16个氨基酸多肽片段对四氯化碳诱导的小鼠肝纤维化的保护作用及机制研究

发布时间：2018-05-24 12:14

本文选题：16个氨基酸多肽片段 + 四氯化碳　；参考：《河北医科大学》2016年硕士论文

【摘要】：目的:酒精、慢性乙型和丙型肝炎病毒感染、肥胖、自身免疫性肝炎、代谢性疾病、药物毒物和胆汁淤积等均可导致肝纤维化,几乎所有的慢性肝病都与纤维化相关,而且易进展为肝硬化、肝衰竭、肝癌。进展性的肝纤维化尚且可逆转,肝硬化则基本不可逆。四氯化碳(Carbon tetrachloride,CCl_4)诱导的小鼠慢性肝损伤模型已较成熟,可用来模拟人类众多慢性肝脏疾病的共同病理过程—肝纤维化。经CCl_4长期刺激可致小鼠肝细胞坏死、炎症、纤维组织增生,伴随血清天冬氨酸转氨酶(aspartate aminotransferase,AST)和丙氨酸转氨酶(alanine aminotransaminase,ALT)升高,并且有大量炎性细胞浸润,细胞外基质大量胶原沉积。为寻找早期肝炎临床诊断的生物标志物,本室前期运用基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF MS)从慢性肝炎病人血清中检测到一个差异性表达的多肽,即16个氨基酸多肽片段,并通过体外实验证实该多肽有促进肝细胞增殖的作用。本室前期研究成果已证实该多肽针对免疫性肝炎肝损伤及脂肪肝具有保护作用,推测其可能在慢性肝脏疾病中发挥同样的保护作用,故进行本研究。本研究利用四氯化碳诱导的小鼠肝纤维化模型研究16个氨基酸多肽片段在肝纤维化中的治疗作用,并对其机制初步探究,为进一步临床肝病治疗提供实验依据。方法:1 16个氨基酸多肽片段对肝纤维化小鼠的保护作用50只雄性,6-8周龄balb/c小鼠随机分为五组,分别为正常组,模型组,秋水仙碱组,16个氨基酸多肽低剂量组(400μg/kg),高剂量组(800μg/kg),每组10只。除正常组在相应给药时点腹腔注射生理盐水(1ml/kg)外,其余各组均分别通过腹腔注射给予25%CCl_4溶液(1.5μl/g),每3天1次,共10次。同时,在第三次注射CCl_4之后,除正常组、模型组分别在相应给药时点通过尾静脉注射给予生理盐水外,其余各组分别经尾静脉注射给予400、800μg/kg体重多肽,每3天1次,持续7次或经灌胃给予秋水仙碱(200μg/kg),每周五天,持续3周后,眼球取血,收集小鼠血液,分离血清,采用全自动生化分析仪测定血清ALT、AST、碱性磷酸酶(Alkaline phosphatase,ALP)水平;ELISA试剂盒测定血清透明质酸酶(Hyaluronic Acid,HA)、四型胶原(Collagen TypeⅣ,Ⅳ-C)水平。通过血清学指标观察16个氨基酸多肽片段对CCl_4诱导肝纤维化的影响。2肝组织病理学检测眼球取血后处死小鼠,剥离肝脏,分别切取肝脏左叶、右叶相同位置合适大小的两块肝组织(一般厚度不超过0.5厘米)制成组织蜡块,行5μm连续切片后分别进行HE染色、Masson染色,于光学显微镜下观察肝组织病理学形态及胶原沉积情况,并采集图像。3小鼠肝纤维化相关基因表达水平的测定自NCBI中获取小鼠源Ⅰ型胶原(Collagen,typeⅠ,alpha 1,Col1a1)、Ⅲ型胶原(Collagen,typeⅢ,alpha 1,Col3a1)、金属蛋白酶组织抑制剂(Tissue Inhibitor of Metalloproteinase,TIMP-1)、基质金属蛋白酶(Matrix Metalloproteinases,MMP-2)、结缔组织生长因子(Connective Tissue Growth Factor,CTGF)的基因序列,设计并合成引物(上海生工生物工程有限公司)。提取小鼠肝组织总RNA,反转录合成cDNA,分别以10倍稀释的Col1a1、Col3a1、TIMP-1、MMP-2、CTGF、GAPDH cDNA为模板进行实时荧光定量PCR检测。反应结束后确认扩增曲线和融解曲线,记录基因相对表达量RQ值进一步分析。探讨16个氨基酸多肽片段对CCl_4诱导的肝纤维化的相关作用机制。4肝细胞内活性氧的测定采用流式细胞术,对各组肝细胞内活性氧(Reactive oxygen species,ROS)含量进行检测,了解氧化应激水平。结果:1各组小鼠血清肝功能及肝纤维化变化情况16个氨基酸多肽片段低剂量组(36.85±3.36,71.03±7.42)、高剂量组(35.27±4.34,73.03±8.13)、秋水仙碱组(34.65±5.57,69.49±11.76)及正常组(31.05±2.91,62.02±9.46)血清ALT、AST水平均低于模型组(45.07±5.32,85.48±3.44)(P0.05);各组血清ALP水平均无差异(P0.05);模型组(286.95±37.82)血清HA水平较正常组(229.15±32.43)升高(P0.05),其他各组较模型组有所降低,但并无差异;16个氨基酸多肽片段低剂量组、高剂量组及正常组血清Ⅳ-C水平(129.68±29.87,109.26±30.16,79.10±30.33)均低于模型组(180.96±40.73)(P0.05),秋水仙碱组(157.12±32.26)较模型组有所降低,但并无差异(P0.05),16个氨基酸多肽片段低剂量组、高剂量组血清Ⅳ-C水平均低于秋水仙碱组(P0.05)。2肝组织病理学检测结果HE结果显示,模型组小鼠肝细胞杂乱无章,中央静脉和门管区有纤维组织生成,并伴有大量炎细胞浸润,出现大片肝细胞坏死区域;秋水仙碱阳性对照组有效改善肝细胞坏死情况,炎细胞浸润减少;16个氨基酸多肽片段两个剂量组均未见明显坏死及炎细胞浸润。Masson染色模型组中央静脉、汇管区可见大量绿色胶原纤维,肝小叶被宽窄不一的纤维间隔分割成假小叶;秋水仙碱阳性对照组肝纤维化程度降低,仅见少量纤维组织;16个氨基酸多肽片段两个剂量组与模型组相比纤维组织显著减少。3各组小鼠肝纤维化相关基因表达水平变化荧光定量PCR结果显示,16个氨基酸多肽片段低剂量组(7.18±1.61,3.44±1.73,4.37±1.56,3.39±3.19,1.45±0.77)、高剂量组(5.66±2.07,3.77±2.64,3.43±1.42,3.43±2.92,1.68±1.21)、秋水仙碱组(9.60±0.52,5.58±0.65,3.97±4.01,5.93±4.84,1.91±1.03)及正常组(1.00)肝组织Col1a1、Col3a1、TIMP-1、MMP-2、CTGF各基因表达水平均低于模型组(20.67±6.60,12.61±1.35,8.25±1.77,11.93±6.82,3.97±1.90)(P0.05)。4各组小鼠肝细胞内活性氧(ROS)水平变化情况16个氨基酸多肽片段低剂量组(431.83±174.80)、高剂量组(404.50±199.43)、秋水仙碱组(523.00±218.61)及正常组(220.33±62.69)ROS水平均低于模型组(859.63±337.81)(P0.05)。结论:1 16个氨基酸多肽片段可显著改善CCl_4诱导的小鼠肝纤维化。2 16个氨基酸多肽片段减轻CCl_4诱导的小鼠肝纤维化损伤其机制可能与减少氧化应激反应,抑制TIMP-1、MMP-2、CTGF因子的表达有关。
[Abstract]:Objective: alcohol, chronic hepatitis B and C virus infection, obesity, autoimmune hepatitis, metabolic diseases, drug toxicants and cholestasis can lead to liver fibrosis, almost all chronic liver diseases are associated with fibrosis, and are easily progressing to cirrhosis, liver failure, liver cancer. Progressive liver fibrosis is still reversible, cirrhosis of the liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis of liver is reversible, cirrhosis It is basically irreversible. Carbon tetrachloride (CCl_4) induced chronic liver injury model in mice is more mature and can be used to simulate the common pathological process of human liver disease - liver fibrosis. The long-term stimulation of CCl_4 can cause necrosis, inflammation, fibroplasia, and serum aspartate aminotransferase (a). Spartate aminotransferase, AST) and alanine aminotransferase (alanine aminotransaminase, ALT) are elevated, and a large number of inflammatory cells are infiltrated and the extracellular matrix is deposited in large quantities. In order to find a biomarker for the clinical diagnosis of early hepatitis, the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has been used in the early stage of the room. In the sera of the patients with sexual hepatitis, a polypeptide of differential expression, that is, 16 amino acid polypeptide fragments, has been proved to have the effect of promoting the proliferation of liver cells in vitro. The results of the previous study in this room have confirmed that the polypeptide has protective effect on liver injury and fatty liver in immune hepatitis and presumably it may be in the chronic liver. This study used the same protective effect in the disease. This study used carbon tetrachloride induced mouse liver fibrosis model to study the therapeutic effect of 16 amino acid polypeptide fragments in liver fibrosis and to explore the mechanism for further clinical liver disease treatment. Methods: 116 amino acid polypeptide fragments were used. The protective effect of liver fibrosis in 50 males and 6-8 weeks old balb/c mice were randomly divided into five groups, which were normal group, model group, colchicine group, 16 amino acid polypeptide low dose group (400 mu g/kg), high dose group (800 g/kg), 10 in each group. The other groups were divided into normal saline (1ml/kg) at the time point of the corresponding administration, the rest of the groups were equally divided. Do not give 25%CCl_4 solution (1.5 mu l/g) by intraperitoneal injection, 10 times a total of 1 times every 3 days. At the same time, after the third injection of CCl_4, the model group passed the tail vein injection to the normal saline, and the other groups were given 400800 g/kg weight polypeptide by the tail vein, each 3 days 1 times, 7 or more times. Gavage was given to colchicine (200 mu g/kg). After every Friday and 3 weeks, blood was collected, blood was collected, blood was collected and serum was collected. Serum ALT, AST, alkaline phosphatase (Alkaline phosphatase, ALP), serum hyaluronidase (Hyaluronic Acid, HA), and type four collagen (Collagen Type IV) were measured by an automatic biochemical analyzer. The effect of 16 amino acid polypeptide fragments on CCl_4 induced liver fibrosis through the serological index..2 liver histopathological examination of the liver, the liver was removed, the liver was stripped, the left lobe of the liver was removed, and the right lobe of the same position of the two block of liver (no more than 0.5 centimeters in thickness) was made to make tissue wax blocks, and 5 mu m was performed. After continuous slice, HE staining and Masson staining were used to observe the pathological morphology and collagen deposition of liver tissue, and the expression level of liver fibrosis related genes in.3 mice was measured. The collagen type I collagen (Collagen, type I, alpha 1, Col1a1) and type III collagen (Collagen, type III, alpha 1) were obtained from NCBI. Col3a1), the gene sequence of Tissue Inhibitor of Metalloproteinase (TIMP-1), matrix metalloproteinase (Matrix Metalloproteinases, MMP-2), connective tissue growth factor (Connective Tissue Growth), and the design and synthesis of primers (Shanghai bioengineering Bioengineering Co., Ltd.). Total RNA, reverse transcriptional synthesis of cDNA, using 10 times diluted Col1a1, Col3a1, TIMP-1, MMP-2, CTGF, GAPDH cDNA as templates for real-time quantitative PCR detection. After the reaction, the amplification curve and melting curve were confirmed, and the RQ value of the relative expression of the gene was further analyzed. The phase of the 16 amino acid polypeptide fragment on the liver fibrosis induced by CCl_4 was discussed. The active oxygen in the hepatocytes of.4 was measured by flow cytometry, and the content of active oxygen (Reactive oxygen species, ROS) was detected in each group of hepatocytes to understand the level of oxidative stress. Results: 1 the serum liver function and the changes of liver fibrosis in each group were 16 (36.85 + 3.36,71.03 + 7.42). High dose group (35.27 + 4.34,73.03 + 8.13), colchicine group (34.65 + 5.57,69.49 + 11.76) and normal group (31.05 + 2.91,62.02 + 9.46) serum ALT, AST level were lower than model group (45.07 + 5.32,85.48 + 3.44) (P0.05), serum ALP level was not different (P0.05), the level of serum HA in the model group (286.95 + 37.82) was higher than that of normal group (229.15 + 32.43). P0.05), the other groups were lower than the model group, but there was no difference. The low dose group of 16 amino acid polypeptide fragments, the high dose group and the normal group serum level IV -C (129.68 + 29.87109.26 + 30.16,79.10 + 30.33) were lower than the model group (180.96 + 40.73) (P0.05), and the autumn Narcissus group (157.12 + 32.26) was lower than the model group, but there was no difference (P0.05). In the low dose group of 16 amino acid polypeptide fragments, the level of serum IV -C in the high dose group was lower than that of the colchicine group (P0.05).2 liver histopathology. The results showed that the liver cells in the model group were disorderly, the central vein and the portal area had fibrous tissue, and a large number of inflammatory cells infiltrated, and the necrotic area of hepatocytes appeared in autumn. The positive control group of the Narcissus alkali positive control group effectively improved the necrosis of hepatocyte and the infiltration of inflammatory cells; there was no obvious necrosis and the central vein in the.Masson staining model group of the 16 amino acid polypeptide fragments, and a large number of green collagen fibers were seen in the sinks, and the hepatic lobules were divided into false lobules by the spaced and narrow fiber interval; The degree of liver fibrosis in the immortal control group was reduced, only a small amount of fibrous tissue was found, and the 16 amino acid polypeptide fragment two dose groups significantly decreased the expression level of liver fibrosis related genes in.3 mice compared with the model group. The results of fluorescence quantitative PCR showed that 16 amino acid polypeptide fragments were low dose group (7.18 + 1.61,3.44 + 1.73,) 4.37 + 1.56,3.39 + 3.19,1.45 + 0.77), high dose group (5.66 + 2.07,3.77 + 2.64,3.43 + 1.42,3.43 + 2.92,1.68 + 1.21), colchicine group (9.60 + 0.52,5.58 + 0.65,3.97 + 4.01,5.93 + 4.84,1.91 + 1.03) and normal group (1) liver tissue Col1a1, Col3a1, which were lower than that of the model group (20.67 + In 5 + 1.77,11.93 + 6.82,3.97 + 1.90) (P0.05).4, the changes of active oxygen (ROS) level in the hepatocytes of each group of mice were low dose group (431.83 + 174.80), high dose group (404.50 + 199.43), colchicine group (523 + 218.61) and normal group (220.33 + 62.69) ROS level were lower than that of model group (859.63 + 337.81) (P0.05). Conclusion: 11 6 amino acid polypeptide fragments can significantly improve the.2 16 amino acid peptide fragments induced by CCl_4 induced liver fibrosis in mice. The mechanism of CCl_4 induced liver fibrosis in mice may be related to the reduction of oxidative stress and the inhibition of the expression of TIMP-1, MMP-2, and CTGF factors.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.2

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