日本血吸虫感染改善糖尿病鼠肝脏胰岛素敏感性及其机制的初探

发布时间：2018-08-16 14:35

【摘要】：血吸虫病是一种人兽共患寄生虫病,其主要病理机制虫卵到达宿主肝脏组织引发的局部慢性炎症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。一直以来,人们认为血吸虫对人体是有害无益的,但相关研究显示,血吸虫除对机体造成一定损伤外,也能降低一些自身免疫性疾病和代谢性疾病的发病率。慢性炎症反应与肥胖等因素引起的胰岛素抵抗和2型糖尿病等代谢性疾病的发生和发展联系紧密。有研究显示,曼氏血吸虫感染或注射其虫卵抗原均可通过诱导2型免疫反应来改善宿主脂肪组织及肝脏中的胰岛素敏感性。日本血吸虫和曼氏血吸虫在发病机制、感染过程、所致的病理变化等方面具有许多相似点。因此,我们推测日本血吸虫感染亦可改善2型糖尿病患者全身及局部的胰岛素敏感性。本研究以BALB/c小鼠、C57BL/6小鼠和Lepr~(db/db)小鼠为研究对象,建立日本血吸虫感染模型,初步探索血吸虫感染对宿主全身及肝脏胰岛素敏感性的改善作用。本研究技术路线如下:以40条日本血吸虫尾蚴感染小鼠来建立感染模型。本研究共设计6个实验组,即BALB/c小鼠对照组(BALB/c-con)、BALB/c小鼠感染组(BALB/c-inf)、C57BL/6小鼠对照组(C57BL/6-con)、C57BL/6小鼠感染组(C57BL/6-inf)以及Lepr~(db/db)小鼠对照组(Lepr~(db/db)-con)、Lepr~(db/db)小鼠感染组(Lepr~(db/db)-inf)。我们采用如下实验方法对葡萄糖耐量、胰岛素敏感性、外周血胰岛素浓度、炎性细胞因子及相关的miRNAs等指标进行了检测,包括GTT检测葡萄糖耐量情况、ITT检测胰岛素敏感性、自动生化分析仪检测高密度脂蛋白(HDL-L)、低密度脂蛋白(LDL-L)、甘油三酯(TG)、总胆固醇(TC)水平、ELISA检测外周血胰岛素浓度、HE染色评价肝脏炎症程度、油红染色判断肝脏脂肪沉积情况、荧光实时定量PCR检测肝脏炎症基因及相关miRNA的表达水平等,以期初步探索血吸虫感染对宿主肝脏胰岛素敏感性的改善作用及相关的机制。本研究主要结果如下:1.肝脏miRNA基因芯片结果显示:与对照组小鼠相比,日本血吸虫感染组小鼠肝脏miR-155、miR-146b、miR-802等多种miRNAs的表达水平发生显著改变。2.RT-PCR检测肝脏miR-802及其靶基因Hnf1b结果显示:感染日本血吸虫后,小鼠肝脏miR-802表达降低,其靶基因Hnf1b表达量升高。由此推测,日本血吸虫感染抑制宿主肝脏miR-802表达,可弱化其对靶基因Hnf1b的抑制作用。3.GTT、ITT结果显示:感染日本血吸虫后,糖尿病小鼠(Lepr~(db/db))的葡萄糖耐量和胰岛素敏感性均有显著改善。4.肝脏油红染色结果显示:Lepr~(db/db)小鼠与正常组小鼠相比,感染组肝脏切片中显示脂滴的红染面积与密度明显减少,提示肝脏中脂肪沉积减少。5.ELISA结果显示:日本血吸虫感染后的Lepr~(db/db)小鼠和C57BL/6小鼠外周血胰岛素浓度均低于各自对照组。6.自动生化分析仪检测结果显示:与对照组小鼠相比,感染小鼠血清HDL-L、LDL-L、TC、TG水平显著下降。7.RT-PCR检测miR-802和靶基因结果显示:感染6周组后,两组小鼠肝脏局部的miR-802表达量低于各自对照组,其靶基因Hnf1b表达量升高,在Lepr~(db/db)小鼠中变化更加显著。8.RT-PCR检测肝脏炎性细胞因子结果显示:感染6周组后,两组小鼠肝脏局部的IL-22、IL-4、TNF-α和IL-6的m RNA水平显著高于对照组,在Lepr~(db/db)小鼠中变化更加显著。9.体外验证IL-22、TNF-α对肝细胞miR-802表达调控的结果显示:TNF-α可使小鼠肝细胞系miR-802表达量升高,IL-22可降低肝细胞miR-802表达水平。综上所述,本研究发现日本血吸虫感染可改善糖尿病鼠的胰岛素敏感性,通过负向调控肝脏局部miR-802的表达,弱化其对靶基因Hnf1b的抑制作用,以此改善肝脏局部胰岛素敏感性,为2型糖尿病的防治研究提供新的思路。
[Abstract]:Schistosomiasis is a zoonotic parasitic disease. Its main pathological mechanism is the local chronic inflammation caused by eggs reaching the host liver and the excessive repair of tissue damage caused by chronic infection, which leads to liver fibrosis. Chronic inflammation is closely related to the occurrence and development of insulin resistance and metabolic diseases such as type 2 diabetes caused by obesity and other factors. Studies have shown that Schistosoma mansoni infection or injection of egg antigens can reduce the incidence of some autoimmune and metabolic diseases. Insulin sensitivity in adipose tissue and liver can be improved by inducing type 2 immune response. Schistosoma japonicum and Schistosoma mansoni have many similarities in pathogenesis, infection process, pathological changes and so on. Therefore, we speculate that Schistosoma japonicum infection can also improve systemic and local pancreas in type 2 diabetes mellitus. In this study, BALB / C mice, C57BL / 6 mice and Lepr ~ (db / db) mice were used to establish a model of Schistosoma japonicum infection, and to explore the effect of Schistosoma japonicum infection on the insulin sensitivity of the whole body and liver of the host. In this study, six experimental groups were designed, namely BALB/c mice control group (BALB/c-con), BALB/c mice infection group (BALB/c-inf), C57BL/6 mice control group (C57BL/6-con), C57BL/6 mice infection group (C57BL/6-inf) and Lepr ~ (db/db) mice control group (Lepr ~ (db/db) - con, Lepr ~ (db/db) mice infection group (db/db) - inf. Methods Glucose tolerance, insulin sensitivity, peripheral blood insulin concentration, inflammatory cytokines and related microRNAs were measured, including GTT, ITT, high density lipoprotein (HDL-L), low density lipoprotein (LDL-L), triglyceride (TG), total. Cholesterol (TC) level, serum insulin concentration detected by ELISA, liver inflammation evaluated by HE staining, liver fat deposition detected by oil red staining, and liver inflammation gene and related microRNA expression detected by real-time fluorescence quantitative PCR were used to explore the role and phase of schistosomiasis infection in improving insulin sensitivity of host liver. The main results of this study are as follows: 1. Compared with the control mice, the expression levels of multiple microRNAs in the liver of mice infected with Schistosoma japonicum, such as microRNAs-155, microRNAs-146b, microRNAs-802 and so on, were significantly changed. 2. Detection of liver microRNAs-802 and its target gene Hnf1b by RT-PCR showed that: after infection with Schistosoma japonicum, the expression levels of microRNAs in the liver of mice infected with Schistosoma japonicum It was speculated that Schistosoma japonicum infection could inhibit the expression of hepatic microRNA-802 and weaken its inhibitory effect on the target gene Hnf1b. 3. GTT, ITT results showed that the glucose tolerance and insulin sensitivity of diabetic mice (Lepr ~ (db / db)) infected with Schistosoma japonicum were both increased. The results of liver oil red staining showed that the area and density of lipid droplets in liver slices of infected mice were significantly reduced compared with those of normal mice, suggesting a reduction of lipid deposition in liver. 5. The results of ELISA showed that the levels of insulin in peripheral blood of Lepr ~ (db / db) mice and C57BL / 6 mice infected with Schistosoma japonicum were high. The results of automatic biochemical analyzer showed that the levels of HDL-L, LDL-L, TC and TG in serum of infected mice were significantly lower than those of control group. 7. RT-PCR detection of microarray-802 and target gene showed that after 6 weeks of infection, the expression of microarray-802 in liver of infected mice in both groups was lower than that of control group, and the expression of Hnf1b was lower than that of control group. The levels of IL-22, IL-4, TNF-alpha and IL-6 m RNA in the liver of the mice infected with Lepr~ (db/db) were significantly higher than those of the control group after 6 weeks of infection. The changes were more significant in Lepr~ (db/db) mice. 9. In vitro validation of IL-22, TNF-alpha on hepatocyte microRNA802 In conclusion, this study found that Schistosoma japonicum infection can improve insulin sensitivity in diabetic mice, and weaken its inhibition of target gene Hnf1b by negatively regulating the expression of local hepatic microRNAs-802. In order to improve the local insulin sensitivity of the liver, it will provide new ideas for the prevention and treatment of type 2 diabetes.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R532.21;R587.1

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