【作者】 张彩娥；

【导师】 吴雄文；

【作者基本信息】 华中科技大学 ， 医学免疫学， 2007， 博士

【摘要】 目的: HPV6对皮肤粘膜的感染往往造成局部组织增生而形成CA,CA的转归与患者的细胞免疫功能密切相关。为了探讨CA患者的T细胞免疫状态,本研究拟:①构建一种能够与HPV6E7抗原特异性T细胞结合的HLA-A2/HPV6E7四聚体;②应用该四聚体来检测不同病程CA患者的抗原特异性T细胞的频率。方法:①通过原核表达、大量提取HLA-A2-BSP、β2m两种蛋白,通过体外稀释复性、与人工合成的HPV6E7抗原肽(GLHCYEQLV)折叠形成相应的HLA-A2/HPV6E7单体,经过Western-blot和ELISA证实其构象正确。在BirA酶的作用下,将生物素结合到HLA重链的BSP位点,实现HLA-A2/HPV6E7单体的特异位点生物素化。通过生物素与亲合素4:1的特异性结合关系,将4个生物素化的HLA-A2/ HPV6E7单体与PE标记的亲合素结合形成HLA四聚体;②以制备的PE标记的HLA-A2/HPV6E7四聚体和FITC-CD8单克隆抗体对HLA-A2阳性、HPV6感染致CA患者的PBL和疣体组织进行双色荧光染色,经流式细胞仪和荧光显微镜观察。③通过T2细胞(HLA-A2阳性,TAP缺陷)负载HPV6E7抗原肽,作为刺激细胞,与CA患者的PBL进行共培养,获得了特异性CTL,经过LDH法进行了不同病程CA患者CTL的杀伤活性检测。结果:①成功构建了有生物学功能的HLA-A2/HPV6E7四聚体;②不同病程CA患者PBL中抗原特异性CTL(即CD8+和HLA-A2/HPV6E7四聚体染色阳性细胞)的检测结果显示:缓解期CA患者双阳性的细胞比例是0.11%,反复发作期CA患者双阳性的细胞比例是0.10%,而正常对照是0.02%。③特异性CTL的体外扩增及杀伤活性检测:经过长期混合淋巴细胞培养后,缓解期CA患者CTL的频数明显增高(0.23% vs 1.19%,P<0.05),反复发作期CA患者CTL的频数增高也很明显(0.23% vs 1.16%,P<0.05),利用LDH方法检测抗原特异性CTL的杀伤活性。缓解期CTL杀伤率为(64.5±2.5)%,复发期为(51.5±1.5)%,不同病程CA的CTL的杀伤率比较有显著差异(P<0.05)。④不同病程CA的疣体组织冰冻切片后四聚体原位染色:选取HLA-A2阳性、HPV6感染导致的不同病程CA患者,其疣体组织经手术切除后,进行40μm冰冻切片,经FITC-CD8单抗及PE-HPV6E7四聚体共染后,荧光显微镜观察发现:缓解期CA患者疣体组织中有一群阳性细胞,此即HPV6特异性的CTL;而反复发作期CA患者疣体组织中未能观察到抗原特异性T细胞浸润。结论:①本研究成功制备了加载HLA-A2限制性HPV6E7抗原肽的四聚体,并用此四聚体对HPV6感染、HLA-A2阳性的患者的PBL进行了HLA-A2/HPV6E7四聚体染色,缓解期CA与反复发作的CA患者的特异性T细胞数量相差不大,即反复发作的CA不存在全身特异性T细胞数量绝对减少。②不同病程CA患者的CTL的杀伤率比较有显著差异(P<0.05)。缓解期CA的CD8+T细胞杀伤活性处于高水平激活状态,从而能够有效地清除HPV,导致疾病的缓解。而反复发作的CA患者的PBL,抗原特异性T细胞虽然数量没有减少,但是杀伤活性不强,即机体不能建立有效的免疫反应,可能是导致CA反复发作的原因。③荧光显微镜观察,缓解期的患者疣体中能观察到CD8+和HLA-A2/HPV6E7四聚体的双阳性细胞浸润,而反复发作的患者疣体中未见此群细胞。提示这些抗原特异性T细胞在疣体中的浸润并发挥抗病毒作用,才导致CA的缓解。而反复发作的CA虽然外周血液中抗原特异性T细胞数量并没有减少,但是这些CTL的杀伤活性较低,并且在疣体局部几乎没有观察到抗原特异性T细胞的浸润。分析原因可能是局部细胞因子、粘附分子分泌障碍,不能有效地将血液中的T细胞趋化到疣体局部组织,所以局部存在免疫缺陷,难以建立有效的细胞免疫反应,造成HPV清除障碍。目的:家族性进行性色素过度沉着症(familial progressive hyperpigmentation, FPH)是一种单基因、常染色体显性遗传病,其致病基因尚未在染色体上定位。本研究的目的是:①在一个三代共17人的FPH家系的样本中,通过检测STR进行FPH基因定位;②为了永久性地保存该家系的具有特定生物学性状的标本,采用EBV转化外周血B淋巴细胞,获得该FPH家系成员的永生化的B淋巴母样细胞系(B lymphoblastoid cell line,B-LCL);③对该致病基因初步定位的染色体区段进行分析,其中存在着与色素代谢相关的基因丝氨酸/苏氨酸蛋白激酶11(Serine/Threonine protein Kinase 11,STK11),为此进行了FPH家系中病人的STK11基因的克隆和序列鉴定。方法:①提取该家系成员的外周血基因组DNA,在每条常染色体上选取8个STR位点进行基因筛查,对基因型与疾病表型间进行连锁分析,筛选可疑染色体;在筛出的染色体相应位置上增加6个STR位点以进行单倍型分析;②采用EB病毒(Epstein-Barr virus,EBV)感染,加入环孢霉素A抑制T淋巴细胞的方法,对该家系17人外周血B淋巴细胞进行了转化;③采用分子克隆技术,构建FPH患者的pcDNA3.0-STK11的克隆载体,测序并与NCBI网上(BC007981)进行了序列比对。结果:①经单倍型分析证实FPH的致病基因定位于19p13.1-19pter。Marshfield遗传图距为45.48cM,UCSC物理图距为17.17Mb,其间有628个基因;②成功建立该家系成员永生化B-LCL15个;③构建了该FPH家系中患者的STK11克隆载体。经NCBI网上比对,测序结果与正常序列(BC007981)完全一致,而未发现突变。结论:①将FPH的致病基因定位于19p13.1-19pter,该致病基因与初步定位区段内的色素代谢相关的基因STK11可能无关。在19p13.1-19pter之间可能存在新的致病基因,导致FPH的发生;②成功建立的该家系的永生化B-LCL为后续精细定位和克隆FPH致病基因、进行皮肤色素代谢障碍的生理生化分子机制研究提供了长期的研究材料。更多还原

【Abstract】 Objectives: MHC tetramer is a powerful tool for detecting the antigen specific T cells with exquisitely antigen-specific and highly sensitive. Since HLA-A*0201 is the most common allele of human MHC I molecules; Human Papillomavirus 6 (HPV6) is the major cause of Condyloma Acuminata (CA), we choose HLA-A*0201 and its restricted peptide HPV6E7 (22-30) to construct sHLA-A2/HPV6E7 tetramer. With the tetramer, we detected the antigen specific T cells in the different courses of CA.Methods: The two subunits of the human leucocyte antigen (HLA-A2) have been expressed at high levels as insoluble aggregates in bacterial cells. They were induced by IPTG to overexpress the target proteins. In the presence of an HLA-A2 restricted antigenic peptide (HPV6E7), refolding was initiated by dilution of the denaturant in the solution of heavy chain andβ2m. After concentrate and buffer exchange of refolding products, the BirA enzyme was used to biotinylate the refolded complex. Biotinylation was performing at the COOH-terminus of the heavy chain named BirA substrate peptide (BSP). The refolded and biotinylated products were detected by ELISA and Western blot with mAbW6/32 and HRP-streptavidin. sHLA-A2/HPV6E7 tetramer was prepared by mixing the biotinylated peptide-HLA complex with phycoerythrin (PE) labeled avidin at a molar ratio of 4:1.Peripheral blood was obtained from HLA-A2 positive healthy individual and different course of CA. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Paque density gradient centrifugation and then placed in sterile tissue culture dishes for separation of adherent from nonadherent cells by 2h incubation at 37℃. The nonadherent cells (PBLs) were plated into tube. With the PE-tetramer and FITC-CD8 mAb, a group of double positive cells can be observed by FACS.Here we use the T2 (HLA-A2+) cell pulsed with HPV6E7 as stimulating cells to stimulate the HLA-A2+ CA with HPV6 infected PBLs to generate the peptide-specific CTLs with the long term mixed lymphocytes culture. After culture, the specific CTLs will proliferate, and are used to monitor our sHLA-A2/ HPV6E7 tetramer. To detect the function of these specific CTLs, T2 cells loading peptide act as target cells, we detect their killing activation by LDH assay.In situ tetramer (IST) staining has opened the door to exploration of antigen specific T cells in tissues. The development of MHC tetramer staining has revolutionized our ability to study antigen specific T cells. This technique permits visualization of antigen-specific T cells in tissue sections along with their spatial relationship to their microenvironment. The quantities and qualities of CTLs infiltrating in tissues reflect the status of cell immunity. All have an effect on prognosis of CA. Condyloma tissues sections were incubated with the tetramers, washed with PBS and frozen in Tissue-Tek OCT compound. To monitor tetramer binding to CD8-positive antigen specific CTL in the cryopreserved tissue, 40μm cryosections were prepared, fixed in acetone for 10 min at RT and pre-incubated for 30 min in 5% (v /v) normal cow serum. Then they were incubated with the relevant mAb CD8+ and analyzed by fluorescence microscope.Results: After initial purification, the aggregates were dissolved in 8M urea. The yields are 450mg and 350mg for theβ2m and heavy chain from 6 liter saturant baterial cells, respectively. The purity of theβ2m and heavy chain is 65.3% and 69.8%, respectively. The western blot result shows monoclonal antibody (mAb) W6/32, which recognizes a determinant present on the heavy chain only when the heavy chain is associated withβ2m, binds to the refolded component. Sandwich ELISA with mAb W6/32 and rabbit anti-β2m antibody demonstrates the success of refolding. The sHLA-A2/ HPV6E7 tetramer were constructed successfully.The frequency of double positive cells in PBL of healthy dividual is 0.02%; but it is 0.11% in paramedic CA and is 0.10% in recurrence CA. We apply our prepared sHLA-A2/ HPV6E7 tetramer to stain the cells after long term mixed lymphocytes culture. The results shows our tetramer is able to discriminate the frequencies of specific CTL induced by HLA-A2/HPV6E7. The frequency of specific CTLs in paramedic CA is very high (0.23% vs 1.19%,P<0.05). The frequencies of specific CTLs in recurrence CA is obvious high too (0.23% vs 1.16%,P<0.05). The CTLs killing frequency in paramedic CA is (64.5±2.5) %. But the CTLs killing frequency in recurrence CA is (51.5±1.5) %. The difference is obvious in the CTLs killing frequency different course CA. Additionally, antigen specific T cells infiltrating in tissues in paramedic CA can be observed, but not observed in recurrence CA.Conclusions: In this study the HLA-A2 / HPV6E7tetramer were successfully constructed. The tetramer can be applied to directly visualize antigen-specific CTLs efficiently and become the critical approach in assessment of T cell immune responses in CA patients.Objectives: In this study, we undertook a linkage analysis with microsatellite polymorphic markers?short tandem repeat (STR) in a three-generation Familial Progressive Hyperpigmentation (FPH, MIM 145250) family with 17 members, including 6 affected individuals residing in Hubei Province. The immortal B Lymphocyte Cell Lines (B-LCLs) of the family were established by EBV transformation and addition of cyclosporin A (CysA) to inhibit the activity of T cells. Additional, the plasmid pcDNA3.0-STK11 was constructed from 3 affected individuals in this FPH family.Methods: DNA was isolated from peripheral blood cells from all 17 individuals. Genome screening was performed using 176 STR microsatellite markers from 22 autosomes, 8 for each chromosome, these STR markers were selected for their polymorphism in Chinese population and their physic locations which were able to represent a given chromosome segment. Additional 6 STR markers were chosen from the short arm of 19th chromosome to define the borders of the cosegregating region. These markers were amplified by polymerase chain reaction (PCR) based on the protocol reported previous. PCR products were analyzed by vertical electrophoresis polyacrylamide gels (PAGE) and silver staining. According to the guideline recommended by the DNA Commission of the International Society for Forensic Haemogenetics (ISFH), every allele of STR locus was named by the repeat number of its repetitive sequence. After purification, PCR products of 2 alleles from every STR locus were directly sequenced for their repetitive numbers on an Applied Biosystems 377 automatic sequencer. PCR products were synelectrophoresed with molecular weight standard and then every allele was named by its repeat number. As FPH was demonstrated to be an autosomal dominantly inherited disease, principles for judging linkage between its genotype and phenotype are as follows: (1) If an allele or haplotype is present in all affected individuals and not present in all unaffected individuals, it is suggested to be linked to the pathogenic gene; (2) If an allele or haplotype is present in some, but not all, affected individuals and not present in all unaffected individuals, it is possible that this allele or haplotype links to the pathogenic gene; (3) If an allele or haplotype is present randomly in affected and unaffected individuals, it is excluded to be linked to the pathogenic gene.The total cell RNA is extracted from the Peripheral blood mononuclear cells; and the cDNA encoding the STK11 was acquired by RT-PCR technique and then inserted into the plasmid pcDNA3.0.Results: the FPH locus is observed to link to the region covering D19S714 and D19S591 which are markers for 19p13.1-19pter. The genetic distance of region in Marshfield is 45.48 cM and physical distance in UCSC is 17.17Mb, harboring 628 defined genes. 15 immortal cell lines were established. The results showed that cellular volume of these immortal cells was enlarged and their morphology was diversity with glitch. There was increasing development of cell aggregates of proliferative lymphocyte cells. The recombinant plasmid pcDNA3.0-STK11 was identified to be correct by restriction endoniclease digestion and sequence analysis.Conclusions: the FPH locus is linked to chromosome 19p13.1-19pter. Interestingly, this region harbors STK11 gene, germline mutations in which were shown to be associated with Peutz-Jeghers Syndrome (PJS). PJS and FPH share the disorder of hyperpigmentation. But construcated STK11 sequences were completely coincidence with Genebank. So, the STK11 was not the virulence gene for FPH. There is a new gene not found for FPH in 19p13.1-19pter. For further fine mapping, it is obvious that more familial or/and sporadic FPH cases are required for the further fine mapping. The fine mapping of FPH gene is expected to lead to a better understanding of the etiology for both FPH and PJS.The immortal B-LCLs acquired successfully provide a long and plentiful DNA material for mapping and cloning causative gene for FPH and studying punctually the molecular mechanism of pigment metabolism in the skin.更多还原