本文選題：PHF8 + 胚胎干細胞　； 參考：《上海交通大學》2012年博士論文

【摘要】：研究目的:研究組蛋白去甲基化酶在胚胎干細胞向心肌細胞分化中的作用及調控機制。內容:通過篩選、鑒定尋找與胚胎干細胞分化為心肌細胞相關的組蛋白去甲基化酶,揭示它們在胚胎干細胞定向分化中的功能,并進一步探討其分子機理。方法:本課題首先通過Q-PCR篩選出PHF8為調控心肌細胞分化的候選蛋白,并采用慢病毒感染對ES細胞進行基因操作;通過堿性磷酸酶染色、多能性標志蛋白表達分析鑒定KO細胞系的自我更新;通過MTT實驗分析KO細胞系在未分化和分化過程中的細胞存活力;通過Brdu摻入法、細胞周期分析鑒定KO細胞系的增殖能力;通過早期凋亡標志蛋白annexin V染色、TUNEL法檢測分析KO細胞的凋亡情況;通過早期胚層分化細胞標志基因和終末端細胞標志基因的表達分析鑒定KO細胞的三個胚層的分化能力。結果:通過對胚胎干細胞向心肌細胞分化中組蛋白去甲基化酶的表達譜篩選,我們發(fā)現(xiàn)PHF8表達在分化第1天驟然上升,之后隨著分化時間推移逐漸下降,這提示我們PHF8可能在胚胎干細胞分化中具有重要的生物學功能。對PHF8敲除細胞系的研究發(fā)現(xiàn),PHF8不調控ES細胞的自我更新,但PHF8的敲除抑制ES細胞向外胚層的分化,促進了向中胚層和內胚層分化,進而促進心肌細胞的分化。在對機制的研究中我們發(fā)現(xiàn)PHF8的敲除抑制分化中的胚胎干細胞的凋亡水平,尤其是早期中胚層前體細胞的凋亡。進一步凋亡機制的研究發(fā)現(xiàn)PHF8敲除雖然促進了經典凋亡蛋白caspase3總蛋白和活性片段的表達卻并未促進凋亡,而是通過抑制凋亡誘導因子AIF(apoptosis induce factor)實現(xiàn)對ES細胞分化凋亡抑制。結論:PHF8的敲除不影響ES細胞自我更新,但抑制ES細胞向外胚層的分化,促進中胚層和內胚層分化,進而促進心肌細胞的分化。同時PHF8的敲除抑制分化中ES細胞的凋亡水平,尤其是早期中胚層前體細胞的凋亡。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329.2

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