MicroRNA-18a在胰腺祖细胞和腺泡细胞中对转录因子Ptf1a的调控研究

发布时间：2018-11-15 13:26

【摘要】：胰腺是人体内仅次于肝脏的第二大腺体器官,也是消化功能最强的脏器。它由内、外两分泌部组成,分别行使维持血糖代谢和消化食物的生理功能。胰腺特异转录因子Ptf1a的调控作用贯穿整个胰腺发育过程,在早期发育中Ptf1a就出现在幼稚的上皮细胞中,调节上皮细胞的生长和增殖,Ptf1a表达丰度的高低也一定程度上控制着胰腺细胞的分化命运。在分化的腺泡细胞中Ptf1a表达丰度极高,并结合在消化酶类基因的启动子上调控消化酶基因的转录。如今,胰腺发育和相关疾病发生中所涉及到的分子调控机制研究的尚不透彻,调控Ptf1a的基因和分子尤甚,因此分子调控机制的研究将对整个胰腺探究领域的意义十分重大。 MicroRNAs (miRNAs)是当今生物科研领域的热点,此类序列短(约22-25nt)、非编码的RNA转录后水平的调节对发育,代谢,疾病肿瘤发生过程中起着至关重要的作用。MiR-18a的miR-17-92家族成员之一,在多种组织中高表达,对细胞的增殖、分化等生理过程均起重要的调控作用。研究通过实时定量RT-PCR分析了miR-18a和Ptfla在小鼠胰腺发育过程和各类胰腺细胞中的表达模式,确定miR-18a对Ptfla的靶向调控作用,并在胰腺祖细胞和腺泡细胞中过表达miR-18a探索其对胰腺的调控作用。结果显示：MiR-18a在小鼠胰腺e12.5表达最高,e18.5表达最低,而Ptf1a在此过程中的表达模式则相反；在小鼠或者大鼠来源的胰腺细胞系中,MiR-18a在祖细胞中表达最高,在MIN6中表达最低,而Ptf1a在AR42J细胞中表达最高,在MIN6中不表达；在3T3细胞系中MiR-18a对Ptfla mRNA和蛋白都有下调作用,双荧光素酶报告系统也从正反向验证了这种靶向作用；在胰腺祖细胞中MiR-18a对内源和外源过表达的Ptfla的抑制作用都很微弱,并对祖细胞的形态数量,基因表达潜影响均不大；在腺泡AR42J细胞中过表达MiR-18a能下调Ptf1a的蛋白表达,并进而抑制一些外分泌酶类基因的转录。综上所示,在胰腺祖细胞和腺泡细胞中Ptf1a均受MiR-18a的调控,并且能影响下游相关基因的表达变化,但MiR-18a主要以一种微调节的方式起作用。
[Abstract]:The pancreas is the second largest glandular organ in the human body after the liver and the most powerful digestive organ. It consists of internal and external secretory parts, respectively, to maintain blood glucose metabolism and digestion of food physiological functions. The regulation of pancreatic specific transcription factor (Ptf1a) throughout the development of pancreas. During the early development of pancreas, Ptf1a appeared in immature epithelial cells and regulated the growth and proliferation of epithelial cells. The abundance of Ptf1a also controlled the differentiation of pancreatic cells to some extent. The expression of Ptf1a in differentiated acinar cells is very high and the transcription of digestive enzyme gene is regulated by the promoter of digestive enzyme gene. At present, the molecular regulation mechanism involved in pancreatic development and related diseases has not been thoroughly studied, especially the genes and molecules regulating Ptf1a. Therefore, the study of molecular regulation mechanism will be of great significance to the whole pancreas exploration field. MicroRNAs (miRNAs) is a hot topic in the field of biological research. These short sequences (about 22-25nt), non-coding RNA posttranscriptional regulation of development, metabolism, One of the members of the miR-17-92 family of MiR-18a is highly expressed in a variety of tissues and plays an important role in the regulation of cell proliferation, differentiation and other physiological processes. In this study, real-time quantitative RT-PCR was used to analyze the expression patterns of miR-18a and Ptfla in mouse pancreatic development and various pancreatic cells, and to determine the targeted regulation of miR-18a on Ptfla. And overexpression of miR-18a in pancreatic progenitor cells and acinar cells to explore its role in the regulation of pancreas. The results showed that the expression of MiR-18a was the highest in mouse pancreas and the lowest in e18.5, but the expression pattern of Ptf1a was opposite. In mouse or rat pancreatic cell lines, the expression of MiR-18a was the highest in progenitor cells, the lowest in MIN6, and the highest in AR42J cells, but not in MIN6. Both Ptfla mRNA and protein were down-regulated by MiR-18a in 3T3 cell line, and the double luciferase reporting system confirmed the targeting effect from positive to negative. In pancreatic progenitor cells, the inhibitory effect of MiR-18a on endogenous and exogenous overexpression of Ptfla was very weak, and had little effect on the number of progenitor cells and the latent expression of genes. Overexpression of MiR-18a in acinar AR42J cells can down-regulate the expression of Ptf1a and inhibit the transcription of some exocrine enzyme genes. In conclusion, Ptf1a is regulated by MiR-18a in pancreatic progenitor cells and acinar cells, and can affect the expression of downstream genes. However, MiR-18a mainly acts in a microregulatory manner.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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