摘要
目的观察SH—SY5Y细胞凋亡，酪氨酸羟化酶（TH）、甲基化CpG结合蛋白2（MeCP2）蛋白表达变化，探讨MeCP2在帕金森病中的作用机制。方法利用真核表达载体构建、细胞转染等技术获得MeCP2表达上调的SH—SY5Y细胞株。免疫荧光技术和Western blot检测转染重组质粒对SH—SY5Y细胞内源性MeCP2蛋白表达的影响以及TH表达的变化；用细胞计数试剂盒（CCK-8）法、流式细胞仪检测正常SH-SY5Y细胞、上调MeCP2表达的SH—SY5Y细胞在6-羟基多巴胺（6-OHDA）诱导下细胞活性及细胞凋亡的变化。结果利用pEGFP—N1-MeCP2靶向MeCP2的上调质粒转染SH—SY5Y细胞来上调MeCP2在SH—SY5Y细胞中的表达，在6-OHDA诱导下，免疫荧光检测MeCP2、TH的表达未下降，Western blot进一步证实MeCP2、TH的表达未下降，MeCP2／β-actin、TH相对表达量分别为0．8865±0．0486、0．4639±0．0266，流式细胞仪检测显示SH—SY5Y细胞细胞凋亡率约为（0．40±0．00）％，CCK-8检测细胞存活率约为（96．25±5．28）％。结论MeCP2帕金森病的发病过程中起重要作用。转染pEGFP—N1-MeCP2重组质粒可抑制6-OHDA诱导的SH—SY5Y细胞凋亡，提高TH的表达，提高细胞存活率。
Objective Observed apoptosis in SH-SY5Y cells and protein expression of tyrosine bydroxylase and methyl cytosine binding protein-2 （ MeCP2）, and explore the role of MeCP2 in parkinson＇ s disease, Methods In this study, to get the up-regulation of MeCP2 in SH-SY5Y cell lines, the eukaryotie expression vector construction, the cells transfeeted technology was used. Recombinant plasmid transfeeted to SH-SY5Y cells, analysis of the MeCP2 protein and endogenous tyrosine hydroxylase （TH） protein expression by Immunofluoreseenee and Western blotting. Detected cell activity and apoptosis in normal SH-SY5Y cells and the SH-SY5Y cell that up-regulation of MeCP2 which pretreated with 6-hydroxydopamine （6-OHDA） by cell counting kit-8 （CCK-8） assay, flow cytometry. Detected the expression of MeCP2 protein and TH protein in each group of SH-SY5Y cells by Imnmnofluoreseenee and Western blotting analysis. Results The plasmid of pEGFP-N1-MeCP2 target the MeCP2 was transfected with SH-SY5Y cells to raise the expression of MeCP2, and then treated with 6-OHDA, the expression of MeCP2 and TH was not decline by the detection of immunofluoreseence and Western blotting, the ratio of MeCP2/β-aetin and TH/β-aetin were 0. 8865±0. 0486 and 0. 4639 ± 0. 0266 respectively. The result of flow eytometry showed that apoptosis of SH-SY5Y ceils was approximately （0, 40 ± 0.00 ） % and CCK-8 detection showed that cell survival rate was approximately （ 96. 25 ± 5.28 ）. Conclusion MeCP2 plays an important role in the pathogenesis of Parkinson＇ s disease. The present data indicate that MeCP2 upregulation can reduce 6-OHDA-induced apoptosis, and increase the protein levels of TH in SH-SY5Y cells, suggesting that MeCP2 may be a potential therapeutic target for the treatment of PD.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第6期1282-1285,共4页
Chinese Journal of Experimental Surgery
基金
湖北省荆门市科技计划资助项目（2013S01）
