【作者】 仇灏；

【导师】 吴士良； 周迎会；

【作者基本信息】 苏州大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 已有大量的文献报道:当细胞恶变或分化时,细胞糖复合物上的糖链结构常发生变化,而这种变化来源于某些合成该糖链的相应的糖基转移酶表达的改变。β1,3-N-乙酰氨基葡萄糖转移酶(β3GnTs)家族参于合成其产物中的β1,3-乙酰氨基葡萄糖连接键,其中β3GnT-2, -4, -8亚型合成糖蛋白上N-或O-连接型糖链中的多聚乙酰氨基乳糖结构。本研究探讨这三种酶的表达和四株白血病细胞分化间的关系,以及转录因子Ets-1对β3GnT-2, -4, -8表达的调控。本论文分为三个部分:一、β3GnT-2, -4, -8和人急性早幼粒白血病细胞株HL60及NB4分化的关系目的:研究糖基转移酶β3GnT-2, -4, -8表达变化和人急性早幼粒白血病细胞株HL60及NB4分化的关系。方法:RT-PCR检测β3GnT-2, -4, -8在六种不同白血病细胞株中的表达情况。其次,使用ATRA处理HL60和NB4两种人急性早幼粒白血病细胞株,使其向粒细胞方向分化;或用TPA(PMA)处理两种细胞株,使其向单核细胞方向分化。用实时定量PCR(Real-time PCR)检测β3GnT-2, -4, -8的mRNA诱导分化前后在两个细胞株中的表达变化,并结合细胞形态学观察鉴定ATRA、TPA处理前后两个细胞株的形态变化。结果:β3GnT-2在白血病细胞株SHI-1,THP-1,K562,HL60,U937,NB4中都有不同程度的表达。β3GnT-4仅在NB4细胞中表达;而β3GnT-8则相反,在NB4细胞中不表达,而在其它5株细胞中有不同程度的表达。在10-7mol/L ATRA或10ng/ml TPA作用2h或3d后,不管HL60和NB4细胞株向何方向分化,其β3GnT-2, -4, -8的mRNA表达与不加分化诱导剂的对照组相比均见不同程度的升高。其中以β3GnT-4的改变最为显著,且有时间依赖性变化。β3GnT-8和β3GnT-2的上升幅度大致相近。ATRA使HL60和NB4细胞向粒细胞分化时,βGnTs的上调较TPA使两种细胞向单核细胞分化时明显;而HL60细胞的β3GnTs对两个分化诱导剂的上调作用较NB4细胞更为敏感。结论:早幼粒白血病细胞HL60和NB4在ATRA或TPA诱导向不同方向分化时,可见β3GnT-2, -4, -8的表达有不同程度的增加。ATRA的作用强于TPA;而HL60细胞对分化诱导剂的敏感性强于NB4细胞。二、β3GnT-2, -4, -8和人单核白血病细胞株SHI-1及THP-1分化的关系目的:研究糖基转移酶β3GnT-2, -4, -8表达变化和人单核白血病细胞株SHI-1及THP-1分化的关系。方法:在第一部分研究的基础上,改用人单核白血病细胞株SHI-1及THP-1为研究对象,用实时定量PCR(Real-time PCR)继续检测β3GnT-2, -4, -8的mRNA在ATRA、TPA处理前后两个细胞株中的表达变化,并结合细胞形态学观察鉴定。结果:SHI-1细胞在10-7mol/L ATRA作用2h后,β3GnT-2的mRNA表达即被下调,但3天后回复;而10ng/ml TPA作用2h或3d后,β3GnT-2均上调,第三天更甚。β3GnT-4在TPA处理SHI-1细胞3d后,才见上调,其升高程度与β3GnT-2相仿。β3GnT-8在ATRA或TPA处理3d后才见轻度上调。THP-1细胞对ATRA、TPA均不敏感,其β3GnT-2, -4, -8的表达未见明显改变。与细胞形态学变化一致。结论:人单核白血病细胞株对ATRA及TPA的反应均不如人急性早幼粒白血病细胞株敏感,其β3GnT-2, -4, -8的mRNA表达变化不明显。尤其是THP-1细胞对本文所用浓度的ATRA及TPA都没有反应,与细胞形态学不改变相一致。三、转录因子Ets-1对β3GnT-2, -4, -8表达的调控研究目的:探讨白血病细胞诱导分化过程中Ets-1的表达及其对糖基转移酶对β3GnT-2, -4, -8的转录调控。方法:采用Real-time PCR检测HL60、NB4、SHI和THP-1四种白血病细胞中转录因子Ets-1的mRNA在ATRA或TPA处理后的表达变化,并进一步采用染色质免疫沉淀(ChIP)结合凝胶迁移实验(EMSA)检测分析研究前三种细胞中Ets-1和β3GnT基因DNA的结合以及Ets-1对β3GnT的转录调控。结果:在ATRA或TPA诱导分化下,HL60细胞中Ets-1 mRNA的表达均有不同程度的升高,且ATRA的作用似乎强于TPA。相反,NB4细胞和SHI-1细胞在ATRA或TPA作用后,总体上,Ets-1的表达都是下降的。THP-1细胞Ets-1的表达基本不变。ChIP检测发现:各个β3GnT的基因DNA检出谱基本上和第一部分用RT-PCR法检出的β3GnT mRNA的表达谱一致,结合EMSA证实有活化的Ets-1结合至β3GnT-2或/和β3GnT-8基因DNA片段,但未能检测到Ets-1对β3GnT-4的表达调控。结论:Ets-1很可能参与HL60和SHI-1细胞株中β3GnT-2和β3GnT-8在ATRA和TPA诱导分化时的表达调控,至少也是调控因素之一。但NB4细胞中的β3GnT不受Ets-1的调节。更多还原

【Abstract】 A lot of literatures have been reported that the structures of sugar chains (glycans) in cell glycoconjugates were altered during cell malignant transformation or differentiation, and these alterations were caused by the changed expressions of some glycosyltransferases responsible for the synthesis of cell glycans.β1, 3 -acetyl-glucosaminyltransferase (β3GnT) is a family of glycosyltransferase which participates in the synthesis of aβ1, 3-GlcNAc linkage in its products. Among them,β3GnT-2, -4, -8 subtypes are known to syntheis the poly-N-acetyllactosamine structure in the N- or O-linked glycans of glycoproteins. The aim of this study is to investigate the relationship between the expressions of these 3 enzymes and the differentiation of 4 leukemic cell lines. In addition, the regulation ofβ3GnT-2, -4, -8 by transcription factor Ets-1 was also studied.This thesis was divided into three parts.1. The relation betweenβ3GnT-2, -4, -8 and the differentiation of human acute promelocytic leukemic cellsPurpose: To study the relation between the expression of glycosyltransferaseβ3GnT-2, -4, -8 and the differentiation of human acute promelocytic leukemic cell lines HL60 and NB4.Methods: RT-PCR was used to determine the expressions ofβ3GnT-2, -4, -8 in six different leukemic cell lines. By using ATRA to induce the differentiateion of HL60 and NB4 cell lines toward myelocytes or TPA to induce the differentiateion of these two cell lines toward monocytes, the expression changes ofβ3GnT-2, -4, -8 mRNAs before and after the induced differentiation were measured with Real-time PCR, and accompanied with morphological observation to check the differentiation.Results:β3GnT-2 was expressed in leukemic cell lines, SHI-1, THP-1, K562, HL60, U937 and NB4 with different magnitudes.β3GnT-4 was only expressed in NB4 cells. Conversely,β3GnT-8 was not expressed in NB4 only, but expressed on other five cell lines with various degrees. After treated with 10-7mol/L ATRA or 10ng/mlTPA for 2h or 3 days, all of the expressions ofβ3GnT-2, -4, -8 mRNAs were up-regulated with various degrees as compared with the control samples without the addition of inducing agents, especiallyβGnT-4 which was highly increased and show a time-dependent alteration. The elevations ofβ3GnT-8 andβ3GnT-2 were similar. When HL60 and NB4 were differentiated by ATRA toward myelocytes, the up-regulation ofβ3GnTs was more obvious than that using TPA to differentiate two cell lines toward monocytes. In addition, HL60 cells were more sensitive than NB4 cells in the up-regulation ofβ3GnTs by these two differentiation inducers.Conclusion: The expressions ofβ3GnT-2, -4, -8 were increased with various degrees in promelocytic leukemic cell lines during diffrentiation toward myelocytes or monocyte with ATRA or TPA. The effect of ATRA was greater than that of TPA; and HL60 cells were more sensitive to the differentiation agents than NB4 cells.2. The relation betweenβ3GnT-2, -4, -8 and the differentiation of human monocytic leukemic cells.Purpose: To study the relation between the expression ofβ3GnT-2, -4, -8 and the differentiation of human monocytic leukemic cell lines SHI-1 and THP-1.Methods: Based on the results of Part-1, two human monocytic leukemia cell lines, SHI-1 and THP-1, were selected. The mRNA expressions ofβ3GnT-2, -4, -8 were further studied in these two cell lines by real-time PCR before and after the treatment of ATRA and TPA, and accompanied with morphological observation.Results:β3GnT-2 mRNA was promptly down-regulated after SHI-1 cells were treated with 10-7mol/L ATRA for 2h, but returned to pre-treated level after 3 days. β3GnT-2 mRNA was also elevated after 10ng/ml TPA treatment for 2h and 3 days, especially after 3 days.β3GnT-4 was up regulated at the similar degree ofβ3GnT-2 only on 3 day after TPA treatment; andβ3GnT-8 was slightly increased after ATRA or TPA treatment for 3 days. On the contrast, THP-1 cells were not sensitive to the concentration of both ATRA and TPA that we used, as indicated by the un-changed expressions ofβ3GnT-2, -4, -8 mRNAs. This is in coincidence with the un-alteration in morphological observation.Conclusion: Human monocytic leukemic cell lines are less sensitive to ATRA or TPA than human pre-myelocytic cell lines, the changes of the expression ofβ3GnT-2, -4, -8 mRNAs were not apparent as in human pre-myelocytic cells. Between two cell lines, THP-1 cells showed no response to the concentration of both ATRA and TPA that we used,and this results is in accordance with the morphological observation.3. Study on the regulation of the expressions ofβ3GnT-2, -4, -8 by transcription factor Ets-1.Purpose: To investigate the expression of transcription factor Ets-1 during the induced differentiation of leukemic cells and the role of Ets-1 on the regulation of the transcription ofβ3GnT-2, -4, -8.Methods: The expression changes of Ets-1 mRNA were estimated using Real-time PCR after the treatment of ATRA or TPA on four leukemic cell lines, HL60, NB4, SHI-1 and THP-1. The binding of Ets-1 to the gene DNA ofβ3GnTs and the regulation ofβ3GnTs by Ets-1 in HL60, NB4 and SHI-1 cells was further determined by chromatin immuno-precipitation ( ChIP ) method combined with PCR amplification of the sequences of target gene and electrophoresis mobility shift assay (EMSA).Results: After the induced differentiation by ATRA or TPA, the expression of Ets-1 mRNA in HL60 cells was up-regulated with different degrees, ATRA was more effective than TPA. Oppositely, Est-1 expression was down-regulated in NB4 and SHI-1 cells after the treatment of ATRA or TPA in general. THP-1 cell line was resistant to both inducer, so its Ets-1 expression was unchanged after the treatment of these inducers. It was found that the detected pattern of the gene DNA ofβ3GnT subtypes using ChIP method combined with PCR amplification and EMSA was consistent to the expressive pattern ofβ3GnT mRNA detected with RT-PCR, and the binding of Ets-1 to the gene fragments ofβ3GnT-2 or/andβ3GnT-8 was certified.Conclusion: Ets-1 is probably participate in the up-regulation,at least is one of the regulatory factors ofβ3GnT-2 andβ3GnT-8, during the induced differentiation of HL60 and SHI-1 cells with ATRA or TPA. However, in NB4 cells, the expression changes ofβ3GnTs were not in accordance with the expression change of Ets-1.更多还原