登革病毒Ⅱ型对人脐静脉血管内皮细胞通透性的研究

目的 研究登革病毒Ⅱ型(DENV-2)对人脐静脉血管内皮细胞(HUVEC)通透性的影响.方法 DENV-2病毒滴定,DENV-2接种HUVEC单层细胞,用间接免疫荧光法动态观察DENV-2感染HUVEC的情况(30 min,l、3、6、12、24、30、36、42、48、72 h),实时定量荧光PCR方法检测病毒载量,transwell法检测DENV-2对HUVEC通透性的影响,透射电镜观察细胞超微结构的改变.结果 DENV-2对HUVEC通透性的影响30 min时作用最为明显,其次为42 h时.且病毒载量与HUVEC通透性改变呈正相关.透射电镜结果显示有细胞微绒毛脱落,胞质溶解,部分核膜间隙增宽,甚至是细胞核中也存在裂隙,部分线粒体嵴消失甚至空化,线粒体髓样变,包膜不完整.结论 DENV-2对HUVEC通透性有影响,为探究登革病毒的发病机制提供一定的理论依据.

Abstract：

Objective To research Dengue virus type 2 (DENV-2) to human umbilical vein endothelial cells (HUVEC) permeability. Methods The titration of DENV-2 was detected and HUVEC infection DENV-2 layer of cells. At different time points after infection (30 min, 1 h, 3 h, 6 h, 12 h, 30 h,36 h, 24 h, 42 h, 48 h,72 h), the permeability in HUVEC were detected after Dengue virus infection. The virus load in HUVEC was detected by quantitative real-time PCR. And ultrastructure in HUVEC was observed by electron microscope. Results The permeability in HUVEC was changed after Dengue virus infection by time lasting. The most permeability in HUVEC was changed after Dengue virus infection at 30 min and 42 h. And at the same time the virus load were most value in all of time. The results of the cell membrane were changed by electron microscope. The deciduous cell microvillus and dissd endochylema were founded. And some of nuclear membrane blank was wided by Dengue virus. Even the leakage was founded in cell nucleus. The other change included that disappearance mitochondrial and vacuolization crista, elder pith change mitochondria. In addition, the complete of cell membrane were dissolved. Conclusion The permeability of HUVEC was changed by DENV-2. To explore the pathogenesis of Dengue virus provide certain theoretical basis.     

体外培养人脐静脉内皮细胞表达一氧化氮合酶

为了证实人脐静脉内皮细胞是否具有合成一氧化氮的能力，用ＮＡＤＰＨ－黄递酶组织化学法和原位杂交对体外的人脐静脉内皮细胞表达一氧化氮合酶进行了观察。结果显示，体外培养人脐静脉内皮细胞的ＮＡＤＰＨ－黄递酶组织化学的阳性百分率为６２％，诱生型一氧化氮合酶的原位杂交的阳性百分率为７４％。阳性反应产物分布在核周及细胞突起中。本研究首次证明体外培养的人脐静脉内皮细胞能表达诱生型一氧化合酶，即人脐静脉内皮细胞在体     

脂多糖对人脐静脉血管内皮细胞的直接损伤作用

血管内皮细胞 (ｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ,ＥＣ)炎症反应是感染性休克向不良方向演变的重要原因 ,有关激活剂 (ＬＰＳ、ＩＬ 1)导致ＥＣ活化的研究是败血症病理反应的中心课题。内毒素引起微循环ＥＣ损伤是内毒素性组织损伤的重要环节之一。本实验探讨了ＬＰＳ对体外培养脐静脉ＥＣ的直接损伤作用。1　材料与方法1 1　材料 :ＲＭＰＩ 16 40培养基、ＬＰＳ(Ｓｉｇｍａ产品 )1 2　细胞培养及鉴定 :内皮细胞传 4代。ＰＭＮ的分离采用Ｐｅｒｃｏｌｌ’ｓ分离液 5 0 0ｇ离心 2 0ｍｉｎ取白细胞层用培养基稀释成2× 10 6 ｍＬ备用。1 3　…     

体外培养人脐静脉内皮细胞的光电镜观察

体外培养人脐静脉内皮细胞的光电镜观察姚忠祥，蔡文琴第三军医大学重庆６２００３８Ｐｕｐｎｉｃｋ等在培养微血管内皮细胞时，观察到内皮细胞具有多种不同的形态，并推测其原因可能是它们来源于不同节段的微血管。本研究取正常人足月产胎儿脐静脉内皮细胞体外培养，第五...     

黄芪注射液对人脐静脉血管内皮细胞的增殖作用研究

目的：观察黄芪注射液对人血管内皮细胞的作用。方法：黄芪注射液与人脐静脉血管内皮细胞(human umbilicalvein endothelial cell line EVC-304)共同培养后，采用MTT法及形态学方法观察黄芪注射液对人脐静脉血管内皮细胞的作用。结果：黄芪注射液对人脐静脉血管内皮细胞有显著增殖效应，具剂量依赖性，且成正相关。     

肿瘤坏死因子对培养的人脐静脉内皮细胞骨架的影响

目的：用肿瘤坏死因子（TNF－α）处理培养的人脐静脉内皮细胞研究不同作用浓度和不同作用时间的TNF－α的刺激对内皮细胞的形态和骨架的影响。结果：2000U的TNF－α处理内皮细胞8小时,细胞形态发生改变,细胞间的紧密接触变的松攻,间隙增宽,骨架失去原有的网状形态,呈块状收缩,24小时后骨架结构开始恢复。细胞形态似成纤维细胞,1000U的TNF－α对内皮细胞影响比较明显,8小时后细胞变圆,细胞间的接触消失,骨架浓缩,网状结构消失,24小时后出现特征性“鸟喙”样改变,72小时后不完全恢复,不同浓度的TNF－α刺激内皮不同时间,均可使内皮细胞内游离钙比对照组明显增加。结论：TNF－α可改变内皮细胞的形态结构。影响其屏障机能,并可能与胞内游离钙浓度相关。     

原代人脐静脉内皮细胞分离和培养方法的改进

目的建立一套高效、方便、可靠的脐静脉内皮细胞的体外分离和培养方法。方法取剖腹产新生婴儿脐带,加入Ⅰ型胶原酶消化分离人脐静脉内皮细胞,内皮专用培养基EGM-2培养传代。应用相差显微镜观察细胞形态。DiL-Ac-LDL染色证实细胞具有内皮功能。免疫荧光染色证明新分离细胞表达FⅧ相关抗原。流式细胞仪检测细胞表面表达CD31。结果原代培养细胞培养10～15d左右长成单层,细胞呈多角形,铺路石样排列。DiL-Ac-LDL染色阳性,证实细胞具内皮吞噬功能。免疫荧光检测证明细胞特异性表达FⅧ相关抗原。流式细胞仪检测证明细胞表面高表达CD31。结论通过酶灌注法消化脐静脉血管获得细胞,操作简便、高效,EGM-2培养基可保证内皮细胞具有较高纯度,细胞形态学观察和细胞生物学鉴定证明获得人脐静脉血管内皮细胞。     

复方降压片和西拉普利对人脐静脉内皮细胞功能的影响

血管内皮功能已经越来越受到人们的重视 ,研究表明高血压时血管内皮功能受到损害。本文旨在通过细胞培养的方法研究和比较目前临床常用的两种抗高血压药物复方降压片和西拉普利对血管内皮细胞功能的影响。1　材料和方法1 1　材料1 1 1　脐带取自协和医院产科病房。1 1 2　Ｍ - 199、胎牛血清、胰酶、Ｌ -谷氨酰胺为ＧＩＢＣＯ公司产品 ;ＥＣＧＦ、Ｈｅｐｅｓ为ＢｏｅｈｒｉｎｇｅｒＭａｎｎｈｅｉｍ公司产品 ;Ｉ型胶原酶为Ｓｉｇｍａ公司产品。氢氯噻嗪纯品由常州制药厂提供 ,西拉普利由罗氏药厂惠赠。ＴＮＦ由邦定公司提供 ,ＡｎｇⅡ为Ｓｉ…     

改良贴壁法结合内皮细胞培养基培养小鼠肺微血管内皮细胞*☆

背景：肺微血管内皮细胞是研究微循环的重要内皮细胞模型之一,众多培养方法中单纯贴壁法操作相对简便,但耗时长,杂质细胞多,是批量培养细胞的最大障碍。 目的：建立优化的小鼠肺微血管内皮细胞体外培养方案,观察细胞生长状态并鉴定细胞性质。 方法：无菌状态下快速剪碎5日龄C57BL/6J小鼠的肺叶外周组织,肺组织颗粒贴壁法获得肺微血管内皮细胞,并用内皮细胞培养基培养。倒置显微镜观察培养细胞生长和行为状态,Ⅷ因子相关抗原免疫组化和免疫荧光进行细胞鉴定。 结果与结论：肺组织块培养24 h内可见梭形细胞爬出,传代后细胞生长迅速,形态规则呈鹅卵石状,纯度高达98%以上,结合Ⅷ因子相关抗原检测证实其为内皮细胞。结果可见联合运用肺组织颗粒贴壁和内皮细胞培养基可高效获得原代小鼠肺微血管内皮细胞。关键词：肺微血管内皮细胞;细胞培养;优化;鉴定;小鼠;血管组织工程 缩略语注释：PMVECs: pulmonary microvascular endothelial cells,肺微血管内皮细胞 doi:10.3969/j.issn.1673-8225.2012.15.007     

Culture of human umbilical vein endothelial cells on immobilized vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) was immobilized on substrata in photoreactive gelatin to control the adhesion and growth of vascular endothelial cells. The gelatin and VEGF were mixed in water and cast on a polystyrene dish or a silane-coated glass plate. The surface was then photoirradiated in the presence or absence of a photomask and washed. Toughness of the immobilized material was confirmed by ethanol treatment. Human umbilical vein endothelial cells (HUVECs) grew on the immobilized VEGF but not on a nontreated surface. Growth of HUVEC increased significantly with an increase in the amount of immobilized VEGF, and the effects were inhibited by treatment with anti-VEGF antibody. Thus, immobilized VEGF specifically interacted with HUVECs to permit growth in culture. Micropatterning of HUVEC cultures was also achieved using micropattern-immobilized VEGF. This patterning technique may be useful for the formation of blood vessel networks in vitro.     

伞枝犁头霉在体外诱导人血管内皮细胞凋亡

目的 研究伞枝犁头霉对人脐静脉血管内皮细胞活性的影响及其机制.方法 采用锥虫蓝染色法分析不同时间段伞枝犁头霉对人血管内皮细胞存活率的影响.细胞凋亡检测试剂盒在荧光显微镜下分析伞枝犁头霉诱导人血管内皮细胞的凋亡情况.流式细胞仪定量检测伞枝犁头霉诱导人血管内皮细胞在不同时间段的凋亡情况,并观察caspase-3抑制剂对凋亡反应的影响.用Western blot法检测伞枝犁头霉引发人血管内皮细胞caspase-3在不同时间段活化情况.结果 锥虫蓝染色法显示伞枝犁头霉以时间依赖的方式抑制人血管内皮细胞的存活率.伞枝犁头霉在共培养第12小时开始抑制人血管内皮细胞存活率(P=0.001).荧光显微镜显示伞枝犁头霉诱导人血管内皮细胞发生凋亡,而并非坏死.流式细胞仪分析显示伞枝犁头霉以时间依赖的方式诱导人血管内皮细胞发生凋亡反应.凋亡细胞在共培养第12小时显著增高(P=0.0036).Caspase-3抑制剂几乎可以完全逆转伞枝犁头霉诱导的凋亡反应.Western blot法显示伞枝犁头霉引起人血管内皮细胞caspase-3活化随时间逐渐增强.结论 伞枝犁头霉在体外试验中可以诱导人血管内皮细胞发生凋亡.该凋亡信号的传导是通过caspase级联反应.     

人脐静脉内皮细胞与表面改性的PHBHHx膜的体外相容性

目的 评价人脐静脉内皮细胞（HUVECs）在含有不同组分中药涂层、碱处理前后的3-羟基丁酸和3-羟基已酸共聚酯（PHBHHx）膜上的生长状况,为研究PHBHHx膜与内皮细胞的生物相容性提供实验依据。 方法 用Baudine改良法分离培养人脐静脉内皮细胞,并用免疫组织化学方法鉴定;将已传至第3代人脐静脉内皮细胞接种于材料表面,培养8、12、24h,用扫描电镜观察人脐静脉内皮细胞在不同表面上黏附形态的变化,同时用细胞免疫荧光标记法比较其在不同膜上的分布情况,最后用四甲基偶氮唑盐(MTT)法检测各组细胞的活力。 结果 成功分离出人脐静脉内皮细胞,Ⅷ 因子及血管内皮细胞受体Ⅱ(FLK-1)染色阳性;人脐静脉内皮细胞培养结果表明,细胞在各组材料表面均呈良好生长,并大量增殖。MTT分析结果显示,碱处理的PHBHHx膜及添加5%、10%中药涂层的材料对人脐静脉内皮细胞的体外生长、增殖有促进作用;扫描电镜和荧光显微镜下可见,碱处理的PHBHHx膜及添加5%、10%中药涂层的材料表面的人脐静脉内皮细胞活力旺盛、形态饱满、分布均匀。 结论 经表面改性处理的、且加入5%、10%中药涂层的PHBHHx膜,具有良好的人脐静脉内皮细胞相容性,在体外细胞培养的环境下,有利于细胞的生长、贴附和增殖。     

内皮样细胞与脐静脉内皮细胞的比较

BACKGROUND: Stem cells are induced to differentiate into endothelial-like cells that can be used for the treatment of diabetic lower extremity vascular disease. However, it is unclear whether these endothelial-like cells can completely replace endothelial cells to improve vascular disease and what are the differences between endothelial-like cells and endothelial cells. OBJECTIVE: To explore the differences and similarities between endothelial-like cells and human umbilical vein endothelial cells in the aspects of morphology, function, and viability. METHODS: Umbilical cord mesenchymal stem cells and umbilical vein endothelial cells were isolated, cultured and identified using flow cytometry and immunohistochemical method. Isolated umbilical cord mesenchymal stem cells were induced in DMEM-LG/F12 containing 10 µg/L vascular endothelial growth factor, 10 µg/L basic  fibroblast growth factor and 2% fetal bovine serum to differentiate into endothelial-like cells followed by immunohistochemical identification. To compare endothelial-like cells with human umbilical vein endothelial cells, cell migration detection, active substance measurement and three-dimensional angiogenesis test were performed. RESULTS AND CONCLUSION: Isolated umbilical cord mesenchymal stem cells strongly expressed the surface markers of mesenchymal stem cells, and human umbilical vein endothelial cells strongly expressed CD31 and VWF. After induction, the umbilical cord mesenchymal stem cells were identified to highly express CD31 and VWF. Through cell migration, active substance and three-dimensional angiogenesis tests, endothelial-like cells were similar to endothelial cells in the function and activity, and superior to endothelial cells.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Effects of intravenous immunoglobulin and methylprednisolone on human umbilical vein endothelial cells in vitro

Endothelial cells (ECs) do more than just play a role in distinguishing blood and tissues. These cells are also influenced by various chemical mediators present in the blood and tissues. In addition, they produce diverse cytokines, chemokines, adhesion molecules, and growth factors. Therefore, ECs are actively involved in the inflammatory and immune response. We investigated the effects of intravenous immunoglobulin (IVIG) and methylprednisolone (MP) on activated human ECs, by examining the individual and combined effects of the drugs. Human umbilical vein ECs (HUVECs) obtained from the umbilical cords of healthy newborns were cultured. After the HUVECs were treated with interleukin (IL)-1beta, the effects of IVIG and/or MP on the activated HUVECs were assessed by cell proliferation, mRNA expression, and the production of vascular cell adhesion molecule (VCAM)-1, IL-1beta, and vascular EC growth factor (VEGF). IVIG and MP inhibited HUVEC proliferation. IVIG and MP significantly down regulated mRNA expression and the production of VCAM-1, IL-1beta, and VEGF. The combination of IVIG and MP generally showed a greater suppressive effect on mRNA expression and on the production of VCAM-1, IL-1beta, and VEGF. Our results suggest that some of the corticosteroid-sparing effects of IVIG observed in patients with severe asthma could be related to a decreased ability of ECs to proliferate, and to a down regulation of the expression of molecules involved in the onset and progression of airway inflammation.     

培养的人脐静脉内皮细胞产生单核细胞趋化因子的研究

The blood monocytes have been well known as one of the origins of foam cells in atherosclerotic plaque, but the mechanism controlling the migration of monocytes into the subendothelial space is still unclear. The chemotactic activity of the conditioned medium from cultured human umbilical vein endothelial cells for human blood monocytes was investigated by micropore filter assay; meanwhile, heat stability and enzyme digestion assays were undertaken. The results showed that the conditioned medium from the cultured endothelial cells was significantly chemotactic rather than chemokinetic for monocytes. The conditioned medium from the cultured endothelial cells still retained the chemotactic activity for monocytes although it was heated at 80C and 100C for 15 min respectively, whereas after digestion with protease, the chemotactic activity vanished. From the above-mentioned facts, it is reasonable to believe that cultured human umbilical vein endothelial cells can secrete a chemotactic factor for monocytes, which is a kind of peptide.     

不同培养基对人脐静脉内皮细胞体外培养效果的影响

目的研究不同培养基对人脐静脉内皮细胞体外培养生长的影响,探讨内皮细胞的最佳体外扩增培养条件。方法取健康产妇分娩后脐带,用胶原酶Ⅰ消化后得脐静脉内皮细胞,进行原代培养,并用免疫荧光染色的方法对细胞进行鉴定,传代培养时则分别采用RPMI-1640培养基和EGM-2培养基,倒置显微镜观察两种培养条件下细胞的生长状态,同时利用流式细胞仪检测其生长周期,对比培养效果。结果 EGM-2组内皮细胞生长良好,2d后贴壁生长的细胞可达90%。EGM-2组S期细胞比例为（29.07±1.48）%,RPMI-1640培养基组S期细胞为（17.58±3.49）%,二者比较差异有统计学意义（P〈0.01）。结论 EGM-2培养基更适合人脐静脉内皮细胞的体外传代扩增培养。     

人脐静脉内皮细胞分离鉴定及其膜分子的表达

背景：肿瘤微环境作用于血管内皮细胞,影响其膜表面分子的表达,与肿瘤的生长、转移及免疫逃逸作用相关,但迄今相关机制尚不完全清楚。 目的：分析肿瘤微环境下血管内皮细胞的膜表面分子表达量变化。 方法：原代分离培养人脐静脉内皮细胞,采用不同肿瘤培养上清,用不同时间进行诱导。记录培养血管内皮细胞形态;利用免疫组织化学方法,鉴别Ⅷ因子相关抗原;荧光实时定量PCR法测定其膜表面分子mRNA表达量。 结果与结论：肝癌smmc7721细胞培养上清处理后,内皮细胞抗原提呈会随着时间延长减弱,黏附白细胞能力随着时间延长而变化。胃癌SGC7901细胞培养上清处理后内皮细胞后,抗原提呈能力无明显变化;黏附能力有关分子中,CD31和细胞间黏附分子1表达降低,CD62E表达增高。说明肿瘤微环境可能通过上述黏附分子和抗原提呈分子表达的变化影响血管内皮细胞的相应功能。     

尼古丁对人脐静脉内皮细胞凋亡及坏死的影响

目的不同浓度尼古丁对人脐静脉内皮细胞（HUVECs）凋亡及坏死的影响。方法用CD34以免疫组织化学法鉴定HUVECs。通过3种不同浓度的尼古丁（3、30、300 ng/ml）刺激HUVECs 24 h后,用流式细胞仪检测其凋亡及坏死率。结果低浓度尼古丁促进细胞凋亡最强,而随着尼古丁浓度的增加,细胞凋亡率反而逐渐降低,各组差异具有统计学意义（P〈0.05）;此外,随着尼古丁浓度增加,细胞坏死率也日益增多,各组差异具有统计学意义（P〈0.05）。细胞坏死率与尼古丁浓度呈正相关（r=0.675）。结论尼古丁对HUVECs凋亡的影响具有浓度依赖性,细胞坏死率与尼古丁浓度呈正相关。