维生素D受体FokⅠ多态性对牙周组织细胞CYP24A1表达的影响

doi:10.3969/j.issn.1671-167X.2018.01.003

摘要/Abstract

摘要： 目的：维生素D受体（vitamin D receptor，VDR）基因的第二外显子存在唯一一个可以影响VDR蛋白结构的多态性位点，其可以由限制性核酸内切酶FokⅠ所识别，分为FF、Ff和ff三型。CYP24A1是维生素D 24羟化酶的编码基因，是常见的维生素D效应基因。本研究将探讨人牙龈成纤维细胞（human gingival fibroblasts，hGF）和牙周膜细胞（human periodontal ligament cells，hPDLC）中VDRFokⅠ多态性对CYP24A1表达的影响。方法：原代培养12名供体的hGF和hPDLC，提取基因组DNA，PCR扩增包含多态性位点的267 bp的片段。根据FokⅠ对片段酶切的结果判断VDR-FokⅠ基因型。确定基因型后，给各基因型hGF和hPDLC以10 nmol/L 1α,25双羟维生素D3（1,25OH2D3）或1 000 nmol/L 25羟维生素D3（25OHD3）刺激48 h，提取RNA，其中10 nmol/L 1,25OH2D3刺激48 h后还提取蛋白。之后给予hGF和hPDLC VDR拮抗剂ZK159222，再以10 nmol/L 1,25OH2D3或1 000 nmol/L 25OHD3刺激48 h，提取RNA。应用Real-time PCR和Western blot的方法检测维生素D24羟化酶CYP24A1和VDR的mRNA和蛋白的表达水平。结果：12名供体中，FF、ff和Ff型分别为4例、3例和5例。1,25OH2D3刺激hGF和hPDLC后，FF型细胞CYP24A1的mRNA表达水平显著高于Ff型或ff型细胞（hGF：F=31.147，P＜0.01；hPDLC：F=23.347，P＜0.01）；FF型细胞CYP24A1的蛋白表达水平同样显著高于Ff型或ff型细胞（hGF：F=12.368，P＜0.01；hPDLC：F=15.749，P＜0.01）。25OHD3刺激hGF和hPDLC后，FF型细胞CYP24A1的mRNA表达水平也显著高于Ff型或ff型细胞（hGF：F=32.061，P＜0.01；hPDLC：F=32.569，P＜0.01）。如1,25OH2D3刺激伴有ZK159222，则三型细胞CYP24A1的mRNA表达水平差异无统计学意义（hGF：F=0.246，P=0.787；hPDLC：F=0.574，P=0.583）。如25OHD3刺激伴有ZK159222，则三型细胞CYP24A1的mRNA表达水平差异也无统计学意义（hGF：F=1.636，P=0.248；hPDLC：F=0.582，P=0.578）。不同刺激条件下，hGF和hPDLC两种细胞比较CYP24A1或VDR的表达水平，差异均无统计学意义。结论：在hGF和hPDLC中，FF型VDR可介导比其他基因型VDR更为显著的CYP24A1上调，提示FF型VDR可能具有更强的转录活性。

关键词: 维生素D受体, 维生素D受体FokⅠ多态性, 维生素D24羟化酶

Abstract: Objective：There is asingle nucleotide polymorphism (SNP) in the exon 2 of the vitamin D receptor (VDR) gene that can be distinguished using the restriction endonuclease FokⅠ, and accordingly divided into three genotypes: FF, Ff and ff. VDR-FokⅠ polymorphism was the only known SNP that could alter the protein structure of VDR. CYP24A1 is the gene encoding vitamin D 24 hydroxylase and is a vitamin D responsive gene. The influence of rs2228570 on transcriptional activation by VDR in human gingival fibroblasts (hGF) and periodontal ligament cells (hPDLC) was investigated in this study. Methods: hGF and hPDLC of 12 donors’ were primarily cultured and genomic DNA was extracted. A part of genomic DNA with the length of 267 bp was obtained using PCR, which contained the SNP. VDRFok Ⅰ genotypes were determined according to the results of restriction fragment length polymorphism. hGF and hPDLC were stimulated with 10 nmol/L 1α,25 dihydroxy vitamin D3 (1,25OH2D3) or 1 000 nmol/L 25 hydroxy vitamin D3 (25OHD3) for 48 h before RNA was extracted. Then VDR antagonist ZK159222 was used or not used during 1,25OH2D3 or 25OHD3 stimulation with hGF and hPDLC. After 1,25OH2D3 stimulation for 48 h, the proteins in hGF and hPDLC were also collected. The protein expressions of CYP24A1 and VDR were detected using Western blot. Results: Among the 12 donors’ cell cultures, the number of FF, ff and Ff genotypes was 4, 3 and 5, respectively.After stimulation with 1,25OH2D3or 25OHD3 for 48 h,CYP24A1 mRNA levels in FF-hGF were significantly higher than those in other hGF genotypes(1,25OH2D3: F=31.147, P<0.01; 25OHD3: F = 32.061，P <0.01), as was in FFhPDLC (1,25OH2D3: F=23.347, P<0.01; 25OHD3: F = 32.569，P < 0.01). When ZK159222 was used before 1,25OH2D3 stimulation, this statistically significant difference disappeared (hGF: F=0.246, P=0.787; hPDLC: F=0.574, P=0.583). When ZK159222 was used before 25OHD3 stimulation, the trend was similar (hGF: F = 1.636, P = 0.248; hPDLC: F =0.582, P=0.578).After stimulation with 1,25OH2D3 for 48 h, CYP24A1 protein levels in FF-hGF were significantly higher than those in the other hGF genotypes (F=12.368, P <0.01), as was in FFhPDLC (F=15.749, P <0.01). In hGF and hPDLC, the mRNA or protein expression of VDR of different genotypes was not significantly different under different stimulation conditions.The paired comparison showed that there was no statistically significant difference between the expression of CYP24A1 in hGF and that in hPDLC under all the stimulation conditions, as was the expression of VDR. Conclusion: In hGF and hPDLC, the FF-VDR genotype is associated with the more remarkable up-regulation of CYP24A1than the other genotypes, indicating that transcriptional activation of FF-VDR might be higher than those of other vitamin D receptors.

Key words: Vitamin D receptor, Rs2228570, Vitamin D3 24-hydroxylase

中图分类号:

R781.4

刘凯宁，孟焕新，侯建霞. 维生素D受体FokⅠ多态性对牙周组织细胞CYP24A1表达的影响[J]. 北京大学学报(医学版), 2018, 50(1): 13-19.

LIU Kai-ning, MENG Huan-xin, HOU Jian-xia. Influence of vitamin D receptor FokⅠ polymorphism on expression of CYP24A1 in periodontal cells[J]. Journal of Peking University(Health Sciences), 2018, 50(1): 13-19.

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