转基因神经干细胞移植在缓解脑缺血性神经血管损害中的作用

目的 评价转染血管内皮生长因子(VEGF)基因的神经干细胞脑内移植后在宿主缺血区的神经血管保护作用。方法 建立大鼠暂时性大脑中动脉梗死模型(tMCAO)并将其随机分成：(1)对照组；(2)细胞悬液磷酸盐缓冲液(PBS)移植组；(3)神经干细胞移植组；(4)转基因神经干细胞移植组，每组10例。立体定向法将BrdU标记的转基因神经干细胞移植到tMCAO大鼠的纹状体缺血半暗区。移植后2～12周进行神经损害严重程度评分(NSS)并与其他3组比较。移植后12周，免疫荧光染色观察转基因神经干细胞在脑内的分化、迁徙情况；免疫组织化学染色观察各组移植区毛细血管结构，并计每例动物以移植点为中心的一个100倍视野下内皮细胞数。结果移植后2～12周(4)组NSS评分均数分别为5，8、5．0、4．6、4．0、4．0、3．8，均低于其他3组。其中第8周显著低于(1)、(2)组(P=0．008)，第12周显著低于(1)、(2)、(3)组(P=0．000)。移植后12周，转基因神经干细胞在宿主脑内存活、迁徙，部分分化成神经元。转基因神经干细胞移植组移植区毛细血管结构相对完整。血管内皮细胞数显著多于其他3组。结论 转染VEGF基因的神经干细胞移植后对宿主局部血管和神经结构具有保护作用。     

骨髓间充质干细胞通过血管内皮细胞生长因子受体2调控神经干细胞的增殖与分化

目的 观察血管内皮细胞生长因子受体2(VEGFR2)在骨髓间充质干细胞(MSCs)调控神经干细胞(NSCs)增殖与分化中的作用.方法 细胞共培养分为5组:NSCs+MSCs、NSCs+MSCs+SU5416(VEGFR2阻断剂)、NSCs+HMVECs、NSCs+HMVECs+SU5416、NSCs+3T3.共培养6 h后,采用逆转录.聚合酶链反应检测各组NSCs标志物nestin、成熟星形胶质细胞标志物GFAP及Notch信号分子Notchl和hesl的表达.结果 在MSCs及HMVECs共培养组中nestin的表达较高,当VEGFR2信号通路被阻断后,nestin的表达明显下降,而成熟神经细胞GFAP的表达却明显增加.同时,在MSCs及HMVECs共培养组中,Notchl、hesl的表达也明显高于3T3对照组及VEGFR2信号通路阻断剂组.结论 VEGFR2在MSCs调控NSCs增殖与分化过程中可能起重要作用.     

Verocytotoxin-induced apoptosis of human microvascular endothelial cells

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.     

神经干细胞移植改善脑缺血大鼠的神经功能研究

目的 探讨大鼠神经干细胞移植改善脑缺血所致神经功能损伤的可行性。方法 制作大鼠大脑中动脉夹闭致脑缺血模型，记录并比较损伤和移植干细胞前后大鼠的神经功能。6周后杀死大鼠，研究干细胞在体内分化和迁移的情况。结果 接受干细胞移植大鼠神经功能的改善显著好于未移植干细胞的对照组。免疫组织化学方法证实移植后干细胞在脑内分化成胶质细胞和少量的神经细胞，并向损伤区域迁移。结论 神经干细胞体内外均具有多向分化潜能。神经干细胞移植能够有效改善脑缺血大鼠的神经功能。     

Cytomegalovirus-mediated modulation of adhesion molecule expression by human arterial and microvascular endothelial cells

BACKGROUND: Cytomegalovirus (CMV), a betaherpesvirus associated with allograft rejection, infects the endothelium, the cellular interface between allograft tissue and the host immune system. Because of recent appreciation of the phenotypic diversity of endothelial cells (EC) from different vascular compartments, controversy now exists on the universality of CMV-mediated adhesion molecule induction previously described on umbilical vein EC. Therefore, we herein extend these previous studies to arterial and microvascular EC, which represent sites of vascular rejection. METHODS: Human coronary artery, aortic, umbilical artery, and microvascular EC were mock or CMV infected and/or treated with tumor necrosis factor-a before flow cytometric and immunohistochemical analysis. RESULTS: CMV directly enhanced intercellular adhesion molecule-1 on all EC isolates but did not induce E-selectin or vascular cell adhesion molecule-1. Furthermore, CMV-infected EC were refractory to tumor necrosis factor-alpha-mediated induction of these molecules. CONCLUSION: CMV-induced modulations of adhesion molecule expression, which may affect allograft immunogenicity, seem common to all EC regardless of vascular origin.     

Survival of transplanted neural progenitor cells enhanced by brain irradiation

OBJECT: Authors of previous studies have reported that adult transplanted neural progenitor cells (NPCs) are suitable for brain cell replacement or gene delivery. In this study, the authors evaluated survival and integration of adult rat-derived NPCs after transplantation and explored the potential impact on transplant survival of various mechanical and biological factors of clinical importance. METHODS: Adult female Fischer 344 rats were used both as a source and recipient of transplanted NPCs. Both 9L and RG2 rat glioma cells were used to generate in vivo brain tumor models. On the 5th day after tumor implantation, NPCs expressing green fluorescent protein (GFP) were administered either intravenously (3.5 x 10(7) cells) or by stereotactic injection (1 x 10(4)-1 x 10(6) cells) into normal or tumor-bearing brain. The authors evaluated the effect of delivery method (sharp compared with blunt needles, normal compared with zero-volume needles, phosphate-buffered saline compared with medium as vehicle), delivery sites (intravenous compared with intratumoral compared with intraparenchymal), and pretreatment with an immunosuppressive agent (cyclosporin) or brain irradiation (20-40 Gy) on survival and integration of transplanted NPCs. RESULTS: Very few cells survived when less than 10(5) cells were transplanted. When 10(5) cells or more were transplanted, only previously administered brain irradiation significantly affected survival and integration of NPCs. Although GFP-containing NPCs could be readily detected 1 day after injection, few cells survived 4 days to 1 week unless preceded by whole-brain radiation (20 or 40 Gy in a single fraction), which increased the number of GFP-containing NPCs within the tissue more than fivefold. CONCLUSIONS: The authors' findings indicate that most NPCs, including those from a syngeneic autologous source, do not survive at the site of implantation, but that brain irradiation can facilitate subsequent survival in both normal and tumor-bearing brain. An understanding of the mechanisms of this effect could lead to improved survival and clinical utility of transplanted NPCs.     

Cyclosporine toxicity on cultured rat microvascular endothelial cells

The present study was undertaken to study the mechanism of cyclosporine-induced nephrotoxicity and hypertension. Cultured rat microvascular endothelial cells were exposed to cyclosporine for up to six days at one of the following concentrations: 10, 50, 250, and 1000 ng/ml of culture medium. Cyclosporine inhibited endothelial cell replication in a dose-dependent manner; at higher concentrations (250 and 1000 ng/ml), cell replication decreased by as much as 70 to 90% of controls at four and six days post-treatment, with no evidence of increased cell death. Drug-treated endothelial cells revealed abnormal morphological changes such as formation of cytoplasmic vesicles and nucleolar changes. Prostacyclin release by endothelial cells was increased by about threefold with the addition of cyclosporine (P less than 0.01). Indomethacin significantly decreased prostacyclin release by endothelial cells in the presence or absence of cyclosporine (P less than 0.01). Our data suggest that cyclosporine-induced nephrotoxicity may be mediated, at least partly, by the inhibitory influence of cyclosporine on the regenerative response of microvascular endothelial cells to injury, and the resultant alterations in prostacyclin production by these cells.     

转染血管内皮生长因子基因神经干细胞移植对大鼠脑缺血损伤的修复作用

目的 探讨转染血管内皮生长因子（VEGF）基因神经干细胞移植对大鼠脑缺血损伤的修复作用。方法 建立大鼠大脑中动脉梗塞（tMCAO）模型，将tMCAO模型随机分为对照组、注射磷酸盐缓冲液（PBS液）的PBS液对照组、接受神经干细胞（NSCs）移植的NSCs组、接受转染VEGF基因的NSCs移植的NSCs-VEGF组，采用立体定向法将相应移植物注射到tMCAO模型的右侧纹状体缺血半暗区。各组移植后2、4、6、8、10、12周进行神经功能损害程度（NSS）评分；移植后7d，采用免疫荧光染色法观察NSCs-VEGF组移植细胞的VEGF基因表达情况；12周时，采用免疫荧光染色法追踪NSCs-VEGF组移植细胞向神经元方向分化情况，并同时行各组移植区周围血管内皮细胞半定量计数。结果 移植后2～12周，NSCs-VEG组的NSS评分均低于其它3组，第12周时的NSS评分与其它3组比较，差异有统计学意义（P〈0．01）；移植后7d，NSCs-VEGF组的移植细胞从移植点向周围迁徙，部分表达VEGF基因；移植后12周，NSCs-VEGF组部分移植细胞分化成神经元，其移植区血管内皮细胞数显著高于其它3组（P〈0．05）。结论 转染VEGF基因的神经干细胞移植对脑缺血所致的损伤具有一定的修复作用。     

Apoptosis of endothelial cells in vessels affected by cerebral vasospasm

BACKGROUND: Cerebral vasospasm after subarachnoid hemorrhage is a prolonged contraction that leads to cerebral ischemia or infarction. Morphological studies of cerebral arteries during vasospasm have shown extensive necrosis of smooth-muscle cells and desquamation and dystrophy of endothelial cells. The mechanism of cellular death is unknown. METHODS: We report an observation of apoptotic changes in the cerebral arteries of a patient who died after suffering severe cerebral vasospasm caused by aneurysmal rupture. Subarachnoid hemorrhage and cerebral vasospasm were confirmed by computed tomography scanning and angiogram. Histological and immunohistological examinations for apoptosis were performed in cerebral arteries. For control, the arteries from another patient, who died of trauma without head injury, were used. RESULTS: Corrugation of the internal elastic lamina and increased amounts of connective tissue was demonstrated by light microscopy. Apoptotic changes, characterized by condensation of chromatin of the nucleus and detachment from the basal membrane, were found on transmission electron microscopy in endothelial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling reaction revealed positive staining of the nuclei of the endothelial cells. CONCLUSIONS: This study demonstrates that apoptosis occurred in the cerebral arteries in a patient who died of cerebral vasospasm. The possible role of apoptosis in cerebral vasospasm is discussed.     

Steen solution protects pulmonary microvascular endothelial cells and preserves endothelial barrier after lipopolysaccharide-induced injury

ObjectivesAcute respiratory distress syndrome represents the devastating result of acute lung injury, with high mortality. Limited methods are available for rehabilitation of lungs affected by acute respiratory distress syndrome. Our laboratory has demonstrated rehabilitation of sepsis-injured lungs via normothermic ex vivo and in vivo perfusion with Steen solution (Steen). However, mechanisms responsible for the protective effects of Steen remain unclear. This study tests the hypothesis that Steen directly attenuates pulmonary endothelial barrier dysfunction and inflammation induced by lipopolysaccharide.MethodsPrimary pulmonary microvascular endothelial cells were exposed to lipopolysaccharide for 4 hours and then recovered for 8 hours in complete media (Media), Steen, or Steen followed by complete media (Steen/Media). Oxidative stress, chemokines, permeability, interendothelial junction proteins, and toll-like receptor 4-mediated pathways were assessed in pulmonary microvascular endothelial cells using standard methods.ResultsLipopolysaccharide treatment of pulmonary microvascular endothelial cells and recovery in Media significantly induced reactive oxygen species, lipid peroxidation, expression of chemokines (eg, chemokine [C-X-C motif] ligand 1 and C-C motif chemokine ligand 2) and cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule 1), permeability, neutrophil transmigration, p38 mitogen-activated protein kinase and nuclear factor kappa B signaling, and decreased expression of tight and adherens junction proteins (zonula occludens-1, zonula occludens-2, and vascular endothelial-cadherin). All of these inflammatory pathways were significantly attenuated after recovery of pulmonary microvascular endothelial cells in Steen or Steen/Media.ConclusionsSteen solution preserves pulmonary endothelial barrier function after lipopolysaccharide exposure by promoting an anti-inflammatory environment via attenuation of oxidative stress, toll-like receptor 4-mediated signaling, and conservation of interendothelial junctions. These protective mechanisms offer insight into the advancement of methods for in vivo lung perfusion with Steen for the treatment of severe acute respiratory distress syndrome.     

Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors

OBJECTIVE: Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another. METHODS: Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles. RESULTS: SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold). CONCLUSION: The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.     

Biological considerations of mesenchymal stem cells and endothelial progenitor cells

SUMMARY: Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) have been demonstrated as an attractive cell source for tissue engineering applications because of their ability to be isolated and expanded. Even though MSCs and EPCs constitute a powerful candidate cell type for regenerative medicine, more knowledge in terms of their biological properties is required before using these cells as a routinely applied therapy in the clinical setting. The nature of their mobilizing, migratory and homing signals and the mechanisms of differentiation and incorporation into the target tissues need to be clarified and further characterized. This paper examines the biological properties of these cells, the animal trials that have been performed so far and highlights their therapeutic potential in the treatment of musculoskeletal and cardiovascular diseases.     

转基因人骨髓间质干细胞在支架中增殖及矿化的研究

目的:观察携带骨形态发生蛋白2基因(BMP-2)的人骨髓间质干细胞(hMSCs)在生物衍生骨支架环境中的增殖及矿化.方法:利用BMP-2腺病毒载体转染hMSCs后种植生物衍生骨支架中,扫描电镜观察细胞生长增殖,能量谱仪测定钙质分泌,RT-PCR检测培养液上清中BMP-2表达.结果:转染后,hBMSC呈ALP、Ⅰ和Ⅱ型胶原阳性表达;细胞在支架中伸展良好,生长旺盛,并产生钙盐沉积;RT-PCR显示培养液上清中存在BMP-2基因表达.结论:转染BMP-2基因的hMSCs在生物衍生骨支架中能够立体生长、增殖,并继续表达目的基因,为基因修饰的组织工程骨修复骨缺损奠定了基础.     

大鼠神经干细胞与小鼠雪旺细胞混合培养的研究

外培目的观察小鼠雪旺细胞体养时对大鼠神经干细胞存活及分化的影响。方法获取Wistar鼠坐骨神经并采用组织块法分离和纯化雪旺细胞；体外分离新生乳鼠脑神经干细胞，将雪旺细胞和神经干细胞分别培养扩增后进行共培养。共分5组进行：实验组1（NSC悬浮＋SC悬浮＋DMEM／F12）；实验组2（NSC悬浮＋SC贴壁＋DMEM／F12）；试验对照组1（SC培养基＋NSC＋DMEM／F12）；试验对照组2（EGF／bFGF＋NSC＋DMEM／F12）；试验对照组3（NSC＋DMEM／F12）。倒置相差显微镜对各组培养细胞每天观察形态和计数，免疫组织化学检测混合培养细胞特异性标记物的表达：SC采用P0和S100，NS采用nestin标记，神经干细胞分化神经元分别采用GFAP、GalC、Tubulin-β染色。结果共培养组NF染色阳性神经元样细胞的百分率明显高干其他各组；实验组1克隆球直径明显高于其他各组，其平均直径为8μm；实验组神经元样细胞突起的长度比对照组的长，3周长度差为26．5-67．3μm。结论大鼠神经干细胞与小鼠雪旺细胞共培养使两者不仅能够共生，而且雪旺细胞能显著促进体神经干细胞分化为神经元样细胞；神经干细胞分化神经元突起增长并且有序排列成轴索样结构。     

Seeding of human microvascular endothelial cells onto polytetrafluoroethylene graft material

Microvascular endothelial cells from human omental samples were isolated and grown. Adhesion to polytetrafluoroethylene vascular graft material was then studied using scanning electron microscopy and adhesion of cells labelled with indium-111. Grafts coated with fibronectin and type I collagen were found to promote the best adhesion of cells at times up to 90 min. A coating of blood clot matrix was less effective but still resulted in a threefold increase in cell adhesion compared with controls.