Stenberg P E, Levin J, Baker G, Mok Y, Corash L
Department of Laboratory Medicine, University of California School of Medicine, San Francisco.
J Cell Physiol. 1991 Apr;147(1):7-16. doi: 10.1002/jcp.1041470103.
Previous studies to examine the effects of thrombocytopenia on thrombopoiesis have generally utilized immune-mediated platelet depletion. We have developed a nonimmune model to exclude the possibility that adverse immune-mediated effects have been misinterpreted as the physiological response to stimulation of thrombopoiesis. Thrombopoiesis was examined in mice after induction of thrombocytopenia with a single injection of the nonimmunologic agent neuraminidase (Ndase). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 8, 12, 24, 48, 72, 96, and 120 hr after administration of Ndase. Eight to 48 hr after induction of acute, severe thrombocytopenia (mean platelet count less than 50,000/microliters), the medians of the platelet sectional area distributions, as measured morphometrically, were significantly greater than the median platelet sectional area of pooled controls. The maximum median value for platelet sectional area was observed at 24 hr. The largest platelets in these samples contained more profiles of endoplasmic reticulum and Golgi cisternae, and a lower concentration of surface-connected canalicular system, as compared with normal platelets. By 72 hr post-injection of Ndase, virtually all platelets exhibited normal size and organelle complement. Mean platelet volumes, determined by electrical impedance analysis, paralleled the serial changes in platelet sectional areas. MK frequency and ploidy, measured by two-color fluorescence activated flow cytometry, were unchanged 12 and 24 hr following Ndase. At 48 hr, total MK frequency increased significantly (P less than 0.01) from 0.11% to 0.17%, and MK ploidy distribution shifted with a reduction in 16N MK (P less than 0.005) and an increase in 32N MK (P less than 0.01). MK ploidy was maximally altered from normal at 72 hr with increased 32N MK frequency (32.0%, P less than 0.001) and increased 64N MK frequency (2.4%, P less than 0.005). Morphologic and morphometric examination of MK at all time points did not reveal detectable changes from normal in cytoplasmic appearance or size, respectively. Therefore, we have demonstrated marked alterations of morphology and size of platelets, and of MK ploidy, using this nonimmunologic model. These studies further support our previous observations that megakaryocyte ploidy and platelet volume are independently regulated in response to depletion of the circulating platelet mass, and they show that these changes are not dependent upon the mechanism of thrombocytopenia.
以往研究血小板减少对血小板生成的影响时，通常采用免疫介导的血小板耗竭方法。我们开发了一种非免疫模型，以排除不良免疫介导效应被误作血小板生成刺激的生理反应的可能性。用非免疫制剂神经氨酸酶（Ndase）单次注射诱导小鼠血小板减少后，检测其血小板生成情况。利用电子显微镜，我们检查了注射Ndase后8、12、24、48、72、96和120小时获得的血小板和巨核细胞（MK）。急性、严重血小板减少（平均血小板计数低于50,000/微升）诱导后8至48小时，通过形态测量法测得的血小板截面积分布中位数显著大于混合对照组的血小板截面积中位数。血小板截面积的最大中位数在24小时时观察到。与正常血小板相比，这些样本中最大的血小板含有更多的内质网和高尔基池轮廓，以及较低浓度的表面连接小管系统。注射Ndase后72小时，几乎所有血小板的大小和细胞器组成均表现正常。通过电阻抗分析测定的平均血小板体积与血小板截面积的系列变化平行。用双色荧光激活流式细胞术测量的MK频率和倍性，在注射Ndase后12和24小时未发生变化。在48小时时，总MK频率从0.11%显著增加（P<0.01）至0.17%，MK倍性分布发生变化，16N MK减少（P<0.005），32N MK增加（P<0.01）。MK倍性在72小时时与正常情况相比变化最大，32N MK频率增加（32.0%，P<0.001），64N MK频率增加（2.4%，P<0.005）。在所有时间点对MK进行的形态学和形态测量检查均未发现细胞质外观或大小与正常情况有可检测到的变化。因此，我们利用这种非免疫模型证明了血小板的形态和大小以及MK倍性有明显改变。这些研究进一步支持了我们之前的观察结果，即巨核细胞倍性和血小板体积在循环血小板量减少时受到独立调节，并且它们表明这些变化不依赖于血小板减少的机制。
