^99Tc^m标记反义肽核酸探针的制备及其荷瘤裸鼠体内分布

Preparation of ^99Tc^m labeled c-myc mRNA antisense peptide nucleic acid and its biodistribution in tumor-bearing nude mice

目的探讨^99Tc^m标记反义肽核酸（PNA）探针的新方法及其在生物体内的分布。方法合成12mer且5′端含有四肽G-（D）-A-G-G的c-myc mRNA反义、无义PNA片段，利用G-（D）-A-G-G形成的N4结构为螯合基团进行^99Tc^m标记，用聚酰胺薄膜层析法和高效液相色谱仪法（HPLC）测定其标记率和标记物的稳定性，并行人结肠癌荷瘤裸鼠体内分布[每克组织百分注射剂量率（％ID／g）]及显像研究。采用SAS6．22软件对数据进行分析。结果反义、无义PNA片段合成物的纯度〉95％。^99Tc^m标记c-myc mRNA反义、无义PNA的标记率〉95％，标记物室温放置18h测定其标记率仍可达95％以上。c-myc mRNA反义、无义PNA片段4-8℃下放置3个月标记率仍〉95％。HPLC测定标记物呈单峰。^99Tc^m标记c-myc mRNA反义PNA主要分布在荷瘤鼠肾、脾、肿瘤、肠道、肝组织中，^99Tc^m标记c-myc mRNA无义PNA在荷瘤鼠血液、脾、肾、肝及肺组织中分布较多；注射后4h两者在荷瘤鼠肿瘤组织中的分布差异有统计学意义[（1．11±0．12）％ID／g和（0．14±0．02）％ID／g；t=14．75，P〈0．01]。^99Tc^m标记c-myc mRNA反义PNA在小鼠体内肿瘤／肌肉、肿瘤／肺的摄取比值较高，肿瘤显像明显。结论用^99Tc^m标记c-myc mRNA反义、无义PNA的方法简单，标记率高，标记物稳定，前者有望成为一种新型肿瘤显像剂。

Objective The aim of this work was to study a ^99Tc^m labeling method for c-myc mRNA antisense peptide nucleic acid （PNA） fragments and the biodistribution of the labeled product in tumorbearing nude mice. Methods A four amino acid sequence Gly-（D）-Ala-Gly-Gly [ G-（D）-A-G-G ] was used as a chelator. N-GAGG-Aba-GCATCGTCGCGG, a chelator-antisense PNA specific for the human c- myc oncogene mRNA initiation region, was synthesized, purified and characterized. N-GAGG-Aba-GCAT- GTCTGCGG, a chelator mismatched PNA, was synthesized and used as a control. G-（D）-A-G-G provided an N4 configuration for strong, efficient chelation of ^99Tc^m. The labeled PNA was identified with high performance liquid chromatography （HPLC） and the labeling yield and radiochemical purity were measured by paper chromatography. The biodistribution was studied with nude mice bearing colon carcinoma and the percentage activity of injected dose per gram of tissue （ % ID/g） was calculated. ASA 6. 12 was used for data analysis. Results The results of HPLC and paper chromatography confirmed that ^99Tc^m was joined to the PNA or the mismatched PNA with a high radiochemical purity （ 〉 95% ）. Both were stable in vitro or incu- bated with human fresh serum and were excreted through urine. Results of biodistribution studies showed that the highest radioactivity levels were in the kidneys and spleen. The radioactivity of ^99Tc^m labeled antisense PNA in tumor was high whereas that of ^99Tc^m labeled mismatch PNA was very low [ （1.11% ±0.12） %ID/g and （0.14±0.02） %ID/g, respectively; t=14.75, P〈0.01]. Conclusions The c-myc mRNA antisense PNA could be labeled effectively with ^99Tc^m by the method presented in this paper, with good yield, radiochemical purity and stability. It was an efficient method to label antisense PNA with ^99Tc^m. The product seemed to be a potential tumor imaging agent.