单次化疗后肿瘤^99Tc^m-rh-Annexin V分布与Survivin、Caspase-3蛋白表达的相关性研究

Distribution of ^99Tc^m-rh-Annexin V and its relationship with expression of Survivin and Caspase-3 in tumors after a single dose of chemotherapy

目的评价^99Tc^m-重组人膜联蛋白V（rh-AnnexinV）显像检测单次化疗后肿瘤细胞凋亡的可行性，探讨其在肿瘤组织的分布与凋亡调控蛋白Survivin、Caspase-3表达的相关性。方法采用^99Tc^m直接标记法标记rh-AnnexinV，作为核素凋亡显像剂。将小鼠肝癌细胞Hca-F25接种于小鼠右腋窝皮下；区组随机化分为A（对照组，9只）和B（化疗组，10只）组，肿瘤生长至直径约1cm时，B组一次性接受环磷酰胺腹腔内注射，剂量为按体质量150mg／kg，A组注射同体积生理盐水；20h后2组同时由鼠尾静脉注入^99Tc^m-rh-Annexin V；4h后行SPECT显像并处死、取肿瘤组织，用井型γ闪烁计数器检测荷瘤小鼠肿瘤组织内放射性分布，以每克组织百分注射剂量率（％ID／g）表示：采用原位末端标记法（TUNEL）检测肿瘤细胞凋亡；采用免疫组织化学SP法检测标本中Survivin、Caspase-3蛋白表达。采用SPSS10．0软件对数据进行统计学处理。结果单次化疗后B组肿瘤组织内^99Tc^m-rh-Annexin V分布[（0．478±0．123）％ID／g]、TUNEL检测阳性细胞数[（18．030±5．600）个／视野]及Caspase-3蛋白表达[（3．266±0．482）％]均明显高于A组[分别为（0．332±0．061）％ID／g，（6．744±2．325）个／视野，（2．387±0．387）％]，A组Survivin蛋白表达[（4．227±0．636）％]明显高于B组[（3．407±0．519）％]，差异有统计学意义（F值依次为10．502，31．507，18．971，9．560，P均〈0．01）。相关性研究表明，肿瘤组织的放射性摄取（％ID／g）与TUNEL阳性细胞数呈明显正相关（r=0．858，P=0．000）；肿瘤组织内Survivin蛋白表达与Caspase-3蛋白表达呈明显负相关（r=-0．818，P=0．000）；肿瘤组织内^99Tc^m-rh—AnnexinV分布与Caspase-3蛋白表达呈明显正相关，与Survivin蛋白表达呈明显负相关（P〈0．01）。结论肿瘤组织内^99Tc^m-rh—AnnexinV的分布可以反映化疗后早期肿瘤组织细胞凋亡的状况以及Survivin、Caspase-3表达水平的变化。

Objective Recently, molecular imaging for detecting cellular apoptosis is developing rapidly. The aim of the study was to determine the effectiveness of imaging with^99Tc^m labelled recombinant human Annexin V （^99Tc^m-rh-Annexin V ） as a reflection of apoptosis in tumor, and related its distribution with expression of Survivin and Caspase-3 after a single dose of chemotherapy. Methods Eight days after being inoculated with allogenic hepatoma cells（ Hca-F25 ） into right axillary fossa, the mice （ purebred 615 ） were randomly divided into two groups （control group A, n =9;and treated group B, n = 10）. Group B was received a single dose of chemotherapy intraperitoneally （cyclophosphamide, 150 mg/kg）. Groups A and B were given^99Tc^m-rh-Annexin V （3.7 MBq ·0.5 μg^-1 per mouse） intravenously 20 h later. Four hours aften^99Tc^m-rh-Annexin V injection, the animals were imaged and sacrificed, and the tumor samples were weighed and the radioactivity was determined in a well-counter. The accumulation of^99Tc^m-rh-Annexin V in tumor was expressed as the percentage activity of injection dose per gram of tissue （ % ID/g）. Tumor cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling （TUNEL） method, and the expression of Survivin and Caspase-3 in tumor were determined with immunohistochemical method. SPSS 10.0 was used for data analysis. Results Single dose chemotherapy tsignificantly increased the tumor uptake of^99Tc^m-rh-Annexin V [ （0.478 ± 0. 123 ） % ID/g vs （0. 332 ±0. 061） % ID/g] and the positive number of TUNEL [ （ 18. 030 ±5. 600） cells/field vs （6. 744 ±2. 325） cells/field], as well as the expression of Caspase-3 [ （3.266±0.482）% vs （2. 387±0. 387）%, F was 10.502, 31. 507, 18. 971, respectively, all P 〈 0. 01 ], but the expression of Survivin was significantly higher in group A than in group B [（4.227±0.636）% vs （3.407±0.519）%, F =9.560, P〈0.01]. The % ID/g in tumor correlated well with the positive number of TUNEL（ n = 19, r = 0. 858, P = 0.000）, and he expression of Survivin correlated negatively with the expression of Caspase-3 （ r = - 0. 818, P = 0. 000）. In group A and B, the distribution of^99Tc^m-rh-Annexin V correlated positively well with the expression of Caspase-3 and negatively with the expression of Survivin （ P 〈 0.01 ）. Conclusion The distribution of^99Tc^m-rh-Annexin V can not only reflect the extent of apoptosis, but also the change of Survivin and Caspase-3 expression in tumor after a single dose of chemotherapy.