端粒酶活性变化在动脉粥样硬化斑块发生发展过程中的作用机制

发布时间：2018-08-12 09:24

【摘要】：研究背景及目的血管钙化,尤其是动脉粥样硬化钙化是心脑血管疾病的重要因素之一。血管钙化是一个由多种细胞启动并与骨发育类似的、主动的、高度可调控的、可预防和可逆转的生物学过程。目前钙化形成理论有骨形成蛋白调节、脂质代谢紊乱、凋亡体基质囊泡和氧化应激等多种机制,但相关的分子机制尚不完全清楚。血管钙化主要是中层血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的钙化。血管平滑肌细胞表达少量端粒酶(telomerase),有研究表明动脉粥样硬化斑块VSMCs的端粒(telomere)长度明显短于正常血管VSMCs,端粒酶活性也明显低,但端粒酶活性变化的调控机制及其在血管钙化中的作用并不清楚。研究表明,NF-κB炎症信号通路促进骨形成蛋白(bone morphogenetic protein 2,BMP2)和成骨相关转录因子(runt-related transcription factor 2,RUNX2)的表达,促进VSMCs 化结节形成。研究发现核不均一核糖蛋白A1(heterogeneous nuclear ribonucleoproteinA1,hnRNPA1)可通过结合IκBα介导其发生蛋白酶体水解,促进NF-κB入核,激活炎症信号通路。hnRNPA1是hnRNPs家族中含量最为丰富的一类蛋白,通过调控mRNA合成过程如mRNA转录、剪接、稳定性以及mRNA从胞核到胞浆转运过程进而调控靶基因的表达。我们的前期工作发现,在血管平滑肌细胞由收缩型向合成型转变的过程中,人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)和hnRNPA1的表达均显著上调。有文献报道hnRNPA1可以延长细胞端粒长度,防止端粒过度损耗引发的细胞早衰。因此,本研究拟阐明hnRNPA1是否可以调控血管平滑肌细胞端粒酶活性和IκBα/NF-κB信号通路从而影响血管平滑肌细胞的钙化过程。研究方法1.分离培养原代人脐动脉血管平滑肌细胞(human umbilical artery smooth muscle cells,HUASMCs),构建 pcDNA3.1-hnRNP A1真核表达载体并转染 HUASMCs,通过端粒重复扩增法(telomeric repeat amplification protocol,TRAP)、细胞增殖实验(MTS法)与流式细胞仪分别检测hnRNP A1对HUASMCs端粒酶活性、增殖和凋亡功能的影响。2.建立HUASMCs钙化模型,通过实时荧光定量PCR和Western blot法检测NF-κB信号通路调控HUASMCs钙化相关的各基因表达,阐明hnRNP A1在血管平滑肌钙化过程中的作用机制。研究结果1.体外培养HUASMCs结果表明,在HUASMCs由收缩型转变为合成型的分化过程中,端粒酶和hnRNP A1的表达水平均显著上调。过表达hnRNP A1显著上调端粒酶活性2.7倍,但对HUASMCs细胞的增殖和凋亡功能无显著影响。2.通过建立HUASMCs钙化模型结果表明,hnRNPA1可促进HUASMCs细胞形成钙化结节,但其促进钙化的分子机制不是通过IκBα/NF-κB信号通路介导,具体作用机制需要进一步阐明。结论本研究发现端粒酶和hnRNP A1在血管平滑肌细胞由收缩型向合成型转化的过程中表达上调,过表达hnRNP A1显著上调端粒酶活性,促进动脉粥样硬化过程中的血管平滑肌细胞钙化,但具体的分子机制有待于进一步阐明。研究背景及目的动脉粥样硬化是衰老相关的炎性疾病。单核/巨噬细胞激活是动脉粥样硬化发生发展的关键核心,斑块内的巨噬细胞活动可以引起活跃的炎症反应、纤维帽平滑肌细胞钙化和脂质堆积等,最终导致斑块破裂,诱发猝死、急性心肌梗死、不稳定型心绞痛和缺血性脑卒中。研究表明,在动脉粥样硬化形成的过程中端粒酶活性显著上调,然而有关端粒酶激活的分子机制尚不清楚。我们在前期工作中发现人颈动脉粥样硬化斑块中巨噬细胞富集区域的人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)表达显著上调,炎症反应活跃。此外,我们还发现在体外诱导单核细胞巨噬细胞分化的过程中,hTERT表达上调的同时,一个特殊的microRNAs(miRNAs)分子,miR-216a的表达也显著上调。已有研究提示,miR-216a在衰老内细胞中表达上调,抑制内皮细胞自噬功能,参与巨噬细胞脂质代谢,可能影响动脉粥样硬化性心血管疾病的发生发展。我们进一步的实验研究结果提示miR-216a介导单核/巨噬细胞端粒酶激活,促进巨噬细胞激活和炎症反应。本研究旨在探索miR-216a调控巨噬细胞端粒酶激活的分子机制,阐明其对巨噬细胞分化和功能的影响,为动脉粥样硬化性心血管疾病的治疗提供新的理论依据。研究方法1.收集颈动脉内膜剥脱术后患者的颈动脉斑块组织,进行免疫组化与免疫荧光染色,研究斑块中单核/巨噬细胞与hTERT的共定位情况;2.体外佛波酯(phorbol-12-myristate-13-acetate,PMA)诱导 THP1 单核细胞向巨噬细胞分化,探寻miR-216a与hTERT的表达变化,同时过表达及抑制miR-216a,研究miR-216a调控单核/巨噬细胞端粒酶激活的分子机制;3.采用ApoE-/-雄性小鼠进行右颈动脉串联结扎术,高脂饮食,构建小鼠动脉粥样硬化模型,研究miR-216a是否可以通过调控单核/巨噬细胞激活来影响动脉粥样硬化斑块的发展进程。研究结果1.人颈动脉粥样硬化斑块组织标本的免疫组化和免疫荧光染色。结果显示与正常血管比较,hTERT在斑块内巨噬细胞富集区域特异性表达,端粒酶活性显著升高。2.诱导THP-1细胞向巨噬细胞转化发现miR-216a与hTERT的表达均明显升高,进一步研究表明miR-216a通过SMAD3/NF-κB信号通路调控单核/巨噬细胞端粒酶激活。3.小鼠动脉粥样硬化模型结果显示,miR-216a促进小鼠颈动脉单核/巨噬细胞向M1型转化,抑制其向M2型转化,同时miR-216a还造成小鼠斑块中Ⅲ型胶原减少,这与斑块的稳定性相关。结论本研究发现miR-216a可以通过SMAD3/NF-κB信号通路调控单核/巨噬细胞端粒酶激活,促进巨噬细胞向M1型转化及炎症因子分泌,进而促进动脉粥样硬化斑块发展进程。
[Abstract]:BACKGROUND AND OBJECTIVE Vascular calcification, especially atherosclerotic calcification, is an important factor in cardiovascular and cerebrovascular diseases. Vascular calcification is an active, highly controllable, preventable and reversible biological process initiated by a variety of cells and similar to bone development. Vascular calcification is mainly the calcification of vascular smooth muscle cells (VSMCs). Vascular smooth muscle cells (VSMCs) express a small amount of telomerase. Studies have shown that atherosclerotic plaques. The telomere length of VSMCs was significantly shorter than that of normal VSMCs, and telomerase activity was also significantly lower, but the regulatory mechanism of telomerase activity and its role in vascular calcification were not clear. The expression of related transcription factor 2 (RUNX2) promotes the formation of VSMCs nodules. It is found that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) can mediate the proteasome hydrolysis by binding to I kappa B alpha, promote the entry of NF-kappa B into the nucleus and activate the inflammatory signaling pathway. hnRNPA1 is the most abundant member of the hnRNPs family. A class of proteins that regulate the expression of target genes by regulating mRNA synthesis processes such as transcription, splicing, stability, and mRNA transport from nucleus to cytoplasm. Transriptase, hTERT) and hnRNPA1 expression were significantly up-regulated. It has been reported that hnRNPA1 can prolong telomere length and prevent premature senescence induced by telomere depletion. Therefore, this study is to clarify whether hnRNPA1 can regulate telomerase activity and I-kappa Balpha/NF-kappa B signaling pathway in vascular smooth muscle cells, thereby affecting vascular smooth muscle fineness. Methods 1. Human umbilical artery smooth muscle cells (HUASMCs) were isolated and cultured, and pcDNA3.1-hnRNP A1 eukaryotic expression vector was constructed and transfected into HUASMCs. The effect of hnRNP A1 on telomerase activity, proliferation and apoptosis of HUASMCs was detected by flow cytometry. 2. Calcification model of HUASMCs was established. The expression of genes related to calcification of HUASMCs was detected by real-time fluorescence quantitative PCR and Western blot. The mechanism of hnRNP A1 in the process of vascular smooth muscle calcification was elucidated. Results 1. HUASMCs cultured in vitro showed that the expression of telomerase and hnRNP A1 were significantly up-regulated during the process of HUASMCs differentiation from contractile to synthetic. Overexpression of hnRNP A1 significantly increased telomerase activity by 2.7 times, but had no significant effect on the proliferation and apoptosis of HUASMCs. 2. Calcification model of HUASMCs was established by overexpression of hnRNP A1. The results showed that hnRNPA1 could promote the formation of calcified nodules in HUASMCs, but the molecular mechanism of hnRNPA1 was not mediated by I-kappa Balpha/NF-kappa B signaling pathway, and the specific mechanism needed to be further clarified. Overexpression of hnRNP A1 significantly up-regulates telomerase activity and promotes calcification of vascular smooth muscle cells during atherosclerosis, but the specific molecular mechanisms need to be further clarified. Macrophage activity in the heart and plaque can cause active inflammation, calcification of fibrous cap smooth muscle cells and lipid accumulation, leading to plaque rupture, sudden death, acute myocardial infarction, unstable angina and ischemic stroke. However, the molecular mechanism of telomerase activation remains unclear. In our previous work, we found that the expression of human telomerase reverse transcriptase (hTERT) in the macrophage-rich region of human carotid atherosclerotic plaques was significantly up-regulated and the inflammation was active. In addition, we also found that monocytes were induced in vitro. In the process of macrophage differentiation, the expression of hTERT is up-regulated, and a special microRNAs (microRNAs) molecule, microRNAs-216a, is up-regulated. It has been suggested that microRNAs-216a may be up-regulated in aging cells, inhibit endothelial autophagy, participate in lipid metabolism of macrophages, and may affect atherosclerotic cardiovascular disease. Our further experimental results suggest that microRNA216a mediates telomerase activation in monocytes and macrophages, promotes macrophage activation and inflammation. The purpose of this study is to explore the molecular mechanism of microRNA216a regulating telomerase activation in macrophages, and to elucidate its effect on macrophage differentiation and function, which is called atherosclerosis. Methods 1. Carotid plaque tissues from patients after carotid endarterectomy were collected for immunohistochemistry and immunofluorescence staining to study the co-localization of monocyte/macrophage and hTERT in carotid plaque. 2. In vitro phorbol-12-myristate-13-acetate (PMA) induced THP1. Monocytes differentiate into macrophages, explore the changes of expression of microRNAs-216a and hTERT, overexpress and inhibit microRNAs-216a, and study the molecular mechanism of microRNAs regulating telomerase activation in monocytes/macrophages; 3. ApoE-/-male mice were used for right carotid artery ligation, high-fat diet, construction of atherosclerosis model, study of microRNAs-21. Results 1. Immunohistochemistry and immunofluorescence staining of human carotid atherosclerotic plaque tissue specimens. The results showed that hTERT was specifically expressed in the macrophage-rich region and telomerase activity in the plaque compared with normal blood vessels. The expression of microRNA-216a and hTERT was significantly increased in THP-1 cells induced to macrophage. Further studies showed that microRNA-216a regulated the telomerase activity of monocytes/macrophages through SMAD3/NF-kappa B signaling pathway. 3. Mice atherosclerosis model showed that microRNA-216a promoted carotid monocytes/macrophages to M1. Conclusion MicroRNA216a can regulate the telomerase activity of monocytes and macrophages through SMAD3/NF-kappa B signaling pathway, promote the transformation of macrophages to M1 and the secretion of inflammatory cytokines, thereby promoting the activation of monocytes and macrophages. The development of atherosclerotic plaque.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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