本文選題：人胚胎生殖細(xì)胞 + 抗壞血酸��； 參考：《蘇州大學(xué)》2011年碩士論文

【摘要】：目的:1.建立以人胚胎成纖維細(xì)胞為飼養(yǎng)層體外培養(yǎng)、擴(kuò)增人胚胎生殖細(xì)胞(human embryonic germ cells, hEGCs,人EG細(xì)胞)的方法。2.利用抗壞血酸誘導(dǎo)人胚胎生殖細(xì)胞向心肌細(xì)胞分化;3.探討人胚胎生殖細(xì)胞向心肌細(xì)胞分化過程中蛋白質(zhì)表達(dá)譜的改變。 方法:取5-10周人胚胎生殖腺嵴,組織塊培養(yǎng)法原代培養(yǎng)人EG細(xì)胞;取同一胚胎來源的人胚胎成纖維細(xì)胞作飼養(yǎng)層,傳代培養(yǎng)人EG細(xì)胞。將生長(zhǎng)良好的人EG細(xì)胞,接種于細(xì)菌培養(yǎng)皿中進(jìn)行懸浮培養(yǎng),7天后將形成的擬胚體置于明膠包被的24孔培養(yǎng)板中,加入含0.1mg/ml抗壞血酸的誘導(dǎo)液進(jìn)行誘導(dǎo)分化培養(yǎng),用免疫熒光法檢測(cè)誘導(dǎo)2周后細(xì)胞中心肌肌鈣蛋白T(cTnT)的表達(dá)。分別提取人EG細(xì)胞和誘導(dǎo)2周后細(xì)胞的蛋白質(zhì),采用iTRAQ試劑對(duì)蛋白樣品進(jìn)行差異標(biāo)記(114標(biāo)記誘導(dǎo)細(xì)胞蛋白組、115標(biāo)記人EG細(xì)胞蛋白組)、液相色譜(LC)分離這2組樣品的總蛋白、串聯(lián)質(zhì)譜(MS/MS)鑒定人EG細(xì)胞和誘導(dǎo)后細(xì)胞差異表達(dá)的蛋白。 結(jié)果:體外培養(yǎng)的人胚胎成纖維細(xì)胞呈長(zhǎng)梭形,生長(zhǎng)狀態(tài)良好。以同源人胚胎成纖維細(xì)胞為飼養(yǎng)層傳代培養(yǎng)的人EG細(xì)胞增殖旺盛,其集落呈典型的“鳥巢狀”,和原代培養(yǎng)形成的克隆大小無明顯區(qū)別,目前已傳到第6代。人EG細(xì)胞經(jīng)抗壞血酸誘導(dǎo)2周后,陽性表達(dá)cTnT,細(xì)胞呈多邊形。質(zhì)譜分析共鑒定出349種蛋白(蛋白置信度95%),其中27種蛋白(21種上調(diào)蛋白、6種下調(diào)蛋白)在人EG細(xì)胞和誘導(dǎo)后細(xì)胞中差異顯著(差異倍數(shù)3)。 結(jié)論:初步建立了以人胚胎成纖維細(xì)胞為飼養(yǎng)層體外擴(kuò)增人EG細(xì)胞的培養(yǎng)方法。人EG細(xì)胞在抗壞血酸的誘導(dǎo)下分化為心肌細(xì)胞。質(zhì)譜分析方法可高通量篩選與人EG細(xì)胞向心肌細(xì)胞分化相關(guān)的重要蛋白。
[Abstract]:Purpose 1. A method was established to amplify human embryonic germ cells (human embryonic germ cells, hEGCsand human EG cells) by using human embryonic fibroblasts as feeder layer in vitro. Human embryonic germ cells were induced to differentiate into cardiomyocytes by ascorbic acid. To investigate the changes of protein expression profile during the differentiation of human embryonic germ cells into cardiomyocytes. Methods: human EG cells were cultured by tissue mass culture method, and human EG cells were subcultured by using the same embryonic fibroblasts as feeder layer from 5 to 10 weeks of human embryonic gonadal ridge. The well-grown human EG cells were inoculated in a culture dish for 7 days. The embryoid was placed in a 24 well culture plate coated with gelatin, and the culture medium containing ascorbic acid (0.1mg/ml) was added to induce the differentiation of EG cells. The expression of cardiac troponin T (cTnT) was detected by immunofluorescence. The proteins of human EG cells were extracted from human EG cells and those of human EG cells 2 weeks after induction. The total proteins of the two groups were separated by liquid chromatography (LC), and the protein samples were labeled with iTRAQ reagent. Tandem mass spectrometry (MS / MS) identified differentially expressed proteins between human EG cells and induced EG cells. Results: the cultured human embryonic fibroblasts were fusiform and in good growth state. Human EG cells cultured on the feeder layer of homologous human embryonic fibroblasts proliferate strongly, and the colony of EG cells is typical "bird nest", which has no obvious difference from the clone size formed by primary culture, and has been transmitted to the sixth generation. Human EG cells were induced by ascorbic acid for 2 weeks. A total of 349 proteins (95%) were identified by mass spectrometry, of which 27 proteins (21 up-regulated proteins and 6 down-regulated proteins) were significantly different between human EG cells and induced EG cells (3 times of difference). Conclusion: human EG cells were cultured in vitro using human embryonic fibroblasts as feeder layer. Human EG cells differentiate into cardiomyocytes induced by ascorbic acid. High throughput mass spectrometry can be used to screen important proteins related to the differentiation of human EG cells into cardiomyocytes.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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