UVRAG基因在自噬及心脏结构功能维持中的作用

发布时间：2018-12-14 22:15

【摘要】：UVRAG(ultraviolet radiation resistance-associated gene)最初发现于着色性干皮病细胞中,它能部分地恢复该细胞对紫外线的耐受。后来UVRAG被鉴定为一个肿瘤抑制基因,并且有多种生物学功能,例如细胞自噬,细胞Qg吞和DNA修复等。近来,大量的研究增加了对UVRAG的结构和多种生物学功能的理解,但是其生理学功能依然所知甚少。利用piggyBac(PB)转座子插入产生的UVRAG基因敲除小鼠模型,我们研究UVRAG在心脏细胞自噬和维持心脏形态和功能中的作用。PB转座子插入位点位于UVRAG基因的第十四个内含子,RT-PCR实验结果显示,PB转座子的插入干扰小鼠心脏组织UVRAG mRNA的表达。免疫印迹实验结果显示,敲除小鼠心脏、肝脏、脾脏、肺、骨骼肌和脑等组织中无UVRAG蛋白表达。但UVRAG缺失(UVRAGPB/PB)小鼠依旧可存活,有繁殖能力,发育正常。免疫印迹结果显示UVRAG缺失小鼠心脏中LC3 II蛋白显著提高,提示其自噬体数量增加。LC3免疫荧光染色结果显示UVRAG缺失心脏细胞中LC3阳性荧光点状结构数量显著增加。透射电镜结果显示,uvrag缺失心脏中自噬体数量显著增加。自噬体合成增加和自噬清除受损均可导致自噬体数量增加。免疫印迹显示uvrag缺失心脏中p62蛋白含量增加,提示自噬流受损。与野生型相比,uvrag缺失小鼠心脏lc3ii蛋白水平增加,氯喹(chloroquine)处理小鼠阻断自噬体溶酶体融合后,野生型小鼠心脏lc3ii显著提高,但是uvrag缺失心脏lc3ii与处理前相比没有显著差异。表明uvrag缺失心脏中自噬流受损,自噬体清除受阻导致自噬体累积。westernblot结果显示uvrag缺失心脏溶酶体分子标记物lamp-1和lamp-2都显著增加。在小鼠胚胎成纤维细胞(mefs)中,uvrag的缺失也导致自噬流受损,自噬体数量增加。在心脏形态和功能方面,两月龄和六月龄uvrag缺失小鼠心脏拥有与野生型小鼠一样的形态和功能。但十月龄uvrag缺失小鼠心脏体积显著大于野生型对照组。心脏组织横截面切片染色显示,uvrag缺失小鼠心脏横截面变大,左心室室腔变大,心室壁变薄。he染色结果揭示,与野生型相比uvrag缺失小鼠单位视野内心肌细胞数量较少,单个心肌细胞横截面显著增加。表明十月龄时uvrag缺失小鼠出现心肌肥厚。天狼猩红染色结果显示,十月龄uvrag缺失小鼠心脏出现间质胶原累积。实时定量pcr结果显示,十月龄uvrag缺失心脏心室重构分子标记物表达量显著升高,例如心钠素(anf)和脑钠肽(bnp)。免疫印迹实验结果显示,十月龄uvrag缺失小鼠心脏组织bax蛋白水平高于野生型对照组。tunel(terminal-deoxynucleoitidyltransferasemediatednickendlabeling)检测小鼠心脏切片,结果显示与野生型相比十月龄uvrag缺失小鼠心脏细胞凋亡数量显著增加。实时定量pcr结果显示,十月龄uvrag缺失心脏促炎性细胞因子表达量显著增加,例如白细胞介素-6(interleukin-6,il-6)和肿瘤坏死因子-α(tumournecrosisfactor-α,tnfα)。免疫组织化学染色检测结果显示uvrag缺失心脏cd45和cd45r阳性信号显著增加,表明心脏组织中有炎性细胞浸润。实时荧光定量pcr显示,十月龄uvrag缺失小鼠心脏树突细胞分子标记物cd11c表达量显著高于野生型,单核细胞趋化因子mcp-1显著增加,而巨噬细胞前体细胞分子标记物f4/80,m1型巨噬细胞分子标记物cd68,m2型巨噬细胞cd163等均与野生型无显著差异。脂多糖(lps)处理实验小鼠,诱导产生脓血症模型,促炎性因子显著提高,但uvrag缺失小鼠与野生型小鼠相比无显著差异,表明在小鼠心脏中,uvrag缺失与lps诱导的促炎性因子的表达无直接关系。超声心动图结果显示,十月龄uvrag缺失小鼠心脏左心室舒张末期内径(lvedd)和左心室收缩末期内径(lvesd)显著增加,左心室后壁收缩后期厚度(lvpws)降低,射血分数(ef%)和左心室短轴缩短率(%fs)减小,表明其心脏功能受损。这些证据表明uvrag缺失小鼠出现年龄相关肥厚性心肌病,并伴随心脏功能下降。总之,本研究结果表明,UVRAG在心脏自噬和维持小鼠心脏结构和功能上起重要作用。
[Abstract]:UVRAG, originally found in pigmented dry skin cells, can partially restore the cell's tolerance to ultraviolet light. UVRAG is then identified as a tumor suppressor gene and has a variety of biological functions, such as cell autophagy, cell Qg endocytosis, and DNA repair. Recently, a large number of studies have increased understanding of the structure and various biological functions of the UVRAG, but its physiological function is still known to be less. The effects of UVRAG in the autophagy and the maintenance of the heart morphology and function of the heart cells were investigated by using the pigyBac (PB) transposon to insert the resulting UVRAG knockout mouse model. The insertion site of the PB transposon is located at the fourteenth intron of the UVRAG gene, and the RT-PCR results show that the insertion of the PB transposon interferes with the expression of the UVRAG mRNA in the heart tissue of the mouse. The results of immunoblotting showed that no UVRAG protein was expressed in the heart, liver, spleen, lung, skeletal muscle and brain of the knockout mice. However, the UVRAG-deficient (UVRAGPB/ PB) mice were still alive, with reproductive capacity and normal development. The immunoblotting showed that the LC3 II protein in the heart of the UVRAG-deficient mice was significantly increased, suggesting an increase in the number of autophagy bodies. The LC3 immunofluorescent staining showed a significant increase in the number of LC3-positive fluorescent dot structures in the UVRAG-deficient cardiac cells. The transmission electron microscopy (TEM) showed that the number of autophagy in uvag was significantly increased in the heart. The increase in autophagy synthesis and the removal of autophagy can result in an increase in the number of autophagy bodies. Immunoblotting showed an increase in the content of p62 protein in the uvag-deleted heart, suggesting that autophagy was impaired. The wild-type mouse heart lc3ii was significantly increased compared to the wild-type mouse heart lc3ii protein, and the wild-type mouse heart lc3ii was significantly increased after the mice blocked the autophagy-lysosome fusion, but the uvag-deleted heart lc3ii did not differ significantly compared to the pre-treatment. It was shown that uvag was damaged from the autophagy in the heart of the missing heart and the autophagy removal was hindered by the accumulation of autophagy. Western blot showed that uvag deletion of the cardiac lysosomal molecular markers lamp-1 and lamp-2 increased significantly. In mouse embryonic fibroblasts (mfs), the deletion of uvag also resulted in a loss of autophagy and an increase in the number of autophagy bodies. In terms of heart morphology and function, the two-month-old and 6-year-old uvag-deficient mice heart had the same morphology and function as wild-type mice. However, the heart volume of the deficient mice in uvag in October was significantly greater than that of the wild-type control group. The cross-sectional slides of the heart tissue showed that the cross-section of the cardiac tissue of the uvag-deficient mice was large, the chamber of the left ventricle was large, and the ventricular wall was thin. As a result of he staining, the number of myocardial cells in the field of visual field of the uvag-deleted mouse was less than that of the wild-type, and the cross-section of a single cardiac muscle was significantly increased. The presence of cardiac hypertrophy in uvag-deleted mice at the age of October was shown. The results showed that there was an accumulation of interstitial collagen in the heart of uvag-deleted mice in October. The real-time quantitative pcr results show a significant increase in the expression of the cardiac ventricular remodeling molecular markers, such as atrial natriuretic peptide (anf) and brain natriuretic peptide (bnp), in the 10-year-old uvag-deficient cardiac ventricular remodeling. The results of the immunoblotting test show that the level of bax protein in the heart tissue of the mouse of uvag in October is higher than that of the wild-type control group. The results showed that the number of apoptosis in the heart of the mouse was significantly increased in the 10-year-old uvag-deficient mice compared with the wild-type. The real-time quantitative pcr shows that the amount of pro-inflammatory cytokine expression was significantly increased in the october age uvag, for example, interleukin-6 (interleukin-6, il-6), and tumor necrosis factor-1 (tnf). The results of the immunohistochemical staining showed that the ccd45 and cd45r positive signals of uvirag were significantly increased, indicating that there was inflammatory cell infiltration in the cardiac tissue. real-time fluorescence quantitative pcr shows that the amount of cd11c expression of the mouse heart dendritic cell molecular marker cd11c is significantly higher than that of the wild type, the monocyte chemokines mcp-1 are significantly increased in the october age uvag, and the macrophage precursor cell molecular marker f4/ 80, the m1-type macrophage molecular marker cd68, There was no significant difference in type 2 macrophage cd163 and the like with wild type. Lipopolysaccharide (lps) treated the experimental mice to induce a sepsis model, and the pro-inflammatory factor was significantly increased, but the uvag-deficient mice were not significantly different from the wild-type mice, suggesting that the absence of uvag in the heart of the mice was not directly related to the expression of lps-induced inflammatory factors. The results of the echocardiography show that the left ventricular end-diastolic diameter (lvedd) and the left ventricular end-systolic diameter (lvesd) of the heart left ventricular end-diastolic diameter (lvpws) of the left ventricular end wall of the 10-year-old uvag-deficient mice are significantly increased, and the post-systolic thickness (lvpws) of the left ventricle is reduced, The reduction of the injection blood fraction (ef%) and the left ventricular short-axis shortening (% fs) indicates that its heart function is impaired. These evidence indicated age-related hypertrophic cardiomyopathy in uvag-deficient mice and accompanied by a decrease in cardiac function. In conclusion, the results of this study show that the UVRAG plays an important role in the heart autophagy and the maintenance of the heart structure and function of the mouse.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R541

【相似文献】