36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性作用及其机制研究

发布时间：2018-06-13 16:07

本文选题：36个氨基酸多肽片段 + 肝纤维化　；参考：《河北医科大学》2016年硕士论文

【摘要】：目的:肝纤维化是一种慢性进展性疾病,其特征是细胞外基质的形成和过度沉积,这些过程可使肝脏结构发生重塑。肝纤维化是多种慢性肝脏疾病(可在早期阶段被逆转)发生进展的最终共同通路,当肝纤维化严重到不可逆转阶段,患者发展为肝硬化和肝癌的危险性增加。因此对纤维化的早期发现和治疗至关重要。目前,虽然在该病的诊断和治疗方面取得了一些进步,但仍缺乏标准治疗方案和特效治疗药物。36个氨基酸多肽片段是本研究室在寻找用于肝炎诊断的生物学标志物时从慢性肝炎病人血清中检测到的,其含量较高。经研究发现此36个氨基酸多肽片段可改变肝细胞周期,抑制细胞凋亡。目前尚无此36个氨基酸多肽片段在肝纤维化小鼠模型中生理和病理作用的研究。本研究室前期发现36个氨基酸多肽片段对急性肝损伤和非酒精性脂肪肝都具有保护性作用,为探讨该多肽片段是否对肝纤维化也具有类似的作用,故进行本项研究。本实验采用腹腔注射四氯化碳的方法,诱导小鼠发生肝纤维化,以研究36个氨基酸多肽片段对该模型小鼠的保护性作用,并探索其发挥作用的靶点和通路,为肝纤维化的治疗提供实验证据和研究方向。方法:1 36个氨基酸多肽片段对纤维化小鼠的保护作用将50只Balb/c雄性小鼠随机分为5组,分别为正常组、模型组、阳性药物组、36个氨基酸多肽片段低剂量组(150μg/kg)和36个氨基酸多肽片段高剂量组(250μg/kg)。正常组通过尾静脉分别在相应给药时点给予注射生理盐水;模型组腹腔注射25%CCl_4,每3天1次,共10次。自第4次腹腔注射CCl_4后,后一天开始给予尾静脉注射生理盐水进行治疗,共7次;阳性药物组造模后,给予秋水仙碱灌胃治疗,5次/周;两个多肽片段治疗组造模后尾静脉注射36个氨基酸多肽片段进行治疗,共7次。最后一次注射治疗药物后处死小鼠并分离血清,分别检测各组小鼠血清ALT、AST、HA、Ⅳ-C水平,从血清学水平观察36个氨基酸多肽片段对肝功能及纤维化程度的保护性作用。2肝组织病理学检测对各组肝组织进行病理学检测(HE染色和Masson染色),观察其病理形态学改变和胶原纤维沉积情况,评价36个氨基酸多肽片段对肝组织的保护作用。3肝纤维化发展过程中相关基因表达水平提取肝组织总RNA,采用实时荧光定量PCR的方法测定Col1A1、Col3A1、MMP-2、TIMP-1和CTGF的表达情况,分析36个氨基酸多肽片段对各组小鼠的影响情况。4肝细胞内活性氧的检测采用流式细胞术,对各组肝细胞内活性氧(ROS)含量进行检测,了解氧化应激水平。结果:1各组小鼠血清酶(ALT、AST)结果与正常组(31.05±2.91)相比,模型组血清ALT(45.07±5.32)水平均升高(P0.01);与模型组相比,秋水仙碱组(34.65±5.57)、36个氨基酸多肽片段低剂量组(36.08±2.07)、高剂量组(40.08±2.84)血清ALT水平均低于模型组(P0.05)。模型组血清AST(85.48±3.44)与正常组(62.05±10.93)相比,其水平升高(P0.01);秋水仙碱组(73.64±11.76)和36个氨基酸多肽片段低剂量组(71.89±11.02)AST水平低于模型组(P0.05)。2各组小鼠纤维化指标(HA、Ⅳ-C)结果模型组小鼠血清HA(291.98±39.16)比正常组(229.15±32.43)升高(P0.05),36个氨基酸多肽片段低剂量组(240.23±25.75)血清HA水平降低(P0.05)。与正常组(79.10±30.33)相比,模型组Ⅳ-C(180.95±40.73)升高(P0.01),36个氨基酸多肽片段低剂量组(104.55±31.77)和高剂量组(117.46±26.92)血清IV-C水平降低,与模型组具有差异(P0.01)。3各组小鼠肝脏病理学变化情况HE染色结果显示,正常组小鼠肝组织小叶结构清晰,细胞排列整齐形成肝索,无坏死区域;模型组小鼠肝脏出现大面积坏死,正常结构破坏,大量炎性细胞浸润;秋水仙碱组、36个氨基酸多肽片段低剂量组和36个氨基酸多肽片段高剂量组坏死面积大大减少,仅有少量炎性细胞浸润,其中低剂量组几乎未见坏死区域。Masson染色结果显示,正常组小鼠肝组织内无胶原沉积,胞浆呈红色,染色均匀;模型组小鼠肝组织正常小叶结构遭到破坏,出现大量异常沉积的胶原,出现假小叶;秋水仙碱组纤维含量减少,假小叶结构不明显;36个氨基酸多肽片段低剂量组和36个氨基酸多肽片段高剂量组纤维异常沉积明显减少,无假小叶出现。4各组小鼠肝纤维化相关基因m RNA表达水平变化各组Col1A1的相对表达量为:1.00,20.67±6.60,9.60±0.52,11.10±4.17,10.47±5.78(P0.05);Col3A1相对表达量为1.00,12.61±1.35,5.58±0.65,6.16±3.23和6.57±1.07(P0.05);MMP-2相对表达量为1.00,11.93±3.62,5.93±2.84,5.26±1.78和5.67±2.46(P0.05);TIMP-1相对表达量为1.00,8.25±1.87,3.97±1.02,3.36±1.83,4.19±2.78(P0.05);CTGF相对表达量:1.00,3.97±2.12,1.91±1.03,1.74±0.88,2.22±1.39(P0.05)。与正常组相比,模型组Col1A1、Col3A1、MMP-2、TIMP-1和CTGF相对表达量均升高(P0.05)。与模型组相比,秋水仙碱组、36个氨基酸多肽片段低剂量组和36个氨基酸多肽片段高剂量组Col1A1m RNA水平均下降(P0.05);秋水仙碱组、36个氨基酸多肽片段低剂量组和36个氨基酸多肽片段高剂量组Col3A1水平降低(P0.05);秋水仙碱组、36个氨基酸多肽片段低剂量组和36个氨基酸多肽片段高剂量组MMP-2水平下降(均P0.05);秋水仙碱组和36个氨基酸多肽片段低剂量组TIMP-1水平降低(P0.05);秋水仙碱组和36个氨基酸多肽片段低剂量组CTGF水平降低(P0.05)。5各组小鼠肝细胞内活性氧(ROS)水平模型组(859.63±337.81)细胞内ROS水平与正常组(220.33±62.69)相比高于(P0.01);与模型组相比,36个氨基酸多肽片段低剂量组(259.60±118.10)和高剂量组(327.20±123.63)ROS水平下降,差别具有统计学意义(P0.01)。结论:1 36个氨基酸多肽片段可以减轻四氯化碳诱导的肝纤维化小鼠肝脏损伤和纤维化程度,使受损的肝细胞恢复功能,逆转肝纤维化。2 36个氨基酸多肽片段对小鼠纤维化的保护作用可能与减少氧化应激、影响MMP-2、TIMP-1和CTGF表达有关。
[Abstract]:Objective: liver fibrosis is a chronic progressive disease characterized by the formation and overdeposition of extracellular matrix, which can remould the structure of the liver. Liver fibrosis is the ultimate common path for many chronic liver diseases (which can be reversed at the early stage). The risk of liver cirrhosis and liver cancer is increasing. Therefore, early detection and treatment of fibrosis is essential. Although some progress has been made in the diagnosis and treatment of the disease, there is still a lack of standard treatment and.36 amino acid polypeptide fragments of special therapeutic drugs in this laboratory to find biology for the diagnosis of hepatitis. The markers were detected in the serum of the chronic hepatitis patients with high content. It was found that the 36 amino acid polypeptide fragments could change the liver cell cycle and inhibit the apoptosis. At present, there is no study on the physiological and pathological effects of the 36 amino acid polypeptide fragments in the model of liver fibrosis in mice. 36 amino acids were found in the early stage of this study. The peptide fragment has a protective effect on acute liver injury and non-alcoholic fatty liver. This study is conducted to investigate whether the polypeptide fragment has a similar effect on liver fibrosis. Therefore, this study was conducted by intraperitoneal injection of carbon tetrachloride to induce liver fibrosis in mice to study the 36 amino acid polypeptide fragment on the model. The protective effect of mice and the target and pathway of its function were explored to provide experimental evidence and research direction for the treatment of liver fibrosis. Methods: the protective effects of 136 amino acid polypeptide fragments on 50 Balb/c male mice were randomly divided into 5 groups, the normal group, the model group, the positive drug group, and the 36 amino acids. A low dose group (150 g/kg) and a high dose group of 36 amino acid polypeptide fragments (250 mu g/kg). The normal group was injected with saline by the tail vein at the time of the corresponding administration, and the model group was injected with 25%CCl_4, 1 times every 3 days, 10 times. After the fourth intraperitoneal injection of CCl_4, the caudal vein was injected into the caudal vein one day later. The treatment was 7 times; after the model of the positive drug group, colchicine was given to the stomach for gavage, 5 times per week; 36 amino acid polypeptide fragments were injected into the tail vein of the two polypeptide segment treatment group for 7 times. After the last injection, the mice were killed and the serum was separated, and the serum ALT, AST, HA, IV -C levels were detected and the levels of blood from the blood were measured from the blood. The protective effect of 36 amino acid polypeptide fragments on the liver function and the degree of fibrosis.2 liver histopathology detection (HE staining and Masson staining), the pathological changes of the liver and the deposition of collagen fiber were observed, and the protective effect of 36 amino acid polypeptide fragments on the liver tissue was evaluated. The total RNA of liver tissue was extracted from the related gene expression level in the development of.3 liver fibrosis. The expression of Col1A1, Col3A1, MMP-2, TIMP-1 and CTGF were measured by real-time fluorescent quantitative PCR, and the effect of 36 amino acid polypeptide fragments on the mice in each group was analyzed. The detection of active oxygen in the liver cells of.4 liver cells by flow cytometry was used. The content of intracellular reactive oxygen species (ROS) was detected to understand the level of oxidative stress. Results: 1 the serum enzyme (ALT, AST) results of mice in each group were higher than that of the normal group (31.05 + 2.91), and the level of serum ALT (45.07 + 5.32) in the model group increased (P0.01). Compared with the model group, the colchicine group was (34.65 + 5.57), and the low dose group of 36 amino acid polypeptide fragments (36.08 + 2.07). The level of ALT in the high dose group (40.08 + 2.84) was lower than that in the model group (P0.05). The level of serum AST (85.48 + 3.44) in the model group was higher than that in the normal group (62.05 + 10.93), and the level of the group (73.64 + 11.76) and 36 amino acid polypeptide fragments (71.89 + 11.02) AST was lower than that of the model group (P0.05).2 in each group of mice (H). The serum HA (291.98 + 39.16) of the mice in the model group (291.98 + 39.16) was higher than that of the normal group (229.15 + 32.43) (P0.05), and the low dose group (240.23 + 25.75) of the 36 amino acid polypeptide fragment decreased (P0.05). Compared with the normal group (79.10 + 30.33), the model group IV -C (180.95 + 40.73) increased (P0.01), 36 amino acid polypeptide fragment low dose group (104.55 + 229.15). .77) and high dose group (117.46 + 26.92) the level of serum IV-C decreased, and there was a difference between the model group and the model group (P0.01) the pathological changes of liver in each group of.3 mice. The result of HE staining showed that the structure of the hepatic lobule in the normal group was clear, the cells arranged neatly to form the hepatic cord, and there was no necrotic region. The liver of the model group had large area necrosis and the normal structure was broken. In the colchicine group, the necrotic area of the high dose group of the 36 amino acid polypeptide fragments and the 36 amino acid polypeptide fragments decreased greatly and only a small amount of inflammatory cells infiltrated, and the low dose group had almost no necrotic region.Masson staining results, and the normal group had no collagen deposition and cytoplasm in the liver tissue. In the model group, the normal lobule structure of the liver tissue of the model mice was destroyed, a large amount of abnormal deposition of collagen and false lobule appeared, and the fiber content of the colchicine group decreased and the structure of the pseudo lobule was not obvious; the abnormal deposition of fibrous abnormal deposition in the low dose group of 36 amino acid polypeptide fragments and the high dose group of 36 amino acid polypeptide fragments decreased obviously. The expression level of M RNA expression of liver fibrosis related genes in every group of.4 mice was found to be 1.00,20.67 + 6.60,9.60 + 0.52,11.10 + 4.17,10.47 + 5.78 (P0.05), and the relative expression of Col3A1 was 1.00,12.61 + 1.35,5.58 + 3.23 and 6.57 + 1.07. 2.84,5.26 + 1.78 and 5.67 + 2.46 (P0.05); the relative expression of TIMP-1 is 1.00,8.25 + 1.87,3.97 + 1.02,3.36 + 1.83,4.19 + 2.78 (P0.05); CTGF relative expression: 1.00,3.97 + 2.12,1.91 + 1.03,1.74 + 1.39. Compared with the colchicine group, the low dose group of 36 amino acid polypeptide fragments and the high dose group of 36 amino acid polypeptide fragments decreased (P0.05), the colchicine group, the low dose group of 36 amino acid polypeptide fragments and the high dose group of 36 amino acid polypeptide fragments decreased (P0.05), the colchicine group, and the 36 amino acid polypeptide fragments in the colchicine group. Low dose group and 36 amino acid polypeptide segment high dose group MMP-2 level decreased (P0.05); colchicine group and 36 amino acid polypeptide fragment low dose group TIMP-1 level decreased (P0.05); colchicine group and 36 amino acid polypeptide fragment low dose group CTGF level decreased (P0.05) mice liver cell active oxygen (ROS) level model group (8). The ROS level in 59.63 + 337.81) was higher than that in the normal group (220.33 + 62.69). Compared with the model group, the low dose group (259.60 + 118.10) of 36 amino acid polypeptide fragments and the high dose group (327.20 + 123.63) ROS decreased, and the difference was statistically significant (P0.01). Conclusion: 136 amino acid polypeptide fragments could reduce the induction of carbon tetrachloride. The liver damage and fibrosis degree of liver fibrosis in mice, the damaged liver cells recover function, and the protective effect of.2 36 amino acid polypeptide fragments on liver fibrosis may be related to reducing oxidative stress and affecting the expression of MMP-2, TIMP-1 and CTGF.
【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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