骨化三醇对人牙周膜细胞维生素D受体、RANKL和骨保护因子表达的调节

Effects of calcitriol on the expression of vitamin D receptor, RANKL and osteoprotegerin in human periodontal ligament calls

目的探讨骨化三醇（又称1α，25-二羟维生素D3，以下简称VD3）对人牙周膜细胞（human periodontal ligament cells，hPDLC）维生素D受体（vitamin D receptor，VDR）、核因子KB受体活化因子配体（receptor activator of nuclear factor—kappa B ligand，RANKL）和骨保护因子（osteoprotegerin，OPG）表达的调节作用及相关机制。方法原代培养12个供体来源的hPDLC群，传代培养至第3代，分别以10～moL／LVD，（VD，组）和0．1％无水乙醇处理（对照组），6d后用实时荧光定量反转录聚合酶链反应（RT—PCR）法检测VDR、RANKL和OPGmRNA的表达水平。分析12个细胞群RANKL基因转录起始位点上游调控区DNA碱基序列。结果VD，组与对照组相比，VDRmRNA表达水平显著上调（P=0．003），为对照组的（3．04±1．06）倍；RANKLmRNA表达水平显著升高（P=0．001），为对照组的9．82（0．75～119．18）倍；OPGmRNA表达水平为对照组的（94．48±39．15）％，P=0．136；OPG／RANKL比值显著下调（P=0．003），为对照组的10．36％（1．01％-138．00％）。未发现RANKL基因上游调控区序列突变位点；-1832（rs7984870，C／G）多态性位点基因型与RANKLmRNA的表达和调节之间无显著相关性。结论在hPDLC中，VD，可以促进VDRmRNA的表达；VD3可显著上调RANKLmRNA的表达从而降低OPG／RANKL比值，而对OPGmRNA的表达水平影响较小。在VD3作用下，细胞群间RANKLmRNA表达的差异性可能与RANKL基因上游调控区碱基序列无关。

Objective To study the effects of 1, 25-dihydroxyvitamin D3 （ VD3 ） on the expression of vitamin D receptor （VDR）, reeeptor aetivator of nuclear factor-kappa B ligand （RANKL） and osteoprotegerin （OPG） in human periodontal ligament eells （hPDLC） populations and to analyze the potential meehanisms. Methods Twelve hPDLC populations were primarily established from 12 donors individually. Two samples of each hPDLC population of passage three were treated respectively with 10-8 mol/L VD3 （V D3 group） or 0. 1% absolute ethyl aleohol as controls （control group）. Six days later, the mRNA expression levels of VDR, RANKL and OPG in the samples were determined with real-time quantitative RT-PCR. The DNA base sequenees upstream to the transcription start site of RANKL gene were also analyzed. Results Compared with the eontrol group, the mRNA expression level of VDR increased signifieantly in the VD3 group （ P = 0. 003 ）, averagely （ 3.04 ± 1.06 ） times of that in the control group ; the mRNA expression level of RANKL was also up-regulated by VD3 （ P = 0. 001 ） , 9. 82 （ 0. 75-119.18 ） times of that in the control group; the OPG expression level was （94.48 ± 39. 15 ）% of the controls （P = 0. 136）; OPG/RANKL ratio was down-regulated in the VD3 group to averagely 10. 36% （1.01%- 138. 00% ） of the controls （ P = 0. 003 ）. No mutation was found in the DNA fragments upstream to the transcription start site in the RANKL gene and the genotypes of the polymorphism at - 1832 （ rs7984870, C/G） were not shown to be significantly related to the RANKL mRNA expression level. Conclusions In hPDLC, VD3 can significantly increase the mRNA expression level of VDR; VD3 can increase RANKL mRNA expression level to decrease OPG/RANKL ratio, but it has little effect on OPG mRNA expression.The big differences of the RANKL mRNA regulation in response to VD3 treatment among hPDLC populations may not be associated with the DNA sequences upstream to the transcription start site in the RANKL gene.