99mTc-联肼尼克酰胺-膜联蛋白V活体检测兔肝癌细胞凋亡的研究

Apoptosis of liver cancer cells in rabbits in vivo by 99mTc-hydrazino nicotinamide-Annexin V

目的探讨99mTc-联肼尼克酰胺（HYNIC）．膜联蛋白V（AnnexinV）活体检测细胞凋亡的可行性。方法99mTc-通过HYNIC标记AnnexinV合成99mTc-HYNIC-AnnexinV。建立兔VX-2肝癌模型。将10只新西兰大白兔随机分为2组，通过治疗剂量的化疗药物紫杉醇（PAC，n=6），同时建立对照组（C，n=4），分别予化疗前，化疗后24、48h静脉注射99mTc-HYNIC—AnnexinV，通过单光子发射计算体层摄影（SPECT）成像，选取肿瘤部位放射性计数（T）及腿部非肿瘤区部位放射性计数（NT），计算其比值（T／NT），进行半定量分析。最后1次显像后处死兔，取肿瘤组织，分别予免疫组织化学原位末端转移酶标记（TUNEL）和流式细胞仪（FCM）分析组织细胞凋亡率，并与核医学检测结果比较，确定核医学检测结果与其是否相关。实验数据以SPSS17．0软件进行t检验、方差和相关分析。结果99mTc-HYNIC—AnnexinV标记率为95％以上，放化纯为95％以上99mTc-HYNIC—AnnexinV放射显像显示两组化疗前肿瘤部位放射性核素聚集T／NT为2．62±0．56、2．53±0．60。化疗后24h及48h对照组放射性核素聚集rr／NT分别为2．72±0．70、2．62±0．66，紫杉醇干预组放射性核素聚集T／NT分别为6．43±0．97、6．64±1．15，与对照组、化疗前比较差异有统计学意义（P〈0．01），24h与48h两组间放射性核素显像差异无统计学意义（P〉0．05）；对照组与紫杉醇组免疫组织化学分析肿瘤组织细胞TUNEL染色阳性率分别为4．00±0．81、13．50±2．74，流式细胞分析肿瘤组织细胞凋亡率分别为（7．83±1．11）％、（20．81±3．33）％。两组间比较差异均有统计学意义（P〈0．01），放射性核素聚集程度与免疫组织化学分析组织细胞TUNEL阳性率和流式细胞分析组织细胞凋亡率的结果显著相关（rTUNEL=0．747，P〈0．05；rFcM=0．909，P〈0．01）。结论紫杉醇可以诱导兔VX-2肝癌细胞凋亡，99mTc-HYNIC．AnnexinV核素显像检测能够反映活体组织细胞凋亡，该方法能够在活体状态下检测细胞凋亡，化疗后24—48h是放射性检测细胞凋亡的窗口时间。

Objective To investigate the feasibility of apoptosis detection in vivo by 99mTc-hydrazino nicotinamide（HYNIC）-Annexin V. Methods Annexin V was labeled with 99mTcthrough HYNIC. Ten New Zealand rabbits implanted with VX-2 were divided into control （ C, n = 4 ） and paclitaxel （ PAC, n = 6 ） groups, given 2 mL/kg of normal saline or 2. 4 mg/kg of PAC intravenously. Images of the liver tumor were examined by single photon emission computed tomography （SPECT） through intravenous injection ofOgmTe- HYNIC-Annexin V before and 24 and 48 h after chemotherapy. Tumor radioactive count proportion to non- tumor sites （T/NT） was calculated. When last imaging finished, the rabbits were sacrificed. The tumor was taken out and divided into two pieces for TdT-mediated dUTP nick end labeling （TUNEL） immunohistochemieal analysis and flow cytometry （FCM） respectively. Data were analyzed by t test, variance analysis and correlation analysis by SPSS 17.0. Results The labeling rate of Annexin V with 99mTc through HYNIC was above 95%, and radiochemical purity was above 95%. The SPECT showed that TINT in control group and PAC group was 2. 62±0. 56 and 2. 53±0. 60 before chemotherapy, and that in control group and PAC group at 24 and 48 h after chemotherapy was 2. 72±0. 70 and 2. 62±0. 66, and 6.43±0. 97 and 6. 64 ± 1.15 respectively. The T/NT in PAC group at 24 and 48 h after chemotherapy was significantly different from that in control group and PAC group prior to chemotherapy （P 〈0. 01 ）. There was no significant difference between 24 h and 48 h in PAC group （P 〉 0. 05 ）. The tunnel positive cells detected by immunohistochemistry and apoptosis rate detected by FCM in PAC group [ （13.50±2.74）% and （20. 81 ±3. 33）% were significantly different from those in control group （4. 00±0. 81 ） % and （7. 83 ±1.11 ） % ,P 〈 0. 01 ]. The T/NT was significantly correlated with TUNEL positive cells and apoptosis rate of the tumor （ rTUNEL = 0. 747, P 〈 0. 05 ; TFCM = 0. 909,P 〈 0. 01 ）. Conclusion PAC can induce apoptosis of VX-2 cells in the liver of rabbits. Apoptotic cells in vivo can be detected by SPECT through 99mTc-HYNIC-Annexin V. 24-48 h after chemotherapy is a window time for detecting apoptosis.