5-Aza-CdR对胰岛β细胞株中miR-375甲基化及表达水平的研究

发布时间：2017-12-27 04:02

本文关键词：5-Aza-CdR对胰岛β细胞株中miR-375甲基化及表达水平的研究　出处：《石河子大学》2016年硕士论文　论文类型：学位论文

更多相关文章： 2型糖尿病 mi R-375 DNA去甲基化 MALDI-TOF MS

【摘要】：目的:探讨5-氮杂-2’-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-CdR)对小鼠胰岛β细胞株(MIN6)中mi R-375基因DNA甲基化及其表达的影响。方法:(1)用不同浓度的5-Aza-Cd R处理MIN6细胞,阴性对照组不加药,根据处理的剂量分为0umol/L组、0.08umol/L组、0.4umol/L组、2umol/L组、10umol/L组、50umol/L组,分别作用24h、48h、72h用MTT比色法检测细胞的生长抑制情况。(2)采用基质辅助激光解析电离时间飞行质谱仪(MALDI-TOF MS)检测不同浓度5-Aza-Cd R处理24h、48h、72h的MIN6细胞株及未经药物处理的NIH3T3细胞株mi R-375基因DNA甲基化状态。(3)运用q RT-PCR检测NIH3T3细胞及5-Aza-Cd R(50umol/L)处理MIN6细胞24h、48h、72h后mi R-375的表达情况。结果:(1)5-Aza-Cd R对MIN6细胞有抑制作用,2umol/L组5-Aza-Cd R作用24h、48h、72h与其余各浓度组比较OD值均有差异(P0.05),且各时间段间比较差异有统计学意义(P0.05)。(2)MIN6细胞经不同浓度5-Aza-Cd R处理24h、48h、72h,检测各组细胞中mi R-375基因DNA呈高甲基化状态,其中50umol/L组mi R-375平均甲基化率均低于其它浓度组。(3)MIN6细胞经50 umol/L组5-Aza-Cd R处理24h、48h、72h后,检测各组mi R-375的表达与0umol/L组比较差异有统计学意义(P0.05),且mi R-375的表达均高于0umol/L组。(4)NIH3T3细胞组mi R-375的表达比MIN6细胞组高,差异有统计学意义(P0.05)。结论:(1)5-Aza-Cd R能够抑制MIN6细胞的增殖。(2)相对于NIH3T3细胞,MIN6细胞中mi R-375基因DNA启动子区呈高甲基化状态,低表达。(3)50umol/L浓度的5-Aza-Cd R能使MIN6细胞中mi R-375基因DNA去甲基化、激活mi R-375的表达,其甲基化与表达呈负相关。
[Abstract]:Objective: To investigate the effect of 5- aza -2 '5-Aza-2 -deoxycytidine (5-Aza-CdR) on MI R-375 gene DNA methylation and its expression in mouse islet beta cell line (MIN6). Methods: (1) 5-Aza-Cd with different concentration of R treated MIN6 cells, negative control group and no drug treatment, according to the dose divided into 0umol/L group, 0.08umol/L group, 0.4umol/L group, 2umol/L group, 10umol/L group, 50umol/L group, 24h, 48h and 72h respectively by MTT inhibition assay, cell growth. (2) matrix assisted laser desorption ionization time flight mass spectrometry (MALDI-TOF MS) was used to detect MIN6 methylation status of MI R-375 gene in 24h cells, 48h 72h MIN6 cells and NIH3T3 cells treated with different concentrations of 5-Aza-Cd R. (3) the expression of MI R-375 after 24h, 48h and 72h in MIN6 cells was detected by NIH3T3 cells and 5-Aza-Cd R (50umol/L) using Q RT-PCR. Results: (1) 5-Aza-Cd R has inhibitory effect on MIN6 cells, 2umol/L group 5-Aza-Cd R role of 24h, 48h, 72h and the rest of the concentration group (P0.05), difference of OD value were statistically significant and the time difference between (P0.05). (2) MIN6 cells were treated with different concentrations of 5-Aza-Cd R for 24h, 48h and 72h. The MI R-375 gene DNA in each group was hypermethylation, and the average methylation rate of MI group in 50umol/L group was lower than that in other concentration groups. (3) after treatment of 24h, 48h and 72h by 5-Aza-Cd R in 50 umol/L group, the expression of MI R-375 in MIN6 cells was significantly different from that in 0umol/L group (P0.05), and the expression of R-375 was higher than that in group C. (4) the expression of MI R-375 in the NIH3T3 cell group was higher than that in the MIN6 cell group, and the difference was statistically significant (P0.05). Conclusion: (1) 5-Aza-Cd R can inhibit the proliferation of MIN6 cells. (2) compared with NIH3T3 cells, the DNA promoter region of the MI R-375 gene in MIN6 cells showed high methylation status and low expression. (3) the 5-Aza-Cd R of 50umol/L concentration can demethylation of the MI R-375 gene DNA in MIN6 cells and activate the expression of MI R-375, and the methylation and expression are negatively correlated.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

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