Molcular Reguration of Parathyroid Cell Function in Chronic Renal Failure

最近的数据表明，在慢性心力衰竭中，紊乱的基因调节是甲状旁腺功能障碍的基础。然而，这种异常的机制尚未在细胞水平上完全阐明，因为没有建立的甲状旁腺细胞系实际可用。为了调节分散的甲状旁腺细胞的功能，我们通过腺病毒载体转移功能基因1。甲状旁腺增生中维生素D受体（VDR）密度和细胞增殖的分析。尿毒症患者手术切除甲状旁腺连续切片免疫组化结果显示，VDR阳性率与增殖细胞核抗原（PCNA）阳性率呈显著负相关。弥漫性甲状旁腺增生小结节形成细胞的VDR密度较低，PCNA阳性率高于周围细胞。这些数据提示慢性肾衰竭患者VDR密度的降低与细胞增殖之间有更直接的联系。手术切除的甲状旁腺经胶原酶分散，24孔培养皿每孔用0.5ml DMEM/F12 （FBS10%）培养5 × 105个细胞。第二天，加入含有CAG启动子驱动的核b-半乳糖苷酶报告基因的复制缺陷腺病毒载体，剂量为1x107pfu/ml或更低。感染48h后，几乎所有细胞的X-gal染色均为阳性。此时，腺病毒感染本身不影响BrdU掺入的细胞数量。甲状旁腺激素分泌对低、高钙离子浓度的基础和生理反应也不受病毒感染的影响。因此，成功的基因转移是通过腺病毒载体在甲状旁腺细胞中完成的，甲状旁腺细胞仍然保持对钙的生理反应。此外，我们还可以通过直接注射病毒溶液在体内成功感染甲状旁腺细胞。在CAG启动子下构建表达人VDR基因的重组腺病毒。该病毒将VDR基因转移到分散的甲状旁腺细胞中，在1/10生理浓度的骨化三醇存在下，PTH分泌明显受到抑制。因此，通过腺病毒载体转移功能基因在体内调节甲状旁腺功能可能很快成为可能。少

Recent data suggest that deranged gene reguration underlie the parathy-roid dysfunction in chronic failure. However, the mechanism of this abnormality has not been fully elucidated at cellular level because no estsblished parathyroid cell line is practically available.To modulate funnction of diapersed parathyroid cells which maintain physiological response to extracellular only for a few days, we transfered functional genes by adenovirus vector.1.Analysis of Vitamin D Receptor (VDR) Density and Cell Prolifiration in Parathyroid Hyperplasia.Immunohistochemistry of serial section of surgically exsized parathyroid glands from uremic patients revealed that positive rate of VDR and proliferating cell nuclear antigen (PCNA) had significant negative coreration. Further more, cells in small nodule forming within diffuse parathyroid hyperplasia had low density of VDR and higher positive rate of PCNA compared with surrounding cells. These data suggest more direct coreration between decreased de … More nsity of VDR and cell proliferation in chronic renal failure.2.Gene Transfer into Parathyroid Cells by AdenovirusSurgically excised parathyroid glands were dispersed by collagenase and 5x105cells in 0.5ml of DMEM/F12 (FBS10%) were cultured in each well of 24-well dish. On the next day, replication-deficient adenoviral vector that contained a reporter gene enconding thenuclear b-galactosidase driven by CAG promoter was added at 1x107pfu/ml or less. X-gal staining was positive in almost all cells at 48hours of infection. At this point, adenovirus infection itsself did not affect the cell number BrdU incorporation. Basal and phisiolosical response of PTH secretionto low and high calcium ion concentration were not affected by viral infection either. Thus, successful gene transfer was accomplished by adenoviral vectors in parathyroid cells which still maintained physiological response to calcium.Furthermore, we could also successfully infect parathyroid cells in vivo by directly injecting virus solution. Next we constracted recommbinant adenovirus which expresses human VDR gene under CAG promoter. Trnsfer of VDR gene into dispersed parathyroid cells by this virus lead to significant suppression of PTH secretion in the presense of 1/10 of physiological concentration of calcitoriol.Thus, it may soon become possible to modulate parathyroid function in vivo by trnsferring functional genes by adenovirus vector. Less