原子力显微镜对中性粒细胞与K562细胞超微结构及机械性能的探测

采用原子力显微镜在纳米尺度下对正常中性粒细胞与白血病细胞株K562细胞的表面形貌及细胞的硬度、粘附力进行定性定量分析.结果表明,相比正常中性粒细胞的平均粗糙度(Ra=5.31±1.52 nm),K562细胞的超微结构更为复杂,细胞表面平均粗糙度显著升高(Ra=26.54±8.01 nm).此外,细胞的生物机械特性也有显著差别:中性粒细胞的硬度为9.5±1.3 kPa,AFM针尖与中性粒细胞的非特异性粘附力为135±23.4 pN;K562细胞的硬度为3.0±0.8 kPa,AFM针尖与K562细胞的非特异性粘附力为95±15.6 pN.AFM在单细胞水平上的探测表明,中性粒细胞和K562细胞的超微结构和机械特性均有明显差异.通过对细胞表面超微结构和力学特性的探测可以诊断慢性粒细胞白血病,原子力显微镜有望成为临床肿瘤诊断的工具.     

不同干燥条件下壳聚糖膜表面的微观结构及微观力学性能

以自然风干(NW)、真空干燥(VD)及红外干燥(ID)三种干燥方式制备了壳聚糖膜.利用原子力显微镜(AFM)研究这三种壳聚糖膜的表面形貌及微观力学性能.实验结果表明VD和ID改善了膜材料的表面平整度,膜表面粗糙度分别为(5.47±1.34)和(2.79±0.93)nm,均显著低于NW膜((30.67±8.06)nm).干燥条件对壳聚糖膜的微观力学性能有较大影响:ID壳聚糖膜的粘附力((2595.0±68.5)pN)显著大于NW壳聚糖膜((982.6±149.3)pN)和VD壳聚糖膜((1817.9±279.2)pN);而ID壳聚糖膜的杨氏模量((158.8±15.2)MPa)则低于NW壳聚糖膜((204.3±22.7)MPa)和VD壳聚糖膜((195.8±14.6)MPa)的.     

正常人眼角膜上皮细胞的原子力显微镜观察

应用原子力显微镜(AFM)在单细胞水平上分析了人眼角膜上皮细胞的形貌和机械性质,为进一步探讨人眼角膜上皮细胞结构与功能的关系奠定了基础.将体外培养的人眼角膜上皮细胞用2.5%戊二醛固定,空气中干燥后用原子力显微镜进行观察.从AFM形貌图可知,细胞呈长梭形,膜表面布满颗粒状物质,由AFM附带软件IP2.1的线分析及面分析功能,得到细胞膜表面结构的几何参数,包括高低差Rp-v、均方根粗糙度Rq、平均粗糙度Ra、平均高度Meant Ht.利用AFM高空间分辨的力位移曲线测量系统,可得出细胞膜的粘弹力、硬度和杨氏模量.AFM能对人眼角膜上皮细胞表面的超微结构清晰地成像并提供更多更确切的表面信息,从另一层面增加对眼角膜上皮细胞的认识.     

不同刺激条件下人外周血淋巴细胞超微结构与粘滞力的AFM分析

应用原子力显微镜（atomic force microscopy,AFM）探测了静息、脂多糖（LPS）或伴刀豆蛋白（ConA）活化的人外周血淋巴细胞的形态结构、超微结构及粘滞力特性。从AFM图像可知,经LPS或ConA刺激活化后的淋巴细胞比静息状态的淋巴细胞有所增大,并且表面分布着大小不一的颗粒状聚合物。利用AFM高空间分辨的力位移曲线测量系统,发现经LPS或ConA刺激活化后淋巴细胞的粘滞力是静息状态淋巴细胞的2~3倍。通过AFM探测淋巴细胞活化状态的可视化数据,可以更好地理解淋巴细胞的行为。     

515例急性淋巴细胞白血病的免疫分型

用抗人白细胞分化抗原单克隆抗体对515例急性淋巴细胞白血病患者进行了免疫分型诊断,区分出T细胞型(T-ALL)和非T细胞型(Non-T-ALL)白血病,后者又分为普通型(C-ALL)、无标志型(Null-ALL)以及B细胞型(B-ALL)等主要亚型,T细胞及非T细胞型白血病细胞根据其表型特点又可进一步划分。本研究所观察到ALL细胞表型基本反映正常T或B淋巴细胞亚群分化阶段或某些类型细胞的表型特征,急性淋巴白血病免疫分型不仅有助于判断白血病细胞的来源,与FAB分型方法配合使用还可以大大提高对白血病的诊断准确率。同时,分型结果显示出与临床表现、治疗反应以及预后转归有一定的相关性。本文对中国急性淋巴患者的发病特点也进行了探讨。     

一组小鼠白血病细胞系的建立及生物学特性的研究

本支建立了一种经PHA-脾条件培养液刺激,在体外形成白血病干细胞克隆后,再经体内传代,发病后在体外连续培养建成白血病细胞系的新方法。采用此方法成功地建立了T抑制淋巴细胞白血病L7811-85,L7212-85细胞系,非T非B淋巴白血病L1210-86,B淋巴白血病P388-86及红白血病FLCL细胞系等。对上述细胞系进行了较系统的生物学特性研究。电子显微镜观察中发现,白血病细胞的病毒颗粒在体外传代时A型颗粒消失,C型颗粒出现。并发现在L7811-85细胞系培养上清液分离的组分中,有白血病相关抑制活性的存在。     

角膜基质细胞在p(HEMA-MMA)改性前后的粘附率、形貌与力学性能变化

采用原子力显微镜(AFM)对改性前后材料表面粘附生长的角膜基质细胞的亲和力、三维形貌和力学性能进行了分析。结果显示,改性后材料的细胞亲和力有较大改善,且材料表面细胞三维形态更加正常,铺展更为舒展,与材料的接触面积较大。细胞力学性能分析也发现改性后材料表面细胞具有更高的粘附力和杨氏模量,以及更低的硬度,说明未改性p(HEMA-MMA)材料具有明显的细胞毒性,这种毒性作用导致在其表面生长的细胞的细胞骨架遭到破坏,细胞健康状态明显不如改性后材料表面细胞。因此,改性后的Col/bFGF-p(HEMA-MMA)更适合作为人工角膜材料使用。     

原子力显微镜在纳米生物材料研究中的应用

在过去的20年里,原子力显微镜(AFM)在纳米生物材料领域有着广泛的应用。AFM可有力地揭示纳米生物材料的表面结构与力学性质,并且可作为纳米加工工具对其进行操作与处理。本文综述了AFM在纳米生物材料中的最新应用进展,包括纳米生物材料的成像与表征,力学性能测量和纳米加工。AFM可用来观察纳米生物材料的表面形貌并对其特征高度和表面粗糙度进行分析,还可对其动态过程进行原位观察。通过AFM相图还可得到有时候高度图无法获取的一些表面特征。AFM力曲线可用于测量针尖与纳米生物材料之间的黏附力及分子内外的相互作用力。AFM纳米压痕技术可用来测量材料的相关力学性质(弹力,杨氏模量,硬度,纳米断裂行为等)。此外,AFM也已经被探索用于精准、可控、可重复地加工纳米生物材料。总之,作为一个强大的纳米技术工具,AFM已成为纳米生物材料相关研究领域的一个理想的表面分析和表面加工工具。     

原子力显微镜研究聚丙烯酸的单链力学性质聚丙烯酸的单分子力谱

自从1986年发明原子力显微镜(AFM)以来,AFM已经发展成为应用最为广泛的扫描探针显微镜[1],它给材料科学家、化学家和生物学家提供了一个极为便利的研究手段.目前,原子力显微镜的空间分辨率已经达到原子尺度,同时又具有非常高的力的敏感性,可以探测10 pN的力,这就为研究单分子的性质提供了可能性[2,3].     

利用AFM和SNOM对淋巴细胞膜表面超微结构及其光学性质的初步研究

利用原子力显微镜(Atomic Force Microscopy，AFM)对淋巴细胞表面形貌进行了形态学的初步研究，观察到了其膜表面其他显微技术所不能发现的超微结构．同时也运用扫描近场光学显微镜(Scanning Near field Optical Microscopy，SNOM)对淋巴细胞进行成像，观察了其对光的透射、吸收等光学性质，并对两种成像方法进行了比较．研究发现：淋巴细胞膜表面凹凸不平，分布着大量直径几十到几百纳米不等的小颗粒；淋巴细胞中央部位有自发荧光现象．结果表明，AFM和SNOM可作为进一步探讨淋巴细胞的结构与功能关系的有力工具．     

Cell Differentiation and Proliferation in the Bone Marrow and Other Organs of 2D2 Mice during Spontaneous Development of EAE Leading to the Production of Abzymes

The exact cellular and molecular mechanisms of multiple sclerosis and other autoimmune diseases have not been established. Autoimmune pathologies are known to be associated with faults in the immune system and changes in the differentiation profiles of bone marrow stem cells. This study analyzed various characteristics of experimental autoimmune encephalomyelitis (EAE) in 2D2 mice. Differentiation profiles of six hematopoietic stem cells of bone marrow were found to significantly differ in 2D2 male and female mice during the spontaneous development of EAE. In addition, we found various properties of B and T cells, CD4+ and CD8+ lymphocytes in blood and several organs (bone marrow, spleen, thymus, and lymph nodes) of 2D2 male and female mice to be considerably different. These changes in hematopoietic stem cells differentiation profiles and level of lymphocyte proliferation in various organs of 2D2 mice were found to induce the production of IgGs against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, increasing the number of autoantibodies hydrolyzing these substrates. We compared the changes of these immunological and biochemical parameters in 2D2 mice with those of mice of two other lines (Th and C57BL/6), also prone to spontaneous development of EAE. Some noticeable and even extreme variations were found in the time-related development of parameters between male and female mice of 2D2, Th, and C57BL/6 lines. Despite some differences, mice of all three lines demonstrated the changes in hematopoietic stem cells profiles, lymphocyte content, and production of catalytic autoantibodies. Given that these changes are harmful to mice, we believe them to cause the development of experimental autoimmune encephalomyelitis.     

Antigen-specific electrophoretic cell separation for immunological investigations

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of Effective Antitumor Immune Response by Combined Administration of hIL-18 and NDV HN

To analyze the antitumor potential and mechanism of action of simultaneous Newcastle disease virus (NDV) hemagglutinin-neuraminidase(HN) and human interleukin 18(hIL-18) gene transfer in C57BL/6 mice with H22 hepatoma,the mouse model with H22 hepatoma was established in C57BL/6 mice, and the antitumor effects of the combined application of NDV HN and hIL-18 were evaluated in vivo. The results show that the growth of established tumors in mice immunized with adenovirus(Ad)-HN in conjunction with Ad-hIL-18 was significantly inhibited compared with that in mice immunized with Ad-HN, Ad-hIL-18 alone, or the empty vector(Ad-mock). Furthermore, the immunization of mice with Ad-HN in conjunction with Ad-hIL-18 elicited strong natural killer activity and H22 tumor-specific cytotoxic T lymphocyte(CTL) responses in vivo. In addition, T cells from the lymph nodes of mice immunized with Ad-hIL-18 or Ad-HN+Ad-hIL-18 secreted high levels of the Th1 cytokine IL-2 and interferon-γ (IFN-γ), indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with NDV HN together with hIL-18 may be a novel and powerful strategy for cancer immunotherapy.     

Resolution of lymphocyte subpopulations derived from rat spleen by polyamine-graft-PHEMA copolymer

The retention behavior of lymphocyte subpopulations, B cell, T cell and null cell, derived from rat spleen to polyamine-graft-poly(2-hydroxyethyl methacrylate) copolymer (HA) surface was investigated, focusing on the conformational transition of the polyamine side chain as well as the protonation of amino groups in the polyamine grafts. Furthermore, the availability of HA was discussed as a column adsorbent for separation of lymphocyte subpopulations derived from spleen. The conformational transition of polyamine grafts significantly influenced the mode of retention of lymphocyte subpopulations. When polyamine grafts existed in an aggregated conformation (protonatin degree α < 0.5), the retention of lymphocyte subpopulations was decreased in the order B cell> null cell> T cell. On the other hand, when polyamine existed in an extended conformation into the aqueous interior from the matrix interface (α > 0.5), T cell retention became greater than null cell retention, resulting in a decreased B cell> T cell> null cell order. These results indicate that the differential retention of spleen lymphocyte subpopulations is attributed to their differential responses to the change in matrix interface accompanied by the protonation of amino groups. Furthermore, spleen lymphocytes were compared with lymph node lymphocytes in terms of resolution efficacy by an HA copolymer column.     

Simultaneous forward and reverse ABO blood group typing using a paper-based device and barcode-like interpretation

A new platform of a paper-based analytical device (PAD) for simultaneous forward and reverse ABO blood group typing has been reported. This platform can overcome the discrepancy results as influenced by the individual haematocrit. The test and the control of non-haemagglutination on each channel were performed in parallel. The PAD was fabricated by printing six parallel channels with wax onto Whatman No. 4 filter paper. An LF1 blood separation membrane was used for the separation of plasma from whole blood for reverse grouping. The blood group was identified by haemagglutination of the corresponding antigen–antibody. For forward grouping, Anti-A, -B and –A,B were treated on the test line of PAD, and inactivated Anti-A, -B and –A,B were immobilized on the control line. For reverse grouping, 30% standard A-cells, B- and O- were added to the test channel after plasma separation, and O-cells were used as a control. Then, 0.9% normal saline (NSS) containing 1% Tween-20 was bi-functionally used for dilution of the blood sample and elution of the non-agglutinated RBCs within the channels. The distance of agglutinated RBCs in each test line was compared with the distance of non-agglutinated RBCs in the parallel control line. The forward and reverse patterns of blood groups A, B, AB and O were a barcode-like chart in which the results can be visually analysed. The PAD has excellent reproducibility when 10 replications of the A, B, AB or O blood groups were performed. The results of both forward and reverse grouping were highly correlated with conventional methods compared with the slide method and tube method, respectively (n = 76). Thus, this ABO typing PAD holds great potential for future applications in blood typing point-of-care testing.     

The Photodynamic Effect of Victoria Blue BO on Peripheral Blood Mononuclear and Leukemic Cells

Abstract— The photodynamic effect of Victoria blue BO (VB-BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB-BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cytometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB-BO photodynamic action than the monocytes (12% vs 80% of Pi-positive cells). The effect of VB-BO and phototreatment on lymphocyte function was studied using a mitogen-induced proliferation assay. A decrease of mitogen response was observed. The VB-BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB-BO photodynamic action. The high VB-BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB-BO for selective depletion of monocytes or sensitive leukemic cells.     

毛细管区带电泳法分析不同来源血竭中龙血素A和龙血素B

在系统优化了电解质溶液的pH、组成、浓度及仪器条件的基础上,建立了一种测定不同来源血竭中龙血素A和龙血素B的毛细管区带电泳(CZE)方法。采用20 kV的分离电压,25 ℃的毛细管柱温,211 nm的检测波长以及5 s的压力(3447 Pa)进样时间,在20 mmol/L的Na2B4O7缓冲溶液(用NaOH调节pH到9.98,含有10%(v/v,下同)乙腈、5.0%乙二醇和1.0%正丁醇)中,龙血素A和龙血素B在15 min内得到了有效分离与检测。方法的线性范围对于龙血素A和龙血素B分别为1.0～100.0 mg/L和0.5～100.0 mg/L。将该方法用于天然血竭及人工诱导血竭中龙血素A和龙血素B的测定,相对标准偏差在0.6%～3.8%之间,加标回收率在95.1%～105.8%之间。方法具有简单、快速、重现性较好和准确度较高的优点,可以用于血竭样品中龙血素A和龙血素B的测定。     

High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels

Human lymphocyte kynureninase activity was assessed in homogenized cells by determination of 3-hydroxyanthranilic acid production as a function of time after addition of the substrate, 3-hydroxykynurenine. The product, 3-hydroxyanthranilic acid, was determined by isocratic high-performance liquid chromatography and fluorescence detection. Mean (+/- S.D.) lymphocyte kynureninase activity in a group (n = 12) of vitamin B6-deficient men was 5.04 +/- 0.81 pmol 3-hydroxyanthranilic acid formed per mg protein per min, which was significantly (p = 0.005) lower than the 6.69 +/- 1.70 pmol 3-hydroxyanthranilic acid formed per mg protein per min in men with a normal vitamin B6 status. This indicates that lymphocyte kynureninase activity is depressed during a vitamin B6 deficiency.     

Thermal Degradation and Kinetics of Poly(3-hydroxybutyrate)/Organoclay Nanocomposites

Poly(3-hydroxybutyrate)/Cloisite30B (PHB/30B) nanocomposites were prepared by solution-intercalation method. The influence of 30B content on the thermal stability of PHB was investigated. With the addition of 3 wt. % of 30B the highest thermal stability of PHB was achieved. The kinetic analysis of the non-isothermal degradation was performed using the isoconversional Friedman method and invariant kinetic parameters method.     

Determination of N-methyl-4-isoleucine-cyclosporin (NIM811) in human whole blood by high performance liquid chromatography-tandem mass spectrometry

A liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of N-methyl-4-isoleucine-cyclosporin (NIM811) was developed and validated over the concentration range 1-2500 ng/mL in human whole blood using a 0.05 mL sample volume. NIM811 and the internal standard, d(12)-cyclosporin A (d(12)-CsA), were extracted from blood using MTBE via liquid-liquid extraction. After evaporation of the organic solvent and reconstitution, a 10 microL aliquot of the resulting extract was injected onto the LC-MS/MS system. Chromatographic separation of NIM811 and internal standard was performed using a Waters Symmetry RP-8 (50 x 4.6 mm, 3 microm particle size) column. The mobile phase consists of 10 mm ammonium acetate in water (A) and acetonitrile (B), with 45% B from 0 to 0.2 min, 45 to 85% B from 0.2 to 0.8 min and 85% B from 0.8 to 2.2 min. The total run time was 3.5 min with a flow rate of 0.8 mL/min. The method was validated for sensitivity, linearity, reproducibility, stability, dilution integrity and recovery. The precision and accuracy of quality control samples at low (2.00 ng/mL), medium (20.0 and 400 ng/mL) and high (2000 ng/mL) concentrations were in the range 1.1-4.3% relative standard deviation (RSD) and -2.5-10.0% (bias), respectively, from three validation runs. The method has been used to measure the exposure of NIM811 in human subjects.