建立^(99)Tc^m标记联肼尼克酰胺多肽“显像剂”纤维膜组合文库

A ^(99)Tc^m labeled HYNIC peptide"tracer"libraries on continuous cellulose membrane supports

目的证明^(99)Tc^m 标记联肼尼克酰胺(HYNIC)多肽"显像剂"文库是^(99)Tc^m 显像剂研制中的有效方法。方法八肽组合文库采用人工点阵方式合成。HYNIC 多肽通过 C 末端以及双β丙氨酸(连接体)共价连接在纤维膜上。用阻断剂阻断非特异结合位点后,将膜文库和人重组热休克蛋白70(HSP70)温育,冲洗。用生物素-亲和素法和抗体法筛选文库。对筛选阳性多肽化合物进行再合成、^(99)Tc^m 标记(以三羟甲基甘氨酸为辅助配体)以及体内分析。将人肺癌细胞株 A549和 H460接种于雌性裸鼠胸壁皮下,用于体内研究。结果 ^(99)Tc^m 标记文库显示,"显像剂"文库的质量较好。由于天冬酰胺-亮氨酸-亮氨酸-精氨酸-亮氨酸-苏氨酸-甘氨酸多肽(NLLRLTG)对 HSP70家族蛋白具有高度特异性,因此,采用^(99)Tc^m -HYNIC-NLLRLTG 为对照组。经筛选获得的15种阳性多肽化合物中,有8种^(99)Tc^m 化合物在体内肿瘤中的分布优于对照组,其中^(99)Tc^m -HYNIC-谷氨酰胺-甘氨酸-缬氨酸-亮氨酸-苏氨酸-甘氨酸-苏氨酸-精氨酸多肽(QGVLTGTR)在体内肿瘤中的分布最佳。活性肽 NLLRLTG替代实验和肿瘤热疗研究表明,^(99)Tc^m -HYNIC-QGVLTGTR 具有 HSP70结合肽活性。结论 ^(99)Tc^m 标记HYNIC 八肽化合物"显像剂"文库的设计可能为多肽显像剂的研制提供一种模式。

Objective： The interference of bifunctional ligands with activities of small peptides has long been recognized. To solve the problem, the hydrazine-nicotinamide （I-IYNIC） conjugated peptide ＂ tracer＂ libraries were synthesized on a continuous cellulose membrane support and the ^99 Tc^m labeled heat shock protein 70 （HSP70） binding peptides were identified by screening libraries with HSP70. Methods Octapeptide libraries were prepared by manual spot synthesis. HYNIC peptides were C terminally attached to cellulose via a （β-Ma）2 spacer. For screening, the cellulose membranes were incubated with human HSP70 （or biotin labeled HSP70） after nonspecific blocking. Mkaline phosphatase labeled streptavidin and Ab against HSP70 were used for the detection of HSP70 binding. Human lung cancer cell lines （A549 and H460） were cultured in RPMI16d0 medium supplemented with 10% fetal calf serum and antibiotics. For in vivo test, 2 ×10^5 cells were subcutaneously transplanted into the chest of female nude mice. Results Quality control of HYNIC peptide libraries was good as carried out by ^99 Tc^m labeling. Because peptide NLLRLTG had high affinity for HSP70 family members, ^99 Tc^m-HYNIC-NLLRLTG was used as the control. Fifteen HYNIC peptides were found with HSP70 binding property. Among them, eight peptides had higher uptake （ percentage activity of injection dose per gram of tissue, % ID/g） values than ^99 Tc^m-HYNIC-NLLRLTG in tumor. ^99 Tc^m-HYNIC-QGVLTGTR had the best distribution in tumors. Six hours after injection, the % ID/g values of ^99 Tc^m-HYNIC-QGVLTGTR and ^99 Tc^m-HYNIC-NLLRLTG in tumor were （ 1.15 ±0.32） % ID/g and （0.75 ±0.24） % ID/g respectively. In vivo replace studies and heat shock stress of tumors demonstrated that ^99 Tc^m-HYNIC-QGVLTGTR was the HSP70 binding peptide compound, but not ^99 Tc^m-HYNIC-NLLRLTG. Conclusions The identification of ^99 Tc^m labeled HSP70 binding peptides from HYNIC conjugated octapeptide libraries facilitated the hypoth- esis of the ＂tracer＂ libraries. The design and synthesis of ＂tracer＂ libraries would make a significant contri- bution to the tracer research and development.