放射损伤对内皮细胞增殖与凋亡的影响

放射损伤在平战时均可发生，合并放射损伤时组织修复常常延缓，创伤愈合延迟，即出现创伤难愈现象，但其机理尚未完全阐明。创伤局部的组织修复细胞，如血管内皮细胞和成纤维细胞等在创伤愈合过程中具有重要作用。合并全身放射损伤时创伤局部内皮细胞生物学特性异常可能是创伤难愈的重要原因。因此，本实验探讨不同照射剂量对内皮细胞增殖、细胞周期和凋亡的影响，并观察bFGF对内皮细胞放射损伤的拮抗作用，为创面修复提供新思路。     

腺病毒介导的VEGF-B基因促血管内皮细胞增殖作用的研究

为观察腺癌毒素介导的血管内皮细胞生长因子B（VEGF－B）基因体外转染对血管内皮细胞的促增殖作用，构建编码人VEGF－B基因的复制缺陷的重组腺病毒载体，体外转染鼠主动脉血管内皮细胞（RAECs），应用RT－PCR和Western blot检测外源VEGF－B的表达，应用四唑盐（MTT）观察转染后RAECs的增殖。结果发现，RAECs可有效地被重组腺病毒载体感染，并能成功转录和表达VEGF－B基因和蛋白，在转染后细胞培养上清中检测到VEGF－B蛋白的表达，对RAECs有显著促增殖作用。提示重组腺病毒载体介导的人VEGF－B基因有血管新生作用，可用于缺血性心脏病的治疗。     

电离辐射对血管内皮细胞纤维肌动蛋白的影响

目的 观察电离辐射对血管内皮细胞纤维肌动蛋白的影响,探讨血管内皮细胞电离辐射损伤的机制。方法 用0、2、4、6、8、10和12 Gy 60Coγ射线对血管内皮细胞进行照射,于照射后6 h用激光共聚焦显微镜观察其细胞膜骨架蛋白,于照射后12和24h收集细胞,用流式细胞仪测定F-actin的含量。结果 细胞膜骨架随着辐射剂量的增加而破坏,这种变化呈剂量依赖性。F-actin的含量在照射后12 h明显减少,24 h后有所回升。结论 电离辐射能使血管内皮细胞骨架破坏和F-actin解聚,导致细胞损伤。     

血管内皮细胞生长因子-海藻酸钙缓/控释微球的制备及其对人脐静脉血管内皮细胞增殖的影响

目的研制血管内皮细胞生长因子(VEGF)-海藻酸钙微球缓/控释系统并观察其对人脐静脉血管内皮细胞(HUVEC)增殖的影响,为VEGF缓/控释促进组织工程骨血管化提供理论依据。方法应用锐孔挤出-离子交联法制备微球,检测其理化及体外释药性质；培养HUVEC,依据培养基添加物不同,设空白对照组、空白微球组、单用VEGF组及VEGF-海藻酸钙微球组,通过细胞计数法、四甲基偶氮唑盐比色法、流式细胞仪测细胞周期考察细胞增殖情况。结果微球球形圆整,粒径(560±50)μm,载药量0．72ng/mg,包封率54%,体外释药平稳,达10d以上。细胞培养初期,VEGF组的促分裂增殖作用最强,中后期,VEGF微球组的促分裂增殖作用最强,差异有统计学意义(P＜0．05),空白微球组与空白对照组间在细胞计数、细胞活性、细胞周期的各时相点差异均无统计学意义。结论海藻酸钙微球可以保存VEGF活性并持续释放VEGF 10d以上,在较长时期内促进HUVEC的增殖。     

低氧时蛋白激酶C对内皮细胞表达血管内皮生长因子的调节作用

目的探讨低氧培养大鼠肺动脉血管内皮细胞生长因子 (VEGF)表达变化与蛋白激酶C(PKC)活性的关系。方法培养大鼠肺动脉血管内皮细胞 ,观察低氧 ( 1 %O2 )培养 0、1、3、6、1 2h大鼠肺动脉血管内皮细胞PKC活性和VEGFmRNA水平变化 ;同时对培养液中VEGF蛋白水平进行测定。培养基中加入PKC抑制剂 (staurosporine)后 ,立即进行低氧培养 ,测定低氧培养不同时间点上述指标的变化。 结果低氧培养 1hPKC活性首先明显升高 (P >0 .0 5 ) ,至 3hVEGFmRNA表达明显升高 (P <0 .0 1 ) ,6h培养液中VEGF蛋白水平显著升高 (P <0 .0 1 )。而加入PKC抑制剂后 ,低氧培养的内皮细胞PKC活性与 0h比较明显下降 (P <0 .0 1 ) ,相应各时间点的VEGFmRNA及蛋白水平与 0h比较无明显变化 (P >0 .0 5 )。结论低氧能够刺激大鼠肺动脉血管内皮细胞VEGF表达升高 ,低氧时PKC活性升高是调节VEGF表达升高的重要因素之一     

不同幅值脉冲电刺激对内皮细胞形态和功能的影响

目的 探求不同幅值脉冲电刺激对内皮细胞的形态、增殖以及活性物质NO和ET-1分泌量的影响.方法 选择在50 Hz频率,幅值分别为25 mV,50 mV,100 mV,200 mV,400 mV,800 mV,1 600 mV,3 200 mV的电刺激6 h作用于内皮细胞.用扫描电镜观察细胞形态.检测MTT,分析细胞增殖.检测内皮细胞NO和ET-1的分泌量.结果 在低幅值脉冲电刺激(25 mV,50 Hz)作用下,内皮细胞明显增殖.同时大量伪足突起出现,分泌物也同时增多.但当电刺激高于100 mV后,脉冲电刺激明显抑制细胞生长.NO的表达在电刺激作用后明显增加,随电刺激幅值增加而提高,在400 mV左右出现峰值.ET-1的表达在25 mV出现峰值后逐渐下降.结论 脉冲电刺激对内皮细胞的形态、增殖、ET-1和NO表达都有影响.同时,预示低幅值脉冲电刺激是有效辅助血管生成的手段.     

一氧化氮损伤内皮细胞的机制研究

体外培养人脐静脉内皮细胞（ＨＵＶＥＣ），主要研究：①内毒素损伤内皮细胞过程中一氧化氮（ＮＯ）的作用；②不同浓度ＮＯ供体ＳＩＮ－１对内皮细胞的作用以及ＳＯＤ、ＣＡＴ对内皮细胞的保护作用。结果提示：ＮＯ在内毒素损伤内皮细胞过程中发挥重要作用；高浓度ＳＩＮ－１可严重损伤内皮细胞；ＳＯＤ、ＣＡＴ对内皮细胞的不同保护作用说明ＯＮＯＯ＾－的产生可能是ＮＯ损伤内皮细胞的重要机制。     

巨噬细胞对血管内皮细胞周期和VEGF受体、HOXB2 mRNA表达的影响

目的　研究巨噬细胞调节内皮细胞参与血管生成的机制。方法　将人脐静脉血管内皮细胞系 (ECV 30 4 )细胞接种培养 ,待细胞 6 0 %融合时 ,以刀豆蛋白A(ConA)作为人巨噬细胞系U937的激活剂 ,将细胞分为 4组进行培养 ,即单纯ECV 30 4培养组 (对照组 ) ,ConA与ECV 30 4共培养组 ,U937与ECV 30 4共培养组 ,ConA、U937与ECV 30 4共培养组。于共培养 4 8h后在倒置显微镜下观察细胞形态学的变化 ;用流式细胞仪检测细胞周期的变化 ;用RT PCR技术检测内皮细胞VEGF受体KDR和同源盒 (homeobox)HOXB2mRNA表达水平的变化。结果　ConA刺激的U937细胞使内皮细胞间隙变大 ,细胞出现明显的类似神经元样的突起 ,形态不一 ,且S期明显增加(P <0 0 1) ,并使内皮细胞VEGF受体KDR和HOXB2mRNA的表达水平明显上调 (P <0 0 1)。结论　ConA活化的巨噬细胞通过影响内皮细胞的形态、细胞周期、KDR及HOXB2mRNA的表达来促进内皮细胞的增殖和迁移 ,从而调节血管的生成     

重组人Flt3配体对微血管内皮细胞的抗辐射作用研究

目的：研究Flt3配体（FL）对人微血管内皮细胞系细胞（ECV）的抗辐射作用及其可能的作用机理。方法：用流式细胞仪分析ECV表面Flt3受体的表达;用MTT法观察FL对受照射ECV增殖的影响,用AnnexinV-PI方法测定细胞凋亡,用RT－PCR方法分析细胞凋亡基因hax和Bcl-2的改变。结果：ECV表达Flt3受体,FL能激发ECV增殖,ECV经15Gy的照射后,FL能有效地拮抗照射对ECV增殖的抑制作用;同时照后ECV的bax基因表达增加,bcl-2基因表达轻度降低。FL通过抑制bax基因的表达,降低辐射引起的ECV细胞凋亡。结论：FL通过抑制细胞凋亡,减轻辐射引起的ECV的损伤,可能在辐射损务患者造血微环境的保护中具有应用价值。     

血管内皮细胞生长因子基因转染皮肤成纤维细胞的实验研究

为改善人工复合皮在临床上的应用效果 ,构建人血管内皮细胞生长因子 16 5 (hVEGF165)基因重组载体pcDNA3,并转染成纤维细胞。检测转基因细胞培养上清液VEGF浓度 ;用转基因细胞培养上清液刺激人脐静脉血管内皮细胞 ,观察内皮细胞增殖速度 ;通过Miles实验检测VEGF的生物学活性。结果显示 ,转基因成纤维细胞能够表达一定浓度的VEGF,且具有加速体外培养人脐静脉血管内皮细胞 (HUVEC)生长和增强血管通透性的生物学活性。提示转hVEGF165基因成纤维细胞可修饰人工皮的真皮面。     

Differential response to acute low dose radiation in primary and immortalized endothelial cells

Abstract

Purpose: The low dose radiation response of primary human umbilical vein endothelial cells (HUVEC) and its immortalized derivative, the EA.hy926 cell line, was evaluated and compared.

Material and methods: DNA damage and repair, cell cycle progression, apoptosis and cellular morphology in HUVEC and EA.hy926 were evaluated after exposure to low (0.05–0.5 Gy) and high doses (2 and 5 Gy) of acute X-rays.

Results: Subtle, but significant increases in DNA double-strand breaks (DSB) were observed in HUVEC and EA.hy926 30 min after low dose irradiation (0.05 Gy). Compared to high dose irradiation (2 Gy), relatively more DSB/Gy were formed after low dose irradiation. Also, we observed a dose-dependent increase in apoptotic cells, down to 0.5 Gy in HUVEC and 0.1 Gy in EA.hy926 cells. Furthermore, radiation induced significantly more apoptosis in EA.hy926 compared to HUVEC.

Conclusions: We demonstrated for the first time that acute low doses of X-rays induce DNA damage and apoptosis in endothelial cells. Our results point to a non-linear dose-response relationship for DSB formation in endothelial cells. Furthermore, the observed difference in radiation-induced apoptosis points to a higher radiosensitivity of EA.hy926 compared to HUVEC, which should be taken into account when using these cells as models for studying the endothelium radiation response.     

内皮型一氧化氮合酶在急性放射性脑水肿中的表达

目的：探讨大剂量＾６０Ｃｏγ刀照射后脑血管内皮细胞内皮型一氧化氮合酶（ｅＮＯＳ）表达变化及其与急性放射性脑水肿的关系。方法单次２００Ｇｙ＾６０Ｃｏγ刀照射大鼠右侧尾状核头部,于照射后１４ｄ内分批活杀动物,采用光镜,电镜、免疫组化及原位杂交技术研究照射后急性放射性脑水肿的发生发展及脑血管内皮细胞ｅＮＯＳ表达变化。结果照射后２ｈ出现急性放射性脑水肿病理改变。照射后３ｄ达高峰;脑血管内皮细胞ｅＮＯＳ于照     

γ射线照射对两种组织修复细胞的直接效应与W11-a12的作用

目的 探讨电离辐射是否对成纤维细胞和血管内皮细胞的增殖游走功能具有直接抑制作用及其抑制程度,以及W11－a12的促愈使用。方法 采用培养的单层3T3细胞（小鼠胚胎FBs株）和培养的单层ECV304细胞（人脐静脉内皮细胞株）划痕伤口模型进行离体照射,观察痕隙闭合速度。结果 在6Gy照射后培养的单层3T3细胞与单层L－ECV304细胞划痕“伤口”闭合明显延缓。单层3T3细胞在伤后10h对照组伤口完全闭合,而照射组仅闭合77％;照射组单层ECV304细胞在伤后12h仅为对照组的83．6％。W11-a12用药组对单层3T3细胞与ECV304细胞“划痕”伤口的闭合均有促进作用。结论 放射损伤对成纤维细胞与血管内皮细胞的增殖游走有直接抑制作用,这是伤口难愈的重要原因之一。促进伤口细胞游走和增殖是W11－a12作用的一个重要途径。     

大剂量低分割照射大鼠局部肝脏的早期实验研究

目的探讨大剂量低分割照射局部肝脏的耐受剂量和早期病理变化特征。方法 70只W istar大鼠随机分为高剂量（48 Gy）组、中剂量（24 Gy）组、低剂量（8 Gy）组、对照组,设定右肝为照射靶区,8 Gy次/,3次/w,隔日1次。照射后第7、14、28、42 d和2个月时分批处死,取肝组织行常规HE染色和超微结构电镜扫描。结果各剂量组肝脏受照射的体积分数差异无统计学意义（P〉0.05）。高剂量组出现肝小静脉内皮细胞损伤及肝小叶中央静脉和肝窦小静脉阻塞性病变等改变,照射后2个月肝星状细胞激活并有胶原纤维合成。中剂量组4 w内呈现与高剂量组类似改变,6 w后无明显病变。低剂量组和对照组未观察到病理改变。结论当大鼠2/3肝脏受到高剂量照射,早期出现典型的肝小静脉损伤病变,其基础是肝小静脉内皮细胞特别是肝窦内皮细胞持续广泛损伤;2个月后肝脏启动纤维化进程,形成放射性肝损伤。     

缝隙连接蛋白43在受照人血管内皮细胞中的改变及其对受照细胞刚性的作用

目的 研究X射线对人脐静脉内皮细胞（HUVEC）中缝隙连接蛋白43（Cx43）的表达、分布和细胞刚性的影响,初步探讨Cx43对受照细胞刚性的调控作用。方法 采用Western blot方法检测10 Gy X射线照射后不同时间（0、6、12、24和48 h）和不同剂量X射线（0、2.5、5、10和20 Gy）照射后12 h HUVEC细胞中Cx43表达水平的改变,以及不同剂量（0、5和10 Gy） X射线照射后不同时间（3、6、24和48 h） Cx43 3个磷酸化位点（Ser279/282、Ser368和Tyr265）磷酸化水平的变化。采用细胞免疫荧光方法检测Cx43蛋白在受照HUVEC细胞中的分布变化。采用原子力显微镜检测受照细胞在探针压入不同深度（50、100和200 nm）时杨氏模量（细胞刚性）的变化,以及Cx43过表达对受照细胞杨氏模量（细胞刚性）的影响。结果 10 Gy X射线照射后6、12、24、48 h,HUVEC细胞中Cx43表达量降低（t=3.262、3.708、3.686、6.825,P<0.05）,且在2.5、5、10和20 Gy照射后24 h Cx43表达水平的降低与受照剂量呈现剂量依赖性（t=3.034、10.720、13.130、13.650,P<0.05）。5、10和20 Gy X射线照射后24 h,HUVEC细胞中Cx43分布由细胞间隙转移入核及核周,照射后24和48 h,Cx43的Ser368位点磷酸化水平升高并且随剂量增加而增加。10 Gy X射线照射HUVEC细胞后24 h,照射组与对照组相比,在探针压入100和200 nm时,杨氏模量均明显降低（t=3.362、5.122,P<0.05）;过表达Cx43的受照组与空载体受照组相比,在探针压入100和200 nm时,杨氏模量增高（t=2.674、4.398,P<0.05）。结论 X射线照射可导致HUVEC细胞内Cx43 Ser368位点磷酸化,促进Cx43降解和分布变化,降低细胞刚性。提高Cx43的表达水平,有助于受照细胞刚性的恢复,提示Cx43可能是调控X射线致血管内皮细胞损伤的作用靶点。     

In vivo radioprotection of mouse brain endothelial cells by Hoechst 33342

Radiation-induced loss of mouse brain endothelial cells has been examined in mice given an intravenous injection of the DNA-binding radioprotector Hoechst 33342 (80 mg kg-1). At the time of irradiation, 10 min after injection, Hoechst fluorescence in the brain was confined to the endothelial cells. Endothelial cell density was measured using a histochemical fluorescence technique that had been used previously to monitor post-irradiation changes in endothelial cell density in rat brain, in which it was shown that a sensitive subpopulation comprising about 15% of the endothelial cells was lost within 24 h of radiation exposure. The present study shows a similar dose-response for the control mice, with depletion of the sensitive subpopulation to 85% being almost complete after a dose of 2.5 Gy gamma-rays. However, in mice irradiated 10 min after Hoechst 33342 administration, doses between 12 Gy and 20 Gy were required to ablate these cells. The kinetics of cell loss and the rather large dose modification factor suggests that Hoechst 33342 may be suppressing an apoptotic response in this subpopulation. Whatever the mechanism involved, Hoechst 33342 clearly provides substantial protection against early radiation-induced endothelial cell loss. Further studies are necessary to determine the extent to which this initial protection translates into an improved long-term survival of the "protected" cells and, especially, to see whether this endothelial cell protection can ameliorate the later consequences of central nervous system irradiation, namely necrosis and paralysis.     

Mononuclear cell adhesion and cell adhesion molecule liberation after X-irradiation of activated endothelial cells in vitro

PURPOSE: Increased expression of cell adhesion molecules on endothelial cells is an important early event in inflammation. Low-dose radiotherapy is very effective anti-inflammatory treatment. The hypothesis that it may act by modulation of cell adhesion molecule expression in activated endothelial cells and the subsequent adhesion of mononuclear cells onto the activated endothelial cells was tested. MATERIALS AND METHODS: EA.hy.926 endothelial cells were irradiated with 0.3-10 Gy X-rays at different times before or after stimulation with TNFalpha. ICAM-1 or E-selectin expression was measured by ELISA and FACS. Isolated peripheral blood mononuclear cells were incubated with an activated and irradiated confluent monolayer of endothelial cells 4 h, 12 h or 24 h after stimulation, and adhesion was determined in dynamic and static adhesion assays. RESULTS: In the static adhesion assay, where integrin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion of mononuclear cells onto EA.hy.926-EC in vitro by 25-40% and 15-25% of the control level 4 h and 24 h after stimulation, respectively, but increased adhesion 12 h after stimulation. In the dynamic adhesion assay, where selectin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion events by 40-50% and 30-40% of the control level 4 h and 24 h after stimulation, respectively, and again increased adhesion 12h after stimulation. X-ray doses of < or =5 Gy did not induce ICAM-1 expression, or modulate TNFalpha-induced ICAM-1 expression. E-selectin expression was, however, increased in a dose-dependent way 6 h after irradiation. In contrast, X-irradiation 2-5 h before stimulation decreased the characteristic transient expression of E-selectin after TNFalpha stimulation. CONCLUSIONS: Modulation of E-selectin liberation on activated endothelial cells may be one mechanism to decrease leukocyte adhesion after low-dose irradiation in vitro, and could be involved in the therapeutic action of anti-inflammatory radiotherapy.     

Mononuclear cell adhesion and cell adhesion molecule liberation after X-irradiation of activated endothelial cells in vitro

Purpose : Increased expression of cell adhesion molecules on endothelial cells is an important early event in inflammation. Low-dose radiotherapy is very effective anti-inflammatory treatment. The hypothesis that it may act by modulation of cell adhesion molecule expression in activated endothelial cells and the subsequent adhesion of mononuclear cells onto the activated endothelial cells was tested. Materials and methods : EA.hy.926 endothelial cells were irradiated with 0.3-10 Gy X-rays at different times before or after stimulation with TNFα. ICAM-1 or E-selectin expression was measured by ELISA and FACS. Isolated peripheral blood mononuclear cells were incubated with an activated and irradiated confluent monolayer of endothelial cells 4 h, 12 h or 24 h after stimulation, and adhesion was determined in dynamic and static adhesion assays. Results : In the static adhesion assay, where integrin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion of mononuclear cells onto EA.hy.926-EC in vitro by 25-40% and 15-25% of the control level 4 h and 24 h after stimulation, respectively, but increased adhesion 12 h after stimulation. In the dynamic adhesion assay, where selectin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion events by 40-50% and 30-40% of the control level 4 h and 24 h after stimulation, respectively, and again increased adhesion 12h after stimulation. X-ray doses of ≤5 Gy did not induce ICAM-1 expression, or modulate TNF α -induced ICAM-1 expression. E-selectin expression was, however, increased in a dose-dependent way 6 h after irradiation. In contrast, X-irradiation 2-5 h before stimulation decreased the characteristic transient expression of E-selectin after TNF α stimulation. Conclusions : Modulation of E-selectin liberation on activated endothelial cells may be one mechanism to decrease leukocyte adhesion after low-dose irradiation in vitro, and could be involved in the therapeutic action of anti-inflammatory radiotherapy.     

血管内皮生长因子转基因细胞ECV-304辐射生物效应的研究

目的 通过建立正反义血管内皮生长因子(VEGF)转基因细胞系,研究VEGF对血管内皮细胞ECV-304的辐射生物学特性的影响。方法 鉴定VEGF正义表达载体,并构建其反义载体,建立转染VEGF正反义表达载体的血管内皮细胞系;应用MTT法、微核法研究转染细胞系的辐射生物学变化。结果 将VEGF正反义表达载体成功转染到ECV-304中,建立稳定细胞系。与正常细胞相比,转染正义表达载体的ECV-304稳定细胞系经过4 Gy γ射线照射后,细胞促生长率提高至(10.8±1.9)%(P＜0.05),细胞微核率(MNF)从(53.5±6.61)%减少到(42.5±2.46)%(P＜0.01),微核细胞率(MNCF)从(31.6±5.23)%减少至(23.7±2.27)%(P＜0.05);而转染反义表达载体的细胞系细胞促生长率降低至(-9.2±2.8)%(P＜0.05),MNF、MNCF分别增加至(62.1±1.96)%(P＜0.01)、(38.1±1.47)%(P＜0.05)。结论 VEGF对血管内皮细胞具有辐射防护作用,为今后进一步研究放射性溃疡的转基因治疗提供了实验和理论依据。