柚皮素治疗斑马鱼急性酒精性脂肪肝机制的研究及小窝蛋白基因缺陷斑马鱼突变体的构建
发布时间：2018-09-02 07:47
【摘要】：背景:酒精性肝病初期表现为脂肪肝,可发展成肝炎、肝纤维化和肝硬化,甚至肝功能衰竭、肝癌。研究表明柚皮素可清除自由基,保护细胞中的核酸、蛋白质、脂肪等的正常形态和功能,在降血脂、改善体内脂肪代谢等方面具有较强的生物活性。柚皮素能改善脂肪代谢,但目前还没有相关研究探讨柚皮素对酒精性脂肪肝的作用。斑马鱼幼鱼急性酒精性脂肪肝模型比传统小鼠模型操作简单、耗时少、易于观察、所需实验样品用量少、用药简单,在实验研究中更具有优势。小窝蛋白(Caveolin-1,Cav-1)在细胞内胆固醇运输及脂质代谢中发挥着重要作用,并参与肝脏疾病的发生发展。CRISPR/Cas9系统是目前最常用的基因编辑技术,各种斑马鱼突变体已广泛用于实验研究,但目前还没有小窝蛋白基因缺陷斑马鱼突变体的相关研究。目的:研究柚皮素及cav-1基因调控对斑马鱼急性酒精性脂肪肝模型的作用,构建cav-1基因缺陷斑马鱼突变体。方法:通过显微注射cas9 mRNA和cav-1向导RNA到斑马鱼单细胞胚胎中,使CRISPR/Cas系统对cav-1基因序列进行靶向切割,构建cav-1基因缺陷斑马鱼突变体。把4天的斑马鱼浸泡于2%酒精中32小时构建急性酒精性脂肪肝斑马鱼模型,造模后加入不同浓度(2.5mg/L、5mg/L、10mg/L)的柚皮素干预48小时。通过整体油红O染色及石蜡切片HE染色评估模型及柚皮素的干预效果;通过石蜡切片TUNEL染色观察各组斑马鱼的细胞凋亡情况;通过Real-time Quantitative PCR 和 Western Blot 检测 cav-1 mRNA 和蛋白表达水平,初步探究柚皮素的作用机制。结果:通过CRISPR/Cas系统成功对斑马鱼cav-1基因序列完成定点切割,导致该基因目标靶点出现突变,建立cav-1基因缺陷斑马鱼突变体;突变基因序列具有遗传性。斑马鱼整体油红O染色及石蜡切片HE染色均显示野生型斑马鱼幼鱼经2%酒精处理32小时后肝脏发生了严重的脂肪变性;再经过不同浓度的柚皮素干预48小时后,肝脏脂肪病变有不同程度减轻,且减轻程度与药物浓度相关。石蜡切片TUNEL染色可见模型组细胞凋亡,而对照组及药物组均未见明显的凋亡信号。模型组中cav-1 mRNA和蛋白水平比对照组明显升高,药物组则较模型组明显下降。结论:通过CRISPR/Cas系统成功构建了 cav-1基因缺陷斑马鱼突变体,突变基因序列具有遗传性。成功复制斑马鱼幼鱼急性酒精性脂肪肝模型并通过该模型发现柚皮素对斑马鱼酒精性脂肪肝有显著治疗作用,治疗效果与浓度相关;初步推断其治疗作用可能与cav-1基因调控和减少细胞凋亡有关。
[Abstract]:Background: alcoholic liver disease is initially characterized by fatty liver, which can develop into hepatitis, liver fibrosis and cirrhosis, even liver failure and liver cancer. The results show that naringin can scavenge free radicals, protect the normal morphology and function of nucleic acid, protein and fat in cells, and has strong biological activity in lowering blood lipid and improving lipid metabolism in vivo. Naringin can improve fat metabolism, but there is no study on the effect of naringin on alcoholic fatty liver. Compared with the traditional mouse model, juvenile zebrafish acute alcoholic fatty liver model has the advantages of simple operation, less time consuming and easy observation. Nest protein (Caveolin-1,Cav-1) plays an important role in intracellular cholesterol transport and lipid metabolism, and is involved in the occurrence and development of liver diseases. CRISPRR / Cas9 system is the most commonly used gene editing technique, and a variety of zebrafish mutants have been widely used in experimental research. However, there are no studies on the mutant of zebrafish with gene deficiency of nest protein. Aim: to study the effect of naringin and cav-1 gene regulation on acute alcoholic fatty liver model of zebrafish and construct the mutant of cav-1 gene deficient zebrafish. Methods: by microinjection of cas9 mRNA and cav-1 guided RNA into zebrafish single cell embryos, the CRISPR/Cas system was used to target the cav-1 gene sequence to construct the cav-1 gene deficient mutant of zebrafish. The acute alcoholic fatty liver zebrafish model was established by immersing 4 days zebrafish in 2% alcohol for 32 hours, and then different concentrations (2.5 mg / L, 5 mg / L, 10 mg / L) of naringin were added to the model for 48 hours. The model and the intervention effect of naringin were evaluated by whole oil red O staining and paraffin section HE staining, the apoptosis of zebrafish was observed by paraffin section TUNEL staining, and the expression of cav-1 mRNA and protein were detected by Real-time Quantitative PCR and Western Blot. To explore the mechanism of naringin. Results: the cav-1 gene sequence of zebrafish was successfully dissected by CRISPR/Cas system, which led to the mutation of the target point of the gene and the establishment of cav-1 gene defective mutant of zebrafish, and the mutation gene sequence was hereditary. The results of oil red O staining and HE staining in paraffin sections of zebrafish showed that the liver of juvenile zebrafish treated with 2% alcohol for 32 hours had severe steatosis, and after 48 hours of different concentrations of naringin, the liver of juvenile zebrafish had severe steatosis, and the liver of juvenile zebrafish was treated with different concentrations of naringin for 48 hours. Hepatic fatty lesions were alleviated in varying degrees, and the degree of relief was related to drug concentration. TUNEL staining of paraffin sections showed apoptosis in model group, but no obvious apoptotic signal was found in control group and drug group. The levels of cav-1 mRNA and protein in the model group were significantly higher than those in the control group, while those in the drug group were significantly lower than those in the model group. Conclusion: the mutant of cav-1 gene deficient zebrafish was successfully constructed by CRISPR/Cas system, and the mutant gene sequence was hereditary. The acute alcoholic fatty liver model of juvenile zebrafish was successfully duplicated and it was found that naringin had a significant therapeutic effect on alcoholic fatty liver of zebrafish. It is inferred that the therapeutic effect of cav-1 gene may be related to the regulation of cav-1 gene and the reduction of apoptosis.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
[Abstract]:Background: alcoholic liver disease is initially characterized by fatty liver, which can develop into hepatitis, liver fibrosis and cirrhosis, even liver failure and liver cancer. The results show that naringin can scavenge free radicals, protect the normal morphology and function of nucleic acid, protein and fat in cells, and has strong biological activity in lowering blood lipid and improving lipid metabolism in vivo. Naringin can improve fat metabolism, but there is no study on the effect of naringin on alcoholic fatty liver. Compared with the traditional mouse model, juvenile zebrafish acute alcoholic fatty liver model has the advantages of simple operation, less time consuming and easy observation. Nest protein (Caveolin-1,Cav-1) plays an important role in intracellular cholesterol transport and lipid metabolism, and is involved in the occurrence and development of liver diseases. CRISPRR / Cas9 system is the most commonly used gene editing technique, and a variety of zebrafish mutants have been widely used in experimental research. However, there are no studies on the mutant of zebrafish with gene deficiency of nest protein. Aim: to study the effect of naringin and cav-1 gene regulation on acute alcoholic fatty liver model of zebrafish and construct the mutant of cav-1 gene deficient zebrafish. Methods: by microinjection of cas9 mRNA and cav-1 guided RNA into zebrafish single cell embryos, the CRISPR/Cas system was used to target the cav-1 gene sequence to construct the cav-1 gene deficient mutant of zebrafish. The acute alcoholic fatty liver zebrafish model was established by immersing 4 days zebrafish in 2% alcohol for 32 hours, and then different concentrations (2.5 mg / L, 5 mg / L, 10 mg / L) of naringin were added to the model for 48 hours. The model and the intervention effect of naringin were evaluated by whole oil red O staining and paraffin section HE staining, the apoptosis of zebrafish was observed by paraffin section TUNEL staining, and the expression of cav-1 mRNA and protein were detected by Real-time Quantitative PCR and Western Blot. To explore the mechanism of naringin. Results: the cav-1 gene sequence of zebrafish was successfully dissected by CRISPR/Cas system, which led to the mutation of the target point of the gene and the establishment of cav-1 gene defective mutant of zebrafish, and the mutation gene sequence was hereditary. The results of oil red O staining and HE staining in paraffin sections of zebrafish showed that the liver of juvenile zebrafish treated with 2% alcohol for 32 hours had severe steatosis, and after 48 hours of different concentrations of naringin, the liver of juvenile zebrafish had severe steatosis, and the liver of juvenile zebrafish was treated with different concentrations of naringin for 48 hours. Hepatic fatty lesions were alleviated in varying degrees, and the degree of relief was related to drug concentration. TUNEL staining of paraffin sections showed apoptosis in model group, but no obvious apoptotic signal was found in control group and drug group. The levels of cav-1 mRNA and protein in the model group were significantly higher than those in the control group, while those in the drug group were significantly lower than those in the model group. Conclusion: the mutant of cav-1 gene deficient zebrafish was successfully constructed by CRISPR/Cas system, and the mutant gene sequence was hereditary. The acute alcoholic fatty liver model of juvenile zebrafish was successfully duplicated and it was found that naringin had a significant therapeutic effect on alcoholic fatty liver of zebrafish. It is inferred that the therapeutic effect of cav-1 gene may be related to the regulation of cav-1 gene and the reduction of apoptosis.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
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