应用分离微血管段方法培养人脑微血管内皮细胞及对血管内皮细胞生长因子基因表达细胞超微结构的观察

背景观察缺氧状态下脑血管内皮细胞的血管内皮细胞生长因子表达及细胞超微结构变化,可为从细胞和分子水平认识血管生成及其相关的细胞因子参与缺血性脑血管疾病的病变过程.目的构建分离微血管段体外培养人脑微血管内皮细胞的方法,并观察血管内皮细胞生长因子基因表达与细胞超微结构变化.设计随机对照的技术方法研究.单位一所军区总医院的神经外科,一所大学医院的神经外科.对象2002年沈阳军区总医院神经外科脑动静脉畸形患者18例(SpetzlerⅡ～Ⅲ级),全部病例术前均经全脑血管造影证实.取材于手术中切除的完整脑动静脉畸形新鲜标本周围粘连的新鲜脑组织,采用匀浆、过滤和酶消化技术分离微血管内皮细胞.将在培养瓶内生长良好的细胞分成缺氧2,4,8 h组和对照组4组,每4瓶为1组.方法缺氧条件模拟体积分数0.95 N2,体积分数0.05 CO2.免疫组化方法检测细胞中第八因子相关抗原(FⅧ-RA)表达.选用RT-PCR技术观察每组内皮细胞血管内皮细胞生长因子mRNA表达,同时以ELISA方法检测每组细胞上清液中血管内皮细胞生长因子蛋白含量,透射电镜观察细胞超微结构的变化.主要观察指标对照组和各缺氧组血管内皮细胞中血管内皮细胞生长因子mRNA表达和细胞上清液中血管内皮细胞生长因子蛋白含量,以及细胞超微结构的变化.结果相差显微镜下,培养的活细胞具有单层"卵石样"排列的典型特征,90%以上的细胞为FⅧ-RA阳性染色.缺氧4 h细胞血管内皮细胞生长因子mRNA和蛋白表达为0.98±0.19,(180.77±20.15)ng/L,较对照组显著升高[0,(26.20±6.33)ng/L,P＜0.01],8 h后表达下调[0.35±0.07,(31.68±8.34)ng/L],并出现线粒体肿胀、内质网扩张及溶酶体多囊泡形成.结论采用分离微血管段方法培养人脑血管内皮细胞,操作简便易行,细胞纯度可靠.缺血缺氧早期血管内皮细胞生长因子表达不足以维持细胞超微结构完整,随缺氧时间延长细胞可发生损伤性变化.     

Tissue factor expression by endothelial cells in sickle cell anemia.

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.     

Histamine induced homologous and heterologous regulation of histamine receptor subtype mRNA expression in cultured endothelial cells.

Histamine has been proposed to play an important role in early inflammatory responses, based on studies documenting the effects of histamine on vasodilation and permeability of microvascular endothelium, as well as histamine-stimulated neutrophil adhesion to endothelial cells. The complex and heterogeneous biological mechanisms of histamine-mediated responses are not yet completely characterized. One of the factors defining inflammatory responses stimulated by histamine might be the distribution and density of receptor subtypes on cell membranes. The aim of this study was to assess whether the regulation of histamine receptor mRNA levels represents a control point in histamine-stimulated processes. Histamine-induced regulation of histamine receptor subtype expression was studied in vitro by semiquantitative RT-PCR analysis of total RNA extracted from stimulated and unstimulated endothelial cells. The time dependency and extent of histamine induced down-regulation varied between the H1 and H2 receptor message. The rapid decrease of H2 receptor mRNA lasted for 24 h. On the other hand, the less-pronounced reduction of H1 mRNA was transient and returned to control values after 12 h of histamine stimulation. The H2 receptor message was mainly down-regulated by activation of the H1 receptor protein. Down-regulation of the H1 receptor message seemed to be a net result of H1 and H2 receptor activation. This is the first report demonstrating a complex pattern of homologous and heterologous regulation of histamine receptor expression. These findings reveal new insight into the potential diversity of the mechanism involved in processing information via stimulation of multiple G-protein-linked pathways.     

Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers

Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72?bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination.     

Increasing use of bronchoscopic needle aspiration to diagnose small cell lung cancer

OBJECTIVE: To review pathology reports to determine whether a temporal change in diagnostic procedures that included bronchoscopic needle aspiration (BNA) in evaluation of small cell lung cancer (SCLC) had occurred. METHODS: A retrospective review of the computerized pathology database of the Wake Forest University Baptist Medical Center from 1990 to 1998 was performed. All pathology reports of patients newly diagnosed with SCLC were reviewed and abstracted. RESULTS: The number of patients newly diagnosed with SCLC during the 9-year study period totaled 277. Of these, 173 underwent bronchoscopy. From January 1990 to December 1991, 32% (8/25) of bronchoscopies done in patients with SCLC included BNA compared with 81% (120/148) (P < .001) from January 1992 to December 1998. In addition to the increased use of BNA in patients with SCLC undergoing bronchoscopy, the overall diagnostic yield for BNA in SCLC significantly increased over the 9-year study period from 50% (4/8) in 1990 and 1991 to 88% (106/120) thereafter (P = .001). Overall sensitivity of BNA during bronchoscopy was 86% for SCLC with only a small increase in sensitivity with use of all procedures (including BNA) to 91%. The use of forceps biopsy and bronchial brushings decreased over this period. CONCLUSION: With progressive experience with BNA, the frequency of its performance and its diagnostic yield in patients with SCLC increased markedly. The SCLC yield may be a worthwhile marker of BNA program development.     

血管内皮细胞与大肠癌细胞粘附后肌动蛋白Actin表达的研究

目的 探讨血管内皮细胞与大肠癌细胞粘附后,内皮细胞内肌动蛋白Actin表达的情况,分析其表达与肿瘤转移的关系.方法 采用细胞培养、免疫组化及免疫荧光染色技术检测单独培养的内皮细胞及与不同转移能力的人大肠癌细胞系LoVo细胞、HT29细胞共培养之内皮细胞内Actin的表达.结果 单独内皮细胞培养组的细胞内Actin主要分布在细胞的周边,分布均匀、排列整齐,细胞间连接紧密,没有间隙形成,荧光强度较强;而内皮细胞与HT29细胞共培养组中内皮细胞周边的Actin基本消失,出现应力纤维,排列呈明显的极向分布,细胞间隙形成,荧光强度减弱;内皮细胞与LoVo细胞共培养组变化则更显著（26.55±15.23和15.78±11.54, P＜0.05）.结论 大肠癌细胞的粘附可使内皮细胞的骨架蛋白发生改变和分布异常,高转移能力的癌细胞对内皮细胞Actin的影响更为显著.     

不同缺氧状态下骨髓间充质干细胞表达血管内皮生长因子的差异

背景:骨髓间充质干细胞在植入心肌缺血,梗死区域后,面临缺氧缺血微环境,其如何通过旁分泌细胞因子来发挥重建侧支微循环、修复心肌的作用,是目前存在争论的焦点之一.目的:探讨骨髓间充质干细胞在不同时程缺氧环境下血管内皮生长因子的表达规律.设计、时间及地点:细胞学体外实验,于2008-03/1 1在南昌大学第二附属医院分子医学实验中心完成.材料:清洁级新西兰大白兔2只,由南昌大学医学院实验动物中心提供.方法:常规差速贴壁法分离培养兔骨髓间充质干细胞.细胞缺氧微环境在AnaeroPack密闭方盒中产生,盒中置入一次性缺氧催化袋GENbox anaer(可吸收O2,产生CO2),指示条监测氧的耗竭,根据缺氧培养时程分为缺氧0,6,12,24 h组.主要观察指标:RT-PCR法检测血管内皮生长因子mRNA的表达,Western Blot法检测血管内皮生长因子蛋白的表达.结果:血管内皮生长因子mRNA及蛋白的表达水平均为缺氧0 h组<6 h组<12 h组<24 h组(P<0.01).结论:缺氧24 h以内的培养环境下,骨髓间充质干细胞表达血管内皮生长因子的水平呈缺氧时程依赖性增加.     

槲皮素对HL-60细胞形态及血管内皮生长因子表达的影响

目的：探讨槲皮素对HL-60细胞的形态、血管内皮生长因子(VEGF)表达的影响。方法：以HL-60细胞为研究对象，细胞形态学观察采用Wright染色法：膜联蛋白Ⅴ(Annexin Ⅴ)标记检测细胞凋亡率；VEGF表达采用ELISA法。结果：①经槲皮素处理后，HL-60细胞形态学上出现凋亡特征性改变。②槲皮素处理后HL-60细胞凋亡率明显增加。③槲皮素处理后HL-60细胞显著降低VEGF表达。结论：槲皮素在体外能抑制白血病细胞HL-60生长并诱导其凋亡；槲皮素可抑制白血病细胞分泌VEGF。     

Certain batches of albumin solutions influence the expression of endothelial cell adhesion molecules

Objective: Increased levels of soluble adhesion molecules, a decreased PO₂/FIO₂ ratio and a tendency to worsened outcome have been reported following the use of human albumin in critical illness. The reasons are not yet understood. Since albumin solutions have previously been shown to contain proinflammatory mediators, a direct upregulation of adhesion molecules by contaminated batches may explain these findings. To examine this, we studied the effects of different albumin preparations on endothelial cell adhesion molecules in vitro. Design: Experimental study. Setting: Laboratory for cell biology. Methods: Human umbilical venous endothelial cell cultures (n = 4) were incubated for 6 h at 5 mg/ml with four different human albumin solutions (HA1–4) from different manufacturers. Medium served as the control. Using flow cytometry, the effects on E-selectin, ICAM-1 and VCAM-1 expression were determined on unstimulated cells and on cells stimulated with tumour necrosis factor α at 0.5 ng/ml for 4 h. Measurements and results: On unstimulated cells, HA1 and HA4, two different batches from the same manufacturer, increased ICAM-1 by 22 % and 15 %, respectively. After stimulation, both solutions resulted in a 19 % increased expression of E-Selectin. In addition, HA4 decreased VCAM-1 on stimulated cells (p≤ 0.05). Two albumin preparations from other manufacturers did not produce significant effects. Conclusions: Some albumin solutions directly modulate adhesion molecule expression on endothelial cells. This may, at least in part, explain the previous finding of increased soluble adhesion molecules and a decreased PO₂/FIO₂ ratio in critically ill patients undergoing volume replacement with human albumin. Received: 28 May 1999*Accepted: 14 September 1999     

Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes

Sustained expression of recombinant proteins is a critical factor for the effectiveness of numerous applications in the biomedical sciences including the treatment of human disease by gene therapy, the large scale production of therapeutic proteins, as well as the investigation of gene function by transgenesis or cell type specific mutagenesis. Although much attention has been paid to the optimisation of regulatory sequences such as promoters, untranslated regions and polyadenylation signals, effective and sustained expression of recombinant genes in vivo is often difficult to achieve. Here we report that the creation of artificial exons, by insertion of two short heterologous introns into open reading frames, is not only compatible with functional expression, but also leads to a 30-fold enhancement of mRNA production for both green fluorescent protein and the bacteriophage P1-derived Cre recombinase. The levels of green fluorescence were increased five-fold in cell lines and sustained long-term expression at increased levels was observed in rat brain after transduction with a herpes simplex virus-based vector. The data presented identify a means by which the expression of recombinant genes can be enhanced considerably, in addition to and independently from the surrounding regulatory sequences. The method should help obtain sustained and effective expression of recombinant proteins in vivo.     

The addition of endothelial cell growth factor and heparin to human umbilical vein endothelial cell cultures decreases plasminogen activator inhibitor-1 expression.

Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid inhibitor of tissue plasminogen activator (tPA) and urokinase. Clinical studies suggest that PAI-1 may play a crucial role in the regulation of fibrinolysis. A number of factors modulate PAI-1 activity in endothelial cell culture, and the isolation of PAI-1 cDNA now allows study of PAI-1 regulation at the mRNA level. We examined the effect of endothelial cell growth factor (ECGF) and heparin on PAI-1 expression in human umbilical vein endothelial cell (HUVEC) culture. The addition of ECGF and heparin to HUVEC cultures results in a 3-10-fold decrease in the PAI-1 activity secreted into the conditioned media. This effect is mediated at the mRNA level. A decrease in PAI-1 is also seen with higher concentrations of ECGF alone, but is greatly enhanced by the addition of heparin. No significant change in tPA antigen or mRNA levels was observed.     

缺氧预适应对缺氧复氧诱导内皮细胞-中性粒细胞黏附的影响

目的：观察缺氧预适应对缺氧／复氧后血管内皮细胞表面黏附分子的表达以及中性粒细胞－内皮细胞黏附的影响。方法：采用β－N－乙酰氨基己糖苷酶比色法检测黏附率;流式细胞术检测内皮细胞表面黏附分子E－选择素、细胞间黏附分子－1（ICAM－1）的表达;苔盼蓝摄取法检测细胞存活率;常规生化法检测乳酸脱氢酶活性。结果：血管内皮细胞经缺氧／复氧处理后,苔盼蓝摄取率,乳酸脱氢酶活性均明显增高,E－选择素、ICAM－1表达明显上调,其表面中性粒细胞的黏附增加,缺氧预适应显著抑制缺氧／复氧的上述作用。结论：缺氧预适应通过调节内皮细胞表面黏附分子的表达,抑制缺氧／复氧诱导的内皮细胞－中性粒细胞黏附。     

脂多糖诱导大鼠主动脉内皮细胞表达血管性血友病因子裂解酶的研究

目的 从剂量-效应和时间-效应关系探讨脂多糖(LPS)对大鼠主动脉内皮细胞(RAECs)血管性血友病因子裂解酶(ADAMTS-13)mRNA和蛋白表达的影响.方法 采用组织贴块法培养Wistar大鼠的RAECs,1周后按1∶3传代至第4～5代,将细胞分为空白对照组和0.01、0.1、1、5 μg/ml LPS刺激组,分别于12、24、48、72 h用半定量逆转录-聚合酶链反应(RT-PCR)测定内皮细胞ADAMTS-13 mRNA表达;用酶联免疫吸附法(ELISA)检测上清液内ADAMTS-13蛋白水平.结果 空白对照组有一定量的ADAMTS-13mRNA和蛋白表达.随着LPS刺激浓度增加和刺激时间延长,ADAMTS-13 mRNA和蛋白表达逐渐下降.与空白对照组(25.22±1.41)比较,0.01μg/ml LPS刺激48 h时ADAMTS-13 mRNA表达(18.78±0.86)即明显下降(P＜0.01);0.1μg/ml和1μg/ml LPS刺激24 h时ADAMTS-13 mRNA表达(23.43±0.63、22.41±0.76)即明显下降(P＜0.05和P＜0.01);5μg/ml LPS刺激12 h时ADAMTS-13 mRNA表达(20.01±2.47)即明显下降(P＜0.01).与空白对照组[(115.76±2.36)ng/ml]比较,0.01 μg/ml LPS刺激12 h时ADAMTS-13蛋白表达[(113.43±1.07)ng/ml]即明显下降(P＜0.05);至5 μg/ml LPS刺激72 h时ADAMTS-13蛋白表达[(7.63±2.64)ng/ml]降至最低(P＜0.01).结论 RAECs有一定量的ADAMTS-13mRNA和蛋白表达;不同浓度LPS刺激内皮细胞不同时间后ADAMTS-13 mRNA和蛋白表达降低,具有剂量依赖性和时间依赖性.