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目的在用硝普钠体外封闭脆性X智力低下一号(FMR1)基因建立脆性X综合症细胞模型的基础上,初步研究腺苷酸环化酶激活剂弗司可林(FSK)对FMR1基因的重启及表达的影响。方法利用硝普钠产生的一氧化氮可以诱导FMR1基因启动子区Cp G岛甲基化的原理,将人类慢性髓源性白血病细胞系(K-562)在加入适宜浓度硝普钠(SNP)的完全培养基中培养,诱导FMR1基因启动子区CpG岛甲基化从而使FMR1基因封闭,并失表达;用普通及Taqman荧光定量PCR、Westblot技术验证腺苷酸环化酶激活剂弗司可林(forskolin,FSK)诱导FMR1基因封闭后的重启及表达。结果浓度1000μmol/L的SNP作用24h后,可使K-562细胞的FMR1基因量降至正常组的0.2%并达到封闭效果,且72h后可抑制脆性X智力低下蛋白(FMRP)的表达,蛋白表达量降至正常组的48%;发现浓度50μmol/L的FSK可以在24h时重新开启封闭的FMR1基因,使其表达量上升到13.3%,最高达13.8%并持续至48h,且72h后体现在FMRP的表达上,其量基本上与正常组相当。结论 FSK可有效地诱导启动子区CpG岛甲基化封闭后的FMR1基因重新启动以及满意的FM-RP表达,FSK对Fra(X)的治疗作用还有待于进一步研究。
OBJECTIVE: To establish a cell model of Fragile X syndrome with nitroprusside in vitro by blocking Fragile X mental retardation (FMR1) gene and to study the effects of adenosine cyclase activator forskolin (FSK) on the expression of FMR1 gene Restart and express the impact. Methods The nitric oxide produced by sodium nitroprusside can induce the methylation of CpG island in the promoter region of FMR1 gene. When human chronic myeloid leukemia cell line (K-562) was treated with appropriate concentrations of sodium nitroprusside (SNP) , Induced the methylation of CpG island in the promoter region of FMR1 gene, blocked the FMR1 gene and lost its expression. The common and Taqman fluorescent quantitative PCR and Westblot technique were used to verify that adenylyl cyclase activator Forskolin Forskolin (FSK) Induces Restoration and Expression of FMR1 Gene after Blocking. Results After treated with 1000μmol / L SNP for 24 hours, the FMR1 gene level in K-562 cells was reduced to 0.2% of the normal group and reached a blocking effect. After 72 hours, the FMR1 gene expression was inhibited. The expression of FMRP protein, The expression level decreased to 48% of that of the normal control group. FSK at a concentration of 50μmol / L showed that the FMR1 gene was reopened at 24h and the expression level increased to 13.3%, up to 13.8% and continued to 48h, The amount of FMRP was substantially comparable to that of the normal group. Conclusion FSK can effectively induce the promoter reactivation of FMR1 gene and satisfactory expression of FM-RP after methylation of CpG island. The therapeutic effect of FSK on Fra (X) remains to be further studied.