Foxp3+Treg淋巴细胞诱导胰腺癌上皮间质转化的机制研究

Due to the low resection rate and poor clinical prognosis, emerging studies have focused on antitumor immunity in pancreatic carcinomas. In our previous research, we observed that regulatory T lymphocyte (Treg cells) density in primary tumor tissue in patients with pancreatic cancer correlates with lymph node metastasis. Interleukin-10 derived from co-clutureTreg cells induced epithelial-mesenchymal transition (EMT), which is a key event during tumor metastasis, in pancreatic cancer cells. Here, we attempt to reveal the critical role of Treg cells in development of EMT in pancreatic cancers both in vitro and in vivo. Utilizing promoter analysis, small RNA interference and ChIP-PCR, we aim to clarify the involvement of the paracrine IL-10-IL10R axis during this process. Furthermore, we try to determine whether targeting the IL-10-IL10R axis in vivo represents an effective approach to prevent distant metastasis in pancreatic cancers. Therefore, further support of this program would provide theoretical basis and experimental evidence for improvement of pancreatic carcinoma treatment and prognosis.

由于低手术切除率和较差临床预后，针对胰腺癌的抗肿瘤免疫研究已成为关注热点。我们在前期研究中初步证实，胰腺癌组织中调节性T淋巴细胞（Treg细胞）浸润水平与肿瘤远处转移程度呈正相关，细胞共培养结合label-free定量及质谱分析技术发现，来源于Treg细胞的IL-10可诱导胰腺癌细胞发生上皮间质转化（EMT）。EMT是恶性肿瘤发生侵袭转移的关键事件。本项目拟利用人胰腺癌细胞系、临床胰腺癌标本和裸鼠原位模型，从体内外两方面证实Treg细胞诱导胰腺癌发生EMT导致肿瘤远处转移；采用启动子分析、小RNA干扰、染色质免疫共沉淀等方法，阐释IL-10-IL10R旁分泌通路可能介导上述过程的分子调控机制；并通过动物实验观察靶向干扰IL-10-IL10R旁分泌通路对抑制胰腺癌远处转移的作用，从而为有效改善胰腺癌诊疗处理和预后提供理论依据和实践基础

肿瘤发生与发展过程与肿瘤微环境密不可分，其中Treg参与了肿瘤免疫逃逸，可能参与了其复发转移等生物学行为；我们回顾性研究胰腺癌标本发现Foxp3+Treg瘤中心密度显著高于周边，与分化显著相关，周边区域低密度者总生存期显著优于高密度者（8.6vs5.4，P=0.02），周边低密度者淋巴结转移率低于高密度者（19%vs36%，P=0.05）。边缘灶内孤立分布的胰腺癌细胞N-cadherin阳性率达到82.53%，显著高于中心灶内肿瘤细胞N-cadherin阳性率2.15%（P<0.01），而E-cadherin则反之，提示存在EMT；仅在低密度共培养是如此，而高密度时未发生明显EMT。与质谱一致，可进一步通过IL10的诱导或阻断来验证。体外培养中IL10作用后， Snail蛋白表达水平明显提高，并不伴随mRNA表达水平的改变，而其它EMT相关转录因子Slug和Twist蛋白水平则无明显改变；阻断或干扰后显著抑制IL10诱导的EMT现象。免疫共沉淀合并质谱技术分析显示CBP是IL10作用后与Snail相互结合的重要成分，正向诱导或反向抑制均验证如此。据此我们得出Foxp3+Treg经由IL10-IL10R通路低密度梯度下，通过CBP调控Snail蛋白的稳定性和活性，进而导致胰腺癌上皮的间质转化EMT，侵袭与转移能力增强。以上研究为胰腺癌的预后评估以及寻找治疗靶点奠定坚实的理论基础。