人胚胎生殖细胞向心肌细胞诱导分化及质谱分析研究

发布时间：2018-06-23 20:10

本文选题：人胚胎生殖细胞 + 抗坏血酸　；参考：《苏州大学》2011年硕士论文

【摘要】：目的:1.建立以人胚胎成纤维细胞为饲养层体外培养、扩增人胚胎生殖细胞(human embryonic germ cells, hEGCs,人EG细胞)的方法。2.利用抗坏血酸诱导人胚胎生殖细胞向心肌细胞分化;3.探讨人胚胎生殖细胞向心肌细胞分化过程中蛋白质表达谱的改变。方法:取5-10周人胚胎生殖腺嵴,组织块培养法原代培养人EG细胞;取同一胚胎来源的人胚胎成纤维细胞作饲养层,传代培养人EG细胞。将生长良好的人EG细胞,接种于细菌培养皿中进行悬浮培养,7天后将形成的拟胚体置于明胶包被的24孔培养板中,加入含0.1mg/ml抗坏血酸的诱导液进行诱导分化培养,用免疫荧光法检测诱导2周后细胞中心肌肌钙蛋白T(cTnT)的表达。分别提取人EG细胞和诱导2周后细胞的蛋白质,采用iTRAQ试剂对蛋白样品进行差异标记(114标记诱导细胞蛋白组、115标记人EG细胞蛋白组)、液相色谱(LC)分离这2组样品的总蛋白、串联质谱(MS/MS)鉴定人EG细胞和诱导后细胞差异表达的蛋白。结果:体外培养的人胚胎成纤维细胞呈长梭形,生长状态良好。以同源人胚胎成纤维细胞为饲养层传代培养的人EG细胞增殖旺盛,其集落呈典型的“鸟巢状”,和原代培养形成的克隆大小无明显区别,目前已传到第6代。人EG细胞经抗坏血酸诱导2周后,阳性表达cTnT,细胞呈多边形。质谱分析共鉴定出349种蛋白(蛋白置信度95%),其中27种蛋白(21种上调蛋白、6种下调蛋白)在人EG细胞和诱导后细胞中差异显著(差异倍数3)。结论:初步建立了以人胚胎成纤维细胞为饲养层体外扩增人EG细胞的培养方法。人EG细胞在抗坏血酸的诱导下分化为心肌细胞。质谱分析方法可高通量筛选与人EG细胞向心肌细胞分化相关的重要蛋白。
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【相似文献】