甲状旁腺全切除后骨髓细胞甲状旁腺素的合成和调节研究

Parathyroid hormone (PTH) is an important hormone to regulate calcium and phosphorus metabolism, and parathyroid glands are the major PTH secretion organs. In addition to parathyroid glands, thymus cells also secrete PTH. Many studies had shown that serum PTH levels of patients after parathyroid total resection was still in a certain level whereas parathyroid missing mouse without thymus merely contained trace of PTH. It is suggested that there are other sources of PTH. Our study had found that bone marrow mesenchymal stem cells (BMSCs) have the expression of Gcm2 and PTH in adult rats. We speculate that bone marrow may be another source of synthesis and secretion of PTH. We consider that the locally synthesized PTH not only have local effects on hematopoietic niche but also play a role in systemic PTH homeostasis that could be done by inducing BMSCs to differentiate into functional parathyroid cell-like cells (PTLCs). Consequently, it is interesting that whether BMSCs have the potential to differentiate to parathyroid precursor cells and PTLCs. .In this study, the concentrations of PTH will be measured by ELISA in 3rd passage BMSCs culture medium at 1,3,7,14,21 days. The parathyroid precursor markers Pax1, Eya1, Six1 and PTLCs markers Gcm2, CaSR and PTH will be detected by immunocytochemistry, RT-PCR and western blot in bone marrow cells(BMCs) and a bone marrow slices after total parathyroidectomy (PTX) in vivo and in 3rd passage BMSCs cultured in vitro. We are also concerned with the mechanism that how BMSCs differentiate to parathyroid precursor cells(PTPCs) and PTLCs. The effects of Shh , activin A, PTH and low calcium in culture medium will be investigated on the cultured BMSCs to explore whether BMSCs are induced into functional PTLCs. It will be detect the expression of PTH, CaSR, Gcm2 and the level of PTH produced by differentiated cells during the processes. We shall observe the effects on cell differentiation and PTH secretion with extracellular low calcium in culture medium and judge the capability of the cells to produce PTH, the response to Shh, Activin A, PTH and low calcium, and the regulation mechanisms. The aim is to observe the reaction characteristics of the bone marrow cells after PTX and find the mechanism of individual differences in hypoparathyroidism or hyperparathyroidism caused by a variety of reasons and provide a new research clue for the treatment hypoparathyroid by mobilizing endogenous parathyroid precursor cells in BM.

病人甲状旁腺全切除（PTX）后血清PTH仍有一定水平，提示有甲状旁腺（PT）外来源的PTH。我们研究见骨髓细胞表达PTH、Gcm2等,推测骨髓是分泌PTH另一来源，对造血微环境和全身PTH浓度维持和适应调节起一定作用。本研究采用免疫细胞化学、RT-PCR和western blot法，以PTX鼠为模型，观察PTX后骨髓切片和骨髓细胞PT前体细胞（PTPCs）标志Pax1、Eya1和Six1及PT细胞样细胞（PTLCs）标志Gcm2、CaSR和PTH表达，探讨PTX对骨髓细胞PTH合成的影响，并用第三代传代培养骨髓间充质干细胞（BMSCs）加入Shh、激活素A、PTH及低钙培养对BMSCs向PTPCs和PTLCs分化过程、分化细胞分泌PTH能力的影响来阐明调节机制，同时测定培养细胞上清PTH浓度, 判断是否分化为功能性PTLCs，为细胞移植或动员骨髓PTLCs治疗甲状旁腺功能减退症提供研究基础

甲状旁腺素是调节钙磷代谢的激素，甲状旁腺是PTH的主要分泌器官，骨髓可能是合成和分泌PTH另一来源，可通过骨髓间充质干细胞(BMSCs)分化为有功能的甲状旁腺细胞样细胞（PTLCs）。本研究以PTX鼠为模型，PTX后1、3、7、14、21天和第三代传代培养BMSCs细胞Shh、激活素A、环杷明及低钙诱导1、3、7、14、21天,免疫组织化学、定量PCR和western blot法检测在体骨髓和培养BMSCs的甲状旁腺前体细胞标志Pax1、Eya1和Six1及PTLCs标志Gcm2、CaSR和PTH表达和SHH信号通路Shh、Ptc、Smooth、Gli1的表达，观察BMSCs向甲状旁腺前体细胞和PTLCs分化过程。PTX后1、3、7、14、21天通过免疫组化法检测各组骨髓切片向甲状旁腺细胞各阶段发育分化的标志蛋白结果表明，骨髓细胞对双侧甲状旁腺全切除会产生增加向甲状旁腺细胞样细胞分化的反应，PTX组SHH、PTCH、SMO基因表达表现为先增高后降低，术后1天、3天PTX组与对照组比较有非常显著差异，PTX组术后早期GCM2、PTH、CASR、Pax1、Eya1和Six1基因表达水平较高，后期逐渐降低。术后１天、３天PTX组与对照组和假手术组比较这些基因表达有非常显著差异。定量PCR结果表明，PTX组骨髓细胞PTCH、GLI1、PAX１、SIX１表达术后3天下调，PTH、PTCH、GLI1、SIX１表达术后14天上调，EYA１表达术后3天上调，PTCH、 GLI1 、CASR表达术后14天下调。PTX组术后前７天PAX１、EYA１基因表达增强，７天后表达受抑制。SIX１基因PTX组术后前１４天表达增强，１４天后表达受抑制。离体诱导BMSCs 分化的定量PCR结果表明， 4种诱导因素均可诱导细胞分化为PTLCs。低钙处理在第3天以及SHH+激活素A和PTH处理在14天可以增强CaSR基因的表达（p＜0.05），低钙处理在第3天、PTH处理在第6天以及SHH+激活素A处理在第14天可明显增强PTH表达（p＜0.01），低钙处理在第3天和SHH+激活素A处理在第6天可明显增强GCM2表达（p＜0.05），低钙处理和SHH+激活素A处理都有较明显的诱导BMSCs向甲状旁腺细胞样细胞分化作用，低钙和Shh通路与BMSCs分化为PTLCs有关,这为探索干细胞诱导分化提供新的方法。