ELISPOT检测人乳头瘤病毒特异性的记忆T细胞

应用ELISPOT方法检测人乳头瘤病毒感染后自发清除者外周血中抗原特异性的记忆T细胞。收集人乳头瘤病毒（HPV）感染后自发清除者外周血（病毒清除后74个月）,分离外周血单个核细胞（peripheralblood mononuclear cells,PBMC）。体外应用已鉴定的表位肽刺激PBMC,10 d后计数细胞,去除表位肽,继续培养。第11天,ELISPOT方法检测PBMC中HPV抗原特异性的记忆T细胞。PBMC经表位肽刺激10 d后,细胞数量有明显增加,由最初的4.1×105增加为4.2×106。第11天,细胞数量增加为4.65×106,为抗原刺激前细胞数的11.3倍。ELISPOT结果显示,PBMC中的记忆T细胞活化后,能够识别抗原递呈细胞递呈的抗原肽,并分泌IFN-γ。此HPV自发清除者外周血中抗原特异性的记忆T细胞的频数为0.007%。人乳头瘤病毒（HPV）感染后自发清除者外周血存在抗原特异性记忆T细胞,抗原肽可激活记忆T细胞,使之数量增加,分泌IFN-γ。ELISPOT可用于检测外周血中HPV特异性的记忆T细胞。     

EBV转化的人乳头瘤病毒感染者B淋巴母细胞系的建立及应用

建立EB病毒(EBV)转化的B淋巴母细胞系(LCL),应用此细胞系作为抗原递呈细胞筛选抗原特异性的T细胞克隆。收集人乳头瘤病毒(HPV)感染者外周血20份,分离外周血单个核细胞(PBMC),利用Pan T细胞试剂盒去除PBMC中的T细胞,获得non-T淋巴细胞,EBV病毒转化non-T淋巴细胞。应用转化的LCL细胞作为抗原递呈细胞,ELISPOT筛选可能的HPV抗原特异性的T细胞克隆。成功建立18个LCLs,建株成功率为90%。转化成功的LCL细胞体积增大,聚集成团状或簇状。建株失败的两份标本,用于转化的non-T淋巴细胞数少于2×106。应用LCL细胞作为抗原递呈细胞筛选出64个HPV抗原特异性的T细胞克隆。成功建立LCLs,此细胞系细胞可作为体外研究HPV特异性免疫反应的刺激细胞。     

人乳头瘤病毒与宫颈癌关系研究进展

宫颈癌是一种严重危害女性健康的恶性肿瘤,其发病率较高,位居女性恶性肿瘤的第二位,仅次于乳腺癌。自从1977年德国学者ZurHausen等从宫颈癌标本中发现了人乳头瘤病毒(Human papillomavirus HPV)DNA,并推测HPV感染与宫颈癌发生有关后,许多学者对HPV与宫颈癌的相关性进行了大量的研究,并证实HPV感染是宫颈癌发病的必需因素。目前,对于宫颈HPV感染检测有多种手段,其中聚合酶链反应(PCR)和捕获杂交技术在实验室中应用较广泛。在宫颈癌筛查中联合应用HPV检测和细胞学,不仅可以提高敏感性,而且还可以减少随诊频率,从而大大降低了宫颈癌的发生。     

人乳头瘤病毒16型晚期蛋白质基因L1的克隆及表达

人乳头瘤病毒16型(HPV16)含有两个晚期开放读码框(L_1ORF和L_2ORF),L_1ORF编码主要衣壳蛋白,我们用质粒pHPV16、p16L_2BX_5和pATH,采用基因重组技术,制备了含有HPV16个长L_1ORF序列的基因克隆p16L_1BN(5071—253),它能在大肠杆菌中有效表达,产生分子量约90KD的含有E. coli trpE的融合蛋白。Western blot检测,该蛋白可被抗牛乳头瘤病毒的抗体识别,这说明克隆p16L_1BN能有效表达,基因产物具有乳头瘤病毒型的共同抗原的性质。     

Tetramer技术检测人乳头瘤病毒抗原特异性T细胞条件的优化

人乳头瘤病毒(human papillomavirus,HPV)感染是宫颈癌发生的首要因素。针对病毒早期蛋白E6的特异性细胞毒性T淋巴细胞(cytotoxic Tlymphocyte,CTL)在清除HPV感染细胞和病毒转化形成的肿瘤细胞过程中发挥重要作用,因此检测体内抗原特异性CTL的频数和功能有助于了解病毒感染者或宫颈癌患者体内的特异性细胞免疫反应。利用加载HPV16 E6表位抗原肽(E6 133-142;HNIRGRWTGR)的HLA-A6801四聚体(Tetramer),即HPV16 E6 133-142/HLA-A6801-PE四聚体,通过检测混有HPV特异性CTL的PBMC标本,优化Tetramer染色的实验条件,探讨染色的最佳温度及Tetramer浓度。结果显示,染色温度(4℃、室温及37℃)对Tetramer与CTL的结合无明显影响。Tetramer稀释度为1∶1 600时,HPV特异性CTL荧光强度保持高水平,且非特异性染色少,为最佳染色浓度。研究结果为进一步检测HPV感染者或宫颈癌患者体内抗原特异性的CTL打下基础。     

一氧化氮、人乳头瘤病毒和宫颈癌之间相互关系的研究进展

人乳头瘤病毒感染是宫颈癌发生的重要始动原因,从HPV感染到宫颈癌发生,需要许多共刺激因子的参与。这些共刺激因子均可引起宫颈局部一氧化氮浓度的增高。而一氧化氮既可影响HPV的转录和翻译,又在肿瘤发生过程中具有重要调节作用。深入研究一氧化氮、人乳头瘤病毒感染及宫颈癌之间的关系,可为宫颈癌的防治提供新的重要理论基础和药物研制实验平台,通过使用一氧化氮合酶抑制剂降低宫颈局部NO浓度将为全面有效防治宫颈癌带来新的希望。     

人乳头瘤状病毒分型检测技术研究进展

HPV病毒有100多个亚型,反复持续的高危型人乳头瘤状病毒(Human papillomavirus,HPV)的感染是子宫颈癌发生的必要条件.对其进行分型检测,对子宫颈病变的早期发现和预防有很重要的意义.近年来,HPV分型检测技术发展十分迅速,本文对其相关研究新进展进行综述.     

人乳头瘤病毒治疗性疫苗的研究进展

人乳头瘤病毒(Human papillomavirus,HPV)感染是宫颈癌的主要致病因素,多年来人们努力通过研发HPV疫苗来控制HPV相关疾病。虽然预防性HPV疫苗的接种代表了重大的公共卫生突破,但它仅能预防HPV部分型别的感染,并不具有治疗效果,无法防止预先存在的HPV感染进展为恶性肿瘤。而以致癌蛋白E6、E7等为靶点,诱导特异性细胞毒性T细胞的HPV治疗性疫苗可以减轻甚至消除HPV相关高度病变和癌症,因此其研究具有重要意义。本文旨在阐述近年来HPV治疗性疫苗的研究进展,并为以后的治疗性疫苗研究提供指导。     

Preservation of cell-based immunotherapies for clinical trials

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5–10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.     

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self‐renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large‐scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor‐intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel‐coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF‐microcarriers was less than that on MEF‐plates, the doubling time of hESCs on Matrigel‐microcarriers was indistinguishable from that of hESCs expanded on Matrigel‐coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier‐based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Presence of side-population cells in an immortalized nontumorigenic human liver epithelial cell line

Side-population (SP) cells have been shown to be highly enriched stem cells. We investigated whether an immortalized, nontumorigenic human liver cell line, THLE-5b, contains SP cells. Flow cytometry analysis after Hoechst 33342 staining demonstrated that the THLE-5b line contained a small component of SP cells. These SP cells were essentially eliminated by treatment with verapamil and expressed higher levels of ABCG2 mRNA than non-SP cells. In addition, the level of these SP cells detected by Hoechst 33342 staining was affected by the experimental conditions including the incubation medium. This is the first report of the presence of SP cells in the immortalized, nontumorigenic human liver cell line.     

Rapid ex vivo expansion of highly enriched human invariant natural killer T cells via single antigenic stimulation for cell therapy to prevent graft-versus-host disease

Background aims

CD1d-restricted invariant natural killer (iNK) T cells are rare regulatory T cells that may contribute to the immune-regulation in allogeneic stem cell transplantation (ASCT). Here, we sought to develop an effective strategy to expand human iNK T cells for use in cell therapy to prevent graft-versus-host disease (GVHD) in ASCT.

Expression of some hepatocyte-like functional properties of WRL-68 cells in culture

Summary Some morphologic and functional characteristics of an hepatic fetal human epithelial cell line (WRL-68 cells) were determined to validate the use of these cells as an in vitro hepatic model. WRL-68 cells have a morphologic structure similar to hepatocytes and hepatic primary cultures. They secrete alpha-feto protein and albumin and exhibit a cytokeratin pattern similar to other hepatic cultures. WRL-68 cells preserve the activity of some characteristic or specific liver enzymes or both used in clinical chemistry for the diagnosis of hepatic disorders, i.e. alanine amino transferase, aspartate amino transferase, gamma-glutamyl transpeptidase, and alkaline phosphatase.