新型预测分子标识等位基因变异指数的建立及其高效敏感实时定量均相检测方法研究

Detection of the important allele variations provides key prognostic evidence for the monitor of the development and personalized therapy of cancer. Now, allele variations genotype and its absolute quantity is the molecular marker. However, the prognostic value of allele variations genotypes and its absolute quantity is low. There is no reasonable, sensitive, specific and precision molecular marker and detection method for the monitor of the development and personalized therapy of cancer. It has been reviewed that there is a heterogeneity and dose effect in allele variations. Both allele wild-type and multiplex variations control individual phenotype and clinical characteristics. So we believe that the relative quantity of allele variations or the ratio of allele variations and wild-type is much more related with the individual disease development and response to drugs. The relative quantity or the ratio of allele variations and wild-type has a higher prognostic value for the monitor of development and personalized therapy of cancer than allele variations genotype and its absolute quantity does. However, until now, the ratio of allele variations and wild-type can not be detected and applied in clinic because of the lacking of detection method and diagnosis theory. This study names the ratio of allele variations and wild-type as allele variations index. On the basis of our previous research of molecular markers detection with fluorescence polarization assay, this study will investigate the prognostic value of allele variations index in vitro, in vivo and in clinical samples. This study will also explore a novel multiplex allele variations amplification-hybridization reaction in a closed-tube and a matched realtime quantity fluorescence polarization assay. A novel molecular marker, allele variations index, and its sensitive, specific, efficient, standard, quantitative realtime homogeneous detection method will be established. This study will provide a novel scientific, reasonable, practical and precision theory for allele variations detection and application in the monitor of the development and personalized therapy of cancer.

等位基因变异检测为肿瘤监测及个体化治疗提供预测依据。现行的分子标识等位基因变异型别和其绝对量检测的预测诊断价值低，导致肿瘤监测及个体化治疗缺乏可靠特异的预测分子标识。研究表明，等位基因变异存在异质性和剂量效应，变异与野生型共同决定个体表型特征，故反映两者性质数量的综合指标即等位基因变异与野生型相比的相对数量水平较单一的变异型和其绝对量与疾病转归和药物反应性更显著相关，检测涵盖两者的等位基因变异相对数量水平将能获得准确的预测依据。但目前缺乏相应检测方法和诊断应用理论。本题命名等位基因变异与野生型相比的相对数量水平为等位基因变异指数，拟在前期分子标识与检测方法研究基础上，在体内外及临床实验中系统量化地探索其预测诊断价值，探索其高效、灵敏特异、标准量化的荧光偏振实时均相检测方法。该研究将为肿瘤监测及个体化治疗提供全面可靠、准确实用的新型分子标识和检验方法，建立等位基因变异科学合理检测和应用新理论

为建立全面客观、适合临床应用的疗效预测指标，本研究分别探索了等位基因变异指数、免疫微环境、化疗损伤微环境监测在肿瘤预见性个体化治疗分子诊断中的评价价值。重要等位基因变异检测为肿瘤个体化治疗提供预测依据，是目前通用的分子标志，但肿瘤等位基因变异存在异质性，等位基因变异与野生型相比的相对数量水平较变异型和绝对数量与疾病转归及药物反应性更显著相关，对肿瘤监测与个体化治疗具更高临床诊断价值。但目前缺乏等位基因变异与野生型相比的相对数量水平分子诊断理论及检测方法，只检测等位基因变异型别或绝对量。本研究建立、评估了等位基因变异指数在肿瘤监测及预见性个体化治疗分子诊断中的评价价值，建立了等位基因野生及多重变异定性定量检测反应和实时定量荧光偏振液相检测分析技术，发现等位基因变异指数与药物反应性理论预测值并不一致，提示在突变与野生细胞共存的异质肿瘤中，突变与野生细胞间存在更根本的基因信号相互沟通交流影响，从而使耐药性呈现并不与等位基因野生与变异型比值成线性比例的改变，虽并未为肿瘤个体化治疗提供预期的实用及标准量化的疾病转归及药物反应性分子标识，但揭示了肿瘤内异质细胞间存在基因变异信号相互沟通影响，为下一步肿瘤治疗策略机制研究提供了新依据和新线索；本研究评价了免疫细胞、免疫微环境调节对不同等位基因变异指数肿瘤的药物反应性影响，发现异质肺癌中，免疫调节与药物治疗协同促凋亡、抑制增殖，EMT参与了基因变异瘤细胞药物敏感性调节，提示在异质肿瘤中，免疫微环境监测对基因变异耐药肿瘤个体化治疗具诊断评价价值；本研究评估了化疗损伤微环境监测对基因变异耐药肿瘤药物反应性影响，发现化疗损伤微环境DAMP分子释放与药物及免疫治疗协同促进肿瘤细胞免疫原性死亡及凋亡、抑制增殖，提示化疗损伤微环境DAMP分子在等位基因变异耐药肿瘤个体化治疗疗效预测中具分子诊断评价价值。该研究将为肿瘤个体化综合治疗疗效预测提供新策略.