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

目的 评价99Tcm-重组人膜联蛋白Ⅴ(rh-Annexin Ⅴ)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99Tcm直接标记法标记rh-Annexin Ⅴ,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1 cm时,B组一次性接受环磷酰胺腹腔内注射,剂量为按体质量150 mr,/kg,A组注射同体积生理盐水;20 h后2组同时由鼠尾静脉注入99Tcm-rh-AnnexinⅤ;4 h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SPSS 10.0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99Tcm-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99Tcm-rh-Annexin Ⅴ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99Tcm-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.

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

Objective Recently,molecular imaging for detecting cellular apoptosis is developing rapidly.The aim of the study was to determine the effectiveness of imaging with 99Tcm labelled recombinant human Annexin Ⅴ(99Tcm-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 99Tcm-rh-Annexin Ⅴ(3.7 MBq·0.5μg-1 per mouse)intravenously 20 h later.Four hours after 99Tcm-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 99Tcm-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 99Tcm-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 99Tcm-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 99Tcm-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.