正常人外周血淋巴细胞凋亡受体的表达与细胞凋亡的关系

本研究检测凋亡受体Fas、TNFRⅠ、TNFRⅡ在人外周血淋巴细胞（PBL）增殖过程中的表达情况及其细胞周期特异性,并探讨其与凋亡受体途径介导的细胞周期特异性细胞凋亡的关联性。应用双参数流式细胞术检测人外周血淋巴细胞在G0期和经植物凝集素刺激（PHA）进入细胞周期后Fas、TNFRⅠ、TNFRⅡ的表达情况以及细胞周期特异性,同时检测肿瘤坏死因子-α、抗Fas抗体诱导人外周血淋巴细胞的凋亡。结果表明：经植物凝集素刺激24小时后的外周血淋巴细胞Fas、TNFRⅠ、TNFRⅡ表达率较G0期外周血淋巴细胞分别增加了（35.55±6.63）%,（30.63±2.66）%,（26.62±5.14）%,均具有显著性差异（P〈0.01）,而且主要表达在G1期;未经植物凝集素刺激的外周血淋巴细胞停留在G0期,TNF-α和anti-Fas诱导后没有发生凋亡,而刺激后进入细胞周期的淋巴细胞经TNF-α和anti-Fas诱导后发生凋亡,其凋亡主要在G1期。结论：凋亡受体在外周血淋巴细胞中的表达与凋亡受体途径介导的细胞凋亡有明显的量效关系,凋亡受体途径介导细胞凋亡的细胞周期特异性与凋亡受体表达的细胞周期特异性有关,细胞凋亡的发生与细胞是否进入细胞周期有关。     

IL-6受体α亚单位mRNA和蛋白在人白血病细胞中的表达

为了探讨IL－6R的α亚单位基因与蛋白在人白血病细胞中的表达，为临床利用IL－6／IL－6R系统介导重组IL－6－PE40外毒素融合蛋白靶向杀伤白血病细胞提供可靠依据，采用RT－PCR半定量技术、直接免疫荧光标记及流式细胞术检测了IL－6R的α亚单位基因及蛋白在多种人白血病细胞细胞系中的表达。结果表明，粒系、单核系、红白血病细胞系HL－60，KG－1，U937和TF－1均高表达IL－6R的α亚单位基因和蛋白；淋巴系白血病细胞系Raji亦有一定量的基因表达；而淋巴系细胞等CEM和HuT28以及慢性粒细胞白血病细胞系K562无论是IL－6Rα亚单位的基因还是蛋白均为阴性。相对表达丰度依次为KG－1＞TF－1＞U266＞U937＞HL－60＞Raji。鉴于粒系、单核系、红白血病细胞高表达IL－6Rα亚单位的基因和蛋白，而IL－6Rα亚单位蛋白在正常人外周血细胞均为阴性表达这一事实，提示可以利用IL－6／IL－6R系统介导重组IL－6－PE40外毒素融合蛋白进行靶向杀伤和治疗这些白血病，而且不会对正常血细胞产生毒副作用。     

Specificity and Sensitivity of Cortisol-Induced Changes in Alpha Aminoisobutyric Acid Transport in Human Leukemic Small Lymphocytes and Leukemic Myeloblasts

We have examined the in vitro effect of glucocorticoid and nonglucocorticoid steroids on the transport of [3-(14)C]alpha aminoisobutyric acid (AIB) in lymphocytes from patients with chronic lymphocytic leukemia (CLL), and myeloblasts from patients with acute granulocytic leukemia (AGL). AIB uptake by CLL lymphocytes was markedly inhibited at 1.0 muM (52+/-2.1%) and slightly inhibited at 0.1 muM (17+/-3.0%) cortisol. A similar degree of inhibition developed at 50-fold lower concentrations of dexamethasone, indicating that the effect of these steroids on AIB accumulation parallels their glucocorticoid activity in vivo. In contrast, minimal or no inhibition was observed with steroids devoid of glucocorticoid activity (progesterone, testosterone, cortisone). 11-deoxycortisol, a nonglucocorticoid known to impede the binding of cortisol to cellular receptors in animal lymphocytes, failed to inhibit AIB uptake by CLL lymphocytes appreciably, but reduced the effect of cortisol to a statistically significant degree. Hence, cortisol-induced inhibition of AIB transport in CLL lymphocytes is related to its glucocorticoid activity and appears to require initial interaction with glucocorticoid-specific cellular receptors.In contrast, 1.0 muM cortisol enhanced the accumulation of AIB in AGL myeloblasts from each of five patients studied (mean = 19%, range 7-43%). Neither cortisone nor 11-deoxycortisol stimulated AIB uptake, and cortisol-mediated stimulation was not seen during simultaneous treatment with 11-deoxycortisol, suggesting that this effect of cortisol also represents a specific glucocorticoid effect. The divergent effects of cortisol on amino acid transport in CLL lymphocytes and AGL myeloblasts may explain, in part, the contrasting clinical effects of glucocorticoids administered to patients with these lymphoid and granulocytic hematopoietic malignancies.     

The Human Rosette-Forming Cell as a Marker of a Population of Thymus-Derived Cells

Sheep red blood cells can surround, in vitro, some human peripheral blood lymphocytes in a formation called a rosette. The number of rosetteforming cells (RFC) in 50 normal persons had a wide range (4-40%).The organs of 13 human fetuses (11-19 wk conceptional age) were examined for the presence of RFC. The thymus possessed the highest percentage of RFC, the maximum being 65% of total thymocytes in two 15-16 wk fetal specimens. Blood RFC were always present and their number slightly increased in the oldest fetuses. The bone-marrow showed 0-8% in the six fetuses studied. RFC were found in the spleen around the 13th wk and in the liver around the 17th wk of gestation. These observations lead to the hypothesis that human blood RFC may be chiefly thymic derived. Studies of patients with immunological disorders support this hypothesis: one patient with Nezelof syndrome had no blood RFC and four patients with Wiskott-Aldrich syndrome had a low number of blood RFC (1 and 1.5%). Patients with acquired hypogammaglobulinemia showed a normal percentage of RFC. With the fetal thymocytes, the percentage of inhibition with anti-mu serum increased with the fetal age to become complete in the oldest fetuses studied. Incubation of the oldest fetal thymocytes or the blood lymphocytes with anti-gamma serum of anti-mu serum completely inhibited the rosette formation. These results suggest that mu-chain determinants are present on human fetal thymocytes and blood RFC. The significance of the presence of gamma-chain determinants on these cells is unclear.     

Glucocorticoid-Binding Proteins in Human Acute Lymphoblastic Leukemic Blast Cells

The first known step in steroid hormone action is the association of the steroid with specific cytoplasmic steroid-binding proteins (SBP). Using a competitive binding assay, we detected, quantified, and partially characterized such a SBP in cytosol from glucocorticoid-sensitive human lymphoblastic leukemic blasts. The affinity of steroids for the SBP was directly related to their known killing potency. For example, steroids without glucocorticoid effect such as androstenedione, etiocholanolone, and tetrahydrocortisol were unable to displace radiolabeled dexamethasone from the SBP in the binding reaction. The dose-response curve for in vitro inhibition of [(3)H]thymidine uptake in leukemic blasts correlated closely with the binding affinity of glucocorticoids to the SBP, providing additional support for an essential physiologic role for SBP in steroid action. SBP activity was either greatly diminished or absent in glucocorticoid-resistant cells. Six patients who intially had SBP in their blasts and were responsive to combinations of drugs including glucocorticoids no longer had SBP activity detectable at a time when they no longer responded to combinations of drugs including glucocorticoids. In vitro [(3)H]thymidine uptake was not inhibited by steroids in leukemic blast cells lacking SBP activity. Other patients who had received some antileukemic therapy including glucocorticoids and who still had SBP in their leukemic blasts, were still responsive to drug combinations that included glucocorticoids. This appears to be the first study demonstrating glucocorticoid receptors in a human tissue.     

红细胞生成素受体在白血病细胞的表达和红细胞生成素水平与白血病贫血关系的探讨

本研究探讨红细胞生成素受体（EPOR）在白血病细胞上的表达及其意义，以及急性白血病患者血清红细胞生成素（sEPO）水平与贫血的关系，为急性白血病（AL）贫血的细胞因子治疗提供新的理论依据。用RT—PCR法检测30例急性白血病患者的白血病细胞EPOR表达，以化学发光法测定sEPO含量，采用全自动血细胞分析仪测定Hb水平。结果表明：30例AL患者中18例表达EPOR，表达率为60％，其中急性髓系白血病（AML）的EPOR表达率为61．9％（13／21），急性淋巴细胞白血病（ALL）的EPOR表达率为55．6％（5／9），AML与ALL的EPOR表达率之间无显著差异（P〉0．05），AL的EPOR表达率明显低于对照组（86．7％）（P〈0．05）。AML的EPOR表达量高于ALL，AL的EPOR表达量明显低于对照组（P〈0．01）。30例AL患者的sEPO水平均明显高于对照组（P〈0．01），与Hb水平呈负相关关系（r=-0．658，p〈0．01）。结论：急性白血病细胞有EPOR的表达，但表达水平较低，AML与ALL的EPOR表达率无显著差异。AL时EPO对贫血的负反馈机制未受损害，AL贫血不完全是由于机体EPO产生不足所致，推测主要是由于骨髓红系生成受抑制引起。重组人红细胞生成素（rh—EPO）在治疗急性白血病贫血仍被广泛应用，对白血病贫血的病人使用rh—EPO治疗是否可能促进白血病细胞异常增殖尚需进一步分析与研究。     

Comparative Study of 6-Mercaptopurine Metabolism in Human Leukemic Leukocytes and L1210 Cells

Leukocytes from patients with leukemia and L1210 cells from mice were examined for the rate of formation and cellular concentration of phosphoribosylpyrophosphate, the rate of thioinosinic acid formation, and a number of selected enzymes involved in purine nucleotide synthesis. The amount of thioinosinic acid formed in L1210 cells was much higher than that in human leukemic leukocytes. In cell extracts, the synthesis of thioinosinic acid was similar in both cell types, and the amount of purine phosphoribosyltransferase was not rate limiting in either case. Much higher concentrations and rates of formation of phosphoribosylpyrophosphate were found in L1210 cells than in human leukemic leukocytes. The difference in response to 6-mercaptopurine between L1210 cells and human leukemic leukocytes might be attributed to their difference in supply of phosphoribosylpyrophosphate. Phosphoribosylpyrophosphate-amidotransferase was found to be high in L1210 cells, but was not detected in human leukemic leukocytes.     

Studies of Cellular Proliferation in Human Leukemia. I. Estimation of Growth Rates of Leukemic and Normal Hematopoietic Cells in Two Adults with Acute Leukemia Given Single Injections of Tritiated Thymidine

Two adults with rapidly progressive acute myeloblastic and myelomonoblastic leukemia were given single injections of tritiated thymidine, and measurements were made of the growth rates of their leukemic and normal hematopoietic cells by radioautographic methods. Although almost all leukemic blasts in both marrow and blood were metabolically active as shown by their ability to incorporate tritiated uridine and leucine in vitro, only 5.6% and 6.1% of the blasts in the marrow and even fewer in the blood incorporated tritiated thymidine. The mitotic indexes of the marrow blasts were 0.66% and 0.52%; no circulating blasts were dividing. The mean generation times of the actively proliferating blasts were estimated to be 49 and 83 hours. This cannot be equated with the doubling time of the total leukemic population as there is evidence that many blasts fail to continue dividing and die. The mean durations of the phases of the blasts' mitotic cycles were as follows: DNA synthesis (S) = 22 and 19 hours, premitosis (G(2)) = 3 hours, mitosis (M) = 0.47 and 0.62 hour (minimal estimates), and postmitosis (G(1)) = 24 and 61 hours. In both patients the maximal mean transit time of the blasts in the blood was 36 hours, and the minimal numbers of actively dividing blasts present were 1.6 and 2.6 x 10(9) per kg of body weight.Estimates were also made of the rates of proliferation and maturation of the residual normal erythrocytic and granulocytic cells in these two patients. Although total production was markedly diminished because of reduction in the number of normal elements, the relatively few remaining normal cells appeared to be dividing and maturing at rates that are about the same or only slightly slower than those found in normal subjects.We conclude that main reason leukemic blasts displace normal hematopoietic precursors in acute leukemia is that the blasts largely fail to differentiate. Many die but many others persist in the marrow and elsewhere as primitive cells and continue to proliferate. As the blasts accumulate, they gradually displace the normal hematopoietic cells, most of which continue their normal course of differentiation and leave the marrow as nondividing mature cells. It is not known why the over-all production of normal cells is not adequately increased to compensate for the anemia, granulocytopenia, and thrombocytopenia that develop, but apparently the leukemic cells somehow interfere with the proliferation or differentiation or both of normal stem cells.     

Recognition of Hapten-Modified Cells In Vitro by Human T Lymphocytes

Clearer definition of the recognitive structures of human T lymphocytes for antigens will be required to elucidate the molecular basis of diseases and immunological responses induced or regulated by normal or abnormal T-cell function. For this purpose we have investigated the cellular requirements for immune responses in vitro to trinitrophenyl-conjugated peripheral blood mononuclear cells. The responding cell was characterized as a T cell on the basis of rosetting with sheep erythrocytes. T-cell recognition of hapten in proliferative responses depended upon presentation of antigen in an appropriate stimulator-cell context. Neither autologous hapten-modified erythrocytes nor T cells restimulated responses of in vitro-primed lymphocytes. Moreover, hapten-conjugated non-T cells were more effective than modified unfractionated cells in restimulating proliferative responses. Both macrophages and non-T lymphocytes effectively restimulated hapten-conjugate responses.Cell-mixing experiments indicated that the failure of haptenated T cells to stimulate proliferative responses was not because of a lack of fresh macrophages; these experiments suggested instead that T cells do not express appropriate structures necessary to present haptenic determinants in an immunogenic form. Hapten-modified T cells, however, were capable of inducing primed lymphocytes to become efficient cytotoxic effector cells, indicating that T-cell recognitive units for stimulation of proliferative and cytotoxic responses are different. These data support the concept that for induction of proliferative responses, human T cells recognize conventional antigens in association with HLA-D-region-encoded Ia-like molecules.     

二磷酸氯喹对K562细胞增殖与凋亡的影响

本研究探讨二磷酸氯喹对K562细胞增殖与凋亡的影响及其可能的作用机制。用细胞增殖试验（MTT法）检测不同浓度二磷酸氯喹对K562细胞的增殖抑制作用；应用细胞形态学检查、流式细胞术、DNA琼脂糖凝胶电泳观察和检测细胞凋亡；以罗丹明123（Rhodamine 123）为细胞染色剂，采用流式细胞术检测不同浓度二磷酸氯喹处理后K562细胞线粒体膜电位（△Ψm）的变化。结果表明：用不同浓度二磷酸氯喹（1.5625、3．125、6．25、12．5、25、50、100μmol／L）分别作用于K562细胞24、48和72小时后，细胞生长活力明显降低，具有剂量依赖性；典型的细胞形态学改变、亚G1峰的测出、梯形条带的显现共同证实了二磷酸氯喹能诱导K562白血病细胞凋亡；Rhodamine 123染色显示二磷酸氯喹处理的K562细胞线粒体膜电位强度下降。结论：二磷酸氯喹对K562细胞有生长抑制、诱导凋亡作用，其作用可能与细胞线粒体膜电位（△Ψm）下降有关。     

Studies of the Transferrin Receptor on both Human Reticulocytes and Nucleated Human Cells in Culture: COMPARISON OF FACTORS REGULATING RECEPTOR DENSITY

The transferrin receptor, present on reticulocytes and nucleated cells in tissue culture, has been measured with both immunoassay techniques and transferrin binding studies. The total cellular immunoreactive receptor is rapidly lost from erythrocytes during the process of reticulocyte maturation (from as many as 400,000 molecules to <20,000 molecules/reticulocyte). This event parallels the loss of cell surface transferrin binding sites and RNA content, and correlates with previous studies that have measured the decline in hemoglobin synthesis.     

Immunoglobulin Secreting Cells in Normal Human Bronchial Lavage Fluids

Immunoglobulin secreting cells were quantitated in the bronchial lavage fluids of 12 normal volunteers and compared with immunoglobulin secreting cells in peripheral blood, by a reverse hemolytic plaque assay. The mean number of cells secreting immunoglobulin (Ig)G in bronchial lavage fluids was 489 per million lymphocytes vs. a mean of 175 IgG secreting cells per million lymphocytes in peripheral blood (P < 0.02). The mean number of IgA secreting cells in bronchial lavage fluids was 633 per million lymphocytes as compared to 100 per million lymphocytes in peripheral blood (P < 0.005). Thus, compared to peripheral blood, cells from the lavage fluids were relatively enriched for both IgG and IgA secreting cells. However, IgA secreting cells were the major class of immunoglobulin secreting cells in bronchial lavage fluids, whereas IgG secreting cells predominated in peripheral blood. The prominence of IgA secreting cells in bronchial lavage fluids was further demonstrated by a mean ratio of IgA/IgG secreting cells in bronchial lavage fluids of 1.26 compared to a ratio in peripheral blood of 0.57 (P < 0.02). Cells secreting IgM were identified in only four of seven bronchial lavage fluid samples studied but in all peripheral blood samples. IgE secreting cells were not present in normal peripheral blood but could be demonstrated in 5 of 11 lavage fluid specimens. Thus, cells actively secreting immunoglobulins can be identified in the lower bronchial-alveolar tree of normal human subjects. Cells secreting IgG, IgA, or IgM may function in local lung defenses against infection; cells secreting IgE may contribute to hypersensitivity reactions in the lung.     

Fenretinide诱导白血病细胞凋亡的机理研究

Fenretinide（4-HPR）是人工合成的全反式维甲酸衍生物,能够通过诱导凋亡抑制多种肿瘤细胞生长和增殖,但是其机理尚不很清楚.本研究考察4-HPR对几种白血病细胞的影响,并用U937为对象研究其作用机制.在研究中进行了细胞生长和增殖实验,以膜联蛋白检测细胞凋亡,测定活性氧（ROS）和线粒体跨膜电位（△ψm）及用Western blot检测蛋白表达.研究结果表明,4-HPR能够剂量依赖性地抑制多种白血病细胞株的增殖,进一步发现4-HPR能够诱导U937细胞发生凋亡,并且此凋亡可以被维生素C所抑制.此凋亡过程伴有活性氧的升高,线粒体跨膜电位的下降,以及酶原型胱冬酶-8（caspase-8）,-3的蛋白水平表达下降.结论：4-HPR可能通过升高细胞内ROS的水平,造成线粒体膜损伤,引发由caspase家族成员介导的凋亡,提示4-HPR可能是一种线粒体靶向的药物.     

Serum-Red Cell Interactions at Low Ionic Strength: Erythrocyte Complement Coating and Hemolysis of Paroxysmal Nocturnal Hemoglobinuria Cells

Complement coating and hemolysis were observed when erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were incubated in isotonic sucrose solution in the presence of small amounts of serum. Normal cells were likewise coated with complement components but did not hemolyze. Both normal and PNH erythrocytes reduced the hemolytic complement activity of the serum used in this reaction.Experience with other simple saccharides and related compounds suggests that the low ionic strength of the sucrose solution is the feature that permitted complement coating of red cells and hemolysis of PNH erythrocytes. Isotonic solutions of other sugars or sugar alcohols that do not readily enter human erythrocytes could be substituted for sucrose.The mechanism for these reactions may possibly relate to the agglutination observed with erythrocytes tested in the serum-sucrose system. Even though PNH hemolytic activity could be removed by prior heating of serum or barium sulfate treatment of plasma, the agglutination phenomenon still persisted.The in vitro conditions necessary for optimal sucrose hemolysis of PNH erythrocytes were described and compared with those of the classical acid hemolysis test. The requirement for less serum in the sucrose hemolysis system than needed in the standard acid hemolysis reaction makes certain experiments, especially those using large amounts of autologous PNH serum, much more feasible. Additional advantages of the sucrose hemolysis test are that it can be carried out at room temperature in the presence of oxalate and citrate and that critical pH control is not essential. To date, the sucrose hemolysis test has been a sensitive and specific one for PNH. A modified test used for screening purposes, the "sugar water" test, is very easy to perform.     

Flexible Programs of Chemokine Receptor Expression on Human Polarized T Helper 1 and 2 Lymphocytes

Chemokines and their receptors are important elements for the selective attraction of various subsets of leukocytes. To better understand the selective migration of functional subsets of T cells, chemokine receptor expression was analyzed using monoclonal antibodies, RNase protection assays, and the response to distinct chemokines. Naive T cells expressed only CXC chemokine receptor (CXCR)4, whereas the majority of memory/activated T cells expressed CXCR3, and a small proportion expressed CC chemokine receptor (CCR)3 and CCR5. When polarized T cell lines were analyzed, CXCR3 was found to be expressed at high levels on T helper cell (Th)0s and Th1s and at low levels on Th2s. In contrast, CCR3 and CCR4 were found on Th2s. This was confirmed by functional responses: only Th2s responded with an increase in [Ca²⁺]_i to the CCR3 and CCR4 agonists eotaxin and thymus and activation regulated chemokine (TARC), whereas only Th0s and Th1s responded to low concentrations of the CXCR3 agonists IFN-γ–inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig). Although CCR5 was expressed on both Th1 and Th2 lines, it was absent in several Th2 clones and its expression was markedly influenced by interleukin 2. Chemokine receptor expression and association with Th1 and Th2 phenotypes was affected by other cytokines present during polarization. Transforming growth factor β inhibited CCR3, but enhanced CCR4 and CCR7 expression, whereas interferon α inhibited CCR3 but upregulated CXCR3 and CCR1. These results demonstrate that chemokine receptors are markers of naive and polarized T cell subsets and suggest that flexible programs of chemokine receptor gene expression may control tissue-specific migration of effector T cells.     

手术后硬膜外镇痛对老年患者红细胞补体受体Ⅰ活性及丙二醛水平的影响

目的 :探讨老年患者手术后硬膜外镇痛 (PCEA)对红细胞免疫功能及丙二醛水平的影响。方法 :38例择期前列腺摘除手术老年患者随机分为PCEA组 (2 0例 )和对照组 (18例 )。PCEA组病人手术后采用硬膜外自控镇痛 ,维持视觉模拟评分 (VAS)<3分。对照组根据需要间断肌注哌替啶。分别在麻醉前 ,手术后 1、2、3、5d采静脉血样检测红细胞C3b受体花环率 (RBC -C3bRR) ,红细胞免疫复合花环率 (RBC -ICR) ,红细胞免疫粘附肿瘤细胞功能 (RBC -CaR)、红细胞免疫粘附促进因子 (RFER)、红细胞免疫粘附抑制因子 (RFIR)、丙二醛 (MDA)。结果 :麻醉前各指标两组间无显著差异 (P >0 .0 5 )。手术后 1、2、3d :PCEA组RBC -C3bRR、RFER比麻醉前明显上升 (P <0 .0 5 ) ,RBC -ICR、RFIR、MDA比麻醉前明显下降 (P <0 .0 5 )。而对照组RBC -C3bRR、RBC -CaR、RFER明显低于麻醉前 (P <0 .0 5～ 0 .0 1) ,RBC -ICR、RFIR、MDA明显高于麻醉前 (P <0 .0 5～ 0 .0 1) ,两组间有极显著差异 (P <0 .0 1)。手术后 5d时各值无显著差异 (P >0 .0 5 )。结论 :手术后PCEA能提高老年病人红细胞免疫功能和降低MDA水平 ,有助于病人康复     

Studies on Lipolysis in Human Adipose Cells

Epinephrine stimulated lipolysis and the uptake of oxygen by subcutaneous adipose cells of man. When glucose-(14)C was present in the medium, its utilization was not increased by epinephrine, although lipolysis was accelerated. Insulin did not reduce the production of fatty acids that had been stimulated by epinephrine.The combination of human growth hormone and cortisol stimulated the production of fatty acids by isolated human adipose cells to a lesser extent than epinephrine. When human growth hormone or cortisol was used singly, or when bovine growth hormone was added in combination with cortisol, no effect on fatty acid production was observed. Furthermore, an acetone-dried preparation of human pituitary glands, which was shown to stimulate lipolysis in rat adipose cells, had no effect on fatty acid formation in human adipose cells. This suggested that the human pituitary gland contained no more potent lipolytic agents than growth hormone and was supported by the lack of response of human adipose cells to purified corticotropin.