慢性炎症通过活化胰腺星状细胞Hedgehog-Gli1通路促进KRAS诱导的胰腺癌前恶性转化

Mutated KRAS is the driving gene of pancreatic neoplastic initiation. However, cells only dominated by mutated KRAS can hardly undergo carcinogenesis, in which may need other factors to participate. Chronic pancreatitis, a widely accepted pre-malignant lesion, characterized by abundant desmoplasia, may vitally influence the initiation of pancreatic cancer. According to our previous studies, we hypothesize that pancreatic stellate cells (PSCs) could be activated by the inflammation injury through Hedgehog-Gli1 pathway. The activated PSCs-derived cytokines promote the “dedifferentiation” and “reprogramming” of KRAS-mutated acinar cells and induce the sequential malignant transformation process of “acinar-to-ductal metaplasia - ductal intraepithelial neoplasia - ductal adenocarcinoma”, leading to malignancy eventually. In order to verify this hypothesis, clinical pathology informations, cell experiment in vitro and transgenic mice would be designed. A variety of technology will be conducted, including molecular biology technology, Laser capture microscopic cutting, 3D cell co-culture, laser confocal microscopy, live-cell imaging, and optical in vivo Imaging technology and so on. This important research will help us to explore the vital role of activated PSCs in the sequential malignant transformation process of pancreatic acinar cells under the inflammation microenvioment, and give a new-insights to understand the promoting effects of pancreatic stellate cells and provide potential therapeutic targets.

胰腺癌发生的驱动性基因改变是KRAS突变，但仅有KRAS突变不足以使细胞癌变，还可能需要其它因素共同作用。公认的癌前病变慢性胰腺炎，在炎症阶段就有丰富间质增生，可能对肿瘤发生有重大影响。我们根据前期研究推测：炎症通过损伤胰腺间质星状细胞并活化其Hedgehog-Gli1通路，活化的星状细胞旁分泌细胞因子作用于KRAS突变的腺泡细胞，促使腺泡细胞“重编程”与“去分化”，启动“腺泡-导管化生”–“导管内上皮瘤变”–“导管腺癌”的连续性恶性转化，最终导致肿瘤发生。为证实该假说，我们将从临床病理分析、体外细胞、转基因鼠模型三个层次，采用分子生物学、显微切割捕获、3D细胞共培养、激光共聚焦、活细胞动态摄像、动物成像等技术，探究在炎症微环境下，星状细胞Hedgehog-Gli1通路在胰腺恶性转化的连续性事件中的作用，从新角度阐明星状细胞促进胰腺癌发生的机制，并为相关靶点应用于转化医学奠定理论基础。

胰腺癌发生的驱动性基因改变是KRAS突变，但仅有KRAS突变不足以使细胞癌变，还可能需要其它因素共同作用。公认的癌前病变慢性胰腺炎，在炎症阶段就有丰富间质增生，可能对肿瘤发生有重大影响。我们推测：炎症通过损伤胰腺间质星状细胞并活化其Hedgehog-Gli1通路，活化的星状细胞旁分泌细胞因子作用于KRAS突变的腺泡细胞，促使腺泡细胞“重编程”与“去分化”，启动“腺泡-导管化生”–“导管内上皮瘤变”–“导管腺癌”的连续性恶性转化，最终导致恶性肿瘤发生。为证实该假说，我们从（1）通过胰腺手术切除标本，应用HE染色和天狼星红、阿尔辛蓝等特殊染色检测间质增生和PanIN发生程度，应用Western blot、qRT-PCR、免疫组化、免疫荧光等方法检测星状细胞活化标志，胰腺腺泡标志，胰腺导管标志，Hh 通路成员，ADM相关细胞因子等的表达量与定位情况，结果揭示炎症性间质增生、Hedgehog通路活化与胰腺ADM-PanIN-PDAC病变之间存在正相关性；（2）通过体外细胞学实验，选用伴有/不伴有炎症损伤的不同胰腺星状细胞，再与不同KRAS状态的胰腺腺泡细胞进行共培养；随后通过Caerulein刺激、上下调节SHH蛋白表达、上下调节Gli1活性、添加KRAS抑制剂等各种干预措施，在不同层次调节实验假说中各关键分子的水平或功能；结果揭示炎症刺激星状细胞并活化其Hedgehog通路后，可促进胰腺ADM及恶性转化；（3）通过转基因动物模型，由Hedgehog抑制剂干预胰腺特异性转基因小鼠及其胰腺炎模型，结果明确炎症刺激胰腺星状细胞活化后促进胰腺ADM及恶性转化，而抑制星状细胞Hedgehog通路活化可以阻断炎症刺激促进胰腺癌变的过程。本项目的成果揭示炎症引发的间质增生可促进胰腺癌发生，其机制与胰腺星状细胞Hedgehog通路活化有关，并为相关靶点应用于临床转化医学奠定基础。