本文選題：CX3CR1 + 血吸蟲肉芽腫��； 參考：《長江大學》2014年碩士論文

【摘要】：血吸蟲病目前依然廣泛流行于亞洲、非洲、拉丁美洲。具世界衛(wèi)生組織統(tǒng)計,全世界受血吸蟲感染的國家或地區(qū)一共有74個。全球估計有2億人感染,6億人受到血吸蟲感染的威脅。血吸蟲病是一種免疫性疾病的的概念已經(jīng)被廣泛認可。血吸蟲病是一種嚴重威脅人類身體健康的寄生蟲病,而我國則是日本血吸蟲病流行最嚴重的國家之一。血吸蟲病對機體的損害主要是肝臟,而主要致病因子是沉積于肝臟的蟲卵釋放的可溶性蟲卵抗原(soluble egg antigen, SEA)。宿主對SEA產(chǎn)生免疫應答,形成蟲卵肉芽腫、繼發(fā)肝纖維化。因此在血吸蟲病中,肝纖維化、門脈高壓及靜脈曲張出血是最主要的病理改變,并造成嚴重的肝、脾疾病。 從免疫學的角度來講,血吸蟲病是一種細胞和體液免疫共同參與的蟲卵肉芽腫性疾病。招募大量的巨噬細胞到達肉芽腫病變部位對于血吸蟲病肉芽腫的形成和發(fā)展起到了重要的作用。而趨化因子受體CX3CR1在巨噬細胞的趨化和粘附過程中發(fā)揮著重要作用,盡管在不同的臨床疾病和動物模型中其機制可能有所不同。最近的免疫學研究證明：在慢性炎癥反應中,阻斷CX3CR1的表達是一個非常有前途的治療手段,在應用CX3CR1的拮抗劑后,在人和小鼠身上都顯示出了抗炎的作用。通過阻斷CX3CR1在巨噬細胞表面的表達,可以有效的達到減少巨噬細胞在組織炎癥部位的浸潤�？梢灶A見,阻斷和破壞CX3CR1信號通路,將成為治療血吸蟲病肉芽腫的一個新策略。 本研究中,研究者觀察到小鼠在感染日本血吸蟲后,肝臟CX3CR1水平有明顯的升高。于是,研究者利用CX3CR1基因缺失的小鼠建立的血吸蟲病模型,來觀察CX3CR1缺失后對小鼠肝臟肉芽腫形成的影響。在感染日本血吸蟲8周后,與野生型小鼠組相比,CX3CR1基因缺失小鼠體重沒有明顯丟失,肝臟腫大、脾臟腫大、結腸腫脹均減輕,同時肝臟功能也得到了有效的保護。病理學研究發(fā)現(xiàn),CX3CR1基因敲除小鼠,肉芽腫周圍的巨噬細胞浸潤數(shù)量明顯減少,肉芽腫的面積減小。在體外細胞實驗中,同等條件下,使用血吸蟲可溶性蟲卵抗原刺激CX3CR1基因缺失巨噬細胞與野生型巨噬細胞,96小時后,發(fā)現(xiàn)CX3CR1基因缺失的巨噬細胞腫瘤壞死因子-a (TNF-a)、TGF-β誘導型一氧化氮(iNOS)的表達均減少,而與此同時,精氨酸酶-1(Arg-1)和白細胞介素-10(IL-10)的表達增加,并使巨噬細胞更多的向M2型巨噬細胞(F4/80+CDllb+CD206+)轉化。在細胞信號通路的機制研究中,研究者發(fā)現(xiàn),CX3CR1基因的缺失加強了巨噬細胞STAT-6的磷酸化和上調了其下游分子PPAR-r的表達。研究者的結果顯示,CX3CR1基因的敲除,可以有效的減輕血吸蟲肉芽腫的病理過程,其主要作用機制是CX3CR1基因的缺失減少了巨噬細胞在肉芽腫周邊的侵入以及促進炎癥部位的巨噬細胞更多的向M2型巨噬細胞轉化。上述結果不僅為我們闡明CX3CR1在日本血吸蟲肉芽腫形成中的作用提供了重要的依據(jù),同時也為臨床治療血吸蟲病提供了一個新思路。 目的：研究小鼠感染日本血吸蟲后,趨化因子受體CX3CR1的缺失,對肝臟肉芽腫形成的影響。通過動物及細胞實驗,觀察動物標本的病理改變,從免疫學的角度闡明其分子生物學發(fā)生機制,并為今后進一步以評估以CX3CR1為基礎的后續(xù)實驗和臨床研究提供依據(jù)。 方法：使用鼠齡6周的C57BL/6小鼠和CX3CR1基因缺失的C57BL/6小鼠,通過經(jīng)腹壁皮膚感染血吸蟲尾蚴的方法感染血吸蟲。感染8周后,取肝組織行HE染色,計算肝內血吸蟲肉芽腫的面積。應用免疫熒光雙染技術定性分析巨噬細胞在肉芽腫周邊浸潤的數(shù)量。提取野生型C57BL/6小鼠和CX3CR1基因缺失的C57BL/6小鼠的腹腔原代巨噬細胞,經(jīng)日本血吸蟲蟲卵中提取的血吸蟲蟲卵抗原刺激96小時后,應用酶聯(lián)免疫吸附測定方法(ELISA)測定上清中TNF-a, IL-4,IL-10, TGF-β含量。應用蛋白質免疫印跡方法(Western blot)檢測了細胞中CX3CR1, iNOS, Arg-1的表達,以及巨噬細胞信號通路上的蛋白質分子STAT-6、磷酸化的STAT-6及其下游蛋白質分子PPAR-r的表達。應用流式細胞術檢測巨噬細胞亞型M1,M2的表面標記并計算其在總細胞群中的比例。結果使用平均值±標準誤差(SEM)。統(tǒng)計學分析的數(shù)據(jù)使用t檢驗。p0.05具有統(tǒng)計學意義。 結果：(1)在日本血吸蟲感染8周后,通過活體樣本表型的觀察發(fā)現(xiàn),CX3CR1基因缺失的小鼠在感染后機體獲得有效的保護：體重沒有下降,肝臟腫大、脾臟腫大、結腸腫脹均減輕,同時有效的保護了肝臟的功能,具有統(tǒng)計學意義(P0.05)。(2) CX3CR1基因缺失后,使得招募炎性細胞的信號通路受損,降低了血吸蟲蟲卵沉積后形成的肉芽腫部位炎性細胞的浸潤數(shù)目,特別是減少了巨噬細胞的浸潤。在野生型小鼠組和CX3CR1基因缺失小鼠組中具有統(tǒng)計學意義(P0.05)。(3)在日本血吸蟲感染后,CX3CR1基因敲除的小鼠的肝臟肉芽腫明顯減小,其主要特點是促使炎癥部位的免疫反應更多的向Th2極化。在野生型小鼠組和CX3CR1基因缺失小鼠組中具有統(tǒng)計學意義(P0.05)。(4)在日本血吸蟲蟲卵抗原的刺激實驗中,趨化因子受體CX3CR1的缺失,與野生型巨噬細胞相比,使更多的巨噬細胞向M2型極化。在野生型小鼠組和CX3CR1基因缺失小鼠組中具有統(tǒng)計學意義(P0.05)。(5) CX3CR1基因缺失的巨噬細胞與野生型巨噬細胞相比,上調了信號通路IL-4(CD206)-STAT6-PPAR-r分子蛋白質的表達,兩組蛋白質的表達具有顯著性差異(P0.05)。 結論：與野生型小鼠相比,CX3CR1的敲除,使感染日本血吸蟲后小鼠血吸蟲肉芽腫的病理過程明顯減輕,其主要作用機制是減少了巨噬細胞在肉芽腫周邊的侵入以及促進炎癥部位的巨噬細胞更多的向M2型巨噬細胞極化。這些數(shù)據(jù)不僅使我們在理解CX3CR1在日本血吸蟲肉芽腫形成中的作用提供了重要的數(shù)據(jù),同時也為治療日本血吸蟲病提供了一個重要的思路。
[Abstract]:Schistosomiasis is still widely prevalent in Asia, Africa, and Latin America. With WHO statistics, there are 74 countries and regions in the world infected by Schistosoma. It is estimated that 200 million people are infected and 600 million people are threatened by schistosomiasis. The concept of schistosomiasis is widely recognized as a disease free disease. Insect disease is a serious threat to human health, and China is one of the most serious countries in Japan. Schistosomiasis is mainly the liver, and the main pathogenic factor is the soluble egg antigen (soluble egg antigen, SEA) released from the eggs of the liver. The host is free from the SEA. Immune response, the formation of granuloma, secondary hepatic fibrosis in schistosomiasis. Therefore, hepatic fibrosis and portal hypertension and varices bleeding is the main pathological changes, and cause severe liver, spleen disease.
From an immunological point of view, schistosomiasis is an egg granulomatous disease associated with cell and humoral immunity. The recruitment of a large number of macrophages to the site of granulomatosis plays an important role in the formation and development of schistosomiasis granuloma, and chemokine receptor CX3CR1 is chemotactic and adhered to macrophages. The process plays an important role, although the mechanism may vary in different clinical diseases and animal models. Recent immunological studies have shown that blocking the expression of CX3CR1 in chronic inflammatory reactions is a very promising treatment. After the application of the antagonist of CX3CR1, the anti inflammation is shown in both human and mice. Effect by blocking CX3CR1 expression in macrophage, can effectively reduce the infiltration of macrophages in inflammatory sites. Predictably, blocking and destroy the CX3CR1 pathway, will become a new strategy for the treatment of schistosomiasis granuloma.
In this study, the researchers observed a significant increase in the liver CX3CR1 level in mice infected with Schistosoma japonicum. The researchers used the schistosomiasis model established in mice with CX3CR1 gene deletion to observe the effect of CX3CR1 deletion on the formation of liver granuloma in mice. After 8 weeks of infection with Schistosoma japonicum, it was associated with the wild type mice. CX3CR1 gene deletion mice had no significant loss of weight, liver enlargement, swelling of the spleen, colonic swelling all lessened, and the liver function was also effectively protected. Pathological study found that the number of macrophages around granuloma in CX3CR1 gene knockout mice decreased significantly, and the area of granuloma decreased. In vitro cell experiments, Under the same condition, 96 hours after the use of schistosomiasis soluble egg antigen to stimulate CX3CR1 gene missing macrophages and wild type macrophages, the CX3CR1 gene deletion of macrophage tumor necrosis factor -a (TNF-a) and the expression of TGF- beta inducible nitric oxide (iNOS) were reduced. At the same time, arginase -1 (Arg-1) and white blood cells were found. The expression of -10 (IL-10) increased and made macrophages more converted to M2 type macrophage (F4/80+CDllb+CD206+). In the mechanism of cell signaling pathway, the researchers found that the deletion of the CX3CR1 gene enhanced the phosphorylation of STAT-6 in macrophages and up the expression of the downstream molecule PPAR-r. The results of the researchers showed that the CX3CR1 gene was the CX3CR1 gene. The knockout can effectively reduce the pathological process of Schistosoma granuloma, the main mechanism of which is the deletion of CX3CR1 gene that reduces the invasion of macrophages around granuloma, and promotes the transformation of macrophages to M2 type macrophages in the inflammatory site. The results not only elucidate CX3CR1 in the granuloma of Schistosoma japonicum, but also of the granuloma of the Schistosoma japonicum. It provides an important basis for the formation and provides a new idea for clinical treatment of schistosomiasis.
Objective: To study the effect of the deletion of chemokine receptor CX3CR1 on the formation of liver granuloma after infection with Schistosoma japonicum in mice. Through animal and cell experiments, the pathological changes of animal specimens were observed and the mechanism of molecular biology was elucidated from the immunological point of view, and the follow-up experiments on the basis of CX3CR1 were evaluated for the future step. It provides a basis for clinical research.
Methods: C57BL/6 mice of 6 weeks of age and C57BL/6 mice with CX3CR1 gene deletion were infected with Schistosoma japonicum through the infection of schistosoma cercariae through the abdominal wall skin. After 8 weeks of infection, the liver tissue was stained with HE to calculate the area of Schistosoma granuloma in the liver. The immunofluorescence double staining technique was used to qualitatively analyze the infiltration of macrophages in the granuloma surrounding the granuloma. The primary peritoneal macrophages of the wild type C57BL/6 mice and the CX3CR1 gene deleted C57BL/6 mice were extracted. After 96 hours of stimulation of schistosomiasis egg antigen extracted from the eggs of Schistosoma japonicum, the content of TNF-a, IL-4, IL-10, and TGF- beta in the supernatant was determined by enzyme linked immunosorbent assay (ELISA). The application of protein immunoblotting was used. Method (Western blot) was used to detect the expression of CX3CR1, iNOS, Arg-1, protein molecule STAT-6, phosphorylated STAT-6 and the expression of the downstream protein molecule PPAR-r in the macrophage signaling pathway. The flow cytometry was used to detect the M1 of macrophage subtype, the surface marker of M2, and the proportion of the protein in the total cell group. Using mean value of + standard error (SEM). Statistical analysis of data using t test.P0.05 has statistical significance.
Results: (1) after 8 weeks of infection of Schistosoma japonicum, it was found that the mice with CX3CR1 gene deletion were effectively protected after the infection: the body weight did not decline, the liver was enlarged, the spleen was swollen and the colonic swelling was reduced, and the function of the liver was effectively protected (P0.05). (2) CX3CR1 After gene deletion, the signal pathway of recruitment of inflammatory cells was damaged, the number of infiltration of inflammatory cells in the granulomatous site of the schistosomiasis eggs was reduced, especially the infiltration of macrophages. In the wild type mice and the CX3CR1 gene deletion mice (P0.05). (3) infection in Schistosoma japonicum After the CX3CR1 gene knockout mice, the liver granuloma was significantly reduced, the main feature of which was to promote the immune response in the inflammatory site to more Th2 polarization. In the wild type mice and the CX3CR1 gene deletion mice (P0.05). (4) the chemokine receptor CX3CR1 in the stimulation experiment of the egg antigen of Schistosoma japonicum. More macrophages were polarized to M2 type than wild type macrophages. In wild type mice and CX3CR1 gene deletion mice (P0.05). (5) the expression of IL-4 (CD206) -STAT6-PPAR-r molecular protein in the signal pathway was up regulated by the macrophages missing from the CX3CR1 gene compared with the wild type macrophages. There was a significant difference in protein expression between the two groups (P0.05).
Conclusion: compared with the wild type mice, the knockout of CX3CR1 significantly alleviated the pathological process of the granuloma of Schistosoma japonicum infected by Schistosoma japonicum. The main mechanism was to reduce the invasion of macrophages around granuloma and to promote the polarization of macrophages to M2 type macrophages. Our role in understanding the formation of CX3CR1 in Schistosoma japonicum granuloma provides important data, but also provides an important method for the treatment of schistosomiasis.
【學位授予單位】：長江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R532.21

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