基于遗传和表观遗传协同作用研究GSTP1对结直肠癌患者奥沙利铂治疗过程证素动态变化的影响

GSTP1, which directly participates in the detoxification of platinum compounds, is an important mediator of both intrinsic and acquired resistance to oxaliplatin, a mainstsy in the treatment of colorectal cancer. Our preliminary work suggested that the genetic and epigenetic mechanisms of GSTP1 gene may account for obvious individual difference in efficacy and toxicity of oxaliplatin-based treatment. We propose to examine 150 colorectal cancer patients receiving oxaliplatin-based treatment to understand whether GSTP1 is one of the congenital factors determining the dynamic change patterns of TCM pathology and sydrome during chemotherapy. TCM diagnostic information and 2ml blood of patients will be collected at 5 observation points from one day before the treatment to one week after the whole treatment. The method of Syndrome Elements Differentiation then will be applyed for TCM diagnosis and pathological grading for each patients in order to reveal the dynamic pattern of TCM pathology and syndrome.The GSTP1 Ile105Val polymorphism will be genotyped using genomic DNA isolated from blood for understanding the genetic basis of individual susceptibility to TCM syndrome and personal characteristic of TCM pathology. Finally the impact of interaction among SNP, DNA methylation and 3 GSTP1-targeted microRNAs on TCM patterns of oxaliplatin treatment will be analyzed using the data from tests of cancerous tissue samples from surgery and blood samples colleted at 5 observation points. The dynamic TCM patterns during chemothrapy and related genetic factors found this study will provide the basis for the joint use of chemotherapy and TCM, therefore contribute to reducing toxicity and improving efficacy of chemotherapy.

奥沙利铂是结直肠癌化疗的基本药物。GSTP1是该药减毒和耐药的重要基因，前期研究表明其遗传和表观遗传调控可导致药物疗效和毒性的个体差异。为了探讨GSTP1是否为奥沙利铂化疗过程影响证素变化规律的"先天因素"，本课题拟以150例术后用奥沙利铂治疗的结直肠癌患者为观察对象进行研究。在化疗前、中、后（共5个观察点），采集患者的四诊资料和血样，用证素辨证法进行诊断和病理分级，阐明化疗不同阶段中医证的动态变化特征；检测GSTP1 的Ile105Val遗传多态性分析不同基因型患者证的易感性和病理差异，部分揭示证候形成和演变的遗传学基础；分析GSTP1mRNA、3种调控miRNA和DNA甲基化水平并结合血清miRNA的动态变化，明确GSTP1遗传和表观遗传协同作用对化疗过程证的变化特征的影响。本研究通过系统性地研究化疗过程证的动态变化及其遗传基础，为中西医联合用药提供依据，促进肿瘤化疗增效减毒。

本项目在化疗前和化疗过程的不同阶段，采集结肠癌患者的四诊资料和血样，用证素辨证法进行诊断和病理分级，阐明化疗不同阶段中医证的动态变化特征。检测GSTP1 的Ile105Val遗传多态性分析不同基因型患者证的易感性和兼夹演变。结合GSTP1多态性、DNA甲基化水平和血清microRNA，明确GSTP1遗传和表观遗传协同作用对化疗过程证的变化特征的影响。结果如下：.（1）证素分布。在未化疗组和化疗组中，气虚、阳虚、阴虚、血虚、气滞、痰、湿、热、肝、脾、大肠和肺证素均为结肠癌患者主要证素（频率≥10%）。气滞、肝和肾证素的频率在化疗组显著增加，而热证素则在化疗组中显著降低（P<0.05）。.（2）化疗过程的证素动态变化。对结直肠癌患者不同化疗阶段的持续观察，比较不同阶段证素变化，肾和脾证素的频率，随着化疗的推进，药物剂量的增加而显著地升高；脾、肾、血瘀和痰的积分随着化疗周期增加而明显升高（P<0.05）。.（3）GSTP1基因多态性对证素的影响。不同GSTP1基因型的患者血瘀证严重程度和变化趋势有显著差异。在随着化疗推进，至少带有一个G的患者血瘀证的严重程度显著高于A/A型患者。气虚兼夹脾证素的频率在A/A，A/G和G/G三种基因型患者中依次为54.9%、36.8%和14.3%，呈下降趋势，差异具有显著性。.（4）遗传和表观遗传的协同作用。采用logstic二元回归分析发现，GSTP1甲基化水平、mir-133a和mir-152表达高低对痰证素的诊断有协同作用；mir-1260、mir-152和mir-4286对气滞证素的诊断具有显著性影响；miR-152和患者的化疗阶段对湿证素的诊断有显著性影响。.（5）证素与预后的关系。对不同证素与血清肿瘤标志物CEA和CA19-9的关系进行分析，发现痰证组中CEA阳性率明显高于非痰证组中CEA阳性率（P＜0.05），提示痰证患者可能有更高的复发转移的可能性。