合成一個新型NF-κB相關啟動子藉此增加在腫瘤細胞中的基因表現

Abstract

利用腫瘤專一性起動子可以專一性的在腫瘤細胞中表現轉殖基因。但是因其活性太弱，所以治療效果有限。在此我們建構一個能快速得到具有高表現量或專一性的啟動子庫平台。近年來研究發現，NF-κB與腫瘤細胞的轉移、生長、抗細胞凋亡的機制有關。因此，本實驗以NF-κB為主軸並選用與NF-κB相關能直接或間接加強基因表現的轉錄因子，Sp1、CREB和MEF-2將之隨機黏合配對，做出一個NF-κB相關啟動子庫。再者，低效率的啟動子會妨礙高效率啟動子的篩選，所以我們嘗試利用已知的磁珠分離技術搭配競爭劑以降低表現量的啟動子。實驗證明在Balb/3T3利用這種改良的磁珠分離技術，的確可降低低效率啟動子1.59% 左右，而中效率啟動子的佔有率提高了7.99%。所以此種改良的磁珠分離技術的確可降低質體中，低效率啟動子的比率。 此技術可以在Balb/3T3，A549細胞株上挑選到高表現量，或是具腫瘤專一性的啟動子。雖然目前還有很多序列未定出 (長度大於1 kb)，但是可以看出在腫瘤細胞中具高表現量的兩個啟動子其中有一個有兩個NF-κB結合部位。另一個則無。這說明了腫瘤雖然有高活性的NF-κB，但是其它的轉錄因子或許也是很重要的。利用此種策略不但可以了解腫瘤細胞的最佳啟動子組成，也可以觀測出哪些轉錄因子在腫瘤細胞是重要的。藉此可以更深入的了解腫瘤細胞，進而找到治療癌症更好的方法。

Tumor-specific promoters are useful tools to accomplish specific expression in targeted tumor cells. However, a low level of gene expression is the chief defect of tumor-specific promoters. We have set up a platform to quickly gain high active and specific promoter library. Recently, many researches have discovered that NF-κB is associated with cancer, including metastasis, tumor proliferation and anti-apoptosis. Therefore, we created a novel NF-κB based promoter library by randomly assembling Sp1, CRE, and MEF-2 binding sites. Furthermore, low expressive promoters may interfere with the selection for the promoter with high expression activity. I used the techniques magnetic beads separation combing with the competitor (MBSCS) to solve this problem. In this experiment, promoter library transfected into Balb/3T3 from this modified separation system could decrease the expression of low expressive promoters by 1.59 % overall. Meanwhile, it increased the expression of medium expressive promoters by 7.99% overall. Therefore, the method can reduce the expression ratio of the low expressive promoters in plasmid. Using this technology, high expressive or tumor-specific promoters were selected from A549 and Balb/3T3 cells. Although their sequences are not fully (over 1kb), there were sequenced still observed some results. There are two high and tumor-specific promoters from separation. One has two binding sites; the other has none. Let us which means other transcription factors may play an important role in cancers except for NF-κB. This strategy not only let us understand the context of high efficient promoter in tumor cells, but also let us knows which factors in the tumor cells are important. This may take advantage of it to more thorough understanding about tumor cells, and find a better treatment for cancer.