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| 成人源性甲状腺类器官的构建、鉴定及体外培养方案的优化 |
| 盛琪轩,李蕾,李伟,张伟,王强,查斯洛,马冠君,徐昕昀,单成祥* |
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(海军军医大学(第二军医大学)第二附属医院甲乳疝外科, 上海 200003
*通信作者) |
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| 摘要: |
| 目的 构建成人源性甲状腺类器官模型，并优化其体外培养方案。方法 选取成人甲状腺乳头状癌（PTC）患者癌旁正常甲状腺组织，经消化、分离得到甲状腺细胞，以基质胶包埋细胞，设计不同甲状腺类器官条件培养基进行培养，在显微镜下观察第1代和第2代类器官形成效率和出芽情况。采用H-E染色观察类器官模型的组织学形态，免疫组织化学染色检测类器官模型中甲状腺特异性标志物NK2同源框蛋白1（NKX2.1）、配对盒基因8（PAX8）和甲状腺球蛋白（TG）表达情况，评估类器官模型与正常甲状腺组织的一致性。采用ELISA法检测类器官模型上清液中甲状腺素（T4）和三碘甲状腺原氨酸（T3）水平，验证其是否具有激素分泌功能。结果 从7例PTC患者的手术标本中成功建立了8例甲状腺类器官模型。在甲状腺类器官培养体系中，提高Noggin和表皮生长因子浓度及联用多种小分子抑制剂后，第1代和第2代类器官模型出芽更多、球体更大，类器官形成效率更高。H-E染色结果显示，甲状腺类器官模型中可见大量由单层立方上皮细胞围成的滤泡结构，与人体甲状腺组织结构特征相似。免疫组织化学染色结果显示，类器官模型中甲状腺标志物NKX2.1、PAX8和TG阳性，与来源组织生物标志物特征一致。ELISA检测结果显示，类器官模型上清液中可见T3、T4分泌，表明其具有甲状腺激素分泌能力。结论 采用成人甲状腺组织构建的类器官模型稳定，为甲状腺功能减退症的再生医学治疗提供了新的方向。 |
| 关键词: 类器官 模型 甲状腺 培养 甲状腺功能减退症 |
| DOI：10.16781/j.CN31-2187/R.20230119 |
| 投稿时间：2023-03-17修订日期：2023-11-09 |
| 基金项目: |
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| Construction, identification, and optimization of in vitro culture protocol of adult-derived thyroid organoid model |
| SHENG Qixuan,LI Lei,LI Wei,ZHANG Wei,WANG Qiang,ZHA Siluo,MA Guanjun,XU Xinyun,SHAN Chengxiang* |
(Department of Thyroid, Breast and Hernia Surgery, The Second Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200003, China
*Corresponding author) |
| Abstract: |
| Objective To construct an adult-derived thyroid organoid (ADTO) model and optimize its culture protocol in vitro. Methods Normal thyroid tissue from adult patients with papillary thyroid cancer (PTC) was digested and separated to obtain thyroid cells. The cells were embedded with Matrigel. Different conditioned culture media were designed and added. The forming efficiency and budding number of the first- and second-generation organoid were observed under microscope. Hematoxylin-eosin (H-E) staining was used to observe the histological morphology of the organoid model; immunohistochemical staining was used to detect the expression of thyroid specific markers, including NK2 homeobox protein 1 (NKX2.1), paired-box 8 (PAX8), and thyroglobulin (TG), to evaluate the consistency between the organoid model and normal thyroid tissue. Thyroxine (T4) and triiodothyronine (T3) levels in the supernatant of the ADTO models were detected by enzyme-linked immunosorbent assay (ELISA) to verify whether they have hormone secretion function or not. Results Eight ADTO models were successfully established from the surgical specimens of 7 PTC patients. In the ADTO culture system, after increasing the concentrations of Noggin and epidermal growth factor and combination of multiple small molecule inhibitors, the first- and second-generation organoid models had more buds, larger spheres, and higher efficiency of organoid formation. H-E staining showed that a large number of follicular structures surrounded by monolayer cubic epithelial cells could be seen in the ADTO model, which was similar to the structural characteristics of human thyroid tissue. Immunohistochemical staining showed positive NKX2.1, PAX8, and TG, which was consistent with the primary tissue. The results of ELISA showed T3 and T4 in the supernatant of the ADTO models, indicating that it had thyroid hormone secretion function. Conclusion The ADTO model established from human thyroid tissue is stable, which provides insights into regenerative medicine to treat hypothyroidism. |
| Key words: organoid model thyroid culture hypothyroidism |
