【摘要】：乙型肝炎病毒（Hepatitis B virus, HBV）的感染不僅可引起急、慢性病毒性肝炎（Acute and chronic hepatitis），而且還與肝硬化（Liver cirrhosis, LC）和原發(fā)性肝細(xì)胞癌（Hepatocellularcarcinoma, HCC）的發(fā)生、發(fā)展密切相關(guān)。我國是HBV感染的高發(fā)區(qū)，目前我國HBV感染者約有9300萬人，其中慢性乙型肝炎患者約2000萬人。乙型肝炎病毒可分為A到J共10個基因型，我國以B、C基因型占優(yōu)勢�；蛐筒町惒粌H影響e抗原的血清學(xué)轉(zhuǎn)換、前C區(qū)啟動子和前C區(qū)的突變位點、疾病的發(fā)展進(jìn)程，還與抗病毒療效有關(guān)�；蚍中驮\斷有助于臨床醫(yī)生針對患者制定個體化的治療方案。 為了克服目前HBV基因分型法存在的檢測時間長、靈敏度低、特異性差等缺點，我們將微流控芯片、巨磁阻傳感器、磁性納米團(tuán)簇、環(huán)介導(dǎo)等溫擴(kuò)增（Loopmediatedisothermal amplification, LAMP）以及線性探針雜交技術(shù)相結(jié)合，建立了一種快速區(qū)分中國HBV優(yōu)勢基因型B和C的微流控芯片檢測方法，，它集樣品混合、核酸擴(kuò)增和信號采集等多功能于一體，具有檢測時間短、靈敏度高、特異性以及抗干擾能力強(qiáng)等優(yōu)點。 本論文首先建立了一種集成巨磁阻傳感器、微流控芯片、磁性納米團(tuán)簇、PCR擴(kuò)增和核酸雜交于一體的乙型肝炎病毒基因分型檢測方法。172nm左右的磁性納米團(tuán)簇表面修飾有鏈霉親和素，可與“PCR產(chǎn)物——核酸探針”雜交復(fù)合物末端的生物素發(fā)生特異性結(jié)合，GMR傳感器可對停留其表面的磁性納米團(tuán)簇進(jìn)行檢測，檢測靈敏度初步達(dá)到200IU·mL-1（103copies·mL-1）。隨后，又對該檢測方法進(jìn)行了優(yōu)化升級：我們將環(huán)介導(dǎo)等溫擴(kuò)增技術(shù)集成在微流控芯片中，進(jìn)行核酸樣品的高效擴(kuò)增。同時，GMR傳感器不再集成在微流控芯片中，而是作為獨立檢測器以供重復(fù)使用。最終可在一小時內(nèi)實現(xiàn)最低10copies·mL-1HBV DNA的基因分型檢測。此后，為了在微流控芯片上實現(xiàn)核酸擴(kuò)增前核酸樣本與反應(yīng)試劑的快速高效混合，我們基于仿生學(xué)的原理，設(shè)計了一種由高深寬比的直通道單元與高寬深比的圓形室單元重復(fù)交替相連而成的微混合器。 此外，為了評估氧化石墨烯作為HBV疫苗免疫佐劑的可能性，我們通過體外細(xì)胞實驗分別研究了氧化石墨烯（Grapheneoxide,GO）和經(jīng)過PVP修飾的氧化石墨烯（PVP-GO）對樹突狀細(xì)胞、T淋巴細(xì)胞、巨噬細(xì)胞等主要免疫細(xì)胞的影響。結(jié)果表明，氧化石墨烯經(jīng)PVP修飾后，其免疫學(xué)毒性明顯降低，并具有一定的免疫增強(qiáng)作用，因此，PVP-GO可以作為候選的免疫佐劑。
[Abstract]:Hepatitis B virus (Hepatitis B virus, HBV) infection can not only cause acute and chronic viral hepatitis (Acute and chronic hepatitis), but also be closely related to the occurrence and development of liver cirrhosis (Liver cirrhosis, LC) and primary hepatocellular carcinoma (Hepatocellularcarcinoma, HCC). China is a high incidence area of HBV infection. At present, there are about 93 million people infected with HBV in our country, including 20 million patients with chronic hepatitis B. Hepatitis B virus can be divided into 10 genotypes (A to J). Genotypic differences not only affect the serological transformation of e antigen, the mutation sites of pre C region promoter and pre C region, and the progression of disease, but also relate to the antiviral effect. Genotyping diagnosis helps clinicians develop individualized treatments for patients. In order to overcome the disadvantages of HBV genotyping, such as long detection time, low sensitivity and poor specificity, we used microfluidic chips, giant magnetoresistive sensors and magnetic nanoclusters. A microfluidic chip method for rapid identification of dominant genotypes B and C of Chinese HBV was developed by combining the ring mediated isothermal amplification of (Loopmediatedisothermal amplification, LAMP) with linear probe hybridization. Nucleic acid amplification and signal acquisition have many advantages, such as short detection time, high sensitivity, specificity and strong anti-interference ability. In this paper, an integrated giant magnetoresistive sensor (GMR), a microfluidic chip, is proposed. Detection of Hepatitis B virus genotyping by PCR Amplification and nucleic Acid Hybridization of Magnetic Nanoclusters. The surface of magnetic nanoclusters, about 172nm, was modified with streptavidin. The biosensor can be used to detect magnetic nanoclusters at the end of the hybrid complex "PCR product-nucleic acid probe". The detection sensitivity is up to 200IU mL-1 (103copies mL-1). Subsequently, the detection method was optimized and upgraded: we integrated the ring mediated isothermal amplification technique into microfluidic chip to amplify the nucleic acid samples efficiently. At the same time, the GMR sensor is no longer integrated in the microfluidic chip, but is used as an independent detector for reuse. Finally, genotyping of the lowest 10copies mL-1HBV DNA can be achieved within an hour. Since then, in order to achieve rapid and efficient mixing of nucleic acid samples and reaction reagents on microfluidic chips, we have based on the principle of bionics. A micromixer consisting of a straight channel unit with a high aspect ratio and a circular cell unit with a high aspect ratio is designed. In addition, in order to evaluate the possibility of graphene oxide as an immune adjuvant for HBV vaccine, we studied the effects of graphene oxide (go) and PVP modified graphene oxide (PVP-GO) on dendritic cell T lymphocytes in vitro. The effect of major immune cells such as macrophages. The results showed that the immunological toxicity of graphene oxide modified by PVP was obviously decreased and its immunological enhancement was also found. Therefore, PVP-GO could be used as a candidate immune adjuvant.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.62

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