脾多肽对化疗所致血小板减少症雌鼠的疗效及促血小板生成的机制研究

目的 研究脾多肽注射液对化疗所致血小板减少症的作用及其血小板生成机制。方法 将85只健康昆明雌鼠随机分为正常组、模型组、重组人血小板生成素（rhTPO）阳性对照组、脾多肽注射液低剂量组、脾多肽注射液高剂量组。模型组、给药组第1天单次腹腔注射70 mg/ kg 卡铂复制血小板减少症模型，正常组注射生理盐水。第2天起，不同药物连续干预15 d，并于化疗前以及化疗后第2～16天隔天尾部取血计数血小板；化疗后第8天采用瑞氏-吉姆萨染色骨髓巨核细胞，显微镜观察其形态和数量，采用流式细胞术检测骨髓巨核细胞百分率，酶联免疫吸附试验检测血清干细胞因子（SCF）、促血小板生成素（TPO）水平。结果 各组雌鼠化疗前及化疗后2 d、4 d、6 d、8 d、10 d、12 d、14 d、16 d血小板计数比较，经重复测量设计的方差分析，结果 ①不同时间点血小板计数变化有差异（F =22.413，P =0.000）；②各组血小板计数有差异（F =6.822，P =0.006）；③各组血小板计数变化趋势有差异（F =6.326，P =0.008）。镜下观察巨核细胞细胞核及胞体较其他细胞大，且随着巨核细胞的成熟，核多为不规则形态，胞质愈丰富。细胞染色后，深紫色部分为核，浅紫色部分为胞质。模型组骨髓巨核细胞数量和百分率低于正常组（P <0.05），rhTPO阳性对照组高于模型组（P <0.05），脾多肽注射液高剂量组高于模型组（P <0.05）。模型组SCF、rhTPO水平高于正常组（P <0.05），脾多肽注射液高剂量组SCF水平低于模型组（P <0.05），rhTPO阳性对照组rhTPO水平低于模型组（P <0.05）。结论 60 mg/（kg·d）脾多肽注射液能有效改善卡铂化疗所致雌鼠血小板减少症，可能与调节血清SCF因子恢复至正常水平及上调骨髓巨核细胞数量有关。     

Quantitation of pulmonary megakaryocytes and fibrin thrombi in patients dying from burns

Pulmonary megakaryocytes and fibrin microthrombi were counted in lung sections from 22 patients dying from extensive burns. There was a significant correlation between numbers of megakaryocytes and fibrin microthrombi, supporting a relationship between disseminated intravascular coagulation (DIC) and numbers of pulmonary megakaryocytes. No correlation was found between antemortem platelet counts and either fibrin microthrombi or megakaryocytes. Possible explanations for this are forwarded and the nature of pulmonary megakaryocytes discussed.     

Expression of an anti-CD33 single-chain antibody by Pichia pastoris

CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the alpha-mating factor secretory signal sequence (alpha-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the alpha-MF remaining at the N-terminus. Despite the aberrations in alpha-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively.     

人巨细胞病毒体外感染巨核系细胞加重凋亡

为了研究人巨细胞病毒 (HCMV)感染对巨核系细胞加快凋亡的作用机制及HCMV感染引起血小板减少症的机制 ,采用巨核细胞株CHRF 2 88 11和HCMVAD16 9株共同培养 ,用PCR检测HCMVIEA ,用形态学观察、DNALadder形成及AnnexinV/PI流式细胞仪检测分析细胞凋亡情况。结果显示 :HCMVAD16 9株的不同浓度病毒组(10 -3 ,10 -2 ,10 -1)均能显著抑制CHRF细胞的生长。在感染 7天后 ,3组细胞的活率水平分别是 77% ,73%和 6 8% ,而对照组为 98%。用流式细胞仪检测AnnexinV/PI,在感染 7天后 ,10 -3 ,10 -2 和 10 -1病毒组凋亡细胞的百分数是(2 1.3± 2 .4 9) % ,(2 5 .8± 3.6 5 ) %和 (31.4± 3.91) % ,对照组则为 (3.6 8± 1.4 7) %。凋亡率随培养液中病毒浓度的加大和感染后时间的延长而增高 ,二者呈依赖关系。形态学观察和DNALadder的形成进一步证实了凋亡细胞的存在 ,用PCR确定了在CHRF细胞内有HCMVIEA的表达。结论 :HCMV可直接感染巨核系细胞并加重它的凋亡。     

Recombinant human c-Mpl ligand is not a direct stimulator of proplatelet formation in mature human megakaryocytes

To evaluate the effect of the c-Mpl ligand on platelet production by megakaryocytes, we investigated proplatelet formation in isolated human megakaryocytes cultured in serum-free medium, with or without the c-Mpl ligand, interleukin-6 and erythropoietin. When interleukin-6 was added to the culture medium, the percentage of megakaryocytes displaying proplatelets was approximately 1.5-fold the control value; whereas, in the presence of the c-Mpl ligand, the percentage of megakaryocytes displaying proplatelets decreased in a dose-dependent manner and did not increase compared to control values at any dose tested. However, the viability of megakaryocytes after a 4 d culture in the presence of the c-Mpl ligand was significantly higher than that of the cells cultured without it. The c-Mpl ligand did not stimulate the proplatelet formation in megakaryocytes in vitro     

Complementary and antagonistic effects of IL-3 in the early development of human megakaryocytes in culture

The effect of IL-3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41⁺ MK from pre-MK, the number of cells produced was greatly augmented by the addition of IL-3 and SCF. IL-3 was also able to support the growth of MK colonies in semi-solid media (CFU-MK). The CD41⁺ cells that developed in suspension cultures containing IL-3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL-3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb-IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL-3. The inhibitory effects of IL-3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL-3 did not stimulate MK growth. The results obtained in these cultures suggest that IL-3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL-3 results in the appearance of cells that do not mature normally.     

The birth of the platelet

Summary.  Platlets are small subcellular fragments that are formed from the cytoplasm of bone marrow megakaryocytes, which circulate in blood with characteristic discoid shapes. To assemble and release platelets, megakaryocytes follow a maturation program that accumulates in the conversion of the bulk of their cytoplasmic into multiple long processes called proplatelets. A megakaryocyte may protrude as many as 10–20 proplatelets, each which begins as a blunt protrusion that is driven out by microtubule-based forces. With time, these protrusions thin and branch repeatedly. Platelets form only at the ends of proplatelets. As the nascent platelet matures, its content of granules and organelles are delivered as a stream of individual particles moving from the megakaryocyte cell body to the proplatelet tip. Once the platelet has been filled with its content of intracellular materials, a single microtubule ∼ 100 µm in length is rolled into a coil, and the platelet releases into the medium. Platelet formation can be divided into two phases. In the first phase, there is nuclear proliferation to 16–32×N and the enlargement of the megakaryocyte cytoplasm as it is filled with cytoskeletal proteins, platelet specific granules and granule contents and membranous systems. This phase occurs over a period of days and requires induction by megakaryocyte specific growth factors. Proplatelets are extended in the second phase and platelets are released. This phase is completed in hours.     

γ线全身照射对小鼠骨髓巨核细胞的影响

采用骨髓细胞乙酰胆碱酯酶染色、^3sS体内掺入骨髓巨核细胞(MK)和图象分析方法.观察了小鼠经2、4、6、8和l0Gy⁶⁰Coy线照射后2,4、6,8,10、14、18、24、30、40、60天骨碡MK的动态变化.结果表明:照后早期,各剂量组MK数均明显下降.较小的MK对射线敏感性较高,10Gy照射后,成熟的MK的功能也失常.在恢复期中较小的MK增加迅速,数目超常,大MK也有增多的趋势.在整个恢复期中较低百分比的MK数可维持较高水平的血小板生成量.受到2Gy以上照射后MK的大小即发生显著变化.并贯穿在MK变化的全过程中.不同时期可表现不同特点.