Molcular Reguration of Parathyroid Cell Function in Chronic Renal Failure

最近的数据表明，基因调控的错乱是慢性衰竭中副甲状腺功能障碍的基础。然而，由于目前尚无甲状旁腺细胞系的建立，这种异常的细胞机制在细胞水平上尚未完全阐明。为了调节对细胞外仅有几天生理反应的甲状旁腺细胞的功能，我们用腺病毒载体转导功能基因。1.甲状旁腺增生组织中维生素D受体(VDR)密度和细胞增殖的分析。弥漫性甲状旁腺增生内形成小结节的细胞与周围细胞相比，VDR密度低，增殖细胞核抗原阳性率高。这些数据表明，De…的下降与2.腺病毒转导甲状旁腺细胞用胶原酶分散甲状旁腺，每孔培养5x105个细胞，加入0.5ml DMEM/F12(FBS10%)。第二天，以1x107pfu/ml或更低的浓度加入含有报告基因的复制缺陷型腺病毒载体，该报告基因编码由CAG启动子驱动的核β-半乳糖苷酶。感染后48h，几乎所有细胞X-Gal染色均为阳性。此时，腺病毒感染本身并不影响细胞数量BrdU的掺入。病毒感染也不影响甲状旁腺激素分泌对低和高钙离子浓度的基础反应和生理反应。因此，腺病毒载体在甲状旁腺细胞中成功地完成了基因转移，并且仍然保持了对钙的生理反应。此外，我们还可以通过直接注射病毒液的方法在体内成功地感染甲状旁腺细胞。接下来，我们构建了在CAG启动子下表达人VDR基因的重组腺病毒。该病毒将VDR基因转入分散的甲状旁腺细胞，使甲状旁腺激素的分泌在生理浓度为1/10的情况下受到显著抑制，从而有望在体内通过腺病毒载体转导功能基因来调节甲状旁腺功能。较少

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