Quantification of Proliferative and Dead Cells in Enteroids.
Quantification of Proliferative and Dead Cells in Enteroids.
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肠类中增殖和死亡细胞的定量
DOI：
10.3791/60501
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发表时间：
2020-01-31
期刊：
影响因子：
--
通讯作者：
Zha JM
中科院分区：
文献类型：
--
作者：
Li HS;Xu SF;Sheng JY;Jiang ZH;Wang J;Ding N;Wang T;Odenwald MA;Turner JR;He WQ;Xu H;Zha JM
肠上皮作为屏障，防止肠道内容物，如病原微生物群和毒素，进入身体的其他部分。上皮屏障功能需要肠上皮细胞的完整性。虽然上皮细胞增殖维持了一层连续的细胞形成屏障，但上皮损伤导致屏障功能障碍。因此，肠道内容物可以通过不受限制的途径穿过肠道屏障。肠道屏障功能障碍与许多肠道疾病有关，如炎症性肠病。离体小鼠肠隐窝可以培养并维持为隐窝绒毛样结构，称为类肠器官或“类肠”。肠样细胞是体外研究肠上皮细胞增殖和细胞死亡的理想材料。在本方案中，我们描述了一种简单的方法来量化培养肠中增殖细胞和死亡细胞的数量。用5-乙基-2′-脱氧尿苷（EdU）和碘化丙啶标记肠样细胞的增殖细胞和死亡细胞，流式细胞术分析增殖细胞和死亡细胞的比例。这是测试药物治疗对肠上皮细胞增殖和细胞存活影响的有用工具。肠上皮细胞的一个基本功能是保护肠道内容物（如致病菌和毒素）的进入。为了实现这一功能，肠道干细胞不断增殖分化为各种上皮细胞，包括肠细胞和分泌细胞，它们通过紧密连接形成屏障。肠上皮细胞的快速更新需要细胞增殖、细胞分化和细胞死亡的严格协调。细胞增殖减少或细胞过度死亡导致上皮损伤和屏障功能受损。肠屏障功能障碍与炎症性肠病有关。一种培养肠隐窝的方法已经被开发出来。使用这种技术，分离的小鼠隐窝生长为肠道类器官（enteroid），具有隐窝绒毛样结构并包含所有肠上皮细胞谱系。5-乙基-2 ' -脱氧尿苷（EdU）是一种胸腺嘧啶类似物，能够在细胞增殖过程中取代DNA中的胸腺嘧啶(T)。EdU染色可以快速准确地标记增殖细胞。碘化丙啶（PI）是溴化乙啶的类似物，在插入双链DNA时释放红色荧光。PI专门检测死细胞，因为它只穿过受损的细胞膜。
The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or “enteroids”. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2’-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival. A fundamental function of intestinal epithelial cells is to protect the entry of luminal contents such as pathogenic bacteria and toxins. To perform such a function, intestinal stem cells continuously proliferate and differentiate into a variety of epithelial cells, including enterocytes and secretory cells, which form a barrier by forming tight connections. The rapid renewal of intestinal epithelial cells requires strict coordination of cell proliferation, cell differentiation, and cell death. Reduced cell proliferation or excessive cell death leads to epithelial damage and compromised barrier function. Dysfunction of intestinal barrier has been associated with inflammatory bowel diseases. A method to culture intestinal crypts has been previously developed. Using this technique, isolated mouse crypts grow into intestinal organoids (enteroids), which have crypt-villus like structures and contain all intestinal epithelial cell lineage. 5-Ethynyl-2’-deoxyuridine (EdU) is a thymidine analog that is capable of replacing thymine (T) in DNA that is undergoing replication during cell proliferation. The proliferative cells can be quickly and accurately labeled by EdU staining. Propidium iodide (PI) is an analog of ethidium bromide that releases red fluorescence upon insertion into double-stranded DNA. PI specifically detects dead cells, since it only passes through the damaged cell membrane.
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影响因子：
3.9
作者：
Vancamelbeke M;Vermeire S
通讯作者：
Vermeire S
影响因子：
82.9
作者：
Graham, W. Vallen;He, Weiqi;Turner, Jerrold R.
通讯作者：
Turner, Jerrold R.
DOI：
10.1038/nrgastro.2016.169
发表时间：
2017-01
期刊：
Nature reviews. Gastroenterology & hepatology
影响因子：
--
作者：
Odenwald MA;Turner JR
通讯作者：
Turner JR
确定肠上皮细胞渗透性的体外和体内方法
影响因子：
1.2
作者：
Li, Ban-Ruo;Wu, Jia;He, Wei-Qi
通讯作者：
He, Wei-Qi
影响因子：
64.8
作者：
Sato, Toshiro;Vries, Robert G.;Clevers, Hans
通讯作者：
Clevers, Hans