Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

一个与化学因素致鼻咽癌相关的硝基还原酶基因的克隆与鉴定(英文)

在运用cDNAmicroarray分析鼻咽癌细胞系CNE1与正常鼻咽上皮细胞差异表达基因的基础上 ,发现ESTW 95 442在细胞系CNE1中存在明显表达下调 .随后采用生物信息学的方法克隆出了该EST所代表的硝基还原酶基因NOR1(GenBank登录号为AF4 6 2 348) .Northern印迹分析表明 ,该基因在脑、心脏、肺等正常组织中均有 2个转录产物 (1.6kb ,1.2kb) .RT PCR分析显示 ,NOR1基因在鼻咽癌活检组织中也存在表达下调 .但酶活性测定实验表明 ,它在鼻咽癌细胞系CNE1中的活性比正常鼻咽上皮细胞高 .通过基因转染实验发现NOR1基因具有与细菌硝基还原酶NTR相似的功能 ,能够将单功能烷基化试剂 2 硝基苯氮丙啶类化合物CB195 4的第 4位硝基还原成亚硝基从而生成细胞毒性物质 .研究结果表明 ,NOR1基因可能通过它的亚硝化作用及高活性而参与化学性因素致鼻咽癌的过程     

一种新型的重组单链三特异抗体的构建与表达

双特异抗体由于其缺乏共刺激信号 ,激活的T细胞往往会导致凋亡。为了更有效地杀伤肿瘤细胞 ,构建了抗卵巢癌单链×抗CD3单链×抗CD2 8单域重组三特异抗体的表达载体。利用大肠杆菌中的分子伴侣FkpA与三特异抗体共表达 ,提高了三特异抗体的在BL2 1Star菌中的可溶性原核表达。ELISA结果显示 ,可溶性的重组单链三特异抗体 (sTRI)与SK OV 3细胞的膜抗原 ,Jurkat细胞上的CD3分子以及重组CD2 8抗原均有较强的结合活性。桥连试验证明该抗体能同时与SK OV 3细胞和Jurkat细胞结合。体外杀伤试验表明该抗体能激活外周血T细胞来杀伤肿瘤细胞。形态学观察也进一步说明该抗体具有良好的结合活性以及体外杀伤活性。这种新型的重组单链三特异抗体为基于T细胞的癌症免疫治疗建立了一个新的技术平台。     

子宫内膜癌组织中GDF-15和p-mTOR蛋白的表达及临床意义

目的:研究子宫内膜癌组织中生长分化因子15(GDF-15)和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达,并分析其临床意义,为临床治疗提供依据。方法:选取2011年1月到2016年2月我院子宫内膜癌组织75例为研究组,另选取同期正常子宫内膜组织75例为对照组,应用免疫组化法检测GDF-15和p-mTOR蛋白的表达。结果:研究组GDF-15和p-mTOR蛋白的表达阳性率显著高于对照组,比较差异具有统计学意义(P0.05);子宫内膜癌分期Ⅲ-Ⅳ期、淋巴结转移者、病理分级G2+G3、肌层浸润深度≥1/2的GDF-15和p-mTOR蛋白的表达阳性率均较高(P0.05);子宫内膜癌组织GDF-15与p-mTOR蛋白表达呈正相关关系(P0.05)。结论:子宫内膜癌组织中GDF-15与p-mTOR蛋白表达升高,且与肿瘤转移、临床分期、肌层浸润深度有关。     

Caspase-8及Caspase-9在肝细胞癌组织中的表达及意义

目的:观察凋亡相关因子半胱氨酸天冬氨酸蛋白酶8(caspase-8)和半胱氨酸天冬氨酸蛋白酶9(caspase-9)在肝细胞癌中的表达及临床病理意义。方法:选择我院自2012年7月~2013年4月的53例肝细胞肝癌患者作为观察组,选择同一时期在医院体检的50例正常肝组织患者作为对照组,采用原位分子杂交方法对两组患者的Caspase-8和Caspase-9mRNA进行检测。结果:观察组与对照组中caspase-8、caspase-9的阳性表达率分别为84.91%(45/53)、88.68%(47/53)和60.00%(30/50)、82.00%(41/50),观察组与对照组比较均有升高趋势,比值有显著性差异(P0.05)。结论:肝癌细胞中caspase-8、caspase-9的较高表达显示在肝癌的发生发展过程中起到重要作用,在肝癌组织细胞增殖凋亡中具有一定作用,肝癌中caspase-8和caspase-9蛋白的表达均对于判断肝癌预后具有一定的临床价值。     

Autophagy inhibition radiosensitizes in vitro,yet reduces radioresponses in vivo due to deficient immunogenic signalling

Clinical oncology heavily relies on the use of radiotherapy, which often leads to merely transient responses that are followed by local or distant relapse. The molecular mechanisms explaining radioresistance are largely elusive. Here, we identified a dual role of autophagy in the response of cancer cells to ionizing radiation. On one hand, we observed that the depletion of essential autophagy-relevant gene products, such as ATG5 and Beclin 1, increased the sensitivity of human or mouse cancer cell lines to irradiation, both in vitro (where autophagy inhibition increased radiation-induced cell death and decreased clonogenic survival) and in vivo, after transplantation of the cell lines into immunodeficient mice (where autophagy inhibition potentiated the tumour growth-inhibitory effect of radiotherapy). On the other hand, when tumour proficient or deficient for autophagy were implanted in immunocompetent mice, it turned out that defective autophagy reduced the efficacy of radiotherapy. Indeed, radiotherapy elicited an anti-cancer immune response that was dependent on autophagy-induced ATP release from stressed or dying tumour cells and was characterized by dense lymphocyte infiltration of the tumour bed. Intratumoural injection of an ecto-ATPase inhibitor restored the immune infiltration of autophagy-deficient tumours post radiotherapy and improved the growth-inhibitory effect of ionizing irradiation. Altogether, our results reveal that beyond its cytoprotective function, autophagy confers immunogenic properties to tumours, hence amplifying the efficacy of radiotherapy in an immunocompetent context. This has far-reaching implications for the development of pharmacological radiosensitizers.     

骨桥蛋白通过调控MMP-2和VEGF参与肝癌细胞侵袭的机制研究

目的：骨桥蛋白（Osteopontin,OPN）在肝癌细胞侵袭中的作用机制。方法：采用siRNA干涉的方法处理人肝癌细胞,用PCR和Western-blot法检测OPN的表达;用transwell小室检测不同处理后的HepG2和MHCC97H细胞的侵袭能力;采用Western．b1．ot和ELISA方法检测基质金属蛋白酶-2（matrixmetalloproteinase-2,MMP-2）和血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）蛋白表达和活力的变化情况。结果：在不同肝癌细胞系中,随着肝癌细胞系侵袭能力的增强,OPN的表达逐渐增高。siRNA可以降低HepG2和MHCC97H细胞中OPN的表达,并且能够降低HepG2和MHCC97H细胞的侵袭能力;抑制OPN的表达能够降低MMP-2和VEGF蛋白表达和蛋白活性。结论：OPN在肝癌侵袭过程中起着重要作用,其作用机制可能是通过调控MMP-2和VEGF蛋白表达和活性来参与肝癌的侵袭,OPN可作为肝癌侵袭转移治疗的新靶点。     

结直肠癌中DNA甲基化研究进展

CpG岛是人类基因组中富含CpG二核苷酸的DNA序列,主要位于基因启动子区,大小约为100-1000bp,与约60％编码基因相关。DNA中CpG岛甲基化可导致抑癌基因的表观遗传学转录失活,直接参与肿瘤的发生机制。近年来,甲基化已成为表观遗传学研究的焦点。我们简要综述了DNA甲基化在结直肠癌中的研究进展。     

鼻咽癌癌变的分子机理

鼻咽癌是一种多基因遗传性肿瘤,在中国南方和国外中国南方移民及后裔中发病率极高,严重危害了我国南方地区人民生命健康.对于这样一种具有明显民族聚集现象和地域差异的恶性肿瘤,目前认为其病理发病机制与遗传和环境因素的共同作用密切相关.现主要阐述鼻咽癌作为一种多基因遗传性肿瘤的病因发病机制,初步建立鼻咽癌易感基因群的概念和鼻咽癌发生发展过程中易感基因群主导的多阶段性多米诺骨牌效应分子机制假说,为寻找鼻咽癌遗传易感风险因子,筛选鼻咽癌高危人群以及探索鼻咽癌靶向性及个体化的诊断和治疗手段奠定实验与理论基础.     

肿瘤相关候选基因在舌鳞癌中的筛选

目的：寻找舌癌特异性相关基因,阐明舌癌发病的分子机制,为舌癌的诊断、治疗和预后提供分子靶标。方法：采用半定量RT-PCR方法检测了所选9个肿瘤相关基因DLC1、ANXA1、BCAT1、KRAS2、KCNJ8、LDHB、DNM1L、ETNK1、PTX1在舌鳞癌中的表达情况。结果：DLC1、ANXA1、LDHB、DNM1L、ETNK1、PTX16个基因在舌鳞癌组织与配对正常组织中无明显表达差异;而BCAT1、KRAS2、KCNJ8在舌鳞癌组织中表达上调,上调频率分别为45%（9/20）、50%（10/20）和33.3%（4/12）。结论：BCAT1、KRAS2和KCNJ8参与了舌鳞癌的发生发展,也为进一步在舌鳞癌中分析3个基因的功能提供实验依据。