单细胞转录组测序揭示肠上皮 Becn2 特异性敲除对小鼠肠上皮细胞稳态的影响

Single-cell RNA sequencing revealed the impact on intestinal epithelial cell homeostasis in mice with intestinal epithelial-specific knockout of Becn2

摘要:

目的 构建自噬蛋白基因Becn2的肠上皮特异性敲除小鼠，观察肠上皮敲除Becn2对肠上皮细胞稳态 的调控作用。方法 将 Becn2flox/-/Villin-Cre小鼠与Becn2-/-/Villin-Cre + 小鼠交配繁育、多轮筛选后得到基因型为 Becn2flox/flox/Villin-Cre的对照小鼠（Becn2fl）与基因型为Becn2flox/flox/Villin-Cre+ 的敲除小鼠（Becn2ΔIEC ），使用PCR鉴定小鼠基因型，Western blot检测敲除效率；从4周龄开始记录小鼠体质量的变化，直到8周龄，取出小鼠结肠、脾脏，比 较 Becn2fl小鼠与 Becn2ΔIEC小鼠的结肠长度、脾脏重量差异，并通过 HE染色观察小鼠结肠与脾脏组织结构变化；对 小鼠结肠组织进行单细胞转录组测序，采用生物信息学方法分析小鼠结肠组织中肠上皮细胞亚群的变化。结果 PCR与Western blot结果显示成功建立了对照小鼠Becn2fl 和敲除小鼠Becn2ΔIEC模型；脏器数据统计显示，生理状态下敲除小鼠与对照小鼠在体质量、结肠长度、脾脏指数差异无统计学意义（P > 0.05）；HE染色结果表明两组小鼠结 肠、脾脏均结构完整，无炎性病变；单细胞转录组数据分析结果显示，与对照小鼠相比，Becn2敲除小鼠肠上皮细胞 亚群中杯状细胞比例从36.0%降低至26.5%；进一步结肠上皮细胞差异基因分析发现，结肠上皮细胞中MUC2基因 log2FC =-0.51，表达小幅下调。结论 生理状态下，肠上皮特异性敲除Becn2不改变小鼠结肠组织结构，但可降低 肠上皮杯状细胞比例、下调结肠上皮细胞MUC2基因表达，进而重塑肠上皮细胞组成。

Abstract:

Objective To establish intestinal epithelial-specific knockout mice of the autophagy protein gene Becn2, and to investigate the regulatory effect of intestinal epithelial-specific Becn2 deletion on intestinal epithelial cell homeostasis. Methods Becn2flox/-/Villin-Cre− mice were mated with Becn2−/−/Villin-Cre + mice. After multiple rounds of screening, control mice (Becn2fl) with the genotype Becn2flox/flox/ Villin-Cre− and knockout mice (Becn2ΔIEC) with the genotype Becn2flox/flox/Villin-Cre + were obtained. Mouse genotypes were identified by PCR, and protein-level knockout efficiency was detected by Western blot. Body weight changes were recorded from 4 to 8 weeks of age. Mouse organs were harvested to compare colon length and spleen weight between Becn2fland Becn2ΔIEC mice. Histological changes in the colon and spleen were examined by hematoxylin-eosin (HE) staining. Single-cell RNA sequencing was performed on mouse colon tissues, and bioinformatic methods were used to analyze alterations in intestinal epithelial cell subsets and key differentially expressed genes in mouse colon tissues. Results PCR and Western blot confirmed the successful establishment of control Becn2fl and knockout Becn2ΔIEC mouse models. Statistical analysis of organ data showed no significant differences in body weight, colon length, or spleen index between knockout and control mice under physiological conditions. HE staining revealed intact structure and no inflammatory lesions in the colon and spleen of both groups. Single-cell RNA sequencing analysis revealed that, compared to control mice, the proportion of goblet cells among the intestinal epithelial cell subsets decreased from 36.0% to 26.5% in Becn2 knockout mice. Furthermore, differential gene expression analysis in colonic epithelial cells revealed a slight downregulation of the Muc2 gene (log2FC =-0.51). Conclusion Under physiological conditions, intestinal epithelial-specific knockout of Becn2 does not alter the colonic tissue architecture , but reduces the proportion of goblet cells and downregulates Muc2 gene expression in colonic epithelial cells, thereby remodeling the intestinal epithelial cell composition.