细胞骨架结构及其功能(续)

(二) 肌动蛋白纤维的相互作用 Kane于1975年发现将在低温下制备的海胆卵抽提物加温至正常生理温度时,会在体外形成一种结实的凝胶。此后发现从许多种细胞(例如变形虫、组织培养细胞、巨噬细胞和卵     

免疫诊断细胞方法(续一)

<正> 二,抗人的T和B淋巴细胞与其它白细胞抗血清的获得 各淋巴细胞群体和其他型白细胞特异性抗血清,首先必须与相应的细胞起反应。此外,这些抗血清应当除去非特异的抗淋巴细胞作用,从而加强其免疫抑制的特异性。用相应的材料通过免疫动物获得抗血清。因输血姙娠和某些疾病出现的抗淋巴细胞抗体也能用作试剂。 (一)抗T淋巴细胞血清(ATC) 的制备 1.免疫动物的材料。 可用胸腺细胞或无细胞的胸腺组织。作     

甲胎蛋白(AFP)对巨噬细胞的作用

巨噬细胞是一类重要的免疫活性细胞,在机体免疫防御方面起着重要作用,不仅与B 细胞的抗体形成、T 细胞的激活等有密切联系,而且还能合成和释放大量具有重要生理功能的生物高分子以及直接作用于肿瘤细胞等,因此本文选择人和大鼠巨噬细胞为材料,观察了人体AFP(HAFP)对巨噬细胞的作用。(1)HAFP 具有抑制人巨噬细胞吞噬鸡红血球的能力,并改变巨噬细胞的电泳迁移率,HSA 没有这种作用。(2)AFP 阳性肝癌血清同样显示上述抑制作用,当去除AFP 后,抑制作用得到不同程度的解除。(3)大鼠腹腔渗出细胞与HAFP 的结合能力远远超过与HSA 的结合能力,腹腔巨噬细胞吞噬鸡红血球的能力受HAFP 的抑制率远较受HSA 的抑制率高。(4)HAFP 浓度与大鼠腹腔渗出细胞(巨噬细胞占70～80%)的结合率曲线、结合量曲线显示:(a)当HAFP<1毫微克/200微升时,K_D～2.5×10~(-12)M,HAFP 与腹腔细胞的作用符合正协同效应的变化规律;(b)当HAFP>1毫微克/200微升时,HAFP 的结合较松弛。(5)过氧化物酶标记免疫电镜定位观察到HAFP 滞留在大鼠腹腔巨噬细胞的表面。(6)置换实验证明HAFP 与腹腔细胞的结合有一定的特异性。根据以上结果,本文认为巨噬细胞的表面可能有HAFP 受体,HAFP 通过与巨噬细胞的结合抑制巨噬细胞的吞噬能力,推测在胚胎体内和正常成年个体内HAFP 起免疫调节作用,在肝癌患者体内起免疫抑制作用。     

甲胎蛋白(AFP)对巨噬细胞的作用

巨噬细胞是一类重要的免疫活性细胞,在机体免疫防御方面起着重要作用,不仅与B细胞的抗体形成、T细胞的激活等有密切联系,而且还能合成和释放大量具有重要生理功能的生物高分子以及直接作用于肿瘤细胞等,因此本文选择人和大鼠巨噬细胞为材料,观察了人体AFP(HAFP)对巨噬细胞的作用。(1)HAFP具有抑制人巨噬细胞吞噬鸡红血球的能力,并改变巨噬细胞的电泳迁移率,HSA没有这种作用。(2)AFP阳性肝癌血清同样显示上述抑制作用,当去除AFP后,抑制作用得到不同程度的解除。(3)大鼠腹腔渗出细胞与HAFP的结合能力远远超过与HSA的结合能力,腹腔巨噬细胞吞噬鸡红血球的能力受HAFP的抑制率远较受HSA的抑制率高。(4)HAFP浓度与大鼠腹腔渗出细胞(巨噬细胞占70～80%)的结合率曲线、结合量曲线显示:(a)当HAFP<1毫微克/200微升时,K_D～2.5×10~(12)M,HAFP与腹腔细胞的作用符合正协同效应的变化规律;(b)当HAFP>1毫微克/200微升时,HAFP的结合较松弛。(5)过氧化物酶标记免疫电镜定位观察到HAFP滞留在大鼠腹腔巨噬细胞的表面。(6)置换实验证明HAFP与腹腔细胞的结合有一定的特异性。根据以上结果,本文认为巨噬细胞的表面可能有HAFP受体,HAFP通过与巨噬细胞的结合抑制巨噬细胞的吞噬能力,推测在胚胎体内和正常成年个体内HAFP起免疫调节作用,在肝癌患者体内起免疫抑制作用。     

人胚胎干细胞程序降温保存的实验研究

本文采用升降式程序降温仪对人胚胎于细胞进行了程序降温保存,并探讨和比较了降温速率、置核温度、保护剂和投入液氮前温度对冻存复苏后胚胎干细胞的存活率、活力及分化特性的影响。结果表明:采用Me_2SO 血清 DMEM(体积比为1∶3∶6)的保护剂,从0℃开始,以0.5℃/min的速率对细胞悬液降温;至-10℃时对其进行置核,并于-35℃时将其快速投入液氮中保存,复温后效果最佳。冻存复温后细胞存活率可达81.8%,复苏后的胚胎干细胞形态和集落生长方式都与冻前的生长形态相同,且胚胎干细胞标志之一碱性磷酸酶(AKP)反应阳性,同时染色体组型仍正常。     

血栓调节蛋白在胚胎肺及肺癌中的表达

目的研究血栓调节蛋白(thrombomodulin,TM)在胚胎肺、正常肺组织及肺癌组织中的表达。方法以不同周龄的胚胎肺组织、正常成人肺组织、肺癌组织为研究对象,应用免疫组织化学SP法检测TM的存在。结果8、15、18、21、24、27、29周人胎肺组织中,TM在气管纤毛柱状上皮细胞、I型和Ⅱ型肺泡上皮细胞及软骨、结缔组织均呈阴性表达,围绕肺泡上皮细胞团周围的血管内皮细胞阳性表达。正常成人支气管纤毛柱状上皮细胞、肺泡上皮细胞不表达,但在血管内皮细胞呈阳性表达。TM在鳞状上皮不典型增生的细胞膜和细胞问桥表达,在肺鳞癌表达,阳性率为97．3％(34／35),在癌细胞膜和细胞问桥阳性表达,但腺癌、小细胞癌癌细胞不表达。结论TM在胚胎肺以及成人肺仅见于血管内皮细胞,在支气管上皮、肺泡上皮不表达。与其它的血管内皮细胞标记物不同,TM的表达在肺鳞癌与腺癌表扶明显不同．右助于鉴别肺鳞癌与肺腺癌．     

磷酸脂酶C-γ1在人早期胚胎组织细胞中表达的免疫细胞化学研究

采用免疫细胞化学方法探讨了磷酸脂酶 C-γ1(PL C-γ1)在人早期胚胎组织细胞中的表达 ,发现在胚胎龄为 5 2 -76天的多种细胞中均有表达 ,以软骨、软骨膜及肌组织反应最强。结果提示 ,PL C-γ1相关的细胞内信号传递途径对于人早期胚胎细胞的发育、增殖具有重要的生物学意义。     

雄激素通过雄激素受体途径调控垂体肿瘤转化基因(PTTG1)的表达

雄激素能上调大鼠前列腺中垂体肿瘤转化基因1(PTTG1)的表达,在前列腺癌标本中,发现PTTG1的表达明显高于正常组织.因此,用雄激素依赖的前列腺癌细胞LNCaP来研究雄激素调控PTTG1表达的分子机制.在LNCaP细胞中,通过雄激素刺激后,PTTG1的表达明显升高.根据序列分析以及5-缺失体实验(deletion)和突变实验(mutation),发现PTTG1的启动子上游-950到-933的序列上有1个雄激素受体反应元件(ARE),染色体免疫共沉淀实验(CHIP)也证实了雄激素能促进雄激素受体与PTTG1启动子上ARE结合,从而在转录水平调控PTTG1的表达.     

非洲爪蟾PAPC多克隆抗体的制备及其特异性鉴定

非洲爪蟾ParaxialProtocadherin(PAPC)是一个在爪蟾Spemann组织者特异表达的膜蛋白.它在爪蟾原肠运动阶段的汇聚延伸运动和体节发生阶段的体节边界形成,以及早期听泡的形态发生和细胞特化过程中都有重要的作用.为了研究PAPC基因在早期胚胎发育过程中的表达及其生物学功能,需要制备PAPC抗体.应用谷胱甘肽S-转移酶(glutathioneStransferase,GST)表达系统表达GST-PAPC融合蛋白,亲和纯化后用以免疫新西兰大白兔,获得PAPC多克隆抗体.免疫印迹分析发现,以1∶3000稀释的该多克隆抗体为一抗时,能够在转染了全长PAPC质粒的HEK293T细胞的蛋白质抽提物中,特异地识别出150ku的印迹条带.同时,GST-PAPC融合蛋白可以竞争性抑制该抗体对全长PAPC质粒转染细胞的蛋白质抽提物的特异性条带.用1∶500稀释的该抗体为一抗进行免疫荧光分析时,发现,PAPC多克隆抗体能够识别在HEK293T细胞中过表达以及爪蟾动物极细胞中过表达的PAPC蛋白,荧光信号定位在细胞膜上.免疫印迹分析证明,PAPC抗体能够识别爪蟾胚胎中内源表达的PAPC蛋白.     

小鼠胚胎干细胞(ES-8501细胞)建系过程的核型及特性分析

小鼠胚胎性癌(EC)细胞系的细胞核型大多数异常,对用于分析EC细胞与胚胎细胞之间的关系和进行嵌合体研究等都是不利的。人们都期望能有正常核型的胚胎细胞系的建立。近年来Evans和Kaufman以及Martin等人先后用不同方法直接从小鼠的内细胞团(ICM)细胞建立了多潜能的胚胎干细胞(erabryonicstem eells,简称ES细胞),也有人称之为EK     

A novel cytotoxic T-cell clone that exhibits differential modes of recognition in the proliferative and cytolytic phase

A T-cell clone (1G8-H7) cytotoxic to P815Y mastocytoma (H-2d) has been established from spleen cells of a C3H/He mouse (H-2k) primed with P815Y cells by means of in vitro stimulation with irradiated C3H.H-2o(H-2KdDk) spleen cells. The clone 1G8-H7 was an interleukin 2 (IL-2)-dependent and H-2Kd antigen-dependent CTL clone and it killed P815Y cells but not Concanavalin A-induced spleen blast cells bearing H-2Kd antigen. The involvement of H-2Kd antigen in the cytolytic recognition mechanism was shown by the inhibition of lysis by anti-H-2Kd monoclonal antibody and also by the cold inhibition experiment that employed H-2Kd-bearing spleen cells. Comparison of cytotoxic activities between 1G8-H7 and Kd-specific CTL clones showed that the killing of P815Y cells by clone 1G8-H7 was not explained by the susceptibility to cell-mediated cytolysis of P815Y cells. These results suggest that H-2Kd antigen on the stimulating cell is sufficient to deliver a proliferation signal in the proliferative phase of this clone, but in the cytolytic phase an additional interaction with surface structure on the target cell other than that with H-2Kd antigen is required for the induction of cytolysis. Possible elucidations for the differential modes of recognition are discussed.     

Enhancement of antigen-induced interleukin 4 and IgE production by specific IgG1 in murine lymphocytes.

Conalbumin (CA)-specific type 2 helper T cell (Th2) clone, D10G4.1 (D10) produces IL4 when stimulated with varying doses of TNP-CA in the presence of mitomycin C-treated C3H spleen cells or purified B cells as antigen-presenting cells (APC). The production of IL4 was assessed by bioassay and by expression of IL4 mRNA. IL4 production reached maximum at 100 micrograms/ml of TNP-CA, whereas 1 microgram/ml of the antigen induced less than 10% of the maximum level of IL4. This lower level of IL4 production was augmented to the maximum level when monoclonal anti-TNP IgG1 was added to the culture at 0.5-1 microgram/ml. Anti-TNP IgE, but not anti-TNP IgM, was also effective, though IgE was 1/10 as effective as IgG1. IgG1 with an irrelevant specificity and F(ab')2 of anti-TNP IgG1 did not show augmenting effects. Moreover, the enhancement by anti-TNP IgG1 was completely abolished by monoclonal antibody against murine Fc gamma RII, 2.4G2. These results suggest that a low dose of the antigen complexed with IgG1 is focused on APC by means of Fc gamma RII, processed, and presented efficiently to the Th2 clone. On the other hand, the co-culture of D10 with normal C3H B cells in the presence of 1-100 micrograms/ml TNP-CA resulted in polyclonal IgE production. Anti-TNP IgG1 markedly augmented the lower level of IgE production induced by a suboptimal dose of the antigen (1 microgram/ml). This augmentation was shown to be dependent on endogenous IL4 because the enhancement was abolished by monoclonal anti-IL4 (11B11).     

Activation via TCR or IL-2 receptor of a CD8+ suppressor T cell clone: Effect on IL-10 production and on proliferation of the suppressor T cell

In a previous study, we established CD8⁺ suppressor T cell (Ts) clone 13G2 which produced the suppressive lymphokine, interleukin-10 (IL-10). In this study, we examined what physiological activator could induce both production of IL-10 from 13G2 and the proliferation of 13G2. Both the antigenic stimulation mimicked by the anti-CD3 antibody and the T cell growth factor interleukin-2 (IL-2) induced IL-10 production from the 13G2 clone equally well. 13G2 cells proliferated remarkably with IL-2 stimulation, while anti-CD3 only slightly induced proliferation of the clone. 13G2 cells also produced IL-10 in the presence of hydroxyurea which blocked transit of cells from G₁ to S phase. However, cycloheximide blocked the production of IL-10 from the Ts clone. The study demonstrates that both the anti-CD3 antibody and IL-2 induced IL-10 synthesis of the Ts clone equally well, and the proliferative response of Ts cells was induced more by IL-2 than by anti-CD3. IL-2 proved to be a good stimulator for Ts cells to produce suppressive lymphokine and to multiply their population.Abbreviation Ts suppressor T cell - Th helper T cell - Ag antigen - APC antigen presenting cell - IL interleukin - TCR T cell receptor - mAb monoclonal antibody     

Biochemistry and expression of myelomonocytic antigens

Six monoclonal antibodies (MAb) which react with myelomonocytic cells representing various stages of differentiation, and which precipitate six different cell surface molecules, were identified. A 50 to 55 kilodalton (Kd) glycoprotein, restricted in expression to mature cells of the monocyte lineage, was detected by immunoprecipitation with antibody MoS39. By using COS-7 cells transfected with a cDNA clone encoding the MoS39 antigen, various well-described anti-monocyte MAb, including Mo2, My4, Leu-M3 (MoP9), MoP15, MoS1, and 63D3, also bound to MoS39-expressing COS-7 cells, suggesting that this group of antibodies reacted with the same glycoprotein. Immature cells of the myelomonocytic lineage were shown to express two distinct molecules: one with an m.w. of 26 to 28 Kd identified by antibody SG133, and the second, a 130 to 140 Kd glycoprotein identified by MoU26. Mature granulocytes were found to express a 60 Kd molecule identified by antibody SG185 which was absent from other cells of this lineage. Two other molecules were shown to be present on both mature and immature cells of the granulocytic and monocytic lineages: a 130 to 140 Kd glycoprotein identified by antibody SG134, and a 160 to 170 Kd glycoprotein recognized by antibody MoU48.     

Monoclonal antibodies to monoamine oxidase B and another mitochondrial protein from human liver.

A monoclonal antibody has been generated to human liver monoamine oxidase (MAO) B by fusion of mouse myeloma cells with spleen cells from a mouse immunized with a mixture of semi-purified MAO A and MAO B. The antibody, 3F12/G10, an immunoglobulin G1, reacts with its antigen in cryostat sections of human liver, showing an intracellular particulate distribution as demonstrated by immunoperoxidase staining. The antibody indirectly precipitates [3H]pargyline-labelled human MAO B both from liver and platelet extracts but fails to precipitate MAO A from liver extracts. The antibody does not recognise rat liver MAO B, showing that the determinant is not universally expressed on MAO B. The antibody has no effect on the catalytic activity of MAO B. Other monoclonal antibodies were generated but they are directed to a protein with a subunit Mr of 54 000, a contaminant of the MAO preparation. One of these antibodies, A8/C2, an IgG2a, reacts with the same protein in both rat and human liver extracts.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.     

Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies.

A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 X 10(3) to 1:1 X 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins.     

Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis

Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell's sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.     

Biochemical and morphological differentiation of the human colonic epithelial cell line SW620 in the presence of dimethylsulfoxide.

In vitro models of intestinal cell differentiation provide an important adjunct for studying normal and abnormal intestinal epithelial cell differentiation. The studies reported herein describe morphologic and biochemical changes in the colonic epithelial cell line SW620 following dimethylsulfoxide (DMSO) incubation. Cells cultured in the presence of DMSO showed striking changes in morphology characterized by enlargement, elongation, and formation of process-like structures by light microscopy and a propensity to form microvillus-like structures by electron microscopy. These changes were accompanied by significant differences in the expression of the cell surface markers CD4 (HIV gp120 receptor), CD44 (hyaluronate receptor), and KS1 (adenocarcinoma/epithelial specific antigen). There was a marked decrease in CD4 expression (38% to 2%), an increase in CD44 expression (4% to 50%) and a decrease in KS1 expression (98% to 66%) as detected by flow cytometry following incubation of SW620 cells in DMSO. Parallel changes in the expression of these markers were seen by metabolic and surface labeling studies. Although SW620 cells were infected by HIV-1, DMSO-treated SW620 cells could not be infected. DMSO-induced changes in surface expression of CD4, CD44, and KS-1 were reversible over time upon removal of DMSO from the culture medium. Secretory component, sucrase, neuron-specific enolase, chromogranin-A, and mucin were not detectable in SW620 cells with or without DMSO treatment. SW620 cells provide a useful model for studying specific biochemical and molecular events involved in intestinal epithelial cell differentiation and function.     

Infection of a macrophage-like cell line, P388D1 with reovirus; effects of immune ascitic fluids and monoclonal antibodies on neutralization and on enhancement of viral growth

We studied the effect of antibody on the growth of reovirus, serotypes 1 and 3, in P388D1, a continuous mouse macrophage-like cell line. Enhanced growth of virus was observed when cells were infected in the presence of nonneutralizing monoclonal antibodies or subneutralizing concentrations of either immune ascitic fluids or neutralizing monoclonal antibodies. Both enhancement of viral growth and neutralization were accompanied by an antibody-mediated increase in binding of radiolabeled virus to P388D1 cells. Although neutralization was seen only with monoclonal antibodies directed toward the sigma-1 surface protein of the virus, enhancement was observed with two monoclonal antibodies directed toward other surface proteins. Trypsin treatment of P388D1 cells abrogated enhanced growth of virus mediated by a mouse IgG2a antibody; preincubation with P388D1 with human IgG1 but not IgG2 myeloma proteins also abrogated enhancement by immune ascitic fluid or monoclonal antibody. These observations are compatible with known properties of P388D1 Fc receptors and support the role of the Fc receptor in antibody-mediated infection.