【作者】 贺毅；

【导师】 曾星；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2010， 博士

【摘要】 背景保护小肠粘膜屏障对防止肠内细菌及内毒素的移位具有非常重要的意义,小肠粘膜屏障完整性的维持有赖于小肠隐窝干细胞的增殖、分化、移行、粘附等共同作用,在病理性损伤下,小肠隐窝干细胞的快速增殖对粘膜损伤的修复起到了重要作用。现代研究发现健脾益气类中药能够通过调节肠内细胞的增殖能力对脾虚状态下的肠粘膜的损伤起到修复作用。甘草具有健脾益气的功效,同时甘草对以粘膜损伤为病理特征的疾病有显著的治疗作用如消化性溃疡病等。但目前有关甘草对小肠上皮细胞保护作用的细胞和分子水平药理研究的报道甚少。国外研究中常采用大鼠小肠隐窝干细胞IEC-6和DFMO诱导的生长抑制的IEC-6病理细胞模型为载体研究小肠上皮细胞增殖、移行、粘附、分化等功能。在健脾中药对胃肠上皮保护的机制研究中,小肠隐窝干细胞被认为是健脾益气中药的主要药理作用靶点之一,并采用IEC-6细胞模型进行了相关的药理研究,基于MTT法的初步研究发现甘草对正常IEC-6细胞有促进增殖的作用,但对其中潜在的分子机制并未研究,对病理状态下肠上皮细胞的药效作用也未涉及。国外通过DFMO诱导的生长抑制的IEC-6模型细胞发现了许多与肠上皮细胞细胞增殖相关的基因,这些基因表达受转录后水平的调控,即RNA结合蛋白HuR与其中的某些基因mRNA非编码区的AU富集元件(AU-rich elements, AREs)的相互作用使这些mRNA的稳定性受到影响,从而最终调节这些基因终产物——蛋白的表达及其生物学效应。预实验研究初步发现甘草及其活性成分之一甘草酸对IEC-6细胞内的HuR有调节的作用,因此本研究将更深入的研究甘草如何通过RNA结合蛋白HuR对肠上皮细胞p21、p53的mRNA进行转录后调节,明确HuR是甘草促进IEC-6细胞增殖作用的分子药靶之一。目的1.研究甘草对正常IEC-6细胞和DFMO模型细胞增殖的调节作用。2.研究与甘草调节IEC-6细胞增殖相关的基因及其表达。3.研究甘草在调控相关基因表达中可能与HuR有关的转录后调节机制。方法1.细胞模型和用药以正常IEC-6细胞作为生理性细胞模型,DFMO模型细胞作为病理性细胞模型。实验用药采用的甘草为甘草颗粒(商品化的甘草水提物),甘草成分采用的是2005版《中国药典》中规定的甘草两个主要指标成分甘草酸和甘草苷。2.研究甘草、甘草酸、甘草苷对IEC-6细胞增殖的调节作用和药物的有效剂量采用基于流式细胞术的细胞计数法、DAPI嵌染核酸的细胞周期检测以及CFSE标记细胞检测细胞分裂速度三种方法研究甘草颗粒对正常IEC-6细胞和DFMO模型细胞的增殖的影响并确定有效的剂量范围。采用MTT法研究甘草酸对两种模型细胞的增殖的调节作用；采用流式细胞术的细胞计数法研究甘草苷对两种模型细胞增殖的影响。3.研究甘草对IEC-6细胞促增殖作用相关的靶基因表达的影响利用荧光定量PCR技术和western blot蛋白免疫印迹技术,分别对甘草调节IEC-6细胞增殖、并且与细胞周期相关的基因的mRNA水平和蛋白水平进行检测。4.研究甘草通过HuR调控相关基因mRNA稳定性的转录后调节机制首先通过放线菌素D抑制转录,并结合PCR技术研究甘草对相关mRNA稳定性的影响,以证明甘草通过转录后调节途径调控基因表达的可能性；其次以western blot技术分别检测HuR在胞浆、胞核的水平,研究mRNA稳定性的改变和RNA结合蛋白HuR的相关性；接着重点研究甘草对相关mRNA和HuR的共同调节是否通过mRNA和HuR的相互作用产生,分别以mRNP免疫沉淀技术研究细胞内源性HuR-mRNP复合物中目标mRNA的含量以明确甘草对HuR和靶mRNA结合的影响、以biotin-pull down技术研究相关mRNA和HuR的相互作用是否是通过HuR对mRNA非编码区AREs识别产生、以基因重组和荧光素酶报告基因检测技术验证甘草是否是通过相关mRNA的AREs片段对最终的基因表达产生影响。结果1.甘草对DFMO诱导的生长抑制的IEC-6细胞模型具有促进增殖的作用甘草对正常的IEC-6细胞并无促进增殖的作用；而对DFMO模型细胞的生长抑制的状态却有逆转的作用,且该作用与胞周期的影响有关；甘草酸和甘草苷虽为甘草的主要指标成分,但本实验中并未发现两者对于这两种细胞模型的增殖有和甘草颗粒一致的影响。2.甘草可调控DFMO模型细胞p21、p53基因的mRNA和蛋白表达DFMO模型细胞生长受抑的同时伴随p2、p53mRNA和蛋白水平的表达相对增加,但在添加甘草后得以一定程度的逆转,并且在一定范围内具有剂量依赖性。提示p2l和p53可能是本实验条件下甘草药理作用的靶基因。3.甘草通过RNA结合蛋白HuR从转录后水平调控p21和p53mRNA的稳定性甘草对DFMO模型细胞p21和p53 mRNA水平的下调可通过降低p21和p53mRNA的稳定性实现,同时DFMO引起的HuR向胞浆的外移可被甘草逆转。利用mRNP免疫沉淀技术,检测到甘草处理DFMO模型细胞后内源性HuR-mRNP复合物中的p21和p53mRNA含量,相对DFMO模型细胞减少,提示甘草减少了HuR与p21mRNA和p53mRNA的结合以及HuR携带mRNA向胞浆的转移,从而降低了p21和p53mRNA在胞浆内的稳定表达。Biotin-pull down技术检测的结果提示p21mRNA和HuR的结合可通过p21mRNA的3’-UTR内的AREs和HuR特异性的识别和结合实现；通过基因重组和荧光素酶报告基因技术验证了甘草能通过p21mRNA的3’-UTR内的AREs片段调控相应基因表达的水平。结论甘草对DFMO诱导的生长抑制的IEC-6细胞具有促进增殖的作用,该作用可以通过HuR介导的调节p21和p53mRNA稳定性的转录后调控机制实现。本课题通过体外实验为甘草在病理状态下对肠上皮细胞的保护作用提供了实验依据,证实了HuR作为甘草的分子药靶之一在转录后水平调控基因表达的作用。更多还原

【Abstract】 BackgroundIntestinal mucoasal barrier is recognized as an important protective defense against the hacterial translocation and endotoxin penetrating from intestinal lumen. The integrity of of intestinal mucosal barrier largely depends on the proliferation, differentiation, migration and adhesion of the stem cells reside in small intestinal crypt. The rapid proliferative characteristic of intestinal crypt cells greatly contributes to the healing of intestinal mucosal injuries in pathological conditions. Licorice belongs to the category of herbs that tonifies the spleen and strengthens the Qi, in which the herbs are reported to possess treatment properties for the intestinal mucosal lesion due to Spleen Qi Deficiency through promoting the growth of intestinal epithelium cells. Moreover, licorice has a potent healing effect on the mucosal wounds in varied diseases especially in peptic ulcer. However, the cellular and molecular mechanism about the protective action of licorice on small intestinal epithelium cells is still elusive. The rat small intestinal epithelial cell line, IEC-6 is a commonly-used physiological cellular model for studying the proliferation, migration, adhesion and differentiation of intestinal epithelia cells. Correspondingly, IEC-6 cells treated with DFMO, an agent inhibits the growth of cells, are used as a pathological model. Based on the researches into the protective mechanism of herbs for invigorating spleen, small intestinal crypt cells are considered as a pharmacological target of spleen-strengthening and qi-invigorating herbal medicine. A MTT assay preliminarilly revealed that licorice extracts have a pro-proliferative effect on normal IEC-6 cells, but either the underlying molecular mechanism or the action of licorice on the cells under pathological conditions still remain to be uncovered. Based upon the researches on DFMO-treated cells, it was revealed that the expression of some genes that related with the growth of intestinal epithelial cells were regulated at post-transcriptional level. The mRNA stability of these genes are influenced by the interaction between their AU-rich elements within 3’-UTR (3’untranslated region) and HuR, a typical RNA binding protein, consequently, the quantiy of mRNA available for translation decides the production of proteins and the biological function they exert. A preliminary research conducted in our group discovered that the cytoplasmic localization of HuR in IEC-6 cells could be detected in the presence of glycyrrhizic acid, one of active principles of licorice. Therefore, we hypothesize that the post-transcriptional regulation might be involved in the proliferation of IEC-6 triggered by licorice, and HuR could be one of the potential molecular targets during the process.ObjectiveIn the present research, we aim at:1. evaluating the impact of licorice on the proliferation of normal IEC-6 cells and DFMO-induced growth inhibitory IEC-6 cells.2. examining the gene expression that correlated with the pro-proliferative action of licorice on IEC-6 cells.3. investigating the role of HuR within the process of licorice regulating gene expression and the possible post-transcriptional regulatory mechanism mediated by HuR.Methods1. Cellular models and drugsNormal IEC-6 cells and DFMO-treated IEC-6 cells were separately used as physiological and pathological cellular models under corresponding conditions. Licorice granules (a commercial product of locorice aqueous extract), glycyrrhizic acid monoammonium salt and liquiritin (two major marker compounds defined by Pharmacopoeia of the People’s Republic of China) were used as the drug to determine the effect of licorice on the proliferation of IEC-6 cells.2. Measuring the pro-proliferation action of licorice granules, glycyrrhizic acid monoammonium salt and liquiritin on IEC-6 cells and determining corresponding effective dosageDirect cell number measurement, cell distribution detection and CFSE-labeled cell division tracking were employed as three major flow cytometry-based techniques in our research to evaluate the proliferative activity of normal IEC-6 cells and DFMO-treated IEC-6 cells after the administration of licorice. MTT assay and flow cytometry-based cell counting were respectively applied to determine the action of glycyrrhizic acid monoammonium salt and liquiritin on the proliferation of IEC-6 cells.3. Analyzing the effect of licorice on the expression of target genes Quantitative real-time PCR (qRT-PCR) and western blot were performed to analysize transcriptional and translational expression of genes that associated with the pro-proliferative effect of licorice.4. Investigating the post-transcriptional mechanism that licorice regulates mRNA stabitlity through HuRFollowing transcriptional repression induced by actinomycin D, the changes of mRNA stability after licorice treatment were assessed by qRT-PCR. Then the cytoplasmic and nuclear HuR levels were detected by western blot analysis to identify whether the changes of mRNA stability were accompanied with the alterning intracellular localization of HuR. Furthermore, it is necessary to verify whether the synchronous changes of mRNA stability and nucleo-cytoplasmic distribution of HuR after licorice treatment were dependent on the interaction between HuR and its target mRNAs. mRNP immunoprecipitation was used to facilitate analysis of the level of target mRNAs in endogenously formed HuR-mRNA complexes from cellular extracts. In addition, biotin pull-down assay was employed to identify whether AREs within untranslated region contributes to the binding of HuR and its mRNAs. The biological effect of ARE containing-fragment from HuR targeted mRNAs was validated throuth gene recombination and luciferase reporter gene assay.Results1. Licorice can stimulate the proliferation of DFMO-treated IEC-6 cells by reversing the growth-inhibitory effect of DFMO.Within the dosage studied in our research, licorice was found to significantly promote the growth of IEC-6 cells that had been treated with DFMO, while it did not cause similar effect on the normal IEC-6 cells. The pro-proliferative action of licorice on IEC-6 cells was accompanied with the changes in cell cycle progression. Although glycyrrhizic acid and liquiritin are two major mark components of licorice, they failed to stimulate the proliferation of IEC-6 cells as licorice.2. The pharmacological action of licorice on DFMO-treated IEC-6 cells was associated with the expression of p21 and p53.The raised levels of p21 and p53 mRNA and protein in DFMO-treated cells were significantly down-regulated by licorice in a dose-dependant manner within certain concentration range. It suggested that p21 and p53 might be target genes of licorice in our experiments.3. Licorice exerted its pharmacological action through HuR-mediated post-transcriptional regulatory mechanism.The levels of p21 and p53 mRNAs in DFMO-treated IEC-6 cells could be down-regulated by licorice via stabilizing p21 and p53 mRNAs, meanwhile, the cytoplasmic translocation of HuR induced by HuR could be reversed by licorice. Based on mRNP immunoprecipitation, the decreasing p21 and p53 mRNAs within endogenously formed HuR-mRNP complexes were detected in the IEC-6 cells treated with DFMO plus licorice, compared with DFMO-treated cells. The result indicated that licorice abolished the binding of endogenous HuR to p21 and p53 mRNA, and hence destabilized these two mRNAs and consequently decreased their expression in cytoplasm. It was further supported by biotin pull down assay that the binding of HuR and p21mRNA was due to the specific affinity of HuR to the AREs resided in 3’-UTR of p21 mRNA. Luciferase reporter gene assay proved that licorice modulated gene expression through HuR-targeted fragment from p21mRNA.ConclusionsTaken together, our conclusion from this work is that licorice post-transcriptioinally down-regulates the levels of p21 and p53 mRNAs via HuR and therefore stimulates the growth of DFMO-treated IEC-6 cells. This study presents an interpretation that licorice exerts its healing effect on intestinal mucosa through the modulationg of cell kinetics. More importantly, HuR was identified as a molecular drug target of licorice at post-transcriptional level.更多还原