新型细胞凋亡分子探针99Tcm-CP3-peptide的制备及在肺腺癌细胞系体内外的实验研究

目的制备含天冬氨酸-谷氨酸-缬氨酸-天冬氨酸（DEVD）核心的多肽细胞凋亡分子探针99Tcm-CP3-peptide，研究其在肺腺癌细胞系A549化疗后体内外的生物学分布及SPECT显像。方法采用双功能螯合法合成99Tcm-CP3-peptide，分别于标记后0、1、2、4、6 h行高效液相色谱（HPLC）法检测放射性化学纯度。对经紫杉醇化疗的肺腺癌A549细胞进行体外细胞结合实验，对照组未经化疗，测量细胞内外放射性比值（Cin/Cout），并用流式细胞仪检测细胞凋亡率。经紫杉醇化疗的荷瘤裸鼠尾静脉注射99Tcm-CP3-peptide 3.7 MBq，分别于5、15、30、60、120、240 min测其生物学分布，并行SPECT静态显像，勾画感兴趣区（ROI），计算肿瘤/对侧正常肌肉组织的比值（T/NT）。取显像后的肿瘤组织进行苏木精-伊红染色，观察肿瘤组织的形态学变化。两组比较采用t检验，相关性研究采用双变量相关分析。结果99Tcm-CP3-peptide在0、1、2、4和6 h的放射化学纯度均>97%，合成率为（64.5±5.2）%。体外细胞结合实验结果表明，经紫杉醇化疗后24h，肿瘤细胞Cin/Cout为10.27±2.02，对照组Cin/Cout为1.09±0.03，化疗组为对照组的7.3倍。对同状态的肿瘤细胞行流式细胞检测，化疗组细胞凋亡率为（75.62±2.57）%，对照组细胞凋亡率为（3.42±0.32）%，化疗组和对照组Cin/Cout与流式细胞仪检测的凋亡细胞百分比呈明显正相关（r=0.970，P<0.05）。荷瘤裸鼠体内分布实验结果显示，99Tcm-CP3-peptide在血液中清除速度较快，且主要经肾脏代谢；心脏、脾脏、肺等重要器官放射性摄取值均较低；但在肝脏中摄取稍高且代谢速度较慢；肿瘤组织摄取值在注射后1 h达到峰值[（4.26±1.03）%ID/g]。荷瘤小鼠给药后1 h即可获得清晰图像，勾画ROI显示:化疗组T/NT为3.83±0.11，显著高于对照组的1.57±0.09，两者间差异有统计学意义（t=16.19， P<0.05）。病理结果显示化疗组肿瘤组织见大量核固缩、核碎裂及凋亡细胞；对照组仅见少量核固缩、核碎裂及少量凋亡细胞。结论99Tcm-CP3-peptide是一种靶向caspase-3的显像剂，具有较好的生物学分布，可应用于动物模型肿瘤组织凋亡显像，在监测化疗后肿瘤细胞凋亡方面有一定的临床潜在价值。

Preparation of 99Tcm-CP3-peptide and in vitro and in vivo study of lung adenocarcinoma cell lines

ObjectiveTo preparation a99Tcm labeled caspase -3 polypeptide apoptosis molecular probe(99Tcm-CP3-peptide) based on aspartic glutamic acid-valine-aspartic acid(DEVD) as the core and study the biodistribution and SPECT imaging of lung adenocarcinoma cell line(A549) after chemotherapy.Methods99Tcm-cp3-peptide was synthesized by bifunctional chelate method, and the radiochemical purity was detected by HPLC at the time points of 0, 1, 2, 4, 6 hours after the labeling. An in vitro binding assay was performed in A549 cells treated with paclitaxel.In the control group, without chemotherapy, the intracellular and extracellular radioactive ratio(Cin/Cout) was measured, and the cell apoptosis rate was detected by flow cytometry. 99Tcm-CP3-peptide was injected via tail vein of tumor bearing nude mice treated with paclitaxel(3.7 MBq for each mouse), to detect the biological distribution of 5, 15, 30, 60, 120, 240 min respectively. Static SPECT imaging was performed, ROI outlined, and the ratio of tumor/contralateral normal muscle tissue was calculated. HE staining was performed to observe the morphological changes of tumor tissues after imaging. Test was used for comparison between the two groups, and bivariate correlation analysis was used for correlation study, P<0.05 was considered statistically significant.ResultsThe radiochemical purity of 99Tcm-CP3-peptide at 0, 1, 2, 4 and 6 h was greater than 97%. The synthesis rate was(64.5+5.2)%. An in vitro investigation showed that the Cin/Cout in the tumor cell chemotherapy group was 10.27±2.02 vs. 1.09±0.03 in control group 24 h after paclitaxel chemotherapy. The chemotherapy group of Cin/Cout was 7.3 times as high as that of the control group. Flow cytometric detection of tumor cells in the same state, the rate of apoptosis in the chemotherapy group was(75.62±2.57)% vs.(3.42±0.32)% in contorl group. The ratio of Cin/Cout in the chemotherapy group and the control group was positively correlated with the percentage of apoptotic cells detected by flow cytometry(r=0.970, P<0.05). Invivo distribution of tumor bearing mice showed that: 99Tcm-CP3-peptide is rater fast in blood clearance, and mainly metabolized through kidney. The values of radioactive uptake were low for important organs such as heart, spleen and lung etal, but it is slightly higher in the liver and slower in metabolism. The value of tumor tissue uptake reached a peak(4.26±1.03)%ID/g after injection of 1 h. Clear images of the tumor bearing mice can be obtained 1h after injection of drugs. The ROI showed that the ratio of T/NT in the chemotherapy group was 3.83±0.11, which was significantly higher than that of the control group(1.57±0.09, t=16.19, P<0.05). After imaging, HE staining of tumor tissue showed that a large number of nuclear retraction, nuclear fragmentation and apoptotic cells were found in the tumor tis-sue of the chemotherapy group and only a small amount of that were in control group.Conclusions99Tcm-CP3-peptide, a kind of imaging agent targeting caspase-3 activity,which has good biological distribution, and can be applied to apoptosis imaging of animal models, it has a potential clinical value in monitoring tumor cell apoptosis after chemotherapy.