基于脱氧核酶分子探针检测早期非小细胞肺癌的研究

About 85% of lung cancer cases are non-small cell lung cancer (NSCLC) worldwide. The importance of early detection for the control of the mortality rate associated with NSCLC has been well demonstrated. However, traditional methods are either time-consuming, expensive, invasive or lack of specificity. The RNA-cleaving fluoregenic DNAzyme (RFD) performances some characters like high sensitivity and specificity, cost-saving, detection of unknown substances. RFD probe for cancer cell detection is already developed in our previous study. There are plenty metabolites in sputum, which are considered as potential RFD probe targets for noninvasive detection. In this study, in order to achieve noninvasive detection of early stage NSCLC, we choose sputum from I-II stage NSCLC patients as target for positive selection of RFD probes. The selected DNA pool revealed significant specificity to NSCLC has been obtained based on the difference between NSCLC and other lung diseases. For future study, it is proposed to 1) Selection of best probes from candidate probes based on their specificities as well as sensitivities; 2) Optimization of chosen probes for clinical sample detection; 3) Analysis with proteomics, bioinformatics and molecular biology to get bio-marker of early stage NSCLC. It is expected to provide a new noninvasive method for early stage NSCLC detection as well as new biomarkers of NSCLC.

非小细胞肺癌（NSCLC）占肺癌发生率的85%，早期诊断对于降低病患死亡率至关重要。传统检测方法费时、有创、成本高、标志物特异性不足。可剪切RNA的荧光脱氧核酶（RFD）灵敏、特异、价廉，可检测未知样本。我们已成功利用RFD探针检测肿瘤细胞。因痰液中含有NSCLC产生的代谢物，推测可以针对痰液筛选RFD探针，无创地诊断早期NSCLC。本项目以NSCLC患者代谢物为筛选对象，利用SELEX体外筛选技术，针对早期NSCLC患者与其它肺部疾病患者代谢物的差异性，已初步获得针对早期NSCLC患者代谢物的RFD探针库，拟1）通过结合力与特异性鉴定，选出效果最好的RFD探针；2）优化RFD探针，使其更适于作为临床诊断试剂盒使用；3）结合蛋白质组学、生物信息学与分子生物学，获得早期NSCLC的生物标记物。本项目的实施将有望为NSCLC的早期诊断提供新的无创检测方法及新的生物标志物。

非小细胞肺癌（NSCLC）占肺癌发生率的85%，早期诊断对于提高病患生存率至关重要。传统检测方法准确性差、检测周期长、具有创伤性或对健康具有潜在危险、需要其他手段的联合确诊、标志物检测存在特异性差、假阳性高等缺点。可剪切RNA的荧光脱氧核酶分子探针（RFD探针）灵敏、特异、价廉，且筛选过程避免了特定生物标记物确认和分离纯化。..本项目利用非小细胞肺癌患者肺部灌洗液对核酸分子库进行SELEX筛选，同时利用除非小细胞肺癌患者外其他肺相关疾病患者肺部灌洗液对筛选出的分子库进行反向筛选，得到可特异性针对NSCLC的RFD探针分子L-NSC02。通过对候选探针的特异性验证，筛选出探针L-NSC02具有较好的反应活性及灵敏度；通过优化选取反应性最好的各参数条件，分别为反应温度25°C，pH为7.0-8.0，二价金属阳离子为Mg2+，离子浓度分别为1.0 mM，进一步提高了探针的灵敏度。以5个碱基为一个单位进行碱基删除实验，根据剪切率的变化情况，得到了RFD探针L-NSC02的核心区域，这些区域与L-NSC02的结合能力及催化活性密切相关。利用酶解实验，证明RFD探针L-NSC02的靶标为蛋白质；分子筛截留实验显示其靶标分子量大于50 kDa。本项目得到第一个针对癌症的临床样本筛选出的RFD探针分子，可用于NSCLC的临床检测。该探针将有潜力为NSCLC的早期诊断提供新的无创检测方法。