【作者】 廖文婷；

【导师】 柴逸峰； 潘卫；

【作者基本信息】 第二军医大学 ， 药物分析学， 2012， 博士

【摘要】 HIV-1Tat蛋白是病毒产生的重要调控蛋白和致病因子，在HIV-1的复制、扩散和致病中起重要作用。HIV-1感染靶细胞后，细胞质中合成的Tat可进入细胞核反式激活HIV-1RNA的转录，从而激活HIV-1的增殖。此外，Tat还可被感染细胞分泌到胞外发挥多种细胞外功能在致病中起重要作用，如抑制免疫细胞的增值、分化、诱导其凋亡；促进病毒播散；促进卡波式肉瘤肿瘤细胞的生长；诱发神经损伤等。鉴于其多种生物学活性，Tat被认为是“病毒毒素”，是药物干预和治疗性疫苗的重要靶标。多个研究发现，在HIV感染者中代谢紊乱综合症的发生率显著高于正常人群，如脂代谢异常、胰岛素抵抗、心脏代谢紊乱、磷酸代谢异常和损伤的葡萄糖耐受等。尽管已有研究发现多种危险因素，但其病因仍然不清楚。而有多种生物学功能的HIV-1Tat蛋白在其中所起的作用值得研究和探讨。代谢组学是自上而下的系统生物学方法，它是对一个生物体系中所有小分子代谢物进行定性定量分析的整体性方法，已广泛应用于疾病诊断及作用机理、药物毒理学、药效评价、基因功能及中药质量控制等领域。相对于转录组和蛋白组来说，其分析对象的理化性质更加多样，是一个非常复杂的分析体系。代谢物组中的化合物在性质（从强极性到非极性）和浓度（从mM到pM）上的巨大差异对现有的分析技术是一个重大的挑战。在当前的技术条件下仅凭一种分析技术很难建立无偏的全局的代谢组学分析方法。因此为了获得更加广泛的代谢物信息，近年来多种互补的分析方法相结合广泛应用到代谢组学研究中，如质谱（MS）和核磁共振（NMR）结合；气相色谱-质谱（GC-MS）和液相色谱-质谱结合（LC-MS）；亲水相互作用色谱-质谱（HILIC-MS）和反相液相色谱-质谱（RPLC-MS）相结合等。本研究以GC–MS、RPLC-MS和HILIC–MS联用技术结合化学计量学多元方法为手段，以HIV-1Tat为研究对象，从整体动物和细胞水平阐明代谢组学方法在HIV-1Tat致病机制中的应用。主要内容如下：一、 HIV-1Tat在大肠杆菌中的表达、纯化及生物活性鉴定在本课题组构建的HIV-1HXB2株Tat质粒的基础上，通过PCR方法合成HIV-1IIIB株BH10克隆Tat DNA序列，并构建其原核表达质粒tat-pET21b，转化到表达宿主菌BL21(DE3)。经IPTG诱导，得到的重组蛋白的表达量占细菌总蛋白的约15%；重组表达的Tat蛋白经Ni-NTA亲和柱和SP-Sepharose离子交换柱纯化，得到了纯度约90%的Tat蛋白。经Western鉴定在约15kD得到阳性的蛋白表达条带。鲎试剂盒检测内毒素LPS的含量约为0.05EU/μg，满足LPS限量要求。细胞模型上鉴定纯化的Tat蛋白保留了生物学活性。二、基于GC-MS技术的HIV-1Tat诱导的ICR小鼠的血清代谢组学研究Tat蛋白由感染的细胞释放，发挥多种生物学作用如破坏免疫系统、促进病毒播散和致病性。目前Tat蛋白对代谢物的影响还未见报道。本课题以对照小鼠血清和HIV-1Tat处理的小鼠血清为分析对象，研究建立了内源性代谢物谱的GC-MS分析方法，并利用NIST2002质谱数据库对检测到的代谢物进行快速鉴定。在综合考察供试品制备方法、衍生化条件和色谱分析条件的基础上，获取了一个峰容量大、分离效果佳的GC-MS分析条件，并对其进行了方法学验证。研究结果表明，所建立的GC-MS方法可较好地表征生物样本的代谢物谱特征，分析结果稳定可靠，可用于代谢组学研究。采用建立的GC-MS联用技术检测Tat诱导的小鼠血清中的代谢物，并用主成分分析（PCA）和偏最小二乘法分析（PLS-DA）法对所获得的代谢组数据进行模式识别。研究结果显示，Tat可诱导小鼠血清16种代谢物显著变化，它们涉及氨基酸代谢、三羧酸循环、脂肪酸代谢等多条代谢通路，为Tat致病机制的阐释和艾滋病的治疗提供了新的线索。三、基于LC/GC-MS和定量PCR技术的HIV-1Tat诱导Jurkat细胞的代谢组学研究HIV-1Tat蛋白由感染的细胞释放，作用于邻近未感染的T淋巴细胞诱导多种病理效应。上一章我们已经在小鼠模型上用基于GC-MS的代谢组学方法揭示了Tat可诱导体内多种代谢物发生变化，为了深入研究其干扰机制，本章我们在体外以HIV-1Tat处理的Jurkat T淋巴细胞为研究对象，采用基于GC-MS、RPLC-MS和HILIC-MS相结合的代谢组学方法检测Tat处理的Jurkat T淋巴细胞胞内及胞外代谢物，并用PCA和PLS-DA对所获得的代谢组数据进行模式识别。通过信息处理得到了37种差异代谢物，根据标志物，我们构建了Tat调节网络示意图，并采用实时定量PCR的方法对代谢通路中关键调节酶进行了测定，得到了7种显著改变的调节酶且与代谢物变化相符，从酶的调控验证了Tat诱导的代谢变化。本研究结合上章的结果提示Tat可能是HIV相关的代谢综合症的重要致病因子。我们的研究结果为Tat药物靶点的发现和疫苗的设计提供科学依据。四、我国HIV感染者中抗Tat抗体的反应特点研究在HIV-1感染者中，Tat抗体的出现与艾滋病的发病及病呈进展的关系出现了两种相反的结论。因此，全面深入的Tat抗体检测有助于揭示Tat和HIV发病及病程的关系，对Tat的致病性研究及开发有效的Tat疫苗具有重要意义。为了鉴定Tat抗体反应类型，我们测定了326份HIV感染者中抗Tat抗体反应，100份健康献血员血浆作为阴性对照。我们首先用全长Tat作为检测抗原从326份HIV阳性血浆样本中筛选抗Tat抗体阳性样本，进一步用我们设计合成的6个Tat肽段作为分析抗原将抗体反应进行分类，并对每个样本的Tat反式激活的中和能力进行评价。结果显示，326份HIV阳性血浆样本检测出42份为抗Tat抗体阳性和6份Tat相关抗体，根据与不同分析抗原的反应特点将其分为以下6种反应类型：全阳性反应、综合的反应、N-特异性反应、C-特异性反应、全长Tat反应和Tat相关反应。中和实验显示Tat反式激活的中和能力与抗体反应类型及CD8+T细胞计数显著相关。我们的结果为更好地理解Tat在HIV致病中的重要作用和设计有效的Tat疫苗提供重要信息，也为进一步用代谢组学方法研究Tat在HIV感染者体内的生物活性奠定基础。本文是首次通过基于LC-MS和GC-MS相结合的代谢组学方法在整体动物和细胞水平对Tat活性进行系统的代谢组学研究，通过多变量数据分析得到了Tat活性相关的差异代谢物，涉及糖酵解、三羧酸循环、脂代谢和氨基酸代谢等多条代谢通路。这些发现有助于揭示Tat“病毒毒素”复杂的致病机制，提示Tat可能作为治疗性干预的重要靶标，阻断或修饰这些异常的代谢通路对于治疗由HIV感染引起的代谢紊乱综合症具有潜在的应用价值。此外，我们的结果表明基于整合的GC-MS和LC-MS的代谢组学方法在细胞代谢物组分析中发挥重要作用。更多还原

【Abstract】 As one of the six accessory proteins of HIV-1, Tat is critical for viral replication andplays an important role in viral dissemination and pathogenesis. Intracellular Tat, recruitscellular proteins to relieve the repression of the viral long-terminal repeat (LTR), andthereby the viral promoter can induce the expression of viral genes. Extracellular Tat,secreted from infected cells, was found to play an important role in the pathogenesis ofproliferation inhibition and apoptosis of CD4+T cells, viral dissemination, AIDSassociated Kaposi’s sarcoma (KS) and HIV-1associated dementia. These findings supportthe notion that Tat acts as a “viral toxin” and obligate it as an “important target” for drugintervention and therapeutic vaccines.A number of chronic metabolic abnormalities including disorders of lipid metabolismwith or without lipodystrophy, insulin resistance, cardiometabolic syndrome, alteredphosphate metabolism, and an increased prevalence of impaired glucose tolerance arefound prevalent in HIV patients with or without receiving highly active antiretroviraltherapy (HAART). Although multiple risk factors have been proposed, the etiology ofthese metabolic abnormalities remains unclear. It would be interesting to see whetherHIV-1Tat plays a critical role in HIV metabolic abnormalities.Metabonomics is a top-down systems biology approach whereby metabolic responsesto biological interventions or environmental factors are analyzed and modeled.Metabonomics provides insights into the global metabolic status of the entire organism bymonitoring the entire pattern of low molecular weight compounds rather than focusing onan individual metabolic intermediate (MI). It has been widely applied into diseasediagnosis, pathophysiology, toxicology, pharmacodynamic assessment, gene function andquality control of Traditional Chinese Medicines. Compared to the transcriptome andproteome, the physical and chemical properties of the objects are more complex andmetabolome are diverse in nature (range from highly polar to non-polar) and concentration(range from mM to pM). An extensive metabonomics study needs non-discriminatoryanalytical techniques with each analytical instrument being selective to certain metabolites,and therefore parallel use of multiple analytical methods would be advantageous in identifying a broader spectrum of metabolites relevant to physiopathological alteration.The objectives of this study are to determine how HIV-1Tat influences metabolicenvironments both in mice and CD4+T cells to elucidate its complex pathogenicmechanism using gas chromatography-mass spectrometry (GC-MS), reversed-phase liquidchromatography–mass spectrometry (RPLC-MS) and hydrophilic interaction liquidchromatography-mass spectrometry (HILIC-MS)-based metabonomic methods. The maincontents are:1、 Expression, purification and identification of bioactive Tat proteinThe DNA encoding for Tat protein (1–86aa) from the BH-10clone of the IIIB strainof HIV-1(clade B) was amplified by PCR and inserted into pET21b vector to constructprokaryotic expression plasmids tat-pET21b. The recombinant plasmid was transformedinto E.coli BL21(DE3) for expression. The fusion protein Tat was expressed with relativemolecular weight (MW)15kD under induction of IPTG, which accounts for about15%oftotal bacterial proteins. The expressed Tat protein was purified by Ni-NTA column and SPSepharose fast flow Ion-exchange chromatography, and the purity was about90%.Endotoxin level of purified recombinant Tat protein was determined using the Limulusamebocyte lysate test and the result showed the final concentration of endotoxin in theeluted Tat was approximately0.05EU/μg of protein which is within the acceptable limit.Then the purified endotoxin-free Tat protein was testified with specific anti-Tatmonoclonal antibody by Western-blot and was verified to be bioactive using theLTR-transactivation assay.2、 Metabolic profiling in HIV-1Tat induced ICR mice serum using GC-MSThe HIV-1Tat protein is released by infected cells and has numerous biologicalactivities which might contribute either to the impairment of the immune response or toviral dissemination and pathogenesis. To date, the effects of Tat protein on metabolitesremain unclear. In this study, a metabolomic study on serum of HIV-1Tat-induced ICRmice was performed to research the pathologic mechanism of Tat protein by using gaschromatography coupled to mass spectrometry (GC-MS). NIST2002, a commercial massspectral database, was used for rapid identification of the detected metabolites. The procedures of sample preparation, derivatizative conditions and chromatographicconditions were optimized, and validation of the method was carried out. The resultsshowed that GC-MS analytical method was stable and reliable. It was capable to describethe biochemical composition of biological samples. The established GC-MS analyticalmethod was applied to the metabonomic study on Tat-induced mice serum. Principalcomponents analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) wereused for recognizing the different metabolic patterns between the Tat and control groups.Sixteen significantly changed metabolites in HIV-1Tat-induced mice were identified thatare involved in amino acid metabolism, tricarboxylic acid (TCA) cycle and lipidmetabolism, which contributed to the elucidation of the complex pathogenic mechanism ofTat protein and may shed new lights on future improvement of HIV-1therapy.3、 Combined LC/GC-MS and quantitative real-Time PCR analyses reveal systemsmetabolic changes in Jurkat T-cells induced by HIV-1Tat proteinHIV-1Tat protein is released by infected cells and can affect bystander uninfected Tcells and induce numerous biological responses which contribute to its pathogenesis. Toelucidate the complex pathogenic mechanism, we conducted a comprehensiveinvestigation on Tat protein-related extracellular and intracellular metabolic changes inJurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS),reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilicinteraction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomicsapproach. Quantitative real-time PCR (qRT-PCR) analyses were further employed tomeasure expressions of several relevant enzymes together with perturbed metabolicpathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1Tat causedsignificant and comprehensive metabolic changes, as represented by significant changes of37metabolites and7relevant enzymes in HIV-1Tat-treated cells, which are mainlyinvolved in glycolysis, citrate cycle, lipid metabolism and amino acid metabolism. Theseresults provide metabolic evidence of the complex pathogenic mechanism of HIV-1Tatprotein as a “viral toxin”, and would help obligate Tat protein as “an important target” fortherapeutic intervention and vaccine development. 4、 Characterization of Tat antibody responses in Chinese individuals infected withHIV-1There are two converse conclusions about the relationship between the anti-Tatantibody in HIV infected patients and the development and progression of AIDS. Therefore,wider and deeper anti-Tat responses analyses are very important. To define theseimmunoprofiles, we investigated Tat antibody responses in plasma samples from326Chinese individuals infected with HIV-1as well as100samples from healthy blood donorsas controls. We first screened for anti-Tat-seropositive samples using ELISA withrecombinant full-length Tat protein. Next, the immunoprofiles of the anti-Tat antibodyresponses were determined by ELISA using a synthetic N-terminal domain of Tat (Tat1-21)and six recombinant Tat peptides as the analytic antigens. The neutralizing activities of theanti-Tat-seropositive samples were evaluated using an LTR-transactivation neutralizingassay. Out of326HIV-1-seropositive individuals, only42were found to be positive foranti-Tat antibodies and6for Tat-related antibodies. Among the anti-Tat-seropositive andTat-related samples we found six different immunological profiles of anti-Tat antibodyresponses: full-potential response, combined response, N-specific response, C-specificresponse, full-length Tat-specific response and Tat-related response. These responses weredefined based on differential reactivity with several analytic antigens, and they representtwo types of anti-Tat responses—the major complete response and the alternative C-proneresponse. The anti-Tat-seropositive samples showed significantly higher Tat-neutralizingactivities compared with samples from both anti-Tat-negative HIV-1patients or healthyblood-donor samples. Tat-neutralizing activities were found to significantly correlate withthe reactivities of antibodies against specific Tat antigens and the CD8cell counts. Siximmunoprofiles of anti-Tat responses in Chinese patients infected with HIV-1were definedand were found to have significant Tat-neutralizing activities. The data we present herecould contribute to a better understanding of the significance of anti-Tat responses inpreventing HIV pathogenesis and could be useful for designing more effective vaccines inthe future. It also provided important information for studying the role of Tat in HIVinfected patients using metabonomic approach.In conclusion, this is the first metabonomic study to determine HIV-1Tat-induced biochemical alterations both in ICR mice and Jurkat T-cells using combined LC-MS andGC-MS. We identified16and37significantly changed metabolites in Tat-induced miceserum and Jurkat T-cells, respectively, which are mainly involved in glycolysis, citratecycle, lipid metabolism and amino acid metabolism, which were helpful for revealing thecomplex pathogenic mechanism of HIV-1Tat as a “viral toxin” and may present importanttargets for therapeutic intervention. Blocking or modifying these points of HIV-1Tatperturbed pathways are attractive approaches to the treatment of HIV-related chronicmetabolic abnormalities. Further studies are needed to deepen the understanding about thebiological function and regulation mechanism of HIV-1Tat protein. In addition, ourfindings suggest that metabonomics based on integration of GC-MS and LC-MStechniques could be a potent tool for cell metabolomes.更多还原