肝素对脂多糖诱导内皮细胞通透性增高的保护作用

目的 研究肝素对脂多糖(LPS)诱导入脐静脉内皮细胞(HUVECs)单层通透性和细胞骨架形态的影响.方法 HUVECs细胞株传代培养后随机分为空白对照组、LPS组、肝素组、LPS+肝素组,n=8.用四甲基偶氮唑盐(MTT)比色法测定内皮细胞活性;用Transwell小室法测定单层内皮细胞通透性;用免疫荧光染色法测定内皮细胞纤维状肌动蛋白(F-actin)形态.结果 与空白对照组比较,LPS 10 mg/L及100 mg/L对细胞活性起到明显的抑制作用(0.695±0.015、0.476±0.030比0.860±0.053,P＜0.05和P＜0.01),肝素100 kU/L对内皮细胞活性产生抑制作用(0.675±0.030比0.840±0.023,P＜0.05).LPS10mg/L可增加单层内皮细胞通透性,于4h时达峰值,与空白对照组比较差异有统计学意义(5.882±0.101比4.489±0.015,P＜0.05),并能增加细胞骨架应力纤维的形成;而LPS+肝素刺激2～12h时单层内皮细胞的通透性较LPS组明显降低(2 h:4.382±0.053比5.084±0.129,4 h:4.528±0.044比5.882±0.101,6 h:4.381±0.089比5.479±0.125,12 h:4.447±0.054比4.719±0.080,均P＜0.05),并能减少内皮细胞骨架的重排及应力纤维的形成.结论 肝素可以减轻LPS诱导单层内皮细胞通透性增高及细胞骨架重排,对内皮细胞屏障功能起到保护作用.     

靶向封闭CapG基因对胰腺癌细胞运动侵袭能力的影响

目的观察靶向封闭CapG基因对胰腺癌细胞株BxPC3运动侵袭能力的影响。方法设计并体外合成靶向CapG的小干扰RNA,将其转染到BxPC3细胞中,蛋白质印迹法检测转染前后细胞中CapG蛋白的表达,采用Transwell小室检测转染前后细胞运动侵袭能力的改变。结果CapGsiRNA能有效降低BxPC3细胞中CapG的表达,CapG在蛋白质水平的表达抑制率达80％左右。抑制BxPC3细胞中CapG的表达后,BxPC3细胞的运动侵袭能力明显下降,穿过人工基底膜的细胞数显著低于阴性对照组和空白对照组（P〈0．05）。结论封闭CapG表达能明显抑制胰腺癌细胞BxPC3在体外的运动侵袭能力。     

软骨脱细胞基质多孔支架与骨髓基质干细胞体外构建组织工程软骨的研究

目的 探讨关节软骨脱细胞基质多孔支架(CEDIS)与骨髓基质干细胞(BMSCs)体外培养组织工程软骨的可行性.方法 粉碎人关节软骨,脱细胞处理后差速离心法收集细胞外基质悬液,采用冷冻干燥技术制备三维多孔支架.扫描电镜观察其微观结构,并进行组织学观察,生化成分定量检测其胶原、氨基葡聚糖(GAG)、DNA含量,牛物力学方法测量其干性及覆水状态下压缩弹性模量;分离培养犬骨髓基质干细胞,TGF-β1成软骨诱导,PKH26标记,接种到支架上体外继续诱导培养,荧光显微镜及扫描电镜观察培养3 d后细胞在支架内的黏附、分布情况.结果 制备的CEDPS支架无细胞碎片残留,软骨细胞外基质特异性染色阳性,具有相互贯通的三维孔隙结构;支架生化成分定量检测:总胶原含量为(708.2±44.7)μg/mg,GAG含量为(254.7±25.9)μg/mg,DNA含量为(0.021±0.007)μg/mg;支架纵向压缩弹性模量E=(1.226 ±0.288)MPa,覆水后压缩弹性模量E=(0.052±0.007)MPa.荧光显微镜、电镜检查结果表明细胞广泛均匀的分布在支架内部,呈圆形或椭圆形,在支架上增殖显著,细胞基质分泌明显.结论 CEDPS支架在生化组成和结构上与软骨细胞外基质成分类似,去细胞彻底,具有良好生物力学特性,是一种较为理想的软骨组织工程支架载体;成软骨诱导的BMSCs与CEDPS支架在体外可初步构建类软骨样组织.

Abstract：

Objective To develop a novel cartilage ECM-derived porous scaffold(cEDPs)and investigate the attachment,proliferation and distribution of bone marrow mesenchymal stem cells(BMSCs) cultured in vitro within the scaffolds.Methods Cartilage microfilaments were prepared after pulverization and gradient centrifugation and prepared into suspension after acellularization treatment.The scaffolds were examined by histological staining,scanning electron microscope(SEM),biochemical and biomechanical analysis.After labeling with PKH26,the canine BMSCs were seeded onto the scaffolds.The attachment,proliferation and differentiafion of cells were observed by inveaed fluorescent microscope and SEM.Results On histology,most extracellular matrices were retained in the scaffold after the removal of cell fragments.Safranin O staining and immunfluorescence examination with collagen Ⅱ antibodies provided positive results.Biochemical analysis showed that the collagen content was(708.2 4±44.7)μg/mg,glycosaminoglycan(254.7 ±25.9)μg/mg and DNA(0.021±0.007)μg/mg.Mechanical testing showed the compression moduli(E)were(1.226±0.288)and(0.052±0.007)MPa ander dry and wet conditions respectively.Inverted fluorescent microscope and SEM showed moderate cell adhesion.chondrocyte-like morphology and matrix synthesis around cells. Conclusion The CEDPS retains most extracellular matrices after a thorough decellularization so as to possess an excellent microstructure with ideal biomechanical characteristics and a good biocompatibility. Thus it is a suitable candidate as an alternative cell-carrier for cartilage tissue engineering. Chondrogenic BMSCs and CEDPS may be used to construct cartilage-like tissue in vitro.     

抑郁模型大鼠再应激后海马细胞支架蛋白的改变

目的 研究抑郁模型大鼠接受再次急性和慢性应激后细胞支架微管系统的动态性改变,并探讨可能的机制.方法 将40只大鼠按随机数字表法分为5组:对照组(空白对照+生理盐水),CUMS组(CUMS+生理盐水),氟西汀组(CUMS+氟西汀),急性再应激组(CUMS+氟西汀+药物清洗期+急性游泳应激),CUMS再应激组(CUMS+氟西汀+药物清洗期+CUMS).实验结束后进行行为学观察,并使用免疫印迹法( western blotting)检测大鼠海马乙酰化微管蛋白(Acet-Tub),酪氨酸化微管蛋白(Tyr-Tub),微管结合蛋白2(MAP-2)及磷酸化微管结合蛋白2(phospho-MAP-2).结果 (1)CUMS再应激组糖水偏好[ (43.38±7.84)％],总行程[(859.21±653.62)cm],运动平均速度[(2.05±0.60)cm/s]及直立次数[(0.12±0.30)次]均减少,与对照组及CUMS组相比均差异有显著性(P＜0.01).急性再应激组行为与对照组比较差异无显著性.氟西汀组糖水偏好和旷场实验相关指标与对照组差异无显著性,与CUMS组差异有显著性(P＜0.01).(2) CUMS再应激组Acet-Tub表达升高[(244.24±8.90)％],Tyr-Tub表达降低[ (30.92±11.00)％],与对照组及CUMS组差异均有显著性(P＜0.01).MAP-2的表达与对照组及CUMS组比较差异无显著性,phospho-M AP-2的表达减少[(24.75±8.83)％],与对照组及CUMS组均差异有显著性(P＜0.01).急性再应激组各蛋白水平与对照组比较差异无显著性.氟西汀组各蛋白的表达与对照组比较差异无显著性(P＞0.05),与CUMS组比较差异有显著性(P＜0.01).结论 动物再次暴露于CUMS后,其行为和微管动态性损害更严重,同时伴随微管相关蛋白磷酸化的变化,提示临床抑郁症的发生以及复发的町能机制.     

Cytoskeleton of cells in vocal fold macula flava unphonated for a long period

Cells in the maculae flavae (MFe) are inferred to be involved in the metabolism of extracellular matrices of the human vocal fold mucosa. The latest research has supported the hypothesis that the tension caused by phonation (vocal fold vibration) regulates the behavior of these cells in the MFe of the human vocal fold. Tensile and compressive strains have direct effects on cell morphology and structure including changes in cytoskeletal structure and organization. Cytoskeletons are one of the structures which play a role as mechanoreceptors for the cells. The microstructure of the intermediate filaments and the expression of their proteins were investigated regarding the cells in the MFe of the human vocal fold unphonated over a decade. The adult vocal fold mucosa of a 64-year-old male with cerebral hemorrhage unphonated for 11 years was investigated by immunohistochemistry and electron microscopy. The intermediate filaments in the cytoplasm of the cells had become fewer in number. And the expression of their characteristic proteins (vimentin, desmin, GFAP) was also reduced. The results of this study are consistent with the hypothesis that mechanotransduction caused by vocal fold vibration could possibly be a factor in regulating the function and fate of the cells in the MFe.     

包埋后的几丁质与软骨细胞体外培养的实验研究

目的 探讨几丁质作为组织工程技术中细胞培养支架的可行性。方法 采用聚乳酸、卵磷脂及多聚赖氨酸分别或工同包埋几丁质与软骨细胞体外培养,观察其产亲水性的改变、对细胞吸附力和细胞功能的影响。结果 以聚乳酸包埋的几丁质对细胞的生长有抵制作用;以卵磷脂包埋的几柄质亲水性增强;以多聚赖氨酸包埋的几丁质对细胞吸附力增强;以卵磷脂和多聚赖氨酸共同包埋的几丁质具有良好的亲水性和对细胞吸附力,并可使细胞更好地发挥功能     

Monoclonal antibodies to cytoskeletal proteins: An immunohistochemical investigation of human colon cancer

Monoclonal antibodies raised to a number of microfilament-associated proteins were shown to recognize the appropriate proteins in extracts from human colon tissue. They were then used in an immunohistochemical study of normal colonic mucosa, adenomas, and adenocarcinomas. A strong reaction was seen in stromal cells within the tumours (both adenomas and adenocarcinomas) when frozen sections were stained with antibodies to filamin and caldesmon. In addition, a similar reaction was seen in the adenocarcinomas when stained with antibodies to talin and gelsolin. We believe that immunohistochemical staining with these antibodies reveals a tumour-induced process in the surrounding cells, possibly related to a host response to tumours.     

Decreased gap junctional communication in neurobiotin microinjected lens epithelial cells after taxol treatment

The aim of the study was to examine gap-junction-mediated intercellular communication after experimentally induced aggregations of microtubules in cultured bovine lens epithelial cells. Intercellular communication between lens cells appears to be crucial for normal lens homeostasis. However, investigations on the maintenance of direct ion and metabolite exchange via gap junctions and its quantified dependency of cytoskeletal microtubules have not been available under conditions leading to bundling of microtubules. Thus, metabolic coupling of neighboring lens epithelial cells was quantified following microinjections of neurobiotin into single cells under various conditions. In controls, intensive gap-junction-mediated intercellular communication could be documented by dye-spreading of microinjected neurobiotin. In contrast, taxol treatment for 1–3 days impaired, but did not completely block gap-junction-mediated intercellular communication. After depletion of taxol, a complete recovery of intercellular communication was achieved. In addition, confocal laser scanning microscopy and rapid-freeze deep-etch electron microscopy revealed a displacement of actin-filaments from the perinuclear cytoplasm, accompanied by an abnormal aggregation of microtubules after taxol treatment, including impeded translocation of connexin 43 from the cytoplasm into the plasma membrane. Incubation of cells with nocodazole destroyed the microtubule network, accompanied by a clear reduction of plasma-membrane-integrated connexin 43 and significant impairment of dye spreading. Thus, in lens epithelial cells intercellular communication at gap junctions made by connexin 43 depends on the integrity of the microtubule network through the translocation of connexins to the plasma membrane.     

Cellular Fluid Mechanics and Mechanotransduction

Mechanotransduction, the transformation of an applied mechanical force into a cellular biomolecular response, is briefly reviewed focusing on fluid shear stress and endothelial cells. Particular emphasis is placed on recent studies of the surface proteoglycan layer (glycocalyx) as a primary sensor of fluid shear stress that can transmit force to apical structures such as the plasma membrane or the actin cortical web where transduction can take place or to more remote regions of the cell such as intercellular junctions and basal adhesion plaques where transduction can also occur. All of these possibilities are reviewed from an integrated perspective.     

Alzheimer's disease: A new evidence for common epitopes between microtubule associated protein Tau and paired helical filaments (PHF): Demonstration at the electron microscope level by a double immunogold labelling

Summary Paired helical filaments (PHF) are neuronal landmarks of Alzheimer's disease. These pathological filaments are antigenically related to proteins present in the normal cytoskeleton, particularly to microtubule associated protein Tau. The evidence for these common epitopes was studied on sections of cortex from Alzheimer brains after Araldite embedding. Two rabbit immunsera were used: one was raised against PHF isolated from Alzheimer cortex; the other against Tau proteins extracted from bovine cortex. The comparison of adjacent semi-thin sections alternatively treated with anti-PHF and anti-Tau immunesera reveals that both stained degenerating neurofibrils in pyramidal perikaria and in neurites surrounding senile plaques. On ultra-thin sections, double immunogold labelling of PHF was obtained. These results are in accordance with the hypothesis that Tau proteins are major antigenic components of PHF.