本文選題：36個(gè)氨基酸多肽片段 + 肝纖維化�。� 參考：《河北醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:肝纖維化是一種慢性進(jìn)展性疾病,其特征是細(xì)胞外基質(zhì)的形成和過(guò)度沉積,這些過(guò)程可使肝臟結(jié)構(gòu)發(fā)生重塑。肝纖維化是多種慢性肝臟疾病(可在早期階段被逆轉(zhuǎn))發(fā)生進(jìn)展的最終共同通路,當(dāng)肝纖維化嚴(yán)重到不可逆轉(zhuǎn)階段,患者發(fā)展為肝硬化和肝癌的危險(xiǎn)性增加。因此對(duì)纖維化的早期發(fā)現(xiàn)和治療至關(guān)重要。目前,雖然在該病的診斷和治療方面取得了一些進(jìn)步,但仍缺乏標(biāo)準(zhǔn)治療方案和特效治療藥物。36個(gè)氨基酸多肽片段是本研究室在尋找用于肝炎診斷的生物學(xué)標(biāo)志物時(shí)從慢性肝炎病人血清中檢測(cè)到的,其含量較高。經(jīng)研究發(fā)現(xiàn)此36個(gè)氨基酸多肽片段可改變肝細(xì)胞周期,抑制細(xì)胞凋亡。目前尚無(wú)此36個(gè)氨基酸多肽片段在肝纖維化小鼠模型中生理和病理作用的研究。本研究室前期發(fā)現(xiàn)36個(gè)氨基酸多肽片段對(duì)急性肝損傷和非酒精性脂肪肝都具有保護(hù)性作用,為探討該多肽片段是否對(duì)肝纖維化也具有類(lèi)似的作用,故進(jìn)行本項(xiàng)研究。本實(shí)驗(yàn)采用腹腔注射四氯化碳的方法,誘導(dǎo)小鼠發(fā)生肝纖維化,以研究36個(gè)氨基酸多肽片段對(duì)該模型小鼠的保護(hù)性作用,并探索其發(fā)揮作用的靶點(diǎn)和通路,為肝纖維化的治療提供實(shí)驗(yàn)證據(jù)和研究方向。方法:1 36個(gè)氨基酸多肽片段對(duì)纖維化小鼠的保護(hù)作用將50只Balb/c雄性小鼠隨機(jī)分為5組,分別為正常組、模型組、陽(yáng)性藥物組、36個(gè)氨基酸多肽片段低劑量組(150μg/kg)和36個(gè)氨基酸多肽片段高劑量組(250μg/kg)。正常組通過(guò)尾靜脈分別在相應(yīng)給藥時(shí)點(diǎn)給予注射生理鹽水;模型組腹腔注射25%CCl_4,每3天1次,共10次。自第4次腹腔注射CCl_4后,后一天開(kāi)始給予尾靜脈注射生理鹽水進(jìn)行治療,共7次;陽(yáng)性藥物組造模后,給予秋水仙堿灌胃治療,5次/周;兩個(gè)多肽片段治療組造模后尾靜脈注射36個(gè)氨基酸多肽片段進(jìn)行治療,共7次。最后一次注射治療藥物后處死小鼠并分離血清,分別檢測(cè)各組小鼠血清ALT、AST、HA、Ⅳ-C水平,從血清學(xué)水平觀察36個(gè)氨基酸多肽片段對(duì)肝功能及纖維化程度的保護(hù)性作用。2肝組織病理學(xué)檢測(cè)對(duì)各組肝組織進(jìn)行病理學(xué)檢測(cè)(HE染色和Masson染色),觀察其病理形態(tài)學(xué)改變和膠原纖維沉積情況,評(píng)價(jià)36個(gè)氨基酸多肽片段對(duì)肝組織的保護(hù)作用。3肝纖維化發(fā)展過(guò)程中相關(guān)基因表達(dá)水平提取肝組織總RNA,采用實(shí)時(shí)熒光定量PCR的方法測(cè)定Col1A1、Col3A1、MMP-2、TIMP-1和CTGF的表達(dá)情況,分析36個(gè)氨基酸多肽片段對(duì)各組小鼠的影響情況。4肝細(xì)胞內(nèi)活性氧的檢測(cè)采用流式細(xì)胞術(shù),對(duì)各組肝細(xì)胞內(nèi)活性氧(ROS)含量進(jìn)行檢測(cè),了解氧化應(yīng)激水平。結(jié)果:1各組小鼠血清酶(ALT、AST)結(jié)果與正常組(31.05±2.91)相比,模型組血清ALT(45.07±5.32)水平均升高(P0.01);與模型組相比,秋水仙堿組(34.65±5.57)、36個(gè)氨基酸多肽片段低劑量組(36.08±2.07)、高劑量組(40.08±2.84)血清ALT水平均低于模型組(P0.05)。模型組血清AST(85.48±3.44)與正常組(62.05±10.93)相比,其水平升高(P0.01);秋水仙堿組(73.64±11.76)和36個(gè)氨基酸多肽片段低劑量組(71.89±11.02)AST水平低于模型組(P0.05)。2各組小鼠纖維化指標(biāo)(HA、Ⅳ-C)結(jié)果模型組小鼠血清HA(291.98±39.16)比正常組(229.15±32.43)升高(P0.05),36個(gè)氨基酸多肽片段低劑量組(240.23±25.75)血清HA水平降低(P0.05)。與正常組(79.10±30.33)相比,模型組Ⅳ-C(180.95±40.73)升高(P0.01),36個(gè)氨基酸多肽片段低劑量組(104.55±31.77)和高劑量組(117.46±26.92)血清IV-C水平降低,與模型組具有差異(P0.01)。3各組小鼠肝臟病理學(xué)變化情況HE染色結(jié)果顯示,正常組小鼠肝組織小葉結(jié)構(gòu)清晰,細(xì)胞排列整齊形成肝索,無(wú)壞死區(qū)域;模型組小鼠肝臟出現(xiàn)大面積壞死,正常結(jié)構(gòu)破壞,大量炎性細(xì)胞浸潤(rùn);秋水仙堿組、36個(gè)氨基酸多肽片段低劑量組和36個(gè)氨基酸多肽片段高劑量組壞死面積大大減少,僅有少量炎性細(xì)胞浸潤(rùn),其中低劑量組幾乎未見(jiàn)壞死區(qū)域。Masson染色結(jié)果顯示,正常組小鼠肝組織內(nèi)無(wú)膠原沉積,胞漿呈紅色,染色均勻;模型組小鼠肝組織正常小葉結(jié)構(gòu)遭到破壞,出現(xiàn)大量異常沉積的膠原,出現(xiàn)假小葉;秋水仙堿組纖維含量減少,假小葉結(jié)構(gòu)不明顯;36個(gè)氨基酸多肽片段低劑量組和36個(gè)氨基酸多肽片段高劑量組纖維異常沉積明顯減少,無(wú)假小葉出現(xiàn)。4各組小鼠肝纖維化相關(guān)基因m RNA表達(dá)水平變化各組Col1A1的相對(duì)表達(dá)量為:1.00,20.67±6.60,9.60±0.52,11.10±4.17,10.47±5.78(P0.05);Col3A1相對(duì)表達(dá)量為1.00,12.61±1.35,5.58±0.65,6.16±3.23和6.57±1.07(P0.05);MMP-2相對(duì)表達(dá)量為1.00,11.93±3.62,5.93±2.84,5.26±1.78和5.67±2.46(P0.05);TIMP-1相對(duì)表達(dá)量為1.00,8.25±1.87,3.97±1.02,3.36±1.83,4.19±2.78(P0.05);CTGF相對(duì)表達(dá)量:1.00,3.97±2.12,1.91±1.03,1.74±0.88,2.22±1.39(P0.05)。與正常組相比,模型組Col1A1、Col3A1、MMP-2、TIMP-1和CTGF相對(duì)表達(dá)量均升高(P0.05)。與模型組相比,秋水仙堿組、36個(gè)氨基酸多肽片段低劑量組和36個(gè)氨基酸多肽片段高劑量組Col1A1m RNA水平均下降(P0.05);秋水仙堿組、36個(gè)氨基酸多肽片段低劑量組和36個(gè)氨基酸多肽片段高劑量組Col3A1水平降低(P0.05);秋水仙堿組、36個(gè)氨基酸多肽片段低劑量組和36個(gè)氨基酸多肽片段高劑量組MMP-2水平下降(均P0.05);秋水仙堿組和36個(gè)氨基酸多肽片段低劑量組TIMP-1水平降低(P0.05);秋水仙堿組和36個(gè)氨基酸多肽片段低劑量組CTGF水平降低(P0.05)。5各組小鼠肝細(xì)胞內(nèi)活性氧(ROS)水平模型組(859.63±337.81)細(xì)胞內(nèi)ROS水平與正常組(220.33±62.69)相比高于(P0.01);與模型組相比,36個(gè)氨基酸多肽片段低劑量組(259.60±118.10)和高劑量組(327.20±123.63)ROS水平下降,差別具有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論:1 36個(gè)氨基酸多肽片段可以減輕四氯化碳誘導(dǎo)的肝纖維化小鼠肝臟損傷和纖維化程度,使受損的肝細(xì)胞恢復(fù)功能,逆轉(zhuǎn)肝纖維化。2 36個(gè)氨基酸多肽片段對(duì)小鼠纖維化的保護(hù)作用可能與減少氧化應(yīng)激、影響MMP-2、TIMP-1和CTGF表達(dá)有關(guān)。
[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.
【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(lèi)號(hào)】：R575.2

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