99Tcm—survivinmRNA反义肽核酸制备及荷瘤裸鼠基因显像研究

目的制备99Tcm标记的survivinmRNA反义肽核酸（PNA）探针，并进行荷瘤裸鼠体内基因显像，研究其对肿瘤早期诊断的价值。方法化学合成survivinmRNA的反义、无义PNA，在5’端连上4个氨基酸Gly一（D）Ala—Gly—Gly]，形成1个类似N。结构的强螯合基团；为消除空间阻碍，引入1个1一氨基丁酸（Aba）作为隔离物，利用配体交换法进行99Tcm标记。用HPLC及ITLC对标记物的标记率、放化纯进行鉴定。并对反义、无义组各5只人肺癌A549荷瘤裸鼠行99Tcm一survivinmRNA反义、无义PNA体内显像。注药后1，2和4h分别显像，利用ROI测得肿瘤与对侧组织的rr／NT比值。结果分析采用SPSS13．0软件进行成组t检验。结果99Tcm一survivinmRNA反义PNA即刻标记率为（95．48±1．92）％，放化纯〉95％，且标记物体外稳定性好，与血清保温24h后标记率仍〉85％。无义PNA标记率与之基本一致。99Tcm一survivinmRNA反义PNA在肿瘤组织内特异性浓聚，随时间延长，浓聚程度逐渐增加，1，2和4hT／NT值分别为2．70±0．28，3．44±0．35和4．21±0．63。无义PNA在肿瘤组织中稍有浓聚，但在4h内变化不大，4h时T／NT值（3．12±0．50）与反义组差异有统计学意义（t=2．918，P：0．019）。结论99TcmsurvivinmRNA反义PNA即刻标记率高，稳定性好，无需纯化即可用于显像，在肿瘤组织中特异性浓聚，对肿瘤的早期诊断有潜在价值。

Preparation of 99Tcm labeled survivin mRNA antisense PNA and gene imaging in nude mice bearing lung carcinoma A549 xenografts

Objective To prepare the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA)and investigate its value as a gene imaging agent in tumor bearing mice and early diagnosis in tumor.Methods Survivin mRNA antisense PNA and mismatch PNA were synthesized.Four amino acids (Gly- (D)Ala-Gly-Gly) and Aba (4-aminobutyric acid) were linked to the 5' end of PNA.Gly- (D)Ala-Gly-Gly served as a chelating moiety for strong chelation of 99Tcm and Aba acted as a spacer to minimize the steric hindrance.PNAs were labeled with 99Tcm by the ligand-exchange method.The labeling efficiency and radiochemical purity were measured by HPLC and ITLC methods.There were five BALB/c nude mice bearing human lung carcinoma ( A549 ) in each of antisense PNA and mismatch PNA groups.Gene imaging of 99Tcm-survivin mRNA antisense and mismatch PNAs were performed at 1,2 and 4 h post the injection,respectively,and the T/NT ratio was measured by the method of ROI.The statistical comparisons of average values were performed with the two-group t-test for independent sample by SPSS 13.0.Results The product kept stable in vitro.The labeling efficiency of 99Tcm-survivin mRNA antisense PNA was (95.48 ±1.92)％ and more than 85％ after the incubation for24 h in serum.The radiochemical purity was ＞ 95％.The labeling efficiency of mismatch PNA was similar to the antisense PNA.99Tcm-survivin mRNA antisense PNA was especially uptaken by tumor lesion,and its accumulation reached the top at 4 h post the injection.T/NT ratios at 1,2,and 4 h were 2.70 ± 0.28,3.44 ± 0.35,4.21 ± 0.63,respectively.In the comparison,the T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4 h (3.12 ±0.50) was significantly lower (t =2.918,P =0.019).Conclusions 99Tcm-survivin mRNA antisense PNA has high labeling efficiency,good stability and no need of purification.Its characteristic of especial uptake by tumor lesion provides the potential value in early diagnosis of tumor.