MTS比色法检测嗜中性粒细胞产生的活性氧

目的 建立一种简便可靠的吞噬细胞“呼吸爆发”检测方法。方法 以MTS为受氢体检测嗜中性粒细胞受激发后产生的超氧离子，从而间接反映其“呼吸爆发”发生情况。结果 该法与常用的高铁细胞色素C还原法有良好的相关性，Y（MTS法）＝2．307X－0．291，r=0.9941。19例反复感染患者的嗜中性粒细胞的活性氧产量明显低于健康人。结论 该法是一种简便可靠的吞噬细胞“呼吸爆发”检测方法，可用于吞噬细胞功能的检测。     

2,7—二氨基芴作色源检测血浆游离血红蛋白

在有过氧化氢的条件下 ,血红蛋白 (Hb)催化用 2 ,7 二氨基芴 (2 ,7 diaminofluorene ,DAF)氧化生成芴蓝 ,芴蓝在42 0～ 5 0 0nm间有一吸收峰。选用 44 0nm作检测波长时 ,吸光度值与Hb含量在一定范围 (5～ 45 0mg/L)内线性关系 ,Y =12 6 48X 0 0 39,r =0 996 3;批内CV1 89% ～ 2 91% ;批间CV2 99% ～ 3 6 4% ;平均回收率 10 1 7% ;与直接分光光度法比较Y =0 987X 0 14,r=0 975 2 ;参考范围 7～ 2 5mg/L。本法操作简便 ,结果可靠 ,试剂保存期长 ,适用于一般临床实验室     

DAF比色试验在检测血清补体依赖性细胞毒作用中的应用

以2,7-二氨基芴(2,7-diaminofluorene,DAF)为色源,检测被溶血素致敏的绵羊红细胞在血清补体作用后的溶解程度,从而间接反映血清中补体的溶细胞能力(细胞毒作用)。该法不仅比传统方法操作简便、灵敏度高,且安全、环保,同时也可用于补体结合试验的检测。     

肺癌患者化疗后血清IL-2、IL-8和中性粒细胞吞噬功能的研究

目的探讨肺癌患者化疗前后血清白细胞介素（IL）-2、IL-8和中性粒细胞吞噬功能水平的变化及临床意义。方法应用放免法、酶联免疫法和普通酶母菌检测法对32例肺癌者进行了血清IL-2、IL-8和中性粒细胞吞噬功能水平的检测,并与35例正常健康人作比较。结果肺癌患者在化疗前,血清IL-2水平和中性粒细胞吞噬功能明显低于正常人组（P〈0.01）,血清IL-8水平明显高于正常人组（P〈0.01）,化疗6个月后与正常人比较无显著性差异（P〉0.05）。结论肺癌患者血清IL-2、IL-8和中性粒细胞吞噬功能的变化与患者的病情和预后密切相关。     

烟雾吸入性损伤大鼠肺泡巨噬细胞吞噬功能与炎症消散关系研究

目的 :探讨烟雾吸入性损伤大鼠肺泡巨噬细胞吞噬功能与炎症消散关系。方法 :建立大鼠烟雾吸入损伤模型后于伤后 2、6、12、2 4h及 2、3、4、5d各时相点动态观察 (1)肺泡巨噬细胞体外对鸡红细胞吞噬功能 ;(2 )肺泡巨噬细胞体内对凋亡中性粒细胞吞噬功能。结果 :(1)致伤后 2～ 6h肺泡巨噬细胞对鸡红细胞吞噬率、吞噬指数下降 ,12h后逐渐恢复正常 ,2～ 5d仍保持较高的吞噬功能。 (2 )肺泡巨噬细胞对凋亡中性粒细胞吞噬在伤后 2h即开始逐渐升高 ,2 4h达峰值 ,然后逐渐下降。结论 :(1)烟雾吸入伤早期 (2～ 6h)肺泡巨噬细胞吞噬功能受损害 ,然后逐渐恢复正常 ,并保持较高吞噬活性 ,它有利于清除异物、细菌等。 (2 )肺内中性粒细胞发生凋亡 ,然后被巨噬细胞吞噬参与了恢复期肺内炎症消散的过程     

RDW、NLR、D-二聚体联合检测对急性胰腺炎病情的预测价值

目的 探讨红细胞分布宽度(RDW)、中性粒细胞与淋巴细胞比值(NLR)、D-二聚体联合检测对急性胰腺炎(AP)病情的预测价值.方法 本研究纳入210例在消化内科确诊为AP的患者,126例为轻症组、42例为中度重症组、22例为重症组、20例为危重组,比较4组基本资料及相关参数,评估RDW、NLR、D-二聚体及联合检测对A...     

外周血EOS、NLR联合检测诊断慢性鼻-鼻窦炎价值分析

目的 探讨外周血嗜酸性粒细胞(EOS)、中性粒细胞与淋巴细胞计数比值(NLR)联合检测诊断慢性鼻-鼻窦炎(CRS)价值.方法 选取2017年6月-2020年6月成都医学院第三附属医院80例CRS患者纳入CRS组,按照患者手术以后病理检测组织内EOS的计数水平来分型分为嗜酸粒细胞性慢性鼻-鼻窦炎组(ECRS组,n=59)...     

COPD患者痰液中性粒细胞氧化吞噬功能及相关受体变化的临床意义

目的探讨慢性阻塞性肺疾病(COPD)患者中性粒细胞氧化吞噬功能及相关受体[趋化因子C-C基序受体-1(CCR1)和Toll样受体(TLR)2、TLR4]变化的临床意义。方法建立用于检测COPD患者痰液中性粒细胞氧化吞噬功能及相关受体的流式细胞术方法。采用流式细胞术检测30例COPD患者(COPD组)、30例肺炎患者(肺炎组)和30名体检健康者(正常对照组)外周血及痰液(正常对照组除外)中性粒细胞氧化吞噬功能及其表面CCR1、TLR2和TLR4的表达水平。结果肺炎组和COPD组外周血中性粒细胞氧化吞噬功能均显著低于正常对照组(P0.05),肺炎组与COPD组之间差异无统计学意义(P0.05)。COPD组、肺炎组及正常对照组之间外周血中性粒细胞表面CCR1表达差异均有统计学意义(P0.05),而TLR2、TLR4表达3组之间差异均无统计学意义(P0.05)。COPD组痰液中性粒细胞氧化吞噬功能显著低于肺炎组(P0.05),CCR1表达显著高于肺炎组(P0.05),TLR2和TLR4表达2个组之间差异均无统计学意义(P0.05)。Pearson相关分析结果显示,COPD组痰液中性粒细胞氧化吞噬阳性率与CCR1表达呈负相关(r=-0.548,P0.01),与TLR2、TLR4表达均无相关性(r值分别为-0.206、-0.219,P0.05)。结论 COPD患者痰液中性粒细胞氧化吞噬功能显著下降,其表面CCR1表达升高。COPD患者气道中性粒细胞的氧化吞噬功能受损可能是COPD发病的机制之一。     

IL-2/GM-CSF体外处理的自体外周血单个核细胞治疗再生障碍性贫血49例长期随访

本研究回顾分析2001年4月至2007年12月期间采用IL-2和GM-CSF体外处理的外周血单个核细胞治疗再生障碍性贫血(aplastic anemia,AA)的安全性及远期疗效。取自体外周血单个核细胞,在IL-2和GM-CSF作用下培养48小时后进行静脉回输,细胞总剂量为6×106-1×108,每周1次,连用4-22个月。外周血常规检查、骨髓细胞涂片和骨髓活检评价造血恢复,流式细胞术测定输注细胞的组成以及治疗前后T细胞亚群的变化,多聚酶链反应检测外周血T细胞TCRVβ克隆性。结果表明:49例患者中痊愈37例,随访至今无1例出现复发;5例部分缓解,3例明显进步,4例无效,总有效率91.8%(45/49)。治疗前外周血T细胞亚群CD4/CD8比例倒置者39例,治疗后31例(79.5%)恢复正常。11例患者行外周血TCRVβ克隆性分析,受抑制的亚群重新得到恢复,出现多克隆的表达图像。未发现晚期克隆性疾病及其它远期不良反应。结论 :IL-2和GM-CSF体外处理的外周血单个核细胞疗法疗效肯定,其机制可能与T细胞免疫功能的恢复有关。     

Modification of the Colorimetric Assay for Chloramphenicol in the Presence of Bilirubin

We recently found that bilirubin interferes with the colorimetric chloramphenicol assay of Kakemi et al. (K. T. Kakemi, T. Arita, and S. Ohashi, Yakugaku Zasshi 82:342-345, 1962). Levels of serum bilirubin alone (4 to 6 mg/dl) resulted in apparent concentrations of chloramphenicol which appear to be in the therapeutic range. Concentrations of serum bilirubin greater than 8 mg/dl resulted in levels of apparent chloramphenicol associated with toxicity (>50 mug/ml). Small amounts of activated charcoal added to the isoamyl acetate extraction step of the assay eliminated this interference by bilirubin.     

化学比色法测定血清碳酸氢根的研究

目的建立一种新的血清碳酸氢根(HCO3-)测定方法。方法应用自配试剂建立两步终点测定法,可满足全自动和手工半自动测定,并对该方法进行方法学评价。结果该法比色液吸收峰556~558 nm,线性范围1~50 mm o l/L,批内CV 2.56%,批间CV 3.55%,回收率96%~103%,与血气分析仪(X)相关良好,Y=0.48 0.989X,r=0.982(n=40),脂血、溶血、黄疸标本对结果无影响,健康人静脉HCO3-浓度x-±s=25.76±2.26 mm o l/L。结论该法操作简便,结果准确,试剂稳定,成本低廉,适合各级医院常规和急诊使用。     

刃天青微孔板显色法在抗念珠菌药敏实验中的应用

目的 探讨刃天青微孔板显色法在抗念珠菌药敏实验中的应用.方法 采用NCCLS之M27-A方案测定伊曲康唑、氟康唑和两性霉素B对87株临床分离的念珠菌属真菌的最低抑菌浓度(MIC),终点判定同时采用NCCLS推荐的直接观察法和刃天青微孔板显色法.结果 直接观察法和刃天青微孔板显色法判定所得的MIC结果具有高度的一致性.结论 刃天青微孔板显色法具有结果客观、灵敏度高、准确等特点,因而可替代直接观察法用于真菌药敏实验终点的判定.     

Role for Endogenous and Acquired Peroxidase in the Toxoplasmacidal Activity of Murine and Human Mononuclear Phagocytes

Oxygen products generated by the respiratory burst of mononuclear phagocytes are microbicidal to intracellular pathogens including Toxoplasma gondii. The toxicity of one of these products, H₂O₂, is markedly amplified by the granule peroxidase of circulating phagocytes in the presence of a halide. Eosinophil peroxidase (EPO) binds firmly to the surface of T. gondii and such organisms remain viable as determined by vital staining, uptake of 2-deoxyglucose, and survival and replication in human fibroblasts. They are, however, rapidly killed by the addition of H₂O₂ and iodide under conditions in which control organisms are unaffected. We have used EPO bound to T. gondii to explore the role of peroxidase in the toxoplasmacidal activity of mononuclear phagocytes. Resident mouse peritoneal macrophages lack a granule peroxidase and have a weak respiratory burst; toxoplasma survive and replicate within these cells. However, these cells acquire significant toxoplasmacidal activity, as assessed microscopically and by the inhibition of uracil uptake, when organisms are coated with EPO before ingestion, an effect which is decreased by the hemeprotein inhibitors, aminotriazole and azide. EPO on the surface of Toxoplasma does not increase their ingestion by macrophages or the associated respiratory burst. Monocytes from patients with hereditary myeloperoxidase deficiency have a significant toxoplasmacidal defect that is abolished when EPO-coated organisms are used. In contrast, the toxoplasmacidal defect of monocytes from chronic granulomatous disease patients is unaffected by surface-bound EPO. In these studies, replication of surviving intracellular organisms varied inversely with the magnitude of the respiratory burst: replication was greatest in fibroblasts, slightly less in resident macrophages, and least in monocytes; it was significantly greater in chronic granulomotous disease than in normal or myeloperoxidase-deficient monocytes. These studies support a role for oxygen products and endogenous peroxidase in the optimal killing of T. gondii by monocytes and demonstrate that peroxidase-negative phagocytes can utilize peroxidase on the surface of ingested organisms to augment microbicidal activity.     

Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.     

MTT法药物敏感试验和鼻咽癌临床

目的尝试建立鼻咽癌（NPC）化疗药敏药物的体外筛选方法。方法利用四甲基偶氯唑蓝（MTT）法体外药敏试验检测48例NPC对7种临床化疗药物的敏感性，并且与临床化疗效果进行对比。结果NPC细胞对DDP、5-Fu、VCR、CTX、BLM、HHA、VM-26的敏感性存在明显个体差异，其敏感率分别为52％、56％、61％、48％、48％、35％、74％。与临床化疗结果对比结果显示：阳性符合率89．4％，阴性符合率80．0％；敏感性94．4％，特异性66.7％。结论MTT可应用于NPC临床筛选药物。     

检测白血病细胞增殖力的MTS/pms比色分析法的建立

为了探讨建立一种更为快速、安全、灵敏的白血病细胞增殖力的检测方法，本研究应用HL—60和K562细胞可转化MTS／pms形成水溶性待测产物，其光密度值在492nm波长下半自动测定的特点，研究了MTS／pms比色分析法取代MTT和INT用于白血病细胞增殖力的检测。结果表明，只有活的白血病细胞才能转化MTS／pms形成待测产物，且白血病细胞数与所测光密度值(OD)之间具有良好的相关性(γ＝0．963)；与MTT和INT法相比较，MTS／pms比色分析法中所形成的最终待测产物为水溶性，省略了上清波去除和加入有机溶剂的步骤。结论：MTS／pms比色分析法对白血病细胞增殖力的检测更具有快速、安全、准确、灵敏的优点。     

An Automated Colorimetric Method for the Estimation of Lactate Dehydrogenase Activity in Serum

An automated method for estimating lactate dehydrogenase (LDH) in serum is presented. Fe³⁺ is reduced to Fe²⁺ by NADH formed when lactate is oxidized to pyruvate. Fe²⁺ complexed with 2,2-bipyridyl is measured colorimetrically. Solutions of Fe²⁺ salt are used as standards. Nicotinamide is used to stabilize NAD+ in solution and is shown to enhance enzyme activity. The method is less expensive than most automated methods published and reagents are stable for at least 4 weeks. Results correlate well with a kinetic spectro-photometric method.     

细胞色素酶CYP2C19介导的沙利度胺体外抗血管生成作用研究

本研究探讨人肝细胞微粒体代谢系统对治疗多发性骨髓瘤的沙利度胺体外抗血管生成的影响及细胞色素酶CYP2C19在其中的作用。采用沙利度胺原药或与人肝细胞微粒体在体外共孵育后用MTY法检测人脐带静脉内皮细胞（human umbilical cord vein endothelial cells，hUCVEC）增殖活力，用流式细胞术测定hUVCEC细胞周期和细胞凋亡，以改良的Boyden小室法检测hUCVEC细胞迁移力，以体外小管形成实验检测hUCVEC分化。结果表明：沙利度胺原药对hUCVEC活力无明显抑制作用，细胞凋亡比例也无明显增加，轻度影响细胞迁移，无抗小管形成作用；当与人肝细胞微粒体共孵育后hUCVEC增殖活力明显受抑。100μg／ml沙利度胺与肝细胞微粒体共孵育后hUCVEC增殖活力的抑制率达（11．7±3．9）％，凋亡细胞增加达27．2％，明显下调细胞迁移力并抑制体外小管形成。在共孵育体系中加入CYP2C19特异性抑制剂奥美拉唑，可减弱沙利度胺抑制hUCVEC增殖活力和诱导凋亡的作用，减低细胞迁移力和部分逆转抗小管形成的作用。结论：沙利度胺的体外抗血管生成作用依赖于人细胞微粒体的作用，细胞色素酶系中的CYP2C19可能参与了这一过程。