Crypt Organoid Culture as an in Vitro Model in Drug Metabolism and Cytotoxicity Studies
Crypt Organoid Culture as an in Vitro Model in Drug Metabolism and Cytotoxicity Studies
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DOI：
10.1124/dmd.117.075945
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发表时间：
2017-07-01
影响因子：
3.9
通讯作者：
Chen, Shujuan
中科院分区：
文献类型：
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作者：
Lu, Wenqi;Rettenmeier, Eva;Chen, Shujuan
胃肠道富含异生物质加工蛋白，其在异生物质生物活化、代谢和解毒中起重要作用。转基因小鼠模型的应用有助于表征异生物质加工基因（XPG）及其蛋白在药物代谢中的功能。在这里，我们报告了利用这些动物模型的三维隐窝类器官培养来研究肠道药物代谢和毒性。随着隐窝类器官的成功培养，我们分析了I期和II期XPG表达、药物转运蛋白基因表达和异生物质核受体（XNR）基因表达的丰度。通过用XNR原型激动剂处理隐窝细胞来检查XNR的功能。实时荧光定量聚合酶链反应证实了具有代表性的下游靶基因被诱导。这些发现从XNR缺失小鼠培养物中得到验证。在分离自Pxr（-/-）小鼠的隐窝培养物中，双烯醇酮16 α-甲腈未能诱导Cyp 3a 11基因表达;类似地，WY 14643未能诱导Ppara（-/-）隐窝中的Cyp 4a 10。用喜树碱-11处理对照（Ugt 1（F/F））和肠上皮细胞（IEC）特异性Ugt 1缺失小鼠（Ugt 1（Delta IEC））的隐窝培养物，喜树碱-11是一种具有严重肠毒性的抗癌前药，其来源于其活性代谢产物SN-38的UGT 1A 1依赖性葡萄糖醛酸化不足。在不存在Ugt 1基因表达的情况下，Ugt 1（Delta IEC）隐窝培养物显示SN-38葡糖苷酸的产生非常有限，与Ugt 1（F/F）隐窝培养物相比，这与细胞凋亡增加一致。这项研究表明，隐窝类器官培养物作为一种有效的体外模型，研究肠道药物代谢和毒性。
The gastrointestinal tract is enriched with xenobiotic processing proteins that play important roles in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models has been instrumental in characterizing the function of xenobiotic processing genes (XPG) and their proteins in drug metabolism. Here, we report the utilization of three-dimensional crypt organoid cultures from these animal models to study intestinal drug metabolism and toxicity. With the successful culturing of crypt organoids, we profiled the abundance of Phase I and Phase II XPG expression, drug transporter gene expression, and xenobiotic nuclear receptor (XNR) gene expression. Functions of XNRs were examined by treating crypt cells with XNR prototypical agonists. Real-time quantitative polymerase chain reaction demonstrated that the representative downstream target genes were induced. These findings were validated from cultures developed from XNR-null mice. In crypt cultures isolated from Pxr(-/-) mice, pregnenolone 16 alpha-carbonitrile failed to induce Cyp3a11 gene expression; similarly, WY14643 failed to induce Cyp4a10 in the Ppara(-/-) crypts. Crypt cultures from control (Ugt1(F/F)) and intestinal epithelial cell (IEC) specific Ugt1 null mice (Ugt1(Delta IEC)) were treated with camptothecin-11, an anticancer prodrug with severe intestinal toxicity that originates from insufficient UGT1A1-dependent glucuronidation of its active metabolite SN-38. In the absence of Ugt1 gene expression, Ugt1(Delta IEC) crypt cultures exhibit very limited production of SN-38 glucuronide, concordant with increased apoptosis in comparison with Ugt1(F/F) crypt cultures. This study suggests crypt organoid cultures as an effective in vitro model for studying intestinal drug metabolism and toxicity.