巨噬细胞经细胞融合参与肠上皮电离辐射损伤再生修复的研究

Continuous and integrative intestinal epithelium provides the fundamental barrier to maintain homeostasis of body's internal environment. However, intestinal epithelium is also a highly sensitive tissue to radiation injury. Bone marrow cells (BMC) have been identified to have the capacity to participate in intestinal repair and regeneration process, but the specific cell types and mechanisms remain to be controversial. Previously, using a radiation and transplantation model, we have observed a scattered distribution of bone marrow derived intestinal epithelial cells (IEC) mainly in the villous region and there also exists proliferative BMC and marrow derived IEC, which could be hardly detected in the crypt. Recently it is reported that macrophages of BMC can fuse with intestinal stem cells (ISC) to form epithelial cells in a mouse intestinal adenocarcinoma model. According to another previous report that E-Cadherin can promote the fusion of macrophages, we draw a new hypothesis that macrophages can migrate into the radiation injuried intestinal epithelium, and under the stimulation of local E-Cadherin they can fuse with matured epithelial cells to participate in the repair and regeneration of epithelium. Therefore, we would establish both animal models and cellular models to investigate the validity of this hypothesis and its mechanisms by using macrophages with specific markers. Two photon imaging, intestinal organoid culture and other methods will be employed in this study. All the results from this project may bring us more knowledge for roles of macrophages in the repair and regeneration process of radiation induced intestinal damage.

完整的肠上皮结构是机体维持内环境稳态的重要屏障，而肠上皮又是电离辐射损伤的敏感组织。骨髓细胞可参与肠上皮损伤的再生修复过程，但具体细胞成份和机制尚未完全明确。本课题组前期发现在骨髓移植受体内分化成熟的肠上皮区域存在增殖性骨髓细胞及其形成的肠上皮细胞，而肠隐窝部位则很少见到这一现象。文献报道肠肿瘤内骨髓巨噬细胞能够同肠道干细胞发生细胞融合而形成肠上皮细胞，此外E-Cadherin可以促进巨噬细胞发生细胞融合，因此我们提出电离辐射损伤后巨噬细胞可在局部E-Cadherin的作用下同分化成熟的肠上皮细胞发生细胞融合，进而参与受损肠上皮再生修复过程的理论假说。本项目拟利用双光子成像、肠类器官体培养等方法，在动物模型和细胞模型中研究细胞融合在巨噬细胞参与电离辐射损伤后肠上皮再生修复过程中的作用，以验证这一假说。本研究的相关结果有助于深入认识巨噬细胞对电离辐射损伤后肠上皮再生修复的作用及机制。

胃肠道上皮细胞对射线高度敏感。核爆炸和核事故时，射线暴露造成的肠粘膜屏障损伤是患者死亡的重要原因。肿瘤放射治疗过程中射线也可通过损伤肠粘膜而引发腹泻等放疗副作用。因此研究放射损伤后肠上皮的再生修复过程具有重要临床意义。在研究过程中，课题组主要聚焦于骨髓细胞（特别是巨噬细胞）在电离辐射和氟尿嘧啶所致肠道修复过程中的作用，围绕巨噬细胞能否与肠上皮细胞发生细胞融合及相关机制进行研究。主要取得了如下发现：（1）明确了骨髓细胞在放射损伤肠道内的迁移和定植特点，发现全身放射损伤后供体骨髓细胞同时参与骨髓造血功能重建和肠道放射损伤修复，两者不存在先后顺序而是同步进行。早期植入的骨髓来源细胞促进肠道放射损伤修复，且早期供体骨髓细胞主要定植于肠道固有层内，未见到其直接形成肠隐窝或肠绒毛内的上皮细胞。（2）使用GFP标记骨髓细胞建立嵌合鼠后，免疫荧光染色可见肠上皮当中存在GFP阳性肠上皮细胞，但其并不表达巨噬细胞标记物F4/80，因此并未见到巨噬细胞形成肠上皮细胞的直接证据。（3）课题组研究发现大剂量5-FU注射后，在缺少骨髓来源循环细胞补充的条件下，定植于肠道固有层内的骨髓细胞可作为肠上皮修复的补充来源促进再生。（4）课题组对CD11b-DTR小鼠预先用白喉毒素处理后，可见实验组小鼠在8Gy全身照射后的平均存活时间显著低于对照组，证实巨噬细胞可参与肠道放射损伤修复。（5）mTORC1-自噬通路参与了胃酶细胞的去分化调节过程。课题组在和国外合作研究后发现胃酶细胞高表达mTORC1，而在注射高剂量Tamxifen后，胃酶细胞迅速关闭mTORC1表达并启动细胞自噬过程。（6）课题组在研究巨噬细胞参与放射损伤肠道再生修复的过程中发现，射线暴露后伴随着Lgr5+肠道干细胞的凋亡，潘氏细胞内自噬水平迅速增加，参与肠道干细胞的再生过程。总之，本课题研究发现巨噬细胞在肠道放射损伤修复中具有重要作用。