透明质酸钠在甘油冷冻保存角膜中的应用

目的 评价甘油冷冻保存角膜的效果以及透明质酸钠对保存角膜内皮细胞(CEC)的保护作用.方法 20只兔眼角膜片随机分为两组.对照组角膜直接放入纯甘油中,-25℃保存,实验组则在内皮面均匀黏附透明质酸钠后再放入甘油中低温保存.保存2个月后两组均接受CEC锥蓝-茜素红活性染色检查、电镜检查以及穿透性角膜移植(PKP)实验,以检验保存效果.结果 两组CEC均能保持细胞活性,实验组的保存效果要好于对照组,其CEC密度为3 067.9±127.6/mm2,死亡率为4.2%±3.4%,且行PKP较对照组有更高的成功率和CEC密度.结论 甘油冷冻能够保持CEC的活性,透明质酸钠能进一步增进保存效果,改良后的甘油冷冻保存方法有希望成为一种方便、经济、有效的角膜保存方法.     

血液低温保存国内外研究进展

自194 9年英国学者Polge发现了甘油的低温保护作用以来,血细胞冰冻保存技术取得了较快的发展并逐步应用于临床。目前,血细胞深低温保存已发展为红细胞、干细胞及血小板的深低温保存。近年来,随着我国临床用血的快速增长和无偿献血制度的实施,血液供需矛盾日趋突出,血细胞深低温保存技术对调节临床合理用血、稀有血型输血、骨髓干细胞移植、肿瘤治疗及战备贮血等均有着重要意义。本文对近几年来血细胞深低温保存的原理与方法、低温保存剂的种类及作用机制等作一浅述。1　血细胞深低温保存的原理和方法血细胞离体后,可以在一种类似人体的生理…     

心脏保存液种类及保存时间对离体鼠心低温保存效果的对比研究

目的研究3种心脏保存液[HTK液、FWM液和St.Thomas-Ⅱ(ST-Ⅱ)]在低温条件下保存离体鼠心的效果。方法54只雄性SD大鼠随机均分为HTK组、FWM组、ST-Ⅱ组(n=18),各组再根据低温保存时间分为4、6、8h3个亚组(n=6)。采用Langen-dorff装置进行心脏保存前后的灌注,各实验组分别使用不同保存液灌停心脏并在低温条件下保存4、6、8h,于灌停前及复灌后测定心率(HR)、冠脉流量(CF)、左心室收缩压(LVSP)、左心室内压最大上升速率(+dp/dtmax)、左心室内压最大下降速率(-dp/dtmax)等指标,并计算各指标的恢复率。灌注结束后,取心肌组织制作光镜标本,观察心肌组织结构变化。结果在相同的低温保存时间下,与ST-Ⅱ组相比,HTK组与FWM组心功能各指标恢复率明显较高,光镜下心肌组织结构变化较小。随保存时间延长,使用HTK液保存的鼠心心功能恢复率下降,组织结构发生改变,8h亚组与4、6h亚组间比较差异显著(P<0.05),而4h亚组与6h亚组间无显著差异(P>0.05)。结论在保存时间相同的条件下,HTK液及FWM液的心肌保存效果明显优于ST-Ⅱ液。HTK液保存效果随低温...     

大鼠肝细胞球形体低温保存条件的初步探索

目的探索有效的肝细胞球形体低温保存方法,促进生物人工肝及其他研究的应用。方法采用两步法分离大鼠肝细胞,经无血清培养基(SFM)摇摆培养48h形成肝细胞球形体。将球形体继续培养(对照组),或置于4℃SFM,SFM+1mmol/L去铁胺(Def),SFM+1μmol/L环孢霉素A(Cs A),SFM+1mmol/L Def+1μmol/L Cs A保存液中24h或48h,继续培养4d或5d后观察低温保存后肝细胞球形体的存活率、超微结构变化,以及白蛋白和尿素合成情况。结果Def和Cs A能很好地保护球形体肝细胞,在4℃SFM+Def+Cs A保存液中保存24h,肝细胞球形体功能、结构等变化及尿素合成水平与对照组比较差异无统计学意义,而单独放在SFM中进行低温保存后,细胞活性明显下降。在4℃保存48h后,球形体肝细胞超微结构发生明显变化,死亡细胞增多,但SFM+Def+Cs A组和SFM+Def组细胞结构优于SFM组和SFM+Cs A组,细胞存活率及尿素合成水平明显高于后两组(P<0.05),但合成白蛋白量均处于较低水平,各组间差异无统计学意义(P>0.05)。结论低温状态下肝细胞球形体可保持良好的存活率和功能24h,低温保存能为细胞治疗提供良好的细胞材料。     

青海省稀有血型红细胞深低温保存血库的建立及运行

目的：探讨青海省稀有血型红细胞深低温保存血库的建立及运行的必要性和意义。方法：将合格的Rh（D）阴性的红细胞制备为冰冻红细胞置-86℃深低温储血冰箱保存,建立青海省稀有血型红细胞深低温保存血库。结果：我们已建立了117U的稀有血型深低温保存血库,39U已应用到临床,Rh（D）阴性血液的阳性使用率由2003年的60.84%降至2007年的16.55%。结论：稀有血型红细胞深低温保存血库的建立大大降低了稀有血型血液资源浪费,提高了我省血液储备和应对公共卫生突发事件血液供应能力,对保障西宁地区、六州一地一市乃至周边省份稀有血型急救用血的及时供给具有积极的现实意义。     

犬肺移植中供体肺组织形态学与超微结构计量学研究

目的：观察改进EC液低温灌注犬肺离体保存肺组织随保存时间变化而产生的结构变化。方法：4℃EC液灌注6只健康成年杂种犬肺并离体低温保存，灌注结束后保存30、60、120、180、240min取材制作组织标本，观察不同时点肺组织形态学变化，测试肺组织湿干重比(W／D)与肺泡上皮细胞核的体积密度(vv)、表面积密度(Sv)、表面积与体积比(Rsv)，并以灌注前为对照组。结果：随保存时间延长肺组织细胞水肿渐趋明显，肺组织W／D、肺泡上皮细胞核的Vv、Sv、Rsv实验组与对照组相比差异无显著性(P＞0．05)。结论：Vv、5v、Rsv可作为肺保存过程中评价损伤程度的客观指标，改进后的EC保存液可用于供体肺保存。     

附加二氮嗪与1,6-二磷酸果糖心麻痹液对冷保存大鼠心脏缺血再灌注损伤的保护作用

目的 观察附加二氮嗪（DZ）与1,6-二磷酸果糖（FDP）心麻痹液对冷保存大鼠心脏缺血再灌注损伤的作用。方法 SD大鼠16只,随机分为2组（n=8）,STH组（St．ThomasⅡ号液）和DF组（St．ThomasⅡ号液中附加DZ50μmol／L、FDPSmmol／L）。心脏在4℃条件下保存6小时后,采用离体左心做功模型再灌注30分钟,测定冠脉流出液中乳酸脱氢酶（LDH）、磷酸肌酸激酶（CK）、一氧化氮（NO）及心肌组织丙二醛（MDA）的含量,观察心肌和内皮细胞超微结构的改变。结果DF组再灌注后冠脉流出液中CK、LDH、MDA含量明显低于STH组（P＜0．01）,NO含量则明显高于STH组（P＜0．05）,心肌细胞和内皮细胞的超微结构损伤较STH组轻。结论附加DZ与FDP的心麻痹液对冷保存大鼠心脏缺血再灌注损伤具有较好的保护作用。     

中医温病温热证动物模型实验的研究

目的：制备中医温病湿热症的动物模型。方法：根据中医有关温病湿热证的病因病理理论及临床湿热病的发病症状，主要从“湿”、“热”和“邪毒”三个方面模拟湿热病发病条件，对大鼠施加各种处理因素。结果：给大鼠高脂高糖、高温高湿及病原微生物后，模型大鼠红细胞超氧化物岐化酶（SOD）活性显降低，而丙二醛（MDA）含量显升高，与临床报道结果相符合。而单纯改变饮食环境条件，大鼠SOD活性改变，MDA水平无显变化。在三种因素同时作用下，模型大鼠红细胞免疫功能显改变，大鼠红细胞C3b受体数降低，而免疫复合物花环数升高。结论：湿热模型的研究结果表明，临床湿热证与多因素同时作用有关，其造模方法简单，重复性好。     

细胞间粘附分子-1在细菌性角膜炎角膜组织表达的实验研究

为探讨细胞间粘附分子 1(ICAM 1)在细菌性角膜炎发病机制中的作用 ,笔者研究了大鼠细菌性角膜炎角膜组织ICAM 1的表达及分布。SD大鼠 2 2只随机分为细菌性角膜炎组和对照组。应用抗ICAM 1单克隆抗体及免疫组化染色技术观察角膜上皮细胞、基质层角膜细胞及内皮细胞的特异性染色。结果显示 ,对照组角膜上皮细胞及内皮细胞染色程度为 - + ,基质层角膜细胞染色为 + ;角膜炎组角膜上皮细胞、基质层角膜细胞及内皮细胞染色程度 。细菌性角膜炎组角膜上皮细胞、基质层角膜细胞及内皮细胞ICAM 1的表达较对照组明显增强 (P <0 .0 1)。提示ICAM 1在细菌性角膜炎的炎细胞游走、浸润等病理改变中发挥重要作用。     

长期甘油保存角膜的光镜和电镜观察及临床应用

1　角膜材料及保存　人尸角膜 7只 ,供体年龄 1834岁。于供体猝死后 1ｈ内无菌操作摘除眼球 ,湿房保存送回眼科。保存前 ,用含有庆大霉素的生理盐水 (10 0 0Ｕ/ｍｌ)冲洗眼球 1次 ,再用生理盐水冲洗 2次。然后无菌制作角膜片 ,将角膜片置入 95 %的灭菌甘油 5ｍｌ瓶内脱水 2 4ｈ ,然后转移至另一瓶同样的甘油中 ,胶纸封口 ,4℃冰箱保存。2　手术方法　取角膜 ,置室温 1ｈ ,经生理盐水冲洗后 ,置庆大霉素生理盐水 (10 0 0Ｕ/ｍｌ)溶液中复水 30ｍｉｎ。根据植床的大小及形状 ,取比植床直径大 0 2 0 2 5ｍｍ的植片 ,植片厚度与植床深度吻合。…     

晚期氧化蛋白产物导致血管内皮细胞氧化应激损伤

目的 研究慢性肾功能衰竭病人循环晚期氧化蛋白产物(AOPP)对人血管内皮细胞活性的影响及机制。方法 用次氯酸(HOCl)氧化牛血清白蛋白(BSA)体外制备AOPP-BSA,将人血管内皮细胞株ECV304与不同浓度、不同氧化修饰程度的AOPP-KSA共同培养,用MTT法测定细胞活性,用VICTOR Wallac 1420多标记分析系统动态检测细胞内活性氧的产生量。结果 AOPP-BSA使血管内皮细胞活性降低,其对内皮细胞活性的影响与BSA氧化程度及AOPP-BSA的浓度有关;AOPP-BSA刺激内皮细胞生成活性氧,细胞内活性氧的生成量随KSA氧化程度和AOPP-BSA浓度增加而升高。用硒谷胱甘肽过氧化物酶小分子模拟物ebselen预处理细胞,可使AOPP-BSA诱导产生的活性氧减少53％,并可保护细胞免受AOPP-BSA造成的细胞损伤。结论 晚期氧化蛋白产物能通过氧化应激引起血管内皮细胞损伤。     

乙酰胆碱对人表皮细胞冷存的影响

目的：探讨乙酰胆碱（Ach）对人表皮细胞冷存活力的影响。方法：应用人表皮细胞的分离、培养技术，并进行MTT检测、病理切片以及电镜检测等手段进行观察。结果：不同浓度的Ach对冷存的表皮细胞均有一定的保存活力作用，其中以10^-1-10^-10mol/L的浓度为最佳。结论：认为Ach对冷存中的人表皮细胞有一定的保存作用，它可提高表皮细胞的保存活力。     

氯吡格雷对动脉粥样硬化兔血管内皮细胞功能的影响

目的：用高脂饲喂建立家兔动脉粥样硬化硬化模型,预防性给予氯吡格雷治疗,连续12周。方法：观察血清细胞间黏附分子（ICAM-1）、一氧化氮（NO）、内皮素（ET）及血管内皮细胞生长因子（VEGF）的水平,并观察其主动脉血管病理变化。结果：与单纯高脂组比较,氯吡格雷治疗后NO明显升高,ICAM-1、ET降低,VEGF的表达量显著下降（P〈0.01）;血管内膜增生减弱,斑块面积明显减小（P〈0.01）。结论：提示氯吡格雷具有保护血管内皮细胞的功能,可抑制斑块进展。     

Relative biological effectiveness (RBE) of 210 Po alpha-particles versus X-rays on lethality in bovine endothelial cells

Purpose : Alpha-radiation from polonium-210 (210 Po) can elevate background radiation dose by an order of magnitude in people consuming large quantities of meat and seafood, particularly caribou and reindeer. Because up to 50% of the ingested 210 Po body burden is initially found in the blood, a primary target for the short range alpha-particles is the endothelial cells lining the blood vessels. This study examined the relative biological effectiveness (RBE) of 210 Po alpha-particles versus 250 kVp X-rays in producing injury to cultured bovine aortic endothelial cells. Materials and methods : Radiation effects on cells were measured in four different ways: the percentage viable cells by trypan blue dye exclusion, the number of live cells, the lactate dehydrogenase (LDH) release to medium and the ability to form colonies (clonogenic survival). Results : Comparison of dose-response curves yielded RBE values of 13.1 ±2.5 (SEM) for cell viability, 10.3 ±1.0 for live cell number and 11.1 ±3.0 for LDH activity. The RBE values for clonogenic survival were 14.0 ±1.0 based on the ratio of the initial slopes of the dose-response curves and 13.1, 9.9 and 7.7 for 50, 10 and 1% survival rate, respectively. At X-ray doses <0.25 Gy, a pronounced stimulatory effect on proliferation was noted. Conclusions : Exposure to 210 Po alpha-particles was seven to 14 times more effective than X-ray exposure in causing endothelial cell damage.     

In vitro response of human and porcine vascular cells exposed to high dose-rate γ-irradiation

Aim : The objective of this study was to compare the in vitro response of human and pig endothelial cells, smooth muscle cells and fibroblasts exposed to conventional high dose-rate γ-irradiation. Materials and methods : Clonogenic cell survival and growth responses were obtained after irradiation of plateau-phase cells with a 60 Co source at a dose-rate of 1.5 Gy/min. DNA single-strand breaks were also evaluated using an alkaline filter elution technique. Results : Overall, both the pig and human cell lines showed a similar response to conventional high dose-rate irradiation. Using clonogenic assays, the human aortic smooth muscle cell line was more sensitive than the fibroblast and endothelial cell lines, whereas the pig endothelial cell line was more sensitive than smooth muscle cells and fibroblasts. Shortly after irradiation (10 days) there was a temporary growth arrest, which was similar for endothelial, smooth muscle cells and fibroblasts with doses above 6 Gy. There was also a non-linear, dose-dependent growth delay up to 4 weeks after irradiation. This effect was also consistent between the different cell lines. Using alkaline filter elution, there was no significant difference in relative elution between endothelial cells, smooth muscle cells and fibroblasts, indicating similar DNA damage among the different cell lines. Conclusion : The in vitro response of human and pig endothelial cells, smooth muscle cells and fibroblasts exposed to high doserate irradiation appeared similar. The pig model seems well suited to evaluate the short- and long-term effects of ionizing radiation in the prevention of restenosis after vessel injury.     

Calcium-dependent injury of human microvascular endothelial cells induced by a variety of iodinated radiographic contrast media

RATIONALE AND OBJECTIVES: The aim of the present study was to determine the possible mechanisms underlying the endothelial cell damage induced by iodinated radiographic contrast materials (RCM). METHODS: The cultured human skin microvascular endothelial cells (HMVECs) were exposed to various contrast media, and the cell viability was measured by mitochondrial enzyme activity. Nuclear damage was assessed by Hoechst 33342 staining and a fluorescent single-cell gel electrophoresis. The effects of contrast materials on the cellular ATP content and intracellular free Ca2+ concentration were subsequently examined. RESULTS: Although the iodinated RCM tested all caused the cell injury in HMVECs, ionic RCM including amidotrizoate and ioxaglate were more potent in producing the cell damage than nonionic RCM. It is unlikely that the contrast material-induced cell damage is associated with hyperosmolality, since hyperosmolar solution of mannitol or NaCl had no marked influence on the endothelial cell viability. Nuclear damage was noted in cells exposed to amidotrizoate. Amidotrizoate lowered cellular ATP content while elevating the intracellular free Ca2+ concentration. It was notable that the RCM-induced endothelial cell damage was reversed by the chelation of intracellular Ca2+ with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the removal of extracellular Ca2+. CONCLUSIONS: Both ionic and nonionic contrast materials caused nuclear damage of endothelial cells. The decrease in tissue ATP content and elevation of intracellular Ca2+ are likely to contribute to the contrast materials-induced endothelial cell damage.     

Relative biological effectiveness (RBE) of alpha radiation in cultured porcine aortic endothelial cells

PURPOSE: Northern peoples can receive elevated radiation doses (1- 10 mSv/y) from transfer of polonium-210 (210Po) through the lichen-caribou-human food chain. Ingested 210Po is primarily blood-borne and thus many of its short range alpha particles irradiate the endothelial cells lining the blood vessels. The relative biological effectiveness (RBE) of alpha particles vs. x-rays was examined in porcine aortic endothelial cells as a surrogate for understanding what might happen to human endothelial cells in northern populations consuming traditional foods. MATERIALS AND METHODS: Cultured porcine aortic endothelial cells were exposed to x-ray and 210Po alpha particle radiation. Alpha irradiation was applied to the cell cultures internally via the culture medium and externally, using thin-bottomed culture dishes. The results given here are based on the external irradiation method, which was found to be more reliable. Dose-response curves were compared for four lethal endpoints (cell viability, live cell fraction, release of lactate dehydrogenase [LDH] and clonogenic survival) to determine the relative biological effectiveness (RBE) of alpha radiation. RESULTS: The alpha RBE for porcine cells varied from 1.6-21, depending on the endpoint: 21.2+/-4.5 for cell viability, 12.9+/-2.7 for decrease in live cell number, 5.3+/-0.4 for LDH release to the medium but only 1.6 +/-0.1 for clonogenic survival. The low RBE of 1.6 was due to x-ray hypersensitivity of endothelial cells at low doses.     

Targeting liposomes to tumor endothelial cells for neutron capture therapy.

The aim of our work is to target (10)B to the tumor vasculature for neutron capture therapy (NCT). Alpha (v)-integrin specific RGD-peptides were coupled to liposomes that encapsulated dodecahydrododecaborate. These RGD-liposomes strongly associated with human umbilical vein endothelial cells (HUVEC) expressing this integrin and were internalized. Proliferating HUVEC proved sensitive to treatment with gamma-irradiation resulting in decreased cell viability and pronounced inhibition of DNA-synthesis with increasing dose. Irradiation of RGD-(10)B-liposome incubated HUVEC with neutrons strongly inhibited endothelial cell viability. These results suggest that efficient NCT can be achieved by targeting (10)B-liposomes to angiogenic endothelium in tumors.     

Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging

PURPOSE: To evaluate the effect of using the ferumoxides-poly-l-lysine (PLL) complex for magnetic cell labeling on the long-term viability, function, metabolism, and iron utilization of mammalian cells. MATERIALS AND METHODS: PLL was incubated with ferumoxides for 60 minutes, incompletely coating the superparamagnetic iron oxide (SPIO) through electrostatic interactions. Cells were coincubated overnight with the ferumoxides-PLL complex, and iron uptake, cell viability, apoptosis indexes, and reactive oxygen species formation were evaluated. The disappearance or the life span of the detectable iron nanoparticles in cells was also evaluated. The iron concentrations in the media also were assessed at different time points. Data were expressed as the mean +/- 1 SD, and one-way analysis of variance and the unpaired Student t test were used to test for significant differences. RESULTS: Intracytoplasmic nanoparticles were stained with Prussian blue when the ferumoxides-PLL complex had magnetically labeled the human mesenchymal stem and HeLa cells. The long-term viability, growth rate, and apoptotic indexes of the labeled cells were unaffected by the endosomal incorporation of SPIO, as compared with these characteristics of the nonlabeled cells. In nondividing human mesenchymal stem cells, endosomal iron nanoparticles could be detected after 7 weeks; however, in rapidly dividing cells, intracellular iron had disappeared by five to eight divisions. A nonsignificant transient increase in reactive oxygen species production was seen in the human mesenchymal stem and HeLa cell lines. Labeled human mesenchymal stem cells did not differentiate to other lineage. A significant increase in iron concentration was observed in both the human mesenchymal stem and HeLa cell media at day 7. CONCLUSION: Magnetic cellular labeling with the ferumoxides-PLL complex had no short- or long-term toxic effects on tumor or stem cells.     

Relative biological effectiveness (RBE) of 210Po alpha-particles versus X-rays on lethality in bovine endothelial cells

PURPOSE: Alpha-radiation from polonium-210 ((210)Po) can elevate background radiation dose by an order of magnitude in people consuming large quantities of meat and seafood, particularly caribou and reindeer. Because up to 50% of the ingested (210)Po body burden is initially found in the blood, a primary target for the short range alpha-particles is the endothelial cells lining the blood vessels. This study examined the relative biological effectiveness (RBE) of (210)Po alpha-particles versus 250 kVp X-rays in producing injury to cultured bovine aortic endothelial cells. MATERIALS AND METHODS: Radiation effects on cells were measured in four different ways: the percentage viable cells by trypan blue dye exclusion, the number of live cells, the lactate dehydrogenase (LDH) release to medium and the ability to form colonies (clonogenic survival). RESULTS: Comparison of dose-response curves yielded RBE values of 13.1+/-2.5 (SEM) for cell viability, 10.3+/-1.0 for live cell number and 11.1+/-3.0 for LDH activity. The RBE values for clonogenic survival were 14.0+/-1.0 based on the ratio of the initial slopes of the dose-response curves and 13.1, 9.9 and 7.7 for 50, 10 and 1% survival rate, respectively. At X-ray doses <0.25 Gy, a pronounced stimulatory effect on proliferation was noted. CONCLUSIONS: Exposure to (210)Po alpha-particles was seven to 14 times more effective than X-ray exposure in causing endothelial cell damage.