Autophagy maintains intestinal stem cell integrity. Autophagy maintains intestinal stem cell integrity

The intestinal epithelium is continuously renewed by a pool of intestinal stem cells expressing Lgr5. We show that deletion of the key autophagy gene Atg7 affects the survival of Lgr5+ intestinal stem cells. Mechanistically, this involves defective DNA repair, oxidative stress, and altered interactions with the microbiota. This study highlights the importance of autophagy in maintaining the integrity of intestinal stem cells. Overall design: Intestinal stem cells and their direct transit amplifying progenitors from individual Lgr5-EGFP-IRES-CreERT2 (WT) or Atg7(fl/fl);Lgr5-EGFP-IRES-CreERT2 (Atg7KO) mice treated with tamoxifen were enriched by sorting CD24(low)/EGFP(High) or CD24(low)/EGFP(Low) populations, respectively. RNA was extracted using the RNeasy Micro Kit (Qiagen), reverse transcribed with the Ovation Pico System V2 (Nugen), then the resulting cDNA was fragmented and labeled with biotin using the Encore Biotin Module kit (Nugen). cDNA from 3-4 EGFP(high) and EGFP(low) samples was hybridized to GeneChip® Mouse Gene 2.0 ST (Affymetrix), scanned with a GCS3000 7G, and analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files).

肠道上皮细胞由表达Lgr5的肠道干细胞池持续更新。本研究证实，自噬（autophagy）关键基因Atg7的敲除会影响Lgr5+肠道干细胞的存活。从机制层面而言，该过程涉及DNA修复缺陷、氧化应激，以及与微生物群（microbiota）的相互作用异常。本研究凸显了自噬在维持肠道干细胞完整性中的重要性。总体实验设计：对经他莫昔芬处理的Lgr5-EGFP-IRES-CreERT2（野生型，WT）或Atg7(fl/fl);Lgr5-EGFP-IRES-CreERT2（Atg7敲除型，Atg7KO）小鼠，分别通过分选CD24(低表达)/EGFP(高表达)和CD24(低表达)/EGFP(低表达)群体，富集肠道干细胞及其直接瞬时扩增祖细胞（transit amplifying progenitor）。使用RNeasy Micro Kit（Qiagen）提取RNA，通过Ovation Pico System V2（Nugen）进行反转录，随后利用Encore Biotin Module kit（Nugen）对所得cDNA进行片段化与生物素标记。取3-4份EGFP(高表达)与EGFP(低表达)样本的cDNA，与GeneChip® Mouse Gene 2.0 ST芯片（Affymetrix）进行杂交，经GCS3000 7G扫描仪扫描后，通过Expression Console软件（Affymetrix）分析以获取原始数据（cel文件）。