乙肝、肝硬化、肝癌患者与流感病毒易感性关联的研究

发布时间：2018-01-13 10:34

本文关键词：乙肝、肝硬化、肝癌患者与流感病毒易感性关联的研究　出处：《西北大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景及意义：研究表明,流感病毒感染宿主的过程中,主要是通过血凝素(HA)与宿主细胞表面唾液酸糖链受体进行特异性的结合。人流感病毒HA主要通过识别并结合SA α2-6Gal的糖链受体,而禽流感病毒HA主要识别并结合SA α2-3Gal的糖链受体。已有研究报道,在人呼吸道上皮细胞表面含有着SA α2-6Gal糖链末端结构和少量的SAα2-3Gal糖链末端结构。唾液中含有丰富的糖蛋白,它们具有抗菌、抗病毒以及润滑口腔等作用。近年来有研究显示,疾病的发生涉及到了唾液糖蛋白糖链结构的改变。唾液是防止呼吸道病毒进入人体的首要防线。其中,唾液中的糖蛋白糖链末端结构在抵御病毒中起着决定性的作用,其可以与病毒特异性的结合而达到防御目的。本实验室研究发现,健康志愿者中,老年人与儿童、年轻人相比较,老年人具有更强抵抗流感的能力。其主要分子机制之一,是由于老年人唾液与儿童、年轻人唾液相比较,具有更多的SAα2-3/SA α2-6Gal糖链末端结构。这些糖链末端结构可以结合更多的流感病毒血凝素(HA)。但流感爆发时,临床上发现患者多为患有慢性疾病老年人?其原因是慢性疾病破坏了老年人的免疫系统,使其抵抗力降低。除此原因外,是否有其他生理或病理的因素导致老年人更易感流感病毒?乙肝、肝硬化和肝癌是常见的几种慢性疾病,严重威胁着人类的健康,患有这几种慢性疾病的人群是否更易感流感病毒?感染的特性又是什么?这是本课题的立题依据,也是公共卫生领域亟需解决的一些问题。方法：首先,收集乙肝、肝硬化、肝癌患者和健康志愿者唾液样本,然后通过凝集素芯片技术,对识别SAα2-6Gal糖链末端结构的凝集素SNA和识别SAα2-3Gal糖链末端结构MAL-II所对应的的荧光信号值进行分析、比较,最终得到乙肝、肝硬化、肝癌患者和健康志愿者唾液中SAα2-6Gal和SAα2-3Gal糖蛋白糖链末端结构表达水平的差异性。随后,利用印记技术,将凝集素SNA、MAL-Ⅱ和鸡源H5N1流感病毒、鸭源H5N1流感病毒、鸡源H9N2流感病毒以及甲型H1N1流感病毒裂解疫苗分别与乙肝、肝硬化、肝癌患者和健康志愿者的唾液样本进行印记实验。根据实验结果,对乙肝、肝硬化、肝癌患者与流感病毒易感性关联进行分析讨论。结果：1.应用凝集素芯片技术,对乙肝、肝硬化、肝癌患者和健康志愿者唾液进行检测,分析其中唾液酸化糖蛋白糖链结构表达水平变化的情况。通过分析乙肝、肝硬化和肝癌患者样本的荧光信号值,并将结果与健康志愿者唾液样本荧光信号值进行比对,从而得出比值Ratio值。结果显示,在乙肝、肝硬化和肝癌患者的唾液样本中,凝集素MAL-Ⅱ对应的Ratio值分别为0.423、0.437和0.479。因此,MAL-II所识别的SAα2-3Gal糖链末端结构在乙肝、肝硬化和肝癌患者唾液中的表达水平明显下降。而在乙肝、肝硬化和肝癌患者唾液中,凝集素SNA对应的Ratio值分别为2.294、1.409和1.336,因此,其所识别的SAα2-6Gal糖链末端结构在乙肝、肝硬化和肝癌患者唾液中的表达水平明显上调。 2.应用印记技术,将鸡源H5N1流感病毒、鸭源H5N1流感病毒和鸡源H9N2流感病毒,分别与乙肝、肝硬化、肝癌患者和健康志愿者的唾液样本相结合。通过三种禽流感病毒与唾液糖蛋白结合的印记条带,可以明显观察到：鸭源H5N1流感病毒、鸡源H5N1流感病毒和鸡源H9N2流感病毒与乙肝、肝硬化以及肝癌患者的唾液糖蛋白的结合能力明显要弱于与健康志愿者的结合能力。并将其结果与凝集素MAL-Ⅱ与乙肝、肝硬化、肝癌患者和健康志愿者的唾液糖蛋白结合的印记条带作对比,发现三种禽流感病毒的印迹条带与其保持一致。说明在乙肝、肝硬化以及肝癌患者唾液糖蛋白中,禽流感病毒HA特异性识别、结合的的SAα2-3Gal糖链末端结构表达水平较低。而凝集素SNA与乙肝、肝硬化以及肝癌患者唾液蛋白的结合能力强于与健康志愿者的结合能力,说明在乙肝、肝硬化以及肝癌患者唾液蛋白中,人流感病毒表面HA特异识别、结合的SAα2-6Gal糖链末端结构表达水平较高。同时,甲型H1N1流感病毒裂解疫苗(甲型H1N1流感病毒株(2009),包含有禽流感、猪流感和人流感三种流感病毒的核糖核酸基因片断,同时识别禽流感病毒糖链受体和人流感病毒糖链受体)与四种唾液蛋白样本的结合能力无显著性差异。因此结果表明,乙肝、肝硬化和肝癌患者属于禽流感病毒易感人群,易感禽流感；对人流感病毒有正常的抵抗力。
[Abstract]:The research background and significance: the study shows that the process of influenza virus infection in the host, mainly through the hemagglutinin (HA) combined with specific host cell surface sialic acid sugar chain receptor. The human influenza virus HA by recognizing and binding to the sugar chain SA receptor alpha 2-6Gal, and avian influenza virus HA mainly recognize and bind sugar chain SA receptor alpha 2-3Gal. It has been reported that on the surface of human airway epithelial cells containing the SA alpha 2-6Gal sugar chain end structure and a small amount of SA alpha 2-3Gal sugar chain structure at the end.
The saliva contains rich glycoprotein, antibacterial, antiviral and lubricating oral effects. In recent years studies have shown that the occurrence of diseases related to salivary glycoprotein glycan structure change. Saliva is to prevent respiratory virus into the first line of the body. The glycoprotein glycan structure at the end of saliva in the decisive role in resisting the virus, the virus can be combined with specific to defensive purposes.
This laboratory study found that in healthy volunteers, the elderly and children, young people compared to older people with stronger ability to resist the flu. One of the major molecular mechanism is due to the elderly and young children saliva, saliva compared with more SA alpha 2-3/SA alpha 2-6Gal sugar chain structure at the end of the sugar chain. Terminal structure can be combined with the influenza virus hemagglutinin (HA). But more of an influenza outbreak, clinically found patients are suffering from chronic diseases in elderly people? The reason is that chronic disease destroys the immune system of the elderly, so that the resistance decreased. In addition to this reason, if there are other factors of physiological or pathological in the elderly people are more susceptible to the flu virus? Hepatitis B, cirrhosis and HCC are several common chronic diseases, serious threat to human health, it suffers from several chronic diseases in populations are more susceptible to influenza virus infection characteristics? What is it? This is the basis of this topic, and it is also an urgent problem to be solved in the field of public health.
Method: first, collect hepatitis B, liver cirrhosis, liver cancer patients and healthy volunteers saliva samples, followed by lectin microarray technology, to identify the SA alpha 2-6Gal sugar chain structure at the end of the lectin SNA and identification of SA alpha 2-3Gal sugar chain structure at the end of MAL-II corresponding to the value of the fluorescence signal for analysis, comparison, finally get hepatitis B, cirrhosis of the liver. The level of expression of SA and SA alpha 2-6Gal alpha 2-3Gal glycoprotein glycan structure at the end of HCC patients and healthy volunteers in the saliva. Then, using imprint technology, the lectin SNA, MAL- II and H5N1 avian influenza virus H5N1 influenza virus, duck, chicken H9N2 influenza virus and influenza a H1N1 and Influenza Split Vaccine were hepatitis B cirrhosis HCC patients and healthy volunteers, saliva samples mark experiment. According to the experimental results of hepatitis B, liver cirrhosis, liver cancer and influenza virus susceptibility association analysis Discuss.
Results: 1. cirrhosis by lectin microarray technology for hepatitis B, liver cancer patients and healthy volunteers saliva were detected, analyzed the expression of sialylated glycoprotein glycan structure. Through the analysis of hepatitis B, cirrhosis and hepatocellular carcinoma samples of fluorescence signal values, and the results of healthy volunteers with a saliva sample fluorescence signal value comparison so that the ratio of Ratio value. The results showed that in hepatitis B, cirrhosis and liver cancer with the saliva samples, the corresponding Ratio II lectin MAL- values were 0.423,0.437 and 0.479. so SA alpha 2-3Gal sugar chain structure at the end of MAL-II identified in the saliva of patients with hepatitis B, the expression level of liver cirrhosis and hepatocellular carcinoma in hepatitis B decreased significantly. Saliva, liver cirrhosis and liver cancer patients, lectin SNA Ratio values were 2.294,1.409 and 1.336, therefore, at the end of the SA alpha 2-6Gal sugar chain recognition The expression of the structure in the saliva of the patients with hepatitis B, liver cirrhosis and liver cancer was obviously up-regulated.
2. application of imprinting, the chicken origin H5N1 influenza virus, influenza virus and duck H5N1 avian H9N2 influenza virus, and hepatitis B cirrhosis, respectively, HCC patients and healthy volunteers saliva samples combined. Through the combination of a mark of three avian influenza virus and salivary glycoprotein bands can be observed clearly: duck origin influenza H5N1 virus, influenza virus and hepatitis B avian H5N1 influenza virus and chicken H9N2, cirrhosis and hepatocellular carcinoma patients with salivary glycoprotein binding ability was weaker than the binding ability and healthy volunteers. And the results of the lectin MAL- II and hepatitis B, liver cirrhosis, liver cancer patients and healthy volunteers with salivary glycoprotein bands as mark contrast, found three bird flu virus was consistent with the band. In hepatitis B, cirrhosis and HCC saliva glycoprotein, HA avian influenza virus specific identification, node SA alpha 2-3Gal sugar chain structure at the end of the low expression of lectins SNA and hepatitis B, cirrhosis and liver cancer patients with salivary protein ability in healthy volunteers with the binding capability in hepatitis B, cirrhosis and HCC saliva protein, people sense virus surface HA specific recognition, SA alpha 2-6Gal sugar end of the chain structure with higher expression level. At the same time, Influenza Split Vaccine (A / H1N1 influenza a H1N1 influenza virus strain (2009), including avian influenza, DNA fragments of three influenza virus swine and human influenza, and identification of avian influenza virus and human influenza virus receptor carbohydrate carbohydrate receptor) had no significant difference and combining ability of four kinds of salivary protein samples. The results showed that hepatitis B, cirrhosis and liver cancer patients belonging to avian influenza virus susceptible, susceptible to avian influenza; a normal flu virus on abortion Resistance.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R511.7;R735.7;R512.62

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