重组人瘦素对慢性特发性血小板减少性紫癜患者骨髓巨核细胞的影响

目的：探讨瘦素对 CITP 骨髓巨核细胞的影响。方法以急性巨核细胞白血病细胞株 M07e 和 CITP 骨髓 CD34＋细胞经诱导分化的巨核细胞为研究对象。运用 RT-PCR 检测 M07e 细胞瘦素受体 mRNA 表达，运用 Brdu-ELISA 法检测瘦素对 M07e 细胞的增殖作用，运用 Annexin V/ PI 双标流式检测瘦素对 M07e 细胞的凋亡作用。经TPO、SCF 诱导磁珠分选后获得的 CITP 骨髓 CD34＋细胞分化为巨核细胞，运用流式测定瘦素对其分化过程中CD41a ＋、CD61＋的表达以及 CD41a ＋细胞的凋亡是否有影响。结果瘦素受体长型 Ob-RL 和短型 Ob-RS 在 M07e细胞均有 mRNA 表达。瘦素对 M07e 细胞的增殖有促进作用，并呈现一定的剂量依赖性，浓度为5 ng/ ml 即显示出促增殖效应（P =0.037），但浓度在5、10、25 ng/ ml 之间无明显统计学差异，浓度为100 ng/ ml 时其增殖效应最大。在时间依赖效应上，瘦素作用48 h，其细胞增殖明显高于24 h，而作用72 h 其细胞增殖介于两者之间（P =0.000）。瘦素对 M07e 细胞具有抑制凋亡作用（P =0.001），而且在作用72小时后凋亡率最高。瘦素对骨髓 CD34＋细胞分化为巨核细胞过程中 CD41a ＋、CD61＋表达的影响，与不加瘦素组相比无明显统计学差异（P ＞0.05）。对 CD41a ＋细胞凋亡的影响与不加瘦素组相比亦无明显统计学差异（P ＞0.05）。结论瘦素通过促进 M07e 细胞的增殖，抑制其凋亡来参与巨核细胞白血病的发生、发展。瘦素在 TPO ＋ SCF 诱导 CITP 骨髓巨核细胞分化中无明显协同作用，对分化产生 CD41a ＋巨核细胞的凋亡亦无明显作用。

Effect of recombinant human leptin on megakaryocyte in patients with chronic idiopathic thrombocytopenic purpura

Objective：To explore whether leptin plays a role in megakaryocyte of CITP. Methods：Megakaryocytic leu-kemia cell line M07e,megakaryocyte differentiated from bone marrow CD34 ＋ cells of CITP were used in this study. The mR-NA expression of leptin receptor（ob-R）in M07e was detected by RT-PCR. The proliferative effect of leptin on M07e was investigated with Brdu-ELISA assay and the apoptosis of cells was measured with flow cytometry after Annexin V labeling and PI staining . Bone marrow CD34 ＋ cells were selected by magnetic cell sorting（MACS）,and TPO,SCF were used in the expansion system of megakaryocyte progenitor. The influences of leptin on the expression of megakaryocytic specific mono-clonal antibodies CD41a ,CD61 and the apoptosis of megakaryocyte were measured with flow cytometry. Results：There was mRNA expression of ob-RL and ob-RS in M07e. Leptin promoted the proliferation of M07e in a dose and time dependent manner. The effect of proliferation was the most significant at the concentration of 100ng/ ml and after cultured for 48h. Lep-tin inhibited the apoptosis of M07e. Compared with the control group,leptin had no effect on the expression of CD41a , CD61 and the apoptosis of megakaryocyte during the period of megakaryocyte differentiation from bone marrow CD34 ＋ cells of CITP. Conclusion：Leptin may play an important role in human megakaryocytic leukemia by promoting the proliferation and inhibiting the apoptosis of M07e. Leptin had no synergistic effect on megakaryocyte differentiation of ITP induced by TPO and SCF. There was also no significant influence on the apoptosis of CD41a ＋ cells.