99 Tcm-HYNIC-Annexin V荷瘤小鼠肿瘤细胞凋亡显像研究

目的研究99Tcm-联肼尼克酰胺-膜联蛋白V(HYNIC-Annexin V)的制备及其在荷瘤小鼠体内生物分布特性和活体显像.方法用双功能螯合剂法,以99Tcm标记Annexin V,经高效液相色谱仪分离纯化并检测产物的标记率、放化纯及稳定性.建立荷S180肉瘤小鼠模型,于20～25g/只昆明种小白鼠右前腋皮下接种S180肉瘤细胞1周.荷瘤小鼠在环磷酰胺腹膜内化疗72h后,尾静脉注射7.4 MBq(200μl)99Tcm-HYNIC-Annexin V,分别于5、30min和1、3、6 h进行显像和体内生物分布测定.实验结果应用SPSS 12.0统计软件进行分析.结果 99Tcm-HYNIC-Annexin V的标记率达95%,放化纯为99%.荷瘤小鼠注射99Tcm-HYNIC-Annexin V后6 h显像,可见肿瘤组织放射性浓聚明显异常.体内生物分布实验示,注射显像剂后6 h肿瘤组织的放射性摄取最高[每克组织百分注射剂量率(%ID/g)为1.59±0.44],与其他时相比较,差异有显著性(P＜0.05).99Tcm-HYNIC-Annexin V主要浓聚于肾、肺和肝,经肾脏排泄;其在血液中清除速度快,注射后30 min,血液放射性摄取[(1.59±0.50)%ID/g]较注射后5 min时[(8.85±2.65)%ID/g]减少80%(P＜0.05).注射显像剂后6 h,肿瘤/肌肉放射性摄取比值(3.73±1.42)高于肿瘤/血液(2.80±0.54).结论 99Tcm-HYNIC-Annexin V活体细胞凋亡显像可应用于荷瘤小鼠模型,其临床应用有待进一步研究.     

99Tcm标重组H-Annexin Ⅴ在动物血栓显像中的研究

目的 评价人膜联蛋白V（H—Annexin V）对血栓的亲和性，对^99Tc^m直接标记H—Annexin V与通过联肼尼克酰胺（HYNIC）螫合标记AnnexinV进行比较。方法 在3种不同的反应条件下，分别用^99Tc^m of直接标记AnnexinV、H—AnnexinV及HYNIC—AnnexinV，然后测定标记率、稳定性及对兔股动脉血栓模型进行γ显像，比较损伤侧（T，血栓）及对侧（NT）的放射性比值。结果 在还原剂足量的情况下，^99Tc^mO4^-直接标记H—Annexin V方法简便，反应条件易于控制，^99Tc^m-H—Annexin V标记率达95％以上，17h后测放化纯仍〉80％，血栓显像示动脉损伤处有明显的放射性浓聚，损伤处平均放射性计数为健侧放射性计数的3．714倍。结论 ^99Tc^m-H—AnnexinV与血栓具有很好的亲和力，标记率及稳定性高，标记方法较为简单。     

放射性核素细胞凋亡显像

放射性核素细胞凋亡显像具有直观、无创伤和实时在体观察细胞凋亡等特点,对于脑、心肌细胞缺血再灌注损伤的观察,器官移植排斥反应的监测,肿瘤放疗和化疗效果的评估等方面的应用前景良好,但仍有许多问题有待于进一步探讨.     

99Tcm-Annexin Ⅴ衍生物的制备及其生物分布和凋亡显像

目的 探讨^99Tc^m直接标记带有10个连续组氨酸的膜联蛋白V（Annexin V）衍生物的可行性，研究其在健康小鼠体内的分布和肺癌裸鼠模型凋亡显像。方法 直接标记法制备^99Tc^m-Annexin V衍生物，并测产物放化纯、胶体含量及健康小鼠体内不同时间点（5，30min和1，2,4，6，12，24h）的生物分布，DAS2．0软件拟合计算药代动力学参数。建立荷H460非小细胞肺癌裸鼠模型，分为紫杉醇诱导化疗组（5只）和未治疗对照组（5只）。紫杉醇诱导化疗后48h，于裸鼠尾静脉注射^99Tc^mAnnexinV衍生物18．5MBq，2和4h时进行凋亡显像，勾画ROI并计算肿瘤与对侧正常组织（T／NT）放射性比值。结果放化纯达（98．01±1．67）％，3h后放化纯仍可达（95．45±1．34）％，胶体含量为（3．31±1．37）％。健康小鼠体内分布实验表明肾脏对该示踪剂摄取最高，肝、脾摄取较少。DAS2．0软件拟合得到时间-放射性曲线符合二室模型特征，分布相半衰期（T1/2α）为（21．18±5．95）min，清除相半衰期（T1/2β）为（69．32±0．10）min。荷瘤鼠显像示，给药后2h即可获得清晰的图像，紫杉醇诱导化疗组2和4h的T／NT比值为3．85±1．10和4．63±0．97，明显高于对照组（1．60±0．29和1．71±0．43，P〈0．01）。结论 该AnnexinV衍生物易于^99Tc^m直接标记，方法简便，标记物在血液中清除迅速，肝、脾摄取少，易得到高清晰图像。     

用99Tcm-rh-Annexin V检测放疗或化疗后肿瘤细胞凋亡

目的 验证99Tcm直接法标记的重组人膜联蛋白V(99Tcm-rh-AnnexinV)用于早期评价放化疗后肿瘤组织细胞凋亡状态的可行性.方法 将小鼠肝癌细胞(Hca-F25)接种于实验小鼠右腋下,8 d后当肿瘤生长至直径约1 cm时,将模型鼠分为A(化疗组,13只)、B(放疗组,10只)、C(荷瘤对照组,12只)3组.其中A、C组按注射示踪剂后不同时间(1、4、6 h)各分为3个亚组(A1组3只,A2组4只,A3组6只;C1组3只,C2组4只,C3组5只);A组接受环磷酰胺单次化疗(按体重150 mg/kg,腹腔内注射),C组腹腔内注射同等量生理盐水,20h后A、C组鼠尾静脉注射99Tcm-rh-Annexin V 3.7MBq(0.5μg)/只.B组按不同吸收剂量(2、5、10 Gy)分为3个亚组(B1组4只,B2组3只,B3组3只),放疗后1 h注射与A、C组相同剂量示踪剂,6 h后显像.小鼠模型显像后即刻处死,取组织(肿瘤、血、肌肉)称重后,测量放射性计数,并计算每克组织百分注射剂量率(%ID/g)及肿瘤(T)/肌肉(M)、T/血液(B)放射性比值,应用流式细胞仪检测细胞凋亡百分率.结果 A、B组小鼠肿瘤组织中凋亡细胞百分率[(11.16±3.83)%、(17.40±5.20)%]均明显高于C组[(2.11±1.51)%,F=56.414、94.748,P＜0.001];A2、A3及B组肿瘤组织%ID/g均明显高于C2及C3组(F值分别为14.307、7.074、28.672,P均＜0.05),且与凋亡细胞百分率呈明显正相关(A2、A3组r=0.813,P=0.002;B组r=0.780,P=0.004),而C组肿瘤组织%ID/g与凋亡细胞百分率无相关性(r=-0.238,P=0.229).结论 接受放疗或化疗后,小鼠肝癌组织对99Tcm-rh-AnnexinV的摄取明显增加,且其摄取程度与肿瘤组织内凋亡细胞量呈明显正相关;应用99Tcm-rh-AnnexinV显像可较准确地反映治疗后早期肿瘤组织细胞凋亡的状态.     

99Tcm-HYNIC-Annexin V的制备及其与多巴胺能神经元凋亡模型的结合特性

目的 研究凋亡显像剂99Tcm-人膜联蛋白V(Annexin V)的制备及其与多巴胺能神经元凋亡模型的体外结合特性.方法 运用双功能螯合剂联肼尼克酰胺(HYNIC)进行99Tcm标记Annexin V,形成99Tcm-HYNIC-Annexin V,经Sephadex G-25柱层析分离纯化,采用快速薄层层析(ITLC)法检测放化纯.将99Tcm-HYNIC-Annexin V与经过1-甲基-4-苯基吡啶离子(MPP+)处理制模的大鼠肾上腺嗜铬细胞瘤PC12细胞进行体外细胞量及时间-温度结合实验、饱和结合实验和竞争结合实验,得到99Tcm-HYNIC-Annexin V与多巴胺能神经元凋亡模型的结合特性,并比较其与正常细胞及不同凋亡水平下细胞模型的结合特性.结果 ①99Tcm-HYNIC-Annexin V标记率(64.56±6.23)%,比活度(3.7～74)×105kBq/mg,放化纯(93.6±2.48)%,室温放置4 h后放化纯仍大于90%.②99Tcm-HYNIC-Annexin V与多巴胺能神经元凋亡模型的结合具有特异性、可饱和性,Scatchard图示平衡解离常数(Kd)为(7.16±1.78)nmol/L,最大结合容量(Bmax)为(178.73±32.62)fmol/106细胞.结论 多巴胺能神经元凋亡模型对99Tcm-HYNIC-Annexin V有良好的摄取,可进一步行动物体内研究.     

99Tcm-Ap4A显像探测动脉粥样硬化斑块的动物实验研究

目的　探讨应用二磷酸腺苷 (ADP)的类似物99Tcm 四磷酸二腺苷 (Ap4A)显像探测动脉粥样硬化斑块的可行性。方法　采用酒石酸亚锡还原99TcmO- 4 标记Ap4A ,柱层析纯化 ,纸层析鉴定放射化学纯度。观察99Tcm Ap4A在昆明小鼠体内的生物分布并测定动脉粥样硬化模型家兔病灶斑块 血液、病灶斑块 无斑块动脉壁的放射性比值 ,进行腹主动脉宏观放射自显影及正常家兔及动脉粥样硬化家兔体外体内显像。结果　99Tcm Ap4A的放射化学纯度为 85 %～ 91%。99Tcm Ap4A在小鼠血液内清除迅速。注射99Tcm Ap4A后 30min粥样硬化组靶 血比值达 3.17± 1.2 7,靶 非靶比值为 5 2 3± 1.87。粥样硬化组自显影胶片上的曝光黑影与肉眼可见的动脉粥样硬化斑块有良好的一致性。体外显像 :正常家兔组腹主动脉未显影 ,粥样硬化组腹主动脉放射性浓聚清晰可见。体内显像 :粥样硬化组家兔的腹主动脉在注射99Tcm Ap4A后 15～ 30min与正常家兔组相比放射性浓聚明显增强 ,并持续到注药后 3h。结论　99Tcm Ap4A是一种有潜力的可快速显示动脉粥样硬化斑块形成的显像剂。     

99Tcm-FM2心肌细胞凋亡显像的实验研究

目的探讨99Tcm标记突触结合蛋白Ⅰ的C2A片段-谷胱甘肽转移酶复合物(FM2)在心肌细胞凋亡显像中的应用价值.方法通过基因工程合成FM2,以2-亚氨基噻吩盐酸盐(IT)修饰,经葡庚糖酸钠(GH)转换标记制备99TcmFM2.99Tcm-FM2经纯化后,纸层析法测定放化纯.破碎红细胞膜磷脂结合实验检测99Tcm-FM2在Ca2+介导下与凋亡细胞表面磷脂结合的能力.以6头小型猪制备心肌凋亡模型,通过心导管将球囊送入并充气阻塞冠状动脉主要分支远端(左前降支、左回旋支或右冠状动脉),20～30 min后,撤除球囊,造成缺血再灌注损伤,引发急性心肌梗死.注射99Tcm-FM2后3 h,进行CT定位和SPECT显像,计算心肌异常高放射性区/本底(T/B)比值.处死动物,体外测定凋亡心肌与正常心肌的单位质量放射性比值.用流式细胞分析和电镜检查验证显像结果.结果99Tcm-FM2稳定,放化纯＞95%.FM2经99Tcm标记后仍保持Ca2+介导的与膜磷脂特异结合的能力.活体显像99Tcm-FM2注射后3 h,凋亡心肌清晰显影,T/B比值为3.36±0.74.体外测定缺血再灌注损伤心肌与正常心肌的单位质量放射性比值为11.68±4.02.流式细胞分析和电镜检查均证实心肌高放射性区凋亡心肌组织形成.结论99Tcm-FM2在活体内与凋亡心肌组织特异结合,对无创性检测心肌细胞凋亡有潜在应用价值.     

幼龄兔缺氧缺血性脑损伤的细胞凋亡显像研究

目的探讨99^Tc^m-联肼尼克酰胺．膜联蛋白Ⅴ（HYNIC-Annexin Ⅴ）在幼龄兔缺氧缺血性脑损伤（HIBD）细胞凋亡显像中的价值。方法制作幼龄兔假手术组、HIBD4h组、HIBD40h组模型，对各组动物行注药后60min平面脑显像和脑组织石蜡切片观察，对HIBD 40h组还进行注药后5，30，120min显像；用ROI技术计算各组动物脑患侧与健侧放射性计数比值（T／NT）；假手术组和HIBD4h组还进行MRI、弥散加权成像（DWI）。结果假手术组、HIBD4h组和HIBD40h组注药后60minT／NT分别为0．94±0．14，1．32±0．11，1．81±0．07（F=82．385，P〈0．01）；HIBD40h组不同时间（5，30，60，120min）显像获得的T／NT分别为1．72±0．04，1．77±0．06，1．81±0．07，1．71±0．03（F=3．994，P〉0．01）。切片观察假手术组脑组织未见凋亡细胞，HIBD4h组与HIBD40h组患侧脑见凋亡细胞，后者较明显；HIBD4h组MRI、DWI和表观弥散系数（ADC）图均未见异常。结论99^Tc^m-HYNIC-AnnexinⅤ显像可以早期发现神经细胞凋亡，在新生儿HIBD细胞凋亡的无创性诊断、抑制凋亡疗效评价和预后估计方面有重要的应用前景。     

99Tcm-Annexin V用于体内细胞凋亡显像的研究进展

细胞凋亡早期,磷脂酰丝氨酸从细胞膜脂双层的内层迁移至外层,利用99Tcm标记的Annexin V对细胞凋亡暴露在细胞膜表面的磷脂酰丝氨酸进行体内显像,可检测早期细胞凋亡.     

<Superscript>99m</Superscript>Tc-Annexin A5 for noninvasive characterization of atherosclerotic lesions: imaging and histological studies in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits

Purpose Apoptosis is commonly observed in advanced atherosclerotic lesions. ^99mTc-annexin A5 (^99mTc-annexin V) has been proposed as a potential tracer for imaging apoptosis in atherosclerotic plaques. Accordingly, we determined the usefulness of ^99mTc-annexin A5 as an atherosclerosis imaging tracer in a rabbit model (myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits; WHHLMI rabbits) of spontaneous atherosclerosis. Methods The WHHLMI and control rabbits were injected intravenously with ^99mTc-annexin A5. After in vivo planar imaging, the radioactivity in the aorta was measured. Autoradiography, TUNEL staining, Azan-Mallory staining and immunohistological studies were performed serially throughout the aorta. Results ^99mTc-Annexin A5 accumulation in the aorta of the WHHLMI rabbits was 5.6-fold higher than in that of control rabbits. Autoradiography showed heterogeneous multifocal accumulation of ^99mTc-annexin A5 in WHHLMI rabbits. ^99mTc-Annexin A5 accumulation was highest in the atheromatous lesions (6.2 ± 2.5, %ID × BW/mm² × 10³), followed in decreasing order by neointimal (4.9 ± 1.3), fibroatheromatous (4.5 ± 1.9), and collagen-rich lesions (3.3 ± 1.4). The regional ^99mTc-annexin A5 accumulation was significantly correlated with the TUNEL-positive cell density, macrophage density and “vulnerability index,” an index of the morphological destabilized characteristics. The in vivo imaging clearly visualized the atherosclerotic lesions in WHHLMI rabbits. Conclusion The present study in WHHLMI rabbits showed higher ^99mTc-annexin A5 accumulation in grade IV atheroma than in other more stable lesions. ^99mTc-Annexin A5 may be useful in identifying atheroma that is at higher risk for rupture and possibly in assessing the response to anti-atherosclerotic therapy.     

99Tcm-HYNIC-Annexin V的制备及其在健康小鼠体内的分布

目的制备人膜联蛋白V（Annexin V），应用联肼尼克酰胺（HYNIC）偶联后进行^99Tc^m标记，评价^99Tc^m-HYNIC-Annexin V在健康小鼠体内的分布。方法采用基因工程方法构建Annexin V的原核载体并诱导其表达，与自行合成的双功能螯合剂HYNIC偶联，并进行^99Tc^m标记。测定^99Tc^m-HYNIC-Annexin V的标记率、放化纯，并研究其在健康小鼠体内的生物分布特性。结果Annexin V在大肠杆菌中获得稳定表达，经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）及Western Blotting检测，得到纯度较高的蛋白质。Annexin V经HYNIC偶联后，其^99Tc^m标记率可达90％左右，放化纯〉90％。小鼠体内分布结果表明，^99Tc^m-HYNIC-Annexin V血液清除迅速，主要从肾脏和肝脏排泄。结论Annexin V可在原核生物中稳定表达。其与HYNIC偶联后，^99Tc^m-标记率和放化纯较高，方法简单，条件温和。     

细胞凋亡的放射性核素分子功能显像应用进展

近几年,细胞凋亡体内检测技术发展迅速,其中最富发展前景的是核素示踪细胞凋亡显像技术,它以无创性、早期性、定量性等优势独树一帜,具有广泛的应用价值。     

~(99m)Tc标记annexin V类显像剂细胞凋亡显像在肿瘤研究中的应用及进展

细胞凋亡在肿瘤的发生发展过程中起着重要的作用,成功的肿瘤治疗将在肿瘤组织内诱导肿瘤细胞产生凋亡。99mTc标记annexin V类显像剂凋亡显像能够在体内早期、无创伤性地检测到治疗所引起的细胞凋亡,这将有助于早期评判肿瘤患者的治疗效果及预后。     

Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis

Either inadequate or excessive apoptosis (programmed cell death) is associated with many diseases. A method to image apoptosis in vivo, rather than requiring histologic evaluation of tissue, could assist with therapeutic decision making in these disorders. Programmed cell death is associated with a well-choreographed series of events resulting in the cessation of normal cell function, and the ultimate disappearance of the cell. One component of apoptosis is signaling adjacent cells that this cell is committing suicide by externalizing phosphatidylserine to the outer leaflet of the cell membrane. Annexin V, a 32-kDa endogenous human protein, has a high affinity for membrane-bound phosphatidylserine. We have coupled annexin V with the bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m HYNIC-annexin V and demonstrated localization of radioactivity in tissues undergoing apoptosis in vivo. In this report we describe the results of a series of experiments in mice and rats to characterize the biologic behavior of ^99mTc-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10–180 min after intravenous injection of ^99mTc-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of ^99mTc-annexin V in vivo, mice were treated with dexamethasone at doses ranging from 1 to 20 mg/kg, 5 h prior to ^99mTc-HYNIC-annexin V administration, to induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each sorted cell population was counted in a scintillation counter. To test ^99mTc-HYNIC-annexin V as a tracer for external radionuclide imaging of apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 h post injection. Rat biodistribution studies demonstrated a blood clearance half-time of less than 10 min for ^99mTc-HYNIC-annexin V. The kidneys had the highest concentration of radioactivity at all time points. Studies in the mouse thymus demonstrated a 40-fold increase in ^99mTc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population. A correlation of r=0.78 was found between radioactivity and flow cytometric and histologic evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed a substantial increase of activity in the liver of wild-type mice treated with anti-Fas, while there was no significant change, irrespective of anti-Fas administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were obtained in wild-type mice 30 min after injection of ^99mTc-HYNIC-annexin V. The imaging results were consistent with histologic analysis in these animals. In conlusion, these studies confirm the value of ^99mTc-HYNIC-annexin V uptake as a marker for the detection and quantification of apoptotic cells in vivo. Received 22 February and in revised from 5 May 1999