發(fā)布時(shí)間：2018-05-24 12:14

  本文選題：16個(gè)氨基酸多肽片段 + 四氯化碳��； 參考：《河北醫(yī)科大學(xué)》2016年碩士論文

【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、代謝性疾病、藥物毒物和膽汁淤積等均可導(dǎo)致肝纖維化,幾乎所有的慢性肝病都與纖維化相關(guān),而且易進(jìn)展為肝硬化、肝衰竭、肝癌。進(jìn)展性的肝纖維化尚且可逆轉(zhuǎn),肝硬化則基本不可逆。四氯化碳(Carbon tetrachloride,CCl_4)誘導(dǎo)的小鼠慢性肝損傷模型已較成熟,可用來模擬人類眾多慢性肝臟疾病的共同病理過程—肝纖維化。經(jīng)CCl_4長(zhǎng)期刺激可致小鼠肝細(xì)胞壞死、炎癥、纖維組織增生,伴隨血清天冬氨酸轉(zhuǎn)氨酶(aspartate aminotransferase,AST)和丙氨酸轉(zhuǎn)氨酶(alanine aminotransaminase,ALT)升高,并且有大量炎性細(xì)胞浸潤(rùn),細(xì)胞外基質(zhì)大量膠原沉積。為尋找早期肝炎臨床診斷的生物標(biāo)志物,本室前期運(yùn)用基質(zhì)輔助激光解吸離子化飛行時(shí)間質(zhì)譜(MALDI-TOF MS)從慢性肝炎病人血清中檢測(cè)到一個(gè)差異性表達(dá)的多肽,即16個(gè)氨基酸多肽片段,并通過體外實(shí)驗(yàn)證實(shí)該多肽有促進(jìn)肝細(xì)胞增殖的作用。本室前期研究成果已證實(shí)該多肽針對(duì)免疫性肝炎肝損傷及脂肪肝具有保護(hù)作用,推測(cè)其可能在慢性肝臟疾病中發(fā)揮同樣的保護(hù)作用,故進(jìn)行本研究。本研究利用四氯化碳誘導(dǎo)的小鼠肝纖維化模型研究16個(gè)氨基酸多肽片段在肝纖維化中的治療作用,并對(duì)其機(jī)制初步探究,為進(jìn)一步臨床肝病治療提供實(shí)驗(yàn)依據(jù)。方法:1 16個(gè)氨基酸多肽片段對(duì)肝纖維化小鼠的保護(hù)作用50只雄性,6-8周齡balb/c小鼠隨機(jī)分為五組,分別為正常組,模型組,秋水仙堿組,16個(gè)氨基酸多肽低劑量組(400μg/kg),高劑量組(800μg/kg),每組10只。除正常組在相應(yīng)給藥時(shí)點(diǎn)腹腔注射生理鹽水(1ml/kg)外,其余各組均分別通過腹腔注射給予25%CCl_4溶液(1.5μl/g),每3天1次,共10次。同時(shí),在第三次注射CCl_4之后,除正常組、模型組分別在相應(yīng)給藥時(shí)點(diǎn)通過尾靜脈注射給予生理鹽水外,其余各組分別經(jīng)尾靜脈注射給予400、800μg/kg體重多肽,每3天1次,持續(xù)7次或經(jīng)灌胃給予秋水仙堿(200μg/kg),每周五天,持續(xù)3周后,眼球取血,收集小鼠血液,分離血清,采用全自動(dòng)生化分析儀測(cè)定血清ALT、AST、堿性磷酸酶(Alkaline phosphatase,ALP)水平;ELISA試劑盒測(cè)定血清透明質(zhì)酸酶(Hyaluronic Acid,HA)、四型膠原(Collagen TypeⅣ,Ⅳ-C)水平。通過血清學(xué)指標(biāo)觀察16個(gè)氨基酸多肽片段對(duì)CCl_4誘導(dǎo)肝纖維化的影響。2肝組織病理學(xué)檢測(cè)眼球取血后處死小鼠,剝離肝臟,分別切取肝臟左葉、右葉相同位置合適大小的兩塊肝組織(一般厚度不超過0.5厘米)制成組織蠟塊,行5μm連續(xù)切片后分別進(jìn)行HE染色、Masson染色,于光學(xué)顯微鏡下觀察肝組織病理學(xué)形態(tài)及膠原沉積情況,并采集圖像。3小鼠肝纖維化相關(guān)基因表達(dá)水平的測(cè)定自NCBI中獲取小鼠源Ⅰ型膠原(Collagen,typeⅠ,alpha 1,Col1a1)、Ⅲ型膠原(Collagen,typeⅢ,alpha 1,Col3a1)、金屬蛋白酶組織抑制劑(Tissue Inhibitor of Metalloproteinase,TIMP-1)、基質(zhì)金屬蛋白酶(Matrix Metalloproteinases,MMP-2)、結(jié)締組織生長(zhǎng)因子(Connective Tissue Growth Factor,CTGF)的基因序列,設(shè)計(jì)并合成引物(上海生工生物工程有限公司)。提取小鼠肝組織總RNA,反轉(zhuǎn)錄合成cDNA,分別以10倍稀釋的Col1a1、Col3a1、TIMP-1、MMP-2、CTGF、GAPDH cDNA為模板進(jìn)行實(shí)時(shí)熒光定量PCR檢測(cè)。反應(yīng)結(jié)束后確認(rèn)擴(kuò)增曲線和融解曲線,記錄基因相對(duì)表達(dá)量RQ值進(jìn)一步分析。探討16個(gè)氨基酸多肽片段對(duì)CCl_4誘導(dǎo)的肝纖維化的相關(guān)作用機(jī)制。4肝細(xì)胞內(nèi)活性氧的測(cè)定采用流式細(xì)胞術(shù),對(duì)各組肝細(xì)胞內(nèi)活性氧(Reactive oxygen species,ROS)含量進(jìn)行檢測(cè),了解氧化應(yīng)激水平。結(jié)果:1各組小鼠血清肝功能及肝纖維化變化情況16個(gè)氨基酸多肽片段低劑量組(36.85±3.36,71.03±7.42)、高劑量組(35.27±4.34,73.03±8.13)、秋水仙堿組(34.65±5.57,69.49±11.76)及正常組(31.05±2.91,62.02±9.46)血清ALT、AST水平均低于模型組(45.07±5.32,85.48±3.44)(P0.05);各組血清ALP水平均無差異(P0.05);模型組(286.95±37.82)血清HA水平較正常組(229.15±32.43)升高(P0.05),其他各組較模型組有所降低,但并無差異;16個(gè)氨基酸多肽片段低劑量組、高劑量組及正常組血清Ⅳ-C水平(129.68±29.87,109.26±30.16,79.10±30.33)均低于模型組(180.96±40.73)(P0.05),秋水仙堿組(157.12±32.26)較模型組有所降低,但并無差異(P0.05),16個(gè)氨基酸多肽片段低劑量組、高劑量組血清Ⅳ-C水平均低于秋水仙堿組(P0.05)。2肝組織病理學(xué)檢測(cè)結(jié)果HE結(jié)果顯示,模型組小鼠肝細(xì)胞雜亂無章,中央靜脈和門管區(qū)有纖維組織生成,并伴有大量炎細(xì)胞浸潤(rùn),出現(xiàn)大片肝細(xì)胞壞死區(qū)域;秋水仙堿陽(yáng)性對(duì)照組有效改善肝細(xì)胞壞死情況,炎細(xì)胞浸潤(rùn)減少;16個(gè)氨基酸多肽片段兩個(gè)劑量組均未見明顯壞死及炎細(xì)胞浸潤(rùn)。Masson染色模型組中央靜脈、匯管區(qū)可見大量綠色膠原纖維,肝小葉被寬窄不一的纖維間隔分割成假小葉;秋水仙堿陽(yáng)性對(duì)照組肝纖維化程度降低,僅見少量纖維組織;16個(gè)氨基酸多肽片段兩個(gè)劑量組與模型組相比纖維組織顯著減少。3各組小鼠肝纖維化相關(guān)基因表達(dá)水平變化熒光定量PCR結(jié)果顯示,16個(gè)氨基酸多肽片段低劑量組(7.18±1.61,3.44±1.73,4.37±1.56,3.39±3.19,1.45±0.77)、高劑量組(5.66±2.07,3.77±2.64,3.43±1.42,3.43±2.92,1.68±1.21)、秋水仙堿組(9.60±0.52,5.58±0.65,3.97±4.01,5.93±4.84,1.91±1.03)及正常組(1.00)肝組織Col1a1、Col3a1、TIMP-1、MMP-2、CTGF各基因表達(dá)水平均低于模型組(20.67±6.60,12.61±1.35,8.25±1.77,11.93±6.82,3.97±1.90)(P0.05)。4各組小鼠肝細(xì)胞內(nèi)活性氧(ROS)水平變化情況16個(gè)氨基酸多肽片段低劑量組(431.83±174.80)、高劑量組(404.50±199.43)、秋水仙堿組(523.00±218.61)及正常組(220.33±62.69)ROS水平均低于模型組(859.63±337.81)(P0.05)。結(jié)論:1 16個(gè)氨基酸多肽片段可顯著改善CCl_4誘導(dǎo)的小鼠肝纖維化。2 16個(gè)氨基酸多肽片段減輕CCl_4誘導(dǎo)的小鼠肝纖維化損傷其機(jī)制可能與減少氧化應(yīng)激反應(yīng),抑制TIMP-1、MMP-2、CTGF因子的表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575.2

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