Software compensation improves the analysis of heterogeneous tumor samples stained for multiparameter DNA flow cytometry

Background: High concentrations of propidium iodide (PI), in combination with fluorescein isothiocyanate (FITC) and R-phycoerythrin (RPE) used for multiparameter DNA flow cytometry (FCM), cause spectral cross-talk into the green fluorescence channel (FL1). We have evaluated the use of post-acquisition software compensation (N-Color Compensation) in order to correct this spectral cross-talk caused by PI. Method: Cell mixtures were prepared consisting of keratin 8/18 FITC labeled, keratin 8/18 RPE labeled, and unlabeled MCF-7 breast carcinoma cells. DNA was stained with PI (100 μM). Post-acquisition software compensation was applied to correct the spectral cross-talk of PI fluorescence. Secondly, the distribution of the Ki-67 (FITC) protein during the cell cycle (PI) of SiHa cervical carcinoma cells (no software compensation) was compared to the Ki-67 expression pattern of SiHa cells, simultaneously stained for keratin 8 (RPE), after applying software compensation. Finally, software compensation was used to compare the relative levels of PCNA and p53 expression in two clinical ovarian cancer ascites specimens, stained for PCNA or p53 (FITC), keratin 8/18 (RPE), and DNA (PI), with a known p53 status (positive and negative, respectively). Results: The Ki-67 cell cycle-dependent pattern of a triply stained sample (Ki-67 (FITC), keratin 8 (RPE), and DNA (PI)) is restored after software compensation and the results are comparable to the Ki-67 distribution of a sample stained solely for Ki-67 and DNA. P53 expression could only be resolved after using software compensation in the p53 positive ovarian ascites (OA) sample. Conclusions: We conclude that software compensation is a robust and reliable post-acquisition method for the correction of RPE/PI spectral cross-talk, permitting better identification of weakly expressed proteins in heterogeneous clinical tumor samples stained for multiple cellular antigens and DNA using PI.     

脑囊虫病患者脑脊液中一氧化氮及肿瘤坏死因子α水平研究

目的　探讨脑囊虫病患者各期脑脊液中的一氧化氮 (NO)、肿瘤坏死因子α(TNF α)的变化规律及它们在脑囊虫病中的作用机制。方法　检测 4 9例明确诊断并依据MR分期 ,单发脑实质内囊虫的脑囊虫病患者和 2 0名对照者脑脊液中NO、TNF α水平。结果　NO在脑囊虫病的Ⅰ期显著降低而TNF α水平呈显著升高 ,NO、TNF α于Ⅱ期、Ⅲ期 (整个退变死亡期 )均表现为高水平 ,Ⅳ期恢复正常 ,NO和TNF α存在高度正相关关系。结论　在单发脑实质内囊虫病中NO、TNF α可能参与杀虫作用 ,其免疫调节与杀虫机制存在着动态平衡 ,参与感染控制     

胆囊结石及癌变过程中肿瘤坏死因子可溶性受体的变化

目的　探讨可容性肿瘤坏死因子受体 (sTNFR)在胆囊结石及癌变过程中的变化。方法　用双抗体夹心酶联免疫法对 5 3例胆囊结石 ,9例胆囊癌及 11例正常对照者血清及胆法中的sTNFR水平进行检测。结果　血清及胆汁中sTNFR水平胆囊癌组为 (2 .63± 0 .5 6) μg/L、(10 .0 2± 3 .2 3 ) μg/L较胆石症组 (1.2 5± 0 .3 6) μg/L、(2 .81± 0 .93 ) μg/L及对照组 (0 .95± 0 .19)μg/L、(1.83± 0 .5 4) μg/L均显著升高 (P <0 .0 1)。胆囊黏膜从典型增生、非典型增生到胆囊癌的发展过程中sTNFR在血清 (1.11± 0 .2 8、1.5 3± 0 .3 2、2 .63± 0 .5 6)及胆汁中 (2 .5 0± 0 .81、3 .42±0 .87、10 .0 2± 3 .2 3 )逐级增高 (各组间P <0 .0 5 )。胆汁中sTNFR水平在胆囊癌Ⅰ～Ⅲ期 (8.3 6±2 .60 )与Ⅳ～Ⅴ期 (13 .3 3± 4.46)间差异有显著性 (P <0 .0 0 1) ,肿瘤直径≥ 2cm组 (12 .10± 2 .3 2 )与 <2cm组 (7.42± 2 .10 )间差异有显著性 (P <0 .0 5 )。胆汁中TNFR水平明显高于其对应的血清水平 ,两者之间呈正直线相关 (r =0 .875 ,P <0 .0 0 1)。胆囊癌术后血清sTNFR水平显著下降。结论　sTNFR参与胆囊结石致癌的过程 ,与胆囊癌的临床生物学特点密切相关。     

参附注射液对肠缺血-再灌注大鼠肿瘤坏死因子α的影响

目的观察肿瘤坏死因子α(TNF-α)在大鼠肠缺血-再灌注损伤过程中的作用及参附注射液对TNF-α的影响,探讨参附注射液防治肠缺血-再灌注损伤机制。方法 SD大鼠随机分为肠缺血-再灌注组(IR组)、参附注射液预处理组(SF组)和假手术组(C组)。采用阻断肠系膜上动脉(SMA)的方法制造肠缺血-再灌注模型。分别测定各组动物血浆、肠组织TNF-α含量及血液动力学变化;光镜观察肠粘膜损伤情况。结果IR组再灌注后MAP下降,与C组和SF组比有显著性差异(P<0.01);SF组肠粘膜损伤程度减轻,与IR组比有显著性差异(P<0.01);SF组血浆及肠组织TNF-α水平降低,与IR组比有显著性差异(P<0.01)。结论参附注射液可明显防治大鼠肠缺血-再灌注导致的肠粘膜损伤,这种作用可能是通过抑制TNF-α的释放实现的。     

树突状细胞负载前列腺癌细胞抗原后的抗肿瘤免疫作用

目的 探讨小鼠树突状细胞(DC)负载前列腺癌细胞株RM-1的裂解产物后，诱导特异性细胞毒T淋巴细胞(CTL)及其抗肿瘤的免疫作用。方法 将RM-1细胞的裂解产物(T-lysate)作为肿瘤抗原负载小鼠骨髓来源的DC，构建DC瘤苗(T-lysate／DC)，采用流式细胞术和四唑氮蓝(MTT)还原法检测其免疫活性；ELISA法检测其诱导细胞因子白介素(IL)-2和γ-干扰素(IFN-γ)的作用；体内实验检测DC瘤苗的抗肿瘤免疫治疗作用和免疫保护作用。结果 DC负载RM-1细胞的裂解产物后，其主要组织相容性复合物(MHC-Ⅰ、Ⅱ)及共刺激分子(B7-1、B7-2)的表达明显增高；T-lysate／DC能够诱导小鼠产生RM-1特异性CTL，使细胞上清液中IL-2和IFN-γ水平升高，对小鼠具有免疫保护作用，并能有效抵抗肿瘤细胞的攻击；经T-lysate／DC治疗的荷瘤小鼠瘤体生长减慢，存活期延长，瘤体出现坏死和炎细胞浸润。结论 DC负载前内腺细胞解产物后，能够有效诱导搞肿瘤免疫反应，为前列腺癌的临床治疗提供了新策略。     

Enzyme-linked Immunosorbent Assay of Pro-gastrin-releasing Peptide for Small Cell Lung Cancer Patients in Comparison with Neuron-specific Enolase Measurement

Our previous study demonstrated that pro-gastrin-releasing peptide(31–98), or ProGRP, is a specific tumor marker in patients with small cell lung carcinoma (SCLC). Using a newly developed, highly sensitive enzyme-linked immunosorbent assay (ELISA) for ProGRP, we analyzed 1,446 samples including those obtained from 478 lung cancer patients to evaluate the clinical usefulness of this ELISA. Several properties indicated that ProGRP is a useful tumor marker for SCLC. First, ProGRP was specifically elevated in SCLC patients. In non-SCLC patients and patients with non-tumorous lung diseases, its serum level was very rarely elevated. Secondly, ProGRP was a reliable marker, in terms of the marked elevation of serum ProGRP levels in SCLC patients. Thirdly, serum ProGRP levels were elevated in SCLC patients even at a relatively early stage of this disease. Fourthly, changes in the serum ProGRP level showed an excellent correlation with the therapeutic responses in SCLC patients. Neuron-specific enolase (NSE) is accepted as a tumor marker of SCLC patients. With the aim of comparing ProGRP and NSE as tumor markers for SCLC patients, we measured serum NSE levels in all samples collected in the present study. We found that ProGRP was superior to NSE in terms of sensitivity, specificity and reliability. Therefore, we consider that ProGRP can play a major role as a clinical tumor marker for SCLC patients.     

恶性肿瘤局部淋巴结树突状细胞免疫组化研究

本文采用S-100蛋白为免疫学标记，对34例恶性肿瘤局部淋巴结内树突状细胞进行免疫组化定量研究。结果按S-100蛋白阳性细胞数目多少分为增多（7例）、减少（20例）及正常（7例）3组，统计学分析增多组均值（164.4个/mm^2)明显高于对照组（58.3个/mm^2)；减少组均值（16.5个/mm^2)组显低于对照组；而正常组均值（69.8个/mm^2)与对照组无显著差异。     

Vascular cell adhesion molecule-1 modulation by tumor necrosis factor in experimental allergic encephalomyelitis

Anti-tumor necrosis factor (TNF) antibodies inhibit passively transferred experimental allergic encephalomyelitis (EAE) in SJL mice. The possibility that this occurs through interference in TNF's upregulation of endothelial cell adhesion molecules was investigated. Expression of both vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on spinal cord vessels increased during EAE. The upregulation of VCAM-1 was markedly reduced or prevented by anti-TNF treatment. Leukocytic infiltration was 15-fold lower in anti-TNF-treated than diseased animals. Spinal cord endothelial expression of VCAM-1, though not ICAM-1 or fibronectin, positively correlated with the extent of T cell, B cell or monocyte infiltration in each animal.     

旋毛虫幼虫分泌物抗原在小鼠体内诱生肿瘤细胞毒因子的研究

本文应用Ｌ９２９细胞杀伤法，对注射旋毛虫肌肉期幼虫分泌物（Ｌ１ＥＳ）的小鼠血清进行了检测，发现Ｌ１ＥＳ对已注射卡介苗（ＢＣＧ）或感染旋毛虫的小鼠，均能诱生肿瘤细胞毒因子（ＴＣＦ），而正常对照鼠则不能发生，表明Ｌ１ＥＳ诱生ＴＣＦ需要首先激活巨噬细胞（Ｍφ）这一基本条件。将Ｌ１ＥＳ置３７℃、１ｈ，５６℃、３０ｍｉｎ或１００℃、２ｍｉｎ处理后，其诱生ＴＣＦ的能力均明显高于对照组（Ｐ＜０．０５）。Ｌ１ＥＳ     

肺癌病人血清铜、锌水平的研究

本研究应用原子吸收分光光度法，检测186例肺癌病人血清铜，锌水平，并以150例正常人和64例良性胸部疾患病人作对照，结果显示：肺癌病人血清铜水平明显高于良性胸部疾患病人和正常人，血清锌水平明显低于良性胸部疾患病人和正常人，铜，锌水平的改变与肺癌的病期有明显的关系（P＜0．04），而与组织学类型无关，本研究结果表明：检测肺癌病人血清铜，锌水平可用于肺癌的辅助诊断和判别肺癌的 病期。