A Quantitative Nitrocellulose Enzyme Immunoassay and its Application to the Screening of Hybrbidomas for the Detection of Uncharacterized Tumor-Associated Antigens in Sera of Cancer Patients

Abstract

An enzyme-linked immunosorbent assay on nitrocellulose based microtiter plates for the detection of uncharacterized tumor associated antigens in squamous cell carcinoma and adenocarcinoma cancer patients' sera is described. Nitrocellulose microtiter plates are more sensitive than the plastic plates of polystyrene and polyvinyl chloride for the detection of antigens in serum. Monoclonal antibodies were selected for their net reactivities toward cancer patients' sera as compared to nomal sera. Sera from benign liver and kidney disease patients and activated human peripheral blood leukocyte supernatant were used to reduce potential false positives toward inflammatory and benign diseases. Using this system, fourteen antibodies were selected out of over eight hundred antibodies for their potential serodiagnostic application.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

MyD88 or TRAM Knockdown Regulates Interleukin (IL)‐6, IL‐8, and CXCL12 mRNA Expression in Human Gingival and Periodontal Ligament Fibroblasts

Background: In a previous report, it was shown that Toll‐like receptor (TLR) 2 knockdown modulates interleukin (IL)‐6 and IL‐8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF‐related adaptor molecule (TRAM), could affect mRNA expression of IL‐6, IL‐8, and CXCL12 in HGF and HPDLF. Methods: After small interfering (si) RNA‐mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL‐6, IL‐8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL‐8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL‐6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non‐stimulated cells. Conclusions: These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL‐6 and IL‐8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.     

慢病毒载体感染人口腔黏膜成纤维细胞稳定表达绿色荧光蛋白的研究

目的：确立慢病毒载体高效感染人口腔黏膜成纤维细胞的感染条件,为进一步应用基因工程技术研究人口腔黏膜成纤维细胞奠定基础。方法：采用组织块法培养原代人口腔黏膜成纤维细胞,免疫组化SP法鉴定细胞类型,将携带绿色荧光蛋白基因的慢病毒载体以不同感染复数（multiplicity of infection,MOI）感染纯化细胞,并设置不同感染条件,感染4 d后于荧光显微镜下观察绿色荧光蛋白表达情况。结果：成功分离纯化得到人口腔黏膜成纤维细胞,当慢病毒感染复数为30,polybrene浓度为8μg/mL,并加入感染增强液时,可达到较高的感染效率,满足后续实验需要。结论：慢病毒载体可高效感染人口腔黏膜成纤维细胞,携带的绿色荧光蛋白基因可在成纤维细胞内稳定表达。     

肿瘤坏死因子-α诱导人牙囊细胞的凋亡作用

目的：研究不同浓度肿瘤坏死因子-α（TNF-α）对体外培养人牙囊细胞凋亡的影响。方法：第五代HDFCs分别与浓度为0（对照组）、50、100、200 ng/mL的TNF-α共孵育24 h,流式细胞仪AnnexinⅤ-FITC/PI双染技术检测牙囊细胞凋亡细胞比率。结果：HDFCs的凋亡率随TNF-α浓度升高而上升,由对照组的2.3%增加到7.1%。结论：TNF-可诱导HDFCs的凋亡,这种作用可能在牙齿发育、萌出过程中起重要作用。     

肿瘤坏死因子-α和半胱氨酸天冬氨酸蛋白酶3在肝泡型棘球蚴周围单核细胞中的表达

目的探讨肿瘤坏死因子-α(TNF-α)和半胱氨酸天冬氨酸蛋白酶3(caspase-3)在肝泡型棘球蚴周围单核细胞中的表达。方法40只雌性昆明小鼠随机分为实验组(n=20)和假手术对照组(n=20),实验组小鼠开腹直视下肝脏穿刺注射0.1 ml泡型棘球蚴混悬液,对照组注射等量生理盐水。感染6个月后处死小鼠,取肝组织,观察泡型棘球蚴的生长和转移情况;用苏木素-伊红(HE)染色法观察组织病理变化;免疫组织化学法检测TNF-α和caspase-3在肝泡型棘球蚴和淋巴转移灶周围组织中的表达;原位末端标记技术(TUNEL)检测肝组织和泡型棘球蚴病灶周围单核细胞的凋亡。结果感染6个月后,实验组小鼠肝脏可见大小不等的结节状或团块状的泡型棘球蚴组织,淋巴结转移率为45.0%(9/20)。HE染色后观察发现,实验组肝泡型棘球蚴和淋巴转移灶周围均有大量单核细胞浸润。免疫组化染色结果显示,实验组TNF-α和cas-pase-3蛋白在肝泡型棘球蚴和淋巴转移灶周围均有不同程度的阳性表达,单核细胞阳性细胞检出率分别为100%、100%和95%、100%,与对照组肝组织中阳性细胞检出率(5%和0)比较均有统计学意义(P<0.01)。TUNEL结果显示,肝泡型棘球蚴和淋巴转移灶周围单核细胞发生凋亡,单核细胞阳性细胞检出率为100%,与对照组比较有统计学意义(P<0.01)。结论泡型棘球蚴在感染宿主过程中,引起TNF-α蛋白高表达,可能参与了诱导肝泡型棘球蚴周围宿主单核细胞的凋亡,从而抑制宿主的免疫功能。     

肿瘤坏死因子-α增加肾入球动脉平滑肌细胞胞内钙离子浓度的机制

目的观察肿瘤坏死因子-α(TNF-α)对肾入球动脉平滑肌细胞(RASMCs)内钙离子浓度(〔Ca2+〕i)的影响。方法应用筛网及酶消化法进行原代雄性SD大鼠RASMCs的分离与培养,将原代培养的RASMCs透射电镜下观察肌丝进行鉴定。实验分组:A1组:内皮素刺激组;A2组:TNF-α+内皮素刺激组;B1组:2-氨基乙基二苯硼酸盐(2-APB)+内皮素刺激组;B2组:TNF-α+2-APB+内皮素刺激组。采用confocal显微镜测定单个活细胞内Ca2+荧光强度,计算细胞内〔Ca2+〕i。结果原代RASMCs呈梭形或三角形等;传代RASMCs多呈梭形或长梭形。透射电镜见细胞胞质内大量纵行束样分布的肌丝。各组静息状态下RASMCs内〔Ca2+〕i比较无显著差异(P>0.05)。A1组内皮素刺激后RASMCs内〔Ca2+〕i与加内皮素前比较显著增高(P<0.01);A2组〔Ca2+〕i较A1组显著增高(P<0.01);B1、B2组内皮素刺激后RASMCs内〔Ca2+〕i较A1组显著降低(P<0.01),与加内皮素前比较无显著差异(P>0.05)。结论 TNF-α可增强内皮素刺激引起的RASMCs内〔Ca2+〕i增加,且通过1,4,5-三磷酸肌醇受体(IP3R)发挥作用。     

胆汁内外引流术对梗阻性黄疸大鼠肺肿瘤坏死因子α、中性粒细胞弹性蛋白酶的影响

目的研究胆汁内外引流方法对梗阻性黄疸大鼠肺肿瘤坏死因子α(TNF-α)、中性粒细胞弹性蛋白酶(NE)水平的影响。方法将64只成年SD雄性大鼠随机分为4组,分别建立梗阻性黄疸(OJ)、胆汁内引流术(ID)、胆汁外引流术(ED)及假手术(SH)4组模型。于2次术后第14天留取肺组织匀浆液标本。采用双抗体夹心酶联免疫吸附法(ELISA)检测10%肺匀浆液TNF-α水平,生化法检测10%肺匀浆液NE水平。结果成功建立了大鼠梗阻性黄疸及内外引流术模型。梗阻性黄疸时大鼠肺TNF-α、NE水平较假手术对照组明显升高(100.893 pg/mL±21.271 pg/mL vs 64.091 pg/mL±13.034 pg/mL,P<0.01;50.396μg/mL±17.388μg/mL vs 39.718μg/mL±9.625μg/mL,P<0.05)。通过胆汁内引流术解除黄疸后,大鼠肺TNF-α浓度(75.141 pg/mL±15.849 pg/mL)与梗阻性黄疸组相比下降明显(P<0.01);而通过胆汁外引流术解除黄疸后,大鼠肺TNF-α浓度仍较高(112.129 pg/mL±36.886 pg/mL),与梗阻性黄疸组相比无差异(P>0.05)。行胆汁内、外引流术后,大鼠肺NE水平均降低(39.390μg/mL±12.410μg/mL、44.790μg/mL±16.681μg/mL),但与梗阻性黄疸组相比内引流明显(P<0.05)、外引流无差异(P>0.05),且内引流恢复至正常水平,与假手术对照组相比无差异(P>0.05)。结论梗阻性黄疸可导致肺组织炎症细胞因子升高,胆汁内引流术可明显改善梗阻性黄疸时肺组织炎症细胞因子水平、甚至接近正常,而胆汁外引流术没有改善肺炎症细胞因子水平,提示术前利用胆汁内引流术解除梗阻性黄疸缓解肺部炎症反应优于外引流术。     

ABCG2与消化系统肿瘤相关研究进展

临床实践中,很多肿瘤因具有了多药耐药性(MDR)而对抗肿瘤药治疗无效,其中一重要的机制是与多药耐药转运蛋白相关。ABCG2是ABC转运超家族的一个成员,可转运包括拓扑替康、伊立替康等细胞毒药物,但ABCG2具体的蛋白结构及如何转运这些结构不同的底物的机制尚不明确。本文综述近期ABCG2基础研究成果及与消化系统肿瘤相关研究的进展。这些研究提示ABCG2在食管癌、胃癌、肠癌中肿瘤细胞产生多药耐药机制发挥了重要作用,并可能与预后相关。同时ABCG2基因的单核苷酸多态性(SNP)影响ABCG2蛋白的表达、分布和功能,从而可能导致肿瘤对相关药物的耐药性及作为提示预后差的指标。     

妊娠期肝内胆汁淤积症患者胎盘组织白细胞介素10和肿瘤坏死因子α及细胞因子信号传导负调控因子3的表达

目的 探讨细胞因子信号传导负调控因子3 (SOCS3)、白细胞介素10 (IL-10)及肿瘤坏死因子α(TNFα)在妊娠期肝内胆汁淤积症(ICP)孕妇胎盘组织中的表达及其意义. 方法 选择2010年10月至2011年5月在新疆医科大学第一附属医院产科剖宫产分娩的ICP孕妇37例为病例组,选择同期健康孕妇35例为对照组.采用Envision免疫组织化学法测定IL-10、TNFα在孕妇胎盘组织中的定位与表达水平,Western blot测定胎盘组织中SOCS3的表达水平.组间比较采用t检验,等级资料采用秩和检验,变量间的相关性采用Spearman相关分析.结果 (1)IL-10、TNFα在两组孕妇胎盘组织中均有表达,在病例组中,IL-10的表达低于对照组(Z=-2.626,P＜0.01),而TNFα的表达则高于对照组(Z=-4.350,P＜0.01).(2)SOCS3在两组孕妇胎盘组织中均有表达,病例组的表达水平低于对照组(t=-2.214,P＜ 0.05).且其下降与IL-10呈正相关(r=0.494,P＜0.01),与TNFα呈负相关(r=-0.472,P＜0.01).结论 ICP患者母胎界面Th1型细胞因子TNFα表达增加,而Th2型细胞因子IL-10表达降低,存在Th1偏移.SOCS3可能通过影响细胞因子平衡而参与了ICP的发病.