Aβ25-35对SH-SY5Y细胞Bcl-2、Bax基因启动子区DNA甲基化的影响  

Effect of Aβ_(25-35) on Bcl-2,Bax gene promoter DNA methylation in SH-SY5Y cell

作　　者：郭敏[1,2] 张宪武[3] 马天宇[1] 张宏[1] 苏苗[1] 武燕[1] 李刚[1] 

机构地区：[1]内蒙古医科大学药学院,内蒙古呼和浩特010110 [2]鄂尔多斯市中心医院药剂科,内蒙古鄂尔多斯017000 [3]内蒙古精神卫生中心,内蒙古呼和浩特010010

出　　处：《中国药理学通报》2016年第8期1158-1164,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81260650,81560685);内蒙古自然科学基金资助项目(No 2014BS0805)

摘　　要：目的探讨Aβ_(25-35)介导SH-SY5Y细胞Bcl-2、Bax基因表达改变是否通过基因启动子区甲基化的机制。方法不同浓度Aβ_(25-35)(0、25、50μmol·L^(-1))分别作用于体外培养的SH-SY5Y细胞48、72 h,MTT法确定Aβ_(25-35)诱导SH-SY5Y细胞凋亡的最佳浓度和时间。Western blot检测不同药物处理组细胞凋亡相关蛋白Bcl-2、Bax表达变化,Real time PCR检测DNA甲基化酶DNMT1、DNMT3a、DNMT3b、Me CP2的mRNA水平。Methylation specific PCR(MSP)法分析Aβ_(25-35)介导的Bcl-2、Bax基因启动子区甲基化水平的变化。结果 25μmol·L^(-1)Aβ_(25-35)暴露SH-SY5Y细胞72 h后,MTT检测细胞存活率达(68.49±9.83)%,与对照组比较明显降低(P<0.05),表明成功建立了AD细胞凋亡模型。Western blot结果显示,Aβ_(25-35)药物处理组与空白组比较,Bcl-2表达明显减少,Bax表达明显增加。Real-time PCR结果显示,不同浓度的Aβ_(25-35)药物组与空白组比较,DNA甲基化酶DNMT1、DNMT3a、DNMT3b、MeC P2表达无变化(P<0.05);MSP结果显示空白对照组Bcl-2和Bax甲基化扩增阴性,非甲基化扩增阳性;Aβ_(25-35)处理组Bcl-2和Bax甲基化引物扩增阴性,非甲基化引物扩增阳性。结论 MSP结果显示Aβ_(25-35)介导SHSY5Y细胞凋亡未引起Bcl-2、Bax启动子区甲基化的改变。Aim To investigate whether the effect of Aβ25-35 on Bcl-2 and Bax gene transcription through DNA methylation in SH-SY5Y cell.Methods Differ-ent concentrations of Aβ25-35 （0, 25, 50 μmol·L-1 ） were treated with SH-SY5Y cells for 48 h or 72 h in vitro.The optimal concentration and time of Aβ25-35 in-duced SH-SY5 Y apoptosis were determined by MTT method.Protein expression levels of Bcl-2 and Bax of Aβ25-35-treated groups were determined by Western blot.Real time PCR was used to detect the mRNA lev-els of DNA methyltransferase including DNMT 1 , DN-MT3a, DMT3b, MeCP2. Methylation specific PCR （ MSP） was used to analyze the effect of Aβ25-35 media-ted Bcl-2 and Bax gene promoter methylation .Results 25 μmol？ L-1 Aβ25-35 was exposed to SH-SY5Y cells for 72 h, MTT assay showed that cell viability was （68.49 ±9.83 ）%, which was significantly reduced compared with the control group （ P 〈0.05 ） , indica-ting AD cell apoptosis model was successfully estab-lished.Bcl-2 expression of Aβ25-35-treated group was significantly reduced compared with the control group , on the contrary , the expression of Bax was significantly increased .Real-time PCR results showed that com-pared with the control group , DNMT1, DNMT3a, DMT3b, MeCP2 mRNA levels of the Aβ25-35-treated groups had no significant difference （ P〉0.05 ）; MSP results showed that Bcl-2 and Bax unmethylated ampli-fication was positive , methylated amplification was neg-ative in control group , Bcl-2 and Bax unmethylated amplification was positive and methylated amplification was negative in Aβ25-35-treated group.Conclusion DNA methylation of Bcl-2 and Bax gene promoter are not affected during Aβ25-35 induced SH-SY5Y cell ap-optosis .

关 键 词：SH-SY5Y细胞 amyloid-β 凋亡 DNA甲基化 DNA甲基化酶 Bcl-2 Bax 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R977.3[医药卫生—基础医学]