Optical Pretargeting of Tumor with Fluorescent MORF Oligomers

Objective Pretargeting with radioactivity has significantly improved tumor to normal tissue radioactivity ratios over conventional antibody imaging in both animal studies and clinical trials. This laboratory is investigating DNA analogues such as phosphorodiamidate morpholinos (MORFs) for pretargeting using technetium-99m (^99mTc) for detection. However, the unique properties of fluorescence activation and quenching combined with oligomers with their unique properties of hybridization may be particularly useful when used together for pretargeting with optical detection. The use of linear fluorophore-conjugated oligomer duplexes have been little used in animals, and to our knowledge, have not previously been considered for pretargeting applications. Methods A MORF/cDNA pair was selected such that when hybridized, the fluorescence of the Cy5.5-conjugated 25 mer MORF (Cy5.5–MORF25) is inhibited with a BHQ3-conjugated 18 mer complementary DNA (BHQ3–cDNA18). The short BHQ3–cDNA18 was selected to dissociate in the presence of a long cMORF25 in the pretargeted tumor, thus releasing the inhibitor from the Cy5.5 emitter. In this manner, the Cy5.5 fluorescence will be inhibited everywhere but in the target. The dissociation was first examined in vitro by adding the duplex to the cMORF25 both in solution and immobilized on polystyrene microspheres and by surface plasmon resonance (SPR). Thereafter, biotinylated cMORF25 immobilized on streptavidin polystyrene microspheres were administered intramuscularly in one thigh of hairless SKH-1 mice as target while an identical weight of the identical microspheres but without the cMORF25 was administered in the contralateral thigh as control. The animals then received IV the Cy5.5–MORF25/BHQ3–cDNA18 duplex or equal molar dosage of single-chain Cy5.5–MORF25 and were imaged. Results The SPR studies showed that the immobilized cDNA18 rapidly captured the flowing MORF25 to provide a duplex with a slow dissociation rate constant. Furthermore, when cMORF25 was next allowed to flow over the now immobilized duplex, the cDNA18 was unable to prevent dissociation of the heteroduplex and the formation and release of the cMORF25-MORF25 homoduplex. Images of animals obtained soon after receiving the Cy5.5–MORF25 singlet showed intense whole body fluorescence obscuring the target thigh. However, only 5 minutes after receiving the Cy5.5–MORF25/BHQ3–cDNA18 duplex, the target thigh was clearly visible along with only the kidneys. Conclusions This first study of optical pretargeting provides a proof of concept that oligomer pretargeting found to be useful with radioactivity detection is applicable with fluorescent detection as well. In addition, our results demonstrate that by using linear oligomers for optical pretargeting, chain lengths (and base sequences) may be manipulated to provide duplexes with stabilities and fluorescence inhibition optimized for pretargeting and other in vivo applications of optical imaging.     

眼蛋白质组学

蛋白质组学是对一个细胞、组织或器官的基因组表达的所有蛋白质分析研究，是新世纪生命科学研究的前沿以及功能基因组学的研究重心。蛋白质组学为研究眼部细胞生命活动规律和眼科疾病病变机制提供了一个新的研究平台。本文就蛋白质组学在眼科的应用作一综述。眼科学报2003；19：67-70     

角膜移植术后角膜在共焦显微镜下的形态学改变

目的：研究角膜移植术后角膜组织在共焦显微镜(Confocal microscopy)下的形态学改变。方法：应用Conoscan2．0共焦显微镜对板层角膜移植术后3～7d患者12例(12只眼)，术后1a患者8例(8只眼)，穿透性角膜移植术后3～7d患者10例(10只眼)，术后1a患者11例(11只眼)进行扫描检查，记录各层角膜图像。结果：板层角膜移植术后3～7d，植片中基质细胞较小，可见裂隙状暗纹和细小神经，层间为大面积高反光区，有点状颗粒沉积，植床水肿，细胞成像不清。移植术后1a，植片中未见神经，层间反光明显减弱，但仍有点状高反光颗粒沉积，植床中出现粗大裂隙状暗纹，内皮细胞密度正常。穿透角膜移植术后3～7d，植片中基质细胞“激活”，可见神经和后基质层的粗大暗纹，内皮细胞密度正常，细胞间可镶嵌有高反光点。术后1a，植片中基质细胞仍较小，未见神经，后基质层仍有裂隙状暗纹，内皮细胞体积增大，密度减少，高反光点消失。结论：Confoscan 2．0共焦显微镜可活体检查角膜移植术后角膜组织结构和细胞的病理改变，这对评估手术效果和临床观察以及跟踪随访具有重大意义。     

外伤性视神经病变的临床治疗分析

目的　探讨大剂量甲基强的松龙冲击疗法治疗外伤性视神经病变的疗效。方法　 2 0例 (2 0只眼 )外伤性视神经病变患者为治疗组 ,应用大剂量甲基强的松龙冲击治疗 ,另 2 0例 (2 0只眼 )外伤性视神经病变患者为对照组 ,使用常规剂量的地塞米松治疗。两组均同时使用高渗剂、血管扩张剂、神经营养剂及 B族维生素治疗 ,对两组病例药物治疗的结果进行总结分析。结果　治疗组经治疗后视力开始恢复的时间早于对照组 ,治疗组的总有效率为 80 % ,对照组的总有效率为 6 0 % ,治疗组的疗效优于对照组 ,且治疗越早疗效越好。两组比较具有显著的统计学差异 (P <0 .0 5 )。结论　大剂量甲基强的松龙冲击疗法治疗外伤性视神经病变疗效显著 ,治疗方便 ,是较理想的治疗方法     

冠心病患者低密度脂蛋白中甘油三酯异常特征

本文检测了不同年龄健康人和冠心病（ＣＨＤ）患者血脂及各脂蛋白组分中甘油三脂（ＴＧ）、胆固醇（ＣＨ）水平，结果示总ＴＧ、低密度脂蛋白（ＬＤＬ）－ＴＧ、ＬＤＬ－ＴＧ／ＬＤＬ－ＣＨ比值升高，为ＣＨＤ最为显著的脂质异常特征。分析了ＣＨＤ患者ＬＤＬ脂质构成异常的特征及成因，认为高ＬＤＬ－ＴＧ／ＬＤＬ－ＣＨ比值为冠心病的危险因素。     

毒黄素对血清乳酸脱氢酶同工酶的影响

目的 探讨毒黄素对某些器官的中毒机理。方法 利用从酵米面椰毒假单胞菌中所分离提取出的毒黄素对１８例人血清中的乳酸脱氢酶（ＬＤＨ）及其同工酶进行测定。结果 乳酸脱氢酶总活力下降，ＬＤＨ１受抑制，活性下降，差异显著。结论 毒黄素可抑制心肌能量供应，毒害心肌。     

50岁以上正常人左室舒张期彩色多普勒血流频谱参数变化的研究

目的探讨５０岁以上正常人左室舒张期脉冲多普勒频谱的参数变化情况。方法选择一年半时间内受检的所有５０岁以上正常人４７５例，按每１０岁为一组分４组进行分析。受检者取左侧卧位，常规显示心尖四腔心切面，取样容积置于二尖瓣尖，使声束与二尖瓣血流方向夹角＜１５°，以连续三个心动周期的平均值分别测量Ｅ峰、Ａ峰、Ｅ／Ａ及Ｅ峰减速时间并统计。结果发现Ｅ峰高于正常值低限的例数、平均Ｅ峰血流速度以及Ｅ／Ａ均随着年龄的增加而下降，Ｅ峰减速时间则随着增龄而增加。结论左室舒张期血流频谱的所有参数与年龄有显著的相关性，明显随着增龄而变化，并且有其规律性。     

成人原发性肾病综合征患者尿视黄醇结合蛋白的测定及意义

采用酶联免疫法检测１００例正常人及７９例成人原发性肾病综合征治疗前后尿视内醇结合蛋白的变化。追踪随访２年，发现经治疗后病理类型为ＥＣＰＧＮ，ＭＣＮ，ＩｇＡ ＧＮ者，尿蛋白及ＲＢＰ均明显减少，其肾功能不全的发生率较低，而ＭＧＮ，ＦＳＧＳ，ＳＧＮ者，尿蛋白及ＲＢＰ无明显减少，肾功能不全的发生率较高，ＭｓＰＧＮ因其增生程度的不同，治疗效果及预后有所不同，轻度增生者治疗效果及预后较好，中，重度增生者，基