CX3CR1基因缺失对日本血吸虫病肝脏肉芽肿的作用及机理的研究

发布时间：2018-06-21 00:12

本文选题：CX3CR1 + 血吸虫肉芽肿　；参考：《长江大学》2014年硕士论文

【摘要】：血吸虫病目前依然广泛流行于亚洲、非洲、拉丁美洲。具世界卫生组织统计,全世界受血吸虫感染的国家或地区一共有74个。全球估计有2亿人感染,6亿人受到血吸虫感染的威胁。血吸虫病是一种免疫性疾病的的概念已经被广泛认可。血吸虫病是一种严重威胁人类身体健康的寄生虫病,而我国则是日本血吸虫病流行最严重的国家之一。血吸虫病对机体的损害主要是肝脏,而主要致病因子是沉积于肝脏的虫卵释放的可溶性虫卵抗原(soluble egg antigen, SEA)。宿主对SEA产生免疫应答,形成虫卵肉芽肿、继发肝纤维化。因此在血吸虫病中,肝纤维化、门脉高压及静脉曲张出血是最主要的病理改变,并造成严重的肝、脾疾病。从免疫学的角度来讲,血吸虫病是一种细胞和体液免疫共同参与的虫卵肉芽肿性疾病。招募大量的巨噬细胞到达肉芽肿病变部位对于血吸虫病肉芽肿的形成和发展起到了重要的作用。而趋化因子受体CX3CR1在巨噬细胞的趋化和粘附过程中发挥着重要作用,尽管在不同的临床疾病和动物模型中其机制可能有所不同。最近的免疫学研究证明：在慢性炎症反应中,阻断CX3CR1的表达是一个非常有前途的治疗手段,在应用CX3CR1的拮抗剂后,在人和小鼠身上都显示出了抗炎的作用。通过阻断CX3CR1在巨噬细胞表面的表达,可以有效的达到减少巨噬细胞在组织炎症部位的浸润。可以预见,阻断和破坏CX3CR1信号通路,将成为治疗血吸虫病肉芽肿的一个新策略。本研究中,研究者观察到小鼠在感染日本血吸虫后,肝脏CX3CR1水平有明显的升高。于是,研究者利用CX3CR1基因缺失的小鼠建立的血吸虫病模型,来观察CX3CR1缺失后对小鼠肝脏肉芽肿形成的影响。在感染日本血吸虫8周后,与野生型小鼠组相比,CX3CR1基因缺失小鼠体重没有明显丢失,肝脏肿大、脾脏肿大、结肠肿胀均减轻,同时肝脏功能也得到了有效的保护。病理学研究发现,CX3CR1基因敲除小鼠,肉芽肿周围的巨噬细胞浸润数量明显减少,肉芽肿的面积减小。在体外细胞实验中,同等条件下,使用血吸虫可溶性虫卵抗原刺激CX3CR1基因缺失巨噬细胞与野生型巨噬细胞,96小时后,发现CX3CR1基因缺失的巨噬细胞肿瘤坏死因子-a (TNF-a)、TGF-β诱导型一氧化氮(iNOS)的表达均减少,而与此同时,精氨酸酶-1(Arg-1)和白细胞介素-10(IL-10)的表达增加,并使巨噬细胞更多的向M2型巨噬细胞(F4/80+CDllb+CD206+)转化。在细胞信号通路的机制研究中,研究者发现,CX3CR1基因的缺失加强了巨噬细胞STAT-6的磷酸化和上调了其下游分子PPAR-r的表达。研究者的结果显示,CX3CR1基因的敲除,可以有效的减轻血吸虫肉芽肿的病理过程,其主要作用机制是CX3CR1基因的缺失减少了巨噬细胞在肉芽肿周边的侵入以及促进炎症部位的巨噬细胞更多的向M2型巨噬细胞转化。上述结果不仅为我们阐明CX3CR1在日本血吸虫肉芽肿形成中的作用提供了重要的依据,同时也为临床治疗血吸虫病提供了一个新思路。目的：研究小鼠感染日本血吸虫后,趋化因子受体CX3CR1的缺失,对肝脏肉芽肿形成的影响。通过动物及细胞实验,观察动物标本的病理改变,从免疫学的角度阐明其分子生物学发生机制,并为今后进一步以评估以CX3CR1为基础的后续实验和临床研究提供依据。方法：使用鼠龄6周的C57BL/6小鼠和CX3CR1基因缺失的C57BL/6小鼠,通过经腹壁皮肤感染血吸虫尾蚴的方法感染血吸虫。感染8周后,取肝组织行HE染色,计算肝内血吸虫肉芽肿的面积。应用免疫荧光双染技术定性分析巨噬细胞在肉芽肿周边浸润的数量。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细胞,经日本血吸虫虫卵中提取的血吸虫虫卵抗原刺激96小时后,应用酶联免疫吸附测定方法(ELISA)测定上清中TNF-a, IL-4,IL-10, TGF-β含量。应用蛋白质免疫印迹方法(Western blot)检测了细胞中CX3CR1, iNOS, Arg-1的表达,以及巨噬细胞信号通路上的蛋白质分子STAT-6、磷酸化的STAT-6及其下游蛋白质分子PPAR-r的表达。应用流式细胞术检测巨噬细胞亚型M1,M2的表面标记并计算其在总细胞群中的比例。结果使用平均值±标准误差(SEM)。统计学分析的数据使用t检验。p0.05具有统计学意义。结果：(1)在日本血吸虫感染8周后,通过活体样本表型的观察发现,CX3CR1基因缺失的小鼠在感染后机体获得有效的保护：体重没有下降,肝脏肿大、脾脏肿大、结肠肿胀均减轻,同时有效的保护了肝脏的功能,具有统计学意义(P0.05)。(2) CX3CR1基因缺失后,使得招募炎性细胞的信号通路受损,降低了血吸虫虫卵沉积后形成的肉芽肿部位炎性细胞的浸润数目,特别是减少了巨噬细胞的浸润。在野生型小鼠组和CX3CR1基因缺失小鼠组中具有统计学意义(P0.05)。(3)在日本血吸虫感染后,CX3CR1基因敲除的小鼠的肝脏肉芽肿明显减小,其主要特点是促使炎症部位的免疫反应更多的向Th2极化。在野生型小鼠组和CX3CR1基因缺失小鼠组中具有统计学意义(P0.05)。(4)在日本血吸虫虫卵抗原的刺激实验中,趋化因子受体CX3CR1的缺失,与野生型巨噬细胞相比,使更多的巨噬细胞向M2型极化。在野生型小鼠组和CX3CR1基因缺失小鼠组中具有统计学意义(P0.05)。(5) CX3CR1基因缺失的巨噬细胞与野生型巨噬细胞相比,上调了信号通路IL-4(CD206)-STAT6-PPAR-r分子蛋白质的表达,两组蛋白质的表达具有显著性差异(P0.05)。结论：与野生型小鼠相比,CX3CR1的敲除,使感染日本血吸虫后小鼠血吸虫肉芽肿的病理过程明显减轻,其主要作用机制是减少了巨噬细胞在肉芽肿周边的侵入以及促进炎症部位的巨噬细胞更多的向M2型巨噬细胞极化。这些数据不仅使我们在理解CX3CR1在日本血吸虫肉芽肿形成中的作用提供了重要的数据,同时也为治疗日本血吸虫病提供了一个重要的思路。
[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.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R532.21

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