【作者】 刘菁；

【导师】 王小宁； 刘新垣；

【作者基本信息】 第一军医大学 ， 分子免疫学， 2002， 博士

【摘要】 肿瘤自杀基因治疗是一种较为有效和具有临床应用潜力的治疗策略。目前肿瘤基因治疗面临的最大问题是基因的靶向性问题，自杀基因治疗也不例外。利用肿瘤特异性表达蛋白的肿瘤专一性基因表达调控序列驱动自杀基因，可望实现自杀基因在肿瘤细胞中的专一性表达，从而提高基因治疗的安全性和有效性。 近年研究表明，端粒酶可能是目前所见到的特异性较好的一种广泛的肿瘤新标志物。在正常体细胞中其活性受严格调控，在生理条件下，正常细胞大都没有端粒酶活性表达；而在90％以上的肿瘤细胞中有高水平表达，并且其活性高低与肿瘤恶化程度有关。人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)是端粒酶复合物的核心成分，是细胞调控端粒酶活性的关键环节。癌变细胞中端粒酶重新激活的一个重要因素是由于hTERT基因在转录水平上被组成型激活造成的，研究表明这是由于癌变细胞中形成了一套不同于正常细胞的基因组表达调控模式，癌细胞表达出肿瘤特异性的转录因子，从而导致hTERT启动子活性在肿瘤细胞和正常细胞中的显著差异。已有的研究发现在转录起始位点上游约181bp片段包含有核心启动子序列，该序列对hTERT基因的表达和端粒酶活性起关键作用，它足以启动下游外源基因的表达同时仍保留其肿瘤专一活性。本研究克隆了包含该核心启动子序列的hTERT调控序列，对其肿瘤细胞专一活性进行了检测，并对其顺式调控元件Ebox和转录因子c-Myc进行了初步探讨；然后用该启动子驱动大肠杆菌 胞啼锭脱氨基酶（E colt cytosine deaminase，CD）基因，通过对人结直肠 癌细胞的体内外杀伤对其靶向治疗肿瘤的可行性进行了探讨。 研究结果表明：克隆的hTERT启动子（－378＋78）活性与端粒酶活 性密切相关，具有显著的肿瘤相关性，其在7种不同来源的肿瘤细胞中 活性升高，而在正常细胞中活性很低，这与国外文献报道的结果一致。 此外，位于l 位点的E bOX缺失（么MyCI）使hTERT启动子在细胞 中的转录效率大大降低，其中 Hela细胞下降了 70％，A549和 LoVo细胞 中下降了60％，提示此E b。x对启动子活性是必不可少的：有趣的是， 位于＋44位点的 E bOX缺失（凸MyCZ）对启动子在 A549和 LOVo细胞中 的转录活性几乎没有影响，然而在Hela细胞中却降低了3倍，表明此E b。X的作用是细胞类型依赖性的。这在类似研究中为首次报道。凝胶滞 后实验显示在 LOVo细胞中 C－MyC与 E bOX 乙 165）结合能力较强，而 与 E box（＋44）结合能力较弱，提示 E box作用与其与 c－Myc的结合能 力有关。Western blot结果发现，hTERT启动子具有高转录活性的肿瘤细 胞Bcapl7、Hela显示高表达的c－Myc，而转录活性相对较低的A549细 胞其 C－MyC表达也较低，正常细胞 WI3 8则只能检测到极微量的 C－MyC 的表达，表明C－MyC的表达与hTERT启动子的转录水平具有相关性。这 些结果提示C－MyC作用于 hTERT启动子的）I顾式调控元件 E bOX参与启动 子活性的调控。 基于上述结果设计构建了hTERT启动于控制下的CD基因表达质粒 phTERT（D，结果显示，hTERT启动子控制下CD基因的表达使肿瘤细 胞LoVo对原药SFC的敏感性提高了800倍以上，同时具有明显的旁杀 伤效应；而正常细胞WISH仅提高了6倍，且对低于血药安全浓度 O70uM）的SFC不敏感。动物实验显示体内具有明显的抑制肿瘤效应。 这些结果表明hTERT－CD／SFC系统在体内外均具有显著的抑瘤效果，且 具有高度的肿瘤特异性。这在国内外为首次报道，为肿瘤靶向基因治疗 提供了一种很有希望的途径。更多还原

【Abstract】 Suicide gene therapy has been recognized as an effective strategy for cancer treatment with potential application in clinic. Nowadays the major problems encountered tumor gene therapy is the specific expression of the therapeutic genes in cancer cells,including suicide gene therapy. Utilizing the expression regulation sequences of a gene encoding a tumor-specific protein to drive the suicide gene may be promising to restrict the expression of the therapeutic gene in tumor cells,thus to improve the safety and effect of gene therapy.Recently,telomerase has been recognized as a new wide-range tumor marker. Telomerase is silent in normal cells but active in more than 90% of cancers and correlates well with the degree of malignancy. The core component of telomerase-human telomerase reverse transcriptase (hTERT) is a key determinant of telomerase activity. Further research has demonstrated that one important reason for telomerase reactivation is that hTERT is constitutively activated at the transcriptional level. It is reported that in cancer cells,there has been forming a set of genomic expression model different from normal cells,leading to activation and expression of some specific transcription factors in cancer cells,thus resulting in the significant discrepancy in hTERT promoter activity between tumor and normal cells. Recent studies also found that the 5’-flanking sequences containing the region of 181bp upstream the transcription start site functions as a core promoter,which is sufficient to drive the expression of downstream exoticgene and retain its tumor specificity. In the present study,the hTERT regulation sequence containing the core promoter region is cloned and its tumor specificity is studied,and the cw-element of E box and the c-Myc transcription factor are also preliminary studied;and then the hTERT promoter was used to induce the expression of cytosine deaminase (CD) suicide gene,and the feasibility was tested in cancer gene therapy.Our results showed that:the hTERT promoter (-378-+78) we cloned correlated with the telomerase activity,both of which showed high tumor specificity. They were upregulated in seven tumor cells tested,but repressed in normal cells,which was consistent with the results reported by other independent studies. Furthermore,the transcription activity of the hTERT promoter containing the deletion of the E box at -165 was greatly decreased,with 70%,60% and 60% decrease in HeLa,A549 and LoVo cells respectively,indicating that this E box (-165) is indispensible for the promoter activity. Interestingly,the activity of the promoter containing the deletion of the E box at +44 site did not show any changes in A549 and LoVo cells,but was 3-fold decreased in Hela cells,indicating that the role of this E box (+44) depends on cell type. These results were first reported in similar studies. EMS A showed that the activity of c-Myc binding to E box (-165) was stronger than that of binding to E box (+44),suggesting that the role of the E box is correlated to the binding activity of c-Myc. By Western blot,higher expression of c-Myc is observed in Bcap-37 Hela cells with higher hTERT promoter activity and lower expression of c-Myc in A549 with lower hTERT promoter,while weak expression of c-Myc is found in normal cells WI38,indicating the high correlation of c-Myc expression and hTERT transcription activity. These results suggested that the effect of c-Myc through its target element E box may involved in the regulation of hTERT promoter.Based on the above results,an expression plasmid,phTERT-CD,was constructed,in which the E. coli. cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and anormal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug,5-fluorocytosine (5FC),over 800 folds,and a strong "bystander effect" was also observed;while the sensitivity of WISH cells to 5FC was increased only 6 folds with no se更多还原