益气解毒颗粒联合盐霉素对鼻咽癌细胞株中类肿瘤干细胞的影响与机制分析

One of the biggest bottle necks of clinical treatment of tumor is the recurrence and metastasis which the real cause is cancer stem cells (CSCs). So it is more important to search for new therapy means for tumor from CSCs. Salinomycin can effectively inhibiting the proliferation of CSCs and the growth of tumor stem cell spheres from breast cancer and prostate cancer. Yiqi Jiedu granules is our group formula and can effectively inhibit nasopharyngeal carcinoma (NPC) cells proliferation in vivo and in vitro. There are remarked clinic effect of Yiqi Jiedu granules for rehabilitation of NPC patients, treatment of nasopharyngeal precancerous lesions and high-risk populations. In this study, tumor-like stem cells will be isolated from CNE2 and CNE1, and prepared their implantation tumors. Then tumor-like stem cells and their implantation tumors are treated with different concentration Yiqi Jiedu granules or/and salinomycin. The treating effects are analyzed by SPSS statistical soft. And more, their mechanisms will be analyzed such as the cell proliferation and apoptosis assayed by flow cytometry, the local micro-environmental proteins for tumor stem cell assayed by immunoflorescence, the changes of ultrostructure and autophagy detected by using transmission electron microscopic examination, differential genes and proteins from different medicine treating groups screened by oligonucletide microarray and protein array. This study will provide scientific basis for the applications of combination Chinese and western medicine, provide a new pathway for tumor prevention and lay a foundation for clinic application of Yiqi Jiedu granules.

肿瘤治疗面临最大瓶颈之一是复发和转移，其原因可能是肿瘤干细胞(CSCs)，以CSCs为靶点寻找肿瘤治疗新方法具有重要意义。盐霉素可有效抑制乳腺癌、前列腺癌CSCs增殖及克隆球生长；益气解毒颗粒对体内外鼻咽癌（NPC）细胞均有较强的抑制作用，用于临床NPC康复治疗、鼻咽癌前病变治疗，效果显著。本研究拟先从NPC细胞株分离出类肿瘤干细胞，用益气解毒颗粒、盐霉素单独和联合干预体外培养NPC细胞株类肿瘤干细胞及其移植瘤模型，分析药物单独和联合应用对体外培养NPC类肿瘤干细胞和其移植瘤的影响，再采用免疫荧光法、电镜观察法、流式细胞术、基因和蛋白质表达芯片技术从细胞增殖、凋亡、自噬、局部微环境、超微结构变化、差异表达基因和蛋白分析药物联合应用作用机制，为中西医联合用药协同增效提供科学依据，从CSCs研究中医药抗癌作用，为肿瘤防治研究提供新途径；也为益气解毒颗粒应用进一步奠定基础。

益气解毒颗粒和盐霉素联用是否具有协同抗鼻咽癌干细胞的作用，本项目采用了HE染色法、电镜观察法、流式细胞术、基因芯片、免疫荧光法、免疫组化法和Western blot法分七部分进行了研究，主要结果如下：（1）从CNE2、5-8F、6-10B分离鉴定得到了鼻咽癌干细胞，分别表示为CNE2-SC、5-8F-SC、6-10B-SC。（2）与普通鼻咽癌细胞比较，鼻咽癌干细胞具有较强的自我更新能力、分化潜能、耐药性，增殖速度低、克隆形成率高和克隆小的特征；细胞周期相关蛋白P53、P21、 CyclinD1、CyclinD3、CDK2、CDK4、CDK6及抑癌蛋白PTEN、E-cadherin表达水平显著降低，而干性相关蛋白CD44、N-cadherin表达水平显著升高。（3）益气解毒方可诱导体外培养鼻咽癌干细胞分化，降低其自我更新能力，抑制增殖，诱导凋亡（可能通过内部线粒体途径）。（4）从miRNA角度研究了益气解毒方对鼻咽癌干细胞的杀灭作用，发现该方通过上调miR-200b表达进而抑制CD133表达来杀灭鼻咽癌干细胞。（5）益气解毒方、西药单用和联合应用均可抑制CNE-2Z鼻咽癌细胞裸鼠移植瘤的生长，药物联用具有协同作用且副作用小，药物作用与下调CD44、CD133表达密切相关。（6）益气解毒方、盐霉素单用和联合应用均能抑制鼻咽癌细胞增殖、克隆形成、迁移，影响细胞周期，诱导细胞自噬。（7）不同浓度益气解毒方水提物和总多糖能促进巨噬细胞RAW264.7的生长，而不同浓度醇提物、总皂苷、总黄酮作用不一；益气解毒方水提物和总多糖均能上调RAW264.7细胞TNF-α、IL-12和NO分泌水平，作用可能与上调NF-κBp65蛋白的表达相关。这些结果提示miR-200b、CD44和CD133可作为鼻咽癌预防和治疗的潜在靶点，初步提示益气解毒方和盐霉素的联合应用可作为治疗鼻咽癌的方案，且值得进一步深入研究。已发表期刊论文15篇，待刊1篇，会议论文2篇，参加国际会议2人次，国内会议16人次，会议报告3次，培养毕业硕士研究生4名，博士研究生1名，获得中国中西医结合学会科技进步三等奖和中华中医药学会科技进步三等奖各1项。