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目的探讨三磷酸肌醇受体Ⅲ型(IP3RⅢ)DNA甲基化对硫酸铍(BeSO4)致人胚肺成纤维细胞(MRC-5细胞)损伤的影响。方法建立离体细胞培养实验模型,以终浓度分别为1.0、10.0、100.0μmol/L的BeSO4染毒MRC-5细胞24 h(设为低、中、高剂量染毒组),对照组不使用BeSO4染毒。采用巢式降落式甲基化特异性聚合酶链式反应(PCR)法检测IP3RⅢDNA甲基化的变化,实时定量PCR检测IP3RⅢmRNA的表达,激光共聚焦显微镜检测细胞钙离子浓度([Ca2+]i)。结果高剂量组IP3RⅢDNA甲基化值分别低于对照组、低剂量组和中剂量组[(0.26±0.07)vs(0.48±0.02)、(0.44±0.04)、(0.37±0.03),P<0.01,P<0.01,P<0.05];低、中、高剂量组IP3RⅢmRNA表达量均高于对照组[(0.35±0.08)、(0.31±0.05)、(0.71±0.09)vs(0.03±0.01),P<0.01],高剂量组IP3RⅢmRNA表达量分别高于低、中剂量组[(0.71±0.09)vs(0.35±0.08)、(0.31±0.05),P<0.01];高剂量组细胞内[Ca2+]i平均荧光强度高于对照组[(6 171.66±601.72)vs(3 618.33±559.26),P<0.05]。结论 IP3RⅢDNA低甲基化致细胞内IP3RⅢmRNA表达增加,[Ca2+]i增加,可能是BeSO4致MRC-5细胞损伤的重要机制之一。
Objective To investigate the effect of IP3R Ⅲ DNA methylation on the injury of human embryonic lung fibroblasts (MRC-5 cells) induced by BeSO4. Methods The experimental model was established in vitro. MRP-5 cells were treated with BeSO4 (1.0,10.0,100.0μmol / L) for 24 h (set as low, medium and high dose), and the control group was not used BeSO4 exposure. The methylation of IP3RⅢ DNA was detected by nested-down methylation-specific polymerase chain reaction (PCR) and the expression of IP3RⅢmRNA was detected by real-time quantitative PCR. The concentration of [Ca 2+] i was measured by confocal laser scanning microscopy . Results The methylation values of IP3RⅢDNA in the high dose group were lower than those in the control group, the low dose group and the middle dose group [(0.26 ± 0.07) vs (0.48 ± 0.02), (0.44 ± 0.04) and (0.37 ± 0.03), P <0.01 , P <0.01, P <0.05]. The expression levels of IP3RⅢ mRNA in low, middle and high dose groups were significantly higher than those in control group [(0.35 ± 0.08), (0.31 ± 0.05), P <0.01]. The expression levels of IP3RⅢmRNA in high dose group were significantly higher than those in low and middle dose group [(0.71 ± 0.09) vs (0.35 ± 0.08), (0.31 ± 0.05), P <0.01] ] i mean fluorescence intensity was higher than the control group [(6 171.66 ± 601.72) vs (618.33 ± 559.26), P <0.05]. Conclusion IP3R Ⅲ mRNA expression induced by IP3R Ⅲ DNA hypomethylation increased [Ca2 +] i may be one of the important mechanisms of BeSO4-induced MRC-5 cell injury.