通过碳纳米粒子辅助甲状旁腺切除术 产生 甲状旁腺功能减退大鼠
Summary
获得性甲状旁腺功能减退症（HypoPT）的动物模型对于了解HypoPT如何影响矿物离子稳态和验证新疗法的有效性至关重要。本文提出了一种使用碳纳米颗粒通过甲状旁腺切除术（PTX）生成获得性甲状旁腺功能减退症（AHypoPT）大鼠模型的技术。
Abstract
甲状旁腺功能减退症（HypoPT）是一种涉及甲状旁腺的罕见疾病，其特征是甲状旁腺激素（PTH）的分泌或效力减少，从而导致高血清磷水平和低血清钙水平。HypoPT 最常见的原因是甲状腺或其他颈部前部手术期间腺体意外损伤或切除。近年来，甲状旁腺/甲状腺手术变得越来越普遍，作为术后并发症的HypoPT的发生率相应增加。迫切需要一个HypoPT动物模型来更好地了解HypoPT对矿物离子稳态影响的机制，并验证新疗法的治疗效果。据报道，一种通过使用碳纳米颗粒进行甲状旁腺切除术（PTX）在雄性大鼠中产生获得性HypoPT的技术。与甲状旁腺功能减退症的小鼠模型相比，大鼠模型显示出很大的前景。重要的是，人PTH受体结合区与大鼠的序列相似度为84.2%，高于小鼠共有的73.7%相似度。此外，雌激素的影响可以影响PTH/PTHrP受体信号通路，尚未在雄性大鼠中得到充分研究。碳纳米颗粒是淋巴示踪剂，可将甲状腺淋巴结染成黑色而不影响其功能，但它们不会染色甲状旁腺，这使得它们易于识别和去除。在这项研究中，PTX后血清PTH水平检测不到，这导致了严重的低钙血症和高磷血症。因此，术后HypoPT的临床状态可以在大鼠模型中得到显着表现。因此，碳纳米颗粒辅助PTX可以作为研究HypoPT的发病机制，治疗和预后非常有效且易于实施的模型。
Introduction
甲状旁腺激素（PTH）由甲状旁腺分泌。它是钙平衡的主要调节剂，维持磷酸盐代谢，并参与骨转换1，2。甲状旁腺功能减退症 （HypoPT） 表现为 PTH 分泌减少或功能丧失。它是一种罕见的内分泌疾病，患病率约为 9-37/100，000 人年3，4，5。低PT的特征是血清PTH和钙水平降低，伴血清磷6，7升高。HypoPT根据其原因进行分类：获得性甲状旁腺功能减退症（AHypoPT）或特发性甲状旁腺功能减退症（IHypoPT）8。AHypoPT在临床实践中更常见;大约75%的AHypoPT病例是由甲状腺手术或其他头颈部手术期间甲状旁腺切除或意外损伤引起的。其他原因包括头颈部肿瘤的放化疗和药物毒性1，8。升级的诊断方法和甲状腺相关疾病筛查的增加增加了甲状腺外科手术的数量。这导致相关的甲状旁腺并发症相应增加9，10。
需要易于建立的具有稳定特征的动物模型来更好地研究AHypoPT并验证新疗法的治疗效果。在以前的研究中已经报道了对大鼠和小鼠进行的甲状旁腺切除术（PTX）6，11;然而，由于甲状旁腺的尺寸极小，其解剖分布的可变性，在实践中成功率相对较低。因此，通常进行甲状腺-甲状旁腺切除术（TPTX）（即完全切除甲状腺和甲状旁腺）以确保切除甲状旁腺12。然而，由此产生的低甲状腺素水平可能会使该动物模型的研究复杂化13。通过其他方法（如药物刺激和基因编辑）建立的HypoPT模型不能正确代表最常见的AHypoPT发病机制。我们小组以前使用敲除小鼠模型来标记甲状旁腺，并允许在不损害甲状腺和周围解剖结构的情况下切除甲状旁腺14，15。然而，该方法利用转基因小鼠模型，由于交配和育种要求，需要更长的开发时间。
因此，我们旨在建立一个易于生成的AHypoPT模型。本研究描述了使用碳纳米颗粒标记的PTX大鼠模型。甲状腺手术中常用的50mg/mL碳纳米颗粒悬浮液在局部注射后均匀分布在甲状腺中16。甲状腺变黑，但甲状旁腺未染色17，从而清楚地区分甲状旁腺和甲状腺，并允许在不影响甲状腺的情况下进行PTX。该方法适用于不同年龄的大鼠。碳纳米颗粒悬浮液的注射是安全的，对甲状腺功能的影响可以忽略不计18。本研究产生的碳纳米颗粒标记的PTX大鼠模型在4周观察期内显示出显着的低钙血症和高磷血症表型。因此，该AHypoPT模型易于建立，并且具有可重复的表型。
Protocol
Representative Results
Discussion
流行病学报告表明，甲状腺疾病的检测显着增加，进行的相关手术数量相应增加19，20。术后甲状旁腺功能减退症的发病率约为7.6%8，21，而获得性甲状旁腺功能减退症发病率的增加使这种罕见疾病受到更多的研究关注。因此，建立合适的动物模型来研究疾病的发病机制以及测试新疗法的结果尤为重要。但是，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金基金81800928、四川大学华西学院/口腔医院的研究资助（RCDWJS2021-1）和口腔疾病国家重点实验室开放基金SKLOD-R013的支持。
Materials
| 0.9% Sodium Chloride Solution | Kelun Co. Sichuan, China | ||
| 10 µL 30G NanoFil Syringe | WPI | ||
| 6-0 polyglactin 910 suture with needle | Ethicon, Inc | J510G | |
| Calcium LiquiColor test | EKF | 0155-225 | For Ca2+ analysis |
| Carbon Nanoparticles Suspension Injection | Lummy, Chongqing, China | H20073246 | 1 mL : 50 mg |
| Creatinine (Cr) Assay kit ( sarcosine oxidase ) | Jiancheng, Nanjing, China | C011-2-1 | For creatinine analysis |
| Disposable Scalpel | Shinva, China | ||
| Dumstar Biology forceps | Shinva, China | ||
| Micro Dissecting Spring Scissors | Shinva, China | ||
| MicroVue Rat intact PTH ELISA | Immunotopics | 30-2531 | For the measurement of PTH in rat serum |
| Needle Holder | Shinva, China | ||
| Phosphorus Liqui-UV test | EKF | 0830-125 | For Pi analysis |
| Ply gauze | Weian Co. Henan, China | ||
| Povidone-Iodine | Yongan pharmaceutical Co.Ltd. Chengdu, China | ||
| Prism 9.0 (statistics and graphing software) | GraphPad Software, Inc., San Diego, CA, USA | https://www.graphpad.com/scientific-software/prism/ | |
| Rat C-telopeptide of type I collagen (CTX-I) ELISA Kit | CUSABIO, Wuhan, China | CSB-E12776r | For CTX-I analysis |
| Rat Osteocalcin/Bone Gla Protein (OT/BGP) ELISA Kit | CUSABIO, Wuhan, China | CSB-E05129r | For osteocalcin analysis |
| Safety Single Edge Razor Blades | American Safety Razor Company | 66-0089 | |
| Sprague-Dawley Rats | 8 to 10 weeks old | ||
| Surgical Incise Drapes | Liangyou Co. Sichuan, China | ||
| Urea Assay Kit | Jiancheng, Nanjing, China | C013-2-1 | For urea analysis |
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