In Vivo Diagnosis of Gene Localization and Expression by Nuclear Medicine Modality

本研究的目的是发展核医学技术，用于基因定位和表达的非侵入性诊断。基因在这里指的是为基因治疗而注射的基因或因疾病而过度表达的基因。利用人表皮样瘤细胞系KB-31及其多药耐药细胞系KB-G2进行了实验研究。通过与马萨诸塞州医学院教授唐纳德·J·哈纳托维奇博士的合作，获得了以下结果：1.合成并修饰了与mdr-1相关的反义(AS)和正义(S)寡核苷酸(均为硫代化)，然后通过MAG_3或HYNIC配体标记~(99m)Tc。结果1.获得了比活性较高的TC-99m-AS和TC-99m-S，且不失其反义结合活性。用AS或S处理KB-31或KB-G2细胞，再与99m-MIBI孵育，评价P-糖蛋白(mdr-1表达)功能。AS处理的KB-G2细胞的P-gp功能受到抑制，而S.KB-31处理的KB-G2细胞未表现出明显的P-gp功能。TC-99m-As在KB-G2细胞中积累，而在KB-31细胞中未被摄取，而在KB-G2和KB-31细胞中均未被摄取。这些结果表明，As是通过反义作用积累到细胞内的。分别给荷KB-G2或KB-31的小鼠注射99m-As或99m-S。KB-G2肿瘤对99m-AS的摄取明显高于KB-31肿瘤。KB-G2肿瘤对TC-99m-AS的摄取显著高于TC-99m-S。这些结果表明，通过反义勃起可以将99m-AS特异性地定位到MDR-1肿瘤中，为反义显像的成功奠定了基础。

The aim of this investigation is to develop nuclear medicine techniques for non-invasive diagnosis of gene localization and expression. Gene here means the one injected for gene therapy or the other over-expressed due to the disease. Using human epidermoid tumor cell line ; KB-31 and its mdr-1 transfected cell line ; KB-G2 carried out the study. By co-working with Dr. Donald J Hnatowich, Professor of Massachusetts Medical School, the following results were obtained;1. Antisense (AS) and sense (S) oligonucleotides related to mdr-1 were synthesized and modified (all phosphothiolated) and then, labeled with Tc-99m via MAG3 or HYNIC ligands. Tc-99m-AS and Tc-99m-S were obtained with high specific activity without loosing their antisense-binding affinity.2. KB-31 or KB-G2 were treated with AS or S, followed by incubation with Tc-99m-MIBI, which is to evaluate P-glycoprotein (mdr-1 expression) function. The KB-G2 treated with AS suppressed the P-gp function, while it was not shown in KB-G2 treated by S. KB-31 did not show any influence by treating with AS or S. These results indicated the antisense effect of AS oligonucleiotied we used.3. Tc-99m-AS was accumulated in KB-G2 but not in KB-31, while Tc-99m-S was not taken up either in KB-G2 or KB-31. These results indicated that AS was accumulated into the cell by antisense effect.4. Tc-99m-AS or Tc-99m-S was injected into the mice bearing KB-G2 or KB-31. The uptake of Tc-99m-AS in the KB-G2 tumor was significantly higher than that in the KB-31 tumors. Tc-99m-AS uptake in KB-G2 tumor was significantly higher than Tc-99m-S. These results indicated that Tc-99m-AS was localized into the mdr-1-tumors specifically by antisense erect.All these results would lead the success for the antisense imaging.