^188Re-cNGQGEQc放射性标记及其对肺腺癌细胞的抑制作用

Radiolabeling of ^188Re-cNGQGEQc and Its Inhibitory Effect on Lung Adenocarcinoma Cells

【目的】探讨放射性核素^188Re间接法标记小分子肽cNGQGEQc的可行性,并观察^188Re-cNGQGEQc对肺腺癌A549细胞的体外抑制作用。【方法】^188Re-cNGQGEQc和^188Re-cNAQAEQc(阴性多肽)的制备采用双半胱氨酸(EC)为双功能螯合剂的间接标记法,测定标记多肽的标记率、比活度、放化纯度、脂水分配系数,并观察在正常人血清中的稳定性。采用CCK-8法测定^188Re-cNGQGEQc对体外培养的肺腺癌A549细胞株的抑制效应,观察不同放射性剂量的^188Re-cNGQGEQc对肺腺癌A549细胞增殖的抑制作用,计算相对抑制率及半数有效抑制浓度(IC50),并与^188Re标记的阴性多肽及^188ReO4-进行比较。【结果】^188Re-cNGQGEQc的标记率为(90.24±1.58)%,比活度为(4.07±0.14)TBq/(mmol/L),C18柱纯化后放化纯度为(98.42±0.32)%,^188Re-cNGQGEQc的脂水分配系数lgP为(-2.90±0.03),在37℃正常人血清中放置24 h其放化纯度为(89.08±0.94)%。抑制性实验结果表明^188Re-cNGQGEQc对肺腺癌A549细胞的增殖具有显著的抑制作用,且与放射剂量呈明显正相关;而且^188Re-cNGQGEQc的IC50值为44.88×10^7 Bq/L,明显低于阴性多肽的93.45×10^7 Bq/L和^188ReO4-的99.60×10^7 Bq/L(P<0.01)。【结论】使用双功能螯合剂EC建立了标记率高、体外稳定的^188Re标记小分子肽的方法,体外实验证实^188Re-cNGQGEQc能有效地抑制肺腺癌A549细胞的增殖,研究结果为后续开展小分子多肽的放射靶向治疗肺癌的体内应用提供实验基础。

【Objective】To investigate the feasibility of using the indirect labeling method to label small molecular peptide cNGQGEQc with ^188Re and to observe the inhibitory effect of 188Re-cNGQGEQc on lung adenocarcinoma cell line A549 in vitro.【Methods】188Re-cNGQGEQc and ^188Re-cNAQAEQc(negative polypeptide radiolabeled with 188Re)were prepared by indirect labeling method with ethylene dicysteine(EC)as the bifunctional chelating agent.The labeling rate,specific activity,radiochemical purity(RCP)and octanol-water partition coefficient were determined,and the stability in normal human serum was evaluated.CCK-8 assay was used to detect the inhibitory effects of 188Re-cNGQGEQc,188RecNAQAEQc and free 188ReO4-with different radioactive doses on A549 lung cancer cell proliferation.Then the relative inhibitory rates and 50%inhibiting concentration(IC50)of the three peptides were calculated and compared.【Results】The labeling rate,specific activity and log P value of ^188Re-cNGQGEQc were(90.24±1.58)%,(4.07±0.14)TBq·mmol/L-1 and(-2.90±0.03),respectively.The RCP of 188Re-cNGQGEQc purified by using a Sep-Pak C18 column and after being placed in normal serum for 24 h at 37℃were(98.42±0.32)%and(89.08±0.94)%,respectively.CCK-8 assay results revealed that ^188Re-cNGQGEQc significantly inhibited the proliferation of A549 lung cancer cells in a dose-dependent and positively-correlated manner.IC50 value of ^188Re-cNGQGEQc was 44.88×10^7 Bq/L,significantly lower than 93.45×10^7 Bq/L for 188Re-cNAQAEQc and 99.60×10^7 Bq/L for 188ReO4-(P<0.01).【Conclusion】It is feasible to use EC as the bifunctional chelating agent to label small molecular peptide with 188Re and this labeling method has high labeling rate and good in vitro stability.The in vitro experiment confirms that 188Re-cNGQGEQc can effectively inhibit the proliferation of lung adenocarcinoma cell line A549.These results provide the experimental basis for further in vivo application of small molecular peptides for the targeted therapy of lung cancer.