本文選題：PHF8 + 胚胎干細(xì)胞　； 參考：《上海交通大學(xué)》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(xì)胞向心肌細(xì)胞分化中的作用及調(diào)控機(jī)制。內(nèi)容:通過篩選、鑒定尋找與胚胎干細(xì)胞分化為心肌細(xì)胞相關(guān)的組蛋白去甲基化酶,揭示它們?cè)谂咛ジ杉?xì)胞定向分化中的功能,并進(jìn)一步探討其分子機(jī)理。方法:本課題首先通過Q-PCR篩選出PHF8為調(diào)控心肌細(xì)胞分化的候選蛋白,并采用慢病毒感染對(duì)ES細(xì)胞進(jìn)行基因操作;通過堿性磷酸酶染色、多能性標(biāo)志蛋白表達(dá)分析鑒定KO細(xì)胞系的自我更新;通過MTT實(shí)驗(yàn)分析KO細(xì)胞系在未分化和分化過程中的細(xì)胞存活力;通過Brdu摻入法、細(xì)胞周期分析鑒定KO細(xì)胞系的增殖能力;通過早期凋亡標(biāo)志蛋白annexin V染色、TUNEL法檢測(cè)分析KO細(xì)胞的凋亡情況;通過早期胚層分化細(xì)胞標(biāo)志基因和終末端細(xì)胞標(biāo)志基因的表達(dá)分析鑒定KO細(xì)胞的三個(gè)胚層的分化能力。結(jié)果:通過對(duì)胚胎干細(xì)胞向心肌細(xì)胞分化中組蛋白去甲基化酶的表達(dá)譜篩選,我們發(fā)現(xiàn)PHF8表達(dá)在分化第1天驟然上升,之后隨著分化時(shí)間推移逐漸下降,這提示我們PHF8可能在胚胎干細(xì)胞分化中具有重要的生物學(xué)功能。對(duì)PHF8敲除細(xì)胞系的研究發(fā)現(xiàn),PHF8不調(diào)控ES細(xì)胞的自我更新,但PHF8的敲除抑制ES細(xì)胞向外胚層的分化,促進(jìn)了向中胚層和內(nèi)胚層分化,進(jìn)而促進(jìn)心肌細(xì)胞的分化。在對(duì)機(jī)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(xì)胞的凋亡水平,尤其是早期中胚層前體細(xì)胞的凋亡。進(jìn)一步凋亡機(jī)制的研究發(fā)現(xiàn)PHF8敲除雖然促進(jìn)了經(jīng)典凋亡蛋白caspase3總蛋白和活性片段的表達(dá)卻并未促進(jìn)凋亡,而是通過抑制凋亡誘導(dǎo)因子AIF(apoptosis induce factor)實(shí)現(xiàn)對(duì)ES細(xì)胞分化凋亡抑制。結(jié)論:PHF8的敲除不影響ES細(xì)胞自我更新,但抑制ES細(xì)胞向外胚層的分化,促進(jìn)中胚層和內(nèi)胚層分化,進(jìn)而促進(jìn)心肌細(xì)胞的分化。同時(shí)PHF8的敲除抑制分化中ES細(xì)胞的凋亡水平,尤其是早期中胚層前體細(xì)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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.2

【共引文獻(xiàn)】

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本文編號(hào)：1928009