羟基喜树碱对系统性红斑狼疮患者单核淋巴细胞中p16基因表达和启动子甲基化修饰的影响

Effects of hydroxycamptothecin on expression and promotor methylation of p16 gene in monocytes in patients with systemic lupus erythematosus

目的:探讨羟基喜树碱(Hydroxycamptothecin,HCPT)对系统性红斑狼疮(systemic lupus erythematosus,SLE)患者外周血单个核细胞(peripheral blood mononuclear cells,PBMC)甲基化敏感基因p16的表达和启动子甲基化修饰的影响。方法:密度梯度离心法分离得外周血单个核细胞,使用浓度为0.25、0.5、1和2 mg·L-1的HCPT处理SLE患者PBMC,24 h后收集细胞。MTT法检测处理后PBMC的活力。定量PCR检测p16及DNA甲基转移酶1(DNA Methyltransferase1,Dnmt1)基因mRNA表达水平,2-ΔΔct法分析结果。甲基化特异性PCR分析p16基因启动子区甲基化水平。结果:(1)HCPT处理24h后,0.25、0.5和1 mg·L-1 HCPT处理组PBMC活力之间比较差异无统计学意义(P>0.05),2 mg·L-1 HCPT处理组PBMC活力与0.25、0.5和1 mg·L-1 HCPT处理组比较差异均有统计学意义(P<0.05)。(2)1 mg·L-1 HCPT处理组与对照组比较p16基因mRNA表达水平显著升高,差异有统计学意义(P<0.05)。(3)1 mg·L-1 HCPT处理组与对照组比较Dnmt1基因mRNA表达水平降低,但差异无统计学意义(P>0.05)。(4)DNA甲基化水平检测显示HCPT处理组p16基因启动子甲基化水平较对照组显著降低,差异有统计学意义(P<0.01)。结论:HCPT通过降低Dnmt1基因表达水平,下调p16基因启动子甲基化水平,从而增加SLE患PBMC中p16表达水平。

OBJECTIVE To investigate effects of hydroxycamptothecin （HCPT） on expression and promotor methylation of p16 gene in peripheral blood mononuclear cell （PBMC） from patients with systemic lupus erythematosus （SLE）. METHODS PBMC were isolated by Ficoll density gradient centrifugation, and then were treated by HCPT at 1 mg· L^-1 for 24 h. MTT method was used to assess cell viability; mRNA expression level was measured by real time-PCR; 2-aact method was used for analyzing the difference on p16 and DNA methyltransferase 1 （Dnmtl） expression. Methylation of PBMC p16 gene promoter was measured by methylation specific PCR. RESULTS No significant changes in cell viability were found in PBMC treated by HCPT at 0. 25,0. 5 and 1 mg· L^- 1 （P〉0. 05）. Compared with another HCTP treatment group, cell viability was significantly decreased at 2 mg·L^- 1. Compared with control, mRNA level was significantly higher for p16 （P〈0. 05）, but lower for Dnmtl without significance （P〉0. 05）. Furthermore, extent of methylation of p16 promoter was decreased in HCPT treated PBMC compared with control group （P〈0. 01）. CONCLUSION HCPT can increase p16 gene expression through reducing DNA methylation of p16 promoter in PBMC from patients with SLE.