雌激素膜受体GPER激活诱导潘氏细胞成熟在维持肠干细胞微环境中的作用及机制研究

As the key component of intestinal stem cell niche, paneth cell determines the fate of intestinal stem cell. However, the target that regulates the function of paneth cell is unclear. Our preliminary experiments showed firstly: ① There was expression of GPER on paneth cell at C57BL/6 mice. Activation of GPER prompted the maturation of paneth cell in vivo and epithelial regeneration in organic culture. ② GPER activation up-regulated the expression of Wnt target gene c-myc. Inhibition of Wnt3 secretion from paneth cell blocked the up-regulation of c-myc expression induced by GPER activation in organic culture. ③ GPER activation enhanced the production of Wnt3 mRNA and MMP-7 mRNA in paneth cell. ④ GPER activation alleviated weight loss and intestinal mucositis induced by 5-Fu in vivo. Furthermore, the number of paneth cell, ß-catenin protein expression and intestinal stem cell marker gene were also up-regulated by GPER activation in intestinal injury model. Thus, we suppose that estrogen combines with GPER to induce the maturation of paneth cell via up-regulating the Wnt effectors. GPER is involved in the maintenance of intestinal epithelial regeneration in physiological and pathophysiological situation via niche dependent pathway. Thus, we design to prove the hypothesis and explore the molecular mechanism with Lgr5-EGFP-IRES-CreERT2 mice and paneth cell deficiency mice at the level of whole body, tissue and cell in physiological/pathophysiological state. The research results will reveal a novel mechanism that maintains the paneth cell function and provide a new target for treatment of disease induced by intestinal stem cell injury.

潘氏细胞是肠干细胞微环境的主要细胞成分，是干细胞命运的决定因素，机体调节潘氏细胞的功能靶点复杂不清。我们发现：小鼠小肠潘氏细胞表达雌激素膜受体GPER，GPER激活促进潘氏细胞成熟及离体肠上皮再生；抑制潘氏细胞Wnt3分泌，显著抑制GPER激活诱导的干细胞增殖基因表达上调；GPER激活上调潘氏细胞Wnt3、Wnt信号靶基因MMP-7表达；在体实验发现GPER激活减轻化疗性肠损伤模型的体重下降和小肠粘膜损伤，增加潘氏细胞数目及ß-catenin表达。我们推测：雌激素结合潘氏细胞GPER，通过调节Wnt信号途径诱导潘氏细胞成熟，调节肠干细胞微环境，参与生理肠上皮再生和病理肠粘膜修复。拟利用Lgr5-EGFP-IRES-CreERT2小鼠、潘氏细胞缺陷小鼠，从组织、细胞、分子水平在生理/病理条件下对假设进行研究。揭示一个全新的潘氏细胞功能调节靶点，为肠上皮再生及损伤治疗提供新思路。

肠干细胞(intestinal stem cell ISC)自我更新、增殖和分化（干性）是肠上皮再生的基础，与多种肠上皮损伤相关疾病有关。在本课题中我们研究了G蛋白偶联雌激素受体（GPER）在肠干细胞干性维持中的作用，利用5-氟尿嘧啶（5-FU）诱导的化疗性肠损伤模型，探究GPER对化疗性肠损伤模型的作用及其机制。.回肠Lgr5+干细胞和潘氏细胞均表达GPER。与对照组相比，GPER选择性激动剂G-1处理4周后，回肠隐窝深度增加；隐窝Ki67和Brdu阳性细胞数目增加；干细胞标记物表达上调。G-1处理5天后，类器官面积和出芽明显增多，EdU阳性细胞数目增加。G-1处理促进回肠组织和隐窝类器官Wnt信号途径配体Wnt3表达，Wnt途径下游靶基因β-catenin、c-Myc表达上调。IWP2抑制隐窝类器官潘氏细胞Wnt3分泌，G-1处理依然上调Wnt3表达，但G-1促进类器官形成及上调β-catenin表达的作用被阻断。G-1处理潘氏细胞成熟相关基因SOX9、MMP7表达上调，潘氏细胞产物溶菌酶、隐窝素表达上调。结论：GPER激活促进潘氏细胞成熟和Wnt3分泌，进而激活Wnt/β-catenin促进干细胞增殖。.G-1处理抑制化疗性肠损伤小鼠的体重减轻和组织学损伤，恢复黏膜屏障功能障碍，且G-1治疗不影响5-FU的抗肿瘤作用。G-1治疗降低了化疗性肠损伤模型促凋亡蛋白、cyclin D1和cyclin B1的表达，逆转隐窝中TUNEL+细胞、Ki67+细胞和Brdu+细胞数量的异常,GPER拮抗剂G15消除G-1对化疗性肠损伤模型的保护作用，单独使用G15增加化疗性肠损伤的严重程度。G-1处理抑制5-FU诱导的隐窝细胞DNA损伤。G-1处理恢复5-氟尿嘧啶诱导的隐窝细胞和离体IEC-6细胞的ERK1/2活性抑制，ERK1/2抑制剂PD0325901取消G-1对ERK1/2活性的保护作用后，G-1抑制5-FU诱导的在体隐窝细胞和离体细胞系DNA损伤的作用被消除。PD0325901处理抑制G-1保护化疗性肠损伤的各项表现，如减少体重下降、缓解组织学损伤、保护粘膜屏障、保护隐窝细胞增殖和抑制隐窝细胞凋亡和等均被抑制。研究提示GPER激活以ERK1/2依赖的方式抑制隐窝细胞DNA损伤对化疗性肠损伤模型发挥保护作用，GPER可能是预防化疗性肠损伤的治疗靶点。