柚皮素治療斑馬魚急性酒精性脂肪肝機制的研究及小窩蛋白基因缺陷斑馬魚突變體的構(gòu)建
發(fā)布時間：2018-09-02 07:47
【摘要】：背景:酒精性肝病初期表現(xiàn)為脂肪肝,可發(fā)展成肝炎、肝纖維化和肝硬化,甚至肝功能衰竭、肝癌。研究表明柚皮素可清除自由基,保護細胞中的核酸、蛋白質(zhì)、脂肪等的正常形態(tài)和功能,在降血脂、改善體內(nèi)脂肪代謝等方面具有較強的生物活性。柚皮素能改善脂肪代謝,但目前還沒有相關(guān)研究探討柚皮素對酒精性脂肪肝的作用。斑馬魚幼魚急性酒精性脂肪肝模型比傳統(tǒng)小鼠模型操作簡單、耗時少、易于觀察、所需實驗樣品用量少、用藥簡單,在實驗研究中更具有優(yōu)勢。小窩蛋白(Caveolin-1,Cav-1)在細胞內(nèi)膽固醇運輸及脂質(zhì)代謝中發(fā)揮著重要作用,并參與肝臟疾病的發(fā)生發(fā)展。CRISPR/Cas9系統(tǒng)是目前最常用的基因編輯技術(shù),各種斑馬魚突變體已廣泛用于實驗研究,但目前還沒有小窩蛋白基因缺陷斑馬魚突變體的相關(guān)研究。目的:研究柚皮素及cav-1基因調(diào)控對斑馬魚急性酒精性脂肪肝模型的作用,構(gòu)建cav-1基因缺陷斑馬魚突變體。方法:通過顯微注射cas9 mRNA和cav-1向?qū)NA到斑馬魚單細胞胚胎中,使CRISPR/Cas系統(tǒng)對cav-1基因序列進行靶向切割,構(gòu)建cav-1基因缺陷斑馬魚突變體。把4天的斑馬魚浸泡于2%酒精中32小時構(gòu)建急性酒精性脂肪肝斑馬魚模型,造模后加入不同濃度(2.5mg/L、5mg/L、10mg/L)的柚皮素干預48小時。通過整體油紅O染色及石蠟切片HE染色評估模型及柚皮素的干預效果;通過石蠟切片TUNEL染色觀察各組斑馬魚的細胞凋亡情況;通過Real-time Quantitative PCR 和 Western Blot 檢測 cav-1 mRNA 和蛋白表達水平,初步探究柚皮素的作用機制。結(jié)果:通過CRISPR/Cas系統(tǒng)成功對斑馬魚cav-1基因序列完成定點切割,導致該基因目標靶點出現(xiàn)突變,建立cav-1基因缺陷斑馬魚突變體;突變基因序列具有遺傳性。斑馬魚整體油紅O染色及石蠟切片HE染色均顯示野生型斑馬魚幼魚經(jīng)2%酒精處理32小時后肝臟發(fā)生了嚴重的脂肪變性;再經(jīng)過不同濃度的柚皮素干預48小時后,肝臟脂肪病變有不同程度減輕,且減輕程度與藥物濃度相關(guān)。石蠟切片TUNEL染色可見模型組細胞凋亡,而對照組及藥物組均未見明顯的凋亡信號。模型組中cav-1 mRNA和蛋白水平比對照組明顯升高,藥物組則較模型組明顯下降。結(jié)論:通過CRISPR/Cas系統(tǒng)成功構(gòu)建了 cav-1基因缺陷斑馬魚突變體,突變基因序列具有遺傳性。成功復制斑馬魚幼魚急性酒精性脂肪肝模型并通過該模型發(fā)現(xiàn)柚皮素對斑馬魚酒精性脂肪肝有顯著治療作用,治療效果與濃度相關(guān);初步推斷其治療作用可能與cav-1基因調(diào)控和減少細胞凋亡有關(guān)。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575
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