T型分叉流槽中前列环素和内皮素分泌的研究

动脉粥样硬化（atherosclerosis）的非随机分布与当地的血流动力环境有关。借助平行平板式平直流槽和以T型分叉流槽为代表的平行平板式异型流槽,可以模拟血管的主要形状特征,首先,在数值模拟的基础上分析了流型特征参数,确定了流槽的设计尺寸。然后,通过实验研究,探讨流型改变对内皮细胞血管活性物质分泌的影响,发现扩张效应流线偏转和驻点效应使得异型流槽前列环素和内皮素的分泌水平与相同入口雷诺数（Re）条件下的平直流槽分别有降低趋势和显著差异。为进一步研究流型对血管内皮细胞的影响提供了实验数据。     

功能蛋白的O-糖基化和p38磷酸化共同参与调控单核细胞对血管内皮的粘附和侵袭

探讨单核细胞在炎症因子刺激下通过功能蛋白O-糖基化和p38 MAPK磷酸化、调控其对血管内皮的粘附和侵袭的分子机制。将IFN-γ与LPS体外共刺激后的THP-1细胞加至单层血管内皮细胞EA.hy926共培养,观察单核细胞对血管内皮的粘附和侵袭;并通过测量电阻变化来反应血管内皮通透性的改变。采用Western blot方法检测单核细胞THP-1中p38 MAPK磷酸化的变化,O-GLcNAc糖基转移酶（OGT）和O-GLcNAc糖基化蛋白表达量的变化。分析验证p38 MAPK抑制剂对IFN-γ与LPS诱导的单核细胞对血管内皮粘附和迁移的影响,同时检测OGT、O-GLcNAc糖基化蛋白差异表达的影响。结果显示,IFN-γ与LPS可以共作用促进THP-1对血管内皮的粘附和侵袭,降低血管内皮通透性。同时激活p38 MAPK,此过程与OGT及O-GLcNAc糖基化蛋白表达降低相关。采用p38抑制剂预处理,可逆转上述IFN-γ与LPS诱导的生物学变化。综上,在炎症反应中,单核细胞对血管内皮的粘附和侵袭力的变化受功能蛋白糖基化和磷酸化的双向调控。     

整合素β2促进人乳腺癌细胞MCF-7的迁移,侵袭与粘附

本研究主要目标为探讨整合素β2 (ITGB2)的高表达对人乳腺癌细胞MCF-7迁移,侵袭与粘附能力的影响。本研究首先构建了ITGB2过表达质粒,实验设阴性对照组(pcDNA-3.1+)与ITGB2基因过表达组(pcDNA-3.1+/ITGB2)。ITGB2过表达质粒转染MCF-7细胞后,采用逆转录PCR与Western blotting方法分别检测ITGB2 mRNA转录水平与蛋白翻译水平;流式细胞术检测细胞周期的改变;划痕实验检测细胞横向迁移能力;Transwell小室实验检测细胞纵向迁移能力及侵袭能力;人脐静脉血管内皮细胞(HUVEC)粘附实验检测癌症细胞与血管内皮细胞之间的粘附能力,Western blotting实验检测侵袭相关指标MMP9,整合素经典通路中FAK蛋白磷酸化水平的改变。研究结果表明:转染ITGB2过表达质粒后,MCF-7细胞中ITGB2的m RNA水平(p<0.01)与蛋白水平(p<0.05)均显著增高;流式细胞术实验中,实验组S期的细胞所占比例与对照组无明显差异;划痕实验与Transwell小室实验中,实验组的迁移侵袭能力显著性增强;人脐静脉血管内皮细胞粘附实验中,实验组乳腺癌细胞与血管内皮细胞的粘附能力强于对照组(p<0.05);且Western blotting结果显示MMP9和p-FAK蛋白水平明显上升。由以上结果可得出结论,过表达ITGB2后会增强人乳腺癌细胞MCF-7的迁移、侵袭与粘附能力,而对其增殖能力无明显影响。     

内皮细胞E—选择蛋白的表达及p53基因的影响

为探讨血管内皮细胞Ｅ－选择蛋白的表达及野生型ｐ５３基因对其表达的影响,采用流式细胞术及ＲＴ－ＰＣＲ法分别测定其Ｅ－选择蛋白及其ｍＲＮＡ水平。结果表明：静息状态的内皮细胞表面检测不到Ｅ－选择蛋白的表达,尽管此时细胞内存在Ｅ－选择蛋白的ｍＲＮＡ。肿瘤坏死因子α（ＴＮＦα）可浓度依赖性地诱导内皮细胞表达Ｅ－选择蛋白,内皮细胞表面Ｅ－选择蛋白表达的增加伴随着细胞内Ｅ－选择蛋白ｍＲＮＡ水平的升高。ＴＮＦα刺     

人血管内皮细胞文库的构建

<正> 人体内大约有10~(12)个内皮细胞,它们均位于血管的内面,其面积约1000m~2,内皮细胞不仅为血液流动、防止血凝提供了一个光滑的表面,而且亦是防止某些细胞因子和细胞成分粘附和迁移的屏障。内皮细胞具有广泛的合成能力,可以产生几十种生物活性物质。另外,内皮细胞与血管平滑肌细胞及多种细胞有着广泛的相互作用。这种相互作用对维持机体     

内皮细胞E选择蛋白的表达及p53基因的影响

为探讨血管内皮细胞Ｅ选择蛋白的表达及野生型ｐ５３ 基因对其表达的影响, 采用流式细胞术及ＲＴＰＣＲ法分别测定其Ｅ选择蛋白及其ｍ ＲＮＡ水平。结果表明： 静息状态的内皮细胞表面检测不到Ｅ选择蛋白的表达, 尽管此时细胞内存在Ｅ选择蛋白的ｍＲＮＡ。肿瘤坏死因子α（ＴＮＦα） 可浓度依赖性地诱导内皮细胞表达Ｅ选择蛋白, 内皮细胞表面Ｅ选择蛋白表达的增加伴随着细胞内Ｅ选择蛋白ｍＲＮＡ 水平的升高。ＴＮＦα刺激内皮细胞后４ ～６ ｈ ,Ｅ选择蛋白的表达达高峰。将ｐ５３ 基因导入内皮细胞后, 经ＴＮＦα诱导的内皮细胞表面Ｅ选择蛋白的表达显著下降, 同时细胞内的Ｅ选择蛋白ｍＲＮＡ 的水平也明显降低。导入ｐ５３ 基因不影响静息状态内皮细胞Ｅ选择蛋白的表达及其ｍ ＲＮＡ 的水平。提示： ｐ５３ 基因至少部分通过降低Ｅ选择蛋白的ｍＲＮＡ 水平而抑制ＴＮＦα诱导的内皮细胞表面Ｅ选择蛋白的表达。     

野生型p53基因对内皮细胞细胞间粘附分子-1表达的影响

细胞间粘附分子－１（ＩＣＡＭ－１）是介导白细胞与内皮细胞粘附的重要粘附分子．为研究野生型ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响,分别采用流式细胞术和ＲＴ－ＰＣＲ／ＨＰＬＣ方法测定ＩＣＡＭ－１蛋白及ｍＲＮＡ水平．静息状态的内皮细胞表面结构性地表达有少量的ＩＣＡＭ－１,在肿瘤坏死因子α（ＴＮＦα,１０～１０００Ｕ／ｍｌ）诱导下,其表达呈剂量依赖性增加．将ｐ５３基因导入内皮细胞,则显著抑制ＴＮＦα诱导的内皮细胞表面ＩＣＡＭ－１的表达．ｐ５３基因的导入对静息状态内皮细胞表面结构性表达的ＩＣＡＭ－１影响较小．ｐ５３基因主要通过降低ＩＣＡＭ－１的ｍＲＮＡ水平而抑制内皮细胞表面ＩＣＡＭ－１的表达,但对蛋白的抑制程度小于对ｍＲＮＡ的抑制程度．提示：ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响除转录水平控制外,还存在转录后水平的调控     

基于分子对接的合欢皂苷J_(8)抑制血管内皮细胞增殖作用靶点及其凋亡相关细胞信号通路研究CSCD

为探讨合欢皂苷J_(8)(julibroside J_(8),J8)抑制肿瘤血管内皮细胞增殖作用靶点及其相关细胞凋亡的信号通路。本文采用HPLC法检测内皮细胞经J8作用后,J8的在细胞内外含量变化;Vina软件将J8与VEGF、FAS、DR3、DR4、DR5、TFR-1进行分子对接分析;Western blot法检测在加药前后,内皮细胞中VEGF、p-JNK、Bax、EnDOG、Caspase-3、Caspase-8以及Caspase-9蛋白表达水平的变化。分子对接结果表明VEGF、FAS对应的靶点蛋白与J8结合性能较好且多位点结合。HUVEC细胞在加入J8作用24 h后,VEGF、p-JNK等蛋白表达明显下调,并可显著上调诱导凋亡相关蛋白Bax和EnDOG的表达,而且对Caspase-3、Caspase-8以及Caspase-9的表达水平无显著的影响。J8可能是通过与血管内皮细胞膜表面的VEGF结合,抑制血管内皮细胞增殖,通过削弱VEGF/JNK通路活性从而引起内皮细胞凋亡。     

血小板活化因子对颈髓损伤后血管内皮细胞 ICAM-1 mRNA、ELAM-1 mRNA 表达的影响

血小板活化因子对颈髓损伤后血管内皮细胞ＩＣＡＭ┐１ｍＲＮＡ、ＥＬＡＭ┐１ｍＲＮＡ表达的影响肖建如曾华武候铁胜邵擎东赵定麟（第二军医大学长征医院骨科,上海２００００３）目前认为,组织损伤可刺激介导中性粒细胞与血管内皮细胞间的粘附作用,这种粘附作用增强系...     

脂可平对实验性早期动脉粥样硬化内皮细胞凋亡及Bax、Bcl-2表达的影响

研究脂可平对大鼠血脂、早期动脉粥样硬化血管内皮细胞凋亡及Bax、Bcl-2表达的影响。应用高脂饲料复制SD大鼠早期动脉粥样硬化模型,分别给以脂可平、辛伐他汀片混悬液灌胃,实验10周后,全自动生化分析仪、流式细胞仪定量分别检测各组血脂、主动脉内皮细胞凋亡率及Bax、Bcl-2蛋白的表达;苏木素伊红(HE)染色观察主动脉组织形态学变化。两治疗组均可降低血脂,不同程度的改善了主动脉组织病理损伤,降低内皮细胞凋亡率,调控Bax、Bcl-2蛋白表达。本实验说明脂可平可降低血脂,干预动脉粥样硬化的始动环节及其发生并从蛋白水平调控早期动脉粥样硬化内皮细胞凋亡。     

Thyroid hormone analog, diiodothyropropionic acid (DITPA), exerts beneficial effects on chamber and cellular remodeling in cardiomyopathic hamsters

Diiodothyropropionic acid (DITPA) is a thyroid hormone analog that is currently in phase II clinical trials. However, there have not been any studies to comprehensively analyze its effect on myocyte morphology. In addition, long-term studies with DITPA have not been done. This study compares the effects of DITPA with L-thyroxine (T4) on chamber remodeling, cardiac function, cellular morphology, cardiac blood flow, and protein expression. Normal and cardiomyopathic hamsters were treated with T4 or DITPA for 2 months. At the end of the treatment, echos, hemodynamics, coronary blood flow, cell morphology, and protein expression data were collected. Both T4 and DITPA treatment reduced chamber diameter during diastole, suggesting attenuated chamber dilatation in cardiomyopathic hamsters. Wall thickness also tended to increase, which was supported by cell morphology data in which DITPA significantly increased cross-sectional growth of myocytes specifically in the minor dimension, which is oriented transmurally. T4 and DITPA also increased myocardial blood flow both at baseline and after maximal dilation. This suggests there was increased angiogenesis or reduced loss of arterioles. Both T4 and DITPA had beneficial effects on chamber remodeling, which was most likely due to beneficial changes in cell shape and improved vascular supply.     

黄芩甙对人肝癌BEL-7402细胞增殖和侵袭转移的影响及机制

目的探讨黄芩甙对人肝癌BEL-7402细胞系增殖、侵袭转移的影响及其机制。方法应用细胞培养技术培养人肝癌BEL-7402细胞,MTT实验、软琼脂克隆形成实验检测黄芩甙对肝癌细胞增殖的影响。通过Boyaen小室模型测定其侵袭力,细胞迁移实验测定细胞运动能力,同时观察细胞形态。流式细胞术测定肝癌细胞MMP2、TIMP2表达,免疫组化测定VEGF表达。结果黄芩甙能明显抑制肝癌细胞增殖,细胞侵袭力及运动能力明显下降,且呈量效关系（P〈0．05）。形态学观察发现,黄芩甙处理组细胞形态较圆,伪足数目较少;MMP2阳性表达细胞减少,TIMP2阳性表达细胞增多,MMP2／TIMP2比值下降;VEGF表达减少。结论黄芩甙能抑制肝癌BEL-7402增殖、侵袭与转移,其机制可能与直接抑制细胞迁移运动,抑制细胞基质溶解相关基因蛋白MMP2表达,促进TIMP2表达;VEGF表达减少有关。     

流动剪切力对鼠脑微血管内皮细胞ICAM—1表达的影响

利用内皮细胞流动小室方法,对大鼠脑微血管内皮细胞的剪切力作用下细胞内粘附分子－1（ICAM－1,intercellular adhesion molecule-1）的表达进行了研究。图像分析结果提示,脑微血管内皮细胞在剪切力作用下ICAM－1的表达呈特异上调,且存在着时间依赖性,与一定范围内的剪切力强度无关,用对细胞施加剪切力作用后提取上清液孵育内皮细胞的方法证明：剪切力对鼠脑微血管内皮细胞ICAM－1表达的影响,是直接作用于内皮细胞引起的细胞内的直接反应,而不是剪切力导致细胞先释放细胞因子,释放的细胞因子再引起ICAM－1变化的间接反应。该工作为进一步开展剪切力对微血管内皮细胞信号转导机制的影响提供了实验数据。     

Operant behavior of the rat can be controlled by the configuration of objects in an animated scene displayed on a computer screen

We developed a novel behavioral task in which rats learn to recognize the configuration of objects in an animated scene displayed on a computer screen. The scene consisted of a moving bar and a stationary rectangle. Rats deprived of food were trained to press a lever for reward in a small chamber located in front of the screen. Lever presses were rewarded only when the bar was at the rectangle. Rats anticipated the reward by gradually increasing frequency of lever pressing as the bar approached the rectangle. Control experiments showed that neither the timing nor the discrimination of rewarded and non-rewarded periods as two discrete conditions explain behavior of the rat. Because the changes in the scene were generated by movement of the object, the presented task could be used for studying neural structures involved in spatial behavior of rats using virtual reality technology.     

Study of local hydrodynamic environment in cell-substrate adhesion using side-view μPIV technology

Tumor cell adhesion to the endothelium under shear flow conditions is a critical step that results in circulation-mediated tumor metastasis. This study presents experimental and computational techniques for studying the local hydrodynamic environment around adherent cells and how local shear conditions affect cell-cell interactions on the endothelium in tumor cell adhesion. To study the local hydrodynamic profile around heterotypic adherent cells, a side-view flow chamber assay coupled with micro particle imaging velocimetry (μPIV) technique was developed, where interactions between leukocytes and tumor cells in the near-endothelial wall region and the local shear flow environment were characterized. Computational fluid dynamics (CFD) simulations were also used to obtain quantitative flow properties around those adherent cells. Results showed that cell dimension and relative cell-cell positions had strong influence on local shear rates. The velocity profile above leukocytes and tumor cells displayed very different patterns. Larger cell deformations led to less disturbance to the flow. Local shear rates above smaller cells were observed to be more affected by relative positions between two cells.     

Computational modeling of left heart diastolic function: examination of ventricular dysfunction

A computational model that accounts for blood-tissue interaction under physiological flow conditions was developed and applied to a thin-walled model of the left heart. This model consisted of the left ventricle, left atrium, and pulmonary vein flow. The input functions for the model included the pulmonary vein driving pressure and time-dependent relationship for changes in chamber tissue properties during the simulation. The Immersed Boundary Method was used for the interaction of the tissue and blood in response to fluid forces and changes in tissue pathophysiology, and the fluid mass and momentum conservation equations were solved using Patankar's Semi-Implicit Method for Pressure Linked Equations (SIMPLE). This model was used to examine the flow fields in the left heart under abnormal diastolic conditions of delayed ventricular relaxation, delayed ventricular relaxation with increased ventricular stiffness, and delayed ventricular relaxation with an increased atrial contraction. The results obtained from the left heart model were compared to clinically observed diastolic flow conditions, and to the results from simulations of normal diastolic function in this model [1]. Cases involving impairment of diastolic function were modeled with changes to the input functions for fiber relaxation/contraction of the chambers. The three cases of diastolic dysfunction investigated agreed with the changes in diastolic flow fields seen clinically. The effect of delayed relaxation was to decrease the early filling magnitude, and this decrease was larger when the stiffness of the ventricle was increased. Also, increasing the contraction of the atrium during atrial systole resulted in a higher late filling velocity and atrial pressure. The results show that dysfunction can be modeled by changing the relationships for fiber resting-length and/or stiffness. This provides confidence in future modeling of disease, especially changes to chamber properties to examine the effect of local dysfunction on global flow fields.     

Normal and shear stresses influence the spatial distribution of intracellular adhesion molecule-1 expression in human umbilical vein endothelial cells exposed to sudden expansion flow

Patterns in cell adhesion molecule expression by endothelial cells may play a role in atherogenesis. Previous studies have shown dependence of intracellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVEC) on shear stress and have indirectly linked ICAM-1 expression to spatial gradients in shear stress. The spatial distribution of ICAM-1 in HUVEC pre-exposed to flow for 8h was determined using fluorescence microscopy and a sudden expansion flow chamber with a 2.66 expansion ratio to simulate gradients in wall shear stress found near arterial branches in vivo. When ICAM-1 expression in the disturbed flow region was compared to theoretical stress distributions obtained from a computational model of sudden expansion flow, a modest trend (R2 = 0.327, p < 0.01)was observed between ICAM-1 and shear stress but the correlation between ICAM-1 and shear stress gradient was insignificant. In contrast, a moderately strong trend (R2 = 0.873, p < 0.01) was evident between ICAM-1 expression and the component of normal stress induced by the expansion. Thus, in this in vitro model, normal stress arising from sudden expansion flow modulates the effect of shear stress on ICAM-1 expression.     

RV instantaneous intraventricular diastolic pressure and velocity distributions in normal and volume overload awake dog disease models

Intraventricular diastolic right ventricular (RV) flow field dynamics were studied by functional imaging using three-dimensional (3D) real-time echocardiography with sonomicrometry and computational fluid dynamics in seven awake dogs at control with normal wall motion (NWM) and RV volume overload with diastolic paradoxical septal motion. Burgeoning flow cross section between inflow anulus and chamber walls induces a convective pressure rise, which represents a "convective deceleration load" (CDL). High spatiotemporal resolution dynamic pressure and velocity distributions of the intraventricular RV flow field revealed time-dependent, subtle interactions between intraventricular local acceleration and convective pressure gradients. During the E-wave upstroke, the total pressure gradient along intraventricular flow is the algebraic sum of a pressure decrease contributed by local acceleration and a pressure rise contributed by a convective deceleration that partially counterbalances the local acceleration gradient. This underlies the smallness of early diastolic intraventricular gradients. At peak volumetric inflow, local acceleration vanishes and the total adverse intraventricular gradient is convective. During the E-wave downstroke, the strongly adverse gradient embodies the streamwise pressure augmentations from both local and convective decelerations. It induces flow separation and large-scale vortical motions, stronger in NWM. Their dynamic corollaries on intraventricular pressure and velocity distributions were ascertained. In the NWM pattern, the strong ring-like vortex surrounding the central core encroaches on the area available for flow toward the apex. This results in higher linear velocities later in the downstroke of the E wave than at peak inflow rate. The augmentation of CDL by ventriculoannular disproportion may contribute to E wave and E-to-A ratio depression with chamber dilatation.     

Fluid flow in a spiral device used for irradiation of biological fluids

The manufacture of plasma‐derived therapeutics includes dedicated viral inactivation steps to minimize the risk of infection. Traditional viral inactivation methods are effective for the removal and inactivation of enveloped viruses, but less effective against small nonenveloped viruses. UV‐C irradiation has been demonstrated to be an effective means of inactivating such viruses. The UVivatec lab system consists of a spiral tube around an UV‐C irradiation source. Flow of a solution through the chamber generates and ensures controlled mixing and uniform exposure to irradiation. A detailed assessment of the effect of flow rate, alternate cross sectional design and scale up of the irradiation chamber on Dean vortices was performed using the smoothed particle hydrodynamics method. The aim was to provide a basis for setting flow rate limits and using a laboratory scale apparatus to model viral inactivation in larger manufacturing scale equipment. The effect of flow rate related changes on the fluence rate was also investigated through chemical actinometry studies. The data were consistent with the simulations indicating that Dean vortices were present at low flow rates, but dissipated at higher flow rates through the spiral chamber. Importantly, this work also allowed a correlation between the small system and large scale system to be established. This will greatly facilitate process development and viral validation studies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 359–367, 2013