葡萄糖降解产物对内皮细胞AQP1及eNOS表达影响研究

目的研究腹膜透析液毒性成份之一-葡萄糖降解产物（GDPs）对内皮细胞水孔蛋白1（AQP1）及eNOS表达影响。方法选用两种相关的GDPs-2-糠醛（Fur）及甲基乙二醛（MGly）,采用与传统腹膜透析液浓度相当的GDPs刺激内皮细胞系tEnd.124h（Fur 0.8 uM;MGly 35 uM）,以培养液DMEM作为对照组,研究GDPs对内皮细胞AQP1及eNOS表达影响。并进一步通过时间依赖性及浓度依赖性分析研究GDPs对AQP1 mRNA表达影响。使用RT-PCR检测AQP1及eNOS基因表达。使用Western blot检测AQP1蛋白表达。结果内皮细胞系tEnd.1经GDPs（Fur 0.8 uM;MGly 35 uM）刺激24h后,Fur及MGly显著上调内皮细胞eNOS mRNA表达（P〈0.05）。GDPs对AQP1 mRNA表达有降低的趋势但无统计学意义。时间依赖性及浓度依赖性研究同样提示GDPs对AQP1 mRNA表达有下降的趋势。蛋白质印迹分析（Western blot）检测有相似的结果。结论GDPs促进内皮细胞eNOS的表达但并不促进AQP1的表达,GDPs的这种作用可能参与长期腹膜透析过程中腹膜血管新生及超滤衰竭的发生。     

体外超声助溶兔股动脉血栓对纤维蛋白降解产物的影响

目的：探讨体外超声（ＥＴＵＳ）助溶血栓的机理。方法：我们对１８只新西兰兔双侧股动血栓模型进行研究,实验对象被随机分为三组;不治疗对照组;重组链激酶（ｒＳＫ）,３０,０００Ｕ／ｋｇ联用和不联用ＥＴＵＳ组,ＥＴＵＳ的频率和强度分别为０．８ＭＨｚ和１．２Ｗ／ｃｍ＾２,。治疗前和治疗后动脉采血测定血清纤维蛋白降解产物（ＦＤＰ）和血浆Ｄ二聚体（Ｄ－Ｄｉｍｅｒ）水平。结果：对照组、ｒＳＫ组和ｒＳＫ联用ＥＴＵＳ     

成型加工条件对聚(D,L-乳酸)及其复合材料力学性能的影响

背景加工条件和加工温度会使聚乳酸[Poly(lactic acid),PLA]降解,损害力学性能. 目的探讨成型加工条件对聚(D,L-乳酸)(PDLLA),二苯基甲烷二异氰酸酯(MDI)扩链PDLLA和MDI扩链PDLLA/羟基磷灰石(HA)复合材料力学性能的影响.设计分组对照研究.地点和方法在华南理工大学材料学院完成实验.利用MDI作为扩链剂对PDLLA和PDLLA/HA复合材料扩链,合成MDI扩链PDLLA和MDI扩链PDLLA/HA复合材料,采用自行设计的模压挤出设备对其进行加工.主要观察指标成型加工条件对PDLLA,MDI扩链PDLLA和MDI扩链PDLLA/HA复合材料力学性能的影响.结果在模压温度165℃,挤出温度100℃、口模长度10 mm的最佳成型工艺条件下,PDLLA和PDLLA/HA复合材料的弯曲强度分别为35.1MPa和31.2 MPa,弯曲模量分别为2 413.6 MPa和1 735.0 MPa;在模压温度100℃,挤出温度100℃、口模长度10mm的最佳成型工艺条件下,PDLLA/MDI和PDLLA/HA/MDI复合材料的弯曲强度分别为51.3 MPa和55.4 MPa,弯曲模量分别为1 830.9 MPa和2 068.5 MPa.结论成型加工条件对PDLLA,MDI扩链PDLLA和MDI扩链PDLLA/HA复合材料的力学性能影响显著,而且MDI扩链可显著提高PDLLA和PDLLA/HA复合材料的力学性能.     

D，L-聚乳酸基形状记忆聚合物的生物安全性和细胞相容性

背景：课题组前期实验研制了输卵管避孕器材料D, L-聚乳酸基形状记忆聚合物,依据国内《生物材料和医疗器材生物学评价技术要求》规定,植入体内的组织工程材料必须进行生物安全评价和细胞相容性实验。 目的：观察D, L-聚乳酸基形状记忆聚合物的生物安全性。 方法：①内毒素实验：在鲎试剂中分别加入聚合物浸提液、内毒素工作标准品溶液和细菌内毒素检查用水。②致敏实验：在昆明小鼠肩胛骨内侧分别注射聚合物浸提液＋弗氏完全佐剂＋生理盐水、弗氏完全佐剂＋生理盐水,通过皮内诱导、局部诱导和激发阶段,观察动物激发部位皮肤红斑和水肿反应程度。③急性毒性实验：分别在昆明小鼠腹腔注射100%,50%,25%聚合物浸提液及生理盐水。④细胞增殖MTT实验：直接法为将人脐静脉内皮细胞分别接种于聚合物膜、聚乳酸与玻璃片上;间接法为将人脐静脉内皮细胞分别接种于聚合物浸提液、丙烯酰胺溶液及1640培养液。 结果与结论：D, L-聚乳酸基形状记忆聚合物材料无细菌污染状况,符合生物安全标准,无致敏性及毒性,并且具有较好的细胞相容性。     

聚乳酸/海藻酸钠/壳聚糖复合材料的体外降解性能

背景：聚乳酸由于其疏水性以及在降解过程中的酸致效应,使其在应用中受到限制。通过静电组装技术在聚乳酸表面引入海藻酸钠/壳聚糖,可望克服上述缺点和不足。目的：观察聚乳酸/海藻酸钠/壳聚糖可降解复合材料的体外降解性能。设计、时间及地点：观察实验,于2007-09/2008-06在武汉理工大学生物中心实验室完成。材料：采用1,6-乙二胺对聚乳酸表面进行胺解反应,形成胺化层,在其表面引入带正电的自由氨基,由静电作用依次组装上聚阴离子海藻酸钠和聚阳离子壳聚糖,获得聚乳酸/海藻酸钠/壳聚糖多层复合材料。方法：将制备好的组装层数为5,10,15,20双层聚乳酸/海藻酸钠/壳聚糖复合材料置于37℃恒温的磷酸缓冲溶液中进行体外降解实验。主要观察指标：定期测定并记录不同组装层数复合材料的pH值变化、失重及相对分子质量变化,用扫描电镜观察材料降解的形貌变化。结果：聚乳酸/海藻酸钠/壳聚糖复合材料降解的pH值基本稳定在7.0左右;通过控制组装层数（5～15层）可有效调节材料降解过程中的pH值,pH值随层数的增加而增加。扫描电镜观察,复合材料降解7周后,材料已明显降解。结论：聚乳酸/海藻酸钠/壳聚糖复合材料具有良好的降解性能。     

纤维蛋白（原）降解产物对凝固法测定纤维蛋白原的影响

目的 探讨血浆纤维蛋白（原）降解产物（FDP）、D－二聚体（D－D）对凝固法（Clauss法）测定纤维蛋白原（Fib）的影响。方法 留取22例体检正常标本和86例FDP、D－D可能增高的患者标本，分别用PT－衍生法（PT－der法）和Clauss法测定Fib浓度，ELISA法测定其FDP、D－D含量。再将正常混合血浆和高FDP、D－D浓度的异常混合血浆以不同比例混合后同上法测其Fib、FDP、D－D的浓度。结果 PT-der法和Clauss法测定正常组血浆的Fib无显著性差异，P＞0．05，而对PDP、D－D增高的异常血浆，用两种方法测得的Fib有显著性差异，Clauss法结果低于PT－der法，两者差值与FDP、D－D的浓度呈正相关。结论 表明FDP、D－D会使Clauss法测定Fib的浓度偏低，其偏低的幅度与FDP、D－D的含量呈正相关。     

成型加工条件对聚（D，L-乳酸）及其复合材料力学性能的影响

背景：加工条件和加工温度会使聚乳酸[Poly(lactic acid)，PLA]降解，损害力学性能。目的：探讨成型加工条件对聚(D，L-乳酸)(PDLLA)，二苯基甲烷二异氰酸酯(MDI)扩链PDLLA和MDI扩链PDLLA／羟基磷灰石(HA)复合材料力学性能的影响。设计：分组对照研究。地点和方法：在华南理工大学材料学院完成实验。利用MDI作为扩链剂对PDLLA和PDLLHA复合材料扩链，合成MDI扩链PDLLA和MDI扩链PDLLA／HA复合材料，采用自行设计的模压挤出设备对其进行加工。主要观察指标：成型加工条件对PDLLA,MDI扩链PDLLA和MDI扩链PDLLA／HA复合材料力学性能的影响。结果：在模压温度165℃，挤出温度100℃、口模长度10mm的最佳成型工艺条件下，PDLLA和PDLLA／HA复合材料的弯曲强度分别为35．1MPa和31．2MPa，弯曲模量分别为2413．6MPa和1735．0MPa：在模压温度100℃，挤出温度100℃、口模长度10mm的最佳成型工艺条件下，PDLLA／MDI和PDLLA／HA／MDI复合材料的弯曲强度分别为51．3MPa和55．4MPa，弯曲模量分别为1830．9MPa和2068．5MPa。结论：成型加工条件对PDLLA，MDI扩链PDLLA和MDI扩链PDLLA／HA复合材料的力学性能影响显著，而且MDI扩链可显著提高PDLLA和PDLLA／HA复合材料的力学性能。     

聚乳酸降解材料的绿色化学合成

背景:聚乳酸具有良好的生物相容性和生物降解性,广泛应用于药物缓释、手术缝合线、组织工程支架及骨修复材料等生物医用领域.但其常规合成方法需使用溶剂,生产效率较低且成本较高.目的:对非溶剂的绿色化学方法-乳酸熔融缩聚/二异氰酸酯熔融扩链,合成聚乳酸降解材料的研究进展进行综述.方法:应用计算机检索SCI-Expanded数据库(1995-01/2010-06),以"Poly(lactic acid),diisocyanate"为检索词;应用计算机检索中国期刊网络出版总库(1999-01/2010-06),以"聚乳酸,异氰酸酯"为检索词.共收集130篇关于乳酸熔融缩聚/二异氰酸酯熔融扩链的文献,中文39篇,英文91篇.排除发表内容重复、实验结果较差的文献,共32篇文献符合标准被纳入.结果与结论:采用非溶剂的绿色化学方法-乳酸熔融缩聚/二异氰酸酯熔融扩链,通过改变异氰酸酯和预聚物的种类和比例,就可以制备具有不同相对分子质量和性能的可降解聚乳酸基聚氨酯材料,有望在生物医用领域和日常生活中取得实际的应用.     

聚乳酸降解材料的绿色化学合成

背景：聚乳酸具有良好的生物相容性和生物降解性,广泛应用于药物缓释、手术缝合线、组织工程支架及骨修复材料等生物医用领域。但其常规合成方法需使用溶剂,生产效率较低且成本较高。目的：对非溶剂的绿色化学方法-乳酸熔融缩聚/二异氰酸酯熔融扩链,合成聚乳酸降解材料的研究进展进行综述。方法：应用计算机检索SCI-Expanded数据库（1995-01/2010-06）,以＂Poly（lacticacid）,diisocyanate＂为检索词;应用计算机检索中国期刊网络出版总库（1999-01/2010-06）,以＂聚乳酸,异氰酸酯＂为检索词。共收集130篇关于乳酸熔融缩聚/二异氰酸酯熔融扩链的文献,中文39篇,英文91篇。排除发表内容重复、实验结果较差的文献,共32篇文献符合标准被纳入。结果与结论：采用非溶剂的绿色化学方法-乳酸熔融缩聚/二异氰酸酯熔融扩链,通过改变异氰酸酯和预聚物的种类和比例,就可以制备具有不同相对分子质量和性能的可降解聚乳酸基聚氨酯材料,有望在生物医用领域和日常生活中取得实际的应用。     

纤维蛋白（原）降解产物对用发色底物法测定抗凝血酶Ⅲ活性的影响

目的：探讨纤维蛋白（原）降解产物（FDP）、D－二聚体（D－D）对用发色底物法测定抗凝血酶Ⅲ活性（AT－Ⅲ：A)的影响。方法：留取31例体检正常和85例FDP、D－D可能增高的患标本，分别用发色底物法测定AT－Ⅲ：A，用免疫比浊法测定抗凝血酶Ⅲ抗原（AT－Ⅲ：Ag），用ELISA法测定其FDP、D－D含量。再将正常混合血浆与高FDP、D－D浓度的异常混合血浆、高D－D浓度的标准品以不同比例混合后，用上法分别测定其AT－Ⅲ：A及AT－Ⅲ：Ag、FDP、D－D的含量。结果：AT－Ⅲ：A除肝硬化组外其余各组与对照组比较均无显性差异，P＞0．05，而AT－Ⅲ：Ag含量各组均显低于对照组；AT－Ⅲ：A与AT－Ⅲ：Ag含量的比值均明显高于对照组，其比值增高的幅度与FDP、D－D的浓度呈正相关。结论：FDP、D－D会使发色底物法测定AT－Ⅲ：A结果偏高，其偏高的幅度与FDP、D－D的含量呈正相关，此时测定AT－Ⅲ：Ag含量更能反映其临床意义。     

In vitro effects of mycophenolic acid on the nucleotide pool and on the expression of adhesion molecules of human umbilical vein endothelial cells

The immunosuppressive drug mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid (MPA) selectively inhibit inosine 5'-monophosphate dehydrogenase (IMPDH), and therefore interfere with cellular guanine nucleotide biosynthesis. IMPDH is additionally involved in the synthesis of membrane glycoproteins, some of which are adhesion receptors known to play an active part in the regulation of cell-cell contacts, which are crucial in the process of recruitment and transendothelial infiltration of activated leucocytes in the transplanted organ. As a consequence, MPA leads to a reduction of cellular infiltrates in the course of transplant rejection. In the present study, the effects of MPA on human umbilical vein endothelial cells (HUVEC) are investigated at both molecular and cellular levels. In our experiments, HUVECs are treated with tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) in order to mimic activation occurring at a rejection crisis. The dose-dependent influence of concomitant incubation with MPA (5-20 micromol/l; 48 h, 37 degrees C, 5% CO2) on their intracellular nucleotide profile is observed by determining the concentrations of purine and pyrimidine nucleotides, using a HPLC method based on solvent generated ion-exchange. The possibility of synergistic effects is investigated by incubating endothelial cells with mixtures of three different immunosuppressants (mycophenolic acid; cyclosporin A, 100 ng/ml; prednisolone, 1 micromol/l)--a combination commonly used after transplantation--varying the amount of MPA (5-20 micromol/l). Stimulation with TNFalpha does not significantly modulate the intracellular levels of nucleotides quantitated. In the presence of MPA concentrations of at least 5 micromol/l, GTP levels (68+/-12%) are significantly decreased compared to controls (100%). At a concentration of 20 micromol/l MPA, the GTP amount is reduced to 58+/-7%. In contrast to these observations, the levels of UDP and UTP are increasing significantly under coincubation with MPA concentrations greater than 5 micromol/l. At 20 micromol/l MPA, UDP and UTP are increased to 147+/-19% and 114+/-11%, respectively. All other nucleotides (CTP, ADP, ATP) reveal no significant alterations in their intracellular concentrations under the conditions applied. Incubation of TNFalpha-treated HUVEC monolayers, with a mixture of three immunosuppressive drugs varying the amount of MPA, show no significant differences compared with the data observed after incubation with MPA alone. In addition, the influence of MPA (10 micromol/l) on a cellular level is observed by measuring the cell surface expression of adhesion molecules on cytokine-stimulated HUVECs, using TNFalpha (10 ng/ml), interferon-gamma (100 ng/ml), interleukin-1beta (10 ng/ml) and interleukin-8 (20 ng/ml). Expression of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) was assessed by flow cytometry. Activation of endothelial cell monolayers with TNFalpha significantly increases the mean fluorescence intensity of VCAM-1 (361+/-14%) and ICAM-1 (429+/-47%) surface expression, compared to controls, and additionally induces E-selectin expression (2919+/-134%). The same tendencies, but in a lesser degree, are observed under stimulation of cells with either IFNgamma or IL-1beta. Incubation with a combination of TNFalpha and MPA leads to a significant reduction in VCAM-1 (329+/-13%) and E-selectin (2613+/-167%) expression, compared to the values obtained for HUVEC incubated with the cytokine alone. Treatment of the cells with IL-1beta/MPA also reduces the expression of VCAM-1 to a level significantly lower than the level observed after stimulation with IL-1beta. Incubation with MPA alone reveals no significant modulation in the expression of all surface molecules tested compared to the values of unstimulated HUVECs. The experiments show that the immunosuppressive action of MPA not only inhibits lymphocyte proliferation but also decreases the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process.     

同型半胱氨酸和晚期糖基化终产物影响血管内皮细胞凋亡的协同效应

目的:观察两种尿毒症毒素同型半胱氨酸和晚期糖基化终产物是否协同影响血管内皮细胞的凋亡程度。方法:实验于2006-01/03在中南大学湘雅三医院中心实验室完成。①参考Makita的报道体外制备晚期糖基化终产物,将牛血清白蛋白50g/L、D-葡萄糖0.5mol/L、磷酸盐缓冲盐液0.2mol/L(pH7.4)和蛋白酶抑制剂用0.22μm滤膜过滤除菌,37℃孵育60d。采用荧光分光光度分析法鉴定。晚期糖基化终产物修饰的牛血清白蛋白中晚期糖基化终产物含量为90.52U/mg。②将不同浓度的同型半胱氨酸和晚期糖基化终产物单独或联合作用于人脐静脉内皮细胞株ECV304细胞,分为空白对照组;同型半胱氨酸干预组(分别加入0.05,0.1,0.5,1.0,5.0mmol/L的同型半胱氨酸);同型半胱氨酸 晚期糖基化终产物联合干预组(分别加入0.05,0.1,0.5,1.0,5.0mmol/L的同型半胱氨酸和终浓度为50mg/L的晚期糖基化终产物)。③采用Hoechst染色和流式细胞技术检测细胞凋亡程度。结果:①Hoechst33258染色检测内皮细胞凋亡结果:同型半胱氨酸 晚期糖基化终产物联合干预组在0.05～5.0mmol/L各个浓度较对照组和单纯同型半胱氨酸组都有明显差别(P<0.01)。②流式细胞仪检测内皮细胞凋亡结果:联合干预组总凋亡率为(15.31±2.14)%,早期凋亡率为(9.16±1.38)%,均显著高于同型半胱氨酸或晚期糖基化终产物单独干预组(P<0.01)。结论:同型半胱氨酸和晚期糖基化终产物对血管内皮细胞的凋亡具有协同诱导作用。     

血管内皮细胞生长因子和碱性成纤维细胞生长因子体外联合诱导外周血单个核细胞定向分化为血管内皮细胞

目的:在体外培养的条件下,应用血管内皮细胞生长因子和碱性成纤维细胞生长因子联合诱导人外周血单个核细胞向血管内皮细胞分化,解决组织工程血管化的种子细胞来源问题。方法:实验于2005-11/2006-05在安徽省立医院中心实验室完成。①血管内皮细胞生长因子(Pepro Tech公司,批号090310);碱性成纤维细胞生长因子(Pepro Tech公司,批号0704CY081);人淋巴细胞分离液Ficoll-paque(天津灏洋生物制品科技有限公司);PE标记的CD31,鼠抗人vWF单克隆抗体(BD Biosciences公司);FITC标记的兔抗鼠IgG(北京中杉金桥公司)。②无菌条件下取健康人外周血20mL,肝素抗凝,Hanks液双倍稀释,按1∶2置于淋巴细胞分离液上层,离心后提取分液层与上层交界部位呈混浊的灰白色层,即为单个核细胞层。③取离心后的细胞,向DMEM-F12培养基中分别加入含体积分数为0.2的胎牛血清、10μg/L血管内皮细胞生长因子、10μg/L碱性成纤维细胞生长因子。以不含血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导剂的DMEM-F12培养基作为空白对照。吹打均匀并计数,按1×1010L-1接种于25cm2培养瓶中,于37℃、体积分数为0.05的CO2饱和湿度培养箱中培养,第3天更换培养基,去除未贴壁的细胞,以后每2d换液1次。④倒置相差显微镜下观察细胞单层排列是否呈"铺路石"样结构。运用流式细胞术检测诱导后的细胞表达CD31和vWF情况。透射电镜观察细胞的超微结构。结果:①诱导分化后细胞形态学变化:经血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导后的细胞形态上呈典型的"铺路石"样外观,经历从小圆→梭形→扁平细胞的过程,符合内皮细胞的演变过程。②诱导分化后细胞的表面标志鉴定:诱导分化20d,贴壁细胞中62.5%表达CD31,58.2%表达vWF,54.3%表达CD31/vWF。③培养细胞的超微结构观察:透射电镜下细胞胞浆内可见特征性W-P小体。结论:外周血单个核细胞在血管内皮细胞生长因子和碱性成纤维细胞生长因子体外联合诱导下,可分化为血管内皮细胞。     

In vitro effects of cyclosporin A on the expression of adhesion molecules on human umbilical vein endothelial cells.

BACKGROUND: Among the different factors playing crucial roles in endothelial cell activation, cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been reported to demonstrate profound effects on this cell type. It has been shown that the increased release of IFN-alpha/gamma and TNF-alpha causes structural and functional modulations of the endothelial cell. These cytokines participate in the recruitment and activation of the immune system. CsA is an immunosuppressive drug that is necessary at high levels in human recipients of vascularised xenografts. This drug could contribute to a prolonged graft survival by modulation of endothelial cell activation. METHODS: The present study deals with the effects of cyclosporin A on adhesion molecule expression (i.e. ICAM-1, VCAM-1, E-selectin, P-selectin, PECAM-1 and the L-selectin ligand CD 34) on the surface of cytokine stimulated HUVECs. The in vitro model described herein mimics the stimulation of endothelial cells by cytokines as seen during inflammatory processes after transplantation. Therefore, HUVECs were activated either with TNF-alpha, IL-1beta or with a cytokine mixture consisting of those stimulants present at an elevated level in sera of patients during allograft rejection (i.e. IL-1beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma). RESULTS: The results obtained show that the immunosuppression of CsA is not only achieved by inhibiting lymphocyte proliferation, but also by decreasing the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process. CONCLUSION: Co-incubation of stimulated endothelial cells with a final CsA concentration of 5 microg/ml revealed a significant down-regulating influence on the surface expression of E-selectin and VCAM-1.     

In vitro analysis of the interactions between preadipocytes and endothelial cells in a 3D fibrin matrix

The volume‐persistent survival of transplanted adipose tissue in vivo relies on early vascularization, due to an otherwise early induction of apoptosis of the centrally located cells. Thus, one way to enable the early formation of a capillary network resulting in a sufficient perfusion of the transplanted construct might be the co‐transplantation of autologous preadipocytes with endothelial cells. To investigate preadipocyte–endothelial cell interaction, three‐dimensional proliferation‐ and angiogenesis assays were performed in vitro. Proliferation rates of co‐cultured endothelial cells and preadipocytes suspended in a fibrin matrix were elucidated by Alamarblue assays. The spheroid angiogenesis model was applied for analyzing the effects of vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) (produced by preadipocytes) as well as the impact of cell‐cell interaction between preadipocytes and endothelial cells and fibrin matrix on endothelial cell migration. Preadipocytes proliferated in fibrin glue, whereas endothelial cells underwent apoptosis. By co‐culturing, both cell types demonstrated an increased proliferation rate. Preadipocytes provoked migration of endothelial cells. Blocking bFGF and/or VEGF led to a significant decrease of migration. Changes in fibrin structure were followed by migration of single cells instead of sprouting. An appropriate fibrin matrix as well as already differentiated endothelial cells are necessary for preadipocytes to develop their angiogenic activity via bFGF and VEGF.     

In vitro analysis of the interactions between preadipocytes and endothelial cells in a 3D fibrin matrix.

The volume-persistent survival of transplanted adipose tissue in vivo relies on early vascularization, due to an otherwise early induction of apoptosis of the centrally located cells. Thus, one way to enable the early formation of a capillary network resulting in a sufficient perfusion of the transplanted construct might be the co-transplantation of autologous preadipocytes with endothelial cells. To investigate preadipocyte-endothelial cell interaction, three-dimensional proliferation- and angiogenesis assays were performed in vitro. Proliferation rates of co-cultured endothelial cells and preadipocytes suspended in a fibrin matrix were elucidated by Alamarblue assays. The spheroid angiogenesis model was applied for analyzing the effects of vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) (produced by preadipocytes) as well as the impact of cell-cell interaction between preadipocytes and endothelial cells and fibrin matrix on endothelial cell migration. Preadipocytes proliferated in fibrin glue, whereas endothelial cells underwent apoptosis. By co-culturing, both cell types demonstrated an increased proliferation rate. Preadipocytes provoked migration of endothelial cells. Blocking bFGF and/or VEGF led to a significant decrease of migration. Changes in fibrin structure were followed by migration of single cells instead of sprouting. An appropriate fibrin matrix as well as already differentiated endothelial cells are necessary for preadipocytes to develop their angiogenic activity via bFGF and VEGF.     

体外诱导脂肪干细胞向内皮细胞及成血管的分化

背景：脂肪干细胞作为组织工程中种子细胞的选择,可以诱导为内皮细胞从而有效解决材料血管化困难的难题。目的：体外观察脂肪干细胞经诱导向内皮细胞转分化的可能性、以及在立体培养基上的血管形成情况。方法：切取人皮下脂肪,用酶消化法分离和培养脂肪干细胞,将传至第3代或第4代的脂肪干细胞用内皮细胞诱导液、同时在Matrigel三维培养基内诱导培养,观察细胞生长情况及变化。对脂肪干细胞和诱导细胞行免疫组织化学检查CD31的表达。结果与结论：免疫组织化学检测脂肪干细胞的CD31表达阴性,诱导细胞CD31可见阳性表达。在三维立体培养基内诱导的细胞24h逐渐迁徙成团,伸出伪足,诱导1周细胞形成网格样交叉,2周形成较长血管,后血管增粗,并出现分叉,CD31阳性表达。因此,脂肪干细胞体外可以被诱导向内皮细胞表型转分化,并形成血管,提示脂肪干细胞可以作为促进组织工程移植物血管化的种子细胞的理想选择。     

Fatty acid incorporation in endothelial cells and effects on endothelial nitric oxide synthase

BACKGROUND: The nature of fatty acids provided by the diet as well as plasma lipid metabolism can modify the composition and properties of plasma membrane and thus the activity of membrane proteins. In humans, as well as in experimental models, diabetes is associated with both an alteration in serum lipid profile and a documented endothelial dysfunction. This in vitro study investigated on an immortalized human endothelial cell line (EA.hy 926) the specific effects of several free fatty acids (FFAs) on the composition of cellular membranes and the regulation of endothelial nitric oxide synthase (eNOS). MATERIALS AND METHODS: 0.1% of lipid deprived serum was added to the incubation medium with 25 mM glucose in order to study the effects of individual fatty acids: myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid at 100 microM bound with albumin. The effects of the FFAs on the endothelial nitric oxide synthase were investigated on mRNA level by quantitative PCR, on protein level and Ser1177 phosphorylation by Western blot and on enzymatic activity on living cells using radiolabelled arginine. RESULTS: Free linoleic acid increased the membrane content in n-6 fatty acids (mainly C18: n-6 and its metabolites) with a decrease in saturated and monounsaturated fatty acids. These conditions decreased the basal eNOS activity and reduced the phosphorylation of eNOS-Ser1177 due to activation by histamine. Free palmitic acid enriched the membranes with 16 : 0 with a slight decrease in monounsaturated fatty acids. These conditions increased eNOS activation without increasing Ser1177 phosphorylation upon histamine activation. The addition of the other FFAs also resulted in modifications of membrane composition, which did not to affect eNOS-Ser1177 phosphorylation. CONCLUSION: Among the fatty acids used, only modification of the membrane composition due to linoleic acid supply disturbed the basal enzymatic activity and Ser1177 phosphorylation of eNOS in a way that limited the role of histamine activation. Linoleic acid might involve the dysfunction of both eNOS basal activity and its phosphorylation status and may then contribute to an impaired vasodilatation in vivo.     

丹参多酚酸盐对外周血内皮祖细胞功能的影响

目的观察丹参多酚酸盐对体外培养的人外周血内皮祖细胞(EPCs)增殖、黏附及一氧化氮(NO)分泌功能的影响。方法从人外周血分离培养获取单个核细胞(MNCs),接种在包被有人纤维连接蛋白的培养板上,在EBM完全培养液中培养。EPCs与不同浓度丹参多酚酸盐及辛伐他汀共培养一段时间,观察其对EPCs增殖、黏附和分泌功能的影响。结果丹参多酚酸盐能促进EPCs增殖,与对照组比较差异具有统计学意义(0.335±0.005 vs.0.292±0.005,P<0.01);并且能增强细胞黏附能力(32±0.8 vs.30±0.8,P<0.05),使NO分泌量增加[(51.3±1.8)μmol/L vs.(22.4±1.1)μmol/L,P<0.01]。结论丹参多酚酸盐可以促进体外培养的EPCs增殖并增强其功能。