本文選題：日本血吸蟲 + 肝纖維化��； 參考：《南京醫(yī)科大學(xué)》2014年碩士論文

【摘要】：日本血吸蟲病是一種人獸共患寄生蟲病,其主要病理損害是沉積于宿主肝臟組織的蟲卵引發(fā)的肉芽腫反應(yīng)以及慢性感染造成的組織損傷過度修復(fù),進而發(fā)展成肝纖維化。傳統(tǒng)的病原學(xué)治療即吡喹酮殺蟲,雖然可以大大降低蟲體對機體的炎性刺激和持續(xù)免疫,而患者業(yè)已存在的肝纖維化病變?nèi)杂欣^續(xù)發(fā)展至晚期病變的趨勢。纖維化是機體對炎癥導(dǎo)致的組織損傷產(chǎn)生的自我修復(fù)反應(yīng),長期反復(fù)的肝臟炎癥反應(yīng)是肝纖維化形成的前提條件。環(huán)氧合酶-2(cyclooxgenase-2, COX-2)作為花生四烯酸合成前列腺素類物質(zhì)重要的限速酶,參與炎癥反應(yīng)過程。近年來,研究發(fā)現(xiàn)COX-2在肝纖維化及肝硬化組織中表達明顯增強,而在正常肝組織中表達甚微。因此,推測COX-2可能在肝纖維化形成過程中起關(guān)鍵作用。持續(xù)有效的抗炎癥治療是阻止纖維化進展的治療方法之一。COX-2抑制劑塞來昔布是目前臨床廣泛應(yīng)用的非甾體類抗炎藥,在眾多動物模型中被證實能抑制肝纖維化的發(fā)展。據(jù)此,本研究以BALB/c小鼠為研究對象,建立日本血吸蟲感染及治療模型,探索COX-2與血吸蟲病肝纖維化的關(guān)系以及COX-2抑制劑在血吸蟲病治療中的作用。本研究技術(shù)路線為：以10條日本血吸蟲尾蚴感染小鼠,建立日本血吸蟲急性感染、慢性感染及治療模型。共設(shè)計5個實驗組,即正常組(uninfected)、感染6周組(6w infected)、感染12周組(12w infected)、吡喹酮治療組(PZQ)、吡喹酮加塞來昔布治療組(PZQ+CLC)。采用如下實驗方法對COX-2、纖維化相關(guān)指標、炎性細胞因子及miRNAs等相關(guān)指標進行了檢測,包括：自動生化分析儀檢測血清谷丙轉(zhuǎn)氨酶(ALT)水平,HE染色評價肝臟炎癥及纖維化程度,Masson染色判斷膠原沉積情況,免疫組化染色評價肝臟纖維化相關(guān)因子表達情況,熒光實時定量PCR檢測COX-2、肝臟炎癥和纖維化相關(guān)基因表達水平以及炎性相關(guān)miRNA表達水平等,以探索COX-2抑制劑塞來昔布對血吸蟲病纖維化的作用及相關(guān)機制。本研究主要結(jié)果如下：1.血清轉(zhuǎn)氨酶檢測結(jié)果顯示：與感染12周組小鼠相比,PZQ+CLC治療組小鼠血清ALT水平顯著下降。與PZQ治療組相比,PZQ+CLC治療組小鼠血清ALT水平雖有下降,但兩者間無統(tǒng)計學(xué)差異。2. RT-PCR檢測肝臟COX-2結(jié)果顯示：感染血吸蟲后,小鼠肝臟COX-2 mRNA表達升高,但是感染12周組較感染6周組有所降低。PZQ+CLC治療組較PZQ治療組小鼠肝臟COX-2的表達受到顯著抑制。由此推測,COX-2可能參與血吸蟲感染引起的肝臟病變。3.肝臟HE染色結(jié)果顯示：感染12周組小鼠肝臟中,大量蟲卵聚集在匯管區(qū)附近,多個蟲卵周圍見成纖維細胞、類上皮細胞和膠原纖維增生,呈同心圓狀包繞,外周有嗜酸性粒細胞、淋巴細胞、巨噬細胞等浸潤,而PZQ+CLC治療組和PZQ治療組則膠原纖維增生不明顯,炎癥反應(yīng)明顯減輕。與感染對照組和PZQ治療組相比,PZQ+CLC治療組的單個蟲六卵肉芽腫面積亦明顯減小。4.肝臟Masson染色結(jié)果顯示：正常組小鼠肝臟切片中未見藍染的膠原纖維。與感染12周組和PZQ治療組相比,PZQ+CLC治療組小鼠肝臟切片顯示蟲卵肉芽腫中藍染明顯減少,提示膠原纖維沉積較少。5.肝臟免疫組化染色結(jié)果顯示：與感染12周組和PZQ治療組相比,PZQ+CLC治療組小鼠肝臟中Collagen Ⅰ、CollagenⅢ、α-SMA、MMP-9和TIMP-1表達減少。6. RT-PCR檢測肝臟纖維化相關(guān)因子結(jié)果顯示：與感染12周組和PZQ治療組相比,IZQ+CLC治療組小鼠肝臟肝纖維化相關(guān)因子Co11α1、Col3α1、α-SMA、 MMP-9、TIMP-1 mRNA水平均顯著下降,且MP-9/TIMP-1比例上調(diào)。7. RT-PCR檢測肝臟炎性細胞因子結(jié)果顯示：感染6周組小鼠肝臟局部的促炎因子TNF-α、IL-1β和IL-6的mRNA水平均顯著高于正常組和感染12周組；與感染12周組和PZQ治療組相比,PZQ+CLC治療組小鼠肝臟局部促炎因子(TNF-α、IL-1β和IL-6)及促肝纖維化細胞因子(TGF-β、IL-4和IL-13) mRNA水平均顯著下降。8. RT-PCR檢測肝臟miRNA結(jié)果顯示：感染6周組小鼠肝臟中起促炎作用的miR-155和起抑炎作用的miR-146a的mRNA水平均顯著高于正常組和感染12周組；與感染12周組和PZQ治療組相比,PZQ+CLC治療組小鼠肝臟miR-155和miR-146a的m RNA水平降低,且趨于正常水平。感染6周組小鼠肝臟中起負向調(diào)控COX-2作用的miR-199a和miR-101a的mRNA水平均顯著低于正常組和感染12周組；與感染12周組和PZQ治療組相比,PZQ+CLC治療組小鼠肝臟miR-199a和miR-101a的mRNA水平升高。綜上所述,本研究明確了COX-2在日本血吸蟲病中的表達特征,并且COX-2抑制劑塞來昔布能夠緩解病原學(xué)治療基礎(chǔ)上的日本血吸蟲病肝纖維化,這為血吸蟲病的防治提供了先導(dǎo)性的實驗論據(jù)。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R532.32;R575.2

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