【作者】 陈晓锋；

【导师】 李薇；

【作者基本信息】 吉林大学 ， 内科学， 2006， 硕士

【摘要】 组蛋白的异常乙酰化或去乙酰化在多种恶性肿瘤和白血病的发生发展中起决定作用。组蛋白去乙酰化酶抑制剂(HDACI)能通过抑制去乙酰化酶发挥抗肿瘤作用。HDACI的抗肿瘤作用主要有诱导细胞分化、细胞周期阻滞和细胞凋亡、免疫调节等。目前对其免疫调节方面的影响报道较少。本文采用NB4、HL-60、U937 3种细胞作为研究对象,观察组蛋白去乙酰化酶抑制剂丁酸钠(SB)对共刺激分子CD86表达的影响,半定量RT-PCR检测SB处理细胞后CD86 mRNA的变化,AUT电泳检测SB对核心组蛋白乙酰化程度的改变,pCREB试剂盒检测SB对活化CREB含量的影响。结果表明: 1、SB能上调急性白血病细胞CD86表达。2、SB使上述各细胞CD86 mRNA转录增多,CD86的上调发生在转录水平。3、SB能使白血病细胞核心组蛋白乙酰化程度明显增强,尤以H4最为明显。4、CREB参与了SB上调急性白血病细胞CD86的表达。本研究证实了HDACI的免疫调节作用,验证了HDACI通过抑制去乙酰化酶,促进组蛋白乙酰化程度,促进转录因子活化并与之结合,从而促进特定基因转录这一作用模式,反证了肿瘤细胞免疫分子表达下调与异常的去乙酰化有关。组蛋白乙酰化修饰是一种重要的基因表型修饰形式,对HDACI的抗肿瘤作用研究不仅有助于诠释肿瘤的发病机制、开发新型抗肿瘤药物,还有助于破译组蛋白密码、认识基因表型修饰。更多还原

【Abstract】 Gene expression is not decided by the DNA sequence, but alsoregulated by epigenetics. Epigenetics modification refers to thechanges of epigenetics. Abnormal epigenetics state plays a criticalrole on the pathogenesis of some human disease, especially somemalignant tumors. A couple of adverse proteases——histoneacetylases (HAT) and histone deacetylases (HDAC) are in charge ofthe core histone acetylation state and act with their associated proteinscalled Coactior(CoA) and Corepressor(CoR) respectively. Studieshave shown that abnormal acetylation and deacetylation may beresponsible for many malignant tumors and leukemia. Histonedeacetylase inhibitors (HDACs) can inhibit the activity of histonedeacetylase (HDAC) and promote the gene expression which hadbeen inhibited by excessive deacetylation. This promoting effect isthe dominant anti-tumor mechanism of HDACI. A lot of studies haveverified that HDACI has several anti-tumor effects such as inducingtumor cell differentiation, blocking cell cycle, apoptosis andimmunomodulation. Among them, potentializing the immunogenicityof tumor cells is a new way with great prospect because it maycompletely eliminate tumor cells as biologic treatment agent.We selected 3 cell strains as the study objects which were NB4,HL-60, and U937. The cells were treated with sodium butyrate (SB)at 0.5, 1.0mM concentrations and the expression change of CD86 wasdetected by FACS at 48h.The results showed that the CD86 wassignificantly increased at both concentrations in 3 cells. Among them,the NB4 cells treated with 0.5mM SB was found with the most raisefold, which reached to 36.8 times compared with the control group.Statistical significance was also observed in the other groups whichwere all more than 10 times compared with their control groups.The semi-quantitative RT-PCR was used to examine the genetranscription of CD86 treated by SB at 48h. The results showed thatthe CD86 mRNA were accumulated in all the cells, the CD86/β-actinof SB treated groups in NB4,HL-60,U937 cells is 5.5,5.0 and 3.4folds compared with the control groups respectly. The histones wereextracted at 6h co-cultured with 0.5mM SB, and the AUT gelelectrophoresis was applied to show whether SB would change theacetylation state of core histone. The results indicated that theacetylation degree was accumulated in SB treated cells. There was nomore than one site that was acetylated in the each of the controlgroups while there were definitely 4 sited that were acetylated in theSB treated groups. In the gel graph, they were manifested as fourstrands closely behind H4.A pCREB detection kit was used to detectthe changes of activated CREB after treated by SB for 48h, the resultsshowed that the contents of pCREB in both NB4 and U937 cells wereenhanced after treated with SB. The 2h, 24h, and 48h groups hadmuch greater pCREB contents than control groups in both cell linewith statistical significance, P<0.01. In NB4 cells, 2h group had morepCREB content than 24h and 48h groups, P<0.05. No statisticaldifference between 24h and 48h group. In U937 cells, 2h group hadmore pCREB content than 48h group, P<0.05. So do 24h group and48h group. No statistical difference between 2h and 24h group.These results verified that: 1, SB can up-regulate the CD86express. CD86 is a co-activator and involved in the activation of CTL.The CD86 is often deficient in leukemia which is one of the mainreasons responsible for the immune escape. Studies have shown thatthe up-regulated CD86 molecules can stimulate lymphocytes, hence,HDACI may induce anti-tumor CTL effect in vivo and may be used inimmune treatment to tumors. 2, SB can promote the transcription ofCD86, indicating that the up-regulation of CD86 happened at thetranscriptional level, not at post-transcriptional level. 3, SB can raisethe acetylation degree of core histones. That may relax the chromatinwhich will make it accessible for TFs and promote the transcription ofCD86. 4, CREB is involved in the up-regulation of CD86 induced bySB. CREB is an important TF involved in many genes transcriptionincluding CD86. Here we show that SB can accumulate the activatedCREB, indicating that it may play important role in the process.This study showed that SB can up-regulate the CD86 expressionin acute leukemia cells, NB4, HL-60, U937;explored the mechanismson the transcriptional level;verified the act model that HDACI caninhibit HDAC, relax the chromatin which is benefit for TFs accessingand promoting transcription;argued that abnormal deacetylation takespart in down-regulation of immune molecules in tumor cells;revealedthat CREB is involved in the CD86 up-regulation induced by SB.The study on immunomodulatory effect and mechanism ofHDACI will contribute to revealing the interrelationship among itsmulti anti-tumor effects. Histone acetylation is an importantepigenetics modification. Study on the anti-tumor effect of HDACIwill contribute not only to explaining the pathogenesis of tumor,developing new drug, but also analyzing the histone code,understanding epigenetics modification.更多还原