本文選題：Islet-1 + 組蛋白乙酰化��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的 組蛋白乙�；癁槌Ｒ�(jiàn)的表觀遺傳方式。本課題組前期研究發(fā)現(xiàn)，心臟發(fā)育相關(guān)轉(zhuǎn)錄因子受到組蛋白乙�；恼{(diào)控，而組蛋白乙�；癄顟B(tài)則通過(guò)組蛋白乙�；福╤istone acetyltransferases，HATs）和組蛋白去乙�；福╤istone deacetylase，HDACs）調(diào)節(jié)。然而，組蛋白乙�；](méi)有特異性，在心臟發(fā)育過(guò)程中是否存在輔助因子，招募并協(xié)助HATs識(shí)別心臟發(fā)育相關(guān)轉(zhuǎn)錄因子以介導(dǎo)其啟動(dòng)子區(qū)組蛋白的乙�；�？Islet-1為心臟發(fā)育早期的重要轉(zhuǎn)錄因子，屬于LIM-Homeodomain家族，其結(jié)構(gòu)符合該輔助因子的需求。本研究擬通過(guò)慢病毒轉(zhuǎn)染干擾Islet-1的表達(dá)，探討Islet-1是否作為此種輔助因子介導(dǎo)組蛋白乙酰化參與心臟發(fā)育相關(guān)轉(zhuǎn)錄因子的調(diào)控。 材料與方法 培養(yǎng)心肌祖細(xì)胞，，以轉(zhuǎn)染Islet-1RNAi慢病毒作為Islet-1抑制組，轉(zhuǎn)染未攜帶任何基因的空載體慢病毒作為空載體對(duì)照組，未轉(zhuǎn)染慢病毒的細(xì)胞作為空白對(duì)照組。通過(guò)流式細(xì)胞計(jì)數(shù)檢測(cè)轉(zhuǎn)染效率，運(yùn)用熒光定量聚合酶鏈反應(yīng)（quantitative polymerase chain reaction,Q-PCR）檢測(cè)心臟發(fā)育相關(guān)轉(zhuǎn)錄因子mRNA的表達(dá)水平。通過(guò)Western blotting檢測(cè)心肌祖細(xì)胞內(nèi)組蛋白H3乙�；�。通過(guò)ChIP抗乙�；疕3抗體和抗p300抗體，運(yùn)用染色質(zhì)免疫共沉淀（Chromatin immunoprecitation,ChIP）及Q-PCR技術(shù)檢測(cè)心臟發(fā)育相關(guān)轉(zhuǎn)錄因子啟動(dòng)子區(qū)乙�；郊芭cHATs亞型p300結(jié)合情況。 結(jié)果 1.流式細(xì)胞計(jì)數(shù)顯示空載體組及Islet-1抑制組的轉(zhuǎn)染效率均大于30%。Q-PCR結(jié)果顯示Islet-1抑制組中心臟發(fā)育相關(guān)轉(zhuǎn)錄因子Islet-1、Mef2c、Tbx5的mRNA相對(duì)表達(dá)量與空白對(duì)照組和空載體對(duì)照組相比有明顯降低（P 0.05）。而Gata4的mRNA相對(duì)表達(dá)量各組間差異不具有統(tǒng)計(jì)學(xué)意義（P0.05）。 2. Western blotting結(jié)果顯示空白對(duì)照組、空載體對(duì)照組及Islet-1抑制組之間組蛋白H3乙�；�?jīng)]有明顯變化差異不具有統(tǒng)計(jì)學(xué)意義（P0.05）。ChIP PCR結(jié)果提示Islet-1抑制組中通過(guò)乙�；疕3抗體和p300抗體募集下來(lái)的Mef2c的啟動(dòng)子DNA含量較空白對(duì)照組和空載體對(duì)照組有降低，并且差異具有統(tǒng)計(jì)學(xué)意義（P 0.05），而Gata4、Tbx5的啟動(dòng)子DNA含量各組間則沒(méi)有明顯統(tǒng)計(jì)學(xué)差異（P 0.05）。 結(jié)論 以上數(shù)據(jù)表明，抑制Islet-1可能降低p300與Mef2c啟動(dòng)子區(qū)的結(jié)合，從而下調(diào)Mef2c啟動(dòng)子區(qū)乙�；�，進(jìn)而影響Mef2c的表達(dá)。但Gata4、Tbx5可能并非通過(guò)該途徑而受到組蛋白乙�；恼{(diào)控，其調(diào)控方式仍需進(jìn)一步研究。
[Abstract]:Objective histone acetylation is a common epigenetic pattern. Our previous study found that cardiac development-related transcription factors are regulated by histone acetylation, and the histone acetylation state is regulated by histone acetyltransferes (HATs) and histone deacetylase (histone deacetylase). However, histone acetylation is not specific. The acetylated Islet-1 (Islet-1) involved in the identification of cardiac developmental related transcription factors by HATs is an important transcription factor in the early stage of cardiac development. It belongs to the LIM-Homeodomain family and its structure meets the needs of this cofactor. In this study, lentivirus transfection interfered with the expression of Islet-1 to investigate whether Islet-1 is involved in the regulation of transcription factors associated with cardiac development as an auxiliary factor mediating histone acetylation. Materials and methods Myocardial progenitor cells were cultured. Islet-1RNAi lentivirus was transfected as Islet-1 inhibition group, empty vector lentivirus without any gene was transfected as control group, and cells without lentivirus were used as blank control group. The transfection efficiency was detected by flow cytometry and the expression level of cardiac development-related transcription factor mRNA was detected by fluorescence quantitative polymerase chain reaction (quantitative polymerase chain reaction- Q-PCR). The level of histone H 3 acetylation in myocardial progenitor cells was detected by Western blotting. The acetylation level of the promoter region of cardiac development-associated transcription factor and its binding to Hats subtype p300 were detected by chromatin immunoprecipitation (chip) and Q-PCR using ChIP anti-acetylated H3 and anti-p300 antibodies. Result 1. Flow cytometry showed that the transfection efficiency of empty vector group and Islet-1 inhibition group was higher than that of 30. Q-PCR group. The results showed that the relative expression of Islet-1Mef2ctbx5 mRNA in Islet-1 inhibition group was significantly lower than that in blank control group and empty vector control group (P 0.05). However, the relative expression of Gata4 mRNA was not significantly different among the groups (P0.05). The results of Western blotting showed that the blank control group, There was no significant difference in the level of histone H3 acetylation between empty vector control group and Islet-1 inhibition group (P0.05). The PCR results showed that Mef2c was initiated by acetylated H3 antibody and p300 antibody in Islet-1 inhibition group. The DNA content of the subunit was lower than that of the blank control group and the empty vector control group. The difference was statistically significant (P 0.05), but there was no significant difference in the promoter DNA content of Gata4 (Tbx5) among the three groups (P 0.05). Conclusion the above data suggest that inhibition of Islet-1 may decrease the binding of p300 to Mef2c promoter, thus down-regulate the acetylation level of Mef2c promoter, and then affect the expression of Mef2c. However, Gata4Tbx5 may not be regulated by histone acetylation through this pathway.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725.4

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王金泉,劉志紅;糖皮質(zhì)激素免疫抑制作用機(jī)理的新認(rèn)識(shí)[J];腎臟病與透析腎移植雜志;1997年02期

2 田文洪,汪明春,李楊秋,楊力健;RT-PCR檢測(cè)血液病中GATA-3基因表達(dá)[J];臨床血液學(xué)雜志;1998年02期

3 陳偉;一種新的轉(zhuǎn)錄因子——EPAS1[J];生理科學(xué)進(jìn)展;2000年04期

4 李揚(yáng)秋,楊力建,陳少華,盧育洪,張洹;急性非淋巴細(xì)胞白血病中轉(zhuǎn)錄因子GATA-2基因插入突變[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2000年04期

5 李曉青,曾水清,徐莉莉,程揚(yáng);血清誘導(dǎo)人視網(wǎng)膜色素上皮細(xì)胞中轉(zhuǎn)錄因子E2F1的表達(dá)及活化[J];中華眼底病雜志;2002年03期

6 陳萍,常才;先天性心臟病相關(guān)轉(zhuǎn)錄因子Csx/Nkx2.5基因的研究[J];國(guó)外醫(yī)學(xué).婦產(chǎn)科學(xué)分冊(cè);2004年03期

7 李武修;孫巖;;Forkhead轉(zhuǎn)錄因子超家族成員——FOXL2[J];口腔醫(yī)學(xué)研究;2006年02期

8 席銳;陳楨s

本文編號(hào)：2073687