Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.
Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP.
Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre+ mice with Rosa-mTmGfl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.
自1880年1在人类和其他几个物种由桑德斯特伦甲状旁腺的第一次系统介绍,了解这个小内分泌器官对人类生理和疾病的重要性不断提高。甲状旁腺分泌甲状旁腺激素(PTH),钙代谢的主要调节剂。 PTH也是磷酸盐稳态和骨代谢1,2的重要激素。颈部手术期间甲状旁腺的意外脱落或损坏是甲状旁腺功能减退症的最常见的原因,这种疾病特征在于,低血钙,低PTH,和升高的磷1。
为了更好地研究收购甲状旁腺功能减退和试验新疗法,可靠,方便小鼠模型是必需的。小鼠被广泛用于研究,因为各种各样的遗传工具是一vailable该物种允许复杂的机理研究在体内 。然而,虽然外科甲状旁腺切除(PTX)可以在大鼠2,3,4,5,而较大的哺乳动物6,7,8使用,它在技术上是在因为腺的小尺寸和其可变解剖分布的小鼠非常具有挑战性的9。因此,甲状腺-甲状旁腺切除(TPTX)在小鼠中,其中甲状腺和甲状旁腺除去一起10通常执行的。然而,低甲状腺激素水平在实验中一个潜在的混杂因素,这种模式复杂化。此外,甲状腺内C-细胞,其产生降钙素,激素钙体内平衡的重要的啮齿动物,也失去了在除去日。yroids 11。
甲状旁腺功能低下症的几种遗传小鼠模型存在,其中包括PTH空鼠标12,GCM2空鼠标器13以及NUF鼠标与钙敏感受体(的CaSR)14,15的活化突变。然而,这些遗传缺陷已经胚胎发育过程中存在,和甲状旁腺的功能,因此在胚胎发生期间已经受损。这可能会影响器官的发育,如骨架。这与患者的术后甲状旁腺功能减退症谁掌握疾病以后的生活中。此外,一些这些小鼠模型显示出早期杀伤力和生育能力下降,从而其使用12,13,14进一步复杂化。
我们开发了两个新的鼠标Models用于收购甲状旁腺功能减退。使用遗传工程小鼠表达GFP特意在甲状旁腺允许甲状旁腺可以很容易地确定用于手术切除不去除甲状腺。被穿越PTH-Cre的鼠标,其中5.5 kb的PTH子16与ROSA mTmG鼠标控制下表达Cre重组酶生成此鼠标。由此产生的PTHcre; mTmG小鼠甲状旁腺细胞中特异性表达绿色荧光蛋白。第二小鼠模型使用相同的PTH-Cre的,此时从诱导DTR鼠标在甲状旁腺除去一个STOP盒,导致白喉毒素受体的表达特异性。 DT的全身给药破坏甲状旁腺细胞,使动物hypoparathyroid无需手术。
在这项研究中提出了两种小鼠模型表明在三个月的观察围稳定的低血钙型OD。这些程序很容易执行,表型是可重复的,而hypoparathyroid小鼠表现出成活率高17。
我们使用转基因小鼠与甲状旁腺GFP表达选择性证明GFP引导下甲状旁腺切除的技术。在PTHcre; mTmG小鼠,双荧光(以Cre重组酶表达的细胞和红番茄非酶Cre绿色GFP表达的细胞),使我们能够清楚地识别,并精确地去除所有甲状旁腺无需拆卸甲状腺。而我们优选使用双荧光mTmG小鼠用于识别荧光红色非甲状旁腺组织的能力,我们的程序也应该工作以及使用单个荧光动物,如番茄红(B6.Cg-CT(ROSA)26Sortm14( CAG-tdTomato)HZE / J)鼠标。程序比较容易进行,需要每鼠约20分钟,并且在严重和持续hypoparathyroid表型结果。
此外,GFP阳性甲状旁腺的另一优点是易于检测异常甲状旁腺的。我们的研究表明在大约B6小鼠( 图1),这是由绿色荧光在我们的小鼠模型容易地检测的四分之一甲状旁腺的错误定位。相反甲状腺-甲状旁腺,我们的技术避免了与随后需要甲状腺激素替代和去除降钙素产甲状腺C细胞的未知作用除去甲状腺。荧光标记的甲状旁腺可也用于需要甲状旁腺的隔离其他调查。这种模式的限制包括对手术的要求。基本显微技术和用荧光光源解剖显微镜是必需的。
论证产生hypoparathyroid小鼠,消除对手术的需要的第二种技术。白喉毒素治疗PTHcre-IDTR小鼠表现出温和的甲状旁腺功能低下型,而仅仅需要腹腔注射DT到鼠标。关键部分FO- [R产生这种模式剂量的优化给药方案。高剂量的DT导致毒性,但低剂量降低了该方法的功效。我们确定,在5微克/公斤,给予2次注射剂量团相隔3天,导致无/死亡率最低可靠和稳定的低钙血症。
最终,我们希望这两种方法提供了获得有用的甲状旁腺功能低下症小鼠模型。使用一种新的长效PTH,我们报道在确定甲状旁腺功能低下症17的新药物的疗效在第一次使用这两种模式的。
The authors have nothing to disclose.
这项工作是由美国国立卫生研究院支持的授予口腔疾病公开赛资金SKLOD2015OF01(RB)的R01-DK100584和中国国家重点实验室。我们感谢文平赵,佐藤忠利和凯利劳特寻求帮助。
ROSAmT/mG mice | The Jackson Laboratory | 7676 | |
PTH-Cre mice | The Jackson Laboratory | 5989 | |
iDTRmice | The Jackson Laboratory | 7900 | |
6-0 polyglactin 910 suture with needle | Ethicon, Inc | J510G | |
Safety Single Edge Razor Blades | American Safety Razor Company | 66-0089 | |
Disposable Scalpel | Feather Safety Razor Co., LTD | 72042-11 | |
Povidone-Iodine Prep Pads | Dynarex Corporation | 1108 | |
Ply gauze | Busse. Inc | BHD707 | |
0.9% Sodium Chloride Solution | HOSPIRA Worldwide, Inc | 07983-09 | |
1mL 29G Insulin Syringe | BECTON DICKINSON | 329622 | |
Surgical Incise Drapes | 3M | 6640EZ | |
Dumstar Biology forceps | Roboz Surgical Instrument Co., Inc | RS-4984 | |
Micro Dissecting Spring Scissors | Roboz Surgical Instrument Co., Inc | RS-5605 | |
Needle Holder | MILTEX.,Inc | V98-42 | |
2,2,2-Tribromethanol | Sigma-Aldrich | T48402 | |
2-Methyl-2-Butanol | Sigma-Aldrich | 152463 | For dissolve the 2,2,2-Tribromethanol |
Diphtheria Toxin Powder | Sigma-Aldrich | D0564 | Dissolve in 0.9% sodium chloride solution at 1mg/mL as the stock solution in -80C |
Multicap Blood Collection Capillary tubes | Siemens Healthcare Diagnostics Ltd | 855578 | For collecting blood in iCa2+ analysis using RapidLab 348 Ca2+/pH analyzer |
RapidLab 348 Ca2+/pH analyzer | Siemens Healthcare | For iCa2+ analysis |