垂宁方下调miRNA-26b拮抗泌乳素瘤对溴隐亭耐药的机制研究

Prolactinoma is the most common type of functional pituitary adenoma, accounting for about 50% of all pituitary adenoma. The Combination of traditional Chinese medicine and bromocriptine on pituitary adenoma is one of the most important methods of clinical treatment. Previous study of our group found that ChuiNing formula (Based on TCM theory of Dissipating Phlegm and Removing Blood Stasis) can assist the synergism of bromocriptine, and significantly reduce toxicity side effect. The latest studies have confirmed that miR-26b is involved in the regulation of pituitary adenomas, a key impact on the formation and development of pituitary adenoma, and also involved in the mechanism of bromocriptine resistance (dopamine receptor subtype expression imbalance). We has found that ChuiNing formula significantly decreased serum miR-26b expression. So the relationship between dopamine receptor is worth for further study. In view of this, we proposed the hypnosis that ChuiNing formula affect D2R different types expression and other relative target gene’s transcription through down regulation of miRNA-26b. In this proposal, miR-26b plasmid transfection, ddPCR, Luciferase, CFSE technologies will have been examined for the combination treatment of Chuining formula and bromocriptine on nude mice xenograft model with human pituitary adenoma cells. The successful validation of the hypothesis will be valuable for integrated traditional Chinese and Western medicine treatment of pituitary adenoma in clinic.

泌乳素瘤是功能性垂体腺瘤中最常见的类型，约占垂体腺瘤的50%。中药联合溴隐亭治疗泌乳素瘤是临床最重要的治疗方法之一。课题组临床长期运用化痰祛瘀之垂宁方可增效溴隐亭的抗肿瘤效应，显著降低毒副作用。研究证实：miR-26b是参与调控垂体腺瘤发生、发展的重要环节，与溴隐亭的耐药形成机制，多巴胺受体（D2R）亚型失衡密切相关。申请者前期采用miRNA芯片发现垂宁方显著下调患者血清miR-26b表达，而该方是否通过miR-26参与调控D2R亚型表达仍不清楚。鉴于此，我们拟分别采用miRNA质粒转染，ddPCR，Luciferase，CFSE荧光标记与裸鼠肿瘤接种等技术，从体内、体外不同角度，验证“垂宁方通过下调miR-26b，影响D2R不同分型及相关靶基因的转录，从而拮抗泌乳素瘤对溴隐亭耐药效应”的科学假说。本项目有可能为中西医结合治疗泌乳素瘤的临床运用提供科学依据，具有潜在的实用价值。

溴隐亭是治疗垂体腺瘤的首选药物之一，但部分患者出现耐药，影响治疗。中药联合溴隐亭治疗泌乳素瘤是中西医结合临床最重要的治疗方法之一。课题组临床长期运用化痰祛瘀之垂宁方可增效溴隐亭的抗肿瘤效应，其协同运用可以降低溴隐亭剂量，从而显著降低毒副作用。申请者前期采用miRNA芯片发现垂宁方显著下调患者血清miR-26b表达，而该方是否通过miR-26参与调控D2R表达仍不清楚。鉴于此，我们在临床研究基础上，分别采用miRNA质粒转染，Realtime PCR，与裸鼠肿瘤接种等技术，从体内、体外不同角度，验证“垂宁方通过miR-26b，下调D2R及其相关蛋白表达，从而拮抗泌乳素瘤对溴隐亭耐药效应”的科学假说。在本项目资助下，课题组主要完成以下研究工作：1）利用中医传承辅助平台系统分析了课题组化痰祛瘀法治疗泌乳素型垂体瘤的组方用药规律，为总结课题组中医药临床用药经验提供客观方法；2）成功制备了垂宁方提取物，并采用LCMS技术初步分析了垂宁方提取物中的化合物；3）成功构建了miR-26b的表达质粒PSIREN-miR-26b，模拟miR26b高表达病理背景下的耐药垂体瘤株，显示在miR26b高表达情况下，多巴胺受体蛋白下调，垂体瘤细胞增殖旺盛；4）体外研究表明，垂宁方在IC50 0.72g/5ml浓度下，24h，48h，72h体外培养均显示细胞增殖与对照组相比增殖显著放缓，但效应弱于溴隐亭，垂宁方干预显著降低了体外培养垂体细胞miR-26b下游信号通路蛋白PETEN和AKT表达。5）将体外培养的耐药垂体瘤细胞接种到裸鼠皮下，并以单纯溴隐亭为对照，联合不同剂量的垂宁方口服进行治疗，结果发现垂宁方不同剂量组均可有效抑制垂体瘤的生长，小剂量与中剂量组也优于PBS空白对照组，皮下垂体瘤的生长显著延缓。采用免疫组化显示，垂宁方上调了肿瘤细胞中的D2R受体蛋白表达水平，提示可能是其增效协同作用途径。