南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (2): 238-243.doi: 10.12122/j.issn.1673-4254.2022.02.10

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继发性甲状旁腺功能亢进症来源的甲状旁腺原代细胞的培养及鉴定

李珮婷，李 刚，柳丽丹，黄 珊，李 俊，吴 唯   

  1. 中南大学湘雅三医院乳甲外科，湖南 长沙 410013
  • 出版日期:2022-02-20 发布日期:2022-03-16

Cultivation and characterization of primary human parathyroid cells from patients with severe secondary hyperparathyroidism

LI Peiting, LI Gang, LIU Lidan, HUANG Shan, LI Jun, WU Wei   

  1. Department of Breast Thyroid Surgery, Third Xiangya Hospital of Central South University, Changsha 410013, China
  • Online:2022-02-20 Published:2022-03-16

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摘要： 目的 分离、培养及鉴定继发性甲状旁腺功能亢进症患者来源甲状旁腺。方法 采用胶原酶消化法提取10例继发性甲状旁腺功能亢进症的甲状旁腺原代细胞进行体外培养，通过显微摄像、细胞计数研究其形态学变化及生长特性，用细胞免疫荧光、qRT-PCR及Western blot检测原代及传代甲状旁腺细胞甲状旁腺素（PTH）、钙敏感受体（CaSR）和神经胶质细胞缺失因子2（GCM2）mRNA及蛋白的表达情况，对所得细胞进行分析鉴定。结果 本研究成功提取甲状旁腺原代细胞，其细胞PTH免疫荧光结果为阳性，细胞增长曲线显示甲状旁腺细胞体外分散培养的群体倍增时间约为71.61 h。P2代细胞分泌PTH较P0、P1代细胞减少（P<0.001）。P1代细胞和P0代细胞间PTH（P=0.572）和GCM2（P=0.892）的mRNA表达差异无统计学意义，PTH（P=0.572）和GCM2（P=0.892）的蛋白表达差异也无统计学意义。但P1代细胞的CaSR mRNA（P=0.017）和蛋白表达（P=0.006）均较P0代甲状旁腺细胞减少。结论 采用胶原酶消化法培养的甲状旁腺细胞一定程度上具有与在体细胞相似的细胞生物学特性。

关键词: 继发性甲状旁腺功能亢进症；原代细胞培养；甲状旁腺素；钙敏感受体；神经胶质细胞缺失因子2

Abstract: Objective To establish an cell model of hyperparathyroidism by isolation, in vitro culture, and identification of parathyroid cells from patients with secondary hyperparathyroidism (SHPT). Methods The parathyroid gland tissues obtained from 10 patients with SHPT were dissociated by collagenase digestion for primary culture of the parathyroid cells. Morphological changes and growth characteristics of the cells were assessed by microscopic imaging and cell counting. The mRNA and protein expression levels of parathyroid hormone (PTH), calcium-sensing receptor (CaSR), and glial cells missing 2 (GCM2) in the primary and passaged cells were determined by immunofluorescence, qRT-PCR, and Western blotting. Results Primary cultures of parathyroid cells were successfully obtained. The cells exhibited a high expression of PTH shown by immunofluorescence assay and had a population doubling time of approximately 71.61 h. PTH secretion in the second-passage (P2) cells was significantly lower than that in the primary (P0) and first-passage (P1) cells (P<0.001). Despite a significant downregulation of CaSR mRNA (P=0.017) and protein (P=0.006) in P1 cells as compared with P0 cells, no significant differences were found in mRNA and protein expressions of PTH or GCM2 between the two cell generations. Conclusion Primary cultures of parathyroid cells isolated from SHPT patients by collagenase digestion show similar biological properties to the cells in vivo.

Key words: secondary hyperparathyroidism; primary cell culture; parathyroid hormone; calcium-sensing receptor; glial cells missing 2