h following thermal injury. One of the mechanisms behind these phenomena is that Ad-MK2(A) causes a significant depression of burn-serum-induced HSP27-phosphorylation, while the adeno-viral transported dominant negative PRAK (Ad-PRAK(A)) does not block. Although the effect of blockade of MK2 through its adeno-viral approach requires further study and investigation of alternatives to know for sure, we may have found a new pathway behind thermal-injury-induced blood vessel hyper-permeability, namely: Rho kinase > p38 > MK2 > HSP27.     

Cell-derived microparticles contain caspase 3 in vitro and in vivo.

BACKGROUND: Microparticles (MP) from endothelial cells (endothelial microparticles; EMP) circulate in disease states, but the processes such as apoptosis or cell activation underlying their release are unclear. OBJECTIVES: We investigated whether adherent (viable) or detached (apoptotic) endothelial cells are the possible source of EMP in vitro, i.e. under control and interleukin (IL)-1alpha activation conditions, and in vivo. METHODS: Adherent and detached endothelial cells, and EMP, were isolated from human umbilical vein endothelial cell cultures (n = 6), treated without or with IL-1alpha (5 ng mL(-1); 24 h). Cell fractions were analyzed by flow cytometry for annexin V binding, propidium iodide (PI) and caspase 3 staining (n = 3). Caspase 3 in EMP was studied using Western blot (n = 6) and flow cytometry (n = 6). Plasma from healthy subjects and systemic lupus erythematosus patients (both n = 3) were analyzed for caspase 3-containing (E)MP. RESULTS: Detached but not adherent cells double-stained for annexin V and PI, confirming the apoptotic conditions of the detached cells and the viable nature of the adherent cells. Caspase 3 was solely present in the detached cells and procaspase 3 in the adherent cells. Caspase 3 was present in EMP from both control and IL-1alpha-treated cultures. Counts of EMP and detached cells, but not adherent cells, highly correlated (r = 0.959, P < 0.0001). In vivo circulating MP from nucleated (endothelial cells, monocytes) and anucleated cells (platelets, erythrocytes) contained caspase 3. CONCLUSIONS: EMP contain caspase 3 and may be mainly derived from detached (apoptotic) endothelial cells in vitro. The presence of caspase 3 in MP from anucleated cell types, however, suggests that its presence may not necessarily be related to apoptosis in vivo but may be associated with caspase 3 activation unrelated to apoptosis.