PMA诱导k562细胞向巨核细胞分化过程中组织因子变化的探讨

发布时间：2018-01-11 21:10

  本文关键词：PMA诱导k562细胞向巨核细胞分化过程中组织因子变化的探讨　出处：《中南大学》2012年硕士论文　论文类型：学位论文

  更多相关文章： k562细胞 佛波酯 组织因子

【摘要】：目的： 组织因子(Tissue Factor)即凝血因子Ⅲ,不仅参与着调节与凝血相关性疾病的病理生理过程,同时更参与识别和调控细胞内外信号、参与信号转导的作用。据文献报道：包括结直肠癌,胰腺癌,白血病和淋巴瘤等多种人类晚期肿瘤中组织因子(TF)的RNA和蛋白水平表达升高,但组织因子表达的特异性及其具体的调控机制不明确。K562细胞是从慢性粒细胞白血病急性变患者的胸水中分离并培养建立起来的,具有骨髓造血干细胞的特征,既能在一般培养条件下分裂增殖,又能在某些特殊培养条件下向红系、粒系及巨核系分化,是当今研究白血病细胞分化的理想模型。上个世纪八十年代人们发现佛波酯(PMA)能够诱导k562细胞向巨核细胞分化。目前,k562细胞表面是否存在有组织因子的表达尚存在争议,佛波酯(PMA)诱导k562细胞分化过程中组织因子的变化情况尚不明确。因此,本文旨在观察k562细胞表面是否存在组织因子的表达以及佛波酯(PMA)诱导K562细胞分化过程中组织因子的表达变化情况,探讨其意义。 方法： (1)培养人红白血病髓系细胞株k562细胞,建立培养模型。 (2)佛波酯诱导k562细胞分化。 (3)取原始k562细胞及诱导后48h细胞行瑞氏染色,观察比较细胞形态的变化。 (4)通过流式细胞学检测诱导后48h细胞表面CD33、CD41的表达,鉴定细胞分化方向。 (5)通过RT-PCR及蛋白印迹技术(Western Blot)检测诱导前及诱导后不同时间点TF的mRNA及蛋白的表达。 结果： (1)瑞氏染色：PMA诱导k562细胞48h后,细胞胞体增大、边缘不规则,染色质呈紫红色,粗颗粒状,排列紧密,核仁不甚清晰,细胞内出现空泡,边缘有伪足,出现巨核细胞的特点。 (2)诱导前CD33、CD41表达分别为80.49±0.7%,0.78±1.28%。诱导48h后CD33、CD41表达分别为为17.92±2.9%,49.82±6.1%。 (3)原始k562细胞组织因子的nRNA及蛋白表达水平均为阳性。 (4)RT-PCR结果：佛波酯诱导k562细胞第6天TF在mRNA水平表达开始下降,而第7天的的表达较第6天更低。 (5)Western Blot结果：佛波酯诱导k562细胞第7天TF在蛋白水平表达下降。 结论： k562细胞表面存在有组织因子的表达,并能够在佛波酯10ng/ml浓度诱导下向巨核系分化,佛波酯诱导k562细胞分化过程中能够降低细胞表面组织因子在RNA及蛋白水平的表达。
[Abstract]:Objective:
Tissue factor (Tissue Factor) is factoriii, not only involved in the pathophysiological process of adjustment and coagulation related diseases, but also participate in the identification and regulation of intracellular and extracellular signals involved in signal transduction pathway. It is reported, including colorectal cancer, pancreatic cancer, a variety of human tumor tissue white blood disease and lymphoma and other factors (TF) RNA and protein expression level increased, but the specificity of the expression of tissue factor and its specific regulatory mechanism is not clear.K562 cells from CML patients with pleural fluid separation and culture set up, with the characteristics of bone marrow hematopoietic stem cells, which can proliferation under normal culture conditions also, in some special conditions of erythroid, granulocytic and megakaryocytic differentiation, is an ideal model for the study of differentiation of leukemia cells. In 80s of last century people found the Buddha Wave ester (PMA) can differentiate into megakaryocyte K562 cells induced by K562 cells. At present, the existence of surface expression of tissue factor is still controversial, phorbol ester (PMA) changes induced by tissue during K562 cell differentiation factor is not clear. Therefore, this paper aims to investigate the expression of K562 cell surface presence of tissue factor and phorbol ester (PMA) induced expression of tissue factor in differentiated K562 cells and to explore its significance.
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