Molcular Reguration of Parathyroid Cell Function in Chronic Renal Failure

最近的数据表明，紊乱的基因调控是慢性衰竭的甲状旁腺功能障碍的基础。然而，这种异常的机制还没有完全阐明在细胞水平上，因为没有estbishilled甲状旁腺细胞系是实际上可利用的。利用腺病毒载体转染功能基因1.维生素D受体（VDR）分析甲状旁腺增生的密度和细胞增殖对尿毒症患者手术切除的甲状旁腺连续切片进行免疫组织化学染色，发现VDR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形成的小结节内细胞VDR密度较周围细胞低，PCNA阳性率较高。这些数据表明，降低的死亡率与 ...更多信息 2.腺病毒介导的甲状旁腺细胞基因转染方法取甲状旁腺组织，用胶原酶消化，在24孔培养皿中每孔培养5 × 105个细胞，加入DMEM/F12（FBS 10%）0.5ml。第二天，以1 × 107 pfu/ml或更低的浓度加入含有CAG启动子驱动的核β-半乳糖苷酶报告基因的复制缺陷型腺病毒载体，感染48小时，几乎所有细胞的X-gal染色均为阳性。此时，腺病毒感染本身并不影响BrdU掺入的细胞数。PTH分泌对低、高钙离子浓度的基础反应和生理反应也不受病毒感染的影响。因此，成功地将腺病毒载体转入甲状旁腺细胞，使其对钙离子仍保持生理反应，并通过直接注射病毒溶液成功地感染体内甲状旁腺细胞。然后，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基因转染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的分泌明显受到抑制，因此，用腺病毒载体转染功能基因在体内调节甲状旁腺功能将成为可能。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