miR-130a敲除小鼠模型的建立及其对脂肪发育的影响

发布时间：2018-05-11 15:43

本文选题：小鼠 + miR-130a　；参考：《华南农业大学》2016年硕士论文

【摘要】：MicroRNAs(miRNA)作为脂肪内稳态的重要调节因子,在脂肪生成中发挥着重要的作用。体外研究表明miR-130a可通过直接靶向PPARγ调控脂肪代谢作用,但其在活体水平上的对脂肪代谢的影响尚未报道。本研究采用Crisper/cas9技术制备miR-130a敲除小鼠,探讨了miR-130a缺失后动物对高能量的利用和代谢的分子机制。试验分为4组,分别用常规饲料(5 kcal%脂质)和高脂饲料(60 kcal%脂质)饲喂野生型小鼠和mi R-130a敲除小鼠,饲喂期间对小鼠进行葡萄糖耐受试验和胰岛素耐受试验。饲喂9-14周后,对小鼠的脂肪组织和肝脏组织进行形态学分析和分子检测,试验结果如下:1.测序结果130KO小鼠的miR-130a基因被成功敲除,qRT-PCR结果表明130KO小鼠的miR-130a表达量显著下降(P0.05)。2.普通饲料饲养条件下,野生型(WT)小鼠和miR-130a敲除小鼠(130KO)的体重并无差异,高脂饲料饲养条件下130KO小鼠体重显著高于WT小鼠(P0.05);解剖小鼠发现,130KO小鼠的皮下脂肪和肝脏都显著重于WT小鼠(P0.05);石蜡切片结果显示130KO小鼠的脂肪细胞较大,油红O染色显示130KO小鼠肝脏中的甘油三酯相比WT小鼠显著增多。3.荧光定量PCR结果显示,无论是普通饲料还是高脂饲料饲养下,130KO小鼠的三种脂肪生成标记基因PPARγ、CEBP/α和LPL的表达量均有显著地增加(P0.05),高脂饲养下130KO小鼠皮下脂肪中IL6地表达量显著高于WT小鼠。Western blot结果显示,在高脂诱导下130KO小鼠的PPARγ的表达量增加(P0.01)。4.葡萄糖和胰岛素耐受试验结果显示,在常规饲料和高脂饲料的两种饲喂条件下,130KO小鼠相比于WT小鼠均表现出葡萄糖代谢能力下降(P0.05)。而在高脂诱导下,130KO小鼠出现明显的胰岛素拮抗现象(P0.05)。综上所述,本研究成功建立了miR-130a基因缺失小鼠模型,发现缺失miR-130a小鼠更容易出现肥胖,同时miR-130a基因敲除小鼠葡萄糖清除能力降低并且出现胰岛素拮抗。
[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 纬, 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 纬 CEBP / 伪 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 纬 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.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.23

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