Culture and characterization of chicken small intestinal crypts
Culture and characterization of chicken small intestinal crypts
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鸡小肠隐窝的培养和表征
DOI：
10.3382/ps/pey010
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发表时间：
2018-05-01
期刊：
影响因子：
4.4
通讯作者：
Zhang, C. Q.
中科院分区：
文献类型：
--
作者：
Li, J.;Li, J., Jr.;Zhang, C. Q.
小肠上皮的完整性和正常功能关键依赖于基底干细胞上皮细胞的快速更新。推动这种自我更新过程的密集增殖仅限于肠道隐窝。建立合适的隐窝分离和培养方案是研究肠道自我更新机制的关键。在本研究中，分离、纯化了鸡小肠隐窝，并在Matrigel三维培养系统中进行了进一步培养。培养4d的生长因子浓度测定显示，C组(50 ng/mLEGF、100 ng/mLNoggin、500 ng/mLR-pondin 1)较A组(5 ng/mLEGF、10 ng/mLNoggin、50 ng/mLR-pondin 1)和B组(10 ng/mLEGF、20 ng/mLNoggin)明显增大细胞器直径；188.4%(P=0.026)和176.9%(P=0.034)。透射电子显微镜、中性红染色和5-乙炔基-2‘-脱氧尿嘧啶核苷掺入实验表明，培养的鸡肠道器官具有完整的结构、较高的存活率和增殖活性。此外，在培养的肠道器官中也可以检测到肠道干细胞标志基因(Olfm4、Znrf3、Hopx和Lgr5)。此外，糖原合成酶K3β抑制剂CHIR99021可使鸡肠道器官对外源刺激的反应性分别增加750%(P=0.001)、467%(P<；0.001)、450%(P<；0.001)和333%(P=0.008)。本研究对小鼠培养模型进行了改进和优化，提供了一种可作为体外生理或病理研究平台的鸡肠道器官模型。
The integrity and normal function of the small intestinal epithelium depends critically on the rapid renewal of epithelial cells from basal stem cells. The intensive proliferation that fuels this self-renewal process is confined to the intestinal crypts. Establishment of suitable protocols for crypt isolation and culture is pivotal for the studies of intestinal self-renewal mechanisms. In this study, chicken small intestinal crypts were isolated, purified, and further cultured in a Matrigel 3-D culture system. The growth factor concentration assay on the fourth d of culture showed that Group C (50 ng/mL epidermal growth factor (EGF), 100 ng/mL Noggin, and 500 ng/mL R-spondin 1) supplement in culture medium could significantly enlarge the diameter of organoids when compared with Group A (5 ng/mL EGF, 10 ng/mL Noggin, 50 ng/mL, and R-spondin 1) and Group B (10 ng/mL EGF, 20 ng/mL Noggin, and 100 ng/mL R-spondin 1) by 188.4% (P = 0.026) and 176.9% (P = 0.034), respectively. Transmission electron microscopy, neutral red staining, and 5-ethynyl-2'-deoxyuridine incorporation demonstrated the integrated structure, high viability, and proliferative activity in cultured chicken intestinal organoids. In addition, intestinal stem cell marker genes (Olfm4, Znrf3, Hopx, and Lgr5) also could be detected in cultured intestinal organoids. Furthermore, CHIR99021 (a glycogen synthase kinase 3 beta inhibitor) could enhance the expression of Olfm4, Znrf3, Hopx, and Lgr5 by 750% (P = 0.001), 467% (P < 0.001), 450% (P < 0.001), and 333% (P = 0.008), respectively, indicating the responsiveness of the cultured chicken intestinal organoids to exogenous stimulus. This study modified a murine culture model and optimized it to provide a chicken intestinal organoid model for use as a physiological or pathological research platform in vitro.