^99Tc^m—His10—Annexin Ⅴ探测非小细胞肺癌细胞凋亡的实验研究

In vivo detection of non-small cell lung cancer apoptosis with ^99Tc^m-His10-Annexin Ⅴ

目的探讨^99Tc^m标记带有10个连续组氨酸的膜联蛋白Ⅴ（His10-Annexin Ⅴ）探测荷非小细胞肺癌（NSCLC）裸小鼠模型化疗后肿瘤细胞凋亡的可行性。方法用^99Tc^m直接标记His10-Annexin Ⅴ。荷H460 NSCLC肿瘤裸小鼠模型20只，按体表面积以100mg／m^2剂量紫杉醇化疗诱导，分成未治疗（对照）、化疗诱导后24，48，72h共4组.^99Tc^m-His10-AnnexinⅤ显像探测化疗前后肿瘤组织细胞凋亡，计算肿瘤／对侧正常肌肉组织放射性比值（T／NT），测定每克组织百分注射剂量率（％ID／g）。以^99Tc^m-IgG显像为对照，确定肿瘤的非特异性摄取。用流式细胞术（FCM）测定肿瘤组织活化半胱氨酸天冬氨酸蛋白水解酶（Caspase-3），光学显微镜HE及原位末端标记法（TUNEL）染色测定凋亡指数。采用SPSS12．0软件进行统计学处理，运用单因素方差分析和直线相关分析（Pearson）。结果”^99Tc^m -His10-Annexin Ⅴ放化纯为（98．01±1．67）％。注射^99Tc^m- His10-Annexin Ⅴ后2h即可得到清晰图像，化疗诱导组肿瘤部位可见明显的放射性浓聚，化疗后24，48，72h组的T／NT值分别为2．63±0．76，3．41±0．90，3．85±0．62；对照组T／NT值为1．42±0．19，F值分别为12．064，23．322，70．177，P均〈0．01。未治疗组肿瘤仅有少量放射性摄取，为（1．09±0．18）％ID／g，化疗后肿瘤摄取明显增加，24，48，72h肿瘤摄取分别为（2．55±0．73）、（3．60±1．09）、（3．73±0．97）％ID／g，明显高于未治疗组（F值分别为18．733，20．624，35．626，P均〈0．01）。^99Tc^m-IgG显像化疗组及对照组肿瘤均未见明显放射性浓聚。未治疗组活化Caspase-3为（3．70±0．74）％，化疗后24，48，72h分别为（23．46±2．23）％、（62．85±6．13）％、（70．44±6．09）％，3个化疗诱导组和未治疗组相比差异均有统计学意义（F值分别为354．610，459．438，591．052，P均〈0．01）。光学显微镜HE及TUNEL染色法发现，未治疗组细胞凋亡指数为（3．31±0．61）％，化疗后24，48，72h细胞凋亡指数分别为（32．90±6．64）％、（70．42±7．54）％、（83．23±9．71）％，化疗诱导组与对照组细胞凋亡指数差异均有统计学意义（F值分别为98．627，393．215，337．386，P均〈0．01）。T／NT值、肿瘤组织放射性摄取与活化Caspase-3及细胞凋亡指数均有很好的相关性（r=0．847，0．833，0．774，0．850，P均〈0．01）。结论^99Tc^m-His10-Annexin Ⅴ显像可早期探测NSCLC化疗后细胞凋亡，进而早期预测和评价肿瘤治疗疗效。

Objective Annexin Ⅴ, a human protein with a high affinity for phosphatidylserine, labeled with ^99Tc^m, can detect apoptosis in vivo. To simpiify the preparation and labeling of Annexin V for nuclear medicine studies, we investigated the addition of peptide sequences that would directly form endogenous chelation sites for ^99Tc^m and the feasibility of ^99Tc^m-labeled Annexin V recombinant with ten consecutive histidines （ His10-Annexin Ⅴ ） in detection of apoptosis in non-small cell lung cancer （NSCLC） after chemotherapy. Methods ^99Tc^m-His10-Annexin Ⅴ was prepared with direct-labelling method. Fifteen nude mice bearing H460 non-small cell lung cancer underwent chemotherapy by paclitaxel injection at 24 （ n = 5）, 48 （n =5） and 72 h （n =5）, respectively. Another 5 untreated mice were used as control. ^99Tc^m-Annexin Ⅴ were injected intravenously and planar static images were acquired using a pin-hole collimator. The radioactivity ratio of tumor to non-tumor （T/NT） and tumor uptake （ percentage activity of injection dose per gram of tissue, % ID/g） of ^99Tc^m-His10-Annexin V were calculated. ^99Tc^m-IgG was used as control to determine the non-specific accumulation in tumor. Activated Caspase-3 was analyzed with flow cytometry. HE staining and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling （TUNEL） assay were also performed to confirm the presence of apoptosis. SPSS 12.0 was used for statistical analysis. Analysis of variance and the Pearson correlation coefficient were used. Results The radiochemical purity of ^99Tc^m-His10- Annexin V was （98.01 -＋ 1.67） %. Tumor uptake of 99Tcm-His10-Annexin V was significant 2 h after injection. In the treatment groups, the values of T/NT were 2.63 ± 0.76, 3.41 ± 0.90 and 3.85 ±0.62, respectively at 24, 48, 72 h after chemotherapy, much higher than that of control （ 1.42 ±0. 19）. The difference between treatment and control groups was significant （ F = 12.064, 23. 322, 70. 177, respectively, all P 〈 0.01 ）. Tumor uptake in treatment groups was （2.55± 0.73）, （3.60 ±1.09） and （3.73 ±0.97） % ID/g at 24, 48 and 72 h after chemotherapy, respectively. In the control group, it was （1.09 ±0.18） % ID/g. The tumor uptake in paclitaxel induced groups were significant higher than that of untreated group （ F = 18. 733, 20.624, 35. 626, respectively, all P 〈0.01 ）. There was no conspicuous radioactivity in tumors with ^99Tc^m-IgG imaging. The percentage of activated Caspase-3 in tumor was （3.70 ±0.74） %, （23.46 ± 2.23 ） %, （62.85 ±6.13 ） % and （70.44 ±6.09） % in control and treatment groups respectively. Histological analysis of tumor with HE stained and TUNEL assay revealed that paclitaxel treatment significantly increased the percentage of apoptotic cells. The appearance of apoptotic bodies were seldom in the untreated group with an apoptotic index of （3.31 ±0. 61）%, but elevated to （32. 90 ±6.64）%, （70.42 ± 7. 54） %, （ 83.23 ± 9.71 ） % at 24, 48, and 72 h after paclitaxel inducement, respectively, significantly higher than that in untreated group （F = 98. 627, 393. 215, 337. 386, all P 〈 0.01 ）. Furthermore, the ratio of T/NT and tumor uptake of ^99Tc^m-His10-Annexin Ⅴ correlated well with the activated Caspase-3 and apoptotic index （r=0.847, 0.833, 0.774, 0.850; all P〈0.01）. Conclusions The results demonstrated that ^99Tc^m-His10-Annexin Ⅴ imaging in vivo for detection of apoptosis in NSCLC after chemotherapy is feasible. His10-Annexin Ⅴ has the characteristics of a molecular probe in early prediction of cancer treatment efficacy.