【摘要】：胰腺是人體內(nèi)僅次于肝臟的第二大腺體器官,也是消化功能最強(qiáng)的臟器。它由內(nèi)、外兩分泌部組成,分別行使維持血糖代謝和消化食物的生理功能。胰腺特異轉(zhuǎn)錄因子Ptf1a的調(diào)控作用貫穿整個(gè)胰腺發(fā)育過(guò)程,在早期發(fā)育中Ptf1a就出現(xiàn)在幼稚的上皮細(xì)胞中,調(diào)節(jié)上皮細(xì)胞的生長(zhǎng)和增殖,Ptf1a表達(dá)豐度的高低也一定程度上控制著胰腺細(xì)胞的分化命運(yùn)。在分化的腺泡細(xì)胞中Ptf1a表達(dá)豐度極高,并結(jié)合在消化酶類基因的啟動(dòng)子上調(diào)控消化酶基因的轉(zhuǎn)錄。如今,胰腺發(fā)育和相關(guān)疾病發(fā)生中所涉及到的分子調(diào)控機(jī)制研究的尚不透徹,調(diào)控Ptf1a的基因和分子尤甚,因此分子調(diào)控機(jī)制的研究將對(duì)整個(gè)胰腺探究領(lǐng)域的意義十分重大。 MicroRNAs (miRNAs)是當(dāng)今生物科研領(lǐng)域的熱點(diǎn),此類序列短(約22-25nt)、非編碼的RNA轉(zhuǎn)錄后水平的調(diào)節(jié)對(duì)發(fā)育,代謝,疾病腫瘤發(fā)生過(guò)程中起著至關(guān)重要的作用。MiR-18a的miR-17-92家族成員之一,在多種組織中高表達(dá),對(duì)細(xì)胞的增殖、分化等生理過(guò)程均起重要的調(diào)控作用。研究通過(guò)實(shí)時(shí)定量RT-PCR分析了miR-18a和Ptfla在小鼠胰腺發(fā)育過(guò)程和各類胰腺細(xì)胞中的表達(dá)模式,確定miR-18a對(duì)Ptfla的靶向調(diào)控作用,并在胰腺祖細(xì)胞和腺泡細(xì)胞中過(guò)表達(dá)miR-18a探索其對(duì)胰腺的調(diào)控作用。 結(jié)果顯示：MiR-18a在小鼠胰腺e12.5表達(dá)最高,e18.5表達(dá)最低,而Ptf1a在此過(guò)程中的表達(dá)模式則相反；在小鼠或者大鼠來(lái)源的胰腺細(xì)胞系中,MiR-18a在祖細(xì)胞中表達(dá)最高,在MIN6中表達(dá)最低,而Ptf1a在AR42J細(xì)胞中表達(dá)最高,在MIN6中不表達(dá)；在3T3細(xì)胞系中MiR-18a對(duì)Ptfla mRNA和蛋白都有下調(diào)作用,雙熒光素酶報(bào)告系統(tǒng)也從正反向驗(yàn)證了這種靶向作用；在胰腺祖細(xì)胞中MiR-18a對(duì)內(nèi)源和外源過(guò)表達(dá)的Ptfla的抑制作用都很微弱,并對(duì)祖細(xì)胞的形態(tài)數(shù)量,基因表達(dá)潛影響均不大；在腺泡AR42J細(xì)胞中過(guò)表達(dá)MiR-18a能下調(diào)Ptf1a的蛋白表達(dá),并進(jìn)而抑制一些外分泌酶類基因的轉(zhuǎn)錄。 綜上所示,在胰腺祖細(xì)胞和腺泡細(xì)胞中Ptf1a均受MiR-18a的調(diào)控,并且能影響下游相關(guān)基因的表達(dá)變化,但MiR-18a主要以一種微調(diào)節(jié)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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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