生物素——链霉亲和素放大免疫酶法在鼻咽癌血清学检测中的应用

[目的] 探讨生物素--链霉亲和素放大免疫酶法(BSA)应用于鼻咽癌(NPC)IgA/EA血清学早期筛查效果,提高鼻咽癌早期诊断效率.[方法] 免疫酶法(IE)检测广西鼻咽癌示范基地血清中IgA/VCA(针对EBV壳抗原VCA的IgA型抗体),IgAVCA阳性者再用IE法及生物素--链霉亲和素放大免疫酶法(BSA)分别检测血清IgA/EA(针对EBV早期抗原EA的IgA型抗体),以临床病理检查结果为确诊标准.t检验及X2检验统计分析改进前后两种方法在鼻咽癌血清学早期检测方面的差异.[结果] IE法检测IgA/VCA阳性血清388例,IE法检测IgA/EA阳性率3.6%(14例),BSA法检测IgA/EA阳性率12.9%(50例),BSA法检出阳性率显著高于IE法(X2=22.070,P<0.001).在两种方法检测的血清IgA/EA均阳性的样品中,IgA/EA检测滴度的几何平均值(GMT)分别为1:17.04和1:51.87,BSA法显著高于IE法(t=2.804,P<0.05).进一步的病理检测确诊鼻咽癌患者16例,BSA法提高了NPC检测灵敏度(X2=16_347,P<0.001).[结论] 用生物素--链霉亲和素放大免疫酶法(BSA)检测血清EBV相关抗体特异性较高,并且可以提高鼻咽癌血清学早期检测的灵敏度,为在基层开展更有效的流行病学筛查提供了可行的方法.     

生物素-链霉亲和素介导受体靶向卵巢癌SKOV3细胞量子点体外成像的研究

  目的  研制一种生物素-亲和素系统（BAS）介导叶酸受体（FR）靶向量子点（QD）荧光探针,初步验证其靶向性及信号放大效应。   方法  采用活泼酯法,将链霉亲和素（SA）与QD共价偶联制备QD-SA并对其物理特性进行验证;采用活泼酯法以牛血清蛋白为载体合成生物素化叶酸（FA）。通过生物素化FA与QD-SA结合制备BAS介导叶酸受体（FR）靶向QD探针,比较该探针对卵巢癌SKOV3细胞及FR表达阴性的肺癌A549细胞的识别情况,并验证其靶向特异性;对比该探针在生物素化FA孵育1、4 h时成像的效果;比较该探针与未经BAS介导的QD-FA探针在不同孵育时间对卵巢癌SKOV3细胞成像的差异。   结果  BAS介导FR靶向QD探针可特异性识别FR表达阳性的卵巢癌SKOV3细胞,且孵育4 h较1 h荧光信号明显增强,同等条件下其产生的荧光强度较未经BAS介导的QD-FA探针明显增强。   结论  制备了一种特异性好、灵敏度高的BAS介导FR靶向QD探针,该探针在卵巢癌早期诊断方面具有潜在应用价值。      

重组TFAR19蛋白对鼻咽癌CNE 1细胞端粒酶活性和细胞凋亡的影响

目的：研究重组TFAR19（TF-1 cell apoptsis-related gene 19）蛋白对鼻咽癌细胞株CNE-1端粒酶活性及端粒酶逆转录酶（hTERT）mRNA表达的影响，探讨重组TFAR19蛋白促CNE．1细胞凋亡的作用机制。方法：将不同浓度的高纯度重组TFAR19蛋白分别和人类鼻咽癌细胞株CNE-1共同培养后，通过7-AAD及Annexin V-PE标记进行流式细胞术分析，检测TFAR19蛋白对细胞的促凋亡效应；PT-PCR法比较试验组和对照组CNE-1细胞株端粒酶hTERTmRNA的表达水平；TRAP-银染法检测重组TFAR19蛋白作用下CNE．1细胞株端粒酶活性的改变。结果：（1）一定浓度的TFAR19蛋白在未加载体的情况下，可促进细胞凋亡，加入5、10、15、20、30mg／L的TFAR19蛋白后，CNE-1细胞的凋亡率分别是：15．26％，29．48％，37．11％，54．20％，72．36％。阳性对照组（凋亡诱导剂stuanrosporin处理）与阴性对照组（PBS处理）的细胞凋亡率分别是98．37％、13．40％。（2）试验组与对照组CNE-1细胞株端粒酶hTERTmRNA表达无显著差异。（3）与20mg／L以上浓度的重组TFAR19蛋白共同培养后，CNE-1细胞株端粒酶活性明显降低。结论：重组TFAR19蛋白在较高浓度下可降低鼻咽癌细胞株CNE-1的端粒酶活性，明显促进肿瘤细胞凋亡。     

横纹肌细胞培养液对鼻咽癌CNE2细胞生长抑制作用的实验研究

目的:通过研究横纹肌细胞(心肌和骨骼肌)培养液对人鼻咽癌细胞(CNE2)增殖的影响来观察横纹肌细胞微环境对肿瘤细胞生长的影响,探讨横纹肌内罕见转移瘤这一现象的发生机制.方法:采用酶解法原代培养新生Wistar大鼠心肌细胞和骨骼肌细胞,根据取材部位、形态学和免疫酶染色方法检测横纹肌α-肌动蛋白进行细胞鉴定,采用免疫酶染色方法进行细胞纯度鉴定.原代培养的细胞达对数生长期后更换新鲜培养液,继续培养24小时后取上清液,用MTT法分析骨骼肌和心肌细胞培养液对CNE2的体外生长抑制作用.以顺铂为阳性对照,以DMEM为空白对照,同时以鼠正常肾小球系膜细胞(MCs)为CNE2的阴性对照,观察心肌细胞培养液、骨骼肌细胞培养液和顺铂对CNE2细胞增殖的作用.采用胰酶消化法,热灭活法初步探讨横纹肌细胞培养液的理化性质.结果:CNE2与CMCM、MMCM共同培养后,细胞存活率明显下降,与DMEM相比差异均具有显著性(P<0.05),而MCs细胞存活率无明显影响(P>0.05).DDP对CNE2和MCs细胞均具有显著性抑制作用(P<0.05).心肌细胞培养液和骨骼肌细胞培养液经胰酶消化后活性仍然存在(P<0.05),而经热灭活后活性消失(P>0.05).结论:新生大鼠横纹肌细胞培养液对鼻咽癌细胞增殖有明显的抑制作用;而对正常细胞增殖无明显影响,与顺铂的抑制作用明显不同;由此推测横纹肌细胞培养液中有可能存在一种抑瘤物质,这可能是横纹肌罕见转移瘤的关键因素.     

鼻咽癌患者治疗中CD34和T细胞亚群检测的意义

[目的]探讨鼻咽癌患者治疗过程中T细胞亚群、CD34检测的临床意义.[方法]应用流式细胞仪测定67例鼻咽癌患者治疗前、治疗4周以及治疗后1个月T细胞亚群、CD34和常规检查血液学情况,20例健康人群为对照组.[结果]血液学常规显示鼻咽癌患者白细胞、血红蛋白、血小板以及尿酸在治疗过程中与对照组比较均无显著性差异(P＞0.05).治疗前CD8+和CD4+/CD8+显著性低于正常组(P＜0.05).患者放疗和同期放化疗期间CD34明显下降,CD3+、CD4+、CD8+、CD4+/CD8+升高.鼻咽癌组治疗后1个月CD8+、CD4+/CD8+显著性高于对照组(P＜0.05).鼻咽癌同期放化疗患者CD34治疗4周和治疗后1个月均显著低于单纯放疗组(P＜0.05),且治疗后1个月CD3+、CD4+显著高于单纯放疗组(P＜0.05).[结论]T细胞亚群变化可评估鼻咽癌患者治疗过程中的免疫功能状态,CD34可用于观察放化疗对骨髓功能影响.     

SA-IL-7融合蛋白的制备及其膀胱内灌注对小鼠表浅性膀胱癌的治疗效应

目的：制备链亲和素标记的白介素7（streptavidin-tagged interleukin-7, SA-IL-7）融合蛋白,评价其对小鼠表浅性膀胱癌治疗的效果。方法：构建pET24a-SA-IL-7重组质粒,制备SA-IL-7融合蛋白。流式细胞术检测SA-IL-7融合蛋白与生物素化膀胱癌细胞MB49的结合,胸腺细胞增殖法检测SA-IL-7融合蛋白促进胸腺细胞增殖的生物学活性。应用小鼠表浅性膀胱癌模型评价膀胱内灌注SA-IL-7的治疗效果,观察小鼠生存期,免疫组化法检测各组小鼠肿瘤浸润CD8+T细胞。结果：成功制备的SA-IL-7融合蛋白可锚定于生物素化的MB49细胞表面,结合率达（96.6±1.3）%;同时能够促进胸腺细胞的增殖。SA-IL-7融合蛋白能够锚定于小鼠生物素化的膀胱黏膜表面7 d以上,而未生物素化的膀胱黏膜在始终未检测到IL-7的存在。在小鼠表浅型膀胱癌模型中,MB49细胞接种后的第80天,IL-7灌注治疗组90%小鼠死亡,而SA-IL-7组60%小鼠存活且未出现肿瘤（P<0.05）;且SA-IL-7灌注治疗组小鼠肿瘤组织中浸润CD8+T细胞明显增多(P<0.01)。SA-IL-7治愈的15只表浅型膀胱癌模型小鼠中,11只能够抵抗膀胱内MB49细胞的第二次接种,而对照组15只小鼠仅1只幸存（P<0.01）。结论：SA-IL-7融合蛋白膀胱内灌注能够产生抗肿瘤免疫应答和明显的治疗效果,有可能成为免疫治疗浅表性膀胱癌的新方法。     

应用生物素-链霉亲和素酶联免疫吸附法检测原发性肝癌特异性γ-谷氨酰转移酶

目的 观察生物素-链霉亲和素酶联免疫吸附(ELISA)法检测原发性肝癌(PHC)特异性欧曼陀罗凝集素强结合的γ-谷氨酰转移酶(DSA-GGT)的临床应用价值.方法 对45例正常对照者、58例PHC患者和203例非PHC患者(包括其他肿瘤患者36例,肝良性病变患者167例),应用生物素-链霉亲和素ELISA法检测受试者血清中DSA-GGT的水平,同时用ELISA法检测受试者血清中甲胎蛋白(Are)的水平.结果 58例PHC患者中,DSA-GGT阳性38例,检测DSA-GGT诊断PHC的灵敏度为65.5%;203例非PHC患者中,DSA-GGT阳性18例,检测DSA-GGT诊断PHC的特异度为91.1%.该方法的平均批内及批间变异分别为8.9%和11.5%.58例PHC患者中,AFP阳性40例,检测AFP诊断PHC的灵敏度为69.0%;203例非PHC患者中,AFP阳性19例,检测AFP诊断PHC的特异度为90.6%.DSA-GGT与AFP联合检测诊断PHC的灵敏度和特异度分别为93.1%和85.7%.结论 生物素-链霉亲和素ELISA法检测DSA-GGT和ELISA法检测AFP对PHC诊断的灵敏度和特异度相近,且前者的重复性良好,可作为PHC诊断的较好检测方法;DSA-GGT与AFP联合检测可显著提高PHC诊断的灵敏度.     

蛇床子素对人鼻咽癌CNE2细胞增殖、侵袭、迁移能力的影响

目的研究蛇床子素对人鼻咽癌CNE2细胞增殖、侵袭、迁移能力的影响,并探讨其调节CNE2细胞上皮问质转化（EMT）的分子机制。方法体外培养CNE2细胞,以0、20、40、80μg／ml蛇床子素分别处理CNE2细胞24、48h后,采用四甲基偶氮唑蓝（MTT）法、Transwell实验分别检测其对细胞增殖、侵袭和迁移能力的影响,以0μg/ml蛇床子素处理组为对照组。反转录-聚合酶链反应（RT-PCR）和Western blotting方法分别检测EMT标志物上皮钙黏着蛋白（E-eadherin）、波形蛋白（vimentin）及Writ／β-eatenin信号通路组分B．eatenin、细胞周期蛋白D1（cyclinD1）的转录和蛋白表达水平。结果作用24h和48h后,0、20、40、80μ/ml蛇床子素处理组细胞增殖抑制率分别为0．00％±0．00％、7．45％±0．87％、14．12％±2．29％、27．26％±0．43％和0．00％士0．00％、13．44％±0．84％、29．03％±0．78％、57．49％±1．70％,差异均有统计学意义（F=174．33,P〈0．001;F=1041．40,P〈0．001）,进-步两两比较,差异均具有统计学意义（均P〈0．01）。采用不同浓度的蛇床子素（0、20、40、80μg/ml）处理CNE2细胞48h后,细胞迁移细胞数分别为52．13±4．49、29．00±4．49、18．50±1．93、13．75±2．77,差异有统计学意义（F=200．37,P〈0．001）,进-步两两比较,差异均具有统计学意义（均P〈0．01）。采用不同浓度的蛇床子素（0、20、40、80μg/ml）处理CNE2细胞48h后,细胞侵袭数分别为46．63±2．87、24．13±2．87、16．75±5．29、11．00±1．77,差异有统计学意义（F=131．92,P〈0．001）,进-步两两比较,差异均具有统计学意义（均P〈0．01）。采用不同浓度的蛇床子素（0、20、40、80μg/ml）作用于人鼻咽癌CNE2细胞48h后,β-cadherinmRNA（1．00±0．13、2．61±0．03、3．12±0．09、3．60±0．06;F=20．92,P〈0．001）和E-cadherin蛋白表达（0．22±0．03、0．35±0．01、0．60±0．04、0．82±0．03;F=178．63,P〈0．001）差异有统计学意义,进-步两两比较,差异均有统计学意义（均P〈0．05）。同时,采用不同浓度蛇床子素（0、20、40、80μg/ml）处理CNE2细胞48h后,vimentin的mRNA表达（1．00±0．12、0．68±0．03、0．56±0．01、0．40±0．09;F=9．48,P〈0．010）、B-eatenin的mRNA表达（1．00±0．14、0．78±0．04、0．69±0．07、0．46±0．12;F=4．84,P〈0．050）和eyelinD1的mRNA表达（1．00±0．09、0．82±0．03、0．58±0．09、0．40±0．03;F=9．49,P〈0．010）差异均有统计学意义,各组间进-步两两比较,差异均有统计学意义（均P〈0．05）;vimentin的蛋白表达（0．85±0．02、0．74±0．01、0．34±0．01、0．27±0．01;F=610．58,P〈0．001）、B-eatenin的蛋白表达（0．83±0．00、0．44±0．02、0．39±0．00、0．23±0．03;F：985．74,P〈0．001）、cyelinD1的蛋白表达（0．86±0．02、0．67±0．00、0．35±0．01、0．25±0．0l;F=910．57,P〈0．001）差异均有统计学意义,各组间进-步两两比较,差异均有统计学意义（均P〈0．05）。结论蛇床子素可抑制CNE2细胞的增殖、侵袭和迁移,其机制可能与调节Wnt／β-catenin信号通路进而抑制EMT过程有关。     

T及NK细胞ζ链与恶性肿瘤的研究进展

ζ链是T和NK细胞膜上重要的信号转导分子，能影响T和NK细胞的活化。恶性肿瘤患者T及NK细胞中ζ链的表达水平往往比正常人低甚至缺失，从而导致T和NK细胞免疫功能的缺陷，且ζ链水平与患者肿瘤负荷和预后密切相关，现综述ζ链表达异常与恶性肿瘤发生、发展的相关性以及如何利用‘链对肿瘤患者进行治疗的研究现状。     

线粒体融合蛋白-2基因增强人乳腺癌T47D细胞对小白菊内酯的敏感性

目的:探讨线粒体融合蛋白-2(mitofusin-2,Mfn-2)基因表达对人乳腺癌T47D细胞对小白菊内酯敏感性的影响。方法:Real-time PCR检测不同乳腺癌细胞系(T47D、MDA-MB-231、MCF-7、MDA-MB-435及HCC38)中Mfn-2 mRNA的表达。LipofectamineTM2000体外转染pEGFP和pEGFP-Mfn-2质粒至人乳腺癌T47D细胞,real-time PCR和Western blotting检测T47D细胞中Mfn-2 mRNA和蛋白的表达,MTT法检测T47D细胞的增殖,流式细胞术检测T47D细胞凋亡率及线粒体膜电位。结果:与正常乳腺细胞相比,Mfn-2 mRNA在乳腺癌HCC38细胞系中高表达,在T47D等其他细胞系中均低表达。pEGFP-Mfn-2转染48 h后,T47D细胞中Mfn-2 mRNA和蛋白的表达均明显上调。与pEGFP转染组相比,pEGFP-Mfn-2转染组T47D细胞在小白菊内酯(0.05 mol/L)作用下的存活率明显降低[(47.93±2.21)%vs(56.93±2.05)%,P<0.05]。流式细胞术检测结果显示:50 mmol/L小白菊内酯作用下,pEGFP-Mfn-2转染组与pEGFP转染组相比,T47D细胞凋亡率升高[(71.2±2.1)%vs(38.8±2.6)%,P<0.05],而线粒体膜电位明显降低[(1.6±0.1)%vs(5.0±0.5)%,P<0.05]。结论:pEGFP-Mfn-2转染可增强T47D细胞对小白菊内酯的敏感性。     

食管癌组织及其区域淋巴结中树突细胞CD1a、CD80、CD86的表达

背景与目的:树突细胞(dendriticcell,DC)是重要的抗原提呈细胞,参与抗肿瘤免疫。本研究通过检测人食管癌组织及其区域淋巴结中树突细胞CD1a(特异性标志)、CD80、CD86(共刺激分子)的表达,探讨食管癌发生及其免疫逃逸的机制。方法:采用流式细胞术检测58例食管癌病例(Ⅰ-Ⅱ期27例,Ⅲ-Ⅳ期31例;G1-237例,G321例;鳞癌49例,腺癌9例;有淋巴结癌转移者37例,无淋巴结癌转移者21例)的癌组织及癌旁组织、11例食管炎性组织、12例正常食管组织、31枚有癌转移的区域淋巴结、34枚无癌转移的区域淋巴结中树突细胞CD1a及CD80、CD86的表达。结果:(1)树突细胞CD1a、CD80、CD86的表达在食管癌组织中(6.18±1.47,5.37±1.13,37.35±7.42)明显低于正常食管组织(10.28±2.11,9.67±1.94,48.76±10.23)、食管炎性组织(11.89±2.65,10.46±1.79,51.55±10.60)及癌旁组织(11.79±2.41,10.49±1.89,9.78±12.31)中的表达(P<0.01);(2)树突细胞CD1a、CD80、CD86的表达在食管癌患者有癌转移的区域淋巴结中的表达(8.34±1.66,6.78±1.42,41.70±8.71)明显低于无癌转移的淋巴结中的表达(12.23±2.14,10.82±2.11,59.63±12.52)(P<0.01);(3)癌旁组织及食管炎性组织中树突细胞CD1a、CD80、CD86的表达与正常食管组织中的表达的差异无统计学     

监测肾癌患者外周血CD4~+CD25~+调节性T细胞在术后免疫治疗中的作用

目的:探讨肾癌患者外周血CD4+CD25+T淋巴细胞的监测在术后辅助免疫治疗中的作用。方法:将24例肾癌患者随机分为两组:A组,根治性肾切除术后行IFN-α辅助免疫治疗;B组,单纯行根治性肾切除术;C组,10例健康对照。分别在术前、术后1周以及术后3个月免疫治疗结束时采集外周血样本,流式细胞仪检测CD4+、CD8+、CD4+CD25、CD4+CD25high T淋巴细胞亚群含量,并计算各部分比值。结果:A、B组术前CD4+CD25+、CD4+CD25high T淋巴细胞亚群总体上增加,并且其含量随肿瘤分期的进展而增加(P<0.05),但部分患者并不高于健康对照组。免疫治疗后,肾癌患者CD4+CD25+T淋巴细胞总体略有上升,但术前较高的患者中,约2/3下降。结论:利用流式细胞仪监测肾癌患者外周血CD4+CD25+T淋巴细胞亚群可反映机体抗肿瘤免疫状态,有助于预测免疫治疗的预后,为肾癌尤其是早期肾癌术后辅助免疫治疗的选择提供参考。     

Tat mammaglobin fusion protein transduced dendritic cells stimulate mammaglobin-specific CD4 and CD8 T cells

Summary Proteins can be efficiently introduced into cells when fused to a protein transduction domain, such as Tat from the human immunodeficiency virus. We recently reported that dendritic cells transduced with a Tat fusion protein containing the extracellular domain of Her2/neu (Tat-Her2/neu) induced CD8 cytotoxic T lymphocytes (CTL) that specifically lysed Her2/neu-expressing breast and ovarian cancer cells. In the current study we further investigated the mechanism of protein transduction, utilizing the breast cancer-associated protein, mammaglobin-A, which is expressed in about 80% of breast cancers. Using a Tat-mammaglobin fusion protein, we tested the ability of Tat-mammaglobin transduced dendritic cells to stimulate antigen-specific CD4 and CD8 T cells. Low levels of serum considerably improved protein transduction as determined by Western blot, and also improved presentation of antigenic peptide as evidenced by functional studies using antigen-specific T cells. Confocal microscope analyses of antigen-presenting cells (APC) incubated with Tat-mammaglobin showed localized distribution in addition to diffuse distribution in the cytosol. In contrast, mammaglobin lacking Tat showed only a localized distribution. Simultaneous incubation with both proteins resulted in overlapping localized distributions, suggesting Tat fusion proteins are processed through both the MHC class I and class II pathways. Indeed, stimulation of T cells with Tat-mammaglobin transduced dendritic cells led to an expansion of mammaglobin-specific CD4 T helper-1 lymphocytes along with CD8 CTL. We conclude that Tat-mammaglobin transduced dendritic cells can induce both CD4 and CD8 mammaglobin-specific T cells. These findings could be further exploited for the development of a mammaglobin-based vaccine for breast cancer.     

Frequency of CD45RO+ subset in CD4+CD25(high) regulatory T cells associated with progression of hepatocellular carcinoma

The purpose of this study was to assess the properties of CD4+CD25(high/low/negative) T cell subsets and analyze their relation with dendritic cells (DCs) in patients with hepatocellular carcinoma (HCC). In HCC patients, the prevalence of CD45RO+ cells in CD4+CD25(high) T cells was increased and associated with higher frequencies of plasmacytoid DCs. Larger proportions of this T cell subset were detected in the patients with larger tumor burdens. These results suggest that increased frequencies of the CD45RO+ subset in CD4+CD25(high) Tregs in HCC patients may establish the immunosuppressive environment cooperatively with tolerogenic plasmacytoid DCs to promote disease progression of liver cancer.     

Proportion of CD4+CD25+ regulatory T cell is increased in the patients with ovarian carcinoma

Objective: To study the frequency of the CD4⁺CD25⁺ regulatory T cells (Tregs) in the patients with ovarian carcinoma and its possible mechanism. Methods: The percentages of CD4⁺CD25⁺ Tregs in the peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs) and tumor associated lymphocytes (TALs) from 13 patients with ovarian carcinoma and in the PBLs from 14 healthy women were determined by flow cytometry. The expression of CD69 on CD4⁺PBLs from the patients was detected. PBLs from healthy women were cultured in complete RPMI 1640 containing the supernatant from SKOV3 cell line with or without PHA (phytohemagglutinin) stimulation for 72 hours, then the percentage of CD4⁺CD25⁺ T cells was detected. Results: CD4⁺CD25⁺ Tregs in the PBLs from patients with ovarian carcinoma were significantly increased compared with those from the control. The percentage of CD4⁺CD25⁺ Tregs in TILs was higher than that in PBLs and TALs from the patients, but not significantly. The expression of CD69 on CD4⁺PBLs from the patients was negative. The percentages of CD4⁺CD25⁺ T cells in PBLs cultured with SKOV3 supernatant elevated significantly compared with those without supernatant whether PHA was added or not (P = 0.001 and 0.001, respectively). Conclusion: There is an increasing of the proportion of CD4⁺CD25⁺ Tregs in PBLs, TILs and TALs of the patients with ovarian carcinoma, which probably results from up-regulation of soluble factor secreted by ovarian carcinoma cells.     

CD4+CD25+调节性T细胞与CD4+T、CD8+T细胞在结直肠癌组织中的分布

 目的 分析CD4+CD25+ FOXP3+调节性T细胞（Treg）与CD4+T、CD8+T在结直肠癌（colorectal carcinoma, CRC）组织中的分布及其与临床病理特征之间的关系。方法 收集42例CRC新鲜手术标本,应用冰冻切片、免疫组织化学SP法检测肿瘤组织和癌旁组织中FOXP3+、CD4+T和CD8+T阳性细胞数。结果 CRC患者肿瘤组织中FOXP3表达水平显著升高,与癌旁组织相比差异有统计学意义（P<0.01）;中低分化组Treg细胞数明显高于高分化组（P<0.01）;淋巴结转移组Treg细胞数明显高于无淋巴结转移组（P<0.05）;癌巢内CD4+、CD8+T细胞数及CD4+/CD8+值显著低于间质（P<0.01）;Ⅲ+Ⅳ期、淋巴结转移组癌巢内CD4+/CD8+比值显著低于Ⅰ+Ⅱ期及无淋巴结转移组（P<0.05）;CRC中Treg数量与癌巢内CD4+/CD8+比值显著负相关（r=-0.605, P<0.01）。结论 CRC的发生发展可能与其癌组织局部微环境中Treg数量变化相关,肿瘤局部Treg数量的增多与T淋巴细胞亚群比例失调可能成为肿瘤免疫逃逸的机制之一。     

Specific blockade CD73 alters the “exhausted” phenotype of T cells in head and neck squamous cell carcinoma

The adenosine‐induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto‐5‐nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4⁺ and CD8⁺ T cells was associated with an “exhausted” phenotype. Further, treatment with anti‐CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the “exhausted” phenotype of CD4⁺ and CD8⁺ T cells through downregulation of total expression of PD‐1 and CTLA‐4 on T cells. Whereas the population of CD4⁺CD73^hi/CD8⁺CD73^hi T cells expressed higher CTLA‐4 and PD‐1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC.     

生长抑素受体亚型在人鼻咽癌细胞株CNE2中表达

背景与目的：生长抑素受体（somatostatin receptor SSTR）在许多肿瘤组织均有表达，为生长抑素类似物如奥曲肽用于这些肿瘤的治疗提供了生物学基础，但对生长抑素受体在鼻咽癌中表达情况的研究甚少。本研究旨在了解生长抑素受体基因在鼻咽癌细胞中的表达情况，为生长抑素类似物用于鼻咽癌治疗的进一步研究提供试验基础。方法：采用RT—PCR法和SP免疫组化法联合检测体外培养的人鼻咽癌细胞株CNE2的SSTRs表达，并对PCR mRNA产物测序以鉴定结果。结果：RT—PCR提示人鼻咽癌细胞株CNE2分别表达生长抑素受体亚型SSTR1、SSTR2、SSTR4；免疫组化结果：人鼻咽癌细胞株CNE2表达SSTR1、SSTR2A为强阳性（〉60％），表达SSTR4为弱阳性、SSTR2介于两者之间、SSTR3和SSTR5则无表达。结论：我们的研究证实人鼻咽癌细胞株CNE2有多种生长抑素受体亚型基因表达，且以表达SSTR1、SSTR2较多。     

Targeting high‐grade B cell lymphoma with CD19‐specific T cells

Adoptive T cell therapy is an important additional treatment option for malignant diseases resistant to chemotherapy. Using a murine high‐grade B cell lymphoma model, we have addressed the question whether the B cell differentiation antigen CD19 can act as rejection antigen. CD19^?/? mice inoculated with CD19⁺ B cell lymphoma cells showed higher survival rates than WT mice and were protected against additional tumor challenge. T cell depletion prior to tumor transfer completely abolished the protective response. By heterotypic vaccination of CD19^?/? mice against murine CD19, survival after tumor challenge was significantly increased. To define protective epitopes within the CD19 molecule, T cells collected from mice that had survived the tumor transfer were analyzed for IFNγ secretion in response to CD19‐derived peptides. The majority of mice exhibited a CD4⁺ T cell response to CD19 peptide 27, which was the most dominant epitope after CD19 vaccination. A peptide 27‐specific CD4⁺ T cell line protected CD19^?/? mice against challenge with CD19⁺ lymphoma and also cured a significant proportion of WT mice from recurrent disease in a model of minimal residual disease after chemotherapy. In conclusion, our data highlight CD19‐specific CD4⁺ T cells for adoptive T cell therapy of B cell lymphomas.