發(fā)布時(shí)間：2018-05-11 15:43

  本文選題：小鼠 + miR-130a　； 參考：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：MicroRNAs(miRNA)作為脂肪內(nèi)穩(wěn)態(tài)的重要調(diào)節(jié)因子,在脂肪生成中發(fā)揮著重要的作用。體外研究表明miR-130a可通過直接靶向PPARγ調(diào)控脂肪代謝作用,但其在活體水平上的對脂肪代謝的影響尚未報(bào)道。本研究采用Crisper/cas9技術(shù)制備miR-130a敲除小鼠,探討了miR-130a缺失后動(dòng)物對高能量的利用和代謝的分子機(jī)制。試驗(yàn)分為4組,分別用常規(guī)飼料(5 kcal%脂質(zhì))和高脂飼料(60 kcal%脂質(zhì))飼喂野生型小鼠和mi R-130a敲除小鼠,飼喂期間對小鼠進(jìn)行葡萄糖耐受試驗(yàn)和胰島素耐受試驗(yàn)。飼喂9-14周后,對小鼠的脂肪組織和肝臟組織進(jìn)行形態(tài)學(xué)分析和分子檢測,試驗(yàn)結(jié)果如下:1.測序結(jié)果130KO小鼠的miR-130a基因被成功敲除,qRT-PCR結(jié)果表明130KO小鼠的miR-130a表達(dá)量顯著下降(P0.05)。2.普通飼料飼養(yǎng)條件下,野生型(WT)小鼠和miR-130a敲除小鼠(130KO)的體重并無差異,高脂飼料飼養(yǎng)條件下130KO小鼠體重顯著高于WT小鼠(P0.05);解剖小鼠發(fā)現(xiàn),130KO小鼠的皮下脂肪和肝臟都顯著重于WT小鼠(P0.05);石蠟切片結(jié)果顯示130KO小鼠的脂肪細(xì)胞較大,油紅O染色顯示130KO小鼠肝臟中的甘油三酯相比WT小鼠顯著增多。3.熒光定量PCR結(jié)果顯示,無論是普通飼料還是高脂飼料飼養(yǎng)下,130KO小鼠的三種脂肪生成標(biāo)記基因PPARγ、CEBP/α和LPL的表達(dá)量均有顯著地增加(P0.05),高脂飼養(yǎng)下130KO小鼠皮下脂肪中IL6地表達(dá)量顯著高于WT小鼠。Western blot結(jié)果顯示,在高脂誘導(dǎo)下130KO小鼠的PPARγ的表達(dá)量增加(P0.01)。4.葡萄糖和胰島素耐受試驗(yàn)結(jié)果顯示,在常規(guī)飼料和高脂飼料的兩種飼喂條件下,130KO小鼠相比于WT小鼠均表現(xiàn)出葡萄糖代謝能力下降(P0.05)。而在高脂誘導(dǎo)下,130KO小鼠出現(xiàn)明顯的胰島素拮抗現(xiàn)象(P0.05)。綜上所述,本研究成功建立了miR-130a基因缺失小鼠模型,發(fā)現(xiàn)缺失miR-130a小鼠更容易出現(xiàn)肥胖,同時(shí)miR-130a基因敲除小鼠葡萄糖清除能力降低并且出現(xiàn)胰島素拮抗。
[Abstract]:As an important regulator of adipose homeostasis, MicroRN AsmiRNAs play an important role in adipogenesis. In vitro studies have shown that miR-130a can regulate lipid metabolism through direct targeting of PPAR 緯, but its effect on adipose metabolism at in vivo level has not been reported. In this study, miR-130a knockout mice were prepared by Crisper/cas9 technique, and the molecular mechanism of high energy utilization and metabolism in miR-130a deficient animals was investigated. The mice were fed with 5 kcal% lipids and 60 kcal% lipids, respectively. The mice were fed with wild type mice and MI R-130a knockout mice. During the feeding period, glucose tolerance test and insulin tolerance test were carried out in mice. After 9-14 weeks of feeding, the adipose tissue and liver tissue of mice were studied by morphological analysis and molecular detection. The results are as follows: 1. The results of sequencing showed that the miR-130a gene of 130KO mice was successfully knocked out by qRT-PCR. The results showed that the expression of miR-130a in 130KO mice decreased significantly (P0.05. 2). There was no difference in body weight between wild type WTB mice and miR-130a knockout mice. The body weight of 130KO mice was significantly higher than that of WT mice under high fat diet, and the subcutaneous fat and liver of the mice were obviously focused on the P0.05 of WT mice. The results of paraffin sections showed that the adipocytes of 130KO mice were larger than those of WT mice, and the results of paraffin sections showed that the body weight of 130KO mice was higher than that of WT mice. Oil red O staining showed that triglycerides in the liver of 130KO mice were significantly higher than those of WT mice. Fluorescence quantitative PCR showed that, The expression of PPAR 緯 CEBP / 偽 and LPL in normal diet and high fat diet mice were significantly increased. The expression of IL6 in subcutaneous fat of 130KO mice was significantly higher than that of WT mice. Western blot showed that the expression of IL6 in the subcutaneous fat of 130KO mice was significantly higher than that of WT mice. The expression of PPAR 緯 increased in 130KO mice induced by hyperlipidemia. The results of glucose and insulin tolerance tests showed that compared with WT mice, 130KO mice showed decreased glucose metabolism ability under two kinds of feeding conditions: normal diet and high fat diet. However, the insulin antagonism was observed in 130KO mice induced by hyperlipidemia (P0.05). In conclusion, we successfully established the miR-130a gene deletion mice model, found that miR-130a mice were more prone to obesity, while the miR-130a gene knockout mice reduced glucose clearance and insulin antagonism.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.23

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 Qian Xu;Ying Li;Yong-Fang Shang;Hui-Ling Wang;Min-Xiu Yao;;miRNA-103: Molecular link between insulin resistance and nonalcoholic fatty liver disease[J];World Journal of Gastroenterology;2015年02期

2 Xiao-Chen Wang;Xiao-Rong Zhan;Xin-Yu Li;Jun-Jie Yu;Xiao-Min Liu;;Micro RNA-185 regulates expression of lipid metabolism genes and improves insulin sensitivity in mice with non-alcoholic fatty liver disease[J];World Journal of Gastroenterology;2014年47期

3 方銳;暢飛;孫照霖;李寧;孟慶勇;;CRISPR/Cas9介導(dǎo)的基因組定點(diǎn)編輯技術(shù)[J];生物化學(xué)與生物物理進(jìn)展;2013年08期

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本文編號：1874565