發(fā)布時間：2018-01-11 21:10

  本文關(guān)鍵詞：PMA誘導(dǎo)k562細胞向巨核細胞分化過程中組織因子變化的探討　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： k562細胞 佛波酯 組織因子

【摘要】：目的： 組織因子(Tissue Factor)即凝血因子Ⅲ,不僅參與著調(diào)節(jié)與凝血相關(guān)性疾病的病理生理過程,同時更參與識別和調(diào)控細胞內(nèi)外信號、參與信號轉(zhuǎn)導(dǎo)的作用。據(jù)文獻報道：包括結(jié)直腸癌,胰腺癌,白血病和淋巴瘤等多種人類晚期腫瘤中組織因子(TF)的RNA和蛋白水平表達升高,但組織因子表達的特異性及其具體的調(diào)控機制不明確。K562細胞是從慢性粒細胞白血病急性變患者的胸水中分離并培養(yǎng)建立起來的,具有骨髓造血干細胞的特征,既能在一般培養(yǎng)條件下分裂增殖,又能在某些特殊培養(yǎng)條件下向紅系、粒系及巨核系分化,是當(dāng)今研究白血病細胞分化的理想模型。上個世紀八十年代人們發(fā)現(xiàn)佛波酯(PMA)能夠誘導(dǎo)k562細胞向巨核細胞分化。目前,k562細胞表面是否存在有組織因子的表達尚存在爭議,佛波酯(PMA)誘導(dǎo)k562細胞分化過程中組織因子的變化情況尚不明確。因此,本文旨在觀察k562細胞表面是否存在組織因子的表達以及佛波酯(PMA)誘導(dǎo)K562細胞分化過程中組織因子的表達變化情況,探討其意義。 方法： (1)培養(yǎng)人紅白血病髓系細胞株k562細胞,建立培養(yǎng)模型。 (2)佛波酯誘導(dǎo)k562細胞分化。 (3)取原始k562細胞及誘導(dǎo)后48h細胞行瑞氏染色,觀察比較細胞形態(tài)的變化。 (4)通過流式細胞學(xué)檢測誘導(dǎo)后48h細胞表面CD33、CD41的表達,鑒定細胞分化方向。 (5)通過RT-PCR及蛋白印跡技術(shù)(Western Blot)檢測誘導(dǎo)前及誘導(dǎo)后不同時間點TF的mRNA及蛋白的表達。 結(jié)果： (1)瑞氏染色：PMA誘導(dǎo)k562細胞48h后,細胞胞體增大、邊緣不規(guī)則,染色質(zhì)呈紫紅色,粗顆粒狀,排列緊密,核仁不甚清晰,細胞內(nèi)出現(xiàn)空泡,邊緣有偽足,出現(xiàn)巨核細胞的特點。 (2)誘導(dǎo)前CD33、CD41表達分別為80.49±0.7%,0.78±1.28%。誘導(dǎo)48h后CD33、CD41表達分別為為17.92±2.9%,49.82±6.1%。 (3)原始k562細胞組織因子的nRNA及蛋白表達水平均為陽性。 (4)RT-PCR結(jié)果：佛波酯誘導(dǎo)k562細胞第6天TF在mRNA水平表達開始下降,而第7天的的表達較第6天更低。 (5)Western Blot結(jié)果：佛波酯誘導(dǎo)k562細胞第7天TF在蛋白水平表達下降。 結(jié)論： k562細胞表面存在有組織因子的表達,并能夠在佛波酯10ng/ml濃度誘導(dǎo)下向巨核系分化,佛波酯誘導(dǎo)k562細胞分化過程中能夠降低細胞表面組織因子在RNA及蛋白水平的表達。
[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.
Method錛，

本文編號：1411223