植物血凝素与金色葡萄球菌肠毒素刺激人外周血单个核细胞的比较研究

比较了植物血凝素(PHA)与金色葡萄球菌肠毒素(SEA)对外周血单个核细胞(PBMCs)的刺激效果。取健康人外周静脉血,分离得到PBMCs,分别加入PHA及SEA进行刺激并孵育12 h,利用原子力显微镜和倒置荧光显微镜观察细胞形态变化,利用量子点联合共聚焦显微镜观察活化后细胞膜表面CD3、CD69的分布情况,利用流式细胞术检测淋巴细胞活化早期细胞分化抗原CD69分子表达的变化情况。结果显示,丝裂原PHA刺激的淋巴细胞大多成群聚集,而超抗原SEA刺激的淋巴细胞大多呈分散状,且二者形态有明显差异;两组活化后淋巴细胞的体积均大于静息组,且活化过程中发生极化作用,迁移淋巴细胞,形成膜突起;丝裂原和超抗原刺激淋巴细胞12 h后,使淋巴细胞表达CD69抗原分子,但在量表达上存在差异性(PHA:39.5%±8.7%;SEA:8.3%±1.8%),抗原分子CD3和CD69在膜表面呈不均匀分布,且SEA活化后的T淋巴细胞表面受体CD3和CD69分子在空间上形成了微结构域;外周单个核细胞中其它非T细胞在丝裂原或者超抗原的刺激下也能表达活化抗原分子CD69。     

毛细管气相色谱法测定健康人外周血单个核细胞磷脂脂肪酸组成

采用毛细管气相色谱法对输注脂肪乳剂的健康志愿者外周血单个核细胞磷脂脂肪酸组成进行了分析测定 ,并将定量结果与输注脂肪乳剂前进行了比较 ,结果表明 :与输注脂肪乳剂前相比 ,连续 7d外周静脉输注 2 0 %脂肪乳剂 (2 5 0mL d) ,外周血单个核细胞数无明显变化 ;外周血单个核细胞磷脂酰乙醇胺中棕榈酸(P <0 .0 5 )和油酸 (P <0 .0 1 )明显增加 ,硬脂酸下降 ;磷脂酰胆碱中棕榈油酸 (P <0 .0 5 )和亚麻酸 (P <0 .0 5 )明显增加 ,而两种磷脂中花生四烯酸及其它多不饱和脂肪酸、饱和脂肪酸与不饱和脂肪酸的比值以及脂肪酸的不饱和指数均未发生明显变化。     

Sensitive Sandwich Enzyme Immunoassay of Human Interleukin-2 Produced In Vitro by Peripheral Blood Mononuclear Cells

Abstract

A sensitive sandwich enzyme immunoassay for human inter-leukin-2 (hIL-2) using monoclonal antibody IS described. A monoclonai anti-hIL-2 IgG-coated poiystyrene ball was incubated with hIL-2 and subsequently with affinity-purified rabbit anti-hIL-2 Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. There was no cross-reaction with interleukins 1α and 1bT, epidermal growth factor, insulin and other protein hormones. The detection limit of hIL-2 was 3 pg/tube or 30 ng/1 using 0.1 ml of cuiture supernatant. When peripheral blood mononuclear cells from healthy subjects aged 22-62 yr were cultured in the absence and presence of phytohemagglutinin P for 48 h, hIL-2 levels in the culture supernatants were <0.03-0.10 μg/1 and 0.18-3.7 μg/1, respectively.     

Determination of Gemcitabine Triphosphate in Peripheral Blood Mononuclear Cells by Reverse-Phase HPLC

This paper describes a high-performance liquid chromatographic assay coupled with UV detection at 272 nm, to determine the levels of the nucleotide analogue gemcitabine triphosphate, one of the active metabolites of the antitumoral drug gemcitabine, in peripheral blood mononuclear cells. Isocratic ion-pair chromatography on a C₁₈ column was used. Samples were treated with trichloroacetic acid 40%, cleaned and neutralized with freon:trioctylamine (4:1). The method was linear on the concentration range tested, and the evaluated precision was found satisfactory (RSD < 6.8). The detection limit was 31 pmol, and the quantitation limit 102 pmol. The recovery of gemcitabine triphosphate ranged between 95 and 114%. The procedure was used to provide pharmacokinetic data from cancer patients treated with intravenous gemcitabine.     

Quantification of Saquinavir from Lysates of Peripheral Blood Mononuclear Cells Using Microarrays and Standard MALDI-TOF-MS

Drug monitoring is usually performed by liquid chromatography coupled with optical detection or electrospray ionization mass spectrometry. More recently, matrix-assisted laser desorption/ionization (MALDI) in combination with triple quadrupole or Fourier-transform (FT) mass analyzers has also been reported to allow accurate quantification. Here, we present a strategy that employs standard MALDI time-of-flight (TOF) mass spectrometry (MS) for the sensitive and accurate quantification of saquinavir from an extract of blood peripheral mononuclear cells. Unambiguous identification of saquinavir in the mass spectra was possible because of using internal mass calibration and by an overall low chemical noise in the low mass range. Exact mass determination of the constant background peaks of the cell extract, which were used for recalibration, was performed by an initial MALDI-FT-MS analysis. Fast and multiplexed sample analysis was enabled by microarray technology, which provided 10 replicates in the lower nL range for each sample in parallel lanes on a chip. In order to validate the method, we employed various statistical tests, such as confidence intervals for linear regressions, three quality control samples, and inverse confidence limits of the estimated concentration ratios.

Effect of Cold Atmospheric Plasma Jet and Gamma Radiation Treatments on Gingivobuccal Squamous Cell Carcinoma and Breast Adenocarcinoma Cells

Surgery, chemotherapy and radiotherapy, the conventional treatment modalities of cancer though successful are limited by presence of residual tumor cells, toxic side-effects and treatment resistance, thus raising the need for investigating other novel approaches. Here, we have used a cold atmospheric plasma (CAP) jet and assessed the in vitro efficacy in gingivobuccal squamous cell carcinoma (GB-SCC) – ITOC-03, breast adenocarcinoma—MCF7 and HEK293 cells. Cells lines were subjected to varying doses of ionizing radiation (0, 2, 4, 6, 8 Gy) and CAP jet treatment (0, 60, 180, 240, 300 s). CAP jet treatment showed time dependent increase in H₂O₂ and NO₂^? concentration. Cell viability assay showed potent effect of CAP jet on all three cell lines in comparison to radiation treatment, while helium gas treatment showed minimal inhibitory effect. Irradiated, CAP jet and helium gas treated cells showed loss of nucleic acid features, 788 cm^?1 and 1340 cm^?1 in Raman spectra, indicating DNA damage. Principal Component Analysis (PCA) showed distinct classification of CAP-treated and control cells, while Principal Component – Linear Discriminant Analysis (PC-LDA) based classification of Raman spectra showed ITOC-03 and HEK293 cells to be sensitive to CAP jet and radiation treatment in comparison to MCF7 cells. Collectively, cell viability assay and Raman spectroscopy have shown potent effect of CAP jet in GB-SCC and breast adenocarcinoma cells.

     

基于原子力显微镜的人外周血CD8+T细胞形貌观察与力谱分析

体外分离人外周血CD8+T细胞,用植物凝结素(PHA)刺激活化,利用原子力显微镜(AFM)观察CD8+T细胞的形貌,并结合针尖修饰技术对力谱进行分析,确定了CD8抗原分子的分布.结果表明,与静息的CD8+T细胞相比,活化后的CD8+T细胞直径和高度增大,细胞表面变得更粗糙;CD8抗原-抗体的相互作用力大约是非特异性黏附力的5倍,活化后的CD8+T细胞上特异性黏附力(即CD8抗原分子位置)分布不均匀,其表面的CD8抗原分子分布以纳米团簇分布为主,CD8分子聚集更为明显,CD8+T细胞上CD8抗原-抗体相互作用力不随着活化而发生明显变化,说明CD8抗原-抗体之间具有高选择性.原子力显微镜为特异性T细胞的抗原识别和活化研究提供了一种新手段,能够使T细胞抗原识别和活化的机制得到更好地阐明.     

In Vitro Studies on Ameliorative Effects of Limonene on Cadmium-Induced Genotoxicity in Cultured Human Peripheral Blood Lymphocytes

Applied Biochemistry and Biotechnology - Cadmium (Cd) is a non-redox metal that can indirectly cause oxidative stress by depleting cellular levels of glutathione. It is well-known for the...     

Raman Micro-Spectroscopy of Dental Pulp Stem Cells: An Approach to Monitor the Effects of Cone Beam Computed Tomography Low-Dose Ionizing Radiation

The objective of this study was to determine the molecular and biochemical changes in dental pulp stem cells (DPSCs) due to consecutive low-dose ionizing radiation exposures using label-free Raman micro-spectroscopy (RMS). Ionizing radiation produces biological damage leading to health effects of varying severity. The effects and subsequent health implications caused by exposure to low-dose radiation, such as diagnostic exposure, remain ambiguous. We identified Raman biomarkers characteristic to low-dose cone beam computed tomography (CBCT) irradiation of the DPSCs. The biomarkers were monitored inside the cells using the relative intensity distribution of the 785 and 1734?cm^?1 bands. The control cells presented a higher relative intensity of the nucleic acid specific Raman bands, whereas the irradiated cells revealed an increased intensity of the lipid-induced bands. The results obtained in this study demonstrate the capability of RMS for the detection of cell response to diagnostic radiation dose levels. This may indicate the potential of the technique for future applications such as monitoring the radiation responses in pediatric patients suffering repeated radiological exposures.     

Simple Methodology for the Quantitative Analysis of Fatty Acids in Human Red Blood Cells

In the last years, there has been an increasing interest in evaluating possible relations between fatty acid (FA) patterns and the risk for chronic diseases. Due to the long life span (120 days) of red blood cells (RBCs), their FA profile reflects a longer term dietary intake and was recently suggested to be used as an appropriate biomarker to investigate correlations between FA metabolism and diseases. Therefore, the aim of this work was to develop and validate a simple and fast methodology for the quantification of a broad range of FAs in RBCs using gas chromatography with flame ionization detector, as a more common and affordable equipment suitable for biomedical and nutritional studies including a large number of samples. For this purpose, different sample preparation protocols were tested and compared, including a classic two-step method (Folch method) with modifications and different one-step methods, in which lipid extraction and derivatization were performed simultaneously. For the one-step methods, different methylation periods and the inclusion of a saponification reaction were evaluated. Differences in absolute FA concentrations were observed among the tested methods, in particular for some metabolically relevant FAs such as trans elaidic acid and eicosapentaenoic acid. The one-step method with saponification and 60 min of methylation time was selected since it allowed the identification of a higher number of FAs, and was further submitted to in-house validation. The proposed methodology provides a simple, fast and accurate tool to quantitatively analyse FAs in human RBCs, useful for clinical and nutritional studies.     

柠檬酸镧对人肺癌PG细胞、人胃癌BGC-823细胞的作用

采用噻唑蓝（ＭＴＴ）测定、＾３Ｈ－ＴｄＲ参入、流式细胞术、我偏振和软琼脂培养,观察了柠檬酸镧（ＬａＣｉｔ）对人肺癌细胞（ＰＧ）、人胃癌细胞（ＢＧＣ－８２３）的增殖、ＤＮＡ合成、细胞周期、细胞膜脂流生、软琼脂生长的影响。ＬａＣｉｔ的浓度为０．０５,０．１,０．５,１ｍｍｏｌ．Ｌ＾－１时可抑制肺癌细胞ＰＧ的增殖：浓度为０．５,１ｍｍｏｌ．Ｌ＾－１时可抑制肺癌细胞ＰＧ,ＤＮＡ的合成,其Ｇ１期细胞比例增加     

磺胺多金属氧酸盐对人宫颈癌Hela细胞的抑制作用

对磺胺多金属氧酸盐（C6H9N2O2S）5HP2Mo18O62·15H2O（SPOM-1）,（C6H9N2O2S）H8P2Mo15V3O62·8H2O（SPOM-2）进行了体外毒性实验和抑制宫颈癌Hela细胞作用的研究．当SPOM浓度为50—100mg·L^-1时,对人胚胎成纤维细胞均无细胞毒性作用（P〉0．05）,TC50分别为459mg·L^-1、295mg·L^-1;抑制宫颈癌He—la细胞的EC50分别为88mg·L^-1、24mg·L^-1,治疗指数即分别为5．22,12．29．     

成都市学龄前儿童末梢血微量元素检测结果的分析

为探讨成都市学龄前儿童末梢血微量元素的缺乏现状及其含量与年龄和性别的关系,利用BH 5100型原子吸收光谱仪对23 932例学龄前儿童末梢血中铜、锌、钙、镁和铁元素进行了检测,用SPSS 16.0做了统计分析.结果表明,所测学龄前儿童末梢血铁、锌元素缺乏严重;铜、锌、钙和铁元素含量在年龄上呈现一定变化规律,铜元素在男女...     

Analysis on Trace Elements in Preschoolers' Peripheral Blood in Xi'an City

为探讨西安市学龄前儿童末梢血微量元素现状及其含量与年龄及性别的关系,对2 350例学龄前儿童末梢血中铜、锌、钙、镁、铁5种元素进行了测定,用SPSS 16.0做统计分析.结果表明,受检儿童末梢血中锌、钙、铁缺乏严重,铜和锌含量在年龄上具变化规律,且铜在性别间存在显著差异.提示应充分认识微量元素对儿童健康的重要性,定期对儿童进行微量元素监测,做好防治工作.     

火焰原子吸收光谱法测定人血红细胞中锌、铁、镁

人血红细胞样品经低渗Triton X-100溶液超声振荡溶血处理并稀释100倍后采用火焰原子吸收光谱法测定其中锌、铁和镁的含量。锌、铁和镁的检出限(3S/N)分别为2.44×10~(-3),2.85×10~(-2),1.56×10~(-3)mg·L~(-1),相对标准偏差(n=7)分别为0.69%～1.03%,2.85%～3.84%,0.92%～1.22%。应用该法分析同一混合人血红细胞样品,测定结果与酸消化法测得的结果一致,3种元素的加标回收率为95.0%～110.0%之间。     

应用单细胞微凝胶电泳技术研究氯化镉对人血淋巴细胞DNA的损伤效应

详细介绍了单细胞微凝胶电泳（ＳＣＧ）技术的原理和操作过程，并应用该技术研究了氯化镉（ＣｄＣｌ２）对人血淋巴细胞ＤＮＡ的损伤效应，结果表明，ＣｄＣｌ２能引起细胞ＤＮＡ迁移长度增加，且呈显著的剂量效应关系，对未处理对照细胞的ＤＮＡ迁移的原因及ＳＣＧ实验操作过程中应注意的事项也进行了讨论。     

重组人干扰素—γ的凝胶色谱分离及其变性剂对分离结果的影响

使用十二烷基硫酸钠，盐酸胍和尿素三种不同的变性剂溶解基因重组人干扰素－Ｇａｍｍａ（ｒ－ＩＦＮ－γ）包含体，然后进行凝胶色谱分离。使用活性，电泳和高效疏水作用色谱三种方法，比较了三种变性剂对分离结果的影响，结果为：尿素溶解包含体后ｒ－ＩＦＮ－γ的分子量与杂质相差较大，盐酸胍溶解液经分离后ｒ－ＩＦＮ－γ比活最高，ＳＤＳ对ｒ－ＩＦＮ－γ的构象影响较大。     

Immobilization of Urease on (HEMA/IA) Hydrogel Prepared by Gamma Radiation

In the present study, the copolymeric hydrogels based on 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA) were synthesized by gamma radiation induced radical polymerization, in order to examine the potential use of these hydrogels in immobilization of Citrullus vulgaris urease. Gelation and Swelling properties of PHEMA and copolymeric P (HEMA/IA) hydrogels with different IA contents (96.5/3.5, 94.4/5.6 and 92.5/7.5 mol) were studied in a wide pH range. Initial studies of so-prepared hydrogels show interesting pH sensitivity in swelling and immobilization. C. vulgaris urease was immobilized on HEMA/IA (92.5/7.5) at 6 kGy with 41.3% retention of activity. The properties of free and immobilized urease were compared. Immobilized urease maintained a higher relative activity than free urease at both lower and higher pH levels, indicating that the immobilized urease was less sensitive to pH changes than the free urease. The K_m value of the immobilized urease was approximately 2 times higher than that of the free urease. Temperature stability was improved for immobilized enzyme. The free form exhibited a loss about 80% of activity upon incubation for 15 min at 80°C. The influence of various heavy metal ions at the concentration of l mM was improved after enzyme immobilization. The immobilization of C. vulgaris urease on HEMA/IA (92.5/7.5) at 6 kGy showed a residual activity of 47 % after 4 reuses.