中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2013年
12期
885-888
,共4页
蒋艳玲%赵明%梁功平%王利涛%苏玉文
蔣豔玲%趙明%樑功平%王利濤%囌玉文
장염령%조명%량공평%왕리도%소옥문
骨化三醇%角蛋白细胞%DNA甲基化
骨化三醇%角蛋白細胞%DNA甲基化
골화삼순%각단백세포%DNA갑기화
Calcitriol%Keratinocytes%DNA methylation
目的 探讨1,25-二羟维生素D3[1,25(OH)2D3]对人表皮角质形成细胞株HaCaT细胞增殖活性、基因组DNA总体甲基化水平及增殖相关基因启动子甲基化水平的影响.方法 10-6、10-7、10-8 mol/L1,25(OH)2D3作用HaCaT细胞24 h后,噻唑蓝(MTT)法检测不同浓度1,25(OH)2D3对HaCaT细胞增殖活性的影响;总体DNA甲基化试剂盒检测基因组DNA总体甲基化水平.10-6 mol/L 1,25 (OH)2D3作用HaCaT细胞24h后,实时定量聚合酶链反应(PCR)法检测DNA甲基转移酶(DNMT)和甲基结合蛋白(MBD)mRNA表达水平,甲基化特异性PCR(MS-PCR)检测凋亡相关基因程序化细胞死亡因子5(PDCD5)和基质金属蛋白酶组织抑制因子2 (TIMP2)启动子区DNA甲基化状态.结果 与溶媒对照组比较,10-6 mol/L1,25(OH)2D3处理组HaCaT细胞增殖活性及基因组DNA总体甲基化显著降低(细胞活力:0.152±0.027比0.290±0.017,P＜0.01;总体甲基化:0.187±0.071比0.316±0.049,P＜0.05),DNA甲基转移酶DNMT3a和DNMT3b mRNA水平显著降低(P值＜ 0.01或0.05),甲基结合蛋白MECP2和MBD2、凋亡相关基因PDCD5、TIMP2 mRNA水平显著升高(P值＜ 0.01或0.05).此外,MS-PCR结果显示,1,25 (OH)2D3处理组PDCD5、TIMP2基因启动子区DNA甲基化水平(0.380±0.135,0.460±0.172)较溶媒对照组(0.720±0.121,0.680±0.133)显著降低(均P＜0.05).结论 1,25(OH)2D3可降低HaCaT细胞基因组DNA总体甲基化水平,调控DNA甲基化修饰基因表达.此外,1,25(OH)2D3可降低凋亡相关基因PDCD5、TIMP2特异性启动子区甲基化水平,诱导PDCD5、TIMP2基因表达增加,抑制HaCaT细胞增殖.
目的 探討1,25-二羥維生素D3[1,25(OH)2D3]對人錶皮角質形成細胞株HaCaT細胞增殖活性、基因組DNA總體甲基化水平及增殖相關基因啟動子甲基化水平的影響.方法 10-6、10-7、10-8 mol/L1,25(OH)2D3作用HaCaT細胞24 h後,噻唑藍(MTT)法檢測不同濃度1,25(OH)2D3對HaCaT細胞增殖活性的影響;總體DNA甲基化試劑盒檢測基因組DNA總體甲基化水平.10-6 mol/L 1,25 (OH)2D3作用HaCaT細胞24h後,實時定量聚閤酶鏈反應(PCR)法檢測DNA甲基轉移酶(DNMT)和甲基結閤蛋白(MBD)mRNA錶達水平,甲基化特異性PCR(MS-PCR)檢測凋亡相關基因程序化細胞死亡因子5(PDCD5)和基質金屬蛋白酶組織抑製因子2 (TIMP2)啟動子區DNA甲基化狀態.結果 與溶媒對照組比較,10-6 mol/L1,25(OH)2D3處理組HaCaT細胞增殖活性及基因組DNA總體甲基化顯著降低(細胞活力:0.152±0.027比0.290±0.017,P＜0.01;總體甲基化:0.187±0.071比0.316±0.049,P＜0.05),DNA甲基轉移酶DNMT3a和DNMT3b mRNA水平顯著降低(P值＜ 0.01或0.05),甲基結閤蛋白MECP2和MBD2、凋亡相關基因PDCD5、TIMP2 mRNA水平顯著升高(P值＜ 0.01或0.05).此外,MS-PCR結果顯示,1,25 (OH)2D3處理組PDCD5、TIMP2基因啟動子區DNA甲基化水平(0.380±0.135,0.460±0.172)較溶媒對照組(0.720±0.121,0.680±0.133)顯著降低(均P＜0.05).結論 1,25(OH)2D3可降低HaCaT細胞基因組DNA總體甲基化水平,調控DNA甲基化脩飾基因錶達.此外,1,25(OH)2D3可降低凋亡相關基因PDCD5、TIMP2特異性啟動子區甲基化水平,誘導PDCD5、TIMP2基因錶達增加,抑製HaCaT細胞增殖.
목적 탐토1,25-이간유생소D3[1,25(OH)2D3]대인표피각질형성세포주HaCaT세포증식활성、기인조DNA총체갑기화수평급증식상관기인계동자갑기화수평적영향.방법 10-6、10-7、10-8 mol/L1,25(OH)2D3작용HaCaT세포24 h후,새서람(MTT)법검측불동농도1,25(OH)2D3대HaCaT세포증식활성적영향;총체DNA갑기화시제합검측기인조DNA총체갑기화수평.10-6 mol/L 1,25 (OH)2D3작용HaCaT세포24h후,실시정량취합매련반응(PCR)법검측DNA갑기전이매(DNMT)화갑기결합단백(MBD)mRNA표체수평,갑기화특이성PCR(MS-PCR)검측조망상관기인정서화세포사망인자5(PDCD5)화기질금속단백매조직억제인자2 (TIMP2)계동자구DNA갑기화상태.결과 여용매대조조비교,10-6 mol/L1,25(OH)2D3처리조HaCaT세포증식활성급기인조DNA총체갑기화현저강저(세포활력:0.152±0.027비0.290±0.017,P＜0.01;총체갑기화:0.187±0.071비0.316±0.049,P＜0.05),DNA갑기전이매DNMT3a화DNMT3b mRNA수평현저강저(P치＜ 0.01혹0.05),갑기결합단백MECP2화MBD2、조망상관기인PDCD5、TIMP2 mRNA수평현저승고(P치＜ 0.01혹0.05).차외,MS-PCR결과현시,1,25 (OH)2D3처리조PDCD5、TIMP2기인계동자구DNA갑기화수평(0.380±0.135,0.460±0.172)교용매대조조(0.720±0.121,0.680±0.133)현저강저(균P＜0.05).결론 1,25(OH)2D3가강저HaCaT세포기인조DNA총체갑기화수평,조공DNA갑기화수식기인표체.차외,1,25(OH)2D3가강저조망상관기인PDCD5、TIMP2특이성계동자구갑기화수평,유도PDCD5、TIMP2기인표체증가,억제HaCaT세포증식.
Objective To estimate the influence of 1,25(OH)2D3 on the proliferative ability of and methylation levels of genomic DNA and proliferation-associated gene promoter in human HaCaT keratinocytes.Methods Some cultured HaCaT cells were treated with 1,25 (OH)2D3 of 10-6,10-7 and 10-8 mol/L for 24 hours,then,methyl thiazolyl tetrazolium (MTT) assay was carried out to evaluate the proliferative activity of cells,and a global DNA methylation quantification kit was used to determine the global DNA methylation level.Real-time PCR was conducted to quantify the mRNA expression of DNA methyl transferases (DNMTs) and methyl-DNA binding domain (MBD) proteins,and methylation-specific PCR (MS-PCR) to evaluate the methylation status of promoter region in the programmed cell death 5 (PDCD5) and tissue inhibitor of metalloproteinase 2 (TIMP2) genes,in HaCaT cells after 24-hour treatment with 1,25 (OH)2D3 of 10-6 mol/L.The HaCaT cells receiving no treatment served as the control.Results Compared with the untreated HaCaT cells,those treated with 1,25(OH)2D3 of 10-6 mol/L showed significantly down-regulated proliferative activity (0.152 ± 0.027 vs.0.290 ± 0.017,P ＜ 0.01),global DNA methylation level (0.187 ± 0.071 vs.0.316 ± 0.049,P ＜ 0.05),DNMT3a and DNMT3b mRNA expression levels (P ＜ 0.01 or 0.05),but markedly upregulated mRNA expression levels of MECP2,MBD2,PDCD5 and TIMP2 (P ＜ 0.01 or 0.05).Moreover,the DNA methylation levels within the promoter region of PDCD5 and TIMP2 genes were significantly lower in HaCaT cells treated with 1,25 (OH)2D3 of 10-6 mol/L than in the control cells (0.38 ± 0.135 vs.0.72 ± 0.121,0.46 ± 0.172 vs.0.68 ± 0.133,both P＜ 0.05).Conclusions 1,25(OH)2D3 may down-regulate the global genomic DNA methylation level of,and modulate the expression of DNA methylationmodifying genes in,HaCaT cells.Furthermore,1,25 (OH)2D3 can decrease the promoter methylation levels but induce the overexpression of PDCD5 and TIMP2 genes,and decelerate the proliferation of HaCaT cells.
