前列腺酸性磷酸酶在胃癌中的表达及其体外诱导抗胃癌免疫应答

目的检测前列腺酸性磷酸酶(PAP)在胃癌中的表达水平,探讨PAP作为胃癌主动免疫治疗新靶点的可能性。方法分别用RT-PCR和Western blot方法检测胃癌细胞系中PAP mRNA和PAP蛋白的表达;用免疫组织化学方法检测胃癌组织中PAP的表达。利用PAP表位肽对胃癌患者的PBMCs进行体外诱导,ELISA法检测PAP特异性IFN-γ分泌水平;51Cr释放法检测CTLs的细胞毒活性。结果3种胃癌细胞(MKN-7、MKN-28和MKN-45)表达PAP mRNA,其中MKN-28和MKN-45表达PAP蛋白,胃癌组织中PAP呈阳性表达。从2/4胃癌患者PBMCs中诱导出的PAP多肽特异性CTLs可特异性杀伤HLA-A24+/PAP+的胃癌细胞MKN-45,CTLs的细胞毒活性依赖于CD8+T淋巴细胞。结论PAP有可能成为胃癌特异性免疫治疗的新靶点。     

前列腺特异性膜抗原(PSMA)的概况

近年来,前列腺癌特异性的分子标志物——前列腺特异性膜抗原（PSMA）受到国内外众多学者的关注。主要综述了前列腺特异性膜抗原的结构性质以及其在前列腺癌治疗中的应用。     

转基因树突状细胞激活细胞毒性T细胞特异性杀伤淋巴瘤的体外实验

目的 探讨转基因树突状细胞激活细胞毒性T细胞产生抗淋巴瘤的特异性细胞免疫反应。方法 采用人骨髓来源的髓系前体细胞,在人细胞因子IL-4、GM-CSF和INF-α诱导下,在体外生成大量树突状细胞。将制备好的含有IgVH1核酸质粒,用脂质体法转染树突状细胞。转染成功的树突状细胞与外周血T淋巴细胞共培养,激活特异性CTL细胞,与阳性表达IgVH1的人淋巴瘤Namalwa细胞反应,用3H-TdR掺入法观察CLLs对瘤细胞的特异性杀伤效应。结果 树突状细胞能够用脂质体方法转染IgVH1核酸质粒,并且有效递呈给外周血T细胞,对表达IgVH1的人淋巴瘤Namalwa细胞产生特异性免疫杀伤活性,与对照组相比差异有显著意义（P<0.01）。结论 体外诱导扩增的 DC能够转染 IgVH1核酸质粒,体外激活T淋巴细胞,产生特异性细胞毒效应。     

人精浆前列腺特异抗原的分离纯化及鉴定

用PEG－SephadexG－150两步法，从精浆中分离纯化前列腺特异抗原（PSA）。精浆标本先经PEG粗提，约去除80％的杂蛋白，再经柱层析，进一步提纯，提纯产物经SDS－PAGE及ELISA法证实为免疫纯。     

诱导特异性T细胞免疫反应的HIV疫苗的研究进展

随着HIV特异性T淋巴细胞在控制体内HIV复制方面的作用被认可,HIV疫苗研究的重点越来越多地集中在诱导特异性T细胞免疫反应的HIV疫苗上,这类疫苗可能通过诱导或增强体内针对HIV的特异性T细胞免疫反应,有效控制HIV在体内的复制。本文对近年来诱导特异性T细胞免疫反应的HIV疫苗在健康和感染机体中应用的研究进展作一综述。     

狂犬病疫苗对人外周血B淋巴细胞分泌特异性抗体的体外诱导作用

目的探索在体外用狂犬病疫苗诱导人外周血B淋巴细胞(PBL)分泌特异性抗体,并使B淋巴细胞保持良好增殖和分化状态的方法。方法以狂犬病疫苗免疫志愿者的外周血淋巴细胞进行体外诱导,并对诱导抗原量、诱导时间及IL-2、IL-6和胞壁酰二肽(MDP)等生物因素对诱导产生特异性抗体的影响进行检测,并观察PBL的活化与增殖状态。结果在特异抗原及其他生物因子的共同作用下,经免疫的人PBL体外抗原诱导5d后产生特异性抗体的量达到最大值。MDP、IL-2、IL-6对免疫志愿者的PBL体外特异性抗体的分泌有显著影响,但对未经免疫志愿者无明显影响。显微镜观察显示PBL生长与分化正常。结论已建立了体外诱导人外周血B淋巴细胞分泌特异性抗体的方法,并保持了B淋巴细胞良好的生长增殖和分化状态。     

从精浆前列腺特异抗原的分离纯化及鉴定

用ＰＥＧ－ＳｅｐｈａｄｅｘＧ－１５０两步法，从精浆中分离纯化前列腺特异抗原（ＰＳＡ）。精浆标本先经ＰＥＧ粗提，约去除８０％的杂蛋白，再经柱层析，进一步提纯，提纯产物经ＳＤＳ－ＰＡＧＥ及ＥＬＩＳＡ法证实为免疫纯。     

梅毒螺旋体特异性抗原TpN47基因片段的克隆与表达

目的在大肠埃希菌中表达梅毒螺旋体(Tp)TpN47(68~410 aa)重组蛋白。方法采用PCR法从Tp全基因组中扩增TpN47(202~1230 bp)片段,T-A克隆后构建原核表达质粒pET-22 b(+)-TpN47,经酶切鉴定后转化EcoliBL21(DE3),IPTG诱导表达,表达产物经Ni2+亲和层析柱纯化,Western blot鉴定其免疫反应性。结果PCR法扩增出约1 000 bp的目的片段,原核表达质粒pET-22 b(+)-TpN47经酶切鉴定正确,表达的蛋白约占菌体总蛋白的43%,相对分子质量约为39 000,以包涵体形式存在。经纯化后蛋白纯度达95%以上,Western blot证实该蛋白能与梅毒患者阳性血清发生特异性反应。结论所表达的TpN47重组蛋白具有良好的免疫反应性,为开发临床检测效果更好的梅毒诊断试剂盒奠定了实验基础。     

前列腺特异抗原光激发化学发光免疫检测方法的建立

目的建立前列腺特异抗原(Prostate specific antigen,PSA)光激发化学发光免疫(Light induced chemilumines-cent immunoassay,LICA)定量检测方法。方法用PSA多克隆抗体包被受体微粒,PSA单克隆抗体标记生物素,两者与链霉亲和素包被的供体微粒共同组成检测试剂检测PSA,优化测定条件并对方法进行验证。分别采用本方法与Roche公司电化学发光免疫分析法对82份临床标本进行检测,并进行比较分析。结果本方法的分析灵敏度为0.31 ng/ml;批内变异系数为4.66%～6.75%,批间变异系数为5.68%～8.42%;回收率为95.1%～105.2%。两种方法具有较好的相关性,其R2为0.981 5。结论建立的均相化学发光免疫测定方法能够用于血清PSA的定量测定,且其性能指标符合临床要求。     

抗PD-1/CTLA-4双特异性抗体的质量评价

目的 建立针对抗程序性死亡受体1 (programmed cell death protein 1,PD-1)/细胞毒性T淋巴细胞相关蛋白4(cytotoxic T-lymphocyte-associated protein 4,CTLA-4)双特异性抗体的关键质量属性质控方法 。方法 采用报告基因法测定PD-1靶点的细胞生物学活性,流式细胞荧光分选法测定CTLA-4靶点的竞争结合活性;还原/非还原十二烷基硫酸钠毛细管电泳(capillary electrophoresis-sodium dodecyl sulfonate,CE-SDS)和分子排阻高效液相色谱(size exclusion chromatography-high performance liquid chromatography,SEC-HPLC)法进行抗体分子大小变异体的控制;成像毛细管等电聚焦电泳(imaging capillary isoelectric focusing electrophoresis,iCIEF)法测定电荷异质性;运用肽图对抗PD-1/CTLA-4双特异性抗体进行鉴别;超高效液相色谱法分析糖型...     

治疗用抗人T淋巴细胞分化抗原单克隆抗体与人体组织反应性的体外研究

应用免疫组化酶染色法及免疫细胞荧光染色法体外检测了 WuT_3(抗 CD_3),WuT_4(抗CD_4),WuT_8(抗 CD_8),WuT_9(抗 CD_71 或 TfR),WuTac(抗 CD_(25)或 IL-2R)等5种临床治疗用小鼠抗人 T 淋巴细胞分化抗原单克隆抗体(简称单抗)与25种正常人体组织的反应性。结果显示,WuT_3与髓质胸腺细胞,淋巴结及扁桃体中 T 细胞区细胞,脾白髓中动脉周围淋巴鞘细胞及胃肠道粘膜层淋巴细胞呈阳性反应。WuT_4与皮质胸细胞及大多数髓质胸腺细胞呈阳性反应。WuT_8与皮质胸腺细胞及少数髓质胸腺细胞呈阳性反应。WuT_4 和 WuT_8阳性细胞在淋巴结、扁桃体、脾脏及胃肠道粘膜中的分布与 WuT_3 阳性细胞基本一致,但数量依次减少。淋巴结和扁桃体的生发中心以及脾脏的脾小结中散在分布有少数WuT_3,WuT4及 WuT_8阳性细胞。此外,睾丸中散在分布个别 WuT_8阳性淋巴细胞。WuT_9与26%的骨髓细胞及个别皮质胸腺细胞呈阳性反应。WuT_9 尚与小肠粘膜肠腺底部细胞呈较强染色。WuTac 仅与个别散在的髓质胸腺细胞呈阳性反应。上述5种单抗与其它非淋巴组织及细胞基本呈阴性反应。     

Influence of Polydatin on the Tumor Microenvironment In Vitro: Studies with a Colon Cancer Cell Model

The tumor microenvironment of colon carcinoma, the site at which tumor cells and the host immune system interact, is influenced by signals from tumor cells, immunocompetent cells, and bacterial components, including LPS. A large amount of LPS is available in the colon, and this could promote inflammation and metastasis by enhancing tumor cell adhesion to the endothelium. Polydatin (PD), the 3-β-D-glucoside of trans-resveratrol, is a polyphenol with anti-cancer, anti-inflammatory, and immunoregulatory effects. This study was designed to explore whether PD is able to produce antiproliferative effects on three colon cancer lines, to reduce the expression of adhesion molecules that are upregulated by LPS on endothelial cells, and to decrease the proinflammatory cytokines released in culture supernatants. Actually, we investigated the effects of PD on tumor growth in a coculture model with human mononuclear cells (MNCs) that mimics, at least in part, an in vitro tumor microenvironment. The results showed that PD alone or in combination with MNC exerts antiproliferative and proapoptotic effects on cancer cells, inhibits the production of the immunosuppressive cytokine IL-10 and of the proinflammatory cytokines upregulated by LPS, and reduces E-selectin and VCAM-1 on endothelial cells. These data provide preclinical support to the hypothesis that PD could be of potential benefit as a therapeutic adjuvant in colon cancer treatment and prevention.     

Deciphering Repertoire of B16 Melanoma Reactive TCRs by Immunization,In Vitro Restimulation and Sequencing of IFNγ-Secreting T Cells

Recent advances in cancer immunotherapy have great promise for the treatment of solid tumors. One of the key limiting factors that hamper the decoding of physiological responses to these therapies is the inability to distinguish between specific and nonspecific responses. The identification of tumor-specific lymphocytes is also the most challenging step in cancer cell therapies such as adoptive cell transfer and T cell receptor (TCR) cloning. Here, we have elaborated a protocol for the identification of tumor-specific T lymphocytes and the deciphering of their repertoires. B16 melanoma engraftment following anti-PD1 checkpoint therapy provides better antitumor immunity compared to repetitive immunization with heat-shocked tumor cells. We have also revealed that the most error-prone part of dendritic cell (DC) generation, i.e., their maturation step, can be omitted if DCs are cultured at a sufficiently high density. Using this optimized protocol, we have achieved a robust IFNγ response to B16F0 antigens, but only within CD4+ T helper cells. A comparison of the repertoires of IFNγ-positive and -negative cells shows a prominent enrichment of certain clones with putative tumor specificity among the IFNγ+ fraction. In summary, our optimized protocol and the data provided here will aid in the acquisition of broad statistical data and the creation of a meaningful database of B16-specific TCRs.     

Learning from TCR Signaling and Immunological Synapse Assembly to Build New Chimeric Antigen Receptors (CARs)

Chimeric antigen receptor (CAR) T cell immunotherapy is a revolutionary pillar in cancer treatment. Clinical experience has shown remarkable successes in the treatment of certain hematological malignancies but only limited efficacy against B cell chronic lymphocytic leukemia (CLL) and other cancer types, especially solid tumors. A wide range of engineering strategies have been employed to overcome the limitations of CAR T cell therapy. However, it has become increasingly clear that CARs have unique, unexpected features; hence, a deep understanding of how CARs signal and trigger the formation of a non-conventional immunological synapse (IS), the signaling platform required for T cell activation and execution of effector functions, would lead a shift from empirical testing to the rational design of new CAR constructs. Here, we review current knowledge of CARs, focusing on their structure, signaling and role in CAR T cell IS assembly. We, moreover, discuss the molecular features accounting for poor responses in CLL patients treated with anti-CD19 CAR T cells and propose CLL as a paradigm for diseases connected to IS dysfunctions that could significantly benefit from the development of novel CARs to generate a productive anti-tumor response.     

Modulatory Effects of Biosynthesized Gold Nanoparticles Conjugated with Curcumin and Paclitaxel on Tumorigenesis and Metastatic Pathways—In Vitro and In Vivo Studies

Background: Breast cancer is the most common cancer in women globally, and diagnosing it early and finding potential drug candidates against multi-drug resistant metastatic breast cancers provide the possibilities of better treatment and extending life. Methods: The current study aimed to evaluate the synergistic anti-metastatic activity of Curcumin (Cur) and Paclitaxel (Pacli) individually, the combination of Curcumin–Paclitaxel (CP), and also in conjugation with gold nanoparticles (AuNP–Curcumin (Au-C), AuNP–Paclitaxel (Au-P), and AuNP–Curcumin–Paclitaxel (Au-CP)) in various in vitro and in vivo models. Results: The results from combination treatments of CP and Au-CP demonstrated excellent synergistic cytotoxic effects in triple-negative breast cancer cell lines (MDA MB 231 and 4T1) in in vitro and in vivo mouse models. Detailed mechanistic studies were performed that reveal that the anti-cancer effects were associated with the downregulation of the expression of VEGF, CYCLIN-D1, and STAT-3 genes and upregulation of the apoptotic Caspase-9 gene. The group of mice that received CP combination therapy (with and without gold nanoparticles) showed a significant reduction in the size of tumor when compared to the Pacli alone treatment and control groups. Conclusions: Together, the results suggest that the delivery of gold conjugated Au-CP formulations may help in modulating the outcomes of chemotherapy. The present study is well supported with observations from cell-based assays, molecular and histopathological analyses.     

兔脂肪间充质干细胞胰岛素分泌功能的体外诱导

目的研究兔脂肪间充质干细胞体外诱导分化为具有胰岛素分泌功能的细胞的可能性。方法采用贴壁法分离兔脂肪间充质干细胞,培养、传代后,以胰高糖素样多肽(GLP-1)和尼克酰胺作为诱导剂进行诱导分化。对诱导后的细胞进行双硫腙染色及葡萄糖刺激胰岛素分泌试验,ELISA法检测胰岛素含量。结果细胞诱导后逐渐聚集成细胞团,双硫腙染色呈砖红色,诱导后第14天培养液中可检测到胰岛素,第21天浓度较14d增高,高糖组[(21.5±1.6)μIU/ml]比低糖组[(18.1±2.5)μIU/ml]略高,且差异有显著意义。结论兔脂肪间充质干细胞在体外一定诱导条件下,具有向胰岛素分泌细胞分化的潜能。