细胞外基质凝胶支架混合人脐静脉来源内皮细胞构建组织工程化的内皮细胞片层

背景：由于血管内皮细胞是形成血管网络的主要功能细胞，因此通过在支架复合体中复合血管内皮细胞的方式促进再造心肌的血管化是目前研究的热点。 目的：以细胞外基质凝胶为支架混合人脐静脉内皮细胞，观察内皮细胞在细胞外基质凝胶中的生长过程及发育情况。 设计、时间及地点：观察实验，于2006-11/2008-01在解放军军事医学科学院基础医学研究所组织工程研究室实验室完成。 材料：取健康剖腹产孕妇的脐带分离培养人脐静脉内皮细胞。取新生SD大鼠鼠尾制备Ⅰ型液态胶原溶液。 方法：将2.4 g/L液态Ⅰ型胶原与2×M199培养基等比例混合，用0.1 mol/L NaOH 调为中性，加入分离培养3 d的人脐静脉内皮细胞，使其终浓度为4×109 L-1，再加入200 g/L的Matrigel基质凝胶。最后加入有静态拉伸力的组织工程化片层所需的模具中，构建组织工程化内皮细胞片层。 主要观察指标：体外培养20 d后光学显微镜、常规苏木精-伊红染色、免疫组织化学染色和透射电镜观察。 结果：显微镜下观察可见内皮细胞在细胞外基质凝胶中形成明显的管腔样、分支样、类似血管样结构。通过组织学和免疫组织化学的检测，证实其确为来源于人脐静脉中分离培养的内皮细胞。电镜观察可见随着时间的延长，在组织工程化内皮细胞片层中，内皮细胞可形成管腔，细胞间有紧密连接。 结论：以细胞外基质凝胶为支架混合人脐静脉内皮细胞构建的组织工程化内皮细胞片层中具有类似血管样结构。     

内皮祖细胞在膀胱细胞外基质上的生长及分化

背景：传统的组织工程膀胱支架材料本身无血管结构,植入体内后面临血管化不足的问题。 目的：观察内皮祖细胞与膀胱细胞外基质的生物相容性。 方法：分离培养兔内皮祖细胞,种植于兔膀胱细胞外基质上与其复合培养。将复合生长材料植入兔背部皮下检测其组织相容性。 结果与结论：兔内皮祖细胞能够在膀胱细胞外基质表面正常黏附、生长、增殖,细胞形态良好。内皮祖细胞-膀胱细胞外基质植入兔体内后1周时,可见材料周围炎症反应明显,粘连严重,出血较多;苏木精-伊红染色可见组织中较多炎性细胞浸润,胶原及弹性纤维排列松散。植入后8周时可见材料已降解成碎细丝状,与周围组织融合生长在一起,但质地略脆,易出血;苏木精-伊红染色可见组织中已无明显炎症细胞浸润反应,胶原及弹性纤维排列紧密并且有新生血管长入其中。结果表明内皮祖细胞与膀胱细胞外基质具有良好的相容性,复合培养物与体内组织具有良好的相容性。     

血管内支架置入在缺血性脑血管病的应用及影像学评估

背景：目前对于缺血性脑血管病影像检测手段广泛应用于临床,并且有无创、快速、简便等优点。目的：观察血管内支架置入后影像形态学变化,探讨血管内支架在缺血性脑血管病中应用安全性及不同材料的应用价值。方法：以 “血管内支架,支架材料,再狭窄,影像”为中文关键词;以：“vascular stent,scaffold,restenosis,endothelialization,imaging evaluation”为英文关键词,采用计算机检索1993-01/2009-10相关文章。纳入与不同材料支架置入后颅内靶血管再狭窄的影像形态学评估有关的文章;排除重复研究或Meta分析类文章。以30篇文献为主重点进行讨论不同材料血管内支架在缺血性脑血管病中应用安全性及影像学评价。结果与结论：按置入支架材料不同类型的患者采用影像学技术,观察随访期间靶血管再狭窄形态学变化。可以明确有无血管狭窄、明确病因,并有利于对血管内支架材料的选择进行风险-效益评估。提示：影像判断不同材料支架置入后形态学差异有较高的敏感性、特异性和准确性,有助于选择支架材料及对术中病变部位栓子脱落危险性的评估。     

平滑肌祖细胞在新型细胞外基质支架上的生长特性*☆

背景：血管壁细胞包括内皮细胞和平滑肌细胞,平滑肌细胞合成分泌的胶原蛋白、弹力蛋白和蛋白聚糖等细胞外基质是提供血管塑形及张力的最主要成分,平滑肌细胞在细胞外基质支架上生长可能更接近体内生长环境特性。 目的：观察鼠骨髓来源平滑肌祖细胞在毯状细胞外基质支架上的生长特性。 设计、时间及地点：细胞水平对比观察实验,于2007-07/2008-07在江苏大学临床检验实验室完成。 材料：将纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝coagulation作用下,构建毯状细胞外基质支架。 方法：实验组诱导培养骨髓来源的第2代平滑肌祖细胞种植在毯状细胞外基质支架表面。对照组将骨髓来源的第2代平滑肌祖细胞接种到无菌圆盖玻片上。 主要观察指标：于培养1,4,7,10,14,21 d用电镜观察支架表面结构及平滑肌祖细胞生长情况;Western Blotting法、Real-Time PCR法分别检测α-SMA蛋白及mRNA表达变化。 结果：实验组平滑肌祖细胞贴壁率、增殖数明显高于对照组(P < 0.01);电镜显示细胞在支架表面形成较平整的平面,如典型“平滑肌”形状;实验组平滑肌祖细胞为多层、三维立体、更接近天然血管的结构;实验组α-SMA蛋白及基因表达明显高于对照组(P < 0.05)。 结论：细胞外基质支架能显著促进平滑肌祖细胞黏附、增殖和分化,可作为一种新颖的合成人造血管的生物组织工程支架。     

血管细胞外基质在修复输尿管缺损中释放生长因子增强血管化的研究

背景：血管细胞外基质作为来源于血管组织的天然生物支架材料,在植入宿主体内后能迅速刺激血管生成,加速组织工程化组织血管化,但其确切机制还不清楚。 目的：探讨血管细胞外基质在修复输尿管缺损中释放血管内皮生长因子增强血管化的作用。 设计、时间及地点：细胞学体外观察,于2009-04/2009-08在武汉大学第一临床医学院生物医学工程实验室完成 材料：成年新西兰兔23只（由武汉大学医学院实验动物中心提供）,其中5只供体兔的腹主动脉经手术取出制备血管细胞外基质：腹主动脉置于PBS和叠氮钠的混合溶液中浸泡搅拌过夜;继续用 EDTA溶液、胰蛋白酶浸泡搅拌;甲醛和戊二醛对组织进行交联保护;用NaCl和 DNA酶DNase溶液搅拌;最后用脱氧胆酸钠和叠氮钠溶液浸泡和搅拌。 方法：用ELISA法体外检测血管细胞外基质在PBS孵育液中血管内皮生长因子的含量,应用MTT法体外检测血管细胞外基质对内皮细胞的增殖作用;应用家兔同种异体血管细胞外基质体内修复输尿管缺损,术后不同时间检测兔输尿管缺损修复情况及血管再生情况。 主要观察指标：血管细胞外基质内血管内皮细胞生长因子含量的检测,血管细胞外基质对内皮细胞的增殖作用及对输尿管缺损修复的影响。 结果：体外实验显示血管细胞外基质在PBS孵育液中血管内皮生长因子含量峰值为(124.10±1.42)ng/L,对内皮细胞有促进增殖作用。体内移植修复试验显示术后2周移植物可见新生毛细血管,腔内面边缘部见移行上皮细胞爬行,管壁有平滑肌细胞长入;第8周移植物内大量毛细血管生成,且管腔增大,移行上皮细胞层增厚,平滑肌规则排列成束;术后16周血管细胞外基质修复区已接近正常输尿管组织结构。 结论：血管细胞外基质作为生物支架材料,在降解的同时能释放血管内皮生长因子刺激血管再生,有可能成为一种理想的输尿管重建材料。 关键词：血管细胞外基质;血管内皮生长因子;输尿管;组织工程     

RNA干扰内皮细胞分泌白细胞介素6影响平滑肌细胞的迁移*

背景：血管支架置入后靶血管部位易发生炎症反应。 目的：利用siRNA技术抑制内皮细胞白细胞介素6的生成,观察其对平滑肌细胞迁移的影响。 方法：采用RT-PCR测定脂多糖刺激EA.HY926细胞表达白细胞介素6 mRNA的时间梯度与浓度梯度,针对白细胞介素6构建短发卡状siRNA真核表达载体pGensil-1.1-白细胞介素6,通过lipofectamine 2000转染EA.HY926,抑制其白细胞介素6的产生。 结果与结论：pGensil-1.1-白细胞介素6转染EA.HY926细胞后,脂多糖刺激下EA.HY926细胞表达的白细胞介素6 mRNA及蛋白明显减少。共培养模型中,转染pGensil-1.1-白细胞介素6的EA.HY926细胞作用下,人脐静脉平滑肌细胞表达的基质金属蛋白酶9 mRNA及蛋白明显降低,结晶紫染色显示人脐静脉平滑肌细胞迁移数量减少。说明siRNA技术可抑制内皮细胞白细胞介素6的生成,并通过降低平滑肌细胞基质金属蛋白酶9的表达减弱平滑肌细胞的迁移能力。     

In vitro effects of hyperlipoproteinemic sera on human endothelium: Inhibition of endothelial cell migration by familial hypercholesterolemic sera

Since hyperlipoproteinemia is associated with an increased risk of atherosclerosis we have evaluated the effects of sera of different hyperlipoproteinemic clinical patterns on human endothelial cells in vitro. Cultured human umbilical vein endothelial cells were treated with sera from 2 patients homozygous for familial hypercholesterolemia and 4 patients heterozygous for that disorder. Familial hypercholesterolemic sera inhibited endothelial cell migration by 50% during a 72 hour incubation (p <0.0001) compared to normal pooled human serum or single donor AB serum when measured by an agarose gel technique. The inhibition of migration was not observed when cells were treated with familial combined hyperlipidemic sera (4 patients) or familial hypertriglyceridemic sera (5 patients). Endothelial cell detachment in vitro was not induced by any of the classical patterns of hyperlipoproteinemic sera tested. The development of atherosclerosis in familial hypercholesterolemia may be in part related to an impairment of endothelial repair.     

Platelet interaction with the extracellular matrix produced by cultured endothelial cells: A model to study the thrombogenicity of isolated subendothelial basal lamina

Cultured bovine endothelial cells produce an extensive underlying extracellular matrix (ECM) which closely resemble the vascular subendothelial basal lamina in its organization and chemical composition. This naturally produced ECM was used to study the interaction of platelets with the subendothelium when exposed or covered with vascular endothelial cells. Incubation of platelet rich plasma with the ECM induced a rapid and massive platelet adherence, aggregation, thromboxane formation and release reaction. These were demonstrated using phase and scanning electron microscopy, Indium-111 or (¹⁴C)-serotonin labelled platelets, and a radioimmunoassay for thromboxane B₂. In contrast to the ECM no platelet activation was induced either by uncoated plastic dishes or ECM covered with a confluent endothelial cell monolayer. Aspirinized platelets failed to undergo aggregation and degranulation, when incubated with the ECM. Culture dishes coated with characteristic constituents of the basal lamina such as collagen type IV and type V or fibronectin induced a much lower platelet reactivity as compared with ECM coated dishes. Digestion of ECM components (collagen, fibronectin, hyaluronic acid, and chondroitin sulphate) by specific enzymes was not associated with a substantial decrease in its platelet reactivity. Furthermore, exposure of ECM to sodium dodecyl sulphate or sodium periodate, or freezing and thawing did not decrease its biological activity. In contrast, platelet activation was completely abolished following heat denaturation or glutaraldehyde fixation of the ECM. The availability of a naturally produced ECM provides an appropriate model to study the interaction of platelets with the subendothelium in a controlled system which is isolated from other components of the vessel wall.     

Neuroform EZ支架置入对症状性大脑中动脉重度狭窄患者脑血流灌注、血管内皮功能、认知功能及神经功能缺损的干预效果

目的 探讨Neuroform EZ支架置入辅助治疗症状性大脑中动脉重度狭窄的临床效果。方法 回顾性分析2016年10月至2019年10月该院症状性大脑中动脉重度狭窄患者85例临床资料,按治疗方案不同分为观察组（43例）、对照组（42例）。对照组予以单纯药物治疗,观察组在药物治疗基础上联合Neuroform EZ支架置入术。比较两组术后1年疗效、不良事件发生情况;比较两组及术前、术后6个月、术后1年认知功能评分(MMSE)、神经功能缺损程度评分(NIHSS)、脑血流灌注指标［脑血流量(CBF)、脑血容量(CBV)］、血管内皮功能指标［一氧化氮(NO)、内皮素(ET)］。结果 观察组术后1年总有效率为95.35%,高于对照组78.57%,差异有统计学意义(P<0.05)。两组术后6个月、1年MMSE评分及CBF、CBV、NO水平较术前提高,且观察组高于对照组,差异有统计学意义(P<0.05)。NIHSS评分及ET水平较术前降低,且观察组低于对照组,差异有统计学意义(P<0.05)。观察组不良事件发生率为13.95%,低于对照组33.33%,差异有统计学意义(P<0.05)。结论 Neuroform EZ支架置入辅助治疗症状性大脑中动脉重度狭窄可促进认知与神经功能改善,且在改善脑血流灌注、血管内皮功能、减少不良事件方面具有积极作用。     

胶质细胞诱导内皮细胞系血脑屏障特点的实验研究

目的探索胶质细胞在血脑屏障形成中的重要意义,建立一套可靠、简便的血脑屏障体外研究方法。方法采用内皮细胞系ECV304与星形胶质细胞体外接触共培养的方法,探索星形胶质细胞对内皮细胞系多种血脑屏障特点的诱导作用。结果星形胶质细胞可以诱导内皮细胞系形成紧密连接并产生较高的跨内皮阻抗(transendothelial electricalresistance, TER),同时,内皮细胞系的血脑屏障特征酶γ-谷氨酰转肽酶(γ-glutamyl transpeptidase, γ-GT)与碱性磷酸酶(alkaline phosphatase , ALP)活性明显增强。结论星形胶质细胞可以诱导ECV304细胞产生紧密连接等血脑屏障特点,ECV304细胞与星形胶质细胞的体外共培养可以作为研究血脑屏障结构与功能的一种可靠而简便的体外实验方法。     

Induction of blood-brain barrier properties in cultured brain capillary endothelial cells: comparison between primary glial cells and C6 cell line

The communication between glial cells and brain capillary endothelial cells is crucial for a well-differentiated blood-brain barrier (BBB). It has been suggested that in vitro primary glial cells (GCs) be replaced by the glial C6 cell line to standardise the model further. This study compares directly the structural and functional differentiation of bovine brain capillary endothelial cells (BBCECs) induced by co-culture with rat primary GCs or C6 cells, for the first time. Trans-endothelial electrical resistance (TEER) measurements showed that under no condition were C6 cells able to reproduce TEER values as high as in the presence of GCs. At the same time, permeability of the BBCECs to both radioactive sucrose and FITC-inulin was 2.5-fold higher when cells were co-cultured with C6 than with GCs. Furthermore, immunocytochemistry studies showed different cell morphology and less developed tight junction pattern of BBCECs co-cultured with C6 toward GCs. Additionally, studies on P-glycoprotein (P-gp) showed much lower P-gp presence and activity in BBCECs co-cultured with C6 than GCs. Both VEGF mRNA expression and protein content were dramatically increased when compared with GCs, suggesting that VEGF could be one of the factors responsible for higher permeability of BBB. Our results clearly indicate that, in the presence of the glial C6 cell line, BBCECs did not differentiate as well as in the co-culture with primary GCs at both structural and functional levels.     

降纤酶对大鼠局灶性脑缺血血管内皮细胞超微结构及内皮细胞凋亡的影响

目的 探讨降纤酶对缺血性脑水肿病理过程中血脑屏障（BBB）内皮细胞的保护作用以及其对内皮细胞凋亡的影响。方法 选用Wistar雄性大鼠42只，体重250－300g，鼠龄3－4个月，随机分成降纤酶组、盐水对照组和假手术组，参考Longa等方法建立大鼠大脑缺血动物模型，彩和电观察大鼠BBB的超微结构和TUNEL试剂盒检测内皮细胞的凋亡。结果 通过电镜观察发现降纤酶组的毛细血管内皮细胞膜完整，水肿较轻，其损伤程度明显较对照组轻。降纤酶组在缺血6h和缺血24h随缺血时间延长缺血中心区凋亡细胞数量减少，而缺血半影区凋亡细胞数量增加，其凋讯细胞数量明显少于盐水对照组同一时期值。结论 降纤酶对脑缺血的血管内皮细胞及BBB具有明显的保护作用。     

sEHi对颈动脉狭窄患者晚期内皮祖细胞功能的影响

目的研究不同浓度可溶性环氧化物水解酶抑制剂(soluble epoxide hydrolase inhibitor,s EHi)AUDA对颈动脉狭窄(carotid stenosis,CS)患者外周血来源的晚期内皮祖细胞(late endothelial progenitor cell,late EPC)的影响。方法入选研究对象60例,分成颈动脉狭窄组(n=35)和对照组(n=25)。密度梯度离心法,从外周血获取单个核细胞培养至21 d后鉴定内皮祖细胞;以不同浓度AUDA(0,0.1,1,10μmol/L)和晚期EPC共培养24 h,分别采用MTT法,黏附能力测定实验和Transwell小室来观察其增殖,黏附,迁移能力。同时采用Western blot法观察AUDA处理后其VEGF的表达。结果体外培养21 d时,细胞呈典型长梭形,21 d时,呈铺路石样,并可摄取FITCUEA-I和Dil-ac LDL。与对照组相比,CS患者late EPC的增殖,黏附和迁移能力均显著下降(P0.05);与处理前(0umol/L)相比,AUDA呈剂量依赖性地增强CS患者late EPC增殖,黏附和迁移能力并促进CS患者late EPC表达VEGF。结论 s EHi具有促进late EPC增殖,黏附和迁移等功能的作用,其有望成为一类治疗CS的新型药物。     

Effect of different challenge doses after repeated citalopram treatment on extracellular serotonin level in the medial prefrontal cortex: in vivo microdialysis study

Aims: In order to elucidate the relevance between the delayed onset of clinical efficacy of selective serotonin re‐uptake inhibitors (SSRI) and extracellular 5‐HT levels in the medial prefrontal cortex, the present study compared the ability of low‐dose (3 mg/kg) and high‐dose (30 mg/kg) citalopram to increase extracellular 5‐HT levels in the medial prefrontal cortex following repeated citalopram treatment using in vivo microdialysis. Methods: An SSRI, citalopram, was given 10 mg/kg, s.c. twice daily for 6 days and once on the seventh day in rats. On the eighth day, rats received a single injection of citalopram (3 or 30 mg/kg s.c.), and extracellular 5‐HT levels were assessed in the medial prefrontal cortex of rats using in vivo brain microdialysis. Results: There was no significant difference in basal extracellular 5‐HT levels between the repeated citalopram group and the repeated saline group. The low‐challenge dose of citalopram (3 mg/kg) produced significantly greater increases (170–200% at each time point) in the repeated citalopram group than in the repeated saline group (150%). The high‐challenge dose of citalopram (30 mg/kg), however, increased extracellular 5‐HT levels by 200–250% of basal levels in the repeated citalopram group, which was similar to the increases in the repeated saline group. Conclusions: Repeated SSRI treatment enhances the effect of low‐dose SSRI on extracellular 5‐HT levels but not that of high‐dose SSRI.     

Growth related changes in sugar determinants on the surface of C6 glioma cells in culture: A cytochemical lectin-binding study

The cell surface sugar determinants (CSSD) were examined in C6 glioma cells in cultures at different conditions of growth by peroxidase conjugates of the lectins: peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Helix pomatia agglutinin (HPA), wheat germ agglutinin (WGA), lentil agglutinin (LCA), laburnum bork agglutinin (LABA), and lotus agglutinin (TPA). It was found that the cells bound more intensively WGA, LCA, and RCA compared to PNA, HPA; the weakest staining was provided by LABA and TPA. Binding intensity for PNA significantly increased after pretreatment of the cells with neuraminidase. This indicates that a part of the β-D-galactose residues on the surface membrane of C6 glioma cells is covered by sialic acid. The process of sialization was increased during the culturing of C6 glioma cells. Addition of cis-DDP or dBcAMP to cultures growing in medium with 10% of CS increased the number of Gal residues which are not covered by sialic acid. The expression of β-D-galactose (Gal), N-acetyl-Dgalactosamine (NAcDGal), and fucose (Fuc) residues appeared to be most responsive to changes in growth conditions and degree of cell differentiation. The expressions of N-acetyl-D-glucosamine (NAcDGIc) and mannose (Man) residues were high and seems did not depend on changing of the conditions of culturing. In C6 glioma cells cultures in which the rate of cell division, formation of the cell processes, and adhesiveness of the cells to the substratum were reduced by growing cells in MEM +, expression of β-Gal, NAcDGa1, and Fuc was considerably reduced. The decrease of expression of R-Gal, NAcDGaI, and Fuc on the surface of cell membrane was more pronounced in MEM + with 1% of CS than in MEM + with 10% of CS. In DbcAMP and cis-DDP treated cultures, grown in medium with 1% serum, in which cell division was inhibited without obvious changes in cell adhesiveness to the substratum, binding of PNA and HPA was increased due to higher expression of β-Gal and NAcDGaI. From these observations it was concluded that the pattern of expression of sugar residues on the cell surface varies according to the biological state of the cells and are easily affected by tissue culture conditions. © 1995 Wiley-Liss, Inc.