组蛋白去甲基化酶对胚胎干细胞向心肌细胞分化的作用及机制

发布时间：2018-05-24 06:20

本文选题：PHF8 + 胚胎干细胞　；参考：《上海交通大学》2012年博士论文

【摘要】：研究目的:研究组蛋白去甲基化酶在胚胎干细胞向心肌细胞分化中的作用及调控机制。内容:通过筛选、鉴定寻找与胚胎干细胞分化为心肌细胞相关的组蛋白去甲基化酶,揭示它们在胚胎干细胞定向分化中的功能,并进一步探讨其分子机理。方法:本课题首先通过Q-PCR筛选出PHF8为调控心肌细胞分化的候选蛋白,并采用慢病毒感染对ES细胞进行基因操作;通过碱性磷酸酶染色、多能性标志蛋白表达分析鉴定KO细胞系的自我更新;通过MTT实验分析KO细胞系在未分化和分化过程中的细胞存活力;通过Brdu掺入法、细胞周期分析鉴定KO细胞系的增殖能力;通过早期凋亡标志蛋白annexin V染色、TUNEL法检测分析KO细胞的凋亡情况;通过早期胚层分化细胞标志基因和终末端细胞标志基因的表达分析鉴定KO细胞的三个胚层的分化能力。结果:通过对胚胎干细胞向心肌细胞分化中组蛋白去甲基化酶的表达谱筛选,我们发现PHF8表达在分化第1天骤然上升,之后随着分化时间推移逐渐下降,这提示我们PHF8可能在胚胎干细胞分化中具有重要的生物学功能。对PHF8敲除细胞系的研究发现,PHF8不调控ES细胞的自我更新,但PHF8的敲除抑制ES细胞向外胚层的分化,促进了向中胚层和内胚层分化,进而促进心肌细胞的分化。在对机制的研究中我们发现PHF8的敲除抑制分化中的胚胎干细胞的凋亡水平,尤其是早期中胚层前体细胞的凋亡。进一步凋亡机制的研究发现PHF8敲除虽然促进了经典凋亡蛋白caspase3总蛋白和活性片段的表达却并未促进凋亡,而是通过抑制凋亡诱导因子AIF(apoptosis induce factor)实现对ES细胞分化凋亡抑制。结论:PHF8的敲除不影响ES细胞自我更新,但抑制ES细胞向外胚层的分化,促进中胚层和内胚层分化,进而促进心肌细胞的分化。同时PHF8的敲除抑制分化中ES细胞的凋亡水平,尤其是早期中胚层前体细胞的凋亡。
[Abstract]:Aim: to study the role and regulatory mechanism of protein demethylase in differentiation of embryonic stem cells into cardiomyocytes. Content: through screening, histone demethylase associated with differentiation of embryonic stem cells into cardiomyocytes was identified, their functions in directional differentiation of embryonic stem cells were revealed, and its molecular mechanism was further discussed. Methods: first of all, PHF8 was screened out by Q-PCR as candidate protein to regulate cardiomyocyte differentiation, and gene of es cells was operated by lentivirus infection, and alkaline phosphatase staining was used. The expression of multipotent marker protein was used to identify the self-renewal of KO cell line, the viability of KO cell line during undifferentiation and differentiation was analyzed by MTT assay, and the proliferative ability of KO cell line was evaluated by Brdu incorporation and cell cycle analysis. The apoptosis of KO cells was detected by Tunel staining with annexin V staining, and the differentiation ability of KO cells was evaluated by the expression of marker gene and terminal marker gene. Results: by screening the expression profile of histone demethylase in the differentiation of embryonic stem cells into cardiomyocytes, we found that the expression of PHF8 increased sharply on the first day of differentiation, and then decreased gradually with the development of differentiation. This suggests that our PHF8 may play an important biological role in embryonic stem cell differentiation. Studies on PHF8 knockout cell lines showed that PHF8 did not regulate the self-renewal of es cells, but PHF8 knockout inhibited the differentiation of es cells into ectoderm, promoted the differentiation to mesoderm and endoderm, and further promoted the differentiation of cardiac myocytes. In the study of the mechanism, we found that PHF8 knockout inhibits the apoptosis of differentiated embryonic stem cells, especially in the early mesodermal progenitor cells. Further studies on the mechanism of apoptosis showed that PHF8 knockout did not promote the expression of the total protein and active fragment of classical apoptotic protein caspase3, but inhibited the differentiation and apoptosis of es cells by inhibiting the apoptosis-inducing factor AIF(apoptosis induce factor. Conclusion the knockout of PHF8 does not affect the self-renewal of es cells, but inhibits the differentiation of es cells into ectoderm, promotes the differentiation of mesoderm and endoderm, and further promotes the differentiation of cardiomyocytes. At the same time, PHF8 knockout inhibited the apoptosis of es cells in differentiation, especially in early mesodermal progenitor cells.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329.2

【共引文献】