Novel aptasensor-based assay of sonic hedgehog ligand for detection of portal vein invasion of hepatocellular carcinoma
Novel aptasensor-based assay of sonic hedgehog ligand for detection of portal vein invasion of hepatocellular carcinoma
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基于新型适体传感器的声波刺猬配体检测肝细胞癌门静脉浸润
DOI：
10.1016/j.bios.2020.112738
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发表时间：
2021-02-15
影响因子：
12.6
通讯作者：
Wu, Jian
中科院分区：
文献类型：
--
作者：
Li, Hui-Yan;Yin, Fang-Fei;Wu, Jian
声音刺猬配体（SHH）的高表达与转移，耐药性和肝细胞癌预后不良密切相关。因此，需要对SHH进行敏感，特异性和有效的检测方法来早期诊断和评估预后。 SELEX Technology获得了与SHH（K-D = 25.7 +/- 4.1 nm）特别结合的适体AP32，并进一步优化。体内实验证实，AP32具有HUH-7细胞衍生异种移植的成像探针的潜力。通过分子模拟建立了适体和SHH之间三维构型的相互作用模式，并通过适体关键部位的突变确认。通过将德克萨斯州红标记的AP32连接到Microbeads，成功开发了基于Aptasensor的测定法，并用于分析肝癌细胞裂解液，血清和HCC标本中的SHH含量。该方法表现出广泛的检测范围为0.07至62.5 nm，低检测限为69 pm，恢复率为104.6 +/- 3.9％。当使用该测定法来测量组织裂解物中的SHH含量时，结果表明它具有57.1％的阳性，在区分正常组织的28个HCC标本方面具有100％的特异性，并且是AFP阴性病例中HCC检测的补偿性。此外，SHH水平升高指示门静脉侵袭为77.8％的正率。这种新型基于Aptasensor的SHH测定法可能会提供一种可靠的手段，以预测肝细胞癌早期转移和预后不良。
The high expression of sonic hedgehog ligand (SHh) is closely correlated to the metastasis, drug resistance and poor prognosis of hepatocellular carcinoma (HCC). Therefore, sensitive, specific and efficient detection methods for SHh are needed for the early diagnosis and assessment of prognosis. Herein, an aptamer, AP32 that specifically binds to SHh (K-D = 25.7 +/- 4.1 nM) was obtained by SELEX technology with further optimization. In vivo experiments confirmed that AP32 has the potential to be an imaging probe for Huh-7 cell-derived xenograft. The interaction mode in 3-dimensional configuration between the aptamer and SHh was established by molecular simulation and confirmed by mutations at key sites of the aptamer. An aptasensor-based assay was successfully developed by conjugating Texas-Red-labeled AP32 to microbeads, and was used to analyze SHh content in hepatoma cell lysates, serum and HCC specimens. The method exhibited a broad detection range from 0.07 to 62.5 nM with a low detection limit of 69 pM, and a recovery rate of 104.6 +/- 3.9% in serum. When the assay was used to measure SHh content in tissue lysates, the results demonstrated that it possessed 57.1% positivity, 100% specificity in distinguishing 28 HCC specimens from normal tissues, and was compensatory for detection of HCC in AFP-negative cases. Moreover, elevated SHh levels are indicative of portal vein invasion at 77.8% positive rate. This novel aptasensor-based SHh assay may offer a reliable means in predicting early metastasis and poor prognosis in hepatocellular carcinoma.