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吡喹酮联合塞来昔布延缓小鼠血吸虫病肝纤维化的研究

发布时间:2018-04-21 13:22

  本文选题:日本血吸虫 + 肝纤维化 ; 参考:《南京医科大学》2014年硕士论文


【摘要】:日本血吸虫病是一种人兽共患寄生虫病,其主要病理损害是沉积于宿主肝脏组织的虫卵引发的肉芽肿反应以及慢性感染造成的组织损伤过度修复,进而发展成肝纤维化。传统的病原学治疗即吡喹酮杀虫,虽然可以大大降低虫体对机体的炎性刺激和持续免疫,而患者业已存在的肝纤维化病变仍有继续发展至晚期病变的趋势。纤维化是机体对炎症导致的组织损伤产生的自我修复反应,长期反复的肝脏炎症反应是肝纤维化形成的前提条件。环氧合酶-2(cyclooxgenase-2, COX-2)作为花生四烯酸合成前列腺素类物质重要的限速酶,参与炎症反应过程。近年来,研究发现COX-2在肝纤维化及肝硬化组织中表达明显增强,而在正常肝组织中表达甚微。因此,推测COX-2可能在肝纤维化形成过程中起关键作用。持续有效的抗炎症治疗是阻止纤维化进展的治疗方法之一。COX-2抑制剂塞来昔布是目前临床广泛应用的非甾体类抗炎药,在众多动物模型中被证实能抑制肝纤维化的发展。据此,本研究以BALB/c小鼠为研究对象,建立日本血吸虫感染及治疗模型,探索COX-2与血吸虫病肝纤维化的关系以及COX-2抑制剂在血吸虫病治疗中的作用。本研究技术路线为:以10条日本血吸虫尾蚴感染小鼠,建立日本血吸虫急性感染、慢性感染及治疗模型。共设计5个实验组,即正常组(uninfected)、感染6周组(6w infected)、感染12周组(12w infected)、吡喹酮治疗组(PZQ)、吡喹酮加塞来昔布治疗组(PZQ+CLC)。采用如下实验方法对COX-2、纤维化相关指标、炎性细胞因子及miRNAs等相关指标进行了检测,包括:自动生化分析仪检测血清谷丙转氨酶(ALT)水平,HE染色评价肝脏炎症及纤维化程度,Masson染色判断胶原沉积情况,免疫组化染色评价肝脏纤维化相关因子表达情况,荧光实时定量PCR检测COX-2、肝脏炎症和纤维化相关基因表达水平以及炎性相关miRNA表达水平等,以探索COX-2抑制剂塞来昔布对血吸虫病纤维化的作用及相关机制。本研究主要结果如下:1.血清转氨酶检测结果显示:与感染12周组小鼠相比,PZQ+CLC治疗组小鼠血清ALT水平显著下降。与PZQ治疗组相比,PZQ+CLC治疗组小鼠血清ALT水平虽有下降,但两者间无统计学差异。2. RT-PCR检测肝脏COX-2结果显示:感染血吸虫后,小鼠肝脏COX-2 mRNA表达升高,但是感染12周组较感染6周组有所降低。PZQ+CLC治疗组较PZQ治疗组小鼠肝脏COX-2的表达受到显著抑制。由此推测,COX-2可能参与血吸虫感染引起的肝脏病变。3.肝脏HE染色结果显示:感染12周组小鼠肝脏中,大量虫卵聚集在汇管区附近,多个虫卵周围见成纤维细胞、类上皮细胞和胶原纤维增生,呈同心圆状包绕,外周有嗜酸性粒细胞、淋巴细胞、巨噬细胞等浸润,而PZQ+CLC治疗组和PZQ治疗组则胶原纤维增生不明显,炎症反应明显减轻。与感染对照组和PZQ治疗组相比,PZQ+CLC治疗组的单个虫六卵肉芽肿面积亦明显减小。4.肝脏Masson染色结果显示:正常组小鼠肝脏切片中未见蓝染的胶原纤维。与感染12周组和PZQ治疗组相比,PZQ+CLC治疗组小鼠肝脏切片显示虫卵肉芽肿中蓝染明显减少,提示胶原纤维沉积较少。5.肝脏免疫组化染色结果显示:与感染12周组和PZQ治疗组相比,PZQ+CLC治疗组小鼠肝脏中Collagen Ⅰ、CollagenⅢ、α-SMA、MMP-9和TIMP-1表达减少。6. RT-PCR检测肝脏纤维化相关因子结果显示:与感染12周组和PZQ治疗组相比,IZQ+CLC治疗组小鼠肝脏肝纤维化相关因子Co11α1、Col3α1、α-SMA、 MMP-9、TIMP-1 mRNA水平均显著下降,且MP-9/TIMP-1比例上调。7. RT-PCR检测肝脏炎性细胞因子结果显示:感染6周组小鼠肝脏局部的促炎因子TNF-α、IL-1β和IL-6的mRNA水平均显著高于正常组和感染12周组;与感染12周组和PZQ治疗组相比,PZQ+CLC治疗组小鼠肝脏局部促炎因子(TNF-α、IL-1β和IL-6)及促肝纤维化细胞因子(TGF-β、IL-4和IL-13) mRNA水平均显著下降。8. RT-PCR检测肝脏miRNA结果显示:感染6周组小鼠肝脏中起促炎作用的miR-155和起抑炎作用的miR-146a的mRNA水平均显著高于正常组和感染12周组;与感染12周组和PZQ治疗组相比,PZQ+CLC治疗组小鼠肝脏miR-155和miR-146a的m RNA水平降低,且趋于正常水平。感染6周组小鼠肝脏中起负向调控COX-2作用的miR-199a和miR-101a的mRNA水平均显著低于正常组和感染12周组;与感染12周组和PZQ治疗组相比,PZQ+CLC治疗组小鼠肝脏miR-199a和miR-101a的mRNA水平升高。综上所述,本研究明确了COX-2在日本血吸虫病中的表达特征,并且COX-2抑制剂塞来昔布能够缓解病原学治疗基础上的日本血吸虫病肝纤维化,这为血吸虫病的防治提供了先导性的实验论据。
[Abstract]:Schistosomiasis japonica is a zoonotic parasitic disease. Its main pathological damage is the granulomatous reaction caused by the egg of the host liver tissue and the excessive repair of tissue damage caused by chronic infection, and then to the liver fibrosis. The traditional etiological treatment, namely, pyoquinone, can greatly reduce the body to the body. Inflammatory and continuous immunization, and the existing liver fibrosis in patients still have a trend to continue to develop to advanced lesions. Fibrosis is the body's self repair response to tissue damage caused by inflammation. Long-term repeated liver inflammation is the precondition for the formation of liver fibrosis. Cyclooxygenase -2 (Cyclooxgenase-2, COX-2) ) as an important rate limiting enzyme for the synthesis of prostaglandins from peanut four enes, it is involved in the process of inflammatory reaction. In recent years, the expression of COX-2 in liver fibrosis and liver cirrhosis has been obviously enhanced and expressed very little in normal liver tissue. Therefore, it is presumed that COX-2 may play a key role in the formation of liver fibrosis. Anti inflammatory treatment is one of the treatment methods to prevent the progress of fibrosis, celecoxib, a.COX-2 inhibitor, is a widely used nonsteroidal anti-inflammatory drug, which has been proved to inhibit the development of liver fibrosis in many animal models. Based on this, this study aims to establish a model for the establishment of Schistosoma japonicum infection and treatment model in BALB/c mice. The relationship between COX-2 and schistosomiasis liver fibrosis and the role of COX-2 inhibitors in the treatment of schistosomiasis. The technical route of this study was to establish acute infection, chronic infection and treatment model of Schistosoma japonicum with 10 Schistosoma japonicum cercariae in mice. A total of 5 experimental groups, namely, normal group (uninfected) and 6 weeks of infection (6W infected) were designed. 12 weeks of infection (12W infected), praziquantel treatment group (PZQ), praziquantel plus celecoxib (PZQ+CLC). The following experimental methods were used to detect the related indexes of COX-2, fibrosis related indexes, inflammatory cytokines and miRNAs, including the automatic biochemical analyzer to detect the level of serum alanine transaminase (ALT) and HE staining to evaluate the liver. The degree of inflammation and fibrosis, Masson staining was used to determine the status of collagen deposition, immunohistochemical staining was used to evaluate the expression of liver fibrosis related factors. Fluorescence real-time quantitative PCR was used to detect COX-2, the expression level of liver inflammation and fibrosis related genes and the level of inflammatory related miRNA expression, in order to explore the COX-2 Inhibitor Celecoxib on schistosomiasis. The main results of this study were as follows: 1. the results of serum aminotransferase showed that the serum ALT level of PZQ+CLC treated mice decreased significantly compared with that of the 12 week infected mice. Compared with the PZQ treatment group, the serum ALT level of the mice in the PZQ+CLC treatment group decreased, but there was no statistical difference between the two groups, but there was no statistical difference between the two groups. The results of liver COX-2 test showed that after infection of schistosomiasis, the expression of COX-2 mRNA in liver of mice increased, but the 12 week infection group was lower than that of the 6 week infection group. The expression of COX-2 in the liver of the mice was significantly inhibited than that in the PZQ treatment group. Thus, the COX-2 may be involved in the liver HE staining results of liver lesions caused by Schistosoma infection. The results showed that in the liver of the mice infected with 12 weeks, a large number of eggs were gathered around the pipe area, and fibroblasts were found around the eggs. The epithelioid cells and collagen fibers proliferated in a concentric circle, and there were eosinophils, lymphocytes, macrophages and so on, while the proliferation of collagen fibers was not obvious in the PZQ+CLC treatment group and the PZQ treatment group. Compared with the control group of the infection control group and the PZQ treatment group, the area of the six egg granuloma of the single worm in the PZQ+CLC treatment group was also significantly reduced by the.4. liver Masson staining results showed: no blue stained collagen fibers were found in the liver slices of the normal group. Compared with the 12 week infection group and the PZQ treatment group, the liver slices of the PZQ+CLC group were sliced in the mice. The results showed that the blue staining of the egg granuloma was significantly reduced, suggesting that the.5. liver immunohistochemical staining of less collagen fibrous deposition showed that the results of Collagen I, Collagen III, alpha -SMA, MMP-9 and TIMP-1 expression in the liver of the PZQ+CLC treatment group were compared with the 12 weeks of infection and the PZQ treatment group, and the results showed that the expression of.6. RT-PCR in the liver was reduced to the results of the liver fibrosis related factors. Compared with the 12 week infection group and the PZQ treatment group, the liver fibrosis related factors Co11 alpha 1, Col3 alpha 1, alpha -SMA, MMP-9, TIMP-1 mRNA in the IZQ+CLC treatment group were significantly decreased, and the MP-9/TIMP-1 proportional increase.7. RT-PCR detection of liver inflammatory cytokines showed that the liver local proinflammatory factor TNF- alpha in the liver of the mice was infected for 6 weeks. The levels of mRNA and IL-6 were significantly higher than those of the normal group and the 12 week infection group. Compared with the 12 week infection group and the PZQ treatment group, the liver local proinflammatory factors (TNF-, IL-1 beta and IL-6) and the mRNA levels of liver fibrosis cytokines (TGF- beta, IL-4 and IL-13) were significantly decreased in the PZQ+CLC treatment group. The mRNA levels of miR-155 and miR-146a in the liver of the mice in the week group were significantly higher than those in the normal group and the 12 week group, and the miR-155 and miR-146a m RNA levels of the mice in the PZQ+CLC group were lower than those in the 12 week group and the PZQ treatment group. The liver of the mice infected with the 6 weeks of infection was negative. The mRNA level of miR-199a and miR-101a in the control of COX-2 was significantly lower than that in the normal group and the 12 week group, and the mRNA level of the liver miR-199a and miR-101a in the PZQ+CLC group was higher than that in the 12 week group and the PZQ treatment group. To sum up, the present study clearly defined the expression of COX-2 in the Japanese schistosomiasis, and the COX-2 inhibitor. Celecoxib can alleviate the liver fibrosis of schistosomiasis japonica based on the etiological treatment, which provides a pilot experimental evidence for the prevention and treatment of schistosomiasis.

【学位授予单位】:南京医科大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R532.32;R575.2

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