摘要
目的筛选有效质粒片段,探讨组蛋白乙酰化平衡对MSCs分化的影响。方法提取针对GCN5基因的3条shRNA(ZJ1、ZJ2、ZJ3)重组质粒及阴性对照质粒HK,分别转染入经5-aza诱导的MSCs,24h后荧光显微镜观察细胞、流式细胞仪检测转染效率、Western-blot分析GCN5蛋白表达,ELISA检测组蛋白乙酰化酶活性。结果质粒转染效率大于20%;ZJ3转染组组蛋白乙酰化酶活性及GCN5蛋白表达量均有明显降低,抑制效率分别为[(49.0±0.6)%,(35.7±0.1)%,P<0.05];组蛋白乙酰化水平与MSCs分化进程密切相关,质粒ZJ3可有效抑制不同分化阶段MSCs中乙酰化酶水平,抑制效率分别为[(27.0±0.1)%,(26.7±0.1)%,(25.4±0.1)%,P<0.05]。结论成功筛选出有效质粒片段ZJ3,初步确证GCN5抑制状态可导致组蛋白乙酰化失衡,从而调控MSCs的分化进程,为后期MSCs移植的临床应用奠定实验基础。
Objective To screen the valid plasmid targeted to GCN5 among the constructed recombinant plasmids; and to explore the effects of histone acetylizad modification in regulating MSCs differentiation. Methods Exstract the constructed plasmids and transfect them into MSCs induced by 5-aza. For 24 h, observe MSCs; detect the transfect efficiency by flow cytometry; detect expression of protein GCN5 by Western-blot; detect the HAT activity by ELISA. Results Transfect efficiency was more than 20% ; Expression of protein GCN5 and HAT activity had no difference in group ZJ1 and ZJ2, but had a significant difference to that in group ZJ3. HAT activity of experiment group was significantly lower than that of control groups. Conclusion The inhibition state of histone acetylation results in plasmid ZJ3, it can inhibit the differentiation process of MSCs. The results provide data for the clinical application of MSCs transplantation.
出处
《基础医学与临床》
CSCD
北大核心
2008年第6期557-562,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(30471837)
