肠道隐窝干细胞招募固有层巨噬细胞合作修复放射性肠损伤的机制研究

Radiation therapy of abdominal tumors often arise intestinal tissue damage, which not only seriously hinders the tumor control of patients, but also changes the intestinal function. Our previous study found that: 1) intestinal stem cells settled in the crypt are the key cells of intestinal radiation damage repair, radiation could induce their apoptosis; 2) macrophages were located in the upper part of the intestinal lamina propria (LP), and migrated to the crypt bottom after irradiated. Evenmore, the lack of macrophages would aggravate epithelial barrier injury. The role and mechanism of the cooperation between macrophages and intestinal stem cells in the repair of intestinal injury need to be further explored. In view of this, the project will separate Lgr5+ crypt stem cells from mouse right abdominal bowel exposed to irradiation by flow cytometry sorting and test K+/Cl- flow concentration gradient from stem cells ions pump via atomic absorption spectroscopy and noninvasive measurement, furthermore, we will verify the stem cells secret anti microbial peptide such as Reg3g and IFNL2/3 to maintain macrophage M2 type by ELISA and qRT-PCR; macrophages at the bottom of the stem cells will be obtained with laser capture microdissection system. The stem cell proliferation factors will be detected in the secretion of acquired macrophages. It is suggested that after irradiation macrophages migrate to the intestinal crypt bottom by ion efflux from stem cells, intestinal stem cells and macrophages repair the intestinal radiation injury by the paracrine collaborative mechanism. It has potential application value to explore new interventions to the wound healing of intestinal radiation damage.

腹部肿瘤的放射治疗常致肠道组织损伤，不仅严重阻碍了患者的疗效，而且肠道功能严重改变。我们前期研究发现：1) 定居在隐窝的肠干细胞是放射性肠损伤修复的关键细胞，放射致其凋亡；2) 位于小肠绒毛固有层中上部的巨噬细胞在照射后可迁移至隐窝底，而巨噬细胞的缺失会加重上皮屏障损伤。但巨噬细胞和肠干细胞如何协同修复肠损伤的作用和机制有待进一步探讨。鉴此，本项目通过建立小鼠右腹部照射肠道损伤模型，流式分选Lgr5+隐窝干细胞，原子吸收光谱和非损伤微测技术检测干细胞泵出的K+/Cl-流浓度梯度，酶联免疫法验证干细胞分泌的维持巨噬细胞M2极型的Reg3g、IFNL2/3等抗微生物因子；同时激光捕获显微切割获得干细胞底部的巨噬细胞，发掘其分泌的调控干细胞增殖因子。由此阐明巨噬细胞通过离子流迁移至肠隐窝底部和肠干细胞协同修复肠道放射损伤。这一肠道上皮屏障损伤修复的新机制，对肠型放射损伤的干预具有潜在的应用价值。

本项目全面完成了申请书三个方面的工作：1）母鼠巨噬细胞相比公鼠巨噬细胞在10Gy全身照射或20Gy腹部照射后保存的数量更多（CD68+CD163+ 腹部20Gy/NC母鼠：50%，公鼠：7%），且CD68+CD163+的M2型巨噬细胞都集中在隐窝位置，巨噬细胞吞噬了断裂的染色体后，公鼠巨噬细胞相较母鼠巨噬细胞Grem2分泌少（-100pg/ml），TGFβ1多(+200pg/ml)，促进隐窝干细胞增殖能力弱。因此母鼠受10Gy照射后祛除巨噬细胞（CD11b-DTR小鼠 40ng/g DT处理）比公鼠同样处理后小肠结构更差，绒毛只有公鼠的一半。这一结果的科学意义在于在有DNA损伤的疾病中，治疗上要考虑性别差异，尤其是不同性别免疫功能的差异。2) 10Gy照射后 K+外流可能是肠道外巨噬细胞向肠道内迁移的原因之一；Lgr5+隐窝干细胞分泌Reg3可能是肠道内巨噬细胞尤其是CD68+CD163+分布于隐窝的原因之一。3)免疫细胞对射线非常敏感，射线杀死肿瘤的同时也伤及肿瘤周边和肿瘤内的免疫细胞，免疫细胞数量的不足导致肿瘤免疫反应不足。因此，放疗时如果有肿瘤外部免疫细胞向肿瘤部位的增援，则可以大大增强肿瘤放疗疗效。与文献报道的ATP相比，dGMP可以和10Gy放疗联用，促进巨噬细胞氧化磷酸化增强，更多地向原位结直肠癌肿瘤部位迁移，并更好地协助10Gy放疗控制肿瘤，使得肿瘤更小（大于0.6mm ATP+IR：70±20%，dGMP+IR：32±19% ）更少（ATP+IR：4，dGMP+IR：2）。这一结果使得dGMP可以应用于大剂量（>10Gy）一次性短时间的FLASH放射治疗，借助于FLASH照射对正常组织损伤小，联用后可比传统2Gy分次照射控制肿瘤的效果更好。