本文選題：糖尿病心肌病 + 心肌肥大��； 參考：《上海交通大學(xué)》2015年碩士論文

【摘要】：目的:探討Rac1參與調(diào)控糖尿病心肌病變的上游機制,揭示miRNA在糖尿病心肌病發(fā)生發(fā)展中的作用,為糖尿病心肌病發(fā)病機制及臨床治療的研究提供新視角。方法:第一部分利用生物信息學(xué)方法預(yù)測調(diào)控Rac1的候選miRNAs。在糖尿病組db/db小鼠及對照組db/m小鼠的心肌組織中驗證所有候選miRNAs的表達(dá);并將糖尿病組小鼠心肌中差異表達(dá)的miRNAs與Rac1進(jìn)行Pearson相關(guān)性分析,篩選出與Rac1呈最強負(fù)相關(guān)的miRNA。第二部分原代培養(yǎng)乳鼠心肌細(xì)胞,通過高糖處理心肌細(xì)胞,用real-time RT-PCR技術(shù)進(jìn)一步驗證Rac1、所選miRNA的表達(dá)情況。通過轉(zhuǎn)染所選miRNA的模擬物進(jìn)行干預(yù),實驗分為4組:正常糖組(5mM),高糖組(33mM),正常糖+所選miRNA模擬物組,高糖+所選miRNA模擬物組。通過real-time RT-PCR技術(shù)及Western blot技術(shù)分別檢測各組心肌細(xì)胞Rac1、β-MHC、α-SMA的mRNA及蛋白表達(dá)情況。第三部分8周齡雄性C57/BL-6J小鼠隨機分為正常對照組(n=5),miR-182模擬物對照組(n=5),STZ型糖尿病組(n=15),STZ型糖尿病+所選miRNA模擬物治療組(n=15)。向C57/BL-6J小鼠一次性腹腔注射鏈脲佐菌素(STZ)建立STZ型糖尿病動物模型。8周后實驗達(dá)終點時,記錄小鼠的體重及血糖情況;采用小動物用高分辨超聲儀檢測各組小鼠心臟功能;通過HE染色觀察心肌細(xì)胞大小,通過Masson染色觀察心肌纖維化程度;運用透射電鏡觀察小鼠心肌組織的超微結(jié)構(gòu);利用real-time RT-PCR技術(shù)檢測心肌組織β-肌球蛋白重鏈(β-MHC),α-肌球蛋白重鏈(α-MHC),心房鈉尿肽(ANP),I型膠原(Col I)和III型膠原(Col III)mRNA,待測mRNA及Rac1的表達(dá)情況。結(jié)果:第一部分通過生物信息學(xué)方法,預(yù)測到參與調(diào)控Rac1的候選miRNAs共6個:分別是miR-182、miR-142-3p、miR-140、miR-101a、miR-429、miR-200b。與db/m組比較,db/db組糖尿病小鼠心肌組織中miR-142-3p、miR-182的表達(dá)顯著下調(diào),具有統(tǒng)計學(xué)意義(p0.05)。Pearson相關(guān)性分析顯示miR-182與Rac1表達(dá)水平的負(fù)相關(guān)最顯著,r值為-0.89102。第二部分體外實驗顯示高糖誘導(dǎo)乳鼠心肌細(xì)胞肥大時,mir-182表達(dá)明顯降低,而rac1表達(dá)明顯升高,兩者之間亦呈負(fù)相關(guān)。與正常糖組比較,高糖組心肌細(xì)胞肥大明顯。與高糖組相比,高糖+mir-182模擬物組心肌細(xì)胞肥大程度明顯降低。rt-pcr及westernblot結(jié)果均顯示與高糖組相比,高糖+mir-182模擬物組心肌細(xì)胞rac1及β-mhc的表達(dá)均降低,具有統(tǒng)計學(xué)意義(p0.05),但α-sma的表達(dá)降低不具有統(tǒng)計學(xué)意義。第三部分超聲心動圖檢測顯示:與糖尿病組相比,糖尿病+mir-182模擬物組小鼠的左室射血分?jǐn)?shù)及左室短軸縮短率明顯升高,具有統(tǒng)計學(xué)意義(p0.05)。rt-pcr結(jié)果顯示:與糖尿病組比較,糖尿病+mir-182模擬物組小鼠心肌組織中rac1、anp、coli、coliiimrna的表達(dá)及β/α-mhc比值明顯下降,具有統(tǒng)計學(xué)意義(p0.05)。he染色結(jié)果顯示:正常對照組及mir-182模擬物對照組心肌細(xì)胞排列規(guī)則整齊,細(xì)胞核大小均一;而糖尿病組小鼠心肌排列紊亂,心肌肥大明顯,細(xì)胞核增大,形態(tài)不規(guī)則;糖尿病+mir-182組小鼠心肌肥大程度減低,心肌排列較規(guī)則。masson染色后心肌纖維呈現(xiàn)為深紅色而心臟間質(zhì)膠原呈現(xiàn)為深藍(lán)色:正常對照組及mir-182模擬物對照組小鼠心肌間質(zhì)膠原纖維較少且均勻分布;而糖尿病組小鼠心肌組織膠原纖維明顯增多聚集,分布紊亂不均勻;糖尿病+mir-182組小鼠心肌膠原纖維較糖尿病組明顯減少。透射電鏡結(jié)果顯示:正常對照組及mir-182模擬物對照組小鼠心肌纖維排列整齊有序,肌小節(jié)及明帶暗帶清晰可見,線粒體大小均一,呈圓形或橢圓形沿心肌纖維整齊排列;糖尿病組小鼠心肌纖維排列紊亂,肌絲扭曲、斷裂,心肌線粒體明顯異型增大及增多聚集,線粒體可腫脹、嵴消失甚至溶解,可見糖原顆粒及脂滴增多聚集,心臟間質(zhì)大量膠原纖維聚集,可見較多自噬體;而糖尿病+mir-182模擬物組小鼠心肌超微結(jié)構(gòu)的損傷較糖尿病組明顯減輕。結(jié)論:(1)利用生物信息學(xué)方法預(yù)測、通過動物模型體內(nèi)實驗及pearson相關(guān)性分析,最終篩選得出rac1的候選mirna為mir-182;同時體外實驗進(jìn)一步驗證心肌細(xì)胞中mir-182與rac1表達(dá)呈負(fù)相關(guān)。(2)mir-182模擬物降低高糖誘導(dǎo)心肌細(xì)胞中rac1的表達(dá),減輕高糖誘導(dǎo)心肌細(xì)胞的肥大程度,降低心肌細(xì)胞肥大相關(guān)表型的表達(dá)。(3)mir-182可參與糖尿病心肌病的調(diào)控,mir-182模擬物能可減輕糖尿病小鼠心肌組織rac1表達(dá),改善糖尿病小鼠心臟收縮功能,這與其降低糖尿病小鼠心肌肥大及心肌纖維化程度,減輕糖尿病小鼠心肌超微結(jié)構(gòu)損傷有關(guān)。
[Abstract]:Objective: To explore the upstream mechanism of Rac1 in the regulation of diabetic cardiomyopathy, to reveal the role of miRNA in the development of diabetic cardiomyopathy, and to provide a new perspective for the pathogenesis and clinical treatment of diabetic cardiomyopathy. Method: the first part was pretested by Bioinformatics Method to regulate the candidate miRNAs. of Rac1 in the diabetes group db/db small The expression of all the candidate miRNAs in the myocardium of the mice and the control group db/m mice was verified, and the miRNAs and Rac1 in the myocardium of the diabetic mice were analyzed with the Pearson correlation, and the myocardial cells of the primary cultured milk rat were screened out with the strongest negative correlation with Rac1, and the cardiac myocytes were treated with high glucose, and real-time RT was used. -PCR technique further verified the expression of Rac1 and selected miRNA. The experiment was divided into 4 groups: normal sugar group (5mM), high sugar group (33mM), normal sugar +, miRNA mimic group, high sugar + selected miRNA simulation group. The myocardial fines were detected by real-time RT-PCR technique and Western blot technique respectively. The mRNA and protein expression of cell Rac1, beta -MHC, and alpha -SMA. The third part of 8 weeks male C57/BL-6J mice were randomly divided into normal control group (n=5), miR-182 analogue control group (n=5), STZ type diabetes group (n=15), STZ type diabetes + selected miRNA analogue treatment group. The body weight and blood sugar of the mice were recorded at the end of.8 weeks after the experimental diabetic animal model. The cardiac function was detected by the high resolution ultrasonography of small animals, the size of myocardial cells was observed by HE staining, the degree of myocardial fibrosis was observed by Masson staining, and the ultrastructure of the mice was observed by transmission electron microscopy. Real-time RT-PCR technique was used to detect the myocardial tissue beta myosin heavy chain (beta -MHC), alpha myosin heavy chain (alpha -MHC), atrial natriuretic peptide (ANP), I collagen (Col I) and III type collagen (Col III) mRNA. Results: the first part predicts a total of 6 candidates participating in the regulation by bioinformatics. MiR-182, miR-142-3p, miR-140, miR-101a, miR-429, miR-200b. and db/m group compared with the db/m group, the expression of miR-142-3p and miR-182 in the myocardium of the diabetic mice was significantly down, with a statistical significance (P0.05).Pearson correlation analysis showed that the negative correlation was the most significant with the second parts. In the experiment, the expression of mir-182 was obviously decreased and the expression of Rac1 increased obviously. The hypertrophy of myocardial cells in the high glucose group was significantly higher than that in the normal sugar group. Compared with the high glucose group, the degree of myocardial hypertrophy in the high glucose +mir-182 simulated group significantly reduced the results of.Rt-pcr and Westernblot. Compared with the high glucose group, the expression of Rac1 and beta -mhc in the myocardial cells of the high glucose +mir-182 simulated group decreased and had statistical significance (P0.05), but the decrease of the expression of alpha -sma was not statistically significant. The third part echocardiography showed that the left ventricular ejection fraction in the diabetic group was compared with the diabetic group, and the left ventricular ejection fraction of the diabetic mice was compared with the diabetic group. The short axis shortening rate of the left ventricle was significantly increased, with statistical significance (P0.05).Rt-pcr results showed: compared with the diabetic group, the expression of Rac1, ANP, coli, coliiimrna and the ratio of beta / alpha -mhc in the myocardium of the diabetic +mir-182 mimics group decreased significantly, and the statistical meaning (P0.05).He staining results showed that the normal control group and mir-182 simulation were shown. The myocardial cells in the control group were arranged regularly and the nucleus size was uniform, but the myocardium of the diabetic mice was arranged in disorder, the myocardial hypertrophy was obvious, the nucleus enlarged and the shape was irregular. The degree of myocardial hypertrophy in the diabetic +mir-182 group was reduced, the myocardial arrangement was deep red after the regular.Masson staining, and the cardiac collagen was found in the heart. The collagen fibers in the myocardium of the normal control group and the mir-182 mimic control group were less and evenly distributed, while the collagen fibers in the diabetic group were significantly increased, and the distribution disorder was unevenly distributed. The collagen fibers in the diabetic group +mir-182 mice were significantly lower than that in the sugar urine group. Transmission electron microscopy showed that: The myocardial fibers in the control group and the control group of mir-182 were arranged orderly and orderly. The muscle segments and the dark bands were clearly visible. The size of the mitochondria was uniform, and the size of the mitochondria was round or oval in order. The myocardial fibers in the diabetic mice were arranged in disorder, the muscle filament was twisted, and the myocardial mitochondria were obviously abnormal and increased. The mitochondria can be swollen, the crista disappears or even dissolve, the accumulation of glycogen particles and lipid droplets, the aggregation of mass collagen fibers in the cardiac interstitium and more autophagic bodies, and the damage of the ultrastructure of the diabetic +mir-182 mimics mice is obviously less than that in the diabetic group. Conclusion: (1) the bioinformatics method is used to predict and pass the animal model body. The internal experiment and Pearson correlation analysis showed that the candidate miRNA of Rac1 was mir-182, and in vitro experiments further verified that the expression of mir-182 and Rac1 in cardiac myocytes was negatively correlated. (2) mir-182 mimics reduced the expression of Rac1 in cardiac myocytes induced by high glucose, alleviated the hypertrophy of hyperglycemia induced cardiomyocytes and reduced the hypertrophy of cardiomyocytes. The expression of related phenotypes. (3) mir-182 can be involved in the regulation of diabetic cardiomyopathy. Mir-182 mimics can reduce the expression of Rac1 in myocardial tissue of diabetic mice and improve cardiac contractility in diabetic mice, which is related to reducing myocardial hypertrophy and myocardial fibrosis in diabetic mice and reducing myocardial ultrastructural damage in diabetic mice.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

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