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目的:研究大鼠胚胎发育至第15.5和18.5天胰腺基因表达谱,探讨特异性基因表达在正常内外分泌胰腺发育及糖尿病中所起的作用。方法:①实验于2003-09/2004-06在南京医科大学生化与分子生物学实验室完成。选用SD大鼠,雌雄各10只,将雌雄鼠按1∶2合笼,分别于胚胎发育至第15.5和18.5天,剖腹取出孕子宫,分离胚胎。②应用显微分离及提取技术获得胚胎发育不同阶段胰腺组织并提取RNA,采用高密度寡核苷酸芯片(Affemetrix芯片)对胚胎发育至第15.5天和第18.5胚胎胰腺进行基因转录水平分析,用生物信息学方法分析具体基因的表达情况。结果:胚胎发育至第18.5天与胚胎发育至第15.5天相比,差异表达2倍以上的基因共1319条。其中表达上升的基因664条,表达下降的基因655条,功能已知基因占63%,表达序列标识占37%。控制胰腺细胞分化的转录因子明显下调,与功能代谢相关的转录因子有上升趋势,内外分泌酶类、激素类表达有上升趋势,骨架蛋白有下降趋势,与激素、酶类分泌相关的因子基本无表达。结论:大鼠胚胎胰腺发育至第15.5～18.5天,内分泌典型胰岛结构及外分泌腺泡、导管结构已基本形成,功能细胞分化基因表达下降,而细胞代谢调节功能基因表达上升,因此,在大鼠胚胎发育至第15.5天胰腺,功能细胞的代谢调节功能开始完善。
OBJECTIVE: To study the gene expression patterns of pancreas in rat embryos 15.5 and 18.5 days, and to explore the role of specific gene expression in normal and exocrine pancreas development and diabetes mellitus. Methods: ① The experiment was performed at the Laboratory of Biochemistry and Molecular Biology, Nanjing Medical University from September 2003 to June 2004. SD rats were selected, 10 male and 10 female. The males and females were caged at 1: 2. The embryos were developed to 15.5 and 18.5 days respectively. The pregnant mice were removed by cesarean section and the embryos were isolated. (2) The pancreatic tissues at different stages of embryonic development were obtained by microdissection and extraction, RNA was extracted, and the gene transcription level of the pancreas of embryos developed to 15.5 days and 18.5 embryos was analyzed by high-density oligonucleotide chip (Affemetrix chip) Bioinformatics analysis of specific gene expression. RESULTS: There were 1319 differentially expressed genes that were more than 2-fold higher on the 18.5th embryo than on the 15.5th embryo. Among them, 664 genes were up-regulated, 655 genes were down-regulated, 63% were functionally known genes, and 37% were expressed in sequence. The transcription factors that control the differentiation of pancreatic cells were significantly down-regulated, and the transcription factors related to functional metabolism showed an upward trend. The expression of secretory enzymes and hormones both inside and outside showed an upward trend while that of the skeletal proteins showed a downward trend. The factors related to hormone and enzyme secretion were almost none expression. CONCLUSION: The pancreatic islets of rat embryo developed from 15.5 to 18.5 days. The endocrine typical pancreatic islets and exocrine alveoli were formed. The ductal structure has been basically formed, the expression of functional cell differentiation gene decreased and the expression of cellular metabolic regulation genes increased. Therefore, The embryo develops to the pancreas on the 15.5th day and the metabolic regulation function of the functional cells begins to be improved.