【作者】 侯良芹；

【导师】 德伟；

【作者基本信息】 南京医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 运用高密度寡核苷酸芯片研究了大鼠胰腺不同发育阶段(胚胎(E)12.5,E15.5,E18.5,新生和成年)的基因表达谱,经过生物信息学数据分析发现,许多已经报道过的参与胰岛细胞发育和分化的基因例如:Pdx1、Ngn3、Pax4等在E12.5或E15.5高表达,这与以往的报道相一致;而与胰岛功能相关的基因在E18.5相对高表达,这与胚胎发育后期胰岛功能趋于完善和成熟的发育过程相符;而典型的胰岛结构是胰岛发挥正常生物学功能的基础,这个时期也正是胰岛细胞迁移、聚集形成典型胰岛结构的关键阶段,我们检测到一些参与细胞迁移的黏附分子在E18.5高表达,其中有些基因是已经报道过的参与胰岛细胞的迁移和黏附,而有些基因在胰腺发育过程中尚未见报道,例如黏附分子Mesothelin,本研究从核酸和蛋白两个方面对Mesothelin基因在胰腺发育不同阶段的表达和定位进行研究,并初步探讨该基因在胰腺发育过程中的生物学功能。运用RT-PCR技术进一步验证Mesothelin基因在大鼠胰腺发育不同阶段的mRNA表达水平:在E15.5表达量很低,至E18.5明显升高,到生后14天(P14)表达量达到高峰,然后下降,至成年表达量较低。这与基因芯片中检测到的Mesothelin基因的表达趋势一致;Westernblot检测Mesothelin蛋白在大鼠胰腺发育不同阶段的表达丰度,发现蛋白的表达趋势与mRNA的表达趋势完全相同。用免疫荧光组织化学进行定位研究发现,Mesothelin既与Insulin共表达于胰岛β细胞,又与Vimentin共表达于间充质细胞。最后我们运用实时荧光定量PCR技术对Mesothelin上游基因Wnt-5a在胰腺发育过程中的表达进行检测,发现其表达趋势与Mesothelin的表达趋势相反。实验结果显示,Mesothelin高表达的时期与胰岛结构形成和重塑的发育阶段正好相符,而且定位于内分泌细胞,因此,我们推测黏附分子Mesothelin可能调控胰腺胚胎发育后期胰岛细胞的迁移、聚集、黏附及胰岛结构的形成,并参与出生后胰岛结构重塑的过程,其作用的发挥可能受上游因子Wnt-5a的调控,同时表达Mesothelin的胰腺间充质细胞可能是内分泌细胞的一个新的来源。更多还原

【Abstract】 We expect to use high-density microarray(Affymetrix)to analyze the expression profiles of pancreas development.We generated transcriptional profiles of pancreatic tissue isolated from five biologically significant stages of development:embryonic day(E)12.5,E15.5,E18.5, newborn and adult.These analyses implicated that many genes that have been reported to be implicated in endocrine cytodifferentiation during early pancreas development,such as Pdx1,Ngn3,Pax4 and so on,were significantly elevated between E15.5 and E18.5,consistent with previous reports.Genes involved in islet functions were enriched in E18.5,which is consistent with that later gestation was very important in functional maturation of islets.Many adhesion molecules were enriched in the later gestation when islet cells migration,adhesion and islet architecture are gradually forming.Some of which have been reported to be involved in islets architecture formation and others have not,such as Mesothelin. This study was designed to investigate the expression and function of Mesothelin in development of rat pancreas.The mRNA expression levels of Mesothelin at different developmental stages were examined by RT-PCR.The expression levels of Mesothelin were detectable in E15.5 pancreas,followed by increased levels from E18.5 to P14.The expression levels reached the peak in P14 pancreas,and then decreased until adult rat pancreas.The abundance of Mesothelin protein in the rat pancreas during development were studied using western blot.Mesothelin protein expression tendency was consistent with the Mesothelin mRNA expression.Immunolocalization indicated that Mesothelin not only colocalized with insulin in islet beta-cells but also colocalized with vimentin in mesenchyme of developing rat pancreas.At last,the expression of Wnt-5a,upstream regulator of Mesothelin,was verified by real-time PCR,and showed that its expression tendency was opposite to Mesothelin.The results of our experiments show that the expression of Mesothelin concomitants with islets architecture formation and remodeling and that the Mesothelin is localized in islet cells.This suggested that Mesothelin might play a role in islet cells migration and adhesion during the process of islet architecture formation at the later gestation and in islet architecture remodeling and maturation after birth. The function of Mesothelin might be regulated by Wnt-5a.Mesenchymal cells expressing Mesothelin simultaneously might be a potential source for endocrine cells.更多还原