基于miR-19-p53-mdm2网络调控作用探讨化瘀散结方药干预难治性急性白血病的机制研究

The refractory acute leukemia patients can receive good clinical efficacy with the combination treatment of Huayu and Sanjie recipe therapy and chemotherapy, in our previous studies. Huayu and Sanjie therapy can also reverse the chemo-resistance of leukemia carcinoma cell lines, by down-regulating the expression of miR-19, and by regulating the expression of p53-mdm2 pathway proteins. Therefore, our objective is to study the miR-19a-p53-mdm2 regulatory networks. We plan to evaluate the clinical efficacy of the refractory acute leukemia patients, who receive the combination therapy of Huayu and Sanjie recipe and therapeutics in clinical trial. The level changes of miR-19a-p53-mdm2 regulatory networks proteins will be detected twice from the beginning to end in the trial. High-throughput SELDI-TOF MS protein chip technology will be used to analysis the protein spectra, when abnormal proteins appear in the trial. We plan to induce one kind of K562 leukemia cell lines with miR-19 gene expressed, and induce another kind of K562 leukemia cell lines with miR-19 gene silenced, by genetic engineering techniques in this project. The effects of Huayu and Sanjie recipe to regulate miR-19a-p53-mdm2 regulatory networks in the two kinds of K562 leukemia cellular models will be evaluated. The mechanism of Huayu and Sanjie recipe to regulate miR-19a-p53-mdm2 regulatory networks will be indentified by the outcomes of clinical tiral and cellular experiments. The outcome may also guide future clinical applications and references of Huayu and Sanjie recipe to increase the efficacy of refractory acute leukemia, and potential therapeutic target will be found in the project.

在之前研究发现，化瘀散结方药辅助化疗方案治疗难治性急性白血病能够获得较好疗效，细胞实验也证实该方药能影响p53-mdm2信号通路的蛋白表达，下调miR-19的调控功能而逆转白血病化疗耐药，因此，本课题拟围绕"miR-19-p53-mdm2网络调控作用"而开展，在临床水平，选择难治性急性白血病患者为研究对象，观察化瘀散结方药联合化疗治疗难治性急性白血病的疗效，同时检测治疗前后患者miR-19-p53-mdm2网络调控蛋白的改变，表达异常者，选用高通量的表面增强激光解析电离－飞行时间－质谱蛋白芯片技术分析蛋白质谱；在细胞水平，分别构建miR-19表达和缺失两种类型的K562细胞系，研究在不同miR-19表达情况下，化瘀散结药物血清对p53-mdm2信号通路的影响。通过临床研究和细胞实验，明确化瘀散结方药治疗难治性急性白血病的作用机制，探索难治性急性白血病潜在的治疗靶点，为临床治疗依据和参考。

《基于 miR-19-p53-mdm2 网络调控作用探讨化瘀散结方药干预难治性急性白血病的机制研究》由细胞实验，动物实验和临床研究三部分构成。在细胞实验中，通过建立抑制miR-19表达的细胞模型，明确了化瘀散结方药（huayu and sanjie recipe, HSR）药物血清及单体活性成份对不同miR-19表达的人白血病K562细胞模型的作用机制和分子靶点。首次发现HSR及其活性成份可以通过调控miR-19-MDM2-p53信号通路，诱导人白血病细胞凋亡。同时也发现肿瘤细胞E2的表达可调控Ca2+通道活性，介导肿瘤细胞的化疗敏感性。在移植性人白血病细胞裸鼠动物模型中，发现HSR活性成份腹腔注射（16mg/kg）的抑瘤率为：40.45%，并可抑制瘤块细胞Ki67蛋白表达，增强TUNEL和Caspase 3蛋白的表达，最终诱导瘤块细胞凋亡。在临床研究中，观察了HSR方药对难治性复发性白血病患者血清物质代谢的影响。发现经HSR方药治疗，10例难治性急性白血病患者患者的白细胞（WBC）和中性百分比（NEU%）较治疗前结果有明显下降，HBG较治疗前有升高，结果具有统计差异，p﹤0.05,说明HSR方药对RAL患者降低病理性异常白细胞升高，保护骨髓造血功具有一定效果。经代谢组学技术检测，HSR方药治疗后的10例难治性急性白血病患者的血清中，有9种代谢物（1种氨基酸和8种脂类）在治疗后的血清中都表现为下调，这些代谢物产物的下降可能跟HSR干预难治性急性白血病患者的作用有关，但其具体的分子机制仍需进一步探讨。结合流式细胞术、免疫组化和代谢组学分析作用机制证实，HRS及其活性成份是通过调控白血病细胞miR-19-MDM2-p53信号通路、诱导细胞凋亡、调控能量代谢而发挥直接和间接抗白血病的效应。