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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 the effect of Hydroxycamptothecin (HCPT) on the expression of p16 methylation-sensitive gene in peripheral blood mononuclear cells (PBMC) in patients with systemic lupus erythematosus (SLE) The impact of the modification. Methods: Peripheral blood mononuclear cells were isolated by density gradient centrifugation. PBMC of SLE patients were treated with HCPT at concentrations of 0.25, 0.5, 1 and 2 mg · L-1, and cells were harvested 24 hours later. The activity of PBMC after treatment was detected by MTT assay. Quantitative PCR was used to detect the mRNA expression levels of p16 and DNA Methyltransferase1 (Dnmt1), and the results were analyzed by 2-ΔΔct method. Methylation-specific PCR analysis of p16 gene promoter methylation level. Results: (1) After treated with HCPT for 24 h, there was no significant difference in the activity of PBMC between 0.25, 0.5 and 1 mg · L-1 HCPT treatment groups (P> 0.05). The activity of PBMC in 2 mg · L-1 HCPT treatment group was no significant difference Compared with 0.25, 0.5 and 1 mg · L-1 HCPT treatment groups, the differences were statistically significant (P <0.05). (2) Compared with the control group, the mRNA expression of p16 gene in 1 mg · L-1 HCPT treatment group was significantly increased, the difference was statistically significant (P <0.05). (3) Compared with the control group, the mRNA expression of Dnmt1 in the 1 mg · L-1 HCPT group was lower than that in the control group, but the difference was not statistically significant (P> 0.05). (4) DNA methylation level showed that the methylation level of p16 gene promoter in HCPT group was significantly lower than that in control group (P <0.01). Conclusion: HCPT can reduce the expression of Dnmt1 gene and down-regulate the promoter methylation level of p16 gene, thereby increasing the expression of p16 in PBMC from SLE patients.