【作者】 郭向红；

【导师】 吴士良；

【作者基本信息】 苏州大学 ， 病理与病理生理学， 2011， 博士

【摘要】 第一、二部分pp-GalNAc- Ts在人恶性造血细胞株中mRNA的表达差异及与白血病细胞诱导分化的相关性研究目的:从mRNA水平检测K562细胞株、SHI-1细胞株中多肽:N乙酰氨基半乳糖转移酶家族（pp-GalNAc-Ts）的表达情况。选择其中高表达的pp-GalNAc-T亚型作为研究对象,RT-PCR测定它们在不同浓度的1,25（OH）₂ D3处理后K562和SHI-1细胞中的表达变化,同时进行细胞形态学观察以及细胞周期和凋亡的检测来证实1,25（OH）₂ D3诱导的K562和SHI-1的分化。方法:1.采用RT-PCR方法检测:（1）多肽:N乙酰氨基半乳糖转移酶家族在K562细胞株、SHI-1细胞株中的mRNA表达;（2）分化诱导剂处理后K562细胞株和SHI-1细胞ppGalNAcTs的mRNA表达差异。2.用瑞氏染色细胞后,光学显微镜下观察细胞形态变化。3.流式细胞仪检测加分化诱导剂处理后细胞周期和凋亡情况。结果:1.在K562细胞,只有pp-GalNAc-T2, -T4, -T5和-T7有明显表达,且表达量高低顺序为2> 4 > 7 > 5,而其他的pp-GalNAc-T表达很低或者几乎不表达。在SHI-1细胞,pp-GalNAcT-1, -T2, -T3和-T4表达明显,其中,pp-GalNAc-T4表达最高,其他三个表达几乎相等。2.1,25（OH）₂D3可诱导K562和SHI-1细胞向单核细胞系分化,表现为核染色质浓缩聚集和胞浆空泡增加。且具有剂量依赖性。并以SHI-1变化更为显著。3.在经过不同浓度的1,25（OH）₂D3处理之后,未出现细胞凋亡;也未出现亚G1期细胞,即凋亡峰。这些结果说明,即使在高浓度1,25（O）₂D3的水平(10^-6 mol/L),也不影响K562和SHI-1细胞的凋亡。4.10^-8, 10^-7, 10^-6 mol/L 1,25（OH）₂D3处理K562细胞72 h后,pp-GalNAc-T2, -T4, -T5, -T7表达量均增加,并表现出明显的剂量依赖性模式(pp-GalNAc-T2在低浓度10^-8 mol/L 1,25（OH）₂D3诱导下没有明显增加)。其中,pp-GalNAc-T4的表达明显上调（所有浓度处理组与对照组相比,p<0.01）。pp-GalNAc-T5的表达上调也较明显（中、高浓度处理组与对照组相比,p<0.01）。5.在不同浓度1,25（OH）₂D3处理之后,SHI-1细胞中各型pp-GalNAc-Ts表达量的变化情况与K562细胞存在明显差异。pp-GalNAc-T1,在低浓度,10^-8 mol/L 1,25（OH）₂D3诱导下没有表达变化;在中浓度,10^-7 mol/L 1,25（OH）₂D3诱导下其表达量上调一倍（与低浓度处理组相比p<0.01）;在高浓度,10^-6 mol/L 1,25（OH）₂D3诱导下其表达量上调接近十倍（与中浓度处理组相比p<0.001）。pp-GalNAc-T2,在低浓度,10^-8 mol/L 1,25（OH）₂D3诱导下其表达量上调至对照组表达量的178%（p<0.05）;在中浓度,10^-7 mol/L 1,25（OH）₂D3诱导下其表达量持续上调至对照组表达量的230%（p<0.05）;但在高浓度,10^-6 mol/L 1,25（OH）₂D3诱导下其表达量转而下降。而pp-GalNAc-T3的表达量在经不同浓度1,25（OH）₂D3处理后均无明显改变。pp-GalNAc-T4表达量变化刚好跟TI和T2的结果相反,以一种剂量依赖性方式下调（中、高浓度处理组与对照组相比p<0.01）。结论:本实验中ppGalNAc-Ts在不同白血病细胞株中表达情况不一致,推测不同的ppGalNAc-Ts其多肽底物特异性不同。pp-GalNAc-T2对白血病细胞的分化具有专一的正相关。这是O-糖链合成的起始酶影响白血病细胞株生长、分化的首次报道,以上这些结果都提示了,细胞中糖基转移酶和表面糖链结构的变化可以改变细胞的命运,同时也提出了糖链还具有以往所不知道的新功能。第三、四部分pp-GalNAc-T4正义表达载体的构建及上调pp-GalNAc-T4对白血病细胞分化的影响及机制探讨目的:研究pp-GalNAc-T4在全反式维甲酸诱导的白血病细胞分化中的作用,以及机制的探讨。方法:1,我们构建了pp-GalNAc-T4的正义表达载体,上调白血病细胞NB4中内源性pp-GalNAc-T4的表达.,2,用激光共聚焦显微镜观察细胞形态的变化,3,通过NBT还原实验观察白血病细胞是否向成熟方向分化。4,通过实时PCR,检测了用全反式维甲酸处理NB4-T4稳转细胞株后,其细胞内的癌蛋白PML-RARα基因表达情况。结果:1.首次构建了针对pp-GalNAc-T4的载体; 2.首次研究发现了在pp-GalNAc-T4cDNA转染NB4细胞株后,pp-GalNAc-T4基因的表达增加,观察白血病细胞的形态变化,发现上调pp-GalNAc-T4基因表达后,NB4 -T4细胞组加入ATRA后,细胞核和细胞质没有明显的变化,这说明上调表达pp-GalNAc-T4,并不能促进ATRA诱导NB4细胞的分化,反而抑制了ATRA诱导的NB4细胞分化;3.上调pp-GalNAc-T4基因表达后,用不同浓度的ATRA作用各细胞组72小时后,不能增加NB4-T4细胞的还原能力（P>0.05）,但是可以增加NB4和NB4-0细胞的还原能力（P<0.01）。这说明上调表达pp-GalNAc-T4,抑制了ATRA诱导的NB4细胞的NBT还原能力。pp-GalNAc-T4抑制全反式维甲酸诱导的NB4细胞的分化,而这种作用可能和癌蛋白PML-RARα有关;4.我们对标准型CD44s和变异型v6的RT-PCR结果显示ppGalNAcT4的高表达并不影响CD44s和CD44v6mRNA水平的表达,但这并不能完全排除ppGalNAcT4的这种作用与CD44相关的可能性。可能的情况是:ppGalNAcT4直接修饰CD44,改变了其分子表面的糖链结构从而影响了CD44与透明质酸的结合;从而使得CD44不能与抗体结合,就不能降解PML-RARα蛋白（M3）,从而抑制了NB4细胞的分化。结论:上调表达pp-GalNAc-T4,抑制全反式维甲酸诱导的NB4细胞的分化,而这种作用可能和癌蛋白PML-RARα有关。pp-GalNAc-T4抑制全反式维甲酸诱导的NB4细胞的分化,而这种作用可能和癌蛋白PML-RARα有关。可能的作用机制是:ppGalNAcT4直接修饰CD44,改变了其分子表面的糖链结构从而影响了CD44 ,使得CD44不能与抗体结合,就不能降解PML-RARα蛋白（M3）,从而抑制了NB4细胞的分化。更多还原

【Abstract】 Part1 and Part2 Study on Expressive Spectrum of ppGalNAcTs in Different Leukemia Cells and the Effects of differentiationObjective: To observe the expression of polypeptide : N-acetylgalactosaminyl transferase Ts(ppGalNAcTs) in different leukemia cells including K562 cell line and SHI-1 cell line and to study the variation of acetylglucosaminy transferase expression after 1,25(OH)2 D3 added to the culture medium, to find out the relative function of acetylglucosaminy transferase in the course of leukemias generating and developing, as well as to make out the influence of 1,25(OH)2 D3 on ppGalNAcTs.Methods: 1. Use RT-PCR to detect (1) the mRNA expression of ppGalNAcTs in K562 cell line and SHI-1 cell line;(2)the expression discrepancy of ppGalNAcTs in K562 cell line and SHI-1 cell line after 1,25(OH)2 D3 added to the culture medium. 3.To study the cell circle of leukemia cells induced by 1,25(OH)2 D3 with the flow cytometer. 4. To study the change of cell shape of leukemia cells induced by1,25(OH)2 D3 with the Laser Confocal Scanning Microscope(LCSM). Results:1. The expressions of glycosyltransferase were different in two leukemia cell lines.2. Afte 1,25(OH)2 D3 added to the culture medium, the mRNA expression of ppGalNAcT2、T5 in K562 cell line greatly increased. The cell cycle was arrested in G2/M phase ; No cell death was detected. The expression of glycosyltransferase in SHI-1 cell line has no obvious change than in K562 cell line.Conclusion: This study shows that the expression of glycosyltransferase in different leukemia cells is different. This result suggests that different glycosyltransferases have different particular substances. Leukemia cell differentiation probably has contact with pp-GalNAcT2. T5.Part3 and Part4 Construction of Plus Sense Expression plasmid of pp-GalNAc-T 4 gene and Primary Study on its FunctionObjective: This thesis aims to approach the function of pp-GalNAc-T 4 in the differentiation of leukemia induced by ATRA.Methods:1, Subclone pp-GalNAc-T4 fragment to eukaryotic plasmid and form plus sense plasmids. Transfect the recombinant plasmids separately to NB4. To study the change of cell shape of leukemia cells induced by ATRA with the Laser Confocal Scanning Microscope(LCSM)..Studied the variational characteristics of NBT and PML-RARαexpression on mRNA level with Real Time -PCR.Results:1. Subclone pp-GalNAc-T4 fragment to eukaryotic plasmid and form plus sense plasmids2.Up-regulated expressions of pp-GalNAc-T4,inhibited differentiation of leukemia NB4 induced by ATRA.2. Up-regulated expressions of pp-GalNAc-T4,inhibited differentiation of leukemia NB4 induced by ATRA .PML-RARαmay contribute to this functions. CD44 may contribute to this functions that cannot the degradation of PML-RARαConclusion: Up-regulated expressions of pp-GalNAc-T4,inhibited differentiation of leukemia NB4 induced by ATRA .PML-RARαmay contribute to this functions. CD44 may contribute to this functions that cannot the degradation of PML-RARα.更多还原