【作者】 朱义保；

【导师】 曹新；

【作者基本信息】 南京医科大学 ， 细胞生物学， 2007， 硕士

【摘要】 细胞内乙酰化和去乙酰化是组蛋白翻译后修饰的主要方式，是调节基因表达的一个重要因素，越来越多的实验表明组蛋白乙酰化的失衡与肿瘤发生存在着密切的联系，平衡的打破成为某些肿瘤形成或发展的直接因素。本实验中我们从药物制滴菌素（trichostatin A，TSA）及基因水平阻断组蛋白去乙酰化酶，抑制组蛋白去乙酰化酶活性或下调其基因表达，观察处理因素所致的细胞效应，探讨乙酰化与去乙酰化修饰在MCF-7细胞中的影响。实验中我们发现TSA作用后，MCF-7细胞活性下降，随时间的延长及剂量的增加，细胞效应更加明显，呈现时间和剂量依赖性；Annexin-V／PI凋亡分析显示在不同梯度浓度的TSA作用48h后，MCF-7细胞出现凋亡，随着剂量的增加凋亡率逐步升高；周期实验分析显示0.50βmol几的TSA作用48h、72h后，与空白对照相比，MCF-7细胞呈现明显的G2期阻滞，但无凋亡出现；进一步的的琼脂糖电泳实验显示0.50μmol／L TSA作用无明显的180-200bp的梯度出现；其后的转录调节分析显示除P21基因上调外，ERα、myc-c、cyclin-D及Bcl-2均下调，同朝着增殖抑制、周期阻滞及凋亡的方向发展，表明TSA有效地引起了基因转录的变化，其所致的细胞效应可能与转录调节有关。组蛋白去乙酰化酶（histone deacetylase，HDAC）在药物水平能被阻断，RNA水平是否也可实施阻断呢?我们选基因编号为NM₀04964的HDAC1进行阻断。经RNA提取、反转录及PCR后，成功扩增目的片段，反向插入该片段入pcDNA3.1（-）真核表达载体，经酶切鉴定及测序分析，表明成功构建pcDNA3.1-HDAC1反义质粒；大量扩增、纯化目的质粒DNA，进行瞬时转染。瞬时转染72h后的HDAC1基因分析显示空白及空质粒组目的基因无明显变化，反义组条带强度明显比空白及空质粒组弱，凝胶灰度分析显示反义组相对于空质粒组下降了57％；此外，还发现阳性对照TSA组也下调了HDAC1，强度比反义组更明显。转染后的细胞活性实验也显示反义基因具有细胞增殖抑制作用，反义HDAC1降低了MCF-7细胞的活性，空质粒组与空白组与之相比无明显变化；进一步的细胞流式实验表明反义基因能使细胞周期阻滞，使细胞S期减少，阻滞于G1、G2期。上述实验表明HDAC可在药物及基因水平被阻断，MCF-7细胞的肿瘤形成申可能存在乙酰化与去乙酰化修饰的紊乱，HDAC1是有效的去乙酰化酶，在乙酰化修饰申可能起重要作用；阻断HDAC酶的活性可引起肿瘤细胞增殖抑制、周期阻滞或凋亡，HDAC酶可能用来作为肿瘤治疗的新靶点。更多还原

【Abstract】 It was well known that histone acetylation and deacetylation were the main post-translation modification in cells, they played significant role in gene transcription regulation. Much evidence recently showed that the outbreaks of the balance correlated much more with the formation of tumors, and would be a direct factor contributing to tumorgenesis. In our experiments, we chose histone deacetylation inhibitor trichostatin A and anti-sense HDAC1 as the treatments, inhibited the histone deacetylase activity or down regulated the histone deacetylase gene expression, observed the subsequent cytotoxicity upon the acetylation modification, and further investigated the possible molecular basis.In our observation, we found the proliferation activity of MCF-7 cells decreased after treatment of TSA, as time and dose increased, the inhibition was more manifest, showing a time and dose dependence. Annexin-V/PI double stain analysis showed apoptosis were identified treated with TSA, apoptosis rate increased as the dose rose for 48h. Cell cycle analysis revealed compared to the blank counterpart, MCF-7 cells were arrested in G2 phase at 48h and 72h after treatment of 0.50μmol/L TSA, but no apoptosis identified; semi-quantitate analysis further revealed there was no appearance of 180-200bp DNA ladder, confirming TSA caused a non-late apoptosis; Gel analysis showed ERα, myc-c, cyclin-D and Bcl-2 were all down regulated except P21 gene, all made it to proliferation inhibition, cell arrest and apoptosis, indicating TSA effectively regulated gene transcription, the cytotoxicity may correlate with transcription regulation.The HDAC enzymes could be inhibited by TSA, but would it be done in RNA level? We selected HDAC1 （ID for NM₀04964） as our target gene. After RNA drawn and reverse transcription PCR, gel analysis showed target gene was successfully amplified, exhibiting a clear strap in the electrophoresis, and was the aimed length in contrast to Marks, we took it to construct our plasmid. The plasmid identification displayed the target gene was successfully constructed, electrophoresis showed one was targeted 731bp strap, the other was the pcDNA3.1 vector. Next we amplified it massively in bacteria, and purified them for the subsequent cell transfection. Later transient transfection demonstrated HDAC1 mRNA lever was decreased to 43% in the anti-sense group compaired to negative counterpart for 72h, gel electrophoresis revealed the intensity of HDAC1 gene was apparently weak, suggetting HDAC1 was effectively down regulated. Additionally, we found HDAC1 was also depressed in the positive control of TSA group. Subsequent proliferation activity assay showed anti-sense HDAC1 could decrease activity of MCF-7 cells, inhibit their proliferation, while the blank and negative groups exhibited no markable difference. Further cycle arrest analysis also displayed anti-sense HDAC1 could arrest cells in G1 and G2 phase, indicating HDAC1 gene was an effective histone deacetylase, may play significant role in acetylation modification during the tumorigenesis of MCF-7 cells.Summarize the above experiments, we concluded HDAC were associated with tumorgenesis, they could be inhibited in drugs or RNA levers, resulted gene transcription regulation and subsequent cytocoxicity, suggesting a potential target for the tumor therapy in future.更多还原