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目的:组蛋白乙酰化修饰是表观遗传调控中重要的调控方式之一,组蛋白乙酰化与基因活化关系密切,其中一个重要环节是被特定阅读器结构域所识别,从而招募染色质调控因子到特定区域,协同完成基因表达调控。AF9是这类阅读器之一,其是否参与心脏发育分化还未清楚。因此本研究探讨心脏转录因子联合AF9是否对心脏祖细胞的产生具有促进作用。方法:采用蛋白重组表达方式获得心脏转录因子GATA4、NKX2.5、TBX5(GNT)及AF9,结合纳米转导技术高效转导到目的细胞BMSCs细胞核内,检测细胞毒性,CPCs的生成效率,组蛋白H3K9乙酰化情况,Ch IP-PCR技术检测H3K9ac的作用靶点。结果:心脏转录因子GNT联合AF9能提高BMSCs重编程生成CPCs的效率,在200μg/L/蛋白的工作浓度下,细胞生长良好,与GNT组相比,联合AF9组的H3K9ac表达明显增加,Ch IP-PCR的结果显示H3K9ac富集在MEF2C的启动子区域。结论:心脏转录因子组合GNT联合表观遗传调控因子AF9通过纳米-蛋白转导方式,能高效重编程BMSCs得到CPCs,AF9通过上调H3K9ac来促进重编程过程。
AIM: Histone acetylation is one of the most important regulatory mechanisms in epigenetic regulation. Histone acetylation is closely related to gene activation. One of the important links is recognition by specific reader domain, which recruits chromatin regulatory factors To a specific area, to complete the regulation of gene expression. AF9 is one such reader, its participation in cardiac development and differentiation is not yet clear. Therefore, this study was to investigate whether cardiac transcription factor combined with AF9 can promote cardiac progenitor cells. Methods: Cardiac transcription factors GATA4, NKX2.5, TBX5 (GNT) and AF9 were obtained by recombinant expression of protein, and transduced into the nucleus of BMSCs by Nano-transduction technique. The cytotoxicity, the production efficiency of CPCs, H3K9 acetylation, Ch IP-PCR detection of H3K9ac target. RESULTS: Cardiac transcription factor GNT combined with AF9 increased the efficiency of BMSCs reprogramming to generate CPCs. Under the concentration of 200μg / L / protein, cell growth was good. Compared with GNT group, the expression of H3K9ac in combination with AF9 group was significantly increased The results of PCR showed that H3K9ac is enriched in the promoter region of MEF2C. CONCLUSION: Cardiac transcription factor (GNT) combined with epigenetic regulator AF9 can reprogram BMSCs efficiently to get CPCs through nano-protein transduction. AF9 can promote the reprogramming process by up-regulating H3K9ac.