【摘要】：UVRAG(ultraviolet radiation resistance-associated gene)最初發(fā)現(xiàn)于著色性干皮病細(xì)胞中,它能部分地恢復(fù)該細(xì)胞對(duì)紫外線的耐受。后來(lái)UVRAG被鑒定為一個(gè)腫瘤抑制基因,并且有多種生物學(xué)功能,例如細(xì)胞自噬,細(xì)胞Qg吞和DNA修復(fù)等。近來(lái),大量的研究增加了對(duì)UVRAG的結(jié)構(gòu)和多種生物學(xué)功能的理解,但是其生理學(xué)功能依然所知甚少。利用piggyBac(PB)轉(zhuǎn)座子插入產(chǎn)生的UVRAG基因敲除小鼠模型,我們研究UVRAG在心臟細(xì)胞自噬和維持心臟形態(tài)和功能中的作用。PB轉(zhuǎn)座子插入位點(diǎn)位于UVRAG基因的第十四個(gè)內(nèi)含子,RT-PCR實(shí)驗(yàn)結(jié)果顯示,PB轉(zhuǎn)座子的插入干擾小鼠心臟組織UVRAG mRNA的表達(dá)。免疫印跡實(shí)驗(yàn)結(jié)果顯示,敲除小鼠心臟、肝臟、脾臟、肺、骨骼肌和腦等組織中無(wú)UVRAG蛋白表達(dá)。但UVRAG缺失(UVRAGPB/PB)小鼠依舊可存活,有繁殖能力,發(fā)育正常。免疫印跡結(jié)果顯示UVRAG缺失小鼠心臟中LC3 II蛋白顯著提高,提示其自噬體數(shù)量增加。LC3免疫熒光染色結(jié)果顯示UVRAG缺失心臟細(xì)胞中LC3陽(yáng)性熒光點(diǎn)狀結(jié)構(gòu)數(shù)量顯著增加。透射電鏡結(jié)果顯示,uvrag缺失心臟中自噬體數(shù)量顯著增加。自噬體合成增加和自噬清除受損均可導(dǎo)致自噬體數(shù)量增加。免疫印跡顯示uvrag缺失心臟中p62蛋白含量增加,提示自噬流受損。與野生型相比,uvrag缺失小鼠心臟lc3ii蛋白水平增加,氯喹(chloroquine)處理小鼠阻斷自噬體溶酶體融合后,野生型小鼠心臟lc3ii顯著提高,但是uvrag缺失心臟lc3ii與處理前相比沒(méi)有顯著差異。表明uvrag缺失心臟中自噬流受損,自噬體清除受阻導(dǎo)致自噬體累積。westernblot結(jié)果顯示uvrag缺失心臟溶酶體分子標(biāo)記物lamp-1和lamp-2都顯著增加。在小鼠胚胎成纖維細(xì)胞(mefs)中,uvrag的缺失也導(dǎo)致自噬流受損,自噬體數(shù)量增加。在心臟形態(tài)和功能方面,兩月齡和六月齡uvrag缺失小鼠心臟擁有與野生型小鼠一樣的形態(tài)和功能。但十月齡uvrag缺失小鼠心臟體積顯著大于野生型對(duì)照組。心臟組織橫截面切片染色顯示,uvrag缺失小鼠心臟橫截面變大,左心室室腔變大,心室壁變薄。he染色結(jié)果揭示,與野生型相比uvrag缺失小鼠單位視野內(nèi)心肌細(xì)胞數(shù)量較少,單個(gè)心肌細(xì)胞橫截面顯著增加。表明十月齡時(shí)uvrag缺失小鼠出現(xiàn)心肌肥厚。天狼猩紅染色結(jié)果顯示,十月齡uvrag缺失小鼠心臟出現(xiàn)間質(zhì)膠原累積。實(shí)時(shí)定量pcr結(jié)果顯示,十月齡uvrag缺失心臟心室重構(gòu)分子標(biāo)記物表達(dá)量顯著升高,例如心鈉素(anf)和腦鈉肽(bnp)。免疫印跡實(shí)驗(yàn)結(jié)果顯示,十月齡uvrag缺失小鼠心臟組織bax蛋白水平高于野生型對(duì)照組。tunel(terminal-deoxynucleoitidyltransferasemediatednickendlabeling)檢測(cè)小鼠心臟切片,結(jié)果顯示與野生型相比十月齡uvrag缺失小鼠心臟細(xì)胞凋亡數(shù)量顯著增加。實(shí)時(shí)定量pcr結(jié)果顯示,十月齡uvrag缺失心臟促炎性細(xì)胞因子表達(dá)量顯著增加,例如白細(xì)胞介素-6(interleukin-6,il-6)和腫瘤壞死因子-α(tumournecrosisfactor-α,tnfα)。免疫組織化學(xué)染色檢測(cè)結(jié)果顯示uvrag缺失心臟cd45和cd45r陽(yáng)性信號(hào)顯著增加,表明心臟組織中有炎性細(xì)胞浸潤(rùn)。實(shí)時(shí)熒光定量pcr顯示,十月齡uvrag缺失小鼠心臟樹突細(xì)胞分子標(biāo)記物cd11c表達(dá)量顯著高于野生型,單核細(xì)胞趨化因子mcp-1顯著增加,而巨噬細(xì)胞前體細(xì)胞分子標(biāo)記物f4/80,m1型巨噬細(xì)胞分子標(biāo)記物cd68,m2型巨噬細(xì)胞cd163等均與野生型無(wú)顯著差異。脂多糖(lps)處理實(shí)驗(yàn)小鼠,誘導(dǎo)產(chǎn)生膿血癥模型,促炎性因子顯著提高,但uvrag缺失小鼠與野生型小鼠相比無(wú)顯著差異,表明在小鼠心臟中,uvrag缺失與lps誘導(dǎo)的促炎性因子的表達(dá)無(wú)直接關(guān)系。超聲心動(dòng)圖結(jié)果顯示,十月齡uvrag缺失小鼠心臟左心室舒張末期內(nèi)徑(lvedd)和左心室收縮末期內(nèi)徑(lvesd)顯著增加,左心室后壁收縮后期厚度(lvpws)降低,射血分?jǐn)?shù)(ef%)和左心室短軸縮短率(%fs)減小,表明其心臟功能受損。這些證據(jù)表明uvrag缺失小鼠出現(xiàn)年齡相關(guān)肥厚性心肌病,并伴隨心臟功能下降�？傊�,本研究結(jié)果表明,UVRAG在心臟自噬和維持小鼠心臟結(jié)構(gòu)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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R541

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