人Egr-1启动子调控的自杀基因真核表达载体的构建及其在鼻咽癌细胞株中的放射诱导表达

[目的]构建由人Egr-1放射诱导性启动子调控的自杀基因靶向性真核表达载体，并转染人鼻咽癌细胞株CNE，比较射线诱导前后自杀基因在鼻咽癌细胞中的表达情况。[方法1根据Genebank数据库提供的人Egr—1启动子基因序列，设计-对引物克隆人Egr-1启动子，采用PCR、酶切、连接等分子生物学技术，构建pcDNA3．1（＋）-Egr-1-CD重组质粒表达载体，并利用脂质体转染方法转染人CNE细胞株，经G418筛选后获得阳性细胞克隆。RT—PCR分析鉴定0、5、10、15、20Gy放射剂量进行6MVX线照射对CD基因表达的影响。f结果1经PCR、酶切、测序等证实了pcDNA3．1（＋）-Egr．1．CD重组靶向性质粒表达载体构建成功，G418筛选所得到的CNE细胞阳性克隆经不同放射剂量的诱导后，RT—PCR结果显示体外放射线照射可显著提高CNE细胞中CDmRNA的表达水平，在10Gy及15Gy组CDmRNA的表达水平明显增高，尤以15Gy组CDmRNA的表达水平最高，在20Gy组CDmRNA的表达水平反而下降。[结论]pcDNA3．1（＋）-Egr-1-CD重组质粒表达载体构建成功，并成功转染人鼻咽癌细胞株．建立了基因表达与放射的剂量效应关系．为后续的临床应用研究奠定基础．

Creation of Suicide Gene Eukaryotic Expression Plasmid which Controlled by Human Egr-1 Promoter and Expression in Human Nasopharyngeal Carcinoma Cell Line Induced by Radiation

[Purposel To create eukaryotic expression plasmid with cytosine deaminase gene which controlled by human Egr-1 promoter and transfecting into human nasopharyngeal carcinoma cell line CNE, comparing the expression of CD gene which induced by radio or not. [Methods] A pair of human Egr-1 promoter primers was designed according to the sequence described by Genebank. PcDNA3.1（＋）-Egr-1- CD recombinant expression plasmid was constructed by PCR, enzyme restriction, ligation and other molecular technology, and transfected into human CNE cell line by lipofectamine. After the positive cell clones was selected by G418, the expression of CD gene in positive cell which induced by 0, 5, 10, 15, 20Gy 6MV X-ray was analyzed by RT-PCR. [Results] The recombinant target expression plasmid pcD- NA3.1（＋）-Egr-1-CD was constructed, and identified by sequencing. The expression of CD gene raised in different irradition dose, and the level of CD mRNA notably raised in 10 and 15Gy group, but the level was low in 20Gy group. [ Conclusions ] PcDNA3.1 （＋）-Egr-1-CD recombinant expression plasmid has been successfully constructed and transferred into human nasopharyngeal carcinoma cell line ,and established tumor gene-radiotherapy system for laying a foundation of later experiment and application.